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High Grade CANNABIS BIBLIOGRAPHY SORTED AND ALPHABETIZED BY SUBJECT

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Pest and Disease

Not cannabis specific
A Low transmissibility of hop latent viroid through a generative phase of Humulus lupulus L.
J.Matousek, J. Patzak
Biologica Plantarium 43 (1): 145-148 (2000)
https://bp.ueb.cas.cz/pdfs/bpl/2000/01/33.pdf
A significant reduction of hop latent viroid (HLVd) content during the generative phase of hop was detected using reverse transcriptase-polymerase chain reaction (RT PRC) and molecular hybridisation methods. A low transmissibility of HLVd through seed may be a feature valuable for the selection and maintenance of viroid-free hybrid hops.


A review of Cannabis sativa-based insecticides, Miticides, and repellents
John McPartland, Zahra Sheikh
Journal of Entomology and Zoology Studies 2018; 6(6): 1288-1299
https://www.researchgate.net/publica...and_repellents
Plant-based pesticides are gaining attention as safe, effective, eco-friendly alternatives to synthetic pesticides. We conducted a literature search regarding the use of hemp (Cannabis sativa) as a plant-based insecticide, miticide, or repellent. The search yielded 88 publications, which we grouped into five types of applications: companion planting (17 articles), the use of harvested plant material without any extraction (25 publications), aqueous extracts (20 publications), essential oil extracts (EOs, nine publications), and solvent extracts (17 publications). Few studies chemically analyzed the contents of their extracts, and most studies lacked control comparisons. EO studies were the most rigorous, and yielded the best results. Results with solvent extracts showed moderate efficacy, but little better than aqueous extracts, which lacked tetrahydrocannabinol (THC). Collectively, the studies suggest that EOs (terpenoids) are the primary Cannabis constituents responsible for arthropod deterrence. THC exerts nominal deterrence, but is toxic to insects. Mechanisms of action are discussed

A Study of Different Disease Detection and Classification Techniques using Deep Learning for Cannabis Plant
Kanaad Pathak, Arti Arya, Prakash Hatti, Vidyadhar Handragal and Kristopher Lee
International Journal of Computing and Digital Systems Int. J. Com. Dig. Sys. 10, No.1 (Jan-2021)
DOI: 10.12785/ijcds/100106
https://journal.uob.edu.bh/bitstream...=4&isAllowed=y
In this paper, different models for disease detection and classification are studied for cannabis plants. Cannabis plants are used for medical and recreational purposes with its recent legalization in some places. Cannabis farmers face problems in cultivation of the crop since it’s susceptible to multiple disorders. With early detection of the disease in the crop it is possible to prevent large waste of yield in the crop. A real dataset is considered for disease detection and classification purposes which is a combination of text and image data and that has been collected over a period of one and a half years (Feb 2018-August 2019). The models used in this study are Fast Region Convolutional Neural Network(F-RCNN), MobileNet Single Shot Multibox Detector(MobileNet-SSD), You Only Look Once(YOLO) and Residual Network-50 Layers (ResNet50). It is found that the MobileNet-SSD provided the best accuracy amongst all the object detection models that are studied and has a lesser training time as well. ResNet 50 is used for identifying the number of images required for a good fit without having to label first and then studied for the object detection models.

Acaricidal properties of hemp (Cannabis sativa L.) essential oil against Dermanyssus gallinae and Hyalomma dromedarii
Mohaddeseh Abouhosseini Tabaria, Aref Khodashenasb, Maryam Jafarib, Riccardo Petrellic, Loredana Cappellaccic, Massimo Nabissic, Filippo Maggic, Roman Pavelad, Mohammad Reza Youssef
Industrial Crops and Products Volume 147, May 2020,
DOI: 10.1016/j.indcrop.2020.112238
The use of conventional pesticides in pest management is facing issues such as developing resistance in pests, environmental pollution and impact on human health, together with regulatory hurdles for approval and marketing of new eco-friendly pesticides to comply with the global trend for residue-free foodstuff. In this framework, botanical pesticides represent valuable alternative products to be exploited. Hemp (Cannabis sativa L.) is an eco-friendly, multipurpose crop that is known for its resistance against insects and parasites. This property is assured by the production of bioactive secondary metabolites such as terpenes and cannabinoids. Notably, the hemp essential oil (EO) contains several terpenoid compounds endowed with pesticide properties. On this basis, in the present work we assayed the toxicity of hemp EO on two ectoparasites of veterinary importance, i.e. Dermanyssus gallinae De Geer and Hyalomma dromedarii Koch. In order to identify the EO chemical constituents responsible for the toxicity on the two ectoparasites, the main monoterpenes and sesquiterpenes of hemp EO, namely ?-pinene, myrcene, (E)-caryophyllene and ?-humulene were tested as well. Mite contact toxicity assays were carried out at the concentrations of 5, 10, 20, 50, 100 and 200 ?g/cm3 . Tick larvicidal and ovicidal assays were done testing 0.5, 1, 2, 5, 10, 20, and 50 ?g/mL of the hemp EO and its main components. Results from mite contact toxicity showed that hemp EO was toxic to D. gallinae with LC50 values of 47.1 ?g/mL. Larvicidal and ovicidal assays revealed the promising activity of the EO against tick larvae and eggs. Among all the tested compounds, (E)–caryophyllene and ?-humulene were the most toxic for both ticks and mites, showing LC50 values lower than the whole EO. Myrcene was moderately toxic, with LC50 values higher than the whole EO, whereas ?-pinene showed weak acaricidal activity. Taken together our results remarked the potential of hemp EO as a potential botanical acaricide in pest management programs and food production. The industrial scalability is assured by the great availability of land for the cultivation of hemp and its low cost of production.

NOT CANNABIS SPECIFIC
Advanced methods of plant disease detection. A review

Federico Martinelli, Riccardo Scalenghe, Salvatore Davino, Stefano Panno, Giuseppe Scuderi, Paolo Ruisi, Paolo Villa, Daniela Stroppiana, Mirco Boschetti, Luiz R. Goulart, et al.
Agronomy for Sustainable Development, Springer Verlag/EDP Sciences/INRA, 2015, 35 (1), pp.1-25.
Doi: 10.1007/s13593-014-0246-1
Plant diseases are responsible for major economic losses in the agricultural industry worldwide. Monitoring plant health and detecting pathogen early are essential to reduce disease spread and facilitate effective management practices. DNA-based and serological methods now provide essential tools for accurate plant disease diagnosis, in addition to the traditional visual scouting for symptoms. Although DNA-based and serological methods have revolutionized plant disease detection, they are not very reliable at asymptomatic stage, especially in case of pathogen with syste
mic diffusion. They need at least 1–2 days for sample harvest, processing, and analysis. Here, we describe modern methods based on nucleic acid and protein analysis. Then, we review innovative approaches currently under development. Our main findings are the following: (1) novel sensors based on the analysis of host responses, e.g., differential mobility spectrometer and lateral flow devices, deliver instantaneous results and can effectively detect early infections directly in the field; (2) biosensors based on phage display and biophotonics can also detect instantaneously infections although they can be integrated with other systems; and (3) remote sensing techniques coupled with spectroscopy-based methods allow high spatialization of results, these techniques may be very useful as a rapid preliminary identification of primary infections. We explain how these tools will help plant disease management and complement serological and DNA-based methods. While serological and PCR-based methods are the most available and effective to confirm disease diagnosis, volatile and biophotonic sensors provide instantaneous results and may be used to identify infections at asymptomatic stages. Remote sensing technologies will be extremely helpful to greatly spatialize diagnostic results. These innovative techniques represent unprecedented tools to render agriculture more sustainable and saffe, avoiding expensive use of pesticides in crop protection.

NOT CANNABIS SPECIFIC
Adventive aphids (Hemiptera: Aphididae) of America North of Mexico
R.G. Foottit, Susan Halbert, Gary L. Miller, L.M. Russell
November 2005 Proceedings- Entomological Society of Washington 108(3) SourceOAI
https://www.researchgate.net/publica...orth_of_Mexico
We provide a compilation of 262 species of aphids that are considered as adventive to North America north of Mexico. Included for each species, where applicable, is reference to: the location and date of introduction of the first North American record; pest status in North America; principal economic hosts; and biogeographical origin. Information is also provided for species whose presence in North America is considered erroneous or questionable and for those species that are considered Holarctic or Beringian.

Agdia ImmunoStrips Testing made easy
Virus test strips
https://www.atzlabs.com/pdf/ImmunoSt...tech_India.pdf
Grow with Confidence Agdia is no stranger to understanding how confidence can impact your business. Customers must be confident they are buying quality products; otherwise, they will look for alternatives. In order to ensure customers receive a quality product, the supplier must be confident that they have all the tools necessary to produce it. In the case of crop production, plant disease can have negative impacts on both a grower’s confidence and their reputation. Since 1981, Agdia has been dedicated to helping growers manage plant disease through early and routine diagnosis of plant pathogens. Today, we are the leading provider of plant disease diagnostics in the industry with a comprehensive line of testing products that are used world-wide. We realize you have options when it comes to diagnostic providers. If you want to work with a testing provider that was built and has grown on the principle of confidence, test with Agdia

An Overview of Hemp Diseases
Journal of the Institute of Science and Technology June 2020 (in Turkish)
Mehmet Ali Sevik
DOI: 10.21597/jist.670642
https://www.researchgate.net/publica..._Hemp_Diseases
Industrial hemp (Cannabis sativa var. vulgaris L.), a variant of the C. sativa plant, is an important industrial crop. Industrial hemp is one of the oldest crops plants know to humans. Hemp is very sensitive to environmental conditions, diseases, and pests. Environmentally stressed plants become predisposed to diseases. Diseases of Cannabis are caused by biotic (bacteria, fungi, viruses, viroids, phytoplasmas) or abiotic (nutrient deficiencies) sources. Cannabis species suffer over 100 diseases. Disease prevalence is also varied by geography and climate. Serious fungal diseases on hemp include gray mold, hemp canker, damping off, assorted leaf spots, blights, root rots. Important bacterial diseases include bacterial blight, wilt, crown gall, striatura ulcerosa, and xanthomonas leaf spot. In Cannabis cultivars were reported including the diseases caused by Hemp streak virus (HSV), Hemp mosaic virus (HMV), Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), and Arabis mosaic virus (ArMV). Important diseases affecting the hemp crop along with their symptoms, means of movement and dispersal, and management are briefly summarized in the paper.


An overview of pathogen and insect threats to fibre and oilseed hemp (Cannabis sativa L.) and methods for their biocontrol
Malgorzata Jedryczka, Fatema Bakro, Katarzyna Wielgusz, Marek Bunalski
Integrated Control in Oilseed Crops IOBC-WPRS Bulletin Vol. 136, 2018 pp. 9-20 September 2018
https://www.researchgate.net/publica...eir_biocontrol
Hemp (Cannabis sativa L.) is a treasure trove plant for both sustainable agriculture and industrial usage. It has multi-application properties due to the production of fibre and high quality oil, biomass as a safe source of energy, and numerous compounds for the pharmaceutical sectors, including mainly cannabinoids (THC and CBD) which have a wide range of psychotropic activities. Hemp, like other plants, suffers from a wide range of pests and pathogens. They cause plant damage, huge annual loss of biomass and seed yield as well as the reduction of the quality of the products. With increased demand for hemp products, its production area is anticipated to expand greatly; previously developed tolerance of environmental pressures or defense mechanisms against biotic threats may not meet the demands of new environments and the additional pathogens that will be encountered. In this review we focus on the most common fungal, oomycetes, viral and pest diseases attacking hemp both worldwide and in Poland. We also highlight the methods of biological control that make possible the maintenance healthy plants as well as the high quality of hemp products.

Not Cannabis specific
Analysis of Cryptic, Systemic Botrytis Infections in Symptomless Hosts
Michael W. Shaw, Christy J. Emmanuel, Deni Emilda, Razak B. Terhem, Aminath Shafia, Dimitra Tsamaidi, Mark Emblow and Jan A. L. van Kan
Frontiers in Plant Science, 7 2016
doi: 10.3389/fpls.2016.00625
Botrytis species are generally considered to be aggressive, necrotrophic plant pathogens. By contrast to this general perception, however, Botrytis species could frequently be isolated from the interior of multiple tissues in apparently healthy hosts of many species. Infection frequencies reached 50% of samples or more, but were commonly less, and cryptic infections were rare or absent in some plant species. Prevalence varied substantially from year to year and from tissue to tissue, but some host species routinely had high prevalence. The same genotype was found to occur throughout a host, representing mycelial spread. Botrytis cinerea and Botrytis pseudocinerea are the species that most commonly occur as cryptic infections, but phylogenetically distant isolates of Botrytis were also detected, one of which does not correspond to previously described species. Sporulation and visible damage occurred only when infected tissues were stressed, or became mature or senescent. There was no evidence of cryptic infection having a deleterious effect on growth of the host, and prevalence was probably greater in plants grown in high light conditions. Isolates from cryptic infections were often capable of causing disease (to varying extents) when spore suspensions were inoculated onto their own host as well as on distinct host species, arguing against co-adaptation between cryptic isolates and their hosts. These data collectively suggest that several Botrytis species, including the most notorious pathogenic species, exist frequently in cryptic form to an extent that has thus far largely been neglected, and do not need to cause disease on healthy hosts in order to complete their life-cycles.

Apparent Increase in Biomass and Seed Productivity in Hemp (Cannabis sativa) Resulting from Branch Proliferation Caused by the European Corn Borer (Ostrinia nubilalis)
Ernest Small, David Marcus, Gail Butler, A. R. McElroy
May 2007 Journal of Industrial Hemp 12(1):15-26
DOI: 10.1300/J237v12n01_03
The European corn borer (Ostrinia nubilalis Hübner), or
ECB, is a major pest of hemp (Cannabis sativa L.). During the course of a study of hemp germplasm and cultivar accessions, the ECB attacked the vertical stem leader of hundreds of plants. At the site of invasion the main stem was typically destroyed, and the plant became strongly branched. Although the damaged plants were an average 9% shorter, mean shoot weight was 20% heavier. Seed productivity was also greater, based on a visual scale. The practical significance of such an “overcompensation” response to insect damage, with damaged plants growing more robustly and productively than their undamaged counterparts, is controversial. Certainly, some aspects of productivity, such as fibre quality, are detrimentally affected by the ECB. The insect showed a preference for larger plants, but was indifferent to the level of tetrahydrocannabinol, the chief intoxicant of C. sativa.

Arthropod pests of hemp (Cannabis sativa L.)
Peter A. Edde
In book: Field Crop Arthropod Pests of Economic Importance January 2022 pg 915-952
DOI: 10.1016/B978-0-12-818621-3.00012-4
https://www.researchgate.net/publica...a_L/references
Hemp is a member of the Cannabaceae family in the rose order (Rosales). Cannabaceae currently contains approximately 11 genera and 170 species of plants. Cannabis and hops ( Humulus ) are the most economically important members of the family. The term “hemp,” has been used broadly to describe many different plant species from several genera and families producing bast fibers, all similar in appearance and quality. Among these are sunn hemp, produced from Crotalaria juncea L. (Fabaceae); sisal hemp, obtained from leaves of Agave sisalana Perrine (Asparagaceae); Manila hemp (abaca), derived from the leaf stalks of Musa textilis Née (Musaceae); Mauritius hemp, obtained from green aloe, Furcraea foetida (L.) Haw (Asparagaceae); and India hemp, produced from plants in the genus Corchorus (Malvaceae). The true hemp plant, Cannabis sativa L., is cultivated chiefly for its fiber, but the seed is used for medicinal and narcotic purposes.
There are three distinct types of cultivated hemp, each further subdivided into varieties. One type of hemp is grown for fiber. Depending on variety, hemp grown for fiber are high in cellulose with stalks up to 4.9 m high. A second type is cultivated for fruit (or seed), which is utilized as a source of oil and food due to its high digestible protein and essential fatty acid content. A third type of hemp, Cannabis sativa, var. indica is high in alkaloids called cannabinoids. The cannabinoids are derived from the dried inflorescences and upper leaves of the carpellate plant. The plant has medicinal and narcotic properties ( Small & Marcus, 2002 ). The common names hemp and marijuana are associated with all three forms

Bacterial leaf spot of hemp caused by Xanthomonas campestris pv. cannabis in Japan.
Netsu, O., Kijima, T., & Takikawa, Y.
Journal of General Plant Pathology, 80(2), 164–168. (2013).
doi:10.1007/s10327-013-0497-8
Bacterial leaf spot disease of hemp was observed in Tochigi Prefecture, Japan in 1982 and characterized by necrotic lesions ca. 1–2 mm diameter on leaves with a yellow halo 2–3 mm wide. In this report, we describe the pathological, physiological and genetic properties of the causal bacterium. Our results indicated that this bacterium is identical with Xanthomonas campestris pv. cannabis reported in Romania.
... cannabis is still obscure. When Vauterin et al. (1995) proposed a new species of Xanthomonas as a result of DNA–DNA hybridization studies, pv. cannabis was left unclassified, and the combination of the name X. campestris pv. can- nabis remained (Bull et al. 2010a). .

Bacteriological and cannabinoids analysis
Dr. José Carlos Bouso
https://www.fundacion-canna.es/en/ba...noids-analysis
The main sanitary problem the users of ilegal drugs in general, and Cannabis users in particular, face, is that there are several unknown factors about the product they are consuming, such as: 1. the manufacturing and/or cultivation processes; 2. the purity, agglutinative and other chemical and/or biologic products present; 3. the effects of such products over heath; and 4. how it has been handled, preserved and sold.

Beet Curly Top Virus In Cannabis
YouTube

Most Curtoviruses are one of three Curly Top Virus species
(BCTV) Beet Curly Top Virus
(SpSCTV) Spinach Severe Surley Top Virus
(HrCTV) Horseradish Curly Top Virus
Curtovirus sequences with>77% genome-wide pairwise identity would be classified as belonging to the same species, those sharing >94% identity would be classified as belonging to the strain.

Beneficial Insects for Biological Pest Control in Greenhouse Cannabis Production
Gerasimos Grammenos, Varvara Kouneli, Antonios Kouneli, D Bilalis
Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Horticulture 78(2):85-93 Nov 2021
https://www.researchgate.net/publica...bis_Production (check http)
DOI: 10.15835/buasvmcn-hort:2021.0037
A greenhouse cannabis cultivation took place in Agriculture university of Athens in order to quantify the efficiency of beneficial insects as a main method of pest management. Cannabis plants grown in two greenhouses and beneficial insects were released only in one greenhouse as a means to investigate the efficacy against pests by the comparison with the control greenhouse. Measurements included the visual estimation of infestation, the recording of pest species and populations, and the comparison of infestations and yields amongst greenhouses. Our results indicate that beneficial insects could control pest populations up to 100%. Even though the environmental conditions were not optimal and consecutive pest infestations were observed throughout the duration of our study, the beneficial insects successfully managed the pest populations. In conclusion, biological control with beneficial insects is a very effective method for pest management in greenhouse cannabis production

Bibenzyl synthesis in Cannabis sativa L.
Kelly F. Boddington,Eric Soubeyrand,Kristen Van Gelder,José A. Casaretto,Colby Perrin,Taylor J.B. Forrester,Cameron Parry,M. Sameer Al-Abdul-Wahid,Nicholas G. Jentsch,Jakob Magolan,Gale G. Bozzo,Matthew S. Kimber,Steven J. Rothstein,Tariq A. Akhtar
The Plant Journal Nov 2021
DOI: 10.1111/tpj.15588
https://onlinelibrary.wiley.com/doi/10.1111/tpj.15588
This study focuses on the biosynthesis of a suite of specialized metabolites fromCannabis that are known as the ‘bibenzyls’. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this ‘bibenzyl synthase’ enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing ‘parts prospecting’ inventory that supports the rational metabolic engineering of natural product therapeutics.

Biocontrol Activity of Bacillus spp. and Pseudomonasspp. Against Botrytis cinerea and Other Cannabis Fungal Pathogens
Carole Balthazar, Amy Novinscak, Gabrielle Cantin, David L. Joly, Martin Filion.
Pathology 2021 July 2021
Doi: 10.1094/PHYTO-03-21-0128-R
Gray mold caused by Botrytis cinerea is one of the most widespread and damaging diseases in cannabis crops worldwide. With challenging restrictions on pesticide use and few effective control measures, biocontrol agents are needed to manage this disease. The aim of this study was to identify and characterize bacterial biocontrol agents with wide-spectrum activity against B. cinerea and other major cannabis fungal pathogens. Twelve Bacillus and Pseudomonas strains were first screened with in vitro confrontational assays against ten culturable cannabis pathogens, namely B. cinerea,Sclerotinia sclerotiorum, Fusarium culmorum, F. sporotrichoides, F. oxysporum, Nigrospora sphaerica, N. oryzae, Alternaria alternata, Phoma sp. and Cercospora sp. Six strains displaying the highest inhibitory activity, namely B. velezensis LBUM279, FZB42, LBUM1082, B. subtilis LBUM979, P. synxantha LBUM223, and P. protegens Pf-5, were further assessed in planta where all, except LBUM223, significantly controlled gray mold development on cannabis leaves. Notably, LBUM279 and FZB42 reduced disease severity by at least half compared to water-treated plants and prevented lesion development and/or sporulation up to 9 days following pathogen inoculation. Genomes of LBUM279, LBUM1082 and LBUM979 were sequenced de novo and taxonomic affiliations were determined to ensure non-relatedness with pathogenic strains. Moreover, the genomes were exempt of detrimental genes encoding major toxins and virulence factors that could otherwise pose a biosafety risk when used on crops. Eighteen gene clusters of potential biocontrol interest were also identified. To our knowledge, this is the first reported attempt to control cannabis fungal diseases in planta by direct antagonism with beneficial bacteria.
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Biocontrol agents and their influence on the cannabis testing space
Kevin McKernan, Kristofer Marsh, Steve Cottrell, Sherman Hom
https://osf.io/cn9y4/download
https://osf.io/preprints/cn9y4/
Biocontrol agents are a promising and mature agricultural technology that offer a more environmentally friendly solution to controlling pathogenic microbial risks found on agricultural products than the conventional use of chemical pesticides[1]. Often, the application of nonpathogenic or atoxigenic microbial strains can help to outcompete pathogenic microbes in a given niche[2,3]. The genomes of these biocontrol organisms are often modified to eliminate the production of the toxin[4]. This genome driven approach can reduce or even eliminate the use of pesticides or fungicides[3]. Agricultural markets that utilize extraction techniques to concentrate particular resins or nutrients are often drawn to biocontrol approaches as some pesticides and fungicides are known to become enriched during extraction process while the biocontrol agents are often eliminated[5,6]. Nevertheless, some states have microbial testing regulations that inadvertently ban the use of biocontrol agents through the use of non-specific Total Yeast and Mold (TYM) or Total Aerobic Count (TAC) testing. These tests do not discriminate between commonly used biocontrol agents (like bacillus amyloliquefaciens) and pathogenic risks. Bacillus amyloliquefaciens has even been shown to liquify some petri dish plates, further elevating the plate counts[7]. Many states have retired these tests citing the lack of clinical utility and their arbitrary action limits and actionability. This has led some states where cannabis is regulated to implement species specific testing for Aspergillus, E.coli and Salmonella (Figure 1).

BIOLOGICAL CONTROL ORGANISMS FOR INSECTS AND MITES
Whitney Cranshaw, Austin Broberg, and Wendlin Burns
https://webdoc.agsci.colostate.edu/h...iers%20NEW.pdf
A wide variety of beneficial organisms are offered for sale by several suppliers to assist in management of insects and mites. The following is a listing of most of the US suppliers and it is organized into three sections. First is a brief description of organisms with potential applications followed by reference to sources where they may be purchased. This is followed by a brief summary listing of pest groups and the associated potential biological controls. At the end is a listing of addresses of many suppliers/producers

Biotic stress caused by Tetranychus urticae mites elevates the quantity of secondary metabolites cannabinoids and terpenes in Cannabis sativa L
Elizabeth Kostanda, Soliman Khatib
Industrial Crops and Products ( IF 5.645 ) Pub Date : 2021-12-16 ,
DOI: 10.1016/j.indcrop.2021.114331
Secondary metabolites are known to play a role in the plant's defense system, which can be triggered by biotic or abiotic stress. Cannabis (Cannabis sativa L.) plants and mainly their female flowers, have a variety of bioactive metabolites, predominantly cannabinoids and terpenes, which are synthesized and secreted by the trichomes. Many studies have examined their chemistry and bioactive effects; however, there is insufficient information on the effect of biotic stress on the presence of secondary metabolites in cannabis. The present study examined the effect of a well-known cannabis pest,Tetranychus urticae, on the occurrence and concentration of cannabinoids and terpenes in cannabis leaves and flowers. Six cannabis plants were infested with T. urticae mites (treatment group), and six plants were used as the control group. Cannabinoids and terpenes were analyzed and quantified by liquid chromatograph mass spectrometer and gas chromatograph mass spectrometer, respectively. The contents of several cannabinoids and terpenes increased significantly in the leaves of the treatment group of plants in their late vegetative phase as the mite population increased, compared with the control group. Significantly increased content of almost all terpenes, and the cannabinoids; Δ9-tetrahydrocannabinol, cannabichromene, and cannabigerol, was also seen in mature flowers of the treatment group plants, compared with the control group. Thus, cannabis plant infestation has an impact on its secondary metabolites, cannabinoids and terpenes, reflected by an overall increase in these compounds.
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Not Cannabis Specific
Can UVB Light Control Mold And Powdery Mildew?
California Lightworks September 17, 2019
https://news.californialightworks.co.. .owdery-mildew/
A fungal infestation can be massively devastating to a dedicated grower. After putting in your best efforts to cultivate a quality product under ideal growing conditions, it can be heartbreaking to see your plants destroyed by a relentless fungus.
And for many growers, spraying their grow room with nasty fungicides doesn’t feel like the ideal option, either.
This is why many gardeners are increasingly interested in UV light as a method for controlling mold and powdery mildew. If a simple light could help you save your plants from an untimely end, wouldn’t that be the best option? You would keep toxins out of the air and save yourself the expense and heartache of unusable plants.

Candidatus Phytoplasma asteris’ (group 16SrI) associated with a witches’?broom disease of Cannabis sativa in India
S. K. Raj, S. K. Snehi, M. S. Khan, S. Kumar
Plant Pathology, 57(6), 1173–1173. (2008).
doi:10.1111/j.1365-3059.2008.01920.x
Cannabis sativa, known as hemp, family Cannabaceae, occurs wild through out Himalayas and is cultivated in some provinces of India as a source of narcotic resin, fibre and edible oil (Anonymous, 1992). Symptoms of witches’ broom, similar to those associated with phytoplasmas, were observed in C. sativa plants growing wild along roadsides in Lakhimpur-Kheri, U.P., India during the summer of 2007. The diseased plants exhibited proliferation of branches with shortened internodes and reduced-size leaves , giving rise to the witches’ broom appearance. Total DNA was extracted from leaves of three symptom and three symptomless C. sativa plants. Nested PCR was carried out using P1/P6 (Deng & Hiruki, 1991) and R16F2n/R16R2 (Gundersen & Lee, 1996) universal primers specific to the phytoplasma 16S rRNA gene. PCR products of the expected size, ~1·5 kb and ~1·2 kb, respectively, were obtained from all plant samples with symptoms (3/3) but not from healthy ones. The three amplicons of 1·2 kb were sequenced and sequence data deposited in GenBank (Accession No. EU439257). BLAST search analysis of the 16S rRNA sequence of the C. sativa phytoplasma showed a 99% identity with those of phytoplasma members of 16SrI group, ‘Candidatus Phytoplasma asteris’, associated with periwinkle little leaf (EU375834); onion yellows (AP006628); carrot phytoplasma (EU215426); barley deformation (AY734453); and aster yellows (AY665676). Therefore, the C. sativa phytoplasma was identified as an isolate of the 16SrI group. A witches’ broom disease on a Cannabis sp. has been recently associated with a phytoplasma of elm yellows group (16SrV) in China (Zhao et al., 2007), but this is the first report of a 16SrI phytoplasma associated with witches’ broom on C. sativa in India.

Cannabinoid Receptors Are Absent in Insects
JOHN MCPARTLAND, VINCENZO DI MARZO, LUCIANO DE PETROCELLIS, LISON MERCER, AND MICHELLE GLASS
THE JOURNAL OF COMPARATIVE NEUROLOGY 436:423–429 (2001)
doi: 10.1002/cne.1078
The endocannabinoid system exerts an important neuromodulatory role in mammals. Knockout mice lacking cannabinoid (CB) receptors exhibit significant morbidity. The endocannabinoid system also appears to be phylogenetically ancient—it occurs in mammals, birds, amphibians, fish, sea urchins, leeches, mussels, and even the most primitive animal with a nerve network, the Hydra. The presence of CB receptors, however, has not been examined in terrestrial invertebrates (or any member of the Ecdysozoa). Surprisingly, we found no specific binding of the synthetic CB ligands [3
H]CP55,940 and [3 H]SR141716A in a panel of insects: Apis mellifera, Drosophila melanogaster, Gerris marginatus, Spodoptera frugiperda, and Zophobas atratus. A lack of functional CB receptors was confirmed by the inability of tetrahydrocannabinol (THC) and HU210 to activate G-proteins in insect tissues, utilizing a guanosine-59-O-(3-[35]thio)-triphosphate (GTPgS) assay. No orthologs of human CB receptors were located in the Drosophila genome, nor did we find orthologs of fatty acid amide hydrolase. This loss of CB receptors appears to be unique in the field of comparative
neurobiology. No other known mammalian neuroreceptor is understood to be missing in insects. We hypothesized that CB receptors were lost in insects because of a dearth of ligands; endogenous CB ligands are metabolites of arachidonic acid, and insects produce little or no arachidonic acid or endocannabinoid ligands, such as anandamide

Cannabis Crop Recommendation
Evergreen Growers Supply, LLC
https://www.evergreengrowers.com/med...endations.html
https://www.evergreengrowers.com/fil...1454530813.pdf
Cannabis, or marijuana, is grown for human consumption and therefore every effort should be made to grow the crop without the use of potentially harmful pesticides. Using beneficial insects and natural fungi to eliminate pests is the best way to ensure the cultivation of a clean and quality product for customers and/or patients.
Intensive modern breeding programs for medicinal characteristics have shifted cultivation from traditional, outdoor environments toward protected, indoor environments. Choosing to work indoors gives growers the ability to grow cannabis year-round and at a faster rate, but it also leaves their crops more susceptible to damaging pests. By creating a nurturing environment for their plants and eliminating the possibility of natural pests from outside, indoor growers inadvertently create very inviting breeding grounds for devastating pests.
Because cannabis has been mostly cultivated as a field crop, indoor growers often experience “stressed” plants which attract fungal pathogens and insect pests. Growers should do what they can to minimize stress by being proactive and working to prevent pests before they become a problem.

Cannabis Microbiome Sequencing: Implications for Cannabis Safety Testing
Kyle Boyar
DOI: 10.13140/RG.2.2.18308.17288
Conference: Cannabis Science Conference East April 2019
The cannabis plant and cannabis products are highly varied and complex matrices with each different product and route of administration having its own considerations for microbial testing. In the absence of rigorous study, this immature industry has decided to adopt methods commonly used in food testing to obtain information about the potential microbial hazards present. However, DNA sequencing of both the cannabis microbiome and the conditions before and after culturing tell a tale of inaccurate methodology. Many of the methods that are currently being employed are leading the cannabis industry astray, while blinding them to the real hazards that could be present. This presentation will walk you through the data that shows this and the discoveries we've made along the way that will hopefully open fresh discussions with new perspective on how to tackle microbiological contaminants in cannabis.

Cannabis microbiome sequencing reveals Penicillum Paxilli and the potential for Paxilline drug interactions with Cannabidiol
Kevin McKernan, Jessica Spangler, Yvonne Helbert, Ryan Lynch, Adrian Devitt-Lee, Wendell Orphe, Ted Foss, Chris Hudalla, Matthew Silva, Cindy Orser, Douglas Smith
https://www.medicinalgenomics.com/wp...4.06.15-pm.png
https://www.medicinalgenomics.com/wp...6_edits_v8.pdf
Traditional Cannabis microbial safety testing relies on counting colony forming units (CFU/g) that grow on a petri dish, 3M film or in a culture based system (Marcu, 2013). These systems take 3-5 days to culture fungi or microbes to detectable limits and cannot discern harmful microbes from beneficial microbes and often fail to detect fungi that synthesize compounds contra-indicated in the medicinal use of cannabinoids (McKernan et al.). As a result of this lack of specificity, overuse of fungicides is common in Colorado and California in 2016 (Wurzer, 2016).
An ideal microbial detection system would discern pathogenic from beneficial microbes. This can be challenging to do with selective medias and culturing conditions but can easily be accomplished with DNA based methods. We propose a 2 step method that first depletes or captures beneficial microbial DNA (Bacillus, Trichoderma etc.) onto a magnetic particle.
The supernatant of this particle thus contains the nonbeneficial microbes and can be tested by qPCR using ITS primers, as described in McKernan et al.

Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers
DOI: 10.12688/f1000research.7507.2
https://biorxiv.org/content/biorxiv/e...30775.full.pdf
https://f1000research.com/articles/4-1422/v2
The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products . As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.

Cannabis pathogens XI: Septoria spp. on Cannabis sativa, sensu stricto
John M. McPartland
Sydowia. 47:44-53 (1995)
https://www.zobodat.at/pdf/Sydowia_47_0044-0053.pdf
The genus Septoria Saccardo is quite unwieldy, containing about 2000 taxa. Sutton (1980) notes some workers have subdivided and studied the genus by geographical area. Grouping Septoria spp. by their host range is a more natural way of studying the genus in surmountable subunits. Six previous papers have revised Septoria spp. based on host studies (Punithalingham & Wheeler, 1965; Constantinescu, 1984; Sutton & Pascoe, 1987; Farr, 1991, 1992a, 1992b). Their results suggest Septoria host ranges are limited, and support the continued study of Septoria by host groupings. These compilations and comparisons are especially useful when cultures are lacking

Cannabis sativa as a Host of Rice Root Aphid (Hemiptera: Aphididae) in North America
Whitney Cranshaw, Suzanne Wainwright-Evans
Journal of Integrated Pest Management, Volume 11, Issue 1, 2020, 15,
DOI: 10.1093/jipm/pmaa008
Rice root aphid, Rhopalosiphum rufiabdominale (Sasaki), is a cosmopolitan species widespread in North America. Most records of this insect are of its association with roots of grasses and sedges, but known hosts also include numerous broadleaved plants both grown outdoors and in greenhouses. Indoor grown Cannabis sativa L., particularly when intensively grown for marijuana production, has also emerged as a common host for this insect in the United States and Canada. On this crop, it has an anholocyclic life cycle where it is almost entirely found in association with plant roots. Colonization of new plants is largely by alate forms that may emerge from soil in large numbers as plants near maturity.

Canmed Events Podcasts web talks
https://cannmedevents.com/coffee-tal...ource=hs_email

Characterization and pathogenicity of Fusarium solani causing foot rot on hemp (Cannabis sativa L.) in Southern Italy.

Sorrentino, R., Pergamo, R., Battaglia, V., Raimo, F., Cermola, M., & Lahoz, E.
Journal of Plant Diseases and Protection. (2019).
doi:10.1007/s41348-019-00265-1
Symptoms of foot rot were observed on hemp (Cannabis sativa) plants in Campania region (Southern Italy) in 2018. The symptoms consisted of brownish areas along the main stem followed by wilting and dropping of leaves. The bark was easily removed in correspondence of the lesions since the inner part of the cortex was afected too. The causal agent was isolated on potato dextrose agar and stored at the Research Centre for Cereal and Industrial Crops of Caserta. On carnation leaf agar, the fungus produced long monophialidic conidiogenous cells and both micro- and macroconidia. The frst were oval, ellipsoid and fusiform prevalently 0- and 1-septate with length 9.84–12.68 ?m and width 2.61–4.15 ?m, while falciform macroconidia were prevalently 3- and 4-septate with length and width 23.87–29.23×3.22–4.98, respectively. Chlamydospores were observed intercalated in the hyphae, globose to oval in shape with smooth or rough wall, 5–15 ?m in diameter. Morphologically, the fungus was identifed as Fusarium solani, which was also supported by the BLASTn and the phylogenetic analysis of the sequences of the internal transcribed spacer and elongation factor 1-? genes. Koch’s postulates confrmed F. solani as the etiological agent of the observed disease. Cluster analysis applied to the two genes demonstrated that our isolates belong to the clade 3 of the F. solani species complex.

Characterization of Stolbur (16SrXII) Group Phytoplasmas Associated with Cannabis sativa Witches'-broom Disease in Iran
Fereshteh Vali Sichani, Masoud Bahar and Leila Zirak
Plant Pathology Journal 2011 Volume:10 Issue:4 Page 161-167
DOI: 10.3923/ppj.2011.161.167
A new disease of hemp plants characterized by witches'-brooms and stunting symptoms was appeared in Yazd province in the centre of Iran. Phytoplasma infections were detected in symptomatic hemps by polymerase chain reaction (PCR) amplifications using phytoplasma universal primer pair P1/P7 followed by R16F2n/R16R2 in nested PCR. Restriction fragments length polymorphism (RFLP) analysis results using CfoI restriction enzyme confirmed that the hemp witches'-broom phytoplasma isolates are related to stolbur group. Also, virtual RFLP analysis revealed that hemp witches'-broom phyutoplasmas in Iran are different from members of other 16SrXII subgroups. Sequence analysis of partial 16S rRNA gene indicated that the phytoplasmas associated with hemp in Iran shared high similarity with ‘Candidatus Phytoplasma solani’ and other phytoplasmas related to stolbur group. This research is the first report of hemp infection with phytoplasmas related to stolbur group.


Coffee Talk With Dr David Joly about PM genes and how to breed Cannabis with them.
https://cannmedevents.com/2021/09/29...d-joly-phd/?ut
• How PM infects cannabis plants and how environmental conditions play a role
• The prevention and remediation options cannabis cultivators currently have
• The difference between PM resistance and PM susceptibility and how variations both can affect the severity of infection
• How breeding out PM susceptibility genes provides more robust protection than breeding in PM resistance genes
• What are MLO genes and how do they affect PM susceptibility
• How MLO genes were used to breed our PM susceptibility in other crops
• The importance of having a quality reference genome to investigate genes of interest.

Comparative genomics of a cannabis pathogen reveals insight into the evolution of pathogenicity in Xanthomonas.
Jacobs, J. M., Pesce, Cã©., Lefeuvre, P., & Koebnik, R.
Frontiers in Plant Science, 6. (2015).
doi:10.3389/fpls.2015.00431
Pathogenic bacteria in the genus Xanthomonas cause diseases on over 350 plant species, including cannabis (Cannabis sativa L.). Because of regulatory limitations, the biology of the Xanthomonas-cannabis pathosystem remains largely unexplored. To gain insight into the evolution of Xanthomonas strains pathogenic to cannabis, we sequenced the genomes of two geographically distinct Xanthomonas strains, NCPPB 3753 and NCPPB 2877, which were previously isolated from symptomatic plant tissue in Japan and Romania. Comparative multilocus sequence analysis of housekeeping
genes revealed that they belong to Group 2, which comprises most of the described species of Xanthomonas. Interestingly, both strains lack the Hrp Type III secretion system and do not contain any of the known Type III effectors. Yet their genomes notably encode two key Hrp pathogenicity regulators HrpG and HrpX, and hrpG and hrpX are in the same genetic organization as in the other Group 2 xanthomonads. Promoter prediction of HrpX-regulated genes suggests the induction of an aminopeptidase, a lipase and two polygalacturonases upon plant colonization, similar to other plantpathogenic xanthomonads. Genome analysis of the distantly related Xanthomonas maliensis strain 97M, which was isolated from a rice leaf in Mali, similarly demonstrated the presence of HrpG, HrpX, and a HrpX-regulated polygalacturonase, and the absence of the Hrp Type III secretion system and known Type III effectors. Given the observation that some Xanthomonas strains across distinct taxa do not contain hrpG and hrpX, we speculate a stepwise evolution of pathogenicity, which involves (i) acquisition of key regulatory genes and cell wall-degrading enzymes, followed by (ii) acquisition of the Hrp Type III secretion system, which is ultimately accompanied by (iii) successive acquisition of Type III effectors.

NOT CANNABIS SPECIFIC but this virus is found in Cannabis
Complete Genome Sequence of a Hop Latent Virus Infecting Hop Plants
Yeonhwa Jo, Hoseong Choi, and Won Kyong Chocorresponding author
Genome Announc. 2015 Mar-Apr; 3(2): e00302-15.
doi: 10.1128/genomeA.00302-15
he hop latent virus is a single-stranded RNA virus that mainly infects hop plants. Here, we report the complete genome sequence of a hop latent virus, which was de novo assembled by RNA sequencing (RNA-seq). Our study indicates that transcriptome data are useful for identifying a complete viral genome

Complete sequence of a cryptic virus from hemp (Cannabis sativa)
Angelika Ziegler • Jaroslav Matous?ek • Gerhard Steger • Jorg Schubert
Arch Virol
DOI 10.1007/s00705-011-1168-8
Hemp (Cannabis sativa) was found to be a useful propagation host for hop latent virus, a carlavirus. However, when virus preparations were analysed by electron microscopy, along with the expected filamentous particles, spherical particles with a diameter of around 34 nm were found. RNA from virus preparations was purified, and cDNA was prepared and cloned. Sequence information was used to search databases, and the greatest similarity was found with Primula malacoides virus 1, a putative new member of the genus Partitivirus. The full sequences of RNA 1 and RNA 2 of this new hemp cryptic virus were obtained.

Contrasting Roles of Cannabidiol as an Insecticide and Rescuing Agent for Ethanol–induced Death in the Tobacco Hornworm Manduca sexta.
Park, S.-H., Staples, S. K., Gostin, E. L., Smith, J. P., Vigil, J. J., Seifried, D., … Heuvel, B. D. V.
Scientific Reports, 9(1). (2019).
doi:10.1038/s41598-019-47017-7
Cannabis sativa, also known as marijuana or hemp, produces a non-psychoactive compound cannabidiol (CBD). To investigate the defensive role of CBD, a feeding preference assay was performed with tobacco hornworm Manduca sexta. The larvae clearly show feeding preference towards the Cannabis tissue containing low CBD over high CBD. While the larva avoided the high CBD diet, we investigated detrimental efects of CBD in the insects’ diet. Contrasted to the performance on low CBD-infused artifcial diet (AD), larvae reared on the high CBD diet sufer signifcantly reduced growth and increased mortality. Through testing diferent carriers, we found that the increase of EtOH in the diet is negatively correlated with insect development and behaviors. Notably, CBD treatment signifcantly improved ethanol-intoxicated larval survival rate by 40% and also improved diet searching activity, resulting in increased diet consumption. Electrophysiology results revealed that the CBD-treated ganglia had delayed but much larger response with electric stimuli in comparison to the larvae reared on AD only and EtOH-added diet. Our results show CBDs’ defensive role against pest insects, which suggests its possible use as an insecticide. We also provide evidence that CBD alleviates alcohol-induced stress; consequently, improving the performance and viability of M. sexta larvae.

Cross-Infectivity of Powdery Mildew Isolates Originating from Hemp (Cannabis sativa) and Japanese Hop (Humulus japonicus) in New York.
Weldon, W. A., Ullrich, M. R., Smart, L. B., Smart, C. D., & Gadoury, D. M.
Plant Health Progress, 47–53.(2020).
doi:10.1094/php-09-19-0067-rs
In the recent decade, agricultural production of both hemp (Cannabis sativa) and hop (Humulus lupulus) has expanded throughout the Pacific Northwest, Midwest, and Eastern United States to support the growing industries for which these plants are key components. The significant and rapidly expanding overlap of production regions of these two Cannabaceae plant family members creates a potential dispersal route for organisms that are pathogenic to both hosts. Powdery mildew is a disease of high economic impact in both hemp and hop production systems, yet it was largely unknown whether the powdery mildew fungi commonly associated with hemp could also be pathogenic on hop, and vice versa. We isolated Golovinomyces spadiceusgrowing upon hemp in New York production greenhouses and Podosphaera macularisfrom feral hop (H. japonicus) plantings also in New York. Herein, we report the pathogenicity of P. macularis associated with hop to C. sativa cultivars ‘Anka’ and ‘Wild Horse’ and pathogenicity of G. spadiceus toward hop. The potential for P. macularis to establish, produce viable, infectious conidia, and undergo sexual recombination on hemp could complicate efforts to exclude the MAT1-2 mating type of P. macularis from western North America and could facilitate the spread of races pathogenic toward ‘Cascade’ hop, and hop cultivars with R6-based resistance to P. macularis, including ‘Nugget’. Further assessment of the pathogenicity of diverse P. macularis isolates, in both geographic origin and the range of hop species, is necessary to better understand the dispersal risk of P. macularis on hemp.

Dark Heart Nursery Identifies Major Virus Behind ‘Dud’ Cannabis Plants
https://cannabisnow.com/dark-heart-n...nnabis-plants/
Dark Heart Nursery has identified that the HpLVd virus — said to infect up to 30 percent of crops — causes cannabis plants to grow poorly.
Jimi Devine
In an announcement likely to send shockwaves through the cannabis industry, Dark Heart Nursery announced on Thursday it had positively identified hop latent viroid (HpLVd) as the cause of “dudding” in cannabis plants, resulting in lost vigor.
After identifying HpLVd is the culprit behind poor cannabis plant performance, Dark Heart started testing for it and eliminating it in 2018.
Dark Heart Nursery Founder Dan Grace told Cannabis Now that the research the nursery started in regards to HpLVd would typically be done by universities as a service to industry. “You look at UC Davis and they have a department called Foundation Plant Services,” he said. “The work that we’ve done here with cannabis, they would normally do for grapes, sweet potatoes, almonds and stuff like that. It’s almost always a government service to the industry.”

Dark Heart Nursery identifies pot pathogen BTW, Dark Heart was not the first to ID this in Cannabis
https://www.dailydemocrat.com/2019/0...-pot-pathogen/
OAKLAND — Dark Heart Nursery has announced that it is the first organization to positively identify hop latent viroid as the cause of “dudding” in cannabis.
Since 2018, Dark Heart, which has growing operations in Yolo County, has also been successfully testing for and eliminating HpLVd through a patent-pending clean plant process.
On the heels of this research, the company has also announced that project lead Dr. Jeremy Warren has officially joined DHN as director of Plant Health.
“The positive identification of the HpLVd pathogen and our patent-pending clean plant process to test for and eliminate it represent a significant advance towards keeping cannabis crops healthy and supporting long-term business growth for cultivators,” said Dan Grace, founder and president of Dark Heart Nursery. “With Dr. Warren at the helm, we are incredibly excited to now offer diagnostic and curative services for HpLVd to licensed cannabis businesses in California, as well as continue our research in identifying and eradicating additional cannabis-infecting pathogens.”
“Dudding” is a colloquial name for a variety of symptoms, which include loss of vigor, stunting, reduction in yield, reduction in potency and changes in morphology. The syndrome was codified in 2017 by Dr. Rick Crum who first coined the phrase “Putative Cannabis Infectious Agent” to describe it.
In 2015, Dr. Crum reported that as many as 35 percent of observed plants showed PCIA symptoms.
In 2017, Dark Heart Nursery began working with Dr. Warren to determine the cause of PCIA. Symptomatic and asymptomatic plants were collected, and next generation RNA sequencing was performed to determine a probable cause of the disease.
After analysis of the results in November 2017, it was determined that Hop latent viroid (HpLVd) was the most likely candidate pathogen. A genetic test was then developed to aid in differentiating healthy plants from infected plants.
In 2014, Dark Heart established the cannabis industry’s first tissue culture laboratory. Among other areas of research, this lab has focused on the development of clean plant protocols through which cannabis can be cured of pathogens and cataloged for later use.

Not Cannabis specific
Deep learning models for plant disease detection and diagnosis
Konstantinos P. Ferentinos
Computers and Electronics in Agriculture 145 (2018) 311–318
DOI: 10.1016/j.compag.2018.01.009
https://www.researchgate.net/publica..._and_diagnosis
In this paper, convolutional neural network models were developed to perform plant disease detection and diagnosis using simple leaves images of healthy and diseased plants, through deep learning methodologies. Training of the models was performed with the use of an open database of 87,848 images, containing 25 different plants in a set of 58 distinct classes of [plant, disease] combinations, including healthy plants. Several model architectures were trained, with the best performance reaching a 99.53% success rate in identifying the corresponding [plant, disease] combination (or healthy plant). The significantly high success rate makes the model a very useful advisory or early warning tool, and an approach that could be further expanded to support an integrated plant disease identification system to operate in real cultivation conditions

NOT CANNABIS SPECIFIC but the virus is found in Cannabis
Detection and molecular analysis of Hop latent virus and Hop latent viroid in hop samples from Poland
July 2014Journal fur Kulturpflanzen 66(7):248-254
DOI: 10.5073/JFK.2014.07.04
Angelika Ziegler, Magdalena Kawka, Marcin Przybys, Jl Schubert
Monitoring the occurrence of virus diseases in plants is important for the implementation of early control measures and prevention of further disease spread. In Poland, in 2004 a health programme for hop was started to eliminate viruses and viroids. In 2012/13, in vitro plants, samples from the IUNG-PIB experimental station and commercial hop gardens in Poland were tested for Hop latent virus (HpLV), and Hop latent and Hop stunt viroids (HpLVd and HpSVd). For virus testing, RT-PCR and ELISA methods were used. In order to detect hop viroids, RT-PCR was employed. The overall incidence of HpLV and hop viroids was lower than reported before the start of the programme. Cloning and sequencing revealed that the HpLV and the HpLVd from Polish sources are very similar to the type sequences and the Czech sources.

Developing Insect Pest Management Systems for Hemp in the United States: A Work in Progress
Whitney Cranshaw, Melissa Schreiner, Kadie Britt, Thomas P. Kuhar, John McPartland, and Jerome Grant
Journal of Integrated Pest Management, (2019)
doi: 10.1093/jipm/pmz023
Hemp (Cannabis sativa L.) is now being grown within the United States over a much broader geographic area and for different uses than during its last period of significant production that ended after World War II. Within the past 3 yr, a large number of arthropod species have been documented to feed on hemp in the United States. Among key pest species, corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), has demonstrated greatest potential for crop injury, being particularly damaging to flower buds. Hemp russet mite, Aculops cannibicola (Farkas), and cannabis aphid, Phorodon cannabis Passerini, are the two species observed most damaging among those that suck plant fluids. Eurasian hemp borer, Grapholita delineana Walker, is widely present east of the Rocky Mountains and appears to have potential to significantly damage both flower buds and developing seeds. Numerous species of caterpillars, grasshoppers, and beetles chew hemp foliage; the severity of these defoliation injuries appears to be minimal, but needs further study. Similarly, numerous seed feeding hemipterans, most notably stink bugs and Lygus bugs, are regularly found in the crop but injury potential remains unclear. Some preliminary efforts have been made to develop integrated pest management strategies for these insects, particularly for corn earworm. Future research can be expected to rapidly resolve many of the data gaps that presently restrict advancing pest management on the crop. However, a major confounding issue involves the use of pesticides on hemp. Federal agencies have not yet provided clear direction on this issue, and regulatory decisions have subsequently devolved to the states.

Differentiating Powdery Mildew from False Powdery Mildew
John McPartland, Karl Hillig
April 2008 Journal of Industrial Hemp 13(1):78-87
DOI: 10.1080/15377880801898758
This note continues the “Cannabis Clinic” series, presenting diseases and pests of hemp, featuring color illustrations of signs and symptoms. The fungus Trichothecium roseum produces a white fuzz that covers branches, leaves, and flowering tops of hemp. These symptoms have been confused with true powdery mildew, caused by Sphaerotheca macularis. We illustrate the differences between disease caused by Trichothecium roseum (henceforth dubbed “false powdery mildew”) and disease caused by Sphaerotheca macularis.

While the resistance gene PM1 is an important discovery it is actually more important to eliminate any MLO susceptibility genes in a Cannabis variety, to have no PM. Breeding out PM susceptibility genes provides more robust protection than breeding in PM resistance genes
*Discovery and Genetic Mapping of PM1, a Powdery Mildew Resistance Gene in Cannabis sativa L.
Paul D. Mihalyov and Andrea R. Garfinkel
Front. Agron. 3:720215. (2021)
doi: 10.3389/fagro.2021.720215
https://www.frontiersin.org/articles...170000_ARTICLE
Powdery mildew is among the most common diseases of both hemp- and marijuana-type cultivated Cannabis sativa . Despite its prevalence, no documented studies have characterized sources of natural genetic resistance in this pathosystem. Here we provide evidence for the first resistance (R ) gene in C. sativa , represented by a single dominant locus that confers complete resistance to an isolate of the powdery mildew pathogen Golovinomyces ambrosiae , found in the Pacific Northwest of the United States. Linkage mapping with nearly 10,000 single nucleotide polymorphism (SNP) markers revealed that this R gene (designated PM1 ) is located on the distal end of the long arm of one of the largest chromosomes in the C. sativa genome. According to reference whole genome sequences and Sanger sequencing, the marker was tentatively placed in a cluster of R genes of the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) protein type. PM1 ’s dominant behavior, qualitative penetrance, and a co-segregating qPCR marker to track its inheritance were confirmed in two separate genetic backgrounds totaling 185 recombinant F1 plants. The goal of this study is to provide a foundation for the discovery and characterization of additional sources of genetic resistance to pathogens that infect C. sativa .

Effect of Cannabis sativa L. root, leaf and inflorescence ethanol extracts on the chemotrophic response of entomopathogenic nematodes.
Žiga Laznik & Iztok Jože Košir & Katarina Košmelj & Jana Murovec & Anamarija Jagodi? & Stanislav Trdan & Darja Kocjan A?ko & Marko Flajšman
Plant and Soil. (2020).
doi:10.1007/s11104-020-04693-z
Aims Soils represent the natural habitat of entomopathogenic nematodes (EPNs). When moving in soil, EPNs are oriented to follow a chemical signal (chemotaxis). Cannabis sativa L. is known to secrete a very wide spectrum of secondary metabolites. Ethanol extracts (EE) of different C. sativa L. organs were used to study the effect on EPN chemotaxis. Methods The root, leaf and inflorescence EE of two hemp varieties and two medical cannabis breeding lines were used in laboratory chemotaxis assays with three EPN species (Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora) at 20 and 25 °C. The content of terpenes and cannabinoids in C. sativa L. inflorescences was measured as well. Results Overall, EPNs were most attracted or repelled by inflorescence extracts, followed by leaves and roots. The most abundant terpene in hemp inflorescences was trans caryophyllene (38.2% on average), the highest contents of total cannabidiol, total cannabigerol and total tetrahydrocannabinol were 9.65%, 0.89% and 0.62%, respectively. Conclusions The attraction effect of S. sativa L. EE is a highly interesting outcome and could lead to the development of attractant compounds in EPN biological control. Cannabinoids and terpenes may be responsible for pronounced effects on EPN chemotaxis.

Not Cannabis specific
Effect of UV-exposure on colony formation of Xanthomonas fragariae in vitro
G.J.T. Kessel & M.T. Schilder
https://cleanlight.nl/wp-content/upl...nas-report.pdf

Not Cannabis Specific
Effect of UV - exposure on germination of sporangia of Phytophthora infestans
G.J.T. Kessel & M.G. Förch
https://cleanlight.nl/wp-content/upl...ytophthora.pdf
The oomycete Phytophthora infestans, the cause of late blight in potato and tomato, is considered one of the most important pathogens of potatoes worldwide (Hooker 1981). P. infestans affects foliage and stems, reducing the photosynthetic capacity of the crop and therefore leading to yield reduction. In addition, it affects tubers which reduces both, yield quantity and quality. In the past, crop losses due to late blight have been estimated to account for 10 to 15 percent of the total global annual potato production (Anonymous, 1996). The economic value of the crop lost, plus the cost of crop protection amount to US $ three billion annually (Duncan, 1999). In the Netherlands, the cost of crop protection amounts to
approximately 40 million euro annually on a total of 160 -180 thousand hectares with an average yield of 45 tonnes fresh weight per hectare.
The life cycle of P. infestans can be separated into an asexual cycle and a sexual cycle. The asexual cycle is the driving force behind rapid polycyclic epidemics that can be observed in potato crops during the growing season. Numerous sporangia are produced on infected leaflets and stems. Sporangia are released into the atmosphere under dry conditions or they can be washed into the ridge by rain. When released into the atmosphere sporangia may cause new foliar infections in the same crop or neighbouring crops. When washed into the soil, sporangia may cause tuber infections. In both cases, the ambient temperature determines whether the sporangium germinates directly (optimum at ±23°C) or indirectly (optimum at ±12°C). Direct germination results in formation of a germ tube. Indirect germination results in formation of motile zoospores. When zoospores loose their flagellae, they become cystospores which germinate and infect through a germ tube

Effects of cold plasma, gamma and e-beam irradiations on reduction of fungal colony forming unit levels in medical cannabis inflorescences
Shachar Jerushalmi, Marcel Maymon, Aviv Dombrovsky and Stanley Freeman
Journal of Cannabis Research (2020) 2:12
doi: 10.1186/s42238-020-00020-6
Background: The use of medical cannabis (MC) in the medical field has been expanding over the last decade, as more therapeutic beneficial properties of MC are discovered, ranging from general analgesics to anti-inflammatory and anti-bacterial treatments. Together with the intensified utilization of MC, concerns regarding the safety of usage, especially in immunocompromised patients, have arisen. Similar to other plants, MC may be infected by fungal plant pathogens (molds) that sporulate in the tissues while other fungal spores (nonpathogenic) may be present at high concentrations in MC inflorescences, causing a health hazard when inhaled. Since MC is not grown under sterile conditions, it is crucial to evaluate current available methods for reduction of molds in inflorescences that will not damage the active compounds. Three different sterilization methods of inflorescences were examined in this research; gamma irradiation, beta irradiation (e-beam) and cold plasma to determine their efficacy in reduction of fungal colony forming units (CFUs) in vivo.
Methods: The examined methods were evaluated for decontamination of both uninoculated and artificially inoculated Botrytis cinerea MC inflorescences, by assessing total yeast and mold (TYM) CFU levels per g plant tissue. In addition, e-beam treatment was also tested on naturally infected commercial MC inflorescences.
Results: All tested methods significantly reduced TYM CFUs at the tested dosages. Gamma irradiation reduced CFU levels by approximately 6- and 4.5-log fold, in uninoculated and artificially inoculated B. cinerea MC inflorescences, respectively. The effective dosage for elimination of 50% (ED50)TYM CFU of uninoculated MC inflorescence treated with e-beam was calculated as 3.6 KGy. In naturally infected commercial MC inflorescences, e-beam treatments reduced TYM CFU levels by approximately 5-log-fold. A 10 min exposure to cold plasma treatment resulted in 5- log-fold reduction in TYM CFU levels in both uninoculated and artificially inoculated B. cinerea MC inflorescences.
Conclusions: Although gamma irradiation was very effective in reducing TYM CFU levels, it is the most expensive and complicated method for MC sterilization. Both e-beam and cold plasma treatments have greater potential since they are cheaper and simpler to apply, and are equally effective for MC sterilization.

Not cannabis specific
ELIMINATION OF HOP LATENT VIROID FROM HOP PLANTS BY COLD TREATMENT AND MERISTEM TIP CULTURE
M. Grudzińska, E. Solarska, A. Czubacka, M. Przybyś and A. Fajbuś
http://www.up.poznan.pl/~ptfit1/pdf/...PP40_21-30.pdf
Cold treatment and meristem tip culture were used for elimination of Hop latent viroid from four cultivars of hop (Humulus lupulus). Efficiency of the method was compared for hop cultivars,cold treatments of tested plants and time of meristems excision. HLVd was successfully eliminated from infected plants after one month of cold treatment. Good results were also obtained after cooling tested plants during winter. Excising the meristems as soon as possible after ending of plant cooling was an important factor in HLVd elimination. The effectiveness of viroid elimination depended also on hop cultivar. The use of a very sensitive diagnostic method RT-PCR to confirm complete viroid elimination from infected plants resulted in obtaining a population of HLVd-free hop plants,which remained healthy also after dormancy period.


Not Cannabis Specific
Elimination of Hop Stunt Viroid (HSVd) from Infected Peach and Pear Plants Using Cold Therapy and Chemotherapy
Australian Journal of Basic and Applied Sciences, 4(1): 54-60, 2010
Kh.A. El-Dougdoug, Osman M.E., Abdelkader Hayam S., Dawoud Rehab, A.,and Elbaz Reham M.
https://www.researchgate.net/profile...a97c000000.pdf
Hop stunt viroid (HSVd) was detected in several pear and peach trees collected during summer season by RT-PCR and molecular hybridization assays. Hop stunt viroid disease was very severe in summer; however, the multiplication of viroid decreases drastically in winter. In this study, the shoots cut from Prunus persicae cv. Florida prince and Pierre corneille cv. Balady infected plants treated by cold therapy, thermotherapy and/or chemotherapy was used successfully to eliminate Hop stunt viroid. Results demonstrated that application of 10 to 20 mg/L Virazole. followed by cold
therapy for 30 days at 4ÅãC in vitro gave a survival rate of 63 and 75% for pear and peach respectively. The presence of viroid in recovered plants was evaluated by tissue print hybridization technique. In vitro cold therapy combined with chemotherapy using shoot tip culture eliminates HSVd from infected peach and pear trees and reduces the risk of introducing this pathogen to Egypt.

Elimination of hop latent viroid upon developmental activation of pollen nucleases
Jaroslav Matousek , Lidmila Orctová, Josef Skopek, Karel Pesina, Gerhard Steger
Biol Chem. 2008 Jul.
DOI: 10.1515/BC.2008.096
https://www.academia.edu/19146337/El...op_latent_viro id_upon_developmental_activati on_of_pollen_nucleases
Hop latent viroid (HLVd) is not transmissible through hop generative tissues and seeds. Here we describe the process of HLVd elimination during development of hop pollen. HLVd propagates in uninucleate hop pollen, but is eliminated at stages following first pollen mitosis during pollen vacuolization and maturation. Only traces of HLVd were detected by RT-PCR in mature pollen after anthesis and no viroid was detectable in in vitro germinating pollen, suggesting complete degradation of circular and linear HLVd forms. The majority of the degraded HLVd RNA in immature pollen included discrete products in the range of 230-100 nucleotides and therefore did not correspond to siRNAs. HLVd eradication from pollen correlated with developmental expression of a pollen nuclease and specific RNAses. Activity of the pollen nuclease HBN1 was maximal during the vacuolization step and decreased in mature pollen. Total RNAse activity increased continuously up to the final steps of pollen maturation. HBN1 mRNA, which is abundant at the uninucleate microspore stage, encodes a protein of 300 amino acids (34.1 kDa, isoeletric point 5.1). Sequence comparisons revealed that HBN1 is a homolog of S1-like bifunctional plant endonucleases. The developmentally activated HBN1 and pollen ribonucleases could participate in the mechanism of HLVd recognition and degradation.

Emerging diseases of Cannabis sativa and sustainable management
Zamir K Punja
Pest Management Science (2021)
DOI: 10.1002/ps.6307
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Cultivation of cannabis plants (Cannabis sativa L., marijuana) has taken place worldwide for centuries. In Canada, legalization of cannabis in October 2018 for the medicinal and recreational markets has spurned interest in large‐scale growing. This increased production has seen a rise in the incidence and severity of plant pathogens, causing a range of previously unreported diseases. The objective of this review is to highlight the important diseases currently affecting the cannabis and hemp industries in North America and to discuss various mitigation strategies. Progress in molecular diagnostics for pathogen identification and determining inoculum sources and methods of pathogen spread have provided useful insights. Sustainable disease management approaches include establishing clean planting stock, modifying environmental conditions to reduce pathogen development, implementing sanitation measures, and applying fungal and bacterial biological control agents. Fungicides are not currently registered for use and hence there are no published data on their efficacy. The greatest challenge remains in reducing microbial loads (colony‐forming units) on harvested inflorescences (buds). Contaminating microbes may be introduced during the cultivation and post‐harvest phases, or constitute resident endophytes. Failure to achieve a minimum threshold of microbes deemed to be safe for utilization of cannabis products can arise from organic cultivation methods or application of beneficial biocontrol agents. The current regulatory process for approval of cannabis products presents a challenge to producers utilizing biological control agents for disease management.

Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens
Parijat Kusari & Souvik Kusari & Michael Spiteller & Oliver Kayser
Fungal Diversity (2013) 60:137–151
DOI 10.1007/s13225-012-0216-3
The objective of the present work was isolation, phylogenetic characterization, and assessment of biocontrol potential of endophytic fungi harbored in various tissues (leaves, twigs, and apical and lateral buds) of the medicinal plant, Cannabis sativa L. A total of 30 different fungal endophytes were isolated from all the plant tissues which were authenticated by molecular identification based on rDNA ITS sequence analysis (ITS1, 5.8S and ITS2 regions). The Menhinick’s index revealed that the buds were immensely rich in fungal species, and Camargo’s index showed the highest tissue-specific fungal dominance for the twigs. The most dominant species was Penicillium copticola that could be isolated from the twigs, leaves, and apical and lateral buds. A detailed calculation of Fisher’s log series index, Shannon diversity index, Simpson’s index, Simpson’s diversity index, and Margalef’s richness revealed moderate overall biodiversity of C. sativa endophytes distributed among its tissues. The fungal endophytes were challenged by two host phytopathogens, Botrytis cinerea and Trichothecium roseum, devising a dual culture antagonistic assay on five different media. We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism on changing the media. This revealed the potential chemodiversity of the isolated fungal endophytes not only as promising resources of biocontrol agents against the known and emerging phytopathogens of Cannabis plants, but also as sustainable resources of biologically active and defensive secondary metabolites.

Endophytic microflora harbored in Cannabis sativa
Parijat Kusari, Souvik Kusari, Michael Spiteller, Oliver Kayser
Technische Universitat Dortmund
Cannabis sativa is an annual herbaceous plant of the Cannabaceae family from central Asia. Cannabinoids are one of the major secondary metabolites of this plant, which are known to have important therapeutic benefits like analgesic, anti-inflammatory, neuro-protective, appetite-stimulant and many more. Endophytic microorganisms (endophytes) still remain an unexplored group of very promising organism with diverse potential for exploitation, that are capable of producing bioactive secondary metabolites, sometimes even those natural products considered exclusive to their host plants. Thus, these microorganisms are important not only from molecular and biochemical standpoint but also from the ecological perspectives.
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(In Turkish)
ENDÜSTR?YEL KENEV?RDE HASTALIK, ZARARLI ve YABANCI OT MÜCADELES?/PEST, DISEASE AND WEED CONTROL IN INDUSTRIAL HEMP
Huseyin Onen
https://www.researchgate.net/publica...NDUSTRIAL_HEMP
Bütün kültür bitkilerinde oldu?u gibi kenevir tar?m?nda da bitki koruma etmenleri üretimin en önemli s?n?rlay?c? faktörler aras?nda yer almaktad?r. Ancak ülkemizde kenevir üretimine yakla??k 20 y?ld?r ara verilmesi kenevir üretim sürecinde uygulanan kültürel i?lemlere ili?kin bilgi birikiminde de erozyona neden olmu?tur. Kenevir üretiminin yayg?nla?t?r?lmas?n?n gündemde oldu?u dikkate al?nd???nda; üretim sürecinde meydana gelen bo?luk da göz önünde bulundurularak, bitki koruma sorunlar? ve bunlar?n çözümü konusunda bilgi eksikli?inin giderilmesini zorunluluk haline getirmektedir. Bu dü?ünceden yola ç?k?larak, “kenevir tar?m? için bitki koruma el kitab?” mahiyetinde ki mevcut çal??mayla; öncelikle kenevirde görülen yabanc? ot türleri ve bunlar?n idaresinde izlenecek prosedürlerin ele al?nmas? hedeflenmi?tir. Ancak konu bütünlü?ünün sa?lanmas? amac?yla kenevir bitkisinin genel özelliklerine de?inilmi?, dünya genelinde kenevirde sorun olu?turan ve ekim alanlar?n?n geni?lemesiyle birlikte ülkemizde de kar??la?abilece?imiz muhtemel hastal?k etmenleri ile zararl?lar ve bunlar?n kontrolü konular? da ele al?nm??t?r. Geni? bir derleme mahiyetindeki bu el kitab?n?n ba?ta üreticiler olmak üzere konuyla alakal? teknik personel ve ara?t?r?c?lara faydal? olmas? en büyük sevinç kayna??m olacakt?r. Di?er taraftan herboloji konusunda Türkçe kaynak yetersizli?i dikkate al?nd???nda; haz?rlanan bu el kitab?n?n gelece?in Ziraat Mühendisi adaylar? Ziraat Fakültelerinin çok de?erli ö?rencilerine yard?mc? kaynak olarak i? görebilmesi de amaçlanm??t?r. Daha geni? kitlelere ula??labilmesi için el kitab? elektronik olarak haz?rlanm?? olup ücretlidir. Ücreti; en az bir kez okumak ve varsa öneri/ele?tirilerin yazara iletilmesidir. Bu durum mevcut kayna??n ihtiyaçlar do?rultusunda geni?letilmesi için önem ta??maktad?r. Faydal? olmas? temennisi ile…
Industrial hemp production areas in Turkey have been recently intended to increase, therefore intensive research and development activities are being conducted to achieve the goal. Introducing the hemp, which was removed from the agricultural crop rotation many years ago, into the crop rotation will bring new crop-pest interactions to the agenda. Therefore detailed studies on plant protection (pests, diseases and weeds management) will be needed for a successful industrial hemp production. In this context, this handbook (in Turkish) was compiled from a detailed literature survey and observations on hemp cultivation areas in our country. The handbook is aimed to inform the producers about plant protection (pest, disease and weed management) in industrial hemp production, with a special emphasis on weeds and their management. The study also intended to provide scientific information to researches on integrated pest/weed management in industrial hemp production, and education material for students in agricultural faculties of the country. In the handbook; I) Pests, pathogens and weeds constitute an inseparable complex in the agro-ecosystems. Therefore all problematic agents (diseases and pests) in hemp production areas along with weeds have been discussed in general. II) Problems arising from pests and pathogens in hemp production areas are addressed in broad terms, while the problems caused by the weeds have been discussed in a wider context. III) Weeds causing problems in the hemp fields worldwide have been listed, parasitic weed (dodder and broomrape) species have been determined, and the noxious weeds in hemp production have been documented based on observations in different parts of the country. IV) Strategies that can be used under the “Integrated Pest Management (IPM)” context in industrial hemp fields have been summarized based on the results obtained in different regions of the world. V) The results of scientific studies revealed that the plant is extremely competitive as a consequence of allopathic effect of hemp, and the plant can easily suppress the weeds with cultural measures such as proper site selection, using competitive varieties, early planting, adjusting the plant density, and appropriate fertilization and irrigation etc. Nevertheless, the weed problems that may arise can be easily overcome by soil cultivation. In addition, the plant has been determined also as tolerant to pathogens/pests. Therefore, pesticide use may not be needed in industrial hemp production areas. VI) The volunteer hemps which have similar characteristics with the problematic weeds in agro ecosystems can arise as a weed especially in crops such as cereals and sunflower. The literature review revealed that monitoring the changes in weed population depending on ecological conditions in agricultural areas is needed, and studies on comparison of different hemp varieties, especially local varieties, for weed competitiveness, and regional critical period studies considering the ecological conditions and possible changes in weed populations will be beneficial. In addition, with regard to weed control, studies depending on the purpose of production (fiber and seed) should be carried out to determine the most appropriate sowing time, sowing depth and sowing rate, and the number and timing of the mechanical weed control (hoeing) should be determined. Moreover, studies such as screening pesticides (including herbicides) candidates are needed to determine the potential pesticides that can be used in hemp production. The review/handbook summarizes the current information and status that may be useful to contribute to the establishment of pest/weed management programs in industrial hemp production in Turkey. Sufficient information on cultural measure (soil tillage, sowing, selection of varieties, fertilization, etc.) which may be needed during the hemp production and fiber processing technology, etc. is available in the literature. However, studies on plant protection in general and weed management in particular are very scarce. This reveals that the information presented in the review may need to be revised in the near future. Since there are important data gaps in the literature regarding on weed and pest management, the data and experiences gained in time can allow to have different perspectives.

Epidemiology of the Hemp Borer, Grapholita delineana Walker (Lepidoptera: Oleuthreutidae), a Pest of Cannabis sativa L.
John M. McPartland
Journal of Industrial Hemp 2002 7:1, 25-42
DOI: 10.1300/J237v07n01_04
The hemp borer, Grapholita delineana, is newly described from feral hemp in Vermont, USA. It may pose a serious pest should hemp cultivation resume in the USA. A similar situation occurred in the 1960s, when G. delineana suddenly became a serious pest in southeastern Europe. Evidence suggests the pest was imported from its native range via infested hemp seed. Larvae of G. delineana bore into stalks and destroy fiber, or they infest flowering tops and destroy seed. The larvae and adults are described, along with their life history, geographic range, and host range. Careful phytosanitary measures can prevent the spread of G. delineana into quarantine areas, such as western Europe, Canada, and the entire southern hemisphere. Breeding hemp plants for resistance to G. delineana may prevent future epidemics. Vermont feral hemp appears to be more resistant to G. delineana than feral hemp growing in the Midwestern USA; the Vermont germplasm may have descended from plants imported in the 1830s, called “Smyrna” hemp, a western European landrace devoid of Chinese ancestry. Biological and chemical controls of G. delineana are described.

Endophytes of industrial hemp (Cannabis sativa L.) cultivars: Identification of culturable bacteria and fungi in leaves, petioles, and seeds
Maryanne Scott, Mamta Rani, Mamta Rani, Jamil Samsatly, Suha Jabaji
June 2018 Canadian Journal of Microbiology 64(10):1-17
DOI: 10.1139/cjm-2018-0108
Plant endophytes are a group of microorganisms that reside asymptomatically within the healthy living tissue. The diversity and molecular and biochemical characterization of industrial hemp-associated endophytes have not been previously studied. This study explored the abundance and diversity of culturable endophytes residing in petioles, leaves, and seeds of three industrial hemp cultivars, and examined their biochemical attributes and antifungal potential. A total of 134 bacterial and 53 fungal strains were isolated from cultivars Anka, CRS-1, and Yvonne. The number of bacterial isolates was similarly distributed among the cultivars, with the majority recovered from petiole tissue. Most fungal strains originated from leaf tissue of cultivar Anka. Molecular and phylogenetic analyses grouped the endophytes into 18 bacterial and 13 fungal taxa, respectively. The most abundant bacterial genera were Pseudomonas, Pantoea, and Bacillus, and the fungal genera were Aureobasidium, Alternaria, and Cochliobolus. The presence of siderophores, cellulase production, and phosphorus solubilization were the main biochemical traits. In proof-of-concept experiments, re-inoculation of tomato roots with some endophytes confirmed their migration to aerial tissues of the plant. Taken together, this study demonstrates that industrial hemp harbours a diversity of microbial endophytes, some of which could be used in growth promotion and (or) in biological control designed experiments.

Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens
Parijat Kusari & Souvik Kusari & Michael Spiteller & Oliver Kayser
Fungal Diversity (2013) 60:137–151
DOI 10.1007/s13225-012-0216-3
The objective of the present work was isolation, phylogenetic characterization, and assessment of biocontrol potential of endophytic fungi harbored in various tissues (leaves, twigs, and apical and lateral buds) of the medicinal plant, Cannabis sativa L. A total of 30 different fungal endophytes were isolated from all the plant tissues which were authenticated by molecular identification based on rDNA ITS sequence analysis (ITS1, 5.8S and ITS2 regions). The Menhinick’s index revealed that the buds were immensely rich in fungal species, and Camargo’s index showed the highest tissue-specific fungal dominance for the twigs. The most dominant species was Penicillium copticola that could be isolated from the twigs, leaves, and apical and lateral buds. A detailed calculation of Fisher’s log series index, Shannon diversity index, Simpson’s index, Simpson’s diversity index, and Margalef’s richness revealed moderate overall biodiversity of C. sativa endophytes distributed among its tissues. The fungal endophytes were challenged by two host phytopathogens, Botrytis cinerea and Trichothecium roseum, devising a dual culture antagonistic assay on five different media. We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism (%) on changing the media. This revealed the potential chemodiversity of the isolated fungal endophytes not only as promising resources of biocontrol agents against the known and emerging phytopathogens of Cannabis plants, but also as sustainable resources of biologically active and defensive secondary metabolites.

Not Cannabis specific
Escherichia coli Cells Exposed to Lethal Doses of Electron Beam Irradiation Retain Their Ability to Propagate Bacteriophages and Are Metabolically Active
Front. Microbiol., 10 September 2018
DOI: 10.3389/fmicb.2018.02138
https://www.frontiersin.org/articles...EJ8bOqXDYTcT-0
Reports in the literature suggest that bacteria exposed to lethal doses of ionizing radiation, i.e., electron beams, are unable to replicate yet they remain metabolically active. To investigate this phenomenon further, we electron beam irradiated Escherichia coli cells to a lethal dose and measured their membrane integrity, metabolic activity, ATP levels and overall cellular functionality via bacteriophage infection. We also visualized the DNA double-strand breaks in the cells. We used non-irradiated (live) and heatkilled cells as positive and negative controls, respectively. Our results show that the membrane integrity of E. coli cells is maintained and that the cells remain metabolically active up to 9 days post-irradiation when stored at 4_ C. The ATP levels in lethally irradiated cells are similar to non-irradiated control cells. We also visualized extensive DNA damage within the cells and confirmed their cellular functionality based on their ability to propagate bacteriophages for up to 9 days post-irradiation. Overall, our findings indicate that lethally irradiated E. coli cells resemble live non-irradiated cells more closely than heat-killed (dead) cells.

Not Cannabis specific
Eukaryotic Translation Initiation Factors Shape RNA Viruses Resistance in Plants
Jannat Shopan, Xiaolong Lv, Zhongyuan Hu, Mingfang Zhang, Jinghua Yang
Horticultural Plant Journal (2020)
DOI: 10.1016/j.hpj.2020.03.001
Viruses are representative of a global threat to agricultural production. Genetic resistance is the preferred strategy for the control of viral infection and against loss of crop yield. Viral protein synthesis requires host cellular factors for translating their viral RNAs, and for regulating their replication and cell to cell systemic movement. Therefore, the viruses are dependent on cellular translation factors. Mutations in the gene encoding eIF4E and eIF4G or their isoforms, eIFiso4E, eIFiso4G and eIF2B? have been mapped as a source of plant potyvirus while other genus of plant virus recessive resistance genes in many species are originated from these loci. Some of other plant translation factors, such as eIF3, eIF4A-like helicases, eEF1A and eEF1B, which are required in interacting with viral RNAs and regulating various aspects of the infection cycle, have also been identified. Here, we summarize the mechanisms utilized by RNA viruses of eukaryotic plants and the essential roles of eIFs in virus infection. Moreover, we discuss the potential of eIFs as a target gene in the development of genetic resistance to viruses for crop improvement. This review highlighted newly revealed examples of abnormal translational strategies and provided insights into natural host resistance mechanisms that have been linked to 3’ cap-independent translational enhancer activity.

Evaluating herbicide tolerance of industrial hemp
(Cannabis sativa L.)
January 2020
Crop Science*60(1):419-427
DOI: 10.1002/csc2.20055
Michael L. Flessner Jabari Bryd, Kevin W. Bamber, John H. Fike
Industrial hemp (Cannabis sativa L.) has a wide array of end uses which, when coupled with regulatory reductions in the United States, has spurred renewed interest in its production. Best management practices, including weed control, need to be evaluated. Since little is known about herbicide tolerance of hemp, studies were conducted to identify suitable options for grain or dual?purpose (fiber and grain) production. Greenhouse experiments with pre?emergence and postemergence herbicides were conducted to identify herbicide choices for subsequent field trials. In field studies, S?metolachlor was the safest pre?emergence herbicide, resulting in 0 and ? 15% injury in 2017 and 2018, respectively, and a hemp stand count that was 97% of the nontreated check (pooled across years). All other pre?emergence herbicides tested in the field resulted in ? 25% visible injury in at least one of the years and ? 24% stand reduction relative to the nontreated check (pooled across years). However, no grain yield differences were observed due to pre?emergence herbicides relative to the nontreated check. Postemergence herbicides sethoxydim, quizalofop, bromoxynil, and clopyralid caused < 20% injury across rating timings and years and had similar grain yield as the nontreated check (588 kg ha?1). Our results indicate that S?metolachlor applied pre?emergence or sethoxydim, quizalofop, bromoxynil, and clopyralid applied postemergence are suitable for hemp production, but some of these treatments caused transient visible injury. Future research should be conducted to corroborate results across cultivars, soil types (for pre?emergence herbicides), and environments.

Evaluating the Microbiome of Hemp
Samuel E. Barnett,1 Ali R. Cala,2 Julie L. Hansen,3 Jamie Crawford,3 Donald R. Viands,3 Lawrence B. Smart,4 Christine D. Smart,2,† and Daniel H. Buckley
Phytobiomes Journal • 2020 • 4:351-363
Doi: 10.1094/PBIOMES-06-20-0046-R
Plant microbiomes contribute to plant fitness and crop yields through a variety of mechanisms. Determining variability in microbiome composition among individuals of a species, and identifying core microbiome membership, are essential first steps for exploring host–microbe interactions. Members of a core microbiome are microorganisms that are tightly associated with and are found widespread across individuals of a plant genotype or species. Hemp (Cannabis sativa L.) is an economically important crop that has gained a resurgence following its removal from the list of controlled substances by the U.S. government. Despite renewed interest in this crop, the microbiome of hemp has not been well studied. We analyzed the bacterial and fungal communities associated with four plant compartments (rhizosphere, root tissue, leaf surface, and flowers) of C. sativa ‘Anka’ across six fields in the Finger Lakes region of New York, United States. We found that both bacterial and fungal community composition varied significantly among plant compartments. Rhizosphere communities were largely similar to the bulk soil communities but root tissue, leaf, and flower communities had distinct compositions. We identified candidate core microbiome members of each plant compartment (bacterial core taxa: root tissue [n = 6], leaves [n = 11], and flowers [n = 7]; fungal core taxa: rhizosphere [n = 1], leaves [n = 14], and flowers [n = 2]). Many of these candidate core microbiome members were related to organisms previously associated with plant growth promotion or pathogen resistance in various plants. The core microbiome identified in this study can be further investigated to improve cultivation of this important crop

Evaluation of Biological Insecticides to Control Corn Earworm in Hemp, 2019
Helene Doughty, Kadie Britt, Thomas P Kuhar
Arthropod Management Tests 45(1) January 2020
DOI: 10.1093/amt/tsaa081 https://www.researchgate.net/publica...m_in_Hemp_2019
The objective of this experiment was to assess the efficacy of several biological insecticide products for control of corn earworm (CEW) on grain hemp in Virginia. A field experiment was conducted on planting of ‘Felina 32’ hemp direct seeded with a grain drill at 30 lb. seed per acre on 24 Jun 2019 at the Virginia Tech Eastern Shore Agricultural Research and Extension Center in Painter, VA. The experiment had six treatments: Gemstar (Helicoverpa zea nuclear polyhedrosis virus [HzNPV]), Javelin (Bacillus thuringiensis var. kurstaki), BoteGHA (Beauveria bassiana strain GHA), Entrust (Spinosad), DiPel (Bacillus thuringiensis var. kurstaki), and an untreated check arranged in an RCBD with four replicates. Individual plots were 3 Å~ 10 ft (0.91 Å~ 3.05 m). Hemp plants were sprayed with insecticides in the field using a single-nozzle boom equipped with D3 spray tips powered by a CO2 backpack sprayer at 40 psi. For each treatment, 40.6 fl. oz (1200 ml) was applied to all four replicates, which equates to 58 gallons per acre. All treatments were applied twice with a 7-d interval, except for Gemstar, which was applied three times at 3-d intervals. Treatments were applied on 13 Aug (all treatments), 16 Aug (Gemstar only), 19 Aug (Gemstar only), and 20 Aug (all treatments except Gemstar). On 12 (pre-count), 20, 27 Aug, and 3 Sep, the number of lepidopteran larvae were recorded per inspection of 10 randomly selected plants per plot. On 3 Sep, 10 plants were examined for feeding damage by CEW according to the following rating scale: 0 = no feeding, 1 = some browning/ damage, 2 = advanced browning and feeding damage/holes in seeds, 3 = advanced feeding damage/clipped bud (Table 1). All data were analyzed using ANOVA procedures. Means were separated using Fisher’s LSD at the 0.05 level of significance. CEW was the dominant lepidopteran species observed with an average of 4.2 larvae per 10 plants on 12 Aug (Table 1). Most CEW larvae observed were either second or third instar. Other recorded larvae included yellowstriped armyworm (YSAW) and SMC, but these represented <5% of the total population of lepidopteran larvae. There was a significant treatment effect on CEW counts on 20 Aug with only Entrust resulting in significantly fewer CEW than the untreated check. Although plots treated with Gemstar did not have significantly lower CEW counts, approximately 3–5% of the larvae were diseased with characteristic virus symptoms on 20 and 27 Aug compared with virtually no diseased larvae in the other plots. There was also a significant treatment effect on CEW damage rating, with Entrust resulting in significantly less damage than the untreated check and all other treatments. This is also reflected in the larval count data.

Not Cannabis Specific
Examinations of Fusarium sambucinumon Humulus lupulus and Co-infection with Hop stunt viroid in Commercial Hop Fields
Natasha R. Cerruti THESIS
https://ir.library.oregonstate.edu/c...ions/vq27zs95m
After an unusually high incidence of Fusarium canker was observed in commercial hop fields of the Pacific Northwest, field surveys were conducted and revealed that canker incidence ranged from 20 to 60% of bines sampled in six commercial fields, as well as wide-spread Hop stunt viroid infection in these six fields. A variety of inoculation techniques and incubation conditions were evaluated in laboratory and greenhouse studies to determine whether Fusarium sambucinum incites girdling symptoms on hop bines, which is characteristic of later stage Fusarium canker infection in commercial hop fields. Koch?s postulates were fulfilled, confirming that F. sambucinum incites Fusarium canker and produces girdling, killing the bine. Colonization of detached hop stems with green fluorescent protein-labeled F. sambucinum or F. verticillioides were observed microscopically, but F. sambucinum colonized more aggressively and to a greater extent. Investigation into the effect of relative humidity on colonization of hop stems demonstrated that relative humidities greater than 88% are required for F. sambucinum to colonize green hop stems. Hilling of commercial hop plants was explored as a management strategy to ameliorate canker symptoms or improve yields in commercial fields with wide-spread Hop stunt viroid infection and results indicate that hilling can improve cone yields in commercial hop plantings co-infected with HpSVd and F. sambucinum.

Exploiting Beneficial Pseudomonas spp. for Cannabis Production
Carole Balthazar, David L. Joly and Martin Filion Front. Microbiol. 12:833172.
doi: 10.3389/fmicb.2021.833172 https://www.researchgate.net/publica...ion/references
Among the oldest domesticated crops, cannabis plants (Cannabis sativa L., marijuana and hemp) have been used to produce food, fiber, and drugs for thousands of years. With the ongoing legalization of cannabis in several jurisdictions worldwide, a new high value market is emerging for the supply of marijuana and hemp products. This creates unprecedented challenges to achieve better yields and environmental sustainability, while lowering production costs. In this review, we discuss the opportunities and challenges pertaining to the use of beneficial Pseudomonas spp. bacteria as crop
inoculants to improve productivity. The prevalence and diversity of naturally occurring Pseudomonas strains within the cannabis microbiome is overviewed, followed by their potential mechanisms involved in plant growth promotion and tolerance to abiotic and biotic stresses. Emphasis is placed on specific aspects relevant for hemp and marijuana crops in various production systems. Finally, factors likely to influence inoculant efficacy are provided, along with strategies to identify promising strains, overcome commercialization bottlenecks, and design adapted formulations. This work
aims at supporting the development of the cannabis industry in a sustainable way, by exploiting the many beneficial attributes of Pseudomonas spp.

Expression of Putative Defense Responses in Cannabis Primed by Pseudomonas and/or Bacillus Strains and Infected by Botrytis cinerea
Carole Balthazar, Gabrielle Cantin, Amy Novinscak, David L. Joly and Martin Filion
Front. Plant Sci. 11:572112. 2020
doi: 10.3389/fpls.2020.572112
https://www.frontiersin.org/articles...20.572112/full
Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea’s infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways

*First Insights Into the Virus and Viroid Communities in Hemp (Cannabis sativa)
Judith Chiginsky, Kaitlyn Langemeier, Jacob MacWilliams, Tessa Albrecht, Whitney Cranshaw, Ana Cristina Fulladolsa, Marylee Kapuscinski, Mark Stenglein and Punya Nachappa
Front. Agron. 3:778433. (Dec 2021)
doi: 10.3389/fagro.2021.778433
Hemp (Cannabis sativa L.) production has increased significantly in recent years; however, the crop has been understudied in the U.S. since its production declined in the late 1950s. Disease identification and management is an increasing challenge for hemp growers across the country. In 2019, beet curly top virus (BCTV) was first reported in hemp in Colorado. Hence, we were motivated to understand the diversity and prevalence of BCTV strains infecting hemp in Colorado. We detected BCTV at high incidence rate (81%) in leaf samples from 12 counties. Two different strains of BCTV, Worland (Wor) and Colorado (CO) were present as a single ormixed infection in hemp leaf samples. Phylogenetic analysis revealed BCTV sequences from hemp formed a distinct group along with BCTV strains CO and Wor. To determine other potential viral and viroid pathogens in hemp, we performed next generation sequencing (NGS). Virome analysis revealed the presence of both virus and viroid sequences that had high nucleotide sequence identity with GenBank accessions for cannabis cryptic virus, cannabis sativa mitovirus, citrus yellow vein associated virus, opuntia-like virus and hop latent viroid. In contrast, tobacco streak virus sequences were highly variable compared to sequences in GenBank suggesting a possible new genotype of this virus. The data presented here has important implications for the epidemiology andmanagement of the various diseases of hemp and will lead to the development of integrated pest management strategies designed to interrupt transmission cycles and facilitate efficient crop production.


First report of anthracnose leaf spot caused by Colletotrichum fioriniae on hemp (Cannabis sativa).
Szarka, D., McCulloch, M. J., Beale, J., Long, S., Dixon, E., & Ward Gauthier, N. A.
(2019). Plant Disease.
doi:10.1094/pdis-10-19-2216-pdn
In August 2018, a field hemp sample with leaf spots was submitted to the University of Kentucky Plant Disease Diagnostic Lab from Jackson County, KY. Leaf spots affected all plants in the field, with spots forming on the lower half of plants in early August as plants entered the reproductive stage. Disease
severity on mature leaves was approximately 20%, and incidence was 100% of plants in the field. All three cultivars in the field were equally affected: ‘Cherry x Cherry,’ ‘Trump 1,’ and ‘Sweetened.’ Symptoms included numerous, scattered, round leaf spots that expanded to necrotic blotches. Spots began as light green specks and developed into round or angular spots with tan centers. Spots were circular but occasionally became irregular as they expanded, reaching 3 to 4 mm in diameter with a darker brown margin and yellow halo. Centers of spots cracked or dropped out, causing shothole or
frog-eye symptoms. Spots sometimes coalesced to form necrotic regions up to 20 mm across. Necrotic areas on or near leaf margins resulted in symptoms typical of anthracnose leaf diseases on other hosts, such as of scorching, puckering, and twisting.

First Report of Beet Curly Top Virus Infecting Industrial Hemp (Cannabis sativa) in Arizona.
Hu, J., Masson, R., & Dickey, L.
Plant Disease.(2020).
doi:10.1094/pdis-11-20-2330-pdn
Industrial hemp (Cannabis sativa) is an emerging crop in Arizona, with many uses, including fiber, cosmetic products, and health food. In 2020, severe curly top disease outbreaks were observed in several hemp fields in Yuma and Graham Counties, Arizona, where disease incidence and severity were considerably high, up to 100% crop loss occurring in some fields. A wide range of symptoms have been observed at different infection stages and plant growth stages at the time of infection. Early stage symptoms manifested as light green-to-yellowing of new growth, similar to sulfur or micronutrient deficiency, usually combined with older leaves with dark green “blotchy” mosaic mottling overlaying light green chlorosis. Mosaic mottling of older leaves continued into mid-growth stage, and was coupled with more severe yellowing and witch’s broom (stunted leaves and shortened internode length of stem) of apical meristematic tissue. Curling and twisting of new leaves had also been observed. Symptoms often appeared to be isolated to individual branches, with other branches showing no visual symptoms, often outgrowing and covering affected branches until harvest. Late stage symptoms included severe leaf curling with or without twisting, continued stunting, and necrosis of yellow leaves, resulting in significant yield reduction. Severely affected plants dwarfed by the virus experienced high mortality rates later into the season, most likely attributed to reduced ability to overcome abiotic stress conditions. These
symptoms indicated the likelihood of curly top caused by Beet curly top virus (BCTV), which has been recently reported in Colorado (Giladi et al., 2020).

First Report of Beet Curly Top Virus Infecting Cannabis sativa L., in Western Colorado.
Giladi, Y., Hadad, L., Luria, N., Cranshaw, W., Lachman, O., & Dombrovsky, A.
Plant Disease. (2019).
doi:10.1094/pdis-08-19-1656-pdn
In industrial hemp (Cannabis sativa L.) fields located in North Fork Valley, Delta County Colorado USA, plants bearing symptoms of stunted growth and yellowing leaves were observed in each growing season between 2015-2019. Infected plants display initially fading leaf color to pale green, starting at the leaf base and expanding towards the tips, producing a yellow-green mosaic pattern (Fig. 1). Within ten days, the symptoms spread to the entire plant. In plants with advanced symptoms, newly developing leaves were pale green, narrower and, curled sideways, leading to a stunted, curled plant. Infection was observed in several different hemp cultivars at different developmental stages, from the vegetative to the flowering stages. To identify the infectious agent, leaves were collected from one symptomatic hemp plant and one non-symptomatic hemp plant on 30 July, 2018 and blotted onto FTA cards (Ndunguru et al. 2005). Nucleic acids extracted from FTA cards and subjected to ribosomal RNA depletion, served for library construction as previously described (Luria et al. 2019) using ScriptSeq™ Complete Kit (Plant Leaf, Illumina, San Diego, CA, USA) and sequenced using Illumina Hiseq 2500, at the Technion Genome Center, Israel. The obtained clean reads were searched for viral sequences using VirusDetect software version 1.7. VirusDetect software involved a pipeline combining de novo assembly with mapping to references of plant viruses from Genbank using Velvet (Zerbino and Birney, 2008). This analysis revealed 31 contigs, which aligned to the entire 2,931 nucleotides of Beet curly top virus (BCTV, Geminiviridae Family, Curtovirus genus), sharing 96.5% and 96.4% identity with the genome sequence of isolates AY134867 and KX867020 detected in B. Vulgaris in 2002 and 2006 respectively. In order to validate the NGS findings, the nucleic acids were used as a template for PCR analysis using several specific primer pairs designed to cover most of the BCTV genome. A selected primer set was used for diagnostics: Forward-337-5'…ATGGGACCTTTCAGAGTGGA…3'; Reverse-1,278- Page 2 of 5 5’…TGTATGCCACATTGTTTGGC…3'. Seven symptomatic hemp plants and three nonsymptomatic plants were tested by PCR using the designed BCTV-specific primers. The PCR products were sequenced by Sanger method (HyLabs, Rehovot, Israel) and assembled, resulting in alignment with the majority of BCTV genomes. Importantly, sequences homologous to BCTV were present only in the leaves of the seven symptomatic hemp plants. The complete HTS-derived viral genome sequence was deposited in GenBank (accession No. MK803280) under the name BCTV-Can. There are several strains of BCTV that infect more than 300 plant species and many agricultural crops including beans, sugar beet, cucumber, peppers, spinach and tomatoes (Strausbaugh and Ejayl 2017). BCTV causes symptoms throughout the western United States and sporadic outbreaks occur in western Colorado, where the symptomatic plants described here were located. The virus is solely transmissible by leafhopper vectors and the only known vector of this pathogen in North America is Neoaliturus (=Circulifer) tenellus (Baker) (Hemiptera: Cicadellidae), known as the beet leafhopper (Bennett 1967). BCTV did not appear in the review of hemp diseases by McPartland et al. (2000) and to the best of our knowledge; this is the first report of BCTV infecting hemp and the first report of any leafhopper-vectored pathogen

First Report of Branched Broomrape (Phelipanche ramosa) on Celeriac (Apium graveolens) in Eastern France
Stéphanie Gibot-Leclerc
Plant Disease 98(9):1286 September 2014
DOI: 10.1094/PDIS-02-14-0148-PDN
Branched broomrape, Phelipanche ramosa (L.) Pomel (syn. Orobanche ramosa L.), is a chlorophyll-lacking, obligate root parasitic plant that infests Brassicaceae, Solanaceae, and legumes (3). In western France, P. ramosa has invaded oilseed rape fields since the 1990s, causing significant yield losses (1). This crop has now become the primary host for the parasite, along with buckwheat (Fagopyrum esculentum L.), hemp (Cannabis sativa L.), and tobacco (Nicotania tabacum L.). In September 2013, a field survey indicated that a celeriac (Apium graveolens L. var. Prinlz) crop on clay soil in the Champagne-Ardennes region
(48°20?19? N, 04°01?57? E, 140 m above sea level, eastern France) was infested with branched broomrape where hemp had been grown 4 years before. The celeriac field was planted to wheat (Triticum aestivum L.) in 2012 in rotation with lentils (Lens culinaris Medik.) in 2011. About 2% of the total celeriac field was infested and the estimated yield losses were approximately 25% for this infested area. The host symptoms observed were a slower growth of celeriac, along with leaf chlorosis, lower fruit production, and numerous abortions. The infestation of the celeriac crop was confirmed by verifying the attachment of branched broomrape to the celeriac roots. Broomrape plant heights were between 4.5 and 21 cm. The stems were erect, branched, frail, rather hairy, and bulging. Scale leaves were limited to 4 to 10 mm long, thick, acuminate, alternate scales. The flowers were numerous (between 4 and 51) and were 8.3 to 14.5 mm long. They were borne in the axils of scaly bracts. They had an irregular, curved shape, and a light mauve color. They did not have distinct peduncles and were grouped in rather long floral scapes during advanced flowering. The corolla tube was 10 to 15 mm long and its restricted part stood higher than the divisions of the calyx. It had ciliate, if not hairy, lobes. The calyx was more or less hairy, zygomorphous, with four lobes, and 6 to 8 mm long. Two bracteoles were situated on either side of the calyx. The four stamens observed were didynamous and borne 4 to 5 mm above the corolla base. The dorsifixed bilocularis, longitudinally dehiscent anthers were glabrous or covered with a fine down along sutures. Georges Sallé, (retired) Professor of Botanics at the University Pierre et Marie Curie, Paris, confirmed the identity of P. ramosa based on morphological characteristics (1). Celeriac infection by branched broomrape was confirmed using a developed assay (2). P. ramosa infecting celeriac roots was described by counting the numbers of individuals having reached ontogenic stages according to Gibot-Leclerc et al. (2). To our knowledge, this is the first study reporting P. ramosa infection on celeriac in eastern France. Since celeriac is produced in rotation with lentils, branched broomrape could pose a serious threat to production of these crops.

First report of 'Candidatus Phytoplasma trifolii' associated with a witches' broom disease in Cannabis sativa in Nevada, USA
X. Feng, M. Kyotani, S. Dubrovsky, and A.-L. Fabritius
Plant Disease Vol. 103, No. 7 July 2019
DOI: 10.1094/PDIS-01-19-0098-PDN
In September 2018, symptoms including leaf curling, mottling, chlorosis, witches’ broom, stunting, and node shortening were detected in Cannabis sativa L. plants at two growing sites in Central and Southern Nevada, U.S.A., respectively. Incidence of the disease varied between 5 and 20% at the growing site in Central Nevada, and 30% of plants were affected at the growing site in Southern Nevada. Symptomatic leaves showed “green islands” that were constrained by the veins on the upper leaf surface, and interveinal discoloration on the lower surface, forming a pale “pustule-like” appearance. One infected plant with 80% of leaves exhibiting symptoms was sampled from the Central Nevada site. One infected plant with 50% of leaves exhibiting symptoms was sampled from the Southern Nevada site. Two nonsymptomatic plants were provided by a local grower in California. Total DNA was extracted from the petioles of the most symptomatic leaves. DNA extracted from nonsymptomatic plants was used as the negative control.

First report of Cercospora cf. flagellaris on industrial hemp (Cannabis sativa) in Kentucky
March 2019 Plant Disease
Vinson P Doyle, Hannah Tonry, Bernadette Amsden, Julie Beale, Ed Dixon, Hua Li, Desiree Szarka, Nicole Gauthier
DOI: 10.1094/PDIS-01-19-0135-PDN
In 2015 and 2016, a leaf spot disease was observed in industrial hemp fields in Kentucky during mid-summer as plant canopies became dense. Leaf spots were first visible on older leaves in the lower canopy, but disease quickly spread throughout the canopy. Early symptoms included small individual yellow flecks on upper sides of leaves. Lesions increased in size but retained a round shape. Centers of lesions turned to tan and then to lighter shades of tan or white with age and reached a maximum of 2-6 mm in diameter with raised dark brown to purplish borders and yellow halos; some spots coalesced as disease progressed. Clusters of melanized conidiophores became visible with the unaided eye in centers of spots before the centers disintegrated

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Industrial Hemp in Florida
M. V. Marin, J. Coburn, J. Desaeger and N. A. Peres
Plant Disease Vol. 104, No. 5 May 2020
DOI: 10.1094/PDIS-11-19-2287-PDN
During a greenhouse (July to September) and a field trial (October to December) in 2019, leaf spots were observed on up to 60% of leaves of hemp plants (Cannabis sativa). Symptoms started on older leaves and eventually spread throughout the canopy. Infections began with small yellow, individual flecks. Lesions developed to turn light tan, or even white, with yellow halos and fascicles of conidiophores were visible to naked eye at the center. Severely infected leaves usually developed chlorosis (yellowing), which lead to premature defoliation. Diseased leaves were surface sterilized with 10% bleach solution for 90 seconds and isolation was performed on General Isolation (GI) medium (Forcelini et al. 2016). Resulting colonies were whitish to gray after incubation in a growth chamber at 25°C, 12/12 photoperiod. Isolates were single- spored and resulting colonies were transferred to carrot-agar (CA) and PDA+6% sucrose where they appeared brown- to- dark color due to sporulation (Figure 1) (Leslie and Summerell 2006).

First Report of Chaetomium globosum Causing a Leaf Spot of Hemp (Cannabis sativa) in Tennessee
A. G. Chaffin, M. E. Dee, S. L. Boggess, R. N. Trigiano, E. C. Bernard, and K. D. Gwinn
Plant Disease Vol. 104, No. 5 May 2020
DOI: 10.1094/PDIS-08-19-1697-PDN
Cultivation of hemp (Cannabis sativa), a crop grown for food, fiber, biofuel, and natural medicine worldwide, has increased in the southeastern United States. Pilot and research programs were legalized in 2014, and hemp was removed from the list of Schedule I controlled substances in 2018 (Agriculture Improvement Act of 2018; Mead 2019). Little is known about hemp pathogens, but pathogens of marijuana strains grown in controlled environments were recently described (Punja et al. 2019). Symptomatic plants were found in the North Greenhouse at the University of Tennessee, Knoxville, TN, in September 2017. Leaf spots began as chlorotic lesions near the margins and progressed to necrotic lesions with chlorotic halos. Leaves with chlorotic and necrotic lesions were collected from three 4-month-old plants of two fiber hemp cultivars, Fedora 17 and Futura 75.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp (Cannabis sativa L.) Varieties Cultivated in Southern Spain
S. Casano, A. Hern.ndez Cotan and M. Mar.n Delgado,
Plant Disease 102(8), 1665
DOI: 10.1094/PDIS-02-18-0208-PDN
In June 2015 and July 2016 charcoal rot symptoms were observed in plants of different industrial and medicinal hemp varieties grown in two different fields located in Southern Spain (Los Chapatales and Alcal. del R.o, Seville). In Los Chapatales, disease incidence was 22% in variety Futura 75. In Alcal. del R.o, the medicinal hemp varieties Sara and Aida (disease incidence: 25.5% and 37.1%, respectively) were more susceptible than varieties Theresa (3.8%), Pilar (3.2%), and Juani (2.7%). In both fields, affected plants developed a systemic chlorosis, rapidly wilted, showed necrosis and died. Before the stalk was completely desiccated, internal tissues appeared soft and fluffy. Discoloration of the stalk was detected near the soil line where small black sclerotia were observed. Roots were necrotic with areas of brown-violet color of unprotected vascular cambium.

First Report of Curvularia pseudobrachyspora Causing Leaf Spot on Hemp ( Cannabis sativa ) in Florida
M. V. Marin, N.-Y. Wang, J. Coburn, J. Desaeger and N. A. Peres
Plant Disease October 7 2020
https://apsjournals.apsnet.org/doi/f...03-20-0546-PDN
DOI: 10.1094/PDIS-03-20-0546-PDN
Hemp (Cannabis sativa L.) is an emerging crop in Florida, with potential use in a variety of commercial and industrial products, including rope, textiles, bioplastics, and insulation. During a field trial in 2019 (October to December) in Wimauma, FL, leaf spots were observed on up to 70% of one-month-old hemp plants on several varieties, such as 'Pumma-2', 'Eletta Campana', 'Carmagnola Selezionata', and 'Tygra' with up to 50% leaf damage in the field. Symptoms started on young and old leaves with small yellow spots that eventually turned tan to brown with a yellow halo. Pieces of diseased leaf tissue were surface sterilized with a 10% bleach solution for 90 s, rinsed twice with sterile deionized water, and then placed on General Isolation medium (Forcelini et al. 2016). The plates were kept in a growth chamber at 25°C under a 12/12 photoperiod. Fungal colonies with sparse aerial mycelium, fimbriate margins, and pale light gray zones or alternate gray olivaceous-to-brown zones on the surface were consistently isolated and single-spored. Four isolates were selected for identification and pathogenicity tests.

First Report of Crown and Root Rot Caused by Pythium aphanidermatum on Industrial Hemp (Cannabis sativa) in Arizona
Jiahuai Hu and Robert Masson
Plant Disease March 2021
https://apsjournals.apsnet.org/doi/p...01-21-0065-PDN
During July and August 2020, symptoms of leaf yellowing and browning, sudden wilting, and death were observed on industrial hemp plants (Cannabis sativa L.) in several drip-irrigated fields in Yuma and Graham county, Arizona. About 85% of plants showed severe crown and root rot symptoms. A high percentage of affected plants collapsed under intensive heat stress. Shriveled stem tissue with necrotic lesions can often be seen at the base of the plant, extending upwards more than 5 cm. Internal tissue of main stem and branches was darkened or pinkish brown. Outer cortex of root bark was often completely rotten, exposing the white core. Cottony aerial mycelium was visible on the surface of stalk of some of
the infected plants in two fields in Yuma. To identify the causal agent, a total of twenty symptomatic plants were collected from several fields across the state. Crown and root tissues from affected plants were harvested and rinsed in tap water to remove soils.

First Report of Crown and Root Rot Caused by Pythium myriotylum on Hemp (Cannabis sativa) in Arizona
Jiahuai Hu
Plant Disease 2021
https://apsjournals.apsnet.org/doi/p...12-20-2712-PDN
During August and September 2020, symptoms of leaf chlorosis, stunting, and wilting were observed on
indoor hemp plants (Cannabis sativa L. cv. ‘Wedding Cake’) in a commercial indoor facility located in
Coolidge, Arizona. Plants were grown in soilless coconut coir growing medium (Worm Factory
COIR250G10), watered with 1.5 to 2.1 liters every 24 h through drip irrigation, and supplemented with
18 h of lighting. About 35% of plants displayed symptoms as described above and many symptomatic
plants collapsed. To identify the causal agent, crown and root tissues from four symptomatic plants were
harvested and rinsed with tap water. Tissue fragments (approx. 2 to 4 mm in size) were excised from the
margins of the stem and root lesions, surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed
well in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA) and on oomyceteselective
clarified V8 media containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene
(PARP). Plates were incubated at room temperature (21-24 oC). Five isolates resembling Pythium were
transferred after 3 days and maintained on clarified V8 media. Morphological characteristics were
observed on grass blade cultures (Waterhouse 1967). Grass blades were placed on CV8 inoculated with
the isolate. After a 1-day incubation at 25°C, the colonized blades were transferred to 8 ml of soil water
extract in a Petri dish.

First Report of Diaporthe phaseolorum Causing Stem Canker of Hemp (Cannabis sativa)
M. V. Marin, N.-Y. Wang, J. D. Coburn, J. Desaeger and N. A. Peres
Plant Disease (2021)
https://apsjournals.apsnet.org/doi/p...06-20-1174-PDN
Hemp is an annual herbaceous plant that is used for its fiber and oil in a variety of commercial and industrial products. In Florida, it is currently being explored as a new specialty crop. During a field trial from October to January 2019 in Wimauma, FL, a stem canker was observed on up to 60% of three-month-old plants of 'Eletta Campana', 'Carmagnola Selezionata', and 'Tygra'. Symptoms started on the main stems with light-to-dark brown lesions of different sizes and shapes. Over time, the lesions coalesced into large necrotic areas and bore pycnidia. Isolations were made from diseased stem tissues on General Isolation medium (Amiri et al. 2018) after surface disinfestation (Marin et al. 2020). The plates were placed in a growth chamber at 25°C under a 12/12 photoperiod. A fungus with white, floccose, aerial mycelium and pycnidia producing alpha and beta conidia was consistently isolated. Three single spore isolates were chosen for identification and pathogenicity tests.

First Report of Exserohilum rostratum Causing Foliar Blight of Industrial Hemp (Cannabis sativa L.)
Lindsey Thiessen, Tyler Schappe
January 2019 Plant Disease
DOI: 10.1094/PDIS-08-18-1434-PDN
In the 2017 and 2018 growing seasons (between May and October), industrial hemp plants of several cultivars including those grown for fiber, seed, and flower from numerous counties in North Carolina showed foliar, stem, and floral blight symptoms. Plants were collected from samples submitted to the North Carolina State University Plant Disease and Insect Clinic. Lesions on leaves were round, brown to black, with dark margins. Inside of each lesion, abundant conidia were found. Conidia were rostrate, ellipsoidal to narrowly obclavate, straight or slightly curved, olive-brown, with a protuberant, cylindrical hilum at the base. Conidia were 7 to 12 septate and 75.64±8.31× 15.61±1.41 µm. Conidiophores were cylindrical, olivaceous-brown with swollen conidiogenous cells containing circular conidial scars. Isolates were obtained by transferring single spores to water agar and then transferring to potato dextrose agar.

First Report of Fusarium falciforme (FSSC 3+4) Causing Rot of Industrial 2 Hemp (Cannabis sativa) in California.
K. R. Paugh, J. Del Castillo Múnera, and C. L. Swett
https://apsjournals.apsnet.org/doi/p...08-21-1640-PDN
Industrial hemp (Cannabis sativa) is a newly legal crop in California that is grown for cannabidiol oil, fiber and seed. In August 2019, whole plant decline and root rot were observed affecting <5% of plants in two industrial fields in Fresno County, CA. Symptoms included chlorotic, collapsed foliage, stem vascular discoloration, and root rot with abundant mycelial growth. Stem and root segments (1-2 cm) from three to five diseased plants were agitated in 0.1% tween-20 and soaked in 70% ethanol for 30 s and 1% NaOCl for 2 min. After incubating for 5 to 7 days on 1:10 potato dextrose agar (PDA) amended with tetracycline, Fusarium selective medium (FSM), and PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) medium, white to pale cream aerial mycelium emerged from tissue of all plants on PDA and FSM but not PARP. Isolates cultured on 0.1% potassium chloride agar formed heads of microconidia on long monophialides consistent with the Fusarium solani species complex (FSSC) (Leslie and Summerell 2008). To obtain pure cultures of two isolates (CS529 and CS530), a single-hyphal tip was excised and grown on PDA. FSSC was described as causing foot rot in hemp in Italy (Sorrentino et al. 2019), but these isolates belonged to phylogenetic species 5 (F. solani) not F. falciforme. In addition, F. falciforme was reported as causing root rot in hydroponically grown cannabis (Punja and Rodriguez 2018). These studies provide the foundation for development of management tools for hemp disease.

First Report of Fusarium graminearum Causing Flower Blight On Hemp (Cannabis sativa) in Kentucky
Gabdiel E. Yulfo-Soto, Henry Smith, Desiree Szarka, Ed Dixon, Lisa J. Vaillancourt, Nicole Gauthier
Plant Disease 2021 july
https://apsjournals.apsnet.org/doi/p...06-21-1292-PDN
In October of 2020, a grower in Boyle County, KY, reported mold and blight symptoms on flowers of field-grown hemp. Plants were approaching harvest, and the mold was affecting 100% of the cultivar ‘White CBG’ being grown for cannabinoid (CBD) extraction. Mycelium colonized the flower heads and any seeds within bracts. Affected flower bracts were necrotic, and mycelium and necrosis in the most severe cases also encompassed adjacent (sugar) leaves. Necrotic symptomatic tissue was collected, disinfested in 10% bleach for one minute, and cultured on acidified potato dextrose agar (APDA). Each isolate was single-spored, transferred to PDA, stored in 15% glycerol at -80°C and maintained at room temperature under blacklight blue and fluorescent bulbs on a 12-hour light-dark cycle. Colonies produced white-pink mycelia with a dark red pigment on the undersides. Conidia collected after 7-9 days were falcate and septate (5 to 6). No microconidia were produced. Macroconidia measured 35.4-49.7 µm x 3.4-22 5.8 µm (n=50).

First report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa in Oregon
Michele S. Wiseman, Taylor A. Bates, Andrea R. Garfinkel, Cynthia M. Ocamb, David. H. Gent
Plant Dis. 2021 Feb 2.
doi: 10.1094/PDIS-11-20-2455-PDN
https://apsjournals.apsnet.org/doi/p...11-20-2455-PDN
Oregon is the second largest producer of hemp in the United States with 25,900 ha of hemp licensed to growers in 2019, a nearly six-fold increase over the previous year (Perkowski 2019, Capital Press). Industrial hemp has a wide range of uses including textiles to nutritional supplements; in Oregon, hemp has become one of the most economically promising crops and is mainly cultivated for cannabidiol (CBD) production. Between 2018 and 2019, multiple independent greenhouse growers in western Oregon reported powdery mildew-like signs and symptoms on leaves and buds of several Cannabis sativa cultivars, including ‘Cherry Wine’

First report of Fusarium proliferatum causing crown and stem rot, and pith necrosis, in cannabis (Cannabis sativa L., marijuana) plants
Zamir K. Punja
Canadian Journal of Plant Pathology 2021 Vol. 43, No. 2, 236–255,
DOI: 10.1080/07060661.2020.1793222
https://www.tandfonline.com/doi/pdf/...eedAccess=true
Cannabis (Cannabis sativa L., marijuana) plants grown under greenhouse or controlled environments with symptoms of leaf yellowing, leaf necrosis and defoliation were observed during 2018–2019. Additional symptoms included crown rot and internal browning or blackening of the pith tissues. Stock (mother) plants as well as plants in the vegetative and flowering stages of 15 cannabis strains (genotypes) were affected. In addition, damping-off symptoms were observed on rooted cuttings in propagation rooms. Isolations from diseased tissues yielded predominantly Fusarium proliferatum, with some F. oxysporum also recovered. Phylogenetic analysis of sequences from the translation elongation factor 1α (TEF-1 α) region of 29 isolates of F. proliferatum from eight licenced production facilities in three provinces in Canada (British Columbia, Ontario and New Brunswick), and one cannabis production site in northern California, grouped isolates from cannabis with a large clade of isolates from a wide range of other hosts in different geographic regions. Pathogenicity studies confirmed the ability of F. proliferatum to cause symptoms of wilting, leaf and pith necrosis, and plant death on cuttings, rooted plants and stock plants. Inoculated tomato and cucumber plants developed similar symptoms. Stem colonization was more extensive by F. proliferatum compared to F. oxysporum on cannabis cuttings. Both grew optimally at 25°C on agar media although F. oxysporum grew faster than F. proliferatum at all temperatures tested. The occurrence of F. proliferatum on cannabis plants has not been previously reported, adding to recent reports of F. oxysporum

First report of Golovinomyces cichoracearum sensu lato on Cannabis sativa in Israel
M. Maymon, S. Jerushalmi and S. Freeman
New Disease Reports 42, 11.
DOI: 10.5197/j.2044-0588.2020.042.011
https://bsppjournals.onlinelibrary.w.. .8.2020.042.011
In recent years, the use of cannabis (Cannabis sativa) has gained popularity for medical and other purposes, and its cultivation worldwide has expanded rapidly (Ruchlemer et al., 2015; Jerushalmi et al., 2020).
During April 2020, symptoms of powdery mildew were observed on commercially cultivated medical cannabis in the greenhouses of several farms in Israel, causing serious concerns, since affected material was discarded, deemed unfit for medical consumption. Symptoms initially appeared as small, white circular patches of epiphytic mycelia with conidia on the upper sides of mature leaf surfaces, similar to those described by Pépin et al. (2018). As the disease progressed, colonies expanded in size, coalescing and covering entire leaf surfaces, succulent stems and inflorescences (Fig. 1). Conidia were produced in chains on conidiophores that were single and erect, unbranched and cylindrical, arising from the colonies (Fig. 2). The conidia were hyaline, cylindrical to ellipsoid in shape, measuring 38.8-45.4 µm in length and 15.9-21.1 µm in width (Fig. 3). No chasmothecia were observed.
The pathogen was identified by molecular analyses and sequencing of the internal transcribed spacer (ITS) region of rDNA following amplification by PCR using ITS1 and ITS4 primers. Sequences were deposited in GenBank (Accession Nos. MT791387- MT791389), and BLAST analyses revealed 100% identity to Golovinomyces cichoracearum sensu lato, as recently reported from cannabis plants in Canada (Pépin et al., 2018). Due to its complex taxonomy, G. cichoracearum is considered a species complex, composed of specialised as well as less specialised races, taxa or even cryptic species.

First report of Golovinomyces spadiceus causing powdery mildew on industrial hemp (Cannabis sativa L.) in Ohio.
Farinas, C., & Peduto Hand, F.
Plant Disease. (2020).
doi:10.1094/pdis-01-20-0198-pdn
Industrial hemp cultivation is highly valued in North America for oilseed production as well as cannabidiol (CBD), which are considered having many therapeutic qualities (Cherney et al. 2016). The 2018 Farm Bill’s decriminalization of hemp has expanded the ability of states to cultivate and process hemp and transfer hemp-derived products across state lines, paving the way to new business opportunities for US farmers. At the same time, it has incentivized
universities across the nation to engage in hemp research and provide extension services to farmers. In August 2019, patches of white powdery fungal growth typical of powdery mildew were observed covering the older leaves of two-month old plants of Cannabis sativa cv. Tangerine maintained in the research greenhouse of the Department of Plant Pathology at the Ohio State University in Columbus, OH. Approximately 80% of the plants within the greenhouse were affected.

First Report of Hemp Canker Caused by Sclerotinia sclerotiorum in Alberta, Canada
P. S. Bains, H. S. Bennypaul, S. F. Blade, and C. Weeks
Plant Disease Vol. 84, No. 3 March 2000
DOI: 10.1094/PDIS.2000.84.3.372B
Hemp (Cannabis sativa L.) is a herbaceous annual grown mainly for its blast fiber and seed oil. In 1999, Health Canada issued licenses to plant 12,145 ha of hemp in Canada. Of these, 730 ha were in Alberta. During the last week of August, hemp plants (cv. Fasamo) in a central Alberta field showed the following symptoms and signs: wilting foliage turning light brown; dry tan to gray lesions on stems; shredding and breaking of stems at the lesion; presence of white mycelium in the lesion; and black round, irregular, or oblong sclerotia (up to 5 mm diameter and 2 to 11 mm long) present externally at the lesion on the stem and inside the pith cavity. Lesions were found at the crown, near the inflorescence, and along the entire stem length. Disease incidence in a survey of six commercial fields (40 ha) ranged from 1 to 8%. The organism isolated from lesions on potato dextrose agar produced white aerial mycelia and large numbers of sclerotia characteristic of Sclerotinia sclerotiorum. Pathogenicity was confirmed by inoculating 23-day-old greenhouse-grown hemp plants (cv. Fasamo) with autoclaved wheat grains colonized for 14 days with a S. sclerotiorum culture previously isolated from an infected hemp plant. The grains were placed on soilless growing medium near the plant and covered very lightly. One week after inoculation, grayish lesions appeared on the stems, white mycelia appeared on lesions, and plants wilted. The pathogen was reisolated from the lesions. This is the first report of S. sclerotiorum on hemp in Alberta, Canada. The disease known as hemp canker has been reported to cause severe losses under cool wet conditions in the Netherlands

First Report of Hemp Leaf Spot Caused by a Bipolaris Species on Hemp (Cannabis sativa) in Kentucky.
Szarka, D., Amsden, B., Beale, J., Dixon, E., Schardl, C. L., & Gauthier, N.
Plant Health Progress, 82–84.(2020).
doi:10.1094/php-01-20-0004-br
First Report of Hemp Leaf Spot Caused by a Bipolaris Species on Hemp (Cannabis sativa) in Kentucky
Upon reintroduction of hemp (Cannabis sativa) in 2014, reports of a leaf spot disease became increasingly common in Kentucky. Outdoor-grown plants became severely affected with necrosis and blight symptoms, and many crops were rejected by processors either as a result of leaf and bud necrosis or as a result of reduced levels of cannabidiol. Morphological data and sequences of ITS and partial 28S rDNA identified the pathogen as Drechslera gigantea. Phylogenetic analysis grouped all isolates in a clade within Bipolaris. Dicot and monocot weed hosts within symptomatic fields were also identified. We refer to the disease as Bipolaris leaf spot, but the common reference is hemp leaf spot. This is the first report of a Bipolaris pathogen infecting C. sativa. Widespread distribution, disease severity, and extreme yield losses makes this one of the most important diseases of hemp in Kentucky.

First Report of Meloidogyne enterolobii on Industrial Hemp (Cannabis sativa) in China.
Ren, Z., Chen, X., Luan, M., Guo, B., & Song, Z.
Plant Disease. (2020).
doi:10.1094/pdis-07-20-1451-pdn
Industrial hemp (Cannabis sativa L.) is an important annual herbaceous plant that has great economic value. In March 2020, many small to large galls were observed on the roots of industrial hemp plants growing in a field in Tianya District, Sanya City, Hainan Province, China. The diseased plants did not show obvious aboveground symptoms. Females were obtained by dissecting the galls under a stereomicroscope. Second-stage juveniles (J2s) were collected for 24–48 h from egg masses hatching at 25°C. The morphological characteristics of females and J2s were observed and measured with a Nikon E200 microscope at 100× and 400× magnification. The perineal patterns of females were oval, with coarse and smooth striae, moderately high to high dorsal arches, and lacking distinct lateral lines.

First report of Meloidogyne incognita infecting Cannabis sativa in Alabama
Bisho R. Lawaju, William Groover, Jessica Kelton, Kassie Conner, Edward Sikora and Kathy S. Lawrence
JOURNAL OF NEMATOLOGY e2021-52 | Vol. 53
DOI: 10.21307/jofnem-2021-052
https://www.ncbi.nlm.nih.gov/pmc/art...nem-53-052.pdf
Hemp (Cannabis sativa L.) is a new crop in Alabama. In 2019, symptomatic plants with stunted growth, poor root development, and numerous galls were observed in hemp plants grown in Geneva County, AL. After harvest, soil samples were collected from areas with the symptomatic plants and root-knot nematode (Meloidogyne spp.) were found in the soil. Based on morphological features and the polymerase chain reactions using species-specific primers, it was identified as Meloidogyne incognita. Further, a host differential test in a greenhouse assay confirmed it to be M. incognita race 3. The pathogenicity of the nematode to the hemp was confirmed by a modified version of Koch’s postulates. To our knowledge, this is the first report of M. incognita infecting Cannabis sativa in Alabama.

First Report of Meloidogyne javanica Infecting Hemp (Cannabis sativa) in China.
Song, Z. Q., Cheng, F. X., Zhang, D. Y., Liu, Y., & Chen, X. W.
Plant Disease, 101(5), 842. (2017).
doi:10.1094/pdis-10-16-1537-pdn
Hemp (Cannabis sativa L.) is an important annual herbaceous plant for its bast fiber, seed oil and psychoactive substances. In China, hemp cultivation has more than 6000 years of history, and planting area of seed hemp and fiber hemp accounted for 40-50% of the world total in the last decade (Amaducci et al. 2015). The root-knot nematode Meloidogyne javanica can infect hemp cultivars in South Africa (Pofu and Mashela 2014), but damage to this plant caused by M. javanica has notbeen reported in China until now.

First report of Neofusicoccum parvum causing dieback and canker disease on hemp in the United States
C. Feng, M. I. Villarroel-Zeballos, P. F. Ficheux, H. Zima, B. D.S. Dhillon, J. C. Correll
Plant DiseaseVol. 104, No. 11 November 2020
Doi: 10.1094/PDIS-03-20-0486-PDN
Hemp (Cannabis sativa L.) had been grown as a fiber crop in the U. S. for over 300 years prior to it being banned as schedule 1 drug in 1970 (Small and Marcus 2002). After the farm bill legislation in 2018, hemp was reintroduced as a crop into Arkansas where approximately 700 ha were grown in 2019. In July, on a single farm in Searcy Co., AR, one to two-month old hemp plants (cultivars: Trump and Cherry Wine) were observed with leaf discoloration and curling, and branch dieback with distinct lesion margins on the stem. The dieback was predominantly on the main terminal. Approximately 10% of the plants were observed with dieback symptoms. The infected branches and the whole plants with terminal infections often would die, significantly impacting yield. Small pieces from symptomatic stems and leaves of the field samples were surface sterilized in 10% bleach for one minute, washed in sterile deionized water three times, and then placed on water agar (WA). After three days, mycelia growing out from both leaf and stem tissue was transferred onto potato dextrose agar (PDA) to recover pure cultures. Seventeen isolates recovered from eight separate plants had an identical colony morphology. Seven-day old colonies developed grey aerial mycelia, but no conidia. However, pycnidia, containing a large number of both septate and nonseptate conidia, were found on the WA surface after seven days, or on symptomatic plant tissue incubated in a moist chamber for 48 h. The average size of conidia (n=20) was 22.9±1.6 µM long by 12.8±1.1 µM wide.

First Report of Powdery Mildew Caused by Golovinomyces spadiceus on Industrial Hemp ( Cannabis sativa ) in Kentucky
Desiree Szarka, Lydia Tymon, Bernadette Amsden, Nicole Gauthier, Ed Dixon, Justin Kirk Judy
February 2019 Plant Disease
DOI: 10.1094/PDIS-01-19-0049-PDN
Industrial hemp (Cannabis sativa) was reintroduced to the United States as a pilot research program under the 2014 Farm Bill. By 2017, there were over 25,000 acres of industrial hemp in the United States, with Kentucky having the second highest acreage in the United States (https://www.votehemp.com/resources/publications/). Hemp is processed for fiber (4%), grain/seed (18%), and cannabidiol (CBD, 62%), and grosses $7.5M for Kentucky growers. Between 2014 and 2018, powdery mildew was observed in numerous greenhouses, in multiple locations, and on several varieties including but not limited to Cherry Wine, Endurance, Otto, proprietary CBD varieties, and fiber and grain breeding lines. Symptoms initially appeared as small, inconspicuous white patches on the adaxial side of leaves. Colonies expanded in size, often coalescing and infecting entire leaves and succulent stems. The disease spread readily to asymptomatic plants. Pathogenicity of three isolates collected from hemp specimens was confirmed through touch inoculation where conidia from infected leaves were pressed onto asymptomatic leaves. Inoculated plants were moist chambered for 48 h and maintained in the greenhouse. Symptoms appeared within 6 to 7 days, and morphological features were identical to the original; noninoculated control plants did not develop symptoms. Mycelia were amphigenous and occasionally caulicolous; hyphae were septate with septations 5 to 6 µm apart. Foot cells were cylindrical, measuring (42 to) 57 to 107 (to 120) µm × 9 to 11 µm, followed by one to two shorter cells. Conidiophores were hyaline, singular, and erect, measuring (80 to) 115 to 187 (to 209) µm in length, followed by two to three immature conidia forming a crenate outline. Conidia were catenescent and ellipsoid to ovoid, measuring (29 to) 30 to 39 (to 41) µm × (13 to) 15 to 20 (to 22) µm. Chasmothecia readily formed during autumn, were round and dark brown at maturity, measured (96 to) 109 to 138 (to 159) µm in diameter, and displayed nondescript myceloid appendages. Mature chasmothecia contained five to 15 ovoid-saccate asci, most with short stalks. Asci measured (52 to) 56 to 75 (to 78) µm × (25 to) 29 to 43 (to 50) µm, and each ascus contained two ovoid ascospores measuring (15 to) 18 to 27 (to 32) × (9 to) 11 to 18 (to 19) µm. Morphological characteristics were consistent with descriptions of Golovinomyces spadiceus except that foot cells from these isolates had a wider range of lengths compared with previous reports, 30 to 80 × 9 to 15 µm versus 42 to 116 × 9 to 11 µm (Braun and Cook 2012). Conidial measurements were similar but not identical to G. ambrosiae, which is reported to have a longer conidial length/width ratio (2.0) than G. spadiceus (1.5 to 2.0); these isolates had conidial length/width ratios consistent with G. spadiceus (Braun and Cook 2012). Identification was confirmed by sequencing the 28S and internal transcribed spacer (ITS) regions with primers PM5G/NLP2 for the 3? half of ITS and 28S and ITS5/PM6G for the 5? half of ITS (Bradshaw et al. 2017). Sequence data were deposited into GenBank (MK305282). A GenBank BLAST search resulted in a 100% similarity to G. spadiceus (GenBank accession AB769427) and 97% similarity to G. asterum, G. orontii, and G. cichoracearum. The latter species were eliminated based on strong inconsistences in morphological comparisons. This species was determined to be G. spadiceus based on morphological features and sequence data. This determination is consistent with those of powdery mildew fungi collected from Cannabis in Canada (Pépin et al. 2018). All samples collected during this period were consistent; no other powdery mildew species was identified. This is the first documented report of G. spadiceus causing powdery mildew on hemp in the United States. With recent legalization of hemp in the United States, it is important to document the species and distribution of powdery mildew fungi affecting this crop.

First report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa in Oregon
Michele S. Wiseman, Taylor A. Bates, Andrea R. Garfinkel, Cynthia M. Ocamb, David. H. Gent
Plant Disease (2021)
https://apsjournals.apsnet.org/doi/p...11-20-2455-PDN
Oregon is the second largest producer of hemp in the United States with 25,900 ha of hemp licensed to growers in 2019, a nearly six-fold increase over the previous year (Perkowski 2019, Capital Press). Industrial hemp has a wide range of uses including textiles to nutritional supplements; in Oregon, hemp has become one of the most economically promising crops and is mainly cultivated for cannabidiol (CBD) production. Between 2018 and 2019, multiple independent greenhouse growers in western Oregon reported powdery mildew-like signs and symptoms on leaves and buds of several Cannabis sativa cultivars, including ‘Cherry Wine’.

First Report of Pythium aphanidermatum Crown and Root Rot of Industrial Hemp in the United States
Janna L. Beckerman, Hannah Nisonson, Nicolette Albright, Tom C. Creswell
February 2017 Plant Disease 101(6)
DOI: 10.1094/PDIS-09-16-1249-PDN
During June and July 2015, crown and root rot symptoms were observed on industrial hemp (Cannabis sativa cv. Alyssa and cv. Canda), in research plots in Lafayette, IN. Record setting rainfall in Indiana during June (218.4 mm) and July (162.6 mm), may have factored into this outbreak. Soil type is Crosby-Miami complex alfisol. Symptom development appeared 13 days after sowing with temperatures ranging from 25 to 30°C. Leaves of affected plants were chlorotic, and plants were stunted and often wilted. Brown lesions on roots, and loss of feeder roots were observed when symptomatic plants were removed from soil; symptomatic plants often but not always possessed brown, water-soaked stem lesions. A small percentage of affected plants collapsed, but most persisted in stunted growth. Thin, aerial mycelia were visible on the stem surface of some of the infected plants.

First report of Pythium ultimum causing crown rot in greenhouse grown Cannabis sativa (L.) in California.
Pitman, T. L., Philbrook, R. N., Vetterli, M. R., & Warren, J. G.
Plant Disease. (2020).
doi:10.1094/pdis-10-20-2228-pdn
In April of 2020 cuttings of Cannabis sativa (L.) in a greenhouse in San Mateo County, CA were observed collapsing, and further observation revealed: water-soaked stems, tan discoloration to the cortex, and discolored roots. The greenhouse irrigation system was supplied by a local stream. We collected one liter water samples from: intake pond, reservoir tank, irrigation lines, and local potable water tap. Water samples were filtered and plated as described previously (Rollins et al., 2016). Filter papers were removed after 24 hours. Crown sections from four symptomatic plants and one asymptomatic plant were surfaced sterilized in 10% bleach for five minutes, rinsed in sterile deionized water, cut into fourmillimeter long sections, and plated onto V8 media, then incubated at room temperature for three days. White mycelial growth was observed from foci within the print of the filter paper from all irrigation water samples but not the potable water supply sample. Similar mycelial growth was observed from plated crown tissue from symptomatic plants only. Observation under light microscope revealed characteristics congruent with P. ultimum, including aseptate hyphae and globose sporangia (Watanabe, 2002).

First Report of Pythium ultimum Crown and Root Rot of Industrial Hemp in the United States
Plant Disease
Janna L. Beckerman, Jessica Stone, Gail E. Ruhl, Tom C. Creswell
Plant Disease Apr 2018
DOI: 10.1094/PDIS-12-17-1999-PDN
During June 2016, root rot symptoms were observed on industrial hemp (Cannabis sativacv. ‘Canda’), in research plots in Lafayette, IN. Rainfall in Indiana during June (127.8 mm) of 2016 was similar to previous averages of 116.1, although a severe rainfall event on 4 June (47.2 mm), 2 days after planting, resulted in physical crusting of the soil surface. Symptom development first appeared 14 days after sowing with temperatures ranging from 25 to 30°C. Affected plants were stunted with chlorotic leaves. Most of the symptomatic plants simply persisted in stunted growth. A sampling of 12 symptomatic plants was removed from the soil, and brown, water-soaked root lesions were observed. Plants were rinsed in tap water to remove soil debris and then 1 to 2 in. was clipped from the bottom of each seedling root to use on Pythium selective medium.

First Report of Root Rot and Wilt Caused by Pythium myriotylum on Hemp (Cannabis sativa) in the United States
C. S. McGehee, P. Apicella, R. Raudales, G. Berkowitz, Y. Ma, S. Durocher and J. Lubell
Plant Disease Vol. 103, No. 12 December 2019
DOI: 10.1094/PDIS-11-18-2028-PDN
In September 2018, a hemp plant (Cannabis sativa L. cv. Dinamed) presented severe wilting, root rot, and mortality in a research greenhouse at the University of Connecticut in Storrs, CT. The hemp plant was grown in a 11.36-liter container with soilless peat-based potting mix (SunGro Fafard 3B, Agawam, MA, U.S.A.). The plants were in a polycarbonate greenhouse with a daytime heating set point of 20°C and a ventilation set point of 26°C under 18 h of supplemental lighting. Roots were collected from the symptomatic hemp plant, washed three times with sterile deionized water, blotted dry, and plated on PARP selective medium (Jeffers and Martin 1986). The plates were incubated in the dark at 21°C for 48 h. Mycelia resembling the morphology of Pythium species were observed in all plates.

First Report of Serratia marcescens Causing a Leaf Spot Disease on Industrial Hemp (Cannabis sativa L.).
Schappe, T. L., Thiessen, L., Ritchie, D. F., & Thiessen, L. D.
Plant Disease. (2019).
doi:10.1094/pdis-04-19-0782-pdn
In the 2017 and 2018 growing seasons (between May and October), several cultivars of industrial hemp plants grown for flowers in greenhouse production from four North Carolina counties showed symptoms of an angular leaf spot on leaves, stems, and flower parts. Lesions were initially small, dark brown, 1 to 3 mm in size, and vein-limited. As the disease progressed, lesions coalesced to form larger regions of necrosis that engulfed large portions of leaves, and whole plants were lost to disease. Red bacterial ooze was observed streaming from the plant tissues.

First Report of Sclerotinia Crown Rot Caused by Sclerotinia minor on Hemp
S. T. Koike, H. Stanghellini, S. J. Mauzey and A. Burkhard
Plant Disease Vol. 103, No. 7 July 2019
DOI: 10.1094/PDIS-01-19-0088-PDN
In summer 2018, commercial field grown hemp (Cannabis sativa L.) plants in San Benito County, CA, showed signs of a disease. Leaves initially wilted and turned dark green. Foliage eventually dried up completely. The base of the plant crown in contact with soil supported the growth of white to gray mycelium and small (0.5 to 3 mm diameter), irregularly shaped, black sclerotia. Crown tissue beneath the epidermis was necrotic. However, plant roots appeared symptomless. To estimate disease incidence, eight replicates of 100 plants each were assessed for collapse symptoms and sclerotia on plant crowns. Overall disease incidence was limited and was approximately 1%. To test for pathogens, symptomatic crown pieces were surface sterilized in 0.006% NaOCl for 2 min and plated on acidified (2 ml of 25% lactic acid/liter) corn meal agar. After 2 to 3 days, rapidly growing, colorless, appressed mycelium emerged from crown pieces

First report of Sclerotinia sclerotiorum causing stem canker on Cannabis sativa L. in Oregon
A.R. Garfinke
CBD Oregon
https://apsjournals.apsnet.org/doi/p...10-20-2142-PDN
In August of 2020, plants of Cannabis sativa L. grown in hoop houses at two farms located in Benton County, Oregon exhibited wilting and chlorosis, followed by shoot necrosis. Symptomatic plants had dry, tan-brown lesions or cankers, often accompanied by large, round to irregular or ribbon-shaped, black sclerotia and/or profuse white mycelial growth. Lesions or cankers were observed on the stems at both the plant crown (soil) level and higher in the canopy; flower infections were not observed. Sclerotia were removed from two infected plants and placed on potato dextrose agar (PDA) at room temperature. Fast-growing, pure white, largely appressed, sterile mycelium grew radially from plated sclerotia. Hyphal tips were transferred to obtain a pure culture. Additional sclerotia, solitary and aggregate, approximately 30 to more than 50 per plate, exhibiting identical features to those observed on plant tissue, formed in culture 6-7 days following transfer and ranged in size from 2 to 11 mm in length or width (n=50). Mycelia were aseptically harvested from cultures for DNA extraction (Quick-DNA Plant/Seed Miniprep Kit, Zymo Research). Primers ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer region (ITS) and primers G3PDHfor and G3PDHrev were used to amplify the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Staats et al. 2005) from a single isolate, LAS01. The ITS region from LAS01 (MW079844) shared 100 to >99% homology to several Sclerotinia species isolates in GenBank

First Report of Southern Blight Caused by Sclerotium rolfsii on Hemp (Cannabis sativa) in Sicily and Southern Italy.
Pane A, Cosentino SL, Copani V, Cacciola SO
Plant Dis. 2007 May;91(5):636.
doi: 10.1094/PDIS-91-5-0636A
Hemp (Cannabis sativa L.), family Cannabaceae, is an annual herbaceous plant that is 1.5 to 4.0 m tall and native to the Caucasus Region, northern India, and Iran. It is cultivated in warm to temperate regions worldwide for its fiber, oil, and psychoactive substances. In Europe, commercial plantings have decreased from 52,872 ha in 1989 to 18,716 ha in 2005. Recently however, cultivation of hemp as a natural fiber species has been encouraged by European Union policy (2). During the summer of 2003, patches of dead plants were observed in test plots of 12 monoecious and dioecious hemp cultivars (Beniko, Epsylon 68, Felina 34, Ferimon, Fedora 17, Futura 75, Bialobrzeskie, Dioica 88, Fibranova, Tiborszallasi, Lovrin, and Carmagnola) in an experimental field near Catania (eastern Sicily) previously planted to artichoke (Cynara scolymus L.). Plots were irrigated with a drip irrigation system. Symptoms were first detected in July with day/night temperatures between 35 and 26°C. Infected plants showed a dark brown-to-tan discoloration of the stem near the soil line. As disease progressed, the rot extended down to the crown and taproot, foliage became yellow, and the entire plant eventually collapsed. An extensive white, cottony mycelium and numerous spherical tan-to-dark brown sclerotia (0.5 to 4.0 mm in diameter) developed externally on infected tissues and soil. As much as 60% of the plants were affected in a single plot. Monoecious cultivars that had been planted earlier escaped the disease. Isolations from diseased tissues were performed by plating symptomatic tissues previously disinfected for 1 min in 1% NaOCl and rinsed in sterile water on acidified potato dextrose agar (pH 4.5). Isolations consistently yielded a fungus whose characters corresponded to Sclerotium rolfsii Sacc. (teleomorph Athelia rolfsii (Curzi) Tu & Kimbrough). Pathogenicity of two isolates obtained from infected plants was confirmed by inoculating 120-day-old hemp plants grown in individual pots. Twenty plants for each of the above listed cultivars (10 plants for each isolate) were inoculated by applying toothpick tips (5 mm) colonized by S. rolfsii to the lower part of the stem. Ten noninoculated plants served as controls. Plants were kept in a greenhouse with temperatures between 26 and 32°C and 95% relative humidity. High soil moisture was maintained with frequent watering. All inoculated plants showed blight and basal stem rot after 2 weeks, irrespective of the cultivar. By the third week, plants began to dry up and mycelium and sclerotia developed on the crown. Noninoculated controls remained symptomless. S. rolfsii was reisolated from inoculated plants. Although S. rolfsii has been reported on hemp in India since the 1930s (3), to our knowledge, this is the first report of southern blight caused by this fungus on C. sativa in Sicily and southern Italy. Residues of artichoke, a very susceptible host of S. rolfsii (1), might have been the source of inoculum for this outbreak on hemp. Most likely, high summer temperatures and overirrigation exacerbated the disease severity.

First Report of white root rot of hemp (Cannabis sativa L.) caused by Dematophora necatrix in Campania region (Southern Italy)
R. Sorrentino, G. M. Baldi, V. Battaglia, F. Raimo, G. Piccirillo, E. Lahoz
Plant Dis. 2021 Apr 1
https://apsjournals.apsnet.org/doi/p...07-20-1521-PDN
Industrial hemp (Cannabis sativa L.) was cultivated in Italy until the end of Second World War. Since then, it
has been abandoned and substituted with other crops mainly due to legal restrictions and public concerns.
Public legislation passed in 2016, has allowed for the production of hemp seeds, flowers and fibers (law n.
242/2016). During a 2019 survey on hemp sanitary status in the province of Naples (40°57'6"12 N,
14°22'37"56 E), plants ‘Kompolty’ with symptoms of root rot were observed at a private farm and collected
for further analysis at the phytosanitary laboratory of CREA in Caserta. Death generally occurred within 2-3
weeks after the appearance of the first symptoms, occurring on ca. 10% of plants, consisting of yellowing,
canopy wilt and signs of roots covered with white mycelium and fan-like mycelium under the bark. The causal
agent, was isolated from small root segments were excised from symptomatic plants, the surface was
disinfected with 2% sodium hypochlorite, placed on potato dextrose agar (PDA) amended with streptomycin
sulphate (100mg/L) and incubated in the dark at 25°C for 5 days. Small pieces (2-3 mm) at the edge of the
resulting colonies were sub-cultured onto PDA and incubated at 25°C in the dark for one week. The mycelia
from 15 isolates showed pear-shaped swellings adjacent to the septa.

First Report of Witches'-Broom Disease in a Cannabis spp. in China and Its Association with a Phytoplasma of Elm Yellows Group (16SrV).
Zhao Y1, Sun Q1, Davis RE1, Lee IM1, Liu Q2.
Plant Dis. 2007 Feb;91(2):227.
doi: 10.1094/PDIS-91-2-0227C.
Hemp fiber plants (Cannabis spp.) spread naturally in almost every climate zone in China and have a long history of cultivation in the country (1). While hemp stalks provide high-quality fibers for making ropes, clothes, and paper products, hemp seeds are a rich source of edible oil. During the summer of 2004, a disease characterized by witches'-broom symptoms was observed in wild hemp fiber plants growing in suburban Taian, Shandong, China. The diseased plants developed clusters of highly proliferating branches with much shortened internodes and leaves on the affected branches were significantly reduced in size. Phytoplasma infection was suspected in this hemp fiber witches'-broom (HFWB) disease because of the typical symptoms and because of its geographic location where other phytoplasmal diseases such as jujube witches'-broom (JWB), paulownia witches'-broom (PaWB), paper mulberry witches'-broom (PMWB), and Chinese wingnut witches'-broom (CWWB) diseases were previously reported (3,4). Total DNA was extracted from leaves of four diseased and four nearby healthy looking hemp fiber plants. Nested PCR were carried out on the DNA samples using phytoplasma universal 16S rDNA primers (P1A/16S-SR and R16F2n/R16R2) (2). Results revealed that all examined diseased plants were infected by phytoplasma, whereas nearby healthy looking plants were phytoplasma free. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA R16F2n/R16R2 fragment indicated that the phytoplasma associated with HFWB disease belongs to subgroup 16SrV-B of the elm yellows (EY) phytoplasma group. Nucleotide sequence analysis of the cloned HFWB phytoplasma partial rRNA operon (GenBank Accession No. EF029092), spanning a near full-length 16S rRNA gene and a partial 16S-23S rRNA intergenic spacer, suggested that HFWB phytoplasma is most closely related to JWB and PMWB phytoplasmas, both members of subgroup16SrV-B. To further characterize the HFWB phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F1A/rp(V)R1A (2), cloned, and sequenced (GenBank Accession No. EF029093). The nucleotide sequence of the HFWB phytoplasma rplV and rpsC locus is nearly identical (99.9%) to that of JWB phytoplasma. To our knowledge, this is the first report of a phytoplasmal disease in Cannabis spp. Since HFWB and JWB phytoplasmas share extremely high sequence identity and share the same eco-geographic location, further investigation is warranted to determine whether these two phytoplasmas are actually one species that can infect both plants, an issue having important implications in managing both diseases. References: (1) S. Hong and R. C. Clarke. J. Int. Hemp Assoc. 3:55, 1996. (2) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) Q. Liu et al. Plant Dis. 88:770, 2004. (4) Q. Liu et al. Plant Dis. 89:529, 2005.

Flower and foliage-infecting pathogens of marijuana (Cannabis sativa L.) plants.
Punja, Z. K.
Canadian Journal of Plant Pathology. (2018).
doi:10.1080/07060661.2018.1535467
Flower buds of Cannabis sativa develop as inflorescences (buds) which are harvested and dried prior to sale. The extent to which fungal plant pathogens can colonize the buds prior to harvest has not been previously studied. Flower buds were sampled at various preharvest and harvest time periods during 2015–2017 at locations in British Columbia and Alberta to determine the range of fungi present. Isolated fungi were inoculated onto developing buds to determine the extent of tissue colonization. A pre- and post-harvest internal rot was associated with Botrytis cinerea, causing botrytis bud rot. In addition, two species of Penicillium – P. olsonii and P. copticola – were recovered from pre-harvest flower buds, as well as dried buds, and shown to cause penicillium bud rot. Scanning electron microscopy studies revealed colonization and sporulation on bracts and stigmas of the flower buds by P. olsonii. Several Fusarium species, which were identified using ITS rDNA sequences as F. solani, F. oxysporum and F. equiseti, were isolated from pre-harvest flower buds. These fungi colonized the flower buds following artificial inoculation and caused visible rot symptoms. The most severe symptoms were caused by F. solani, followed by F. oxysporum and, to a much lesser extent, F. equiseti. Powdery mildew infection of the foliage and flower buds was caused by Golovinomyces(Erysiphe) cichoracearum. The pathogen was detected on young vegetatively propagated cuttings and sporulation was abundant on older plants and on flower buds. The various fungi recovered from cannabis flower buds may be present as contaminants from aerially dispersed spores and have the potential to cause various types of pre- and post-harvest bud rot under conducive environmental conditions. Powdery mildew may be spread through aerially disseminated spores and infected propagation materials. Management of these pathogens will require monitoring of the growth environment for spore levels and implementation of sanitization methods to reduce inoculum sources.

Not directly Cannabis related
Fomitopsis betulina (formerly Piptoporus betulinus): the Iceman's polypore fungus with modern biotechnological potential
Małgorzata Pleszczyńska , Marta K Lemieszek , Marek Siwulski , Adrian Wiater , Wojciech Rzeski , Janusz Szczodrak
World J Microbiol Biotechnol (2017) 33:83 . 2017 May;33(5):83.
doi: 10.1007/s11274-017-2247-0
https://www.ncbi.nlm.nih.gov/pmc/art...ticle_2247.pdf
Higher Basidiomycota have been used in natural medicine throughout the world for centuries. One of such fungi is Fomitopsis betulina (formerly Piptoporus betulinus), which causes brown rot of birch wood. Annual white to brownish fruiting bodies of the species can be found on trees in the northern hemisphere but F. betulina can also be cultured as a mycelium and fruiting body. The fungus has a long tradition of being applied in folk medicine as an antimicrobial, anticancer, and anti-inflammatory agent. Probably due to the curative properties, pieces of its fruiting body were carried by Ötzi the Iceman. Modern research confirms the health-promoting benefits of F. betulina. Pharmacological studies have provided evidence supporting the antibacterial, anti-parasitic, antiviral, anti-inflammatory, anticancer, neuroprotective, and immunomodulating activities of F. betulina preparations. Biologically active compounds such as triterpenoids have been isolated. The mushroom is also a reservoir of valuable enzymes and other substances such as cell wall (1→3)-α-D-glucan which can be used for induction of microbial enzymes degrading cariogenic dental biofilm. In conclusion, F. betulina can be considered as a promising source for the development of new products for healthcare and other biotechnological uses.

Fool’s gold: diseased marijuana and cannabis hyperemesis syndrome
Oscar Armando Dorantes
J Investig Med 2021;69:1063–1064.
doi:10.1136/jim-2021-001980
https://jim.bmj.com/content/jim/69/5/1063.full.pdf
I am writing in response to the article by Gajendran et al1 and the editorial by Ron Shay, 'Cannabis hyperemesis syndrome: the conundrum
is here to stay'.2 As an emic cultural investigator (anthropologist) and from my vantage point, the problem of cannabis hyperemesis syndrome could easily go away just as quickly as it has appeared. There is a large disconnect between the professional medical community and the marijuana subculture. The majority of the latter are somewhat in denial that cannabis is the cause of this condition and, on the flip side, doctors feel strongly about their conclusions. Perhaps both can be right, because in the marijuana subculture community there are two types of marijuana: 'the sticky, skunky weed' or the 'the bunk weed', 'the dank or the regs' or otherwise viewed as the 'good stuff versus not good stuff'. Were any of the samples of consumed marijuana analyzed during the marijuana hyperemesis diagnosis, as presented in Gajendran et al's paper?

Fungal Pathogens Affecting the Production and Quality of Medical Cannabis in Israel.
Jerushalmi, S., Maymon, M., Dombrovsky, A., & Freeman, S.
Plants, 9(7), 882. (2020).
doi:10.3390/plants9070882
The use of and research on medical cannabis (MC) is becoming more common, yet there are still many challenges regarding plant diseases of this crop. For example, there is a lack of formal and professional knowledge regarding fungi that infect MC plants, and practical and effective methods for managing the casual agents of disease are limited. The purpose of this study was to identify foliar, stem, and soilborne pathogens affecting MC under commercial cultivation in Israel. The predominant major foliage pathogens were identified as Alternaria alternata and Botrytis cinerea, while the common stem and soilborne pathogens were identified as Fusarium oxysporum and F. solani. Other important fungi that were isolated from foliage were those producing various mycotoxins that can directly harm patients, such as Aspergillus spp. and Penicillium spp. The sampling and characterization of potential pathogenic fungi were conducted from infected MC plant parts that exhibited various disease symptoms. Koch postulates were conducted by inoculating healthy MC tissues and intact plants with fungi isolated from infected commercially cultivated symptomatic plants. In this study, we report on the major and most common plant pathogens of MC found in Israel, and determine the seasonal outbreak of each fungus.

Fusarium and Pythium species infecting roots of hydroponically grown marijuana (Cannabis sativa L.) plants.
Punja, Z. K., & Rodriguez, G.
Canadian Journal of Plant Pathology. (2018).
doi:10.1080/07060661.2018.1535466
An increase in the cultivation of Cannabis sativa (cannabis or marijuana) plants in Canada is becoming associated with increased incidence and severity of various diseases, many of which have not been previously reported. In this study, hydroponically grown C. sativa plants were sampled over a 3-year period (2014-2017) to determine the prevalence of root pathogens. Following isolation, pathogenicity studies were conducted to establish the extent of disease symptoms caused by the recovered microbes. Root rot was found to be caused by two Pythium species - Pythium dissotocum Drechsler and P. myriotylum Drechsler. As well, two Fusarium species were recovered from diseased plants - Fusarium oxysporum Schlecht. emend. Snyder & Hansen and F. solani (Mart.) Sacc. Upon inoculation onto healthy plants, all isolates of Pythium spp. caused browning and a reduction in root mass, accompanied by stunting. Inoculation of plants with F. oxysporum caused browning of roots and crown rot infection, accompanied by pith and vascular discoloration, and in some cases wilting of plants, while root and crown infection was observed with F. solani. Phylogenetic analysis of internal transcribed spacer (ITS) and elongation factor 1 ? (EF-1 ?) sequences revealed that the Fusarium species affecting cannabis plants shared 99-100% sequence homology with isolates causing stem rot and wilt in other hosts, including cumin and tomato, suggesting they were not uniquely adapted to cannabis. The potential for spread of F. oxysporum through the hydroponic system was confirmed by its detection in the recirculating nutrient solution. Furthermore, rooted cuttings obtained from commercial propagators were found to harbor Fusarium root infection that resulted in subsequent stunting, yellowing and occasional death of plants. This demonstrates the potential for long-distance spread of the pathogen. The two Pythium species recovered from cannabis plants have an extremely broad host range and are not unique to this host. An additional species, P. aphanidermatum (Edson) Fitzp., was recovered from diseased plants grown under greenhouse conditions in 2018. The management of these root pathogens on C. sativa will require the evaluation and implementation of sanitization methods, biological
control agents, and chemical products adapted from greenhouse vegetable production practices. The use of pathogen-free propagation materials and identification of potential sources of disease resistance should also become a priority.

Fusarium Oxysporum f. sp. Cannabis Isolated from Cannabis Sativa L.: In Vitro and In Planta Biocontrol by a Plant Growth Promoting-Bacteria Consortium
Marika Pellegrini, Claudia Ercole Carmslo Gianchino, Matteo Bernardi, Loretta Pace and Maddalena Del Gallo
Plants 2021, 10(11), 2436;
DOI: 10.3390/plants10112436
https://mdpi-res.com/d_attachment/pl...0-02436-v2.pdf
Industrial hemp (Cannabis sativa L.) is a multipurpose plant used in several fields. Several phytopathogens attack hemp crops.Fusarium oxysporum is a common fungal pathogen that causes wilt disease in nurseries and in field cultivation and causes high losses. In the present study, a pathogenic strain belonging to F. oxysporum f. sp. cannabis was isolated from a plant showingFusarium wilt. After isolation, identification was conducted based on morphological and molecular characterizations and pathogenicity tests. Selected plant growth-promoting bacteria with interesting biocontrol properties—Azospirillum brasilense,Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Burkholderia ambifaria—were tested against this pathogen. In vitro antagonistic activity was determined by the dual culture method. Effective strains (in vitro inhibition > of 50%) G. diazotrophicus, H. seropedicae and B. ambifaria were combined in a consortium and screened for in planta antagonistic activity in pre-emergence (before germination) and post-emergence (after germination). The consortium counteracted Fusarium infection both in pre-emergence and post-emergence. Our preliminary results show that the selected consortium could be further investigated as an effective biocontrol agent for the management of this pathogen

Fusarium Wilt
John M. McPartland, Karl W. Hillig
Dec 22, 2004 Journal of Industrial Hemp· Vol. 9(2) 2004
DOI :10.1300/J237v09n02_07Copy DOI
Fusarium wilt is caused by two closely-related fungi, Fusarium oxysporum f. sp. vasinfectum and Fusarium oxysporum f. sp. cannabis. The disease was first described on hemp in Eastern Europe about 50 years ago, but is now found throughout the Northern hemisphere. Greenhouse studies demonstrated that all cultivars of Cannabis that were tested are susceptible. Signs and symptoms, life history of the pathogens, and control measures are discussed. Fusarium wilt is a menace to hemp cultivation worldwide because its causal fungi cannot be constrained by known organic control measures. The disease threat is compounded by misguided attempts to spread the fungi around the world to stop illicit marijuana cultivation.

Not Cannabis specific
Genetic Engineering of Terpenoid Metabolism Attracts Bodyguards to Arabidopsis
Iris F. Kappers, Asaph Aharoni, Teun W. J. M. van Herpen, Ludo L. P. Luckerhoff, Marcel Dicke, Harro J. Bouwmeester
SCIENCE 23 SEPT 2005 VOL 309
DOI: 10.1126/science.1116232
Herbivore-damaged plants release complex mixtures of volatiles that attract
natural enemies of the herbivore. To study the relevance of individual components of these mixtures for predator attraction, we manipulated herbivoryinduced volatiles through genetic engineering. Metabolic engineering of terpenoids, which dominate the composition of many induced plant volatile bouquets, holds particular promise. By switching the subcellular localization of the introduced sesquiterpene synthase to the mitochondria, we obtained transgenic Arabidopsis thaliana plants emitting two new isoprenoids. These altered plants attracted carnivorous predatory mites (Phytoseiulus persimilis) that aid the plants’ defense mechanisms

It is actually more important to eliminate any MLO susceptibility genes in a Cannabis variety, to have no PM. While the resistance gene PM1 is an important discovery, elimination of any MLO susceptibility genes will confer better protection to PM. Breeding out PM susceptibility genes provides more robust protection than breeding in PM resistance genes
*Genome-wide characterization of the MLO gene family in Cannabis sativa reveals two genes as strong candidates for powdery mildew susceptibility
Noémi Pépin, Francois Olivier Hebert, and David L. Joly
Biorxiv 2021
DOI: 10.1101/2021.07.16.452661
https://www.biorxiv.org/content/10.1...661v1.full.pdf
Cannabis sativa is increasingly being grown around the world for medicinal, industrial, and recreational purposes. As in all cultivated plants, cannabis is exposed to a wide range of pathogens, including powdery mildew (PM). This fungal disease stresses cannabis plants and reduces flower bud quality, resulting in significant economic losses for licensed producers. The Mildew Locus O (MLO) gene family encodes plant-specific proteins distributed among conserved clades, of which clades IV and V are known to be involved in susceptibility to PM in monocots and dicots, respectively. In several studies, the inactivation of those genes resulted in durable resistance to the disease. In this study, we identified and characterized the MLO gene family members in five different cannabis genomes. Fifteen Cannabis sativa MLO (CsMLO) genes were manually curated in cannabis, with numbers varying between 14, 17, 19, 18, and 18 for CBDRx, Jamaican Lion female, Jamaican Lion male, Purple Kush, and Finola, respectively (when considering paralogs and incomplete genes). Further analysis of the CsMLO genes and their deduced protein sequences revealed that many characteristics of the gene family, such as the presence of 7 transmembrane domains, the MLO functional domain, and particular amino acid positions, were present and well conserved. Phylogenetic analysis of the MLO protein sequences from all five cannabis genomes and other plant species indicated seven distinct clades (I through VII), as reported in other crops. Expression analysis revealed that the CsMLOs from clade V, CsMLO1 and CsMLO4, were significantly upregulated following Golovinomyces ambrosiae infection, providing preliminary evidence that they could be involved in PM susceptibility. Finally, the examination of variation within CsMLO1 and CsMLO4 in 32 cannabis cultivars revealed several amino acid changes, which could affect their function. Altogether, cannabis MLO genes were identified and characterized, among which candidates potentially involved in PM susceptibility were noted. The results of this study will lay the foundation for further investigations, such as the functional characterization of clade V MLOs as well as the potential impact of the amino acid changes reported. Those will be useful for breeding purposes in order to develop resistant cultivars.

Hemp and Cannabis Crop Diseases A guide to field diagnosis and management
Shouhua Wang
Nevada Dept of Agriculture
Power Point https://agri.nv.gov/uploadedFiles/ag...management.pdf and also a Book
www.cabi.org/bookshop/book/9781789246070
Defining Hemp: A Fact Sheet
“Botanically, hemp and marijuana are from the same species of plant, Cannabis sativa, but from different varieties or cultivars. However, hemp and marijuana are genetically distinct forms of cannabis that are distinguished by their use and chemical composition as well as by differing cultivation practices in their production. While marijuana generally refers to the cultivated plant used as a psychotropic drug (whether used for medicinal or recreational purposes), hemp is cultivated for use in the production of a wide range of products, including foods and beverages, personal care products, nutritional supplements, fabrics and textiles, paper, construction materials, and other manufactured and industrial goods. Hemp and marijuana also have separate statutory definitions in U.S. law.”

*Hemp Diseases and Pests Management and Biological Control
J.M. McPartland, Robert Connell Clarke, David Paul Watson
CABI Publishing 251 pages
https://avalonlibrary.net/ebooks/Rob...%20Control.pdf
As the 21st century begins, we are favoured with a worldwide resurgence in hemp cultivation. As global forests dwindle to rampikes and timber limits, we see a glimmer of hope for our future, thanks to a renewed interest in this ancient and humble source of food and fibre. This book is our contribution to hemp's revival. Much of the literature regarding hemp diseases and pests dates back 50 years or more. Further, these publications are frequently buried in obscure agronomy journals. Cultivators of illicit Cannabis have published high-calibre research in the last 25 years, but they published in semi-clandestine "grey journals" such as Sinsenzilla Tips. Our primary effort was to collect this scattered bibliography and assimilate it into a comprehensive and readable format.
Our second effort was to manoeuvre the control of diseases and pests into the 21st century. Most hemp research dates to the days when DDT was considered a glamorous panacea. We must find new control methods for sustainable hemp cultivation. Many "new" pesticides are old, such as pyrethrum, a popular insecticide before the days of DDT. Biological control also is old; the use of biocontrol against hemp pests began around 1886, in France. Etre une tete a Papineau, we see biocontrol and hemp resurging together.

Hemp Diseases in North Carolina
Lindsey Thiessen 2017-2018
https://growingsmallfarms.ces.ncsu.e.. .sen.pdf?fwd=no
• Things learned:
• Potential disease problems for producers in NC
• Good/bad growing conditions for hemp
• Things still unknown:
• Chemical management options
• Can use labeled chemistries under FIFRA 25b list until more added
• Environmental condition impact in 2019

Hemp Pest Spectrum and Potential Relationship between Helicoverpa zea Infestation and Hemp Production in the United States in the Face of Climate Change
Olufemi S. Ajayi and Michelle Samuel-Foo
Insects 2021, 12,940.
DOI: 10.3390/insects12100940
https://www.mdpi.com/2075-4450/12/10/940
Simple Summary: Cultivation of industrial hemp Cannabis sativa in the United States is now being expanded due to the recent legalization of the crop. Multiple insect pests attack the crop. One of the common pests is the corn earworm Helicoverpa zea that causes extensive damage to the marketable parts of hemp. Changing global climate may lead to expansion of the geographic range of insect pests. Thus, growers of this crop in the United States have to face new and intense pest problems now and in the years to come. Here, we assess the potential relationship between corn earworm infestation and hemp production in the US in the face of climate change. We also provide an update on the arthropods associated with hemp cultivation across the US. Climate change can affect aspects of interactions between hemp and corn earworm. Temperature and photoperiod affect the development and diapause process in H. zea. Drought leads to a reduction in hemp growth. Overall, our assessment suggests the selection of varieties resistant to stresses from climate and insects. Host plant diversity may prevent populations of corn earworm from reaching outbreak levels. Ongoing research on effective management of H. zea on hemp is critical.
Abstract: There has been a resurgence in the cultivation of industrial hemp, Cannabis sativa L., in the United States since its recent legalization. This may facilitate increased populations of arthropods associated with the plant. Hemp pests target highly marketable parts of the plant, such as flowers, stalks, and leaves, which ultimately results in a decline in the quality. Industrial hemp can be used for several purposes including production of fiber, grain, and cannabidiol. Thus, proper management of pests is essential to achieve a substantial yield of hemp in the face of climate change. In this review, we provide updates on various arthropods associated with industrial hemp in the United States and examine the potential impact of climate change on corn earworm (CEW) Helicoverpa zea Boddie, a major hemp pest. For example, temperature and photoperiod affect the development and diapause process in CEW. Additionally, drought can lead to a reduction in hemp growth. Host plant diversity of CEW may prevent populations of the pest from reaching outbreak levels. It is suggested that hemp varieties resistant to drought, high soil salinity, cold, heat, humidity, and common pests and diseases should be selected. Ongoing research on effective management of CEW in hemp is critical.


History, Origin, and Diversity of Hop Stunt Disease and Hop Stunt Viroid
T. Sano
Acta horticulturae 1010(1010):87-96 October 2013
DOI: 10.17660/ActaHortic.2013.1010.9
Hop stunt disease first emerged in Japan and reported as “dwarf hop” or “cedar-shaped hop” in the 1940s and the early 1950s. The disease emerged in Korea in 1988 and was confirmed in North America in 2004 and in China in 2007. The diseased plants develop yellowish green leaves and drooped leaf petioles in early to mid-growing season, and results in stunting of main and lateral bines. Depending on hop variety, visual stunting becomes apparent only several years after the infection. More serious is the reduction of the alpha-acid content, which is occasionally accompanied by reduction in the total cone numbers per vine. The alpha-acid content of sensitive cultivars is reduced to less than one half. Viroids are the smallest known pathogens and cause severe to mild diseases in economically important crops. They are single-stranded, circular, and self replicating non-coding RNAs with a size of 250 to 400 nucleotides. Viroid replication is dependent on host transcriptional machinery, and pathogenicity depends entirely on interactions with cellular components of the host. Hop stunt disease (HSD) is caused by the Hop stunt viroid (HpSVd), a member of the Pospiviroidae family. Infection of hops by Apple fruit crinkle viroid also exhibits similar disease symptoms. HpSVd was first discovered from the dwarfed-hop, but soon after, it was found to have infected cultivated grapevines, citrus and stone fruits, including plum, peach, apricot, almond and Jujube. HpSVd is now considered to be a ubiquitous and genetically variable pathogen that has spread among cultivated crops worldwide. Unfortunately, all the HpSVd isolates have a potential to cause hop stunt, and current HSD epidemics in Japan, USA, and China may have originated from inter-specific transmission of HpSVd from cultivated grapevines to hops.

Host-Parasite Relationships in Cannabis
John McPartland, Karl Hillig
October 2005 Journal of Industrial Hemp 10(2):85-104
DOI: 10.1300/J237v10n02_08
Plant taxonomy is primarily based on patterns of morphologicalvariation and geographical distribution. Plant-parasite relationships can also offer clues regarding the phylogeny of the host plant. Many obligate parasites coevolve with their hosts, eventually becoming restricted to an individual taxon. Host restriction may take place at different taxonomic ranks, i.e., parasites may become restricted to one host genus within a plant family, to one species within a genus, or to one infraspecific taxon within a species. This phylogenetic congruence between plants and their parasites may be due to cospeciation or due to co-adaptation (reciprocal adaptive selection). Our study reviewed plantparasite interactions with regard to putative Cannabis taxa. The results suggest that certain parasites may have co-evolved with putative species and biotypes within the Cannabis genus.

Identifying Unknown Microbiological Contaminants in Cannabis
Jack Rudd, Analytical Cannabis
https://www.analyticalcannabis.com/a...annabis-311544
We know microbiological contaminants pose a potential risk to cannabis consumers. In particular, bacteria and fungi may cause opportunistic infections immunocompromised individuals and even dead or dormant organisms may present a threat. As regulations have evolved across the US and Canada, it has become clear that robust, routine microbiological testing is essential in protecting consumer safety for medical patients and recreational users alike. As things stand, a patchwork of different testing regulations mandate a range of testing requirements across the US, and discussions on the best approach to guaranteeing accurate, reliable results are ongoing.
To find out more about the challenges faced and the technology available to the industry, we spoke to Amrita Puri, a Field Marketing Specialist with Bio-Rad, which offers a range of validated molecular- and culture-based
diagnostic tools for the detection of pathogens in cannabis and other products.

Industrial Hemp Crop Diseases What We’ve Seen and What We Know
Shouhua Wang, Ph.D.
State Plant Pathologist Nevada Department of Agriculrure
https://agri.nv.gov/uploadedFiles/ag...203-7-2018.pdf
A plant disease is a result of interaction of three factors:
Host - A susceptible host plant is available
Pathogen - A pathogen is present
Environment - Environmental conditions that favour the host and pathogen to allow disease development

Insecticidal activity and biochemical composition of Citrullus colocynthis, Cannabis indica and Artemisia argyi extracts against cabbage aphid (Brevicoryne brassicae L.).
Ahmed, M., Peiwen, Q., Gu, Z., Liu, Y., Sikandar, A., Hussain, D., Ji, M.
Scientific Reports, 10(1). (2020).
doi:10.1038/s41598-019-57092-5
Plant extracts contain many active compounds, which are tremendously fruitful for plant defence against several insect pests. The prime objectives of the present study were to calculate the extraction yield and to evaluate the leaf extracts of Citrullus colocynthis (L.), Cannabis indica (L.) and Artemisia argyi (L.) against Brevicoryne brassicae and to conduct biochemical analysis by gas chromatography-mass spectrometry (GC-MS). The results suggested that when using ethanol, C. colocynthis produced a high dry yield (12.45%), followed by that of C. indica and A. argyi, which were 12.37% and 10.95%, respectively. The toxicity results showed that A. argyi was toxic to B. brassicae with an LC50 of 3.91mgmL?1 , followed by the toxicity of C. colocynthis and C. indica, exhibiting LC50 values of 6.26 and 10.04mgmL?1 , respectively, which were obtained via a residual assay; with a contact assay, the LC50 values of C. colocynthis, C. indica and A. argyi were 0.22mgmL?1 , 1.96 and 2.87mgmL?1 , respectively. The interaction of plant extracts, concentration and time revealed that the maximum mortality based on a concentration of 20mgL?1 was 55.50%, the time-based mortality was 55% at 72h of exposure, and the treatment-based mortality was 44.13% for A. argyi via the residual assay. On the other hand, the maximum concentration-based mortality was 74.44% at 20mgmL?1 , the time-based mortality was 66.38% after 72h of exposure, and 57.30% treatment-based mortality was aforded by A. argyi via the contact assay. The biochemical analysis presented ten constituents in both the A. argyi and C. colocynthis extracts and twenty in that of C. indica, corresponding to 99.80%, 99.99% and 97% of the total extracts, respectively. Moreover, the detected caryophylleneonides (sesquiterpenes), ?-bisabolol and dronabinol (?9 -THC) from C. indica and erucylamide and octasiloxane hexamethyl from C. colocynthis exhibited insecticidal properties, which might be responsible for aphid mortality. However, A. argyi was evaluated for the frst time against B. brassicae. It was concluded that all the plant extracts possessed signifcant insecticidal properties and could be introduced as botanical insecticides after feld evaluations


IPM for Cannabis Pests
William Quarles
IPM Practitioner, XXXVI (5/6) Published August 2018
https://www.birc.org/IPMPCannabis.pdf
About 35 million people in the U.S. use marijuana on a regular basis. It is the fourth most popular recreational drug after caffeine, alcohol, and tobacco
(Ingraham 2017). Marijuana is legal for medical use in 31 states, and it has been decriminalized in 13 other states. Recreational marijuana is legal for adults in nine states, including Alaska, California, Colorado, Oregon, Maine, Massachusetts, Nevada, Vermont, and Washington. Legalization in Michigan and New Jersey is expected soon. There are only four states where it is totally
illegal according to state law (Wikipedia 2018). Marijuana may be the largest
cash crop in the U.S. with an estimated value of $35.8 billion each year. Its closest rival is soybeans, whose value varies with yields, and recently with tariffs. Hemp also has a lot of economic potential. About $688 million of imported hemp products were sold in the U.S. in 2016 (Strickler 2018). Cultivation of both marijuana and hemp is illegal according to federal law. The federal government lists Cannabis as a Controlled Substances Act Schedule I drug and claims it has no medical value. The federal fate of marijuana is uncertain, but a law legalizing the cultivation of hemp throughout the U.S. was introduced into the U.S. Senate on April 12, 2018 (McConnell
2018).

NOT CANNABIS SPECIFIC
Interactions of insect pheromones and plant semiochemicals
Gadi V.P. Reddy and Angel Guerrero
TRENDS in Plant Science Vol.9 No.5 May 2004
doi:10.1016/j.tplants.2004.03.009
Plant semiochemicals are known to produce a wide range of behavioral responses in insects. Some insects sequester or acquire host plant compounds and use them as sex pheromones or sex pheromone precursors. Other insects produce or release sex pheromones in response to specific host plant cues, and chemicals from host plants often synergistically enhance the response of an insect to sex pheromones. Plant volatiles can also have inhibitory or repellent effects that interrupt insect responses to pheromones and attract predators and parasitoids to the attacking species after herbivory injury. Here, we review different interactions between plant semiochemicals and insect pheromones, paying attention to those that can result in the development of more efficient and reliable programs for pest control

NOT CANNABIS SPECIFIC
Investigation on some biological aspects of Chrysoperla lucasina (Chrysopidae: Neuroptera) on Bemisia tabaci in laboratory conditions
Alinaghi Mirmoayedi
Comm. Appl. Biol. Sci, Ghent University, 77/4, 2012
https://www.researchgate.net/publica...ory_conditions
Bemisia tabaci is one of the most important key pests of many types of cultivated plants. Lacewings (Chrysopidae: Neuroptera) are predatory insects, widely used in biological control programs. Between them green lacewing is a promising biological control agent of pests in green houses and crop fields.
In this study, gravid females of the green lacewing Chrysoperla lucasina (Lacroix) were captured from Sarepolzahab ( altitude 540m, latitude 34°,14´ N 46°,9´ E) in western part of Iran. Collected insects were reared in a growth chamber, under experimental conditions (25±1°C, 70±5% RH and a photoperiod of 16:8 L: D). Different diets were offered to larvae which consisted of a whitefly species B.tabaci, an aphid Myzus persica and also lyophilized powder of drone honeybee (Apis melifera). As different foods were used to nurish larvae, so for each diet, mean larval period were calculated, and finally means were compared to each other. Anova in MSTAT-C was used for analysis of variance, and Duncan multiple range test (DMRT) to compare between means. The results showed that larvae had maximum duration of 27±0.33 days when fed on honeybee lyophilized powder and the minimum value was 17.9±0.3 days for B. tabaci. 25±0.27 day recorded for M. persicae. Food preference of the 3rd instar larvae of green lacewing was surveyed, they showed a food preference to M. persicae, to compare with B. tabaci, as the former has a bigger body size, so more easily to be captured by the predator larvae. The 3rd instar larvae of lacewing were more voracious on preys, than the 1st or the 2nd instar larvae. Statistically speaking, there were a significantly difference when mean of different preys consumed by predator larvae were compared. We found, that when the predator larvae have fed on B.tabaci, their development time was shorter, and when arrived to adult stage, the adults showed, an improved fertility. The results indicated that the suitable prey not only can increase the rate of through accelerating developmental stages of the predator and by means of an increase in its pupal body weight consequently promoting the fecundity of resulting adults, but also can alter predators population density in relation to own production numbers.


Is Irradiation Treatment on Cannabis Too Good to be True
Medicinal Genomics Jan 3 2022
www.medicinalgenomics.com/irradiation-treatment-cannabis/


Isolation of the pathogens causing hemp stem disease
X. He, S. Gang Geng, K. Li, N. Zheng
January 2016
DOI: 10.5013/IJSSST.a.17.29.19
Hemp, with very strong vitality, is a kind of annual tall herbaceous plant. Hemp is naturally hardy and drought tolerant which grows well in a cold climate. Hemp plants can resist various plant diseases. Besides, hemp can fight against insect pests without toxic pesticides and chemical fertilizers and thus effectively improve the soil salinization. In this work, two strains of pathogenic fungi (DN1 and DN2) were isolated from lesion site of the hemp plant, by an observation of hypha and spore morphology, the DN1 and the DN2 are respectively determined as deuteromycotina Alternaria (Alternaria alternata) and Verticillium (M.Verticillium). At pH values between 4 and 14, the two fungi can grow normally and show a great ability to adapt to the environment. Effects of hemp on soil pH were determined by pot experiment. The results showed that hemp could repair the weak alkaline soil.

ISSUES AND MITIGATIONS Hop Latent Viroid
DR. JOHN BRUNSTEIN (SEGRA)
https://mcusercontent.com/205b36c25f...Whitepaper.pdf
Zoom video:
https://us02web.zoom.us/rec/play/5F5...x_zm_rhtaid=19
Large-scale crop loss is every grower’s nightmare, but unfortunately, it is becoming an increasingly common reality. Modern Cannabis pathogens such as Hop Latent Viroid (HLVd) are emerging and accelerating, particularly in California, the most mature cannabis market in the world. While California
has long been an inspiration to the global cannabis community as an established and thriving market, it now also needs to serve as a cautionary tale as global cannabis operators come into contact with a growing list of pathogens causing documented economic losses.

Jahniella bohemica
John McPartland, Karl Hillig
October 2006 Journal of Industrial Hemp 11(2):97-108
DOI: 10.1300/J237v11n02_08
This note continues the “Cannabis Clinic” series, presenting diseases and pests of hemp, featuring colour illustrations of signs and symptoms. The fungus Jahniella bohemica damages hemp stalks in Europe. For 125 years this fungus has been confused with Leptosphaeria acuta, a fungus that infests nettle stalks. The morphology of J. bohemica is described in detail, compared to other Jahniella species, and also compared to L. acuta. The nomenclature of L. acuta is revised.

Jahniella bohemica Part II: Mistaken Identity with Septoria cannabis Infesting Hemp Stalks
John McPartland, Karl Hillig
May 2007 Journal of Industrial Hemp 12(1):63-74
DOI: 10.1300/J237v12n01_07
This note continues the “Cannabis Clinic” series, presenting diseases and pests of hemp, featuring colour illustrations of signs and symptoms. The fungus Jahniella bohemica damages hemp stalks in
Europe. In the previous issue of “Cannabis Clinic” we illustrated Jahniella bohemica and showed how this fungus has been confused with Leptosphaeria acuta, a fungus that infests nettle stalks. Jahniella bohemica has also been confused with Septoria cannabis, a fungus that usually attacks hemp leaves, but sometimes attacks hemp stalks. We now illustrate differences between these species, using novel methods of microchemistry, and emphasize the signs and symptoms of S. cannabis infestation.

Not Cannabis Specific
Keeping Your Environment Clean: Preventative Measures Against Contamination
By Jeff Scheir
https://cannabisindustryjournal.com/tag/uvc/
For years we have heard about and sometimes experienced, white powdery mildew when growing cannabis. It is a problem we can see, and we have numerous ways to combat it. But now more and more states are introducing regulatory testing on our harvests and they are looking for harmful substances like Escherichia coli., Aspergillis Fumigatus, Aspergillis terreus, … just to name a few. Mycotoxins, mold and bacteria can render a harvest unusable and even unsellable- and you can’t see these problems with the naked eye. How much would it cost you to have to throw away an entire crop?
You bring in equipment to control the humidity. You treat the soil and create just the right amount of light to grow a superior product. You secure and protect the growing, harvesting, drying and production areas of your facility. You do everything you can to secure a superior yield… but do you?
Many of the organisms that can hurt our harvest are being multiplied, concentrated and introduced to the plants by the very equipment we use to control the growing environment. This happens inherently in HVAC equipment.

Kenevir Cannabis sativa L Bitkilerinde Gorulen Virus Kaynakli Hastaliklar
Mehmet Ali Sevik
Türkiye Tarımsal Araştırmalar Dergisi 7 (1) Feb 2020 (in turkish)
DOI: 10.19159/tutad.663715
https://dergipark.org.tr/en/download...le-file/970960
https://www.researchgate.net/publica...=re413_x_i_pbf
Hemp (Cannabis sativa L.) belongs to Cannabis genus in Cannabinaceae family. Industrial hemp (C. sativa var. vulgaris L.), a variant of the C. sativa, is an important industrial crop. Industrial hemp is one of the oldest raw material resource crops know to humans. Hemp is cultivated to produce a vast variety of products such as hemp seeds, hemp oil, clothing, rope, paper, insulation, cosmetics, biodegradable plastics, construction material, resin, fuel, etc. Hemp is very sensitive to environmental conditions, diseases, and pests. Plants are more susceptible to diseases in adverse environmental conditions. More than 100 microorganisms (fungi, bacteria, virus, etc.) can cause disease in cannabis. In many studies conducted; hemp streak virus (HSV), hemp mosaic virus (HMV), alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV) and arabis mosaic virus (ArMV) have been reported to cause diseases in hemp varieties. Also, tobacco mosaic virus (TMV),tobacco ringspot virus (TRSV), tobacco streak virus (TSV), tomato ringspot virus (TomRSV), eunoymous ringspot virus (ERSV), elm mosaic virus (EMV), and foxtail mosaic virus (FMV) are isolated from hemp plants. In recent years, studies on cannabis viruses have reported that beet curly top virus (BCTV) and lettuce chlorosis virus (LCV) infect cannabis plants.

Laboratory Bioassays of Biological/Organic Insecticides to Control Corn Earworm on Hemp in Virginia, 2019
Kadie E. Britt1 and Thomas P. Kuhar
Arthropod Management Tests 45(1) January 2020
DOI: 10.1093/amt/tsaa102
https://www.researchgate.net/publica..._Virginia_2019
Hemp acreage in the United States is increasing and outdoor crops are susceptible to corn earworm (CEW) feeding injury. Two separate bioassays were conducted in fall 2019 to evaluate the effects of biological/ organic insecticide products on CEW in hemp.
Bioassay 1 was initiated on 16 Sep 2019 and included the following treatments: Gemstar (Helicoverpa zea nuclear polyhedrosis virus [HzNPV]), Javelin (Bacillus thuringiensis var. kurstaki), DiPel (Bacillus thuringiensis var. kurstaki), XenTari (Bacillus thuringiensis var. aizawai + kurstaki), Venerate (94.5% Heatkilled Burkholderia spp. strain A396 cells and spent fermentation media), Grandevo (30% Chromobacterium subtsugae strain PRAA4-11 and spent fermentation media), Entrust (Spinosad), and an untreated check (Table 1). Third and fourth instar CEW larvae were collected from ears from an untreated field of sweet corn (Zea mays) established at Virginia Tech’s Kentland Farm in Whitethorne, VA (Kentland). Only vigorous larvae with fresh color were used for the experiment. On 16 Sep 2019, hemp seed heads (‘Felina-32’) were collected from field plots at Kentland, brought to the laboratory, and cut into ~9 cm3 sections. Forty hemp seed head sections were dipped into spray-tank concentrations of each treatment (Table 1) and placed individually into 1 oz plastic diet cups with a single CEW larva. A tray of 10 cups represented a replicate and four replicates were established for each treatment and placed in a different stack on the laboratory bench for the duration of the experiment. Diet cups were placed on the laboratory benchtop and held at laboratory ambient light and temperature (20–25°C) for 96 h and checked daily for mortality. Percent mortality data were analyzed with ANOVA procedures and means separated with Tukey’s HSD.
Bioassay 2 was initiated on 2 Oct 2019 and included the following treatments: Agree (Bacillus thuringiensis var. aizawai), Javelin (Bacillus thuringiensis var. kurstaki), Deliver (Bacillus thuringiensis var. kurstaki), XenTari (Bacillus thuringiensis var. aizawai + kurstaki), Pyganic (pyrethrins), Entrust (spinosad), and an untreated check (Table 2). The experiment was conducted using the same aforementioned procedures except that rather than using fieldcollected CEW, which were depleted from the field, we used third instars raised on artificial diet that were purchased from Benzon Research Inc., Carlisle, PA

Not Cannabis Specific
Mildew control with CleanLight technology
https://cleanlight.nl/wp-content/upl...March-2012.pdf
In September 2010, Horticultural News dedicated a few pages to the introduction of an exciting new crop protection technology in East Africa. It is a technology, not based on chemicals, but based on the use of cleansing light.

Mitochondrial COI Sequence Variations within and among Geographic Samples of the Hemp Pest Psylliodes attenuata from China.
Guo, L., Gao, F., Cheng, Y., Gao, C., Chen, J., Li, Z., … Xu, J.
Insects, 11(6), 370.(2020).
doi:10.3390/insects11060370
The hemp flea beetle Psylliodes attenuata (Coleoptera: Chrysomelidae: Psylliodes) is a common pest of Cannabis sativa, including cultivars of both industrial hemp and medicinal marijuana. Both the larval and adult stages of this beetle can cause significant damages to C. sativa, resulting in substantial crop losses. At present, little is known about the populations of this pest, including its genetic diversity. In this study, we obtained 281 P. attenuata samples from nine field sites representing broad industrial hemp productions in China and analyzed their DNA sequences at the mitochondrial COI gene, the insect DNA barcode. Our analyses revealed a total of 48 haplotypes, with 28 being found only in one specimen each while the remaining 20 were shared by two or more specimens each. Of the 20 shared haplotypes, eight were shared among local populations often from far away locations, consistent with recent long-distance dispersals. However, the observed putative long-distance dispersals have not obscured the significant genetic differentiations among the regional populations from northeastern, eastern, central and southwestern China. Interestingly, haplotype network analyses suggest evidence for potential mitochondrial recombination in natural populations of this species. We briefly discuss the implications of our results on its evolution, center of diversity, route of spread, and pest management strategies in hemp fields.

Not Cannabis specific
Molecular sampling of hop stunt viroid (HSVD) from grapevines in hop production areas in the Czech Republic and hop protection
J. Matousek, L. Orctová, J. Patzak, P. Svoboda, I. Ludvíková
PLANT SOIL ENVIRON., 49, 2003 (4): 168–175
https://www.researchgate.net/publication/264234891
Molecular sampling of HSVd in grapevines in the environs of hop gardens was performed. Specific RT PCR primers were designed to unambiguously distinguish between HLVd and HSVd infections. These primers were used for detection and analysis of HSVd cDNAs from individual samples by thermodynamic methods, TGGE and cDNA heteroduplex analysis. We found that at least 70% of grapevine samples from locations close to hop gardens in Northern Bohemia (atec and Útìk hop production areas) were infected with HSVd forming populations containing quasispecies. Particular sequence variants, dominant in grapevines from wine-growing areas like Znojmo, were also found in minor private vineyards. HSVd was experimentally transmissible (80% success) from these samples to Osvald’s clone 72 of Czech hop, where according to the cDNA library screening, one of the dominant HSVdg variants corresponding to AC E01844 was detected in early populations three weeks p.i. HSVd was detected neither in reproduction materials nor in examined hop gardens. However a potential danger for hop cultivation, consisting in the high biological potential of HSVd spread is discussed.

Kentucky Hemp Disease
Hemp Disease Management 101: Back to Basics
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/diseas...nt-basics.html
Diseases are managed first and foremost by prevention of infection by pathogens*. Once host plants are infected, disease** is not curable. Although some measures can suppress pathogens or slow symptom development, disease cannot be reversed or cured. Thus, it is recommended that preventative measures become priority in every system.

Introduction of diseased plant material into a field or greenhouse puts all healthy plants at risk for infection. Careful selection of plant material and adoption of production guidelines is critical. For example, new plants should remain separated for two to three weeks until they are confirmed to be disease (and insect) free. This period of quarantine helps protect existing healthy plant material.

On occasion, pathogens are introduced to production systems. Immediately upon identification/confirmation, protect healthy plants from infection and spread. Destroy, quarantine, and/or apply chemical treatments to infected plants. Nearby plants should be treated as infected and infective (even if symptoms have not yet developed). All diseased plant material is a threat to healthy plants, and thereby should be removed from production areas. As mentioned previously, disease cannot be cured or eradicated. Elimination of pathogens is only possible by extreme measures (e.g. crop destruction or greenhouse disinfestation).

• Disease101: Hemp Disease Management Basics
• ARTICLES:
• Septoria Leaf Spot
• Leaf Spot Diseases, An Overview
• Gray Mold (Botrytis) in the Greenhouse
• Powdery Mildew of Hemp
• Pythium Root Rot
• Sanitation for Disease Management in Greenhouse Hemp
• Sanitation for Disease Management in Field Hemp
• Cultural Calendar
• KY Hemp Disease

Disease Management Guide to Septoria Leaf Spot in the Field
Dr. Nicole Gauthier
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/septoria-leaf-spot.html
Septoria sp.
Hemp (Cannabis sativa) is grown both outdoors and in greenhouses. Outdoor-grown hemp is susceptible to infection by leaf spot fungi that affect other crops and nearby plants, especially during periods of extensive rain or high humidity. There are three primary leaf spot diseases that affect hemp in Kentucky: Cercospora leaf spot, hemp leaf spot, and Septoria leaf spot. Septoria leaf spot is the most commonly reported of the leaf spot diseases.

Symptoms. Septoria leaf spot causes leaf spotting that can result in leaf loss and reduction of photosynthesis. Under extreme conditions, plants can lose 50% to 90% of leaves. If disease becomes severe early in the season, plant stunting and loss of vigor is possible. Plant death can occur under extreme conditions.

Disease symptoms begin in lower leaves and within the inner canopy where leaf wetness and high humidity occur. Disease begins as small irregularly shaped spots with bright yellow margins. Spots expand to about ¼ inch in diameter and appear brown with a prominent yellow halo. As spots enlarge, brown areas become more irregular and develop gray to white centers. Yellow halos expand outward from spots. Advanced symptoms include coalescence of yellow areas and/or coalescence of brown spots. Entire leaves rapidly become yellow and drop from plants.

Causal Agent. The fungus that causes Septoria leaf spot is a species within the genus Septoria. The precise causal species of Septoria leaf spot in Kentucky is currently under investigation. In general, Septoria spp. infect specific hosts or a specific group of hosts. For example, Septoria leaf spot of tomato causes disease on tomato and other solanaceous plants and Septoria tritici blotch of wheat affects only wheat and grass hosts. Neither of these diseases are caused by the same species that causes disease in hemp.

Septoria leaf spot spreads by spores called conidia. Infective conidia develop within capsule-like structures called pycnidia. Pycnidia appear as dark specks that resemble black pepper flakes in centers of spots; they contain large numbers of conidia. When excess moisture is present, conidia are released from pycnidia in a curling, oozing formation. Conidia move short distances via rain splash and long distances by wind-driven rain. Each conidium is capable of creating a new infection, and these new infections develop into leaf spots within 7-10 days.

Leaf Spot Diseases, An Overview
A Scientific Look at Diseases of Kentucky Hemp
Cercospora Spot
Septora Leaf Spot
Hemp Leaf Spot
https://www.kyhempdisease.com/leaf-s...-overview.html


Disease Management Guide to Gray Mold in the Greenhouse
Nicole Gauthier
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/gray-m...reenhouse.html
Botrytis cinerea
Hemp (Cannabis sativa x indica) is grown both outdoors and in greenhouses. High humidity greenhouses and other closed environments are often ideal for fungal diseases such as gray mold. Outdoor-grown hemp is susceptible during periods of high humidity or rain.
Symptoms. Gray mold develops primarily in flower buds and tightly-packed plant parts, and it is recognized by its gray moldy growth. Masses of mycelia (fungal strands) and clusters of conidia (asexual spores that are produced in large numbers) make up the dense gray mats. The fungus is commonly found in and between buds where microclimates are humid and air flow is limited. Severe infections can lead to yield loss as buds and flowers become infected.
While moldy buds are the most common disease phase, stems, petioles, and growing tips can also be affected. Pruning cuts, insect damage, and other openings are ideal for the opportunistic fungus. Once the fungus colonizes, it can girdle stems and cause them to break at the point of infection. Seedling infections and damping off can also result under humid conditions and in greenhouses with high levels of fungal contamination. Damping off in seedlings causes plant collapse.
Causal Agent. The fungus Botrytis cinerea is the causal agent of gray mold. It is ubiquitous, in that it is ever-present. Infection by the Botrytis fungus is dependent upon a wound or opening in plant tissue. Once infection takes place, though, the fungus can move from cell to cell, killing tissue as it spreads.
A single bud can host up to 1 million infective spores as humidity rises above 85%. These spores are carried by air currents to healthy plant material; fans, wind, and mechanical means can move spores, setting off a repeating disease cycle.

Management of Powdery Mildew Begins with Understanding the Causal Fungus
Nicole Gauthier
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/powder...w-of-hemp.html
Golovinomyces (synonym Erysiphe sp.)
Hemp (Cannabis sativa x indica) is grown both outdoors and in greenhouses. The environmental conditions of each system varies extensively, resulting in differing disease pressure. High humidity greenhouses and other closed environments are often ideal for fungal diseases such as powdery mildew. Outdoor-grown hemp is much less susceptible to the disease.
Powdery mildew diseases are caused by a group of fungi that develop fungal strands and chains of fungal spores on leaf surfaces and branch tips. Powdery mildew pathogens are host specific; each species of the fungus is specific to certain hosts. Thus, the species that infects Cannabis are restricted to hemp, hops, and a limited number of host plants. Likewise powdery mildew pathogens of surrounding plants (examples dogwood powdery mildew or rose powdery mildew) will not infect Cannabis spp. Golovinomyces sp. (synonym Erysiphe sp.) remains the most common of the powdery mildew pathogens that infect Cannabis spp.

Occurrence of Powdery Mildew Caused by Golovinomyces cichoracearum sensu lato on Cannabis sativa in Canada.
Pépin, N., Punja, Z., & Joly, D. L.
Plant Disease. (2018).
doi:10.1094/pdis-04-18-0586-pdn
Cannabis sativa, a flowering plant belonging to the Cannabaceae family, increasingly is being grown in Canada for medical purposes. As of April 2018, there was 97 authorized licensed producers and more than 200,000 registered clients, making C. sativa an economically important crop in Canada (Government of Canada 2018). Symptoms of powdery mildew were observed on indoor-grown plants in production facilities in Atlantic Canada and British Columbia. Powdery mildew was present in all production sites sampled between 2013 and 2017, and ranged in incidence from 20 to 35%. Symptoms appeared as white circular patches consisting of epiphytic mycelia and conidia on the adaxial leaf surface. As the disease progressed, the mycelia, conidiophores and conidia covered the entire surface of the leaves and spread to flower bracts and buds. The conidiophores were unbranched, cylindrical, measuring 140-260 µm in height and erected singly from mycelium. Conidiophores produced between one and four conidia in chains

Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.
Zamir K. Punja, Danielle Collyer, Cameron Scott, Samantha Lung, Janesse Holmes and Darren Sutton
Frontiers in Plant Science Vol 10, October 2019
doi: 10.3389/fpls.2019.01120
Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCRbased assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on cannabis plants.

Pythium Root Rot Control in Seedlings and Cuttings Starts with Prevention
Nicole Gauthier
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/pythium-root-rot.html
Pythium spp.
Industrial hemp (Cannabis sativa x indica) in Kentucky is grown both outdoors and in greenhouses. Under wet soil conditions in either system, Pythium root rot and damping off diseases pose threats to seedlings, cuttings, and young plants. Indoor-grown hemp is most often infected, however.
Pythium root rot and damping off are caused by several species of Pythium, a water mold pathogen. Pythium are soilborne, fungus-like oomycetes. The term water mold is often used because these pathogens require free water in order to complete their life cycles (reproduce and spread from plant to plant). Under wet conditions such as rain or irrigation, propagules (particularly swimming zoospores) increase dramatically, disseminate to healthy roots, and infect. Disease often develops within a week or two after infection.
Root rot and lower stem decay cause above-ground symptoms such as marginal leaf scorch, stunting, poor vigor, and/or inconsistent stands. These symptoms are the result of lack of water and nutrient uptake as root and vascular tissue is damaged. Disease is more severe under wet soil conditions, high soluble salts, and plant stress.

Hemp Field Sanitation for Small-Scale Plantings
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/sanita...ield-hemp.html
IMPORTANCE OF SANITATION
Although hemp is considered a disease-free plant, it is unknown whether levels of certain pathogens can build up to problematic levels. If diseases become a significant problem in field-planted hemp, results can include premature leaf drop, bud decay, dieback, decline, and even plant death. When diseases do occur, it is often presumed that fungicides are the most important and effective disease management tools available. However, there are limited fungicides available for use in hemp. Thus, a good sanitation program can help reduce the need for chemical controls and can improve the effectiveness of other practices for managing disease. This often-overlooked disease management tool reduces pathogen numbers and eliminates infective propagules (inoculum such as fungal spores and survival structures) that cause disease.
For example, certain fungal and water molds can become prevalent during rainy or humid growing seasons. When disease management is neglected, pathogen populations build-up and continue to increase as long as there is susceptible plant tissue available for infection and disease development. Infected plant tissue, infested soil, and pathogen propagules all serve as sources of pathogens that can later infect healthy plants.
Reduction of pathogens by various sanitation practices can reduce both active and dormant pathogens. While actively growing plants can provide host tissue for pathogen multiplication, dead plant material (diseased foliage, stems, and/or roots) can harbor overwintering propagules for months or years. These propagules can travel via air/wind currents, stick to shoes or tools, or move with contaminated soil or water droplets. Thus, prevention of spread of pathogens to healthy plants and the elimination of any disease-causing organisms from one season to another are the foundations for a disease management program using sanitation practices.


Hemp Greenhouse Sanitation
A Scientific Look at Diseases of Kentucky Hemp
https://www.kyhempdisease.com/sanita...ouse-hemp.html
Diseases are a major concern for greenhouse growers and can be a key limitation to profitable plant production. Disease management in greenhouses is critical because the warm, humid environment in these structures provides optimal conditions for reproduction of many pathogens. When disease management is neglected, pathogen populations build-up and continue to increase as long as there is susceptible plant tissue available for infection and disease development. Infected plant tissue, infested soil, and pathogen inoculum (such as spores and survival structures) all serve as sources of pathogens that can later infect healthy plants.

Koppert Pest Control Guidelines
Ask me for info

Larvicidal Action of Cannabidiol Oil and Neem Oil against Three Stored Product Insect Pests: Effect on Survival Time and in Progeny.
Mantzoukas, S., Ntoukas, A., Lagogiannis, I., Kalyvas, N., Eliopoulos, P., & Poulas, K.
Biology, 9(10), 321.(2020).
doi:10.3390/biology9100321
Stored product pests can be detrimental to agricultural produce. As much as chemical pesticides are effective control agents, they involve several environmental and health risks. Within the framework of studies on alternative pest management methods, interest has focused on a plethora of plants whose extracts have demonstrated promising action as insecticides. Azadirachta indica and the derived neem oil have been extensively tested against many harmful insect species. In contrast, Cannabis sativa L. and its main compound, CBD, a highly concentrated cannabinoid, have not been investigated much. The present study examined the potential insecticidal activity of CBD and neem oils against 4th instar larvae of Tribolium confusum, Oryzaephilus surinamensis and Plodia interpunctella on wheat, rice and corn seeds. Treatment efficacy was expressed in terms of larval mortality. Mortality was observed in relation to dosage, time exposure intervals and product types. The results showed clear pesticidal activity for both oils, which at high doses induced significant mortality. The treatments produced significantly fewer offspring in the insect species tested than the control. The efficacy of treatment in progeny suppression was, as expected, dose dependent.

LEGAL PEST MANAGEMENT PRACTICES FOR CANNABIS GROWERS IN CALIFORNIA
Department of Pesticide Regulation
https://www.cdpr.ca.gov/docs/county/...attach1502.pdf
PESTS OF CANNABIS IN CALIFORNIA
Cannabis pests vary according to cultivar (variety), whether the plants are grown indoors or outdoors, and where the plants are grown geographically. The pests included in this review are preliminary and based on the following sources: a presentation given in 2013 by
Whitney Cranshaw, an extension entomologist at Colorado State University, and a review article by John M. McPartland, a professor of family medicine at the University of Vermont. We also received input from Kevin Hoffman, Primary State Entomologist, California
Department of Food & Agriculture (CDFA).

Lettuce Chlorosis Virus Disease: A New Threat to Cannabis Production
August 2019 Viruses 11(9):802
DOI: 10.3390/v11090802
Lab: Aviv Dombrovsky's Lab
Hadad, Neta Luria, Smith, Aviv Dombrovsky
In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85–99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisia tabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected ‘healthy’ cannabis, Lactuca sativa, and Catharanthus roseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

Longhorn Beetles and Botryosphaeria
John McPartland, Karl Hillig
November 2007 Journal of Industrial Hemp 12(2):123-133
DOI: 10.1300/J237v12n02_09
This note continues the “Cannabis Clinic” series, presenting diseases and pests of hemp, featuring colour illustrations of signs and symptoms. The hemp longhorn beetle (Thyestilla gebleri) damages
hemp stalks in eastern Asia. The fungus Botryosphaeria marconii causes stalk and twig blight disease of hemp in North America and perhaps western Europe. The life cycles of the beetle and fungus overlap on host plants; if their geographic ranges also overlapped they would likely cause synergistic damage–an epidemic waiting to happen?

Marijuana (Cannabis sativa) toxicosis in cattle
David Driemeier
Veterinary and human toxicology · January 1998
4 of 5 cattle died after consuming bales of dried marijuana leaves.
https://www.researchgate.net/publica...osis_in_cattle

Microbial Contaminants in Cannabis: What are the Dangers?
Reginald Gaudino, Steep Hill July 13, 2018
https://800ezmicro.com/cannabis-test...e-dangers.html
Unless cannabis is grown in a clean room with appropriate air ?ltration and other good growing and laboratory practices, it is inevitable that these microorganisms will be found on cannabis ?owers and products made with them. Most microorganisms do not present a problem at low levels, though some pathogenic microorganisms are harmful even at low levels because they produce toxins that cause a variety of symptoms: from allergy-like symptoms to various types of cancer.
Other microorganisms are harmful at mid to higher levels of contamination, particularly if they are inhaled. Immunocompromised patients including those receiving chemotherapy are at a higher level of risk to all microorganisms, where even low levels of microbial contamination can lead to fatality. Contaminated medical cannabis and incidence of diseases such as Aspergillus nodulosis (a disease caused by the inhalation of Aspergillus niger) has already been noted (1).

Microbiological examination of nonsterile Cannabis products: Molecular Microbial Enumeration Tests and the limitation of Colony Forming Units.
Kevin McKernan, Yvonne Helbert, Heather Ebling, Adam Cox, Liam T. Kane, Lei Zhang
https://osf.io/vpxe5/download
Cannabis microbial testing presents unique challenges. Unlike food testing, cannabis testing has to consider various routes of administration beyond just oral administration. Cannabis flowers produce high concentrations of antimicrobial cannabinoids and terpenoids and thus represent a different matrix than traditional foods. In 2018, it is estimated that 50% of cannabis is consumed via vaporizing or smoking oils and flowers while the other half is consumed in Marijuana Infused Products or MIPs. There are also transdermal patches, salves and suppositories that all present different microbial considerations. Several recent publications have surveyed cannabis flower microbiological communities. These have detected several concerning genus and species such as Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Penicillium paxilli and Penicillium citrinum, Clostridium botulinum, Eschericia coli, Salmonella and Staphyloccus. There are several documented cannabis complications and even fatalities due to Aspergillosis in immuno-compromised patients. A recent paper even demonstrates a case of cannabis derived Aspergillosis in an immune competent patient.

Microbiological Safety Testing of Cannabis
Cannabis Safety Institute May 2015
Mowgli Holmes, Jatin M. Vyas, William Steinbach, John McPartland
https://cannabissafetyinstitute.org/...f-Cannabis.pdf
Cannabis is increasingly becoming legal at the state level in the U.S., for either medical or recreational use. Each of these states has had to wrestle with the question of how to ensure the safety of a new product that is not covered under any existing safety guidelines. Safety testing in other agricultural industries is regulated by the FDA, the USDA, or by other federal agencies, all of which have been unable to assist the states in this case. The few states that have produced safety testing guidelines for Cannabis were forced to develop them from scratch, without the regulatory and scientific support that federal agencies typically provide. In the absence of this federal guidance, regulators in each state have turned to different sources for information, and each state has produced a unique set of rules and regulations (if they have produced one at all). Many of these are in outright conflict with each other, and they are largely not grounded on scientific research. This whitepaper is focused entirely on the question of microbiological safety, and has been written in order to promote the adoption of regulatory guidelines for the Cannabis industry that are rational, consistent, and safe. We have gathered what data there are on this issue and related ones, and assembled a broad collection of experts on the general subjects of plant microbiology, medical microbiology, and safety-testing of agricultural and food products.

Microbiological study and quantification cannabinoids cannabis samples catalonia cscs
Fundación CANNA
https://www.fundacion-canna.es/en/mi...catalonia-cscs
In 2014, Fundación CANNA has carried out a study in which 55 Cannabis samples from 31 social Cannabis clubs from Catalonia were analysed. The aim was to learn about microorganisms that could appear and give information about the average potency of the Cannabis dispensed. The results were shown in the scientific park of the University of Catalonia in October.
Microbiological study of Cannabis samples
Fundación CANNA
https://www.fundacion-canna.es/en/mi...nnabis-samples
Cannabis, like any other plant or food, can contain microbiological contaminants potentially harmful for our health. These contaminants are some types of bacteria and fungi that could end up appearing or developing in the final product. It will depend on the growing, drying, processing, preservation and handling of the plant.
Samples from several clubs from Cataluña were analysed in the laboratory. A total of 55 samples from 31 CSC.
In order to determine the microbiological level of the samples analysed, two factors have been taken into account: the total number of microorganisms present in each sample, and the absence of microorganisms with high pathogenic potential. A high number of microorganisms, irrespective of the kind of microorganism, indicates that the production, preservation and/or handling processes haven't been properly managed. Nowadays, some US states are establishing microbiological limits for the Cannabis provided in their dispensaries.

Microbiological Testing of Cannabis and Derived Products
Microbiology International
https://800ezmicro.com/cannabis-testing.html
Cannabis samples are typically received in the testing laboratory as the flower, topical emulsions & concentrated oils, or edible preparations.
Weighing and Diluting Flower and edibles are processed at a dilution factor of 1:9 or 1:99. Topical emulsions and oils are processed at a ratio of 1:99 or 1:9 with the addition of a surfactant/detergent.
Sample enrichment: Take the guesswork out of weighing and diluting by pairing the EZ-Flow gravimetric diluter and MediaBox ready-to-use sterile broths. Based on the weight of your sample, the EZ-Flow will automatically create the correct dilution factor by drawing liquid media directly from MediaBox and dispensing it into your sample bag. MediaBox connects directly to the EZ-Flow diluter for a completely closed system. The EZ-Flow has a dilution range factor from 1/2 to 1/99. Enrichment broth types: Buffered Peptone Water (BPW), Butterfields Phosphate Buffer, Tryptic Soy Broth with Casamino acids and 8mg/L Novobiocin (mTSB+n), Nutrient Broth, Letheen Broth with Tween 80, and 0.1% BPW. Custom formulations available.

Not Cannabis Specific
Mildew control with CleanLight technology
https://cleanlight.nl/wp-content/upl...March-2012.pdf
In September 2010, Horticultural News dedicated a few pages to the introduction of an exciting new crop protection technology in East Africa. It is a technology, not based on chemicals, but based on the use of cleansing light.

Mitochondrial COI Sequence Variations within and among Geographic Samples of the Hemp Pest Psylliodes attenuata from China.
Guo, L., Gao, F., Cheng, Y., Gao, C., Chen, J., Li, Z., … Xu, J.
Insects, 11(6), 370.(2020).
doi:10.3390/insects11060370
The hemp flea beetle Psylliodes attenuata (Coleoptera: Chrysomelidae: Psylliodes) is a common pest of Cannabis sativa, including cultivars of both industrial hemp and medicinal marijuana. Both the larval and adult stages of this beetle can cause significant damages to C. sativa, resulting in substantial crop losses. At present, little is known about the populations of this pest, including its genetic diversity. In this study, we obtained 281 P. attenuata samples from nine field sites representing broad industrial hemp productions in China and analyzed their DNA sequences at the mitochondrial COI gene, the insect DNA barcode. Our analyses revealed a total of 48 haplotypes, with 28 being found only in one specimen each while the remaining 20 were shared by two or more specimens each. Of the 20 shared haplotypes, eight were shared among local populations often from far away locations, consistent with recent long-distance dispersals. However, the observed putative long-distance dispersals have not obscured the significant genetic differentiations among the regional populations from northeastern, eastern, central and southwestern China. Interestingly, haplotype network analyses suggest evidence for potential mitochondrial recombination in natural populations of this species. We briefly discuss the implications of our results on its evolution, center of diversity, route of spread, and pest management strategies in hemp fields.

Molecular characterization of Aster yellows (16SrI) group phytoplasma infecting Cannabis sativa in eastern Uttar Pradesh
SMRITI MALLÅ, G.P. RAO and P.P. UPADHYAYA
Indian Phytopath. 64 (1) : 75-77 (2011)
During survey of diseases of weeds in and around agricultural fields in eastern UP in 2008, non-specific yellowing, chlorosis of leaves, proliferation of axillary shoots and witches’-broom symptoms on Cannabis sativa plants followed by death of entire plants were recorded. Suspected infected and healthy tissues of C. sativa from different locations at Gorakhpur in eastern UP, India, were examined by nested PCR assay using universal phytoplasma primers P1/P6 and R16F2n/R16R2. C. sativa plant with non-specific yellowing and witches’-broom symptoms yielded phytoplasma-exclusive DNA bands of 1.2 kb when nested PCR was performed. BLAST search analysis of the 16S rRNA sequence of the C. sativa phytoplasma shared 99%
identity with those of phytoplasma members of 16SrI group, ‘Candidatus Phytoplasma asteris’. Therefore, the C. sativa phytoplasma under present study was identified as a member of the 16SrI group.

Molecular characterization, vector identification and sources of phytoplasmas associated with brinjal little leaf disease in India
Manish Kumar, Madhupriya, Govind Pratap Rao,
3 Biotech (2017) 7:7
DOI 10.1007/s13205-017-0616-x
Brinjal little leaf (BLL) is a widespread disease of phytoplasma etiology in India that induces severe economic losses. Surveys were conducted in eight brinjalgrowing states of India during July 2014 to September 2015 and eighteen BLL samples showing little leaf, phyllody and witches’ broom symptoms were collected for phytoplasma identification. Presence of phytoplasmas was confirmed in all the eighteen BLL samples using polymerase chain reaction with phytoplasma-specific primer pairs (P1/P6, R16F2n/R16R2). Pair wise sequence comparison and phylogenetic relationship of 16S rRNA gene sequences of BLL phytoplasma strains confirmed that sixteen out of eighteen BLL strains belonged to clover proliferation phytoplasma (16SrVI) group and two BLL strains (GKP-A and GKP-B) from Gorakhpur, Uttar Pradesh, were classified under 16SrII group. Further virtual RFLP analysis of 16S rDNA sequences allowed finer classification of BLL strains into 16SrII-D and 16SrVI-D subgroups. BLL phytoplasma strains belonging to 16SrVID subgroup were found as the most widespread phytoplasma strains associated with BLL disease in India. 16SrVI-D subgroup phytoplasma association with two symptomatic weed species viz. Cannabis sativa subsp. sativa at Noida, Uttar Pradesh and Portulaca oleracea at IARI fields, New Delhi was also confirmed by nested PCR assays with similar set of phytoplasma-specific primers, pairwise 16S rDNA sequence comparison, phylogeny and virtual RFLP analysis. Out of five identified leafhopper species from BLL-infected fields at Noida, Uttar Pradesh and Delhi, only Hishimonas phycitis was identified as carrier and natural vector of 16SrVI-D subgroup of phytoplasmas by nested PCR assays, sequence comparison, phylogeny, virtual RFLP analysis and transmission assays.

NOT CANNABIS SPECIFIC but many of the viruses are found in cannabis
Molecular variation of viruses infecting hops in Australia and associated studies
October 2010
Damian R CrowleDamian R Crowle
https://www.researchgate.net/publica...ciated_studies
The objectives of this study were to investigate the virus incidence and molecular variation of Apple mosaic virus (ApMV), Hop mosaic virus (HpMV) and Hop latent virus (HpLV) and to examine the Hop latent viroid (HLVd) infection status of Australian hop varieties.
HLVd was found to be ubiquitous in all hop gardens surveyed. This was the first survey of HLVd in Australia. This confirms findings in the Czech Republic where infection was also found to be ubiquitous, while viroid status in other countries also indicates high levels of infection.
A virus survey, primarily to collect viruses for use in molecular analysis, was conducted. The percentage of infected plants detected in this study correlates with those previously undertaken by Pethybridge et al., 2000b. Cultivar ‘Victoria’ had the greatest level of ilarvirus infections (61%) significantly more than ‘Super Pride’ (6%). Cultivar Opal had the greatest incidence of carlavirus infections (38%) but this was not significantly different to other cultivars sampled. Hops from the farm at Bushy Park recorded the highest incidences of Ilarvirus infection (44%) although this was not significantly different to the other sampled farms. However, hops sampled from the Gunns Plains farm showed significantly more carlavirus infections (40%) than the other three sampled farms. Experiments testing transmission capacity of local aphid species (Macrosiphum euphorbiae and Myzus persicae) of the carlaviruses HpMV and HpLV was performed. It was found that both aphid species transmitted both carlaviruses, this being the first study to demonstrate transmission of HpLV by an aphid other than the hop aphid, Phorodon humuli. This study also showed that prior infection by either virus did not significantly affect subsequent the efficiency of transmission of the other which may have explained observations of greater than expected coinfection of both carlaviruses within the field. It was known that two serologically distinct ilarvirus strains infect hop. Prior literature indicated that these were strains of Prunus necrotic ringspot virus (PNRSV) designated –intermediate (PNRSV-I) type and PNRSV-A (apple serotype). This study undertook molecular analysis of hop-infecting ilarviruses to clarify strain diversity and taxonomic relationships. Analyses showed Australian hops are infected with two distinct strains of ApMV (and not PNRSV) these being distinct to ApMV strain commonly found in Apple. It was proposed that hop infecting strains of ilarvirus be termed ApMV-Hop (the former PNRSV-apple serotype) and ApMV-Intermediate (the former PNRSV intermediate serotype). PCR based assays were developed that could be used to distinguish the two strain types.
Suggestions of strains of HpMV had been described due to lethal and non-lethal response following infection in ‘English Golding’ hops. Molecular analysis of HpMV from Australian hop gardens indicated that there were at least two distinct clades of HpMV present with approximately 80% homology. Further work conducted at the conclusion of this study identified a possible third clade of HpMV. All HpLV isolates that were sequenced in this study had a high degree of identity. This was supported by recent publication of several further sequences on GenBank that also show this high degree of identity.

Multiplex qPCR and Cannabis Microbiome sequencing reveals several Bacteria and Fungi Native to Cannabis flowers
Kevin McKernan, Jessica Spangler, Lei Zhang, Vasisht Tadigotla, Yvonne Helbert, Douglas Smith
Color Poster:
https://system.na3.netsuite.com/core...7c1f3&_xt=.pdf
PDF:
https://www.medicinalgenomics.com/wp...owers_sbmt.pdf

New Hemp Diseases and Pests in New Zealand
John McPartland, Birgit Rhode
June 2005 Journal of Industrial Hemp 10(1):99-108
DOI: 10.1300/J237v10n01_08
This article continues the “Cannabis clinic” series, presenting diseases and pests of hemp (Cannabis sativa L.), featuring color illustrations of signs and symptoms. Hemp cultivation is new to New Zealand (NZ). Field trials began in 2001, evaluating cultivars from Europe or North America. Novel crop plants imported into new geographical areas are exposed to new diseases and pests. The imported plants often present less resistance to local problems than do indigenous plants. Many ‘local’ problems in NZ, however, are introduced organisms. Non-native birds cause the greatest crop damage. Rabbits, famous aliens in NZ, also cause problems. Invertebrate pests include the brown garden snail (Helix aspersa), brown field slug (Deroceras panormitanum), orange-soled slug (Arion distinctus), budworm (Helicoverpa armigera), passion vine leaf hopper (Scolypopa australis), caterpillars (Epiphyas postvittana), and melon aphid (Aphis gossypii). The latter species may be the vector of an unidentified virus that infests hemp. In humid regions and wet conditions, the fungi Botrytis cinerea, Trichothecium roseum, and Sclerotinia sclerotiorum rot flowering tops and stalks. For this article, image capture of invertebrate pests and fungi utilized Auto Montage software, to improve the depth of field and produce perfectly focused images.

New species, combinations, host associations and location records of fungi associated with hemp (Cannabis sativa)
July 1997 Mycological Research 101(7):853-857
DOI: 10.1017/S0953756297003584
John McPartland, Marc A. Cubeta
Micropeltopsis cannabis sp. nov. and Orbilia luteola (Roum.) comb. nov. are proposed. New Cannabis host associations include binucleate Rhizoctonia spp., Curvularia cymbopogonis, Sphaerotheca macularis, Glomus mosseae, and Pestalotiopsis sp. The geographic ranges of Pseudoperonspora cannabina, Septoria neocannabina and Fusarium graminearum are expanded.

Not the one, but the only one: about Cannabis cryptic virus in plants showing “hemp streak” disease symptoms.
Righetti, L., Paris, R., Ratti, C., Calassanzio, M., Onofri, C., Calzolari, D., … Grassi, G.
European Journal of Plant Pathology, 150(3), 575–588. (2017).
doi:10.1007/s10658-017-1301-y
Interveinal chlorosis and leaf margin wrinkling are widespread symptoms of Cannabis sativa. They are traditionally attributed to the so-called hemp streak virus (HSV), but its existence has not been demonstrated yet. To our knowledge, no molecular investigation has so far been performed in order to identify the causal agent of this symptomatology, we therefore decided to use traditional and molecular virology techniques to better characterize symptoms and pursue the etiological agent. No pathogenic virus was found by using targeted PCR reactions and by RNA sequencing, whereas we were able to detect the Cannabis cryptic virus (CanCV) with both techniques. We, therefore, developed an RT-qPCR assay based on a CanCVspecific TaqMan probe and applied it to a wide range of symptomatic and symptomless plants, using a twostep (for quantification), or a one-step (for fast detection) protocol. Both symptoms and the virus were only shown to be transmitted vertically and did not pass via mechanical inoculation or grafting, though we could not find any cause-effect correlation between them. In fact, the virus was found in all the tested hemp samples, and its abundance varied greatly between different accessions and individuals, independently from the presence and severity of symptoms. The suggestion that hemp streak is caused by a virus is therefore questioned. Some abiotic stresses seem to play a role in triggering the symptoms but this aspect needs further investigation. For breeding purposes, a selection of parental plants based on the absence of symptoms proved to be efficient in containment of the disease.

Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.
Zamir K. Punja, Danielle Collyer, Cameron Scott, Samantha Lung, Janesse Holmes and Darren Sutton
Frontiers in Plant Science Vol 10, October 2019
doi: 10.3389/fpls.2019.01120
Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCRbased assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on cannabis plants.

PATS Indoor Drone Solutions
Pulone Sabina, Terpenes and Testing Magazine
https://terpenesandtesting.com/pats-...one-solutions/
Pests control in crops can be a laborious and difficult task, often involving the use of chemicals which can leave residues in the final product. Pesticides are a great concern on human health due to their toxic nature, persistency, lipophilicity, and bioaccumulation. Monitoring insect populations can help reducing the quantities of pesticide treatments and limiting the concentrations of harmful chemicals. What if we exploit modern technology to keep track of insect offspring and to mechanically eliminate harmful insect species?
PATS Indoor Drone Solutions offers an innovative method to control insect spreading and to selectively get rid of flying pests. By using drones, the monitoring and prevention of harmful species in crops is less time consuming than employees scouting activities. Additionally, tracking pest populations can be more accurate, leading to less pesticide treatments or avoiding them all together by eradicating harmful pests with the drone itself.

Pesticide Use on Cannabis
RodgerVoelker, Mowgli Holmes
Cannabis Safety Institute June 2015
https://cannabissafetyinstitute.org/...hite-Paper.pdf
Legalized cannabis production is a rapidly growing agricultural industry. However, given that cannabis production has developed and operated in an Unregulated setting various practices have been adopted that are at odds with accepted regulations regarding human safety and environmental impacts. Chief amongst these is the unregulated use of pesticides, which has potentially serious public health and environmental consequences. In the absence of guidance from the Environmental Protection Agency on this subject, it is critical that state regulators enact programs to protect workers, the environment, and Cannabis consumers. This paper presents data indicating that pesticide use is widespread in the Cannabis Industry, and that pesticide residue on retail Cannabis products is often found at levels exceeding the allowable levels on any agricultural product. In addition, a set of basic recommendations is presented that will allow states to move forward safely until more detailed Cannabis-°©?specific pesticide data is a

Phorodon cannabis Passerini (Hemiptera: Aphididae), a newly recognized pest in North America found on industrial hemp
Whitney S. Cranshaw, Susan Halbert, Colin Favret, Gary L. Miller,
September 2018 Insecta Mundi 662:1-12
https://theowl.fsu.edu/mundi/article/view/0662/102363
Phorodon cannabis Passerini (Hemiptera: Aphididae: Macrosiphini) is reported for the first time as a pest of Cannabis L. crops in North America. The insect has been confirmed from fields of industrial hemp in Colorado and Virginia and has been found present within greenhouses in at least several American states and one Canadian province. The generic position of the aphid species is discussed and other known members of the genus are ruled out. Phorodon cannabis is placed in genus Phorodon Passerini and subgenus (Diphorodon Börner). Phorodon persifoliae Shinji is transferred to Hyalopterus Koch as a nomen dubium.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa
Ibrahim Abbasia, Artur Trancoso Lopo de Queirozb, Oscar David Kirsteina, Abdelmajeed Nasereddinc, Ben Zion Horwitza, Asrat Hailud, Ikram Salahe, Tiago Feitosa Motab, Deborah Bittencourt Mothé Fragab, Patricia Sampaio Tavares Verasb, David Pochef, Richard Pochef, Aidyn Yeszhanovg, Cláudia Brodskynb, Zaria Torres-Pochef, and Alon Warburg
PNAS | November 13, 2018 | vol. 115 | no. 46, 11790–11795
DOI: 10.1073/pnas.1810435115
Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and
transmit them while imbibing vertebrates’ blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a nextgeneration sequencing (NGS)–based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa. We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent “absence” of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial
capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases

NOT CANNABIS SPECIFIC
Points to Consider in Prevention of Crown Gall
Janine Hasey, Daniel A. Kluepfel & Lani Yakabe
https://www.sacvalleyorchards.com/wa...ng-crown-gall/
Pre-plant fumigation
Long-term Agrobacterium survival
Contaminated grafting tools and graft wood
Host Resistance
Overall Prevention Strategy
These Crown Gall management “Points to Consider” are based on experimental laboratory, greenhouse and /or field research and observations. For answers to your crown gall prevention questions, consult your local UCCE Farm Advisor.

Pseudomonas cannabina pv. cannabina pv. nov., and Pseudomonas cannabina pv. alisalensis (Cintas Koike and Bull, 2000) comb. nov., are members of the emended species Pseudomonas cannabina (ex ?Sutic? & Dowson1959) Gardan, Shafik, Belouin,Brosch, Grimont & Grimont 1999
Carolee T. Bull, Charles Manceau, John Lydon, Hyesuk Kong, Boris A.Vinatzer, Marion Fischer-LeSaux
Systematic and Applied Microbiology 33 (2010) 105–115
doi:10.1016/j.syapm.2010.02.001
Sequence similarity in the 16S rDNA gene confirmed that crucifer pathogen Pseudomonas syringae pv. alisalensis belongs to P. syringae sensu lato. In reciprocal DNA/DNA hybridization experiments, DNA relatedness was high (69–100%) between P. syringae pv. alisalensis strains and the type strain of P. cannabina (genomospecies 9). In contrast, DNA relatedness was low (below 48%) between P. syringae pv. alisalensis and reference strains from the remaining genomospecies of P. syringae including the type strain of P. syringae and reference strain of genomospecies 3 (P. syringae pv. tomato) although the wellknown crucifer pathogen, P. syringae pv. maculicola, also belongs to genomospecies 3. Additional evidence that P. syringae pv. alisalensis belongs to P. cannabina was sequence similarity in five gene fragments used in multilocus sequence typing, as well as similar rep-PCR patterns when using the BOXA1R primers. The description of P. cannabina has been emended to include P. syringae pv. alisalensis. Host range testing demonstrated that P. syringae pv. alisalensis strains, originally isolated from broccoli, broccoli raab or arugula, were not pathogenic on Cannabis sativa (family Cannabinaceae). Additionally, P. cannabina strains, originally isolated from the C. sativa were not pathogenic on broccoli raab or oat while P. syringae pv. alisalensis strains were pathogenic on these hosts. Distinct host ranges for these two groups indicate that P. cannabina emend. consists of at least two distinct pathovars, P. cannabina pv. cannabina pv. nov., and P. cannabina pv. alisalensis comb. nov. Pseudomonas syringae pv. maculicola strain CFBP 1637 is a member of P. cannabina

PYTHIUM APHANIDERMATUM CROWN ROT OF INDUSTRIAL HEMP
Jennifer Schoener, Russ Wilhelm and Shouhua Wang,
NPDN News Volume 12 Issue 9 2
https://www.npdn.org/system/files/NPDN_September-17.pdf
Cultivation of industrial hemp (Cannabis sativa) was first approved in 2014 for the purpose of research and development. The Federal Farm Bill Section 7606 authorizes state agencies to conduct pilot trials on the crop to assess crop viability for the creation of an industry in prospective states. In Nevada, the Department of Agriculture authorizes the production of hemp crops for research purposes. The acreage of hemp production in Nevada is relatively small in comparison to the acreage in other states. However, plant diseases associated with hemp crops have been occurring in Nevada in recent years. In 2016, the Nevada Department of Agriculture Plant Pathology Lab detected Fusarium root rot and sudden death disease from an industrial hemp crop, and Fusarium wilt from medical marijuana plants. Here we describe a newly detected hemp disease: Pythium aphanidermatum crown rot. Pythium aphanidermatum crown rot occurred in a commercial hemp field, with approximately 5-10 percent of plants affected. Infected plants were noticed by leaf yellowing, curling, necrosis, and the eventual death of entire plants (Fig A). White-colored mold (Pythium mycelium) growth on the surface of the crown area was frequently observed when the plant was pulled from the ground (Fig D). Close examination of the stalk revealed extensive water-soaked lesions and cankers around the crown and basal stalk regions (Fig C). With disease progression, the majority of stalks became completely necrotic or rotted (Fig F). Some affected plants had mild root rot. In the early stage of the disease, only mild internal discoloration of the basal stalk tissue was observed (Fig B). In later stages, cankers spread from the crown area to lower branched stems (Fig E). Affected tissue plated on potato dextrose agar (PDA) medium amended with streptomycin did not yield growth of any pathogens. On selective PARP medium, a fast-growing Pythium was obtained from all pieces of stem tissue plated. This isolate grew into a full plate (100mm diameter) on PDA medium within 24 hours at 22 ºC in the dark (Fig G), and produced oogonia, antheridia, and sporangia on corn meal agar (CMA) medium. Based on both morphology and the DNA sequence of the ITS region of rDNA , the isolate was identified as P. aphanidermatum. This disease can be detected using Agdia’s Phytophthora immunoStrip as it cross reacts with Pythium aphanidermatum. Hemp crown and root rot caused by Pythium aphanidermatum was recently reported in Indiana in June, 2017 (https://doi.org/10.1094/PDIS-09-16-1249- PDN). It was found in a small research plot where hemp seeds were planted. The disease described here occurred in a commercial field during the middle of the growth term, affecting a large number of plants. The disease appears to be more aggressive on crown and stem tissue, even though root rot was noticed on some plants. The disease was prevalent when plants were grown under plastic mulch film. Removal of mulch and reduction of soil moisture appeared to reduce the incidence of disease temporarily, but it did not stop the disease development in plants that had been infected

Quantitative vs Qualitative Hop Latent Viroid Testing
www.medicinalgenomics.com/quantitative-vs-qualitative-hlvd-testing/
There are currently no viable treatment options available for Hop Latent Viroid (HLVd) infected cannabis plants. Meristem tissue culture can be used to rescue infected plants, but the process can take up to 3-9 months and doesn’t always result in viroid-free plants. The success rate of tissue culture remediation is often cultivar-specific. Cultivators will surely put in the time and effort to save valuable heirloom cultivars, but many others will scrap infected plants and start a new phenohunt to replace them.
Prevention is surely the best way to protect your operation. There are a number of HLVd testing kits available that cultivators can use to confirm suspected infection or identify asymptomatic plants. Screening mother plants regularly, especially before taking a round of cuttings, would help to ensure only clean plants make it into production. Cultivators should also use extreme caution when introducing new genetics from another facility. New clones should be quarantined and screened for HLVd and other pathogens before they are allowed to share a space with existing plants.
A Case for Using qPCR for HLVd Testing
Medicinal Genomics was the first company to bring Eiken Chemical Co, Ltd.’s patented Loop-Mediated Isothermal Amplification (LAMP)-based testing to the Cannabis field with our youPCR® Gender and THC/CBD testing. We learned a lot from this endeavor and have a few thoughts to share as to why we have not yet deployed this patented technology to Hop Latent Viroid (HLVd) testing out of the gate.
Find Pdf

Quorum quenching is an antivirulence strategy employed by endophytic bacteria
Parijat Kusari & Souvik Kusari & Marc Lamshöft & Selahaddin Sezgin & Michael Spiteller & Oliver Kayser
Appl Microbiol Biotechnol
DOI 10.1007/s00253-014-5807-3
Bacteria predominantly use quorum sensing to regulate a plethora of physiological activities such as cell-cell crosstalk, mutualism, virulence, competence, biofilm formation, and antibiotic resistance. In this study, we investigated how certain potent endophytic bacteria harbored in Cannabis sativa L. plants use quorum quenching as an antivirulence strategy to disrupt the cell-to-cell quorum sensing signals in the biosensor strain, Chromobacterium violaceum. We used a combination of high-performance liquid chromatography high-resolution mass spectrometry (HPLC-ESI-HRMSn ) and matrix-assisted laser desorption ionization imaging highresolution mass spectrometry (MALDI-imaging-HRMS) to first quantify and visualize the spatial distribution of the quorum sensing molecules in the biosensor strain, C. violaceum. We then showed, both quantitatively and visually in high spatial resolution, how selected endophytic bacteria of C. sativa can selectively and differentially quench the quorum sensing molecules of C. violaceum. This study provides fundamental insights into the antivirulence strategies used by endophytes in order to survive in their ecological niches. Such defense mechanisms are evolved in order to thwart the plethora of pathogens invading associated host plants in a manner that prevents the pathogens from developing resistance against the plant/endophyte bioactive secondary metabolites. This work also provides evidence towards utilizing endophytes as tools for biological control of bacterial phytopathogens. In continuation, such insights would even afford new concepts and strategies in the future for combating drug resistant bacteria by quorum-inhibiting clinical therapies.

Not Cannabis Specific
Reducing Botrytis in greenhouse crops: periodic UV-light treatment in tomato plants
Dr. E. Heuvelink
Wageningen, 26 October 2006
Greenhouse growers in The Netherlands, and indeed throughout Europe, face a dilemma in the control of pathogenic fungi on their crops. Their customers demand a product that is free of fungi, while on the other hand, their customers demand a minimal level of pesticide residue (MLR) on the final product, thus severely limiting the options available to the grower in controlling the fungus. In this light, various systems are being developed in Wageningen to assist the growers in controlling fungal growth with minimal or no use of fungicides. One such method is the use of low dosage ultraviolet light. This patented method, owned by Clean Light BV of The Netherlands (PO Box 271, 6700 AG Wageningen, www.cleanlight.nl ), is based on the principle that both fungi and green plants are sensitive to ultraviolet light, but that there is a vast difference in lethal dose between fungi and green plants: Fungi can be killed with a very low dose, while green plants can tolerate much higher doses of ultraviolet light without showing signs of stress. This stands to reason given the observation that green plants survive, and indeed thrive, in full sunlight, while most fungi species only survive in surroundings that are sheltered from direct sunlight. The method then, is based on supplying a dose of ultraviolet light to the fungus that is a) high enough to kill the fungus, while b) low enough, so as not to harm the plant.

Research on crop protection is a very important topic, at the moment
https://www.mmjdaily.com/article/914...at-the-moment/
Crop protection for medical cannabis production in greenhouses or outdoors is a very important topic at the present time. Regulations around the world prohibit the use of chemical crop protections and the search for alternatives to combat diseases and pests is an important one within cannabis operations. Cameron Scott, a graduate student completing a Master’s degree at Simon
Fraser University in Burnaby, B.C. has been focusing on plant diseases that specifically affect the cannabis plant. “If one wants to grow medical cannabis, there are many insect and disease challenges to produce an acceptable crop. That is why research on crop protection is a very important topic at the moment: growers need to be provided with effective solutions that prevent yield loss and protect their crop from being damaged to produce a high quality product for consumers”.

Researchers Identify the Root Pathogens Affecting Hydroponically Grown Cannabis Plants
https://www.analyticalcannabis.com/a...-plants-311240
Dr. Zamir Punja and his research team at the Simon Fraser University in British Columbia have recently published the full results of their three-year-long study into root diseases affecting hydroponically grown cannabis plants.
By sampling plants that exhibited symptoms of root disease - such as stunted growth, brown root lesions, root rot, and leaf discoloration due to minor chlorosis - the research group was able to isolate and identify two genera of pathogens that had infected the plants.
The pathogens from the diseased plants were identified by plating affected root samples on a suitable agar-based medium and letting the fungal cultures develop. These were then broadly identified as being from the Fusarium genus and the Pythium genus and sent to the Agriculture and Food Laboratory in the University of Guelph Laboratory Services for polymerase chain reaction (PCR) analysis, in order to identify the exact species of each fungus that was present. The PCR testing indicated the presence of two species of Pythium (P. dissotocum and P. myriotylum) and two species of Fusarium (F. oxysporum and F. solani). At the tail-end of the study, isolates of Pythium aphanidermatum were also observed in a sub-sample of the diseased root cuttings.
Pythium is a parasitic root fungus which most commonly affects young plants or seedlings. Most Fusarium fungi are actually harmless soil microbes, however, the two species identified here have been known to cause root rot and wilting in plants. Both the Pythium and Fusarium species observed here have been detected in other crops, so it can be concluded that these root pathogens are not uniquely adapted to cannabis.

Root and crown rot pathogens causing wilt symptoms on field-grown marijuana (Cannabis sativa L.) plants
Zamir K. Punja, Cameron Scott & Sarah Chen
Canadian Journal of Plant Pathology Volume 40, 2018 - Issue 4
DOI: 10.1080/07060661.2018.1535470
Yellowing and wilting symptoms on field-grown Cannabis sativa (cannabis) plants followed by total plant collapse under conditions of extreme hot weather were observed in northern California in 2017. The crown regions of affected plants were dark and sunken and internal tissue discolouration extended 10–15 cm above the soil surface. Isolations made from the pith, vascular and cortical tissues in the crown region yielded Fusarium oxysporum (40% frequency), F. brachygibbosum (28% frequency), Pythium aphanidermatum (22% frequency),Fusarium solani and F. equiseti (5% frequency each). Pathogenicity tests were conducted on rooted plantlets to establish the extent of root and crown decay, as well as on mature stems to determine the extent of stem tissue colonization caused by these species. Extensive reduction in root length was caused by F. solani, F. oxysporum, F. brachygibbosum and P. aphanidermatum and wounding significantly enhanced disease development. Stem tissue colonization by these pathogens at wound sites was similarly extensive. Isolates of F. equisetiwere non-pathogenic. Both F. solani and P. aphanidermatum caused plant mortality within 6–10 weeks following inoculation. In phylogenetic analyses using the internal transcribed spacer (ITS) rDNA region and the elongation factor 1 (EF-1?) region, F. oxysporum isolates from cannabis plants in northern California were grouped separately from all other formae speciales and from isolates previously recovered from British Columbia. Two isolates of F. brachygibbosum were identical to an isolate previously reported to infect almond stems in cold storage and field-grown seedlings in northern California. These findings indicate that a complex of pathogens potentially can cause root and crown rot under field conditions, resulting in wilt symptoms and collapse of cannabis plants.

Sequence resource of bacterial communities associated to hemp in Ohio.
Willman, M., Keener, H. M., & Benitez Ponce, M.-S.
Phytobiomes Journal.(2020).
doi:10.1094/pbiomes-09-20-0062-a
In spite of changes in regulation, and increased hemp cultivation and commercialization, information on hemp best production practices in the US is scarce. Due to hemp’s human consumption, particularly as concentrated oil, hemp is often grown with low synthetic chemical
inputs. Therefore, microbiome management and the use of microbial inoculants are important tools to increase hemp productivity. As a first step to characterize the hemp core microbiome, we completed a survey of bacterial communities, associated with different tissue types of plants
grown in a field in Ohio. This local dataset could be integrated with further hemp microbiome surveys to identify potential beneficial interactions relevant to plant health and productivity, but also to evaluate microbiome impacts on product efficacy and safety.

Not Cannabis Specific
Silencing of CrNPR1 and CrNPR3 Alters Plant Susceptibility to Periwinkle Leaf Yellowing Phytoplasma.
Sung, Y.-C., Lin, C.-P., Hsu, H.-J., Chen, Y.-L., & Chen, J.-C.
Frontiers in Plant Science, 10. (2019).
doi:10.3389/fpls.2019.01183
Phytoplasmas are prokaryotic plant pathogens that cause considerable loss in many economically important crops, and an increasing number of phytoplasma diseases are being reported on new hosts. Knowledge of plant defense mechanisms against such pathogens should help to improve strategies for controlling these diseases. Salicylic acid (SA)-mediated defense may play an important role in defense against phytoplasmas. Here, we report that SA accumulated in Madagascar periwinkle (Catharanthus roseus) infected with periwinkle leaf yellowing (PLY) phytoplasma. CrPR1a expression was induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. NPR1 plays a central role in SA signaling, and two NPR1 homologs, CrNPR1 and CrNPR3, were identified from a periwinkle transcriptome database. Similar to CrPR1a, CrNPR1 expression was also induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. Silencing of CrNPR1, but not CrNPR3, significantly repressed CrPR1a induction in Tobacco rattle virus-infected periwinkle plants. In addition, symptoms of PLY progressed fastest in CrNPR1-silenced plants and slowest in CrNPR3-silenced plants. Consistently, expression of CrNPR1, but not CrNPR3, was induced by phytoplasma infection as well as SA treatment. This study highlights the importance of NPR1- and SA-mediated defense against phytoplasma in periwinkle and offers insight into plant-phytoplasma interactions to improve disease control strategies.

Southern Blight of Hemp caused by Athelia rolfsii detected in Virginia.
Mersha, Z., Kering, M., & Ren, S.
Plant Disease. (2019).
doi:10.1094/pdis-10-19-2178-pdn
Hemp (Cannabis sativa L.) is an annual herb from Cannabaceae family grown for its multitude of uses including fiber, seed and/or oil, and for its medicinal benefits. In August 2019, a hemp farm was visited in Dinwiddie County, Virginia. At one of the locations where the CBD variety ‘Boax’ plants were grown in drip irrigated and plastic mulch covered raised beds, three suspect plants with intermittent but conspicuous yellowing of foliage were clearly discernible. Disease incidence during the time of the visit, including the few symptomatic plants which were already removed after succumbing to the disease, was estimated between 0.5 to 2.0%. Brown to dark lesions, covered for most part by a white fluffy and, at times, by a fan-shaped mycelium that extended from the lower stem to the crown area near the soil-line were visible upon opening up the canopy. The lesions were irregular shaped and extended along the stem perimeter with lesion length ranging between 5 to 62 mm (mean = 24.4 mm). Brownish mustard-seed-like sclerotia of 0.4 to 1.6 mm in diameter (mean = 0.8, n = 34) were seen on the white fluffy mycelium at the soil-line. A sclerotium was aseptically placed at the center of acidified potato dextrose agar (APDA) and incubated at 30°C in the dark. Within 96 h, the white mycelia of the fungus was observed to have clamp connections and completely covered the 85 mm diamter Petri dishes. Slightly bigger sized sclerotia of 1.2 to 4.2 mm in diameter (mean = 2.2, n = 30) were formed 7 days later.

Storage of cannabinoids by Arctia caja and Zonocerus elegans fed on chemically distinct strains of Cannabis sativa.
ROTHSCHILD, M., ROWAN, M. G., & FAIRBAIRN, J. W.
Nature, 266(5603), 650–651. (1977).
doi:10.1038/266650a0
The larva of the warningly coloured tiger moth Arctia caja (L.) (Lepidoptera) and the grasshopper Zonocerus elegans (Thunberg) (Acridoidea) are polyphagous feeders with a predilection for poisonous foods plants. These species can sequester a store various secondary plant substances such as cardiac glycosides and pyrrolizidine alkaloids which presumably function as predator deterrents. We report here that cannabinoids are also stored by both insects. There is good evidence that Cannabis sativa contains at least two chemical races or strains, one rich in the psychoactive substance delta-1-Tetrahydrocannabinol (THC) and the other in the inactive cannabidiol (CBD) and accordingly one of each type was used in our work.

Striatura Ulcerosa
John McPartland, Karl Hillig
June 2004Journal of Industrial Hemp 9(1):89-96
DOI: 10.1300/J237v09n01_10
This is the second in a series of “Cannabis clinic” notes, presenting diseases and pests of hemp, featuring colour illustrations of signs and symptoms. This note concerns Striatura Ulcerosa, the name of a hemp disease caused by the bacterium Pseudomonas syringae pv. mori. The disease was first described in Italy over 100 years ago, but may have originated in China. Greenhouse studies demonstrated that all cultivars of Cannabis are susceptible. Signs and symptoms, life history of the pathogen, and control measures are discussed.

Surveying for Potential Diseases and Abiotic Disorders of Industrial Hemp (Cannabis sativa) Production
Lindsey D. Thiessen, Tyler Schappe, Sarah Cochran, Kristin Hicks, and Angela R. Post
Plant Health Progress 14 Oct 2020
DOI: 10.1094/PHP-03-20-0017-RS
ndustrial hemp (Cannabis sativa L.) has recently been reintroduced as an agricultural commodity in the United States, and, through state-led pilot programs, growers and researchers have been investigating production strategies. Diseases and disorders of industrial hemp in the United States are largely unknowns because record-keeping and taxonomy have improved dramatically in the last several decades. In 2016, North Carolina launched a pilot program to investigate industrial hemp, and diseases and abiotic disorders were surveyed in 2017 and 2018. Producers, consultants, and agricultural extension agents submitted samples to the North Carolina Department of Agriculture and Consumer Services Agronomic Services Division (n = 572) and the North Carolina Plant Disease and Insect Clinic (n = 117). Common field diseases found included Fusarium foliar and flower blights (Fusarium graminearum), Fusarium wilt (Fusarium oxysporum), and Helminthosporium leaf spot (Exserohilum rostratum). Greenhouse diseases were primarily caused by Pythium spp. and Botrytis cinerea. Common environmental disorders were attributed to excessive rainfall flooding roots and poor root development of transplanted clones.

The diverse mycoflora present on dried cannabis (Cannabis sativa L., marijuana) inflorescences in commercial production.
Punja, Z. K.
Canadian Journal of Plant Pathology, 1–13.(2020).
doi:10.1080/07060661.2020.1758959
The objective of this study was to assess harvested dried inflorescences (buds) of cannabis (Cannabis sativa L., marijuana) for fungal presence and diversity. Samples from drying rooms of three licenced facilities in British Columbia were tested repeatedly during 2017–2019. A swab method was used, wherein sterile cotton swabs were gently swabbed over bud surfaces and directly streaked onto potato dextrose agar containing 140 mg L?1 streptomycin sulphate. Petri dishes were incubated at 21–24°C for 5–6 days and the fungal colonies that developed were recorded. The testing was repeated to provide >40 cumulative sampling times over a 2-year period. Representative colonies of each unique morphological type were identified to genus and species by PCR of the ITS1-5.8-ITS2 region of rDNA and sequence analysis. Among 34 different fungal species identified, the most prevalent were Penicillium (comprising 17 different species), followed by species of Cladosporium, Botrytis, Aspergillus, Fusarium, Talaromyces and Alternaria. All samples had several fungal species present and the number and composition varied at different sampling times and within different facilities. The swab method provided a qualitative assessment of viable mould contaminants on cannabis buds and reflected the diversity of mycoflora present, many of which are previously unreported. Fungi on cannabis buds may originate from spores released from diseased or decomposing plant materials, from growing substrates used in cannabis production, or as airborne contaminants in post-harvest trimming and drying rooms. Samples of dried buds exposed to electrobeam (e-beam) radiation treatment had no detectable fungal contamination when assessed using the swab method.

Not Cannabis specific
The Effect of Blue-light-emitting Diodes on Antioxidant Properties and Resistance to Botrytis cinerea in Tomato
Kangmin Kim, Hee-Sun Kook, Ye-Jin Jang, Wang-Hyu Lee, Seralathan Kamala-Kannan, Jong-Chan Chae and Kui-Jae Lee
J Plant Pathol Microb 4: 203.
doi:10.4172/2157- 7471.1000203
In higher plants, blue-light is mainly perceived by cryptochromes and phototropins, which subsequently orchestrates phototropism, chloroplast relocation, stomatal opening, rapid inhibition of hypocotyl elongation and leaf expansion. Blue-light signaling is also known to mediate the plant responses to biotic stresses, but relevant mechanisms are largely unknown. Here, we demonstrated that blue LED (Light Emitting Diode)-driven inhibition of gray mold disease was highly correlated with the increases in cellular protectants like proline, antioxidants and ROS (Reactive Oxygen Species) scavenger activities. After twenty one days of exposure to various wavelengths of LED lights, blue-LED treated tomato displayed significant increases in proline accumulation in the leaves and stems, whereas red- and green-LED treated tomato exhibited the lower proline contents. Similarly, the blue-LED treatment increased the amount of polyphenolic compounds in tomatoes, compared to other wavelength of LED lights. The activities of various ROS (Reactive Oxygen Species) scavenging enzymes were also slightly increased under the blue-LED lighted conditions. Finally, blue-LED significantly suppressed symptom development of tomato infected by gray mold. Combined results suggest that blue LED light inhibits the development of gray mold disease, which can be mechanistically explained by the enhanced proline accumulation and antioxidative processes at least in partial.

NOT CANNABIS SPECIFIC but this virus is found in Cannabis
The elimination of viruses and Hop Latent Viroid from hop (Humulus lupulus L.) in Poland
M. Grudziska, E. Solarska
February 2005Acta horticulturae 668(668):149-152
DOI: 10.17660/ActaHortic.2005.668.19
Hops in Poland are commonly infected with Hop latent viroid (HLVd) and viruses: Prunus necrotic ringspot virus (PNRSV) and Hop mosaic virus (HpMV). Previous experiments were successful in obtaining virus-free hop plants by heat therapy method but appeared to be inefficient for HLVd elimination. In these studies virus and viroid free hop plants were obtained by meristem-tip culture. The meristem tips were excised from lateral shoot tips of naturally infected plants and placed in vitro. In vitro regeneration ability varied between 0 and 46% of regenerated explants depending upon cultivar, depending upon the time at which meristems tips were obtained. The regenerated plants were tested by ELISA for the presence of viruses and by RT-PCR for the presence of HLVd. Plants with no detectable viruses and HLVd were used for further propagation.

Not Cannabis Specific, but found in Cannabis
The experimental transmission of hop latent viroid and its elimination by low temperature treatment and meristem culture
A N ADAMS, D J BARBARA, A MORTON and P DARBYAnnals of Applied Biology 128(1):37 - 44
DOI: 10.1111/j.1744-7348.1996.tb07087.x
Two aspects of hop latent viroid (HLVd) relevant to control were examined:
the production of viroid-free plants from infected material and transmission of HLVd in the field. Plants free from HLVd were obtained by a combination of storing infected source plants at low temperature (2-4°C in the dark) for several months followed by meristem culture using small explants. A total of 77 plants of six cultivars and male pollinator clones were grown from meristems and 28 of these were free from HLVd. Tests showed that the cutting of stems (mimicking the use of tools) was more effective than abrasion (mimicking natural plant to plant contact) for the mechanical transmission of HLVd between hop plants. When field-grown test plants were inoculated, infection occurred more commonly in May before plants had grown large enough for significant contact between neighbouring plants than later in the season. The aphid Phorodon humuli could not be shown to transmit HLVd.
These results indicate that all hop varieties and pollinator clones can be made available to the industry free from HLVd and that the chances of infection can be reduced by avoiding early-season cultural operations that cut into hop shoots.

The Hemp Russet Mite
John McPartland, Karl Hillig
March 2003 Journal of Industrial Hemp 8(2):107-112
DOI: 10.1300/J237v08n02_10
The hemp russet mite Aculops cannabicola (Farkas) is a little-known but potentially dreadful pest. It primarily feeds on leaves, petioles, and meristems. The mites quickly spread between plants grown in proximity. They cause a curling of leaf edges, followed by leaf russeting. The mites feed on the inflorescences of both sexes, and on glandular trichomes, severely reducing resin production. The hemp russet mite constitutes a real menace to world hemp cultivation, because it cannot be controlled easily.

The hemp russet mite Aculops cannabicola (FARKAS, 1960) (ACARI: ERIOPHYOIDEA) FOUND ON CANNABIS SATIVA L. IN SERBIA: SUPPLEMENT TO THE DESCRIPTION
RADMILA PETANOVI, BILJANA MAGUD, and DRAGICA SMILJANI?
Arch. Biol. Sci., Belgrade, 59 (1), 81-85, 2007.
DOI: 10.2298/ABS0701081P
The description of Aculops cannabicola (Farkas, 1960), a new species for the fauna of Serbia, is supplemented. The male and juveniles are described for the first time. This species was found as vagrants on leaves of hemp, Cannabis sativa L. A comparison of characters of the population from Serbia and the originally described samples from Hungary is given.

The Impact of Insect Herbivory in the Level of Cannabinoids in CBD Hemp Varieties
Brandon Jackson, Lenneisha Gilbert, Tigist Tolosa, Shellyann Henry, Victoria Volkis, Simon Zebelo
DOI: 10.21203/rs.3.rs-155271/v1
https://www.researchgate.net/publica...Hemp_Varieties
Background
In the United States, industrial hemp is de􀂦ned as a Cannabis sativa L. plant not containing more than 0.3% delta-9-tetrahydrocannabinol (D9THC) by dry weight. Plants respond to insect herbivore damage by
changing their chemistry to counter the effects of herbivore attack. Here, we hypothesized that the corn earworm (Helicoverpa zea) infestation might impact the level of cannabinoids (Cannabidiol (CBD) and D9THC).
Results
In a laboratory trial, the CBD hemp, Cherry Blossom, and The Wife varieties were subjected to herbivore damage (HD), Mechanical damage (MD), and Control. After 24hrs of the treatments, we found a significant increase in CBD and D9THC in HD plants compared with MD and Control plants. Similar experiments were conducted in the field condition. A substantial increase in CBD and D9THC observed in herbivore damaged hemp plants compared to the control plants. However, in the field trial, the levels of cannabinoids were not significantly higher in The wife variety. Interestingly, the Corn earworm larvae fed with CBD and D9THC spiked diet showed a significant reduction in body mass, as compared to the larvae fed with the control diets.
Conclusions
The level of cannabinoids seems not genetically 􀂦xed somewhat; it is affected by insect herbivory. Our results suggest that CBD hemp plants are exposed to insect herbivory spikes in cannabinoid production and surpass the 0.3 % legal limit of D9THC. The growth and development of Corn earworm, the number one hemp pest in North America affected by cannabinoids. The increased concentration of CBD and D9THC observed in herbivore damaged hemp plants might be associated with the direct deterrence of the corn earworm larvae. Further research underway using different hemp varieties to assess if herbivory and other biotic stressors impact the level of cannabinoids.

NOT CANNABIS SPECIFIC but this virus us found in Cannabis
The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops.
Puchta H1, Ramm K, Sänger HL.
Nucleic Acids Res. 1988 May 25;16(10):4197-216.
DOI: 10.1093/nar/16.10.4197
A new viroid which does not seem to produce any symptoms of disease, and is therefore tentatively named hop latent viroid (HLV) was found to occur worldwide in hops. HLV proved to be infectious when mechanically inoculated onto viroid- and virus-free hops. The viroid nature of HLV was also substantiated by sequence analysis which revealed that HLV is a circular RNA consisting of 256 nucleotides, that can be arranged into the viroid-specific, rod-like secondary structure. HLV also contains the central conserved region typical for most of the presently known viroids. However HLV does not contain the viroid-specific oligo(A) stretch in the upper left part of its rod-like molecule. Because of this feature and a sequence similarity with the prototypes of the other viroid groups below 55%, HLV can be regarded as the first member of a new viroid group.

The occurrence of Hop latent viroid causing disease in Cannabis sativa in California.
Jeremy Warren, Jennifer Mercado, Dan Grace (Dark Heart Nusery report)
Plant Disease
doi.org/10.1094/PDIS-03-19-0530-PDN
https://www.researchgate.net/publica...ldwide_in_hops
In 2017 disease symptoms were noticed on multiple cultivars of Cannabis sativa plants grown in California, including; stunting, malformation or chlorosis of leaves, brittle stems and reduction in yields. Additionally, cuttings taken from symptomatic plants for clonal propagation showed a reduced rooting success rate. Leaf tissues from five symptomatic and five asymptomatic plants were selected for RNA isolation and subsequent sequencing from northern California (37.7567466, -122.1930517). Total RNA was isolated from leaf tissue using TRIzol reagent (Thermo Fisher). Ribosomal RNA was removed using Ribo-Zero rRNA Removal Kit, Plant (Illumina). RNA-seq libraries were synthesized using YourSeq RNA-seq kit for full transcript coverage libraries (Amaryllis Nucleics, Oakland California). Libraries were sequenced on an Illumina NextSeq 500 using single-read 80bp (Amaryllis Nucleics). Each sample resulted in between 12,861,714 and 19,376,549 reads (data available at www.mg-rast.org project HpLVd – Can). The resulting sequences were de novo assembled and the resulting contigs were aligned to the Cannabis sativa draft genome (ASM341772v2). Contigs that matched the Cannabis sativa genome were removed from the analysis and the remaining contigs were compared to sequences in the viral and viroid Genbank databases. All five symptomatic plants had a single 256 nucleotide contig (total reads mapped per contig: ranged from 4,162 to 50,095) that matched Hop latent viroid (HLVd).

The Occurrence of Hop Latent Viroid in Cannabis sativa with symptoms of Cannabis Stunting Disease in California
Ali Bektas, Kayla M. Hardwick, Kristen Waterman, Jessica Kristof
June 2019 Plant Disease
DOI: 10.1094/PDIS-03-19-0459-PDN
In February 2018 we sampled three symptomatic and three asymptomatic Cannabis plants suspected to host a viral agent from a farm in Santa Barbara County, CA. The symptoms included brittle stems, an outwardly horizontal plant structure and reduced flower mass and trichomes. RNA from each flower sample was extracted using a QIAmp Viral Mini Kit (Qiagen, Hilden, Germany). RNA was purified, concentrated, and ribo-depleted before library construction with NEBNext Ultra II Directional RNA kit (NEB, Ipswich, MA). Individual libraries were tagged with unique adapters and pooled for sequencing with a NextSeq High Output 300 cycle kit (Illumina, San Diego, CA). Sequence data were assembled with Trinity (Grabherr et al. 2011), and Blobtools (Laetsch and Blaxter 2017) was used to search for viral sequences. Possible viral transcripts were filtered for at least 10× coverage, 95% BLAST identity, and alignment length greater than 80% of query and subject sequences. One hundred twenty-five transcripts showed significant similarity to hop latent viroid (HLVd) sequence EF613183. We used STAR (Dobin et al. 2013) to align reads to the transcripts and found that 2.4% of read pairs from symptomatic libraries (of a total of 153,001,495 read pairs) and 0.0001% of read pairs from asymptomatic libraries (of a total of 159,608,791 read pairs) mapped to the putative HLVd sequences. We also identified a transcript with high homology to C. sativa mitovirus 1 (BK010428). Reads from all libraries mapped to this transcript in similar numbers; thus, the mitovirus is unlikely to be associated with the disease symptoms. To confirm our findings, we used two HLVd-specific primer pairs in reverse transcription PCR (RT-PCR). The Zeigler et al. (2014) primer pair produced the expected HLVd amplicon size from only the symptomatic plants, whereas the primer pair of Eastwell and Nelson (2007) produced the expected HLVd amplification pattern from all samples collected. The amplicons were Sanger sequenced and produced at least 200 bp of data with 95% or more pairwise identity to HLVd sequences in GenBank. One sample, CV1 (MK791751), produced 98% pairwise identity across the whole 256-bp HLVd genome as published by Puchta et al. (AX07397). In February 2019, we sampled seven symptomatic and three asymptomatic plants from another farm in Alameda County, CA. RT-PCR revealed the presence of HLVd in all symptomatic plants and one asymptomatic plant. Amplicons from four of these samples were Sanger sequenced and confirmed to be HLVd (MK791747 to MK791750). Analysis of the sequences obtained from the two locations revealed two single-nucleotide polymorphisms between them at positions 190 and 225, which include the centrally conserved region (Puchta et al. 1988). The consistent detection of HLVd alone in all symptomatic plants from both locations and its occurrence in only a few asymptomatic plants point to an association of the disease with the stunted Cannabis. HLVd can persist in hops without symptoms, and Cannabis cultivators should take measures to minimize mechanical transmission while keeping in mind that HLVd may also be seed transmissible (Pethybridge et al. 2008). Considering the detrimental effects of HLVd to the commercially valuable secondary metabolites of the species, a thorough investigation of susceptibility as well as range and expression of Cannabis stunting disease should be conducted at this important juncture.

NOT CANNABIS SPECIFIC
The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields
Qian Wang1, Wei-Fang Bao, Fan Yang, Bin Xu1, Yi-Zhong Yang
PLoS ONE 12(6): e0177789.
Doi: 10.1371/journal.pone.0177789
The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions.

The Threat of Viral Cannabis Diseases
Robert Clarke
Cannabis Business Times Aug. 2020 pg 44-48
https://www.cannabisbusinesstimes.c...ns-the-threat-of-viral-cannabis-diseases-cds/

Around the world people are coming to grips with the health issues and economic fallout of COVID-19. Public awareness of viral contagions is reaching unprecedented levels, presenting an opportune moment to address disease problems within the cannabis industry. As we are seeing with health issues and economic fallout of COVID-19. Public awareness of viral contagions is reaching unprecedented levels, presenting an opportune moment to address disease problems within the cannabis industry. As we are seeing with the novel coronavirus pandemic, harmful viruses emerge and adapt, and this is not exclusive to humans. Cannabisgrowers are increasingly experiencing the negative economic outcomes of decreased vigor, lower flower yields and reduced production of primary target compounds including both cannabinoids and terpenoids. What we are calling “Cannabisdisease syndrome” exhibits a suite of consistent symptoms, but with no readily apparent single cause. These symptoms, which do not appear to be caused by nutrient deficiencies or other pathogens, are often collectively referred to as “dudding” or “dudders.” (The term originated when growers would think a plant with decreased vigor or stunted growth was “just a dud.”)
Steadily declining vigor in commercial Cannabisclones is not a new phenomenon. As vegetative reproduction by rooting cuttings became popular in the 1980s, growers would occasionally see a clone that became weaker and less productive each time cuttings were flowered. Apart from lowered yield, there were few other symptoms of infection. We tentatively called this a “photocopy effect” based on our analogy that copying a copy of a copy of a copy, results in a faded image that eventually becomes a mere ghost of the original. We knew that because lost vigor was appearing in asexuallymultiplied serial cuttings the problem could not be explained by “genetic drift,” which is a shift in the frequency of genes within a small sexuallyreproducing population. Growers wondered what the causes might be, and even addressed the possibility that simply making serial cuttings might result in diminished vigor. Soon we realized the symptoms were caused by transmission of an infectious disease that became more and more prevalent through successive rounds of multiplication. (More on this later.) We destroyed clones exhibiting symptoms, carefully sterilized benches, pots and tools, and began to use fresh blades when taking cuttings from each mother plant. There were no known causes, just obvious adverse effects. Yet we found practical solutions, and soon the problem nearly disappeared. Some similarities exist between the Cannabis disease syndrome (CDS) we are experiencing today and COVID-19. Much like the human coronavirus, CDS is difficult to detect at first, as there. is a wide range of symptoms. Through our and other growers’ observations of affected plants during the past few years, we have learned that vegetative plants can transmit CDS, while flowering plants are more likely to suffer the consequences. Because symptoms are not readily visible and are easily confused with other diseases, they both lie hidden within populations, and can very quickly become economically impactful. Another similarity between CDS and COVID-19 is that asymptomatic plants can infect the otherwise healthy, with more serious outcomes for some than others. Molecular testing is required to identify potential infections, and there are few laboratories that can effectively identify the causal organisms. Other than practicing social distancing and establishing quarantines, there are as yet no solutions to stopping their spread. Cannabis disease syndrome cannot be attributed to a single pathogen, although there is a primary candidate for its cause (more on this later). In symptomatic plants, several infectious organisms may be involved, making accurate diagnosis and effective control even more difficult. If CDS killed more of its hosts rather than simply making them sick, then it would have been noticed much earlier, and should not have already spread so widely. As we also have observed while studying affected plants, the cannabis disease syndrome spreads most quickly by taking cuttings from infected plants, using them as mother plants, and thereby multiplying the disease through future generations.


The Use of Cannabis sativa L. for Pest Control: From the Ethnobotanical Knowledge to a Systematic Review of Experimental Studies
Genı´s Ona, Manica Balant, Jose´ Carlos Bouso, Airy Gras, Joan Valle`s, Daniel Vitales, and Teresa Garnatje
Cannabis and Cannabinoid Research Volume X, Number X, 2021
DOI: 10.1089/can.2021.0095
https://www.liebertpub.com/doi/abs/1...ournalCode=can
Background: Despite the benefits that synthetic pesticides have provided in terms of pest and disease control, they cause serious long-term consequences for both the environment and living organisms. Interest in ecofriendly products has subsequently increased in recent years.
Methods: This article briefly analyzes the available ethnobotanical evidence regarding the use of Cannabis sativa as a pesticide and offers a systematic review of experimental studies.
Results: Our findings indicate that both ethnobotanical and experimental procedures support the use of C. sativa as a pesticide, as remarkable toxicity has been observed against pest organisms. The results included in the systematic review of experimental studies (n = 30) show a high degree of heterogeneity, but certain conclusions can be extracted to guide further research. For instance, promising pesticide properties were reported for most of the groups of species tested, especially Arachnida and Insecta; the efficacy of C. sativa as a pesticide can be derived from a wide variety of compounds that it contains and possible synergistic effects; it is crucial to standardize the phytochemical profile of C. sativa plants used as well as to obtain easily reproducible results; appropriate extraction methods should be explored; and upper inflorescences of the plant may be preferred for the production of the essential oil, but further studies should explore better other parts of the plant.
Conclusion: In the coming years, as new findings are produced, the promising potential of C. sativa as a pesticide will be elucidated, and reviews such as the present one constitute useful basic tools to make these processes easier.
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(Not Cannabis specific but virus is found in Cannabis)
The Variability of Hop Latent Viroid as Induced upon Heat Treatment.
Matoušek, J., Patzak, J., Orctová, L., Schubert, J., Vrba, L., Steger, G., & Riesner, D.
Virology, 287(2), 349–358. (2001).
doi:10.1006/viro.2001.1044
We have previously shown that heat treatment of hop plants infected by hop latent viroid (HLVd) reduces viroid levels. Here we investigate whether such heat treatment leads to the accumulation of sequence variability in HLVd. We observed a negligible level of mutated variants in HLVd under standard cultivation conditions. In contrast, the heat treatment of hop led to HLVd degradation and, simultaneously, to a significant increase in sequence variations, as judged from temperature gradient–gel electrophoresis analysis and cDNA library screening by DNA heteroduplex analysis. Thirty-one cDNA clones (9.8%) were identified as deviating forms. Sequencing showed mostly the presence of quadruple and triple mutants, suggesting an accumulation of mutations in HLVd during successive replication cycles. Sixty-nine percent of base changes were localised in the left half and 31% in the right half of the secondary structure proposed for this viroid. No mutations were found in the central part of the upper conserved region. A “hot spot” region was identified in a domain known as a “pathogenicity domain” in the group representative, potato spindle tuber viroid. Most mutations are predicted to destabilise HLVd secondary structure. All mutated cDNAs, however, were infectious and evolved into complex progeny populations containing molecular variants maintained at low levels.

Three Botrytis species found causing gray mold on industrial hemp (Cannabis sativa) in Oregon
A.R. Garfinkel
DOI: 10.1094/PDIS-01-20-0055-PDN
In September and October of 2019, flowers of hemp plants in Polk and Linn counties in Oregon showed symptoms of die-back with necrosis of the tissues, resulting in significant yield reductions. The tops of the inflorescences were often the most severely affected with the infection sometimes moving down into the petiole or stem. Up to 90% of the plants in these fields had at least one flower infection present, however, foliar symptoms (lesions) were not observed. Gray to white mycelium and Botrytis-like conidiophores could often be seen arising from host tissue. The fungus was recovered from colonized plant tissues either by placing conidia directly onto a Petri dish containing potato dextrose agar (PDA) or placing a small piece of surface sterilized plant tissue onto PDA. A total of 23 pure cultures were recovered from three fields. All cultures displayed white to gray, fast-growing mycelium within which conidiophores sometimes developed bearing Botryose clusters of conidia, followed by the formation of black sclerotia in all isolates.

Too Many Mouldy Joints – Marijuana aand Chronic Pulmonary Aspergillosis
Yousef Gargani, Paul Bishop and David W. Denning
Mediterr J Hematol Infect Dis 2011, 3; Open Journal System
DOI 10.4084/MJHID.2011.005
Chronic pulmonary aspergillosis is a progressive debilitating disease with multiple underlying pulmonary diseases described. Here we report the association of chronic pulmonary aspergillosis and long term marijuana smoking in 2 patients and review the literature related to invasive and allergic aspergillosis.

Toxic effects of palpoluck Polygonum hydropepper L. and Bhang Cannabis sativa L. plants extracts against termites Heterotermes indicola (Wasmann) and
Coptotermes heimi (Wasmann) (Isoptera: Rhinotermitidae)

Hayat Badshah, Abdus Sattar Khan, Abid Farid, Alam Zeb
and Amanullah Khan
Songklanakarin J. Sci. Technol., 2005, 27(4) : 705-710
https://thaiscience.info/Journals/Ar...G/10986937.pdf
A research project was carried out aimed at to study the toxic effects of Palpoluck Polygonum hydropipper L. and Bhang Cannabis sativa L. crude extracts against two species of termites i.e. Heterotermes indicola (Wasmann) and Coptotermes heimi (Wasmann) at Nuclear institute for Food and Agriculture (NIFA) Peshawar, Pakistan in April 2002. Results revealed that after ten days of feeding maximum percent mortality in case of Polygonum hydropipper L. leaf and flower extracts was 28.0, 52.0, 28 and 74.7 for H. indicola and Coptotermes heimi respectively, while in control only 10.7 and 12.0% mortality were recorded. Similarly, for the same species of termites the percent mortality in Cannabis sativa L. extracts was 54.7, 64.0, 58.7 and 70.7 for leaf and seed extracts respectively, while in control only 12.0 and 10.7% mortality were observed. In each extract mortality was significantly different from that of control. Toxic effects of both extracts (leaf and flower) were more profound against Coptotermes heimi than Heterotermes indicola during these ten days of feeding. Also the seed extracts caused more mortality than the leaves for both species, suggesting the availability of high contents of toxic materials in seed.

Two Aphid Species, Phorodon cannabis and Rhopalosiphum
rufiabdominal, Identified as Potential Pests on Industrial Hemp, L., in the US Midwest

Doris Lagos-Kutz, Bruce Potter, Christina DiFonzo,Glen L. Hartman, Russell A. Howard
January 2018 Project: Suction Trap Network in the Midwest
DOI: 10.2134/cftm2018.04.0032
Cannabis sativa L. is indigenous to eastern Asia, grown since ancient times for its medicinal and textile uses (Russo et al., 2008). In the USA, production is currently increasing (USDA-NRCS, 2018)
for industrial fiber, seed oil, medicinal and recreational uses. As production increases, there is growing interest in properly identifying and managing pests attacking the crop in both field and greenhouse environments. In this brief we provide new records of distribution of Phorodon cannabis, a recently introduced insect species in the USA, and Rhopalosiphum rufiabdominale, a widely distributed species on grasses and other dicots. In addition, we are including morphological and molecular information of P. cannabis and P. humuli to avoidthe misidentification of these two closely related insect species

Using Ultraviolet Light to Stop Gray Mold, Powdery Mildew & Other Marijuana Plagues
Marijuana Growing Educators April 9, 2019
https://growingmarijuanaperfectly.co.. .ana-grow-room/
This is an important update to an article we posted a while ago, about using ultraviolet light to fight gray mold, powdery mildew and similar marijuana enemies. The photo you see just above this text shows you two marijuana leaves–one treated with ultraviolet light, and the other not treated. The untreated leaf is infested with powdery mildew, but an ultraviolet light device made by a company called CleanLight blocked powdery mildew on the other leaf!
Ultraviolet light can kill molds and fungi, which are some of the biggest causes of crop failures in marijuana grow rooms. Obviously, you want to know how to do that same mildew-killing ultraviolet treatment in your marijuana garden. In the original version of this article, we discussed an ultraviolet device called the Reme Halo. The Halo is a tiny device that has to be installed by an HVAC professional inline in your grow op building’s air handler. It uses a low-watt ultraviolet light and an ionized hydroperoxide generator to scrub air. The manufacturer claims it removes mold, fungi, and odor.

Not Cannabis Specific
UV light offers possibilities against powdery mildew
https://cleanlight.nl/wp-content/upl...ery-mildew.pdf
The latest experiments with UV light indicate that fewer fungicide applications are required when applied in the correct manner. DLV (research group) tested the potential of UV light treatments on several nursery crops and perennials.

Western Plant Diagnostic Network News : Pythium aphanidermatum Crown Rot of Industrial Hemp
September 2017
Jennifer Schoener, Shouhua Wang, Rus Wilhelm
https://www.npdn.org/system/files/NPDN_September-17.pdf
Cultivation of industrial hemp (Cannabis sativa) was first approved in 2014 for the purpose of research and development. The Federal Farm Bill Section 7606 authorizes state agencies to conduct pilot trials on the crop to assess crop viability for the creation of an industry in prospective states. In Nevada, the Department of Agriculture authorizes the production of hemp crops for research purposes. The acreage of hemp production in Nevada is relatively small in comparison to the acreage in other states. However, plant diseases associated with hemp crops have been occurring in Nevada in recent years. In 2016, the Nevada Department of Agriculture Plant Pathology Lab detected Fusarium root rot and sudden death disease from an industrial hemp crop, and Fusarium wilt from medical marijuana plants. Here we describe a newly detected hemp disease: Pythium aphanidermatum crown rot.

White Leaf Spot
John McPartland, Karl Hillig
April 2006 Journal of Industrial Hemp 11(1):43-50
DOI: 10.1300/J237v11n01_06
This note continues the “Cannabis Clinic” series, presenting diseases and pests of hemp, featuring color illustrations of signs and symptoms. White leaf spot is caused by a fungus, Phomopsis ganjae.
Signs and symptoms of the disease, life history of the pathogen, differential diagnosis, and control measures are discussed. Disease symptoms and fungus morphology are presented in a series of photographs, each representing approximately one-tenth the scale of the previous photo, to provide an enhanced perspective of scale and proportion

Zur virusanfälligkeit von hanfsorten (Cannabis sativaL.).
Kegler, H., & Spaar, D. (1997).
Archives Of Phytopathology And Plant Protection, 30(5), 457–464.
doi:10.1080/03235409709383198
ON THE VIRUS SUSCEPTIBILITY OF VARIETIES OF CANNABIS SATIVA L. The growing of hemp (Cannabis sativa L.) as a reproducing raw material is increasing in Europe. Investigations concerning the susceptibility of three hemp genotypes to 8 plantpathogenic viruses of various taxonomic groups showed distinct differences
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Terpenes


A chemotaxonomic analysis of terpenoid variation in Cannabis
Karl W. Hillig
Biochemical Systematics and Ecology 32 (2004) 875–891
doi:10.1016/j.bse.2004.04.004
To determine whether the terpenoid composition of the essential oil of Cannabis is useful for chemotaxonomic discrimination, extracts of pistillate inflorescences of 162 greenhouse grown plants of diverse origin were analyzed by gas chromatography. Peak area ratios of 48 compounds were subjected to multivariate analysis and the results interpreted with respect to
geographic origin and taxonomic affiliation. A canonical analysis in which the plants were pre-assigned to C. sativa or C. indica based on previous genetic, morphological, and chemotaxonomic studies resulted in 91% correct assignment of the plants to their pre-assigned species. A scatterplot on the first two principal component axes shows that plants of accessions from Afghanistan assigned to the wide-leaflet drug biotype (an infraspecific taxon of unspecified rank) of C. indica group apart from the other putative taxa. The essential oil of these plants usually had relatively high ratios of guaiol, isomers of eudesmol, and other unidentified compounds. Plants assigned to the narrow-leaflet drug biotype of C. indica tended to have relatively high ratios of trans-b-farnesene. Cultivars of the two drug biotypes may exhibit distinctive medicinal properties due to significant differences in terpenoid composition.

A Green Extraction Process to Recover Polyphenols from Byproducts of Hemp Oil Processing.
Mourtzinos, I., Menexis, N., Iakovidis, D., Makris, D., & Goula, A.
Recycling, 3(2), 15.(2018).
doi:10.3390/recycling3020015
The valorization of solid waste hemp (Cannabis sativa L.) by a non-conventional method is presented in this article. Hemp polyphenols were extracted using aqueous solutions of 2-hydroxypropyl-β-cyclodextrin as an eco-friendly extraction solvent. Cyclodextrins (CD’s) are known to enhance the extraction of polyphenols in water by forming water soluble inclusion complexes. The process was optimized by implementing a response surface methodology (RSM) that took into consideration the following independent variables: CD concentration (CCD), solid-to-liquid ratio (S/L), and temperature (T). The assessment of the extraction model was based on two responses: the total polyphenol yield (YTP) and the antiradical activity (AAR). The optimum operating conditions were found to be: CD concentration, 32.1% (w/v); solid/solvent ratio, 1/15.2 g/mL; and extraction temperature, 28 ◦C. Different kinetic models were employed to fit with experimental data and the Peleg’s model was successfully developed for describing the mechanism of extraction under different processing parameters.

NOT CANNABIS SPECIFIC
A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway.
Li, H., Ban, Z., Qin, H., Ma, L., King, A. J., & Wang, G.
Plant Physiology, 167(3), 650–659. (2015).
doi:10.1104/pp.114.253682
Bitter acids (?-type and ?-type) account for more than 30% of the fresh weight of hop (Humulus lupulus L.) glandular trichomes and are well-known for their contribution to the bitter taste of beer. These multi-prenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the ?-bitter acid pathway. Here, we employed a yeast system for the functional identification of aromatic prenyltransferase genes (PT). Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific cDNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstreamgenes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Step-wise mutation of the Asp-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in ?-bitter acid biosynthesis: PT1L only catalyzed the first prenylation step; PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two hybrid system, reciprocal co-immunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop.

A Systematic Approach to Developing Terpene Extraction Conditions Utilising Supercritical Carbon Dioxide
Eric Kawka
Chromatography Today Feb/March 2018
https://www.chromatographytoday.com/...-dioxidep/2337
Cannabis sativa plants produce and accumalate terpene-rich resin within the secretory cells of glandular trichomes. Monoterpenes and sesquiterpenes are important components of Cannabis resin as they contribute to the unique attributes of different Cannabis strains. Terpenes are responsible for the plants aroma and flavor.

Accelerated Solvent Extraction of Terpenes in Cannabis Coupled With Various Injection Techniques for GC-MS Analysis
Colton Myers, Jason S. Herrington, Paul Hamrah, Kelsey Anderson,
Frontiers in Chemistry 9 April 2021
DOI: 10.3389/fchem.2021.619770
https://www.researchgate.net/publica...GC-MS_Analysis
The cannabis market is expanding exponentially in the United States. As state-wide legalization increases, so do demands for analytical testing methodologies. One of the main tests conducted on cannabis products is the analysis for terpenes. This research focused on implementation of accelerated solvent extraction (ASE), utilizing surrogate matrix matching, and evaluation of traditional vs. more modern sample introduction techniques for analyzing terpenes via gas chromatography–mass spectrometry (GC-MS). Introduction techniques included Headspace-Syringe (HS-Syringe), HS-Solid Phase Microextraction Arrow (HS-SPME Arrow), Direct Immersion-SPME Arrow (DI-SPME Arrow), and Liquid Injection-Syringe (LI-Syringe). The LI-Syringe approach was deemed the most straightforward and robust method with terpene working ranges of 0.04–5.12 μg/mL; r ² values of 0.988–0.996 (0.993 average); limit of quantitation values of 0.017–0.129 μg/mL (0.047 average); analytical precisions of 2.58–9.64% RSD (1.56 average); overall ASE-LI-Syringe-GC-MS method precisions of 1.73–14.6% RSD (4.97 average); and % recoveries of 84.6–98.9% (90.2 average) for the 23 terpenes of interest. Sample workflows and results are discussed, with an evaluation of the advantages/limitations of each approach and opportunities for future work.


Accumulation of bioactive metabolites in cultivated medical Cannabis.
Richins, R. D., Rodriguez-Uribe, L., Lowe, K., Ferral, R., & O’Connell, M. A.
PLOS ONE, 13(7), e0201119.(2018).
doi:10.1371/journal.pone.0201119
There has been an increased use of medical Cannabis in the United States of America as more states legalize its use. Complete chemical analyses of this material can vary considerably between producers and is often not fully provided to consumers. As phytochemists in a state with legal medical Cannabis we sought to characterize the accumulation of phytochemicals in material grown by licensed commercial producers. We report the development of a simple extraction and analysis method, amenable to use by commercial laboratories for the detection and quantification of both cannabinoids and terpenoids. Through analysis of developing flowers on plants, we can identify sources of variability of floral metabolites due to flower maturity and position on the plant. The terpenoid composition varied by accession and was used to cluster cannabis strains into specific types. Inclusion of terpenoids with cannabinoids in the analysis of medical cannabis should be encouraged, as both of these classes of compounds could play a role in the beneficial medical effects of different cannabis strains.

ADDING TERPENES TO CONCENTRATES - THE SCIENCE AND THE EFFECTS
MMJDOCTORONLINE Staff
https://mmjdoctoronline.com/health-n...nd-the-effects
Before we get into all the wonderful medical and recreational aspects of terpenes, let's briefly describe what a terpene is. What are Terpenes and what is their effect in making Marijuana Tinctures?
The Terpenes are class of small multi-carbon chained molecules found everwhere in the plant kingdom. They give a straint its flavor, boquet its character. Terpene aroma is a means of communication to animals, insects, and other plants, that all have the ability to sense or smell these small molecules. Bees may be attracted to the flower full of pollen (and terpenes) while other insects avoid some types of terpenes, particularly those in bark, which are a deadly toxin.
Terpenes are seen as “balancers and communicators” in cannabis, where they can amplify, moderate or enhance the activity of THC and the other cannabinoids, by reacting with the CB1 and probably the CB2 receptors that effect many physical and psychological functions in the body.

Alkanes of the essential oil of Cannabis sativa.
Hendriks, H., Malingré, T. M., Batterman, S., & Bos, R.
Phytochemistry, 16(6), 719–721. (1977).
doi:10.1016/s0031-9422(00)89239-0
A waxy fraction obtained by column chromatography of the essential oil of Cannabis sativa consists of n-alkanes ranging from Cg to C,,, 2-methyl and 3-methyl alkanes and some dimethyl alkanes. The qualitative and quantitative composition of this fraction has been compared with the alkane fraction obtained by extraction of the herb.

All About Terpinene
Asia Mayfield
https://terpenesandtesting.com/all-about-terpinene/
γ-Terpinene and terpinolene in cannabis
Terpinene isomers may show up in various cultivars. For example, one popular dispensary reports that terpinolene can be found in cultivars including Jack Herer, Durban Poison, and Super Lemon Haze.

Not Cannabis specific
Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases ?-selinene synthase and ?-humulene synthase
Dawn B Little, Rodney B Croteau
Archives of Biochemistry and Biophysics Volume 402, Issue 1, 2002, Pages 120-135
DOI: 10.1016/S0003-9861(02)00068-1
Two recombinant sesquiterpene synthases from grand fir, ?-selinene synthase and ?-humulene synthase, each produce more than 30 sesquiterpene olefins from the acyclic precursor farnesyl diphosphate. These enzymes contain a pair of DDxxD motifs, on opposite lips of the presumptive active site, which are thought to be involved in substrate binding and could promote multiple orientations of the substrate alkyl chain from which multiple families of cyclic olefins could derive.Mutagenesis of the first aspartate of either DDxxD motif resulted in depressed kcat, with lesser effect on Km, for ?-selinene synthase and afforded a much simpler product spectrum composed largely of monocyclic olefins. Identical alterations in ?-humulene synthase produced similar kinetic effects with a simplified product spectrum of mostly acyclic and monocyclic olefins. Although impaired in product diversity, none of the mutant synthases lost entirely the capacity to generate complex structures. These results confirm the catalytic significance of the DDxxD motifs and imply that they also influence permitted modes of cyclization. Deletion of an N-terminal arginine pair in ?-selinene synthase (an element potentially involved in substrate isomerization) altered kinetics without substantially altering product outcome. Finally, mutation of an active-site tyrosine residue thought to play a role in proton exchange had little influence; however, substitution of a nearby active site aspartate dramatically altered kinetics and product outcome.

Analysis of Terpene and Terpenoid Content in Cannabis Sativa Using Headspace with GC/MSD
Abbey Fausett
Agilent
Terpenes and terpenoids are compounds produced by botanical species to flourish in their environment. The compounds often attract pollinators, repel pests, and assist with adaptation throughout a growth cycle. Chemically, terpenes are comprised of carbon and hydrogen atoms, and are built from isoprene (C5 H8) subunits. Terpenoid describes a larger class of molecules that include oxygen in the chemical structure. Both classes of compounds will be generalized to terpenes for this application note, but they are two distinct classes in the broader scope. Terpenes have an associated fragrance, and have historically been isolated from various botanical sources for a wide range of commercial or therapeutic uses.2 D-limonene is a common component of citrus-scented personal care or disinfecting products, eucalyptol contributes to the minty aoma in many therapeutic products, and linalool is largely responsible for the floral fragrance of lavender-scented products. These terpenes, along with others produced by cannabis plants, are of interest as they are commonly marketed to enhance effects in the population consuming cannabis for medicinal or recreational use.

Analysis of Terpenes in Cannabis sativa L. Using GC/MS: Method Development, Validation, and Application
Elsayed Ibrahim, Mei Wang, Mohamed M Radwan, Mahmoud A Elsohly
Planta Medica 85(5) January 2019
DOI: 10.1055/a-0828-8387
Terpenes are the major components of the essential oils present in various Cannabis sativa L. varieties. These compounds are responsible for the distinctive aromas and flavors. Besides the quantification of the cannabinoids, determination of the terpenes in C. sativa strains could be of importance for the plant selection process. At the University of Mississippi, a GC-MS method has been developed and validated for the quantification of terpenes in cannabis plant material, viz., ?-pinene, ?-pinene, ?-myrcene, limonene, terpinolene, linalool, ?-terpineol, ?-caryophyllene, ?-humulene, and caryophyllene oxide. The method was optimized and fully validated according to AOAC (Association of Official Analytical Chemists) guidelines against reference standards of selected terpenes. Samples were prepared by extraction of the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard. The concentration-response relationship for all analyzed terpenes using the developed method was linear with r2 values > 0.99. The average recoveries for all terpenes in spiked indoor cultivated samples were between 95.0 – 105.7%, with the exception of terpinolene (67 – 70%). The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.32 to 8.47%. The limit of detection and limit of quantitation for all targeted terpenes were determined to be 0.25 and 0.75 µg/mL, respectively. The proposed method is highly selective, reliable, and accurate and has been applied to the simultaneous determination of these major terpenes in the C. sativa biomass produced by our facility at the University of Mississippi as well as in confiscated marijuana samples.

Analysis of Terpenes in Cannabis Using Headspace Solid-Phase Microextraction and GC–MS
Katherine K. Stenerson , Michael R. Halpenny
Cannabis Science and Technology February 28, 2018
https://www.cannabissciencetech.com/...ction-and-gcms
Headspace SPME combined with GC–MS for the qualitative and quantitative analysis of terpenes in cannabis offers several advantages compared to other methods. It does not require the use of organic solvents, does not coextract matrix, and provides additional means of peak identification and purity using spectral data. It is also a nondestructive method.
As the legalization of medicinal cannabis continues to sweep across the United States, an urgent need has developed for fast, accurate, and efficient analytical testing. In addition to testing for contaminants and potency, there is also interest in the determination of terpene identity and concentration levels present in different strains of cannabis. Terpenes have been shown to have therapeutic uses for treatment of different medical conditions ranging from cancer and inflammation to anxiety and sleeplessness. It is believed that the combination of terpenes and cannabinoids in cannabis produce a synergistic effect with regards to medical benefits. The traditional testing method for terpenes in plant materials involves a solvent-based extraction followed by gas chromatography (GC) analysis. In this work, headspace solid-phase microextraction (HS-SPME) was used to identify and quantify terpene content in cannabis. The HS-SPME method offered several advantages compared to solvent extraction in that it provided a cleaner analysis, free of interferences from coextracted matrix, and was nondestructive to the sample. A cannabis sample of unknown origin was first analyzed qualitatively by HS-SPME and GC–mass spectrometry (MS). Spectral library matching and retention indices were used to identify 42 terpenes. Quantitative analysis was then performed for several selected terpenes using spiked samples. Method accuracy was >90%, with reproducibility of <5% relative standard deviation (RSD) for analysis of spiked replicates. The HS-SPME results were then compared to an analysis using a conventional solvent extraction method, and the two approaches were found to produce comparable results.

Anticancer and Antioxidant Properties of Terpinolene in Rat Brain Cells.
Aydin, E., Türkez, H., & Taşdemir, Ş.
Archives of Industrial Hygiene and Toxicology, 64(3), 415–424.(2013).
doi:10.2478/10004-1254-64-2013-2365
Terpinolene (TPO) is a natural monoterpene present in essential oils of many aromatic plant species. Although various biological activities of TPO have been demonstrated, its neurotoxicity has never been explored. In this in vitro study we investigated TPO’s antiproliferative and/or cytotoxic properties using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test, genotoxic damage potential using the single-cell gel electrophoresis (SCGE), and oxidative effects through total antioxidant capacity (TAC) and total oxidative stress (TOS) in cultured primary rat neurons and N2a neuroblastoma cells. Dose-dependent effects of TPO (at 10 mg L-1, 25 mg L-1, 50 mg L-1, 100 mg L-1, 200 mg L-1, and 400 mg L-1) were tested in both cell types. Significant (P<0.05) decrease in cell proliferation were observed in cultured primary rat neurons starting with the dose of 100 mg L-1 and in N2a neuroblastoma cells starting with 50 mg L-1. TPO was not genotoxic in either cell type. In addition, TPO treatment at 10 mg L-1, 25 mg L-1, and 50 mg L-1 increased TAC in primary rat neurons, but not in N2a cells. However, at concentrations above 50 mg L-1 it increased TOS in both cell types. Our fi ndings clearly demonstrate that TPO is a potent antiproliferative agent for brain tumour cells and may have potential as an anticancer agent, which needs to be further studied.

Antifungal Activity of the Volatiles of High Potency Cannabis sativa L. Against Cryptococcus neoformans
Amira S. Wanas, Mohammed M. Radwan, Zlatko Mehmedic, Melissa Jacob, Iklas A. Khan, and Mahmoud A. Elsohly
Rec. Nat. Prod. 10.2 (2016) 214-220
https://www.researchgate.net/publica...cus_neoformans
The n-hexane extracted volatile fraction of high potency Cannabis sativa L (Cannabaceae). was assessed in vitro for antifungal, antibacterial and antileishmanial activities. The oil exhibited selective albeit modest, antifungal activity against Cryptococcus neoformans with an IC50 value of 33.1 ?g/mL. Biologically-guided fractionation of the volatile fraction resulted in the isolation of three major compounds (1-3) using various chromatographic techniques. The chemical structures of the isolated compounds were identified as ?-humulene (1), ?-caryophyllene (2) and caryophyllene oxide (3) using GC/FID, GC/MS, 1D- and 2D-NMR analyses, respectively. Compound 1 showed potent and selective antifungal activity against Cryptococcus neoformans with IC50 and MIC values of 1.18 ?g/mL and 5.0 ?g/mL respectively. Whereas compound 2 showed weak activity (IC50 19.4 ?g/mL), while compound 3 was inactive against C. neoformans.

Anti-inflammatory Potential of Terpenes Present in Cannabis sativa L.
Eric J. Downer
ACS Chem. Neurosci. XXXX, XXX, XXX?XXX
Doi: 10.1021/acschemneuro.0c00075
Cannabis sativa L. (C. sativa) contains an array of plant-derived (phyto) cannabinoids and terpenes that are predominantly located in the trichome cavity of the plant. Terpenes, aromatic organic hydrocarbons characterized for their role in plant protection/pollination, are gaining attention for their potential as novel therapeutics in many areas of biomedicine. This Viewpoint will explore the exciting recent evidence that terpenes have anti-inflammatory/antioxidant propensity by targeting inflammatory signaling mechanisms relevant to human disease. Given their anti-inflammatory properties, terpenes may contribute to the effects of current cannabinoid-based therapies.

Antitumor effect of 1, 8-cineole against colon cancer.
MURATA, S., SHIRAGAMI, R., KOSUGI, C., TEZUKA, T., YAMAZAKI, M., HIRANO, A., … KODA, K.
Oncology Reports, 30(6), 2647–2652.(2013).
doi:10.3892/or.2013.2763
Several essential oils possess pharmacological effects. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects such as anti-bacterial and anti-inflammatory effects. The effect of 1, 8-cineole on human colorectal cancer cells, however, has not reported previously. In this study, we have investigated the anti-proliferative effect of 1, 8-cineole on human colon cancer cell lines HCT116 and RKO by WST-8 and BrdU assays. The cytotoxicity of 1, 8-cineole was investigated by LDH activity and TUNEL staining. The mechanism of apoptosis by 1, 8-cineole was determined by western blot analyses. In in vivo study, RKO cells were injected into the SCID mice and the effect of 1, 8-cineole was investigated. Specific induction of apoptosis, not necrosis, was observed in human colon cancer cell lines HCT116 and RKO by 1, 8-cineole. The treatment with 1, 8-cineole was associated with inactivation of survivin and Akt and activation of p38. These molecules induced cleaved PARP and caspase-3, finally causing apoptosis. In xenotransplanted SCID mice, the 1, 8-cineole group showed significantly inhibited tumor progression compared to the control group. These results indicated 1, 8-cineole suppressed human colorectal cancer proliferation by inducing apoptosis. Based on these studies 1, 8-cineole would be an effective strategy to treat colorectal cancer.


NOT CANNABIS SPECIFIC
A raison d’être for two distinct pathways in the early steps of plant isoprenoid biosynthesis?
Andrea Hemmerlin, John L. Harwood, Thomas J. Bach
Progress in Lipid Research 51 (2):95–148( 2012)
doi: 10.1016/j.plipres.2011.12.001
When compared to other organisms, plants are atypical with respect to isoprenoid biosynthesis: they utilize two distinct and separately compartmentalized pathways to build up isoprene units. The co-existence of these pathways in the cytosol and in plastids might permit the synthesis of many vital compounds, being essential for a sessile organism. While substrate exchange across membranes has been shown for a variety of plant species, lack of complementation of strong phenotypes, resulting from inactivation of either the cytosolic pathway (growth and development defects) or the plastidial pathway (pigment bleaching), seems to be surprising at first sight. Hundreds of isoprenoids have been analyzed to determine their biosynthetic origins. It can be concluded that in angiosperms, under standard growth conditions, C20-phytyl moieties, C30-triterpenes and C40-carotenoids are made nearly exclusively within compartmentalized pathways, while mixed origins are widespread for other types of isoprenoid-derived molecules. It seems likely that this coexistence is essential for the interaction of plants with their environment. A major purpose of this review is to summarize such observations, especially within an ecological and functional context and with some emphasis on regulation. This latter aspect still requires more work and present conclusions are preliminary, although some general features seem to exist.

Not Cannabis specific
A Review of Terpenes from Marine-Derived Fungi: 2015–2019.
Jiang, M., Wu, Z., Guo, H., Liu, L., & Chen, S.
Marine Drugs, 18(6), 321.(2020).
doi:10.3390/md18060321
Marine-derived fungi are a significant source of pharmacologically active metabolites with interesting structural properties, especially terpenoids with biological and chemical diversity. In the past five years, there has been a tremendous increase in the rate of new terpenoids from marine-derived fungi being discovered. In this updated review, we examine the chemical structures and bioactive properties of new terpenes from marine-derived fungi, and the biodiversity of these fungi from 2015 to 2019. A total of 140 research papers describing 471 new terpenoids of six groups (monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, and meroterpenes) from 133 marine fungal strains belonging to 34 genera were included. Among them, sesquiterpenes, meroterpenes, and diterpenes comprise the largest proportions of terpenes, and the fungi genera of Penicillium, Aspergillus, and Trichoderma are the dominant producers of terpenoids. The majority of the marine-derived fungi are isolated from live marine matter: marine animals and aquatic plants (including mangrove plants and algae). Moreover, many terpenoids display various bioactivities, including cytotoxicity, antibacterial activity, lethal toxicity, anti-inflammatory activity, enzyme inhibitor activity, etc. In our opinion, the chemical diversity and biological activities of these novel terpenoids will provide medical and chemical researchers with a plenty variety of promising lead compounds for the development of marine drugs.

Beta-caryophyllene, a CB2R selective agonist, protects against cognitive impairment caused by neuro-inflammation and not in dementia due to ageing induced by mitochondrial dysfunction
Urja Kanojia, Shrikant Gyaneshwar Chaturbhuj, Runali Sankhe, Maushami Das, Raviteja Surubhotla,Nandakumar Krishnadas, Karthik Gourishetti, Pawan Ganesh Nayak, Anoop Kishore
CNS & Neurological Disorders - Drug Targets
DOI : 10.2174/1871527320666210202121103
Background: Dementia is a neurodegenerative disorder majorly evidenced by cognitive impairment. Although there are many types of dementia, the common underlying etiological factors in all the types are neuro-inflammation or ageing induced apoptosis. ?-caryophyllene, a cannabinoid type-2 receptor agonist has reported to have promising neuroprotective effects in cerebral ischemia and neuro-inflammation.
Objective: In the present study, we evaluated the effects of ?-caryophyllene, against animal models of dementia whose etiology mimicked neuro-inflammation and ageing.
Method: Two doses (50 and 100 mg/kg of body weight) of ?-caryophyllene given orally were tested against AlCl3-induced dementia in male Sprague Dawley (SD) rats using Morris water maze test. Subsequently, the effect of the drug was assessed for episodic memory in female SD rats using novel object recognition task in doxorubicin-induced neuro-inflammation and male SD rats for chemobrain model. Moreover, its effects were evaluated in D-galactose-induced mitochondrial dysfunction leading to dementia.
Results: ?-caryophyllene, at both the doses, showed significant improvement in memory when assessed using parameters like target quadrant entries, escape latency and path efficiency in Morris water maze test for spatial memory. In the doxorubicin-induced chemobrain model, ?-caryophyllene at 100 mg/kg significantly elevated acetylcholinesterase and catalase levels and lowered lipid peroxidation compared to the disease control. In the novel object recognition task, ?-caryophyllene at 100 mg/kg significantly improved recognition index and discrimination index in the treated animals compared to the disease control, with a significant increase in catalase and decrease in lipid peroxidation in both hippocampus and frontal cortex. However, in D-galactose-induced mitochondrial dysfunction model, ?-caryophyllene failed to show positive effects when spatial memory was assessed. It also failed to improve D-galactose induced diminished mitochondrial complex I and II activities.

Beta-Caryophyllene, a CB2-Receptor-Selective Phytocannabinoid, Suppresses Mechanical Allodynia in a Mouse Model of Antiretroviral-Induced Neuropathic Pain.
Aly, E., Khajah, M. A., & Masocha, W.
Molecules, 25(1), 106. (2019).
doi:10.3390/molecules25010106
Neuropathic pain associated with nucleoside reverse transcriptase inhibitors (NRTIs), therapeutic agents for human immunodeficiency virus (HIV), responds poorly to available drugs. Smoked cannabis was reported to relieve HIV-associated neuropathic pain in clinical trials. Some constituents of cannabis (Cannabis sativa) activate cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors. However, activation of the CB1 receptor is associated with side effects such as psychosis and physical dependence. Therefore, we investigated the effect of ?-caryophyllene (BCP), a CB2-selective phytocannabinoid, in a model of NRTI-induced neuropathic pain. Female BALB/c mice treated with 20 -30 -dideoxycytidine (ddC, zalcitabine), a NRTI, for 5 days developed mechanical allodynia, which was prevented by cotreatment with BCP, minocycline or pentoxifylline. A CB2 receptor antagonist (AM 630), but not a CB1 receptor antagonist (AM 251), antagonized BCP attenuation of established ddC-induced mechanical allodynia. ?-Caryophyllene prevented the ddC-induced increase in cytokine (interleukin 1 beta, tumor necrosis factor alpha and interferon gamma) transcripts in the paw skin and brain, as well as the phosphorylation level of Erk1/2 in the brain. In conclusion, BCP prevents NRTI-induced mechanical allodynia, possibly via reducing the inflammatory response, and attenuates mechanical allodynia through CB2 receptor activation. Therefore, BCP could be useful for prevention and treatment of antiretroviral-induced neuropathic pain.

β-Caryophyllene: A Sesquiterpene with Countless Biological Properties
Fabrizio Francomano, Anna Caruso, Alexia Barbarossa, Alessia Fazio Chiara La Torre, Jessica Ceramella, Rosanna Mallamaci, Carmela Saturnino, Domenico Iacopetta and Maria Stefania Sinicropi
Applied Sciences 2019
DOI: 10.3390/app9245420
β-Caryophyllene (BCP), a natural bicyclic sesquiterpene, is a selective phytocannabinoid agonist of type 2 receptors (CB2-R). It isn’t psychogenic due to the absence of an affinity to cannabinoid receptor type 1 (CB1). Among the various biological activities, BCP exerts anti-inflammatory action via inhibiting the main inflammatory mediators, such as inducible nitric oxide synthase (iNOS), Interleukin 1 beta (IL-1β), Interleukin-6 (IL-6), tumor necrosis factor-alfa (TNF-α), nuclear factor kapp a-light-chain-enhancer of activated B cells (NF-κB), cyclooxygenase 1 (COX-1), cyclooxygenase 2 (COX-2). Peroxisome proliferator-activated receptors alpha (PPAR-α) effects are also mediated by the activation of PPAR-α and PPAR-γ receptors. In detail, many studies, in vitro and in vivo, suggest that the treatment with β-caryophyllene improves the phenotype of animals used to model various inflammatory pathologies, such as nervous system diseases (Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis, stroke), atherosclerosis, and tumours (colon, breast, pancreas, lymphoma, melanoma and glioma cancer). Furthermore, pre-clinical data have highlighted that BCP is potentially useful in Streptococcus infections, osteoporosis, steatohepatitis, and exerts anticonvulsant, analgesic, myorelaxing, sedative, and antidepressive effects. BCP is non-toxic in rodents, with a Lethal dose, 50% (LD50) greater than 5000 mg/kg. Nevertheless, it inhibits various cytochrome P450 isoforms (above all, CYP3A4), which metabolise xenobiotics, leading to adverse effects, due to drug levels over therapeutic window. All the reported data have highlighted that both pharmacological and toxicological aspects need to be further investigated with clinical trials

BETA CARYOPHYLLENE - A TERPENE OR A CANNABINOID?
Noel Palmer
https://www.cbxsciences.com/blog/201...-a-cannabinoid
Most people understand that cannabis is responsible for producing cannabinoids; most notably delta-9 THC and CBD. What many people don’t fully understand is that cannabis produces other phytochemicals (chemicals produced by plants) that are therapeutically active. Terpenes are a class of phytochemicals produced by cannabis (and other plants), and in fact scientists believe that terpenes serve as the building blocks for cannabinoids in the cannabis plant. That being said, terpenes in and of themselves are considered to be therapeutically relevant in many ways.

Beta-caryophyllene attenuates short-term recurrent seizure activity and blood-brain-barrier breakdown after pilocarpine-induced status epilepticus in rats
Michele Pereira Mallmann, Fernanda Kulinski Mello, Bruna Neuberger, Karine Gabriela da Costa Sobral, Michele Rechia Fighera, Luiz Fernando Freire Royes, Ana Flávia Furian, Mauro Schneider Oliveira
Brain Res. 2022 Mar 14;1784:147883.
doi: 10.1016/j.brainres.2022.147883.
Background: Status epilepticus (SE) is a neurological life-threatening condition, resulting from the failure of the mechanisms responsible for seizure termination. SE is often pharmacoresistant and associated with significant morbidity and mortality. Hence, ceasing or attenuating SE and its consequences is of fundamental importance. Beta-caryophyllene is a functional CB2 receptor agonist and exhibit a good safety profile. Besides, it displays beneficial effects in several experimental conditions, including neuroprotective activity. In the present study we aimed to investigate the effects of beta-caryophyllene on pilocarpine-induced SE.
Methods: Wistar rats were submitted to pilocarpine-induced SE and monitored for 24 h by video and EEG for short-term recurrence of seizure activity (i.e. seizures occurring within 24 h after termination of SE). Rats received beta-caryophyllene (100 mg/kg, ip) at 1, 8- and 16-hours after SE. Twenty-four hours after SE we evaluated sensorimotor response, neuronal damage (fluoro jade C staining) and serum albumin infiltration into brain parenchyma.

Beta-caryophyllene is a dietary cannabinoid
Jurg Gertsch, Marco Leonti, Stefan Raduner, Ildiko Racz, Jian-Zhong Chen, Xiang-Qun Xie , Karl-Heinz Altmann, Meliha Karsak, and Andreas Zimmer
PNAS July 1, 2008 vol. 105 no. 26 9099–9104 PH
DOI: 10.1073/pnas.0803601105
The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB1) and CB2 receptors. Although the CB1 receptor is responsible for the psychomodulatory effects, activation of the CB2 receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)--caryophyllene [(E)-BCP] selectively binds to the CB2 receptor (Ki 155 4 nM) and that it is a functional CB2 agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB2 receptor, showing ligand stacking interactions with residues F117 and W258. Upon binding to the CB2 receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB2 receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB2 receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.

Busting the THC Myth: When it Comes to the Best User Experience, Terpenes Reign Supreme
Mark Lange
Cannabis Business Journal March 3 2022

https://cannabisindustryjournal.com/column/busting-the-thc-myth-when-it-comes-to-the-best-user-experience-terpenes-reign-supreme/

We have just begun to scratch the surface of the potential of terpenes in cannabis. With the right alignment across the industry and a stronger focus on genetics in breeding, we will see the rise of completely unique cannabis varieties.
The scent of pine from your Christmas tree. The fragrance of a ripe summer peach at the farmer’s market. The whiff of eucalyptus and lavender that greets you when you enter a spa.
Aroma is a keystone in how we experience the world. In any given environment, aroma can help shape your mood, solidify memories and instantly transport you to another place or time.
I have focused my career on studying the fascinating compounds that are often behind these powerful aromas: terpenes. They form the largest class of natural products (compounds produced by living organisms), found in nearly all living beings. There are around 50,000 currently known terpenes in nature — with potentially thousands yet to be discovered.
Terpene-rich plants you might be most familiar with are lavender, mint, oranges (in the peel), and yes, cannabis. In recent years, terpenes have rightfully become a central discussion in the recreational cannabis world. This is because terpenes — not THC level, not “Indica-Sativa” classification — are a key determinant of cannabis’s effect, both psychoactive and non-psychoactive. But the current lack of prioritization and understanding of the crucial role terpenes play may put the collective quality of U.S. cannabis at risk.
At this crucial inflection point for legal cannabis, on its path to becoming a $70 billion dollar global industry by 2028, we need to ensure that everyone across the cannabis space, from breeders to testers, growers and consumers, understands which traits to prioritize for a cannabis world brimming with diversity and predictable effects.
What the cannabis industry has to lose
What do we lose if the cannabis industry continues to scale without a clear understanding of the compounds that define the uniqueness of each variety?
There is a ripple effect across the ecosystem. For cannabis testing labs, focusing on only twenty of the most dominant terpenes means we are missing out on tapping into potentially over a hundred of less common terpenes in cannabis. For the cannabis consumer, lack of understanding on the breeding and testing side may make it difficult to find cannabis that delivers on its promised effect time and time again. And, most detrimentally for breeders, not understanding the direct correlation between genetics and the formation of terpenes means we will have increasingly fewer terpene profiles and combinations to work with, especially when the industry-dominant focus has been on cannabinoid potency.
Let’s explore some misconceptions related to potency. In recent years, many breeders have prioritized high THC levels over genetic diversity. Consumers often associate high THC levels and that telltale strong “skunky” aroma with a strain’s quality and effect, when in reality, these are poor indicators of potency. (In fact, recent research indicates that this specific cannabis aroma is caused by a family of sulfur compounds.) Terpene profiling is a much more accurate way to determine a variety’s given effect. In focusing too much on increasing THC, breeders miss out on the true potency powerhouse: tapping into the terpene diversity that’s out there. Terpenes are responsible for giving flowers (including cannabis), fruits and spices their distinctive flavors and aromas. Common terpenes include limonene, linalool, pinene and myrcene.

Cannabinoid Receptors Are Absent in Insects
JOHN MCPARTLAND, VINCENZO DI MARZO, LUCIANO DE PETROCELLIS, LISON MERCER, AND MICHELLE GLASS
THE JOURNAL OF COMPARATIVE NEUROLOGY 436:423–429 (2001)
doi: 10.1002/cne.1078
The endocannabinoid system exerts an important neuromodulatory role in mammals. Knockout mice lacking cannabinoid (CB) receptors exhibit significant morbidity. The endocannabinoid system also appears to be phylogenetically ancient—it occurs in mammals, birds, amphibians, fish, sea urchins, leeches, mussels, and even the most primitive animal with a nerve network, the Hydra. The presence of CB receptors, however, has not been examined in terrestrial invertebrates (or any member of the Ecdysozoa). Surprisingly, we found no specific binding of the synthetic CB ligands [3
H]CP55,940 and [3 H]SR141716A in a panel of insects: Apis mellifera, Drosophila melanogaster, Gerris marginatus, Spodoptera frugiperda, and Zophobas atratus. A lack of functional CB receptors was confirmed by the inability of tetrahydrocannabinol (THC) and HU210 to activate G-proteins in insect tissues, utilizing a guanosine-59-O-(3-[35]thio)-triphosphate (GTPgS) assay. No orthologs of human CB receptors were located in the Drosophila genome, nor did we find orthologs of fatty acid amide hydrolase. This loss of CB receptors appears to be unique in the field of comparative
neurobiology. No other known mammalian neuroreceptor is understood to be missing in insects. We hypothesized that CB receptors were lost in insects because of a dearth of ligands; endogenous CB ligands are metabolites of arachidonic acid, and insects produce little or no arachidonic acid or endocannabinoid ligands, such as anandamide.

Cannabinoids and terpenes as chemotaxonomic markers in cannabis.
Elzinga S, Fischedick J, Podkolinski R, Raber JC (2015)
Nat Prod Chem Res 3: 2
DOI: 10.4172/2329-6836.1000181
https://www.researchgate.net/profile...8c85c016aa.pdf
In this paper, we present principal component analysis (PCA) results from a dataset containing 494 cannabis
flower samples and 170 concentrate samples analyzed for 31 compounds. A continuum of chemical composition
amongst cannabis strains was found instead of distinct chemotypes. Our data shows that some strains are much
more reproducible in chemical composition than others. Strains labeled as indica were compared with those labeled
as sativa and no evidence was found that these two cultivars are distinctly different chemotypes. PCA of “OG” and
“Kush” type strains found that “OG” strains have relatively higher levels of ?-terpineol, fenchol, limonene, camphene, terpinolene and linalool where “Kush” samples are characterized mainly by the compounds trans-ocimene, guaiol, ?-eudesmol,myrcene and ?-pinene. The composition of concentrates and flowers were compared as well. Although the absolute concentration of compounds in concentrates is much higher, the relative composition of compounds between flowers and concentrates is similar.

Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes.
Pawe Rodziewicz, Stefan Loroch, Lukasz Marczak, Oliver Kayser
April 2019 Plant Science 284
doi: 10.1016/j.plantsci.2019.04.008
Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes.

Cannabis chemovar classification: terpenes hyper-classes and targeted genetic markers for accurate discrimination of flavours and effects
Philippe Henry 2017
Doi:10.7287/peerj.preprints.3307v1
The classification of Cannabis varieties has been increasingly discussed in the past years, particularly in the wake of emerging legal markets, with implications for intellectual property development, marketing and improvement of the scientific understanding of this contentious plant. While the concept of chemovars has been proposed and has gained popularity of late, the lack of guidance in introducing this concept and the fact that chemovars are based on indirectly assessed traits with a heritable basis has likely impeded the implementation of the concept to a broader audience. Here I propose a simplified version of terpene hyper-classes based on three dominant terpenes that is shown to outperformed the classic indica-sativa-hybrid scheme of classification as well as a recently proposed terpene super-class scheme. This information was used to identify the most informative genetic markers for chemovar classification based on the terpene hyper-classes. I demonstrate the ability of clearly clustering accessions based on their dominant terpene and propose to extent this approach as a benchmark for chemovar classification in lieu of previously proposed models.

Cannabis Essential Oil: A Preliminary Study for the Evaluation of the Brain Effects
Nadia Gulluni , Tania Re , Idalba Loiacono, Giovanni Lanzo, Luigi Gori, Claudio Macchi, Francesco Epifani, Nicola Bragazzi , and Fabio Firenzuoli
Hindawi
Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 1709182, 1-11 pages
doi: 10.1155/2018/1709182
We examined the effects of essential oil from legal (THC <0.2% w/v) hemp variety on the nervous system in 5 healthy volunteers. GC/EIMS and GC/FID analysis of the EO showed that the main components were myrcene and Beta-caryophyllene.The experiment consisted of measuring autonomic nervous system (ANS) parameters; evaluations of the mood state; and electroencephalography (EEG) recording before treatment, during treatment, and after hemp inhalation periods as compared with control conditions. The results revealed decreased diastolic blood pressure, increased heart rate, and significant increased skin temperature. The subjects described themselves as more energetic, relaxed, and calm. The analysis EEG showed a significant increase in the mean frequency of alpha (8–13Hz) and significant decreased mean frequency and relative power of beta 2 (18,5–30Hz) waves.Moreover, an increased power, relative power, and amplitude of theta (4–8Hz) and alpha brain waves activities and an increment in the delta wave (0,5–4Hz) power and relative power was recorded in the posterior region of the brain. These results suggest that the brain wave activity and ANS are affected by the inhalation of the EO of Cannabis sativa suggesting a neuromodular activity in cases of stress, depression, and anxiety.

Cannabis labelling is associated with genetic variation in terpene synthase genes
Sophie Watts, Michel McElroy, Zoë Migicovsky, Hugo Maassen, Robin van Velzen, and Sean Myles
Nature Plants | VOL 1330 7 | Octobe r 2021 | 1330–1334
DOI: 10.1038/s41477-021-01003-y
https://www.nature.com/articles/s41477-021-01003-y.pdf
Analysis of over 100 Cannabis samples quantified for terpene and cannabinoid content and genotyped for over 100,000 single nucleotide polymorphisms indicated that Sativa- and Indica-labelled samples were genetically indistinct on a genome-wide scale. Instead, we found that Cannabis labeling was associated with variation in a small number of terpenes whose concentrations are controlled by genetic variation at tandem arrays of terpene synthase genes

Cannabis Pharmacology:The Usual Suspects and a Few Promising Leads
Ethan Russo
Poster
https://ethanrusso.org/download/cann...omising-leads/
Outline of an Ideal Cannabis Classification Scheme
Combines shape, content and purpose
Basic class based on primary cannabinoid (e.g. Type I for THC)
Plant morphology (e.g., broad-leaf, compact vs. tall, spindly)
Specific cannabinoid content
Specific terpenoid content
Scent
Taste (when vaporized)
Uses/Effects (patient-oriented)

Cannabis Sativa L.: a comprehensive review on the analytical methodologies for cannabinoids and terpenes characterization.
Micalizzi G, Vento F, Alibrando F, Donnarumma D, Dugo P, Mondello L.
J Chromatogr A. 2020 Dec 30;1637:461864.
doi: 10.1016/j.chroma.2020.461864.
Undoubtedly, the enormous interest about cannabis cultivation mainly derives from the well-known pharmacological properties of cannabinoids and terpenes biosynthesized by the plants. ...Lastly, GC GC techniques are also reported for accurate identification and quantificatification of terpenes in complex cannabis matrices.

Cannabis sativa Terpenes are Cannabimimetic and Provide Support for the Entourage Effect Hypothesis
Justin E. LaVigne, Ryan Heckse, Attila Keresztes, John M. Streiche
Nature Scientific Reports
DOI: 10.1101/2020.10.22.350868
https://www.nature.com/articles/s41598-021-87740-8.pdf
Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an “entourage efect” whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral efects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our fndings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage efect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.

Cannabis sativa terpenes are cannabimimetic and selectively enhance cannabinoid activity
Justin E. LaVigne, Ryan Heckse, Attila Keresztes, John M. Streiche
Nature Scientific Reports
DOI: 10.1038/s41598-021-87740-8
Limited evidence has suggested that terpenes found in Cannabis sativa are analgesic, and could produce an “entourage effect” whereby they modulate cannabinoids to result in improved outcomes. However this hypothesis is controversial, with limited evidence. We thus investigated Cannabis sativa terpenes alone and with the cannabinoid agonist WIN55,212 using in vitro and in vivo approaches. We found that the terpenes α-humulene, geraniol, linalool, and β-pinene produced cannabinoid tetrad behaviors in mice, suggesting cannabimimetic activity. Some behaviors could be blocked by cannabinoid or adenosine receptor antagonists, suggesting a mixed mechanism of action. These behavioral effects were selectively additive with WIN55,212, suggesting terpenes can boost cannabinoid activity. In vitro experiments showed that all terpenes activated the CB1R, while some activated other targets. Our findings suggest that these Cannabis terpenes are multifunctional cannabimimetic ligands that provide conceptual support for the entourage effect hypothesis and could be used to enhance the therapeutic properties of cannabinoids.

Challenges and Opportunities for the Analysis of Terpenes in Cannabis
Terry Rodney, Patrisha Pham-Bugayong, Lakshmi C. Kasi Viswanath, Ghalib A Bello, Gerard Dumancas, Bryan John Subong
https://www.researchgate.net/publica...es_in_Cannabis
Cannabis is a complex plant with over 400 chemical entities of which more than 60 of them are cannabinoids. While cannabinoids are the primary psychoactive and medicinal components of cannabis, volatile terpenes contribute to the many significant fragrance attributes that ultimately influence consumer preference for cannabis. There are over 120 different terpene compounds that have been identified in the Cannabis sativa plant alone. Analysis of terpenes in cannabis is extremely important because they contribute to its potency and sensory perceptions. Current methods of quantifying terpenes in cannabis involve the use of chromatographic techniques. However, such techniques require sample preparation, are time-consuming, and the instrument involved can be expensive and requires a skilled operator. The use of Fourier Transform Infrared spectroscopy and chemometrics offer a fast, non-destructive, and affordable means of analyzing terpenes in cannabis. This manuscript will discuss challenges in cannabis terpene analysis using the aforementioned methods including method fragmentation and method multiplicity as well as issues related to its legal use. In general, the cannabis testing industry is poised for a breakthrough in the field of analytical science given the recent laws legalizing its medicinal use as well as advances in the field of spectroscopic miniaturization.

Characterization of cannabis cultivars based on terpene synthase gene profiles (Patent) WO 2022/006019 A1
Jan 2022
Front RangeBiosciences Inc.
ChristopherStephen Pauli, Anthony Torres, Reginald Gaudino, Keith Allen, Thomas Blank, Kymron deCesare https://www.researchgate.net/publica...terization_of_ cannabis_cultivars_based_on_te rpene_synthase_gene_profiles


Characterizing the smell of marijuana by odor impact of volatile compounds: an application of simultaneous chemical and sensory analysis.
Rice Somchai, Koziel JA (2015)
PloS One Published: December 10, 2015
doi:10.1371/journal.pone.0144160
https://journals.plos.org/plosone/ar...type=printable
Recent US legislation permitting recreational use of marijuana in certain states brings the use of marijuana odor as probable cause for search and seizure to the forefront of forensic science, once again. This study showed the use of solid-phase microextraction with multidimensional gas chromatography—mass spectrometry and simultaneous human olfaction to characterize the total aroma of marijuana. The application of odor activity analysis offers an explanation as to why high volatile chemical concentration does not equate to most potent odor impact of a certain compound. This suggests that more attention should be focused on highly odorous compounds typically present in low concentrations, such as nonanal, decanol, o-cymene, benzaldehyde, which have more potent odor impact than previously reported marijuana headspace volatiles.

Chemical Characterization and Evaluation of the Antibacterial Activity of Essential Oils from Fibre-Type Cannabis sativa L. (Hemp).
Iseppi, R., Brighenti, V., Licata, M., Lambertini, A., Sabia, C., Messi, P., … Benvenuti, S.
Molecules, 24(12), 2302. (2019).
doi:10.3390/molecules24122302
Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that ?- and ?-pinene, ?-myrcene and ?-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field

Chemical Composition of Volatile Oils of Fresh and Air-Dried Buds of Cannabis chemovars, Their Insecticidal and Repellent Activities
Amira S. Wanas, Mohamed M. Radwan, Suman Chandra, Hemant Lata, Zlatko Mehmedic, Abbas Ali, KHC Baser, Betul Demirci, and Mahmoud A. ElSohly
Natural Product Communications Volume 15(5): 1–7
DOI: 10.1177/1934578X20926729
The volatile oils of fresh and air-dried buds of 3 different varieties of Cannabis, namely, high cannabidiol (CBD) chemotype, intermediate CBD/tetrahydrocannabinol (THC) chemotype, and high THC chemotype were prepared by hydrodistillation. Gas chromatography analysis of the volatile oils resulted in the identification of 71 compounds, of which 33 were monoterpenes and 38 were sesquiterpenes. The volatile oil obtained from the THC chemotype showed an increase in the ratio of the sesquiterpenes to monoterpenes content. The content of terpinolene was dramatically decreased upon drying of THC chemotype. Moderate increase in ?-caryophyllene and caryophyllene oxide was observed. However, there was no detectable change in the percentage of monoterpenes and sesquiterpenes content in both the intermediate type and CBD chemotype upon drying. The insecticidal activity of the volatile oils was evaluated. The oil obtained from the fresh and dried high CBD cannabis showed good biting deterrent activity at 10 ug/cm2 compared with N,N-diethyl-meta-toluamide at 4.78 µg/cm2 , and good larvicidal activity

Chromatographic Analyses, In Vitro Biological Activities, and Cytotoxicity of Cannabis sativa L. Essential Oil: A Multidisciplinary Study
Gokhan Zengin, Luigi Menghini, Antonella Di Sotto, Romina Mancinelli,
Francesca Sisto, Simone Carradori, Stefania Cesa, Caterina Fraschetti,
Antonello Filippi, Letizia Angiolella, Marcello Locatelli, Luisa Mannina,
Cinzia Ingallina, Valentina Puca, Marianna D’Antonio and Rossella Grande
Molecules 2018, 23, 3266;
doi:10.3390/molecules23123266
Due to renewed interest in the cultivation and production of Italian Cannabis sativa L., we proposed a multi-methodological approach to explore chemically and biologically both the essential oil and the aromatic water of this plant. We reported the chemical composition in terms of cannabinoid content, volatile component, phenolic and flavonoid pattern, and color characteristics. Then, we demonstrated the ethnopharmacological relevance of this plant cultivated in Italy as a source of antioxidant compounds toward a large panel of enzymes (pancreatic lipase, ?-amylase, ?-glucosidase, and cholinesterases) and selected clinically relevant, multidrug-sensible, and multidrug-resistant microbial strains (Staphylococcus aureus, Helicobacter pylori, Candida, and Malassezia spp.), evaluating the cytotoxic effects against normal and malignant cell lines. Preliminary in vivo cytotoxicity was also performed on Galleria mellonella larvae. The results corroborate the use of this natural product as a rich source of important biologically active molecules with particular emphasis on the role exerted by naringenin, one of the most important secondary metabolites.

Classification of Common California Cannabis Cultivars via Secondary Metabolite Characterization
Mark A. Lewis
https://www.medicinalgenomics.com/wp...annMed2016.pdf
POSTER
Several Years to Achieve Several Goals:
1. Validated assays & analysis to communicate data
2. Collect data & valid survey of ‘chemoscape’
3. Analysis 1: Class cultivars based on chemotype
4. Analysis 2: Observe intra-genotypic variability
5. Analysis 3: Asexual vs. hybrid seed comparison

Not Cannabis specific
Comparative Study of Steam Distillation and Soxhlet for the Extraction of Botanical Oils
Chibuzor Onyinye Okonkwo and Obioma Christopher Ohaeri
Asian Journal of Biological Sciences Jan 2020
DOI: 10.3923/ajbs.2020.62.69
https://www.researchgate.net/publica...Botanical_Oils
Background and Objective: Steam distillation and soxhlet extraction are among the most commonly used methods in the laboratory
for the extraction of biological compounds. This study was aimed at comparing the chemical composition of insecticidal oils extracted
via these two methods.
Materials and Methods: Oils were extracted from both plants via steam distillation and soxhlet extraction
methods. Extracted oils were then subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to investigate the chemical
components of oils.
Results: The steam distilled oils from both plants contained fewer compounds relative to the soxhlet extracted oils
which contained a wider array of chemical compounds including; phenols, acyclic olefins, esters, ketones, carboxylic acids and alcohols.
Conclusion: Steam distillation alone may not be sufficient to extract most biological compounds present in plant oils.

Not Cannabis specific
Combinatorial Evolution of a Terpene Synthase Gene Cluster Explains Terpene Variations in Oryza.
Chen, H., Köllner, T. G., Li, G., Wei, G., Chen, X., Zeng, D., … Chen, F.
Plant Physiology, pp.00948.2019.
doi:10.1104/pp.19.00948
Terpenes are specialized metabolites ubiquitously produced by plants via the action of terpene synthases (TPSs). There are enormous variations in the types and amounts of terpenes produced by individual species. To understand the mechanisms responsible for such vast diversity, here we investigated the origin and evolution of a cluster of tandemly arrayed TPS genes in Oryza. In the Oryza species analyzed, TPS genes occur as a three-TPS cluster, a two-TPS cluster, and a single TPS gene in five, one, and one species, respectively. Phylogenetic analysis revealed the origins of the two-TPS and three-TPS clusters and the role of species-specific losses of TPS genes. Within the three-TPS clusters, one orthologous group exhibited conserved catalytic activities. The other two groups, both of which contained pseudogenes and/or nonfunctional genes, exhibited distinct profiles of terpene products. Sequence and structural analyses combined with functional validation identified several amino acids in the active site that are critical for catalytic activity divergence of the three orthologous groups. In the five Oryza species containing the three-TPS cluster, their functional TPS genes showed both conserved and species-specific expression patterns in insect-damaged and untreated plants. Emission patterns of volatile terpenes from each species were largely consistent with the expression of their respective TPS genes and the catalytic activities of the encoded enzymes. This study indicates the importance of combinatorial evolution of TPS genes in determining terpene variations among individual species, which includes gene duplication, retention/loss/degradation of duplicated genes, varying selection pressure, retention/divergence in catalytic activities, and divergence in expression regulation.

Not Cannabis Specific
Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.
Xu, J., Ai, Y., Wang, J., Xu, J., Zhang, Y., & Yang, D.
Phytochemistry, 137, 34–41. (2017).
doi:10.1016/j.phytochem.2017.02.017
S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/ L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a “polar pocket” that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases

Not Cannabis Specific
Crystal Structure of Pentalenene Synthase: Mechanistic Insights on Terpenoid Cyclization Reactions in Biology.
Charles A. Lesburg, Guangzhi Zhai, David E. Cane, David W. Christianson
Science, 277(5333), 1820–1824.(1997).
doi:10.1126/science.277.5333.1820
The crystal structure of pentalenene synthase at 2.6 angstrom resolution reveals critical active site features responsible for the cyclization of farnesyl diphosphate into the tricyclic hydrocarbon pentalenene. Metal-triggered substrate ionization initiates catalysis, and the a-barrel active site serves as a template to channel and stabilize the conformations of reactive carbocation intermediates through a complex cyclization cascade. The core active site structure of the enzyme may be preserved among the greater family of terpenoid synthases, possibly implying divergence from a common ancestral synthase to satisfy biological requirements for increasingly diverse natural products.

Not Cannabis specific
Designed divergent evolution of enzyme function
Yasuo Yoshikuni, Thomas E Ferrin, Jay D Keasling
Nature 440(7087):1078-82 May 2006
DOI: 10.1038/nature04607
https://www.researchgate.net/publica...nzyme_function
It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity). The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues). To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase gamma-humulene synthase. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.

Differentiation of marijuana headspace volatiles from other plants and hemp products using capillary microextraction of volatiles (CMV) coupled to gas-chromatography–mass spectrometry (GC–MS).
Wiebelhaus N, Kreitals NM, Almirall JR (2016)
Forensic Chem 2: 1–8
doi: 10.1016/j.forc.2016.08.004
The ability to rapidly detect illicit drugs, such as marijuana, is critical to policing legislation across the country. However, it is often difficult to distinguish or identify small quantities of drugs in large spaces without the aid of trained canines. A new device, the capillary microextractor of volatiles (CMV), has the potential to provide rapid detection due to its ability to collect and preconcentrate volatile organic compounds (VOCs) directly from air within minutes. Analysis of the captured compounds can then be performed using a gas chromatography–mass spectrometer (GC–MS). This study focuses on the detection of marijuana volatiles using the CMV as a sampling and preconcentration device given the hypothesis that marijuana will have a distinct chemical profile, or collection of VOCs, that distinguishes it from related plants and other products that could emit similar compounds. Volatile compounds from the headspace of marijuana, related plants, and hemp products were extracted using the CMV and analyzed with GC–MS. The compounds identified and the chemical profiles of each sample were then compared to the volatiles found in the headspace of authentic marijuana samples. The findings presented here suggest that marijuana plants emit volatiles that are readily distinguished from the other samples tested in this study. The distinguishing compounds included a-santalene, valencene, and b-bisabolene. In some cases, THC and cannabinol were also present in the headspace of marijuana. Although these findings support the hypothesis that marijuana has a distinct chemical VOC signature, further work to create a larger database of potential plants and materials is recommended prior to routine use of the CMV coupled to a GC–MS in forensic casework.

Not Cannabis specific
Effect of Soil Nutrient on Production and Diversity of Volatile Terpenoids from Plants
E Ormeño, and C Fernandez
Curr Bioact Compd. 2012 Jan; 8(1): 71–79.
DOI: 10.2174/157340712799828188
Terpenoid production (emission and storage) within foliage plays direct and indirect defensive and protective functions for the plant, mediates complex trophic relationships and controls the oxidation capacity of the atmosphere. Both biotic and abiotic conditions alter terpenoid production, with herbivory, light and temperature effects being reasonably well understood. In this manuscript, the state of the science about nutrient effect on terpenoid production is reviewed. The focus is on isoprene emissions and mono- and sesquiterpenoid

Effect of the Distillation Time on the Chemical Composition, Antioxidant Potential and Antimicrobial Activity of Essential Oils from Different Cannabis sativa L. Cultivars
Sara Palmieri, Francesca Maggio, Marika Pellegrini, Antonella Ricci, Annalisa Serio, Antonello Paparella and Claudio Lo Sterzo
Molecules 2021, 26, 4770.
GOI: 10.3390/ molecules26164770
https://www.ncbi.nlm.nih.gov/pmc/art...s-26-04770.pdf
Within the unavoidable variability of various origins in the characteristics of essential oils, the aim of this study was to evaluate the effect of the distillation time on the chemical composition and biological activity of Cannabis sativa essential oils (EOs). The dry inflorescences came from Carmagnola, Kompolti, Futura 75, Gran Sasso Kush and Carmagnola Lemon varieties from Abruzzo region (Central Italy), the last two being new cultivar here described for the first time. EOs were collected at 2 h and 4 h of distillation; GC/MS technique was applied to characterize their volatile fraction. The EOs were evaluated for total polyphenol content (TPC), antioxidant capacity (AOC) and antimicrobial activity against food-borne pathogens and spoilage bacteria. The time of distillation particularly influenced EOs chemical composition, extracting more or less terpenic components, but generally enriching with minor sesquiterpenes and cannabidiol. A logical response in ratio of time was observed for antioxidant potential, being the essential oils at 4 h of distillation more active than those distilled for 2 h, and particularly Futura 75. Conversely, except for Futura 75, the effect of time on the antimicrobial activity was variable and requires further investigations; nevertheless, the inhibitory activity of all EOs against Pseudomonas fluorescens P34 was an interesting result.

Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens
Parijat Kusari & Souvik Kusari & Michael Spiteller & Oliver Kayser
Fungal Diversity (2013) 60:137–151
DOI 10.1007/s13225-012-0216-3
The objective of the present work was isolation, phylogenetic characterization, and assessment of biocontrol potential of endophytic fungi harbored in various tissues (leaves, twigs, and apical and lateral buds) of the medicinal plant, Cannabis sativa L. A total of 30 different fungal endophytes were isolated from all the plant tissues which were authenticated by molecular identification based on rDNA ITS sequence analysis (ITS1, 5.8S and ITS2 regions). The Menhinick’s index revealed that the buds were immensely rich in fungal species, and Camargo’s index showed the highest tissue-specific fungal dominance for the twigs. The most dominant species was Penicillium copticola that could be isolated from the twigs, leaves, and apical and lateral buds. A detailed calculation of Fisher’s log series index, Shannon diversity index, Simpson’s index, Simpson’s diversity index, and Margalef’s richness revealed moderate overall biodiversity of C. sativa endophytes distributed among its tissues. The fungal endophytes were challenged by two host phytopathogens, Botrytis cinerea and Trichothecium roseum, devising a dual culture antagonistic assay on five different media. We observed 11 distinct types of pathogen inhibition encompassing a variable degree of antagonism on changing the media. This revealed the potential chemodiversity of the isolated fungal endophytes not only as promising resources of biocontrol agents against the known and emerging phytopathogens of Cannabis plants, but also as sustainable resources of biologically active and defensive secondary metabolites.

Not Cannabis specific
Engineering Monoterpene Production in Yeast Using a Synthetic Dominant Negative Geranyl Diphosphate Synthase.
Ignea, C., Pontini, M., Maffei, M. E., Makris, A. M., & Kampranis, S. C
ACS Synthetic Biology, 3(5), 298-306. (2014).
doi:10.1021/sb400115e
Monoterpenes have an established use in the food and cosmetic industries and have recently also found application as advanced biofuels. Although metabolic engineering efforts have so far achieved significant yields of larger terpenes, monoterpene productivity is lagging behind. Here, we set out to establish a monoterpene-specific production platform in Saccharomyces cerevisiae and identified the sequential reaction mechanism of the yeast farnesyl diphosphate synthase Erg20p to be an important factor limiting monoterpene yield. To overcome this hurdle, we engineered Erg20p into a geranyl diphosphate synthase and achieved a significant increase in monoterpene titers. To further improve production, we converted the engineered geranyl diphosphate synthase into a dominant negative form, so as to decrease the ability of the endogenous Erg20p to function as a farnesyl diphosphate synthase, without entirely abolishing sterol biosynthesis. Fusion of the synthetic dominant negative Erg20p variant with the terpene synthase, combined with yeast strain engineering, further improved monoterpene yields and achieved an overall 340-fold increase in sabinene yield over the starting strain. The design described here can be readily incorporated to any dedicated yeast strain, while the developed plasmid vectors and heterozygous ERG20 deletion yeast strain can also be used as a plug-and-play system for enzyme characterization and monoterpene pathway elucidation

Essential oils from Cannabis sativa L.
Alessandro Zatta
September 2005 Conference: 36th International Symposium on Essential Oils. At: Budapest (H)
FIND LINK OR DOI then list on IC
https://www.researchgate.net/publica...nabis_sativa_L
Hemp (Cannabis sativa L.) is an annual species, native of central Asia and spread in Europe and Africa, source of hundreds of biological active compounds such as cannabinoids, terpenoids, flavonoids and polyunsaturated fatty acids. Hemp essential oil, with its unique smell is, at present, used in cosmetic and perfume products, aromatherapy and as beer flavouring agent. Moreover it is traditionally employed as anti-inflammatory in the respiratory and digestive tracts and some its components possess recognized biological properties. In particular, myrcene is a potent analgesic (Rao et al., 1990), 1,8-cineole increases cerebral blood flow and enhances cortical activity (Nasel et al., 1994)and limonene inhibits the growth of many species of fungi and bacteria and as well as -pinene, -terpineol and borneol possesses repellent effects against many insects. For these aspects, it has the potential to be more exploited in different applications. In our research, five hemp cultivars, three dioic (Carmagnola, Dioica 88 and Fibranova ) and two monoic ( Epsilon and Futura), cultivated as fibre crops were also evaluated for the essential oil yields and compositions of their inflorescences. The aim of this work was to deep the knowledge on hemp essential oils and to obtain preliminary information on the possible exploitation the inflorescences, unused in fibre production, as source of a value-added for these cultivars. The oils, obtained by steam distillation and characterized by GC-MS, were made up by the same pool of components with ?-pinene ( from 9.5 to 16.3%), myrcene (from 14.6 to 20.9%) and ?-caryophyllene (from 10.3 to 24.6%) as main constituents followed by ?-pinene, limonene, trans ocimene, terpinolene and ?-humulene. The cultivars showed marked quantitative differences. The oil from Carmagnola had the highest content of myrcene, that from Fibranova was characterized by the higher amounts of ?-caryophyllene and ??humulene and that from Epsilon was the richest in ?-pinene and terpinolene and possessed also a high content of myrcene. The cultivar Fibranova showed the highest oil yield (0.28%) whereas Epsilon the lowest one (0.13%).

Essential Oil of Cannabis sativa L: Comparison of Yield and Chemical Composition of 11 Hemp Genotypes
Ylenia Pieracci, Roberta Ascrizzi, Valentina Terreni, Luisa Pistelli, Guido Flamini, Laura Bassolino, Flavia Fulvio, Massimo Montanari and Roberta Paris
Molecules 2021, 26, 4080.
DOI: 10.3390/ molecules26134080
Cannabis sativa L. is an annual species cultivated since antiquity for different purposes. While, in the past, hemp inflorescences were considered crop residues, at present, they are regarded as valuable raw materials with different applications, among which extraction of the essential oil (EO) has gained increasing interest in many fields. The aim of the present study is the evaluation of the yield and the chemical composition of the EO obtained by hydrodistillation from eleven hemp genotypes, cultivated in the same location for two consecutive growing seasons. The composition of the EOs was analyzed by GC–MS, and then subjected to multivariate statistical analysis. Sesquiterpenes represented the main class of compounds in all the EOs, both in their hydrocarbon and oxygenated forms, with relative abundances ranging from 47.1 to 78.5%; the only exception was the Felina 32 sample collected in 2019, in which cannabinoids predominated. Cannabinoids were the second most abundant class of compounds, of which cannabidiol was the main one, with relative abundances between 11.8 and 51.5%. The statistical distribution of the samples, performed on the complete chemical composition of the EOs, evidenced a partition based on the year of cultivation, rather than on the genotype, with the exception of Uso-31. Regarding the extraction yield, a significant variation was evidenced among both the genotypes and the years of cultivation.

Essential oil of Cannabis sativa L. strains
Vito Mediavilla and Simon Steinemann
Journal of the International Hemp Association Vol.4 Issue 2 1997
https://www.internationalhempassocia.. ./jiha4208.html
The aroma of hemp (Cannabis sativa L.) could be of considerable commercial value if evaluation of varieties and development of extraction methods led to a pleasing scent in the resulting essential oils. We compared the composition and smell of some fiber hemp and drug Cannabis essential oils isolated by steam distillation. The essential oil of some hemp strains contained particular monoterpenes and sesquiterpenes that imparted to the specimen a desireable scent. These preliminary one-year results do not take into account the influence that harvest time and the weather "just-before-harvest" could have on the quality of the essential oil. The ?9-tetrahydrocannabinol (THC) concentration in the essential oils was very low and varied between 0.02% and 0.08%. The ratio of this compound to cannabidiol showed only small changes during steam distillation.

Not Cannabis specific
Essential Oil Safety
A Guide for Health Care Professionals
Robert Tisserand, Rodney Young, Elizabeth M Williamson
S E C O N D E D I T I O N
DOI: 10.1016/B978-0-443-06241-4.00001-1
This revised edition took 12 years to complete, and is considerably longer than the previous edition. There are three reasons for the comprehensive revision. First, since the text was first published in 1995, there have been many notable developments in the area of essential oil safety. In addition to new data being published, many guidelines and restrictions have been revised or issued by various authorities, and we have introduced some of
our own. Second, significant changes and improvements have been made to the text, especially in the area of profiles, some of these in response to reader feedback. The structure of both the Essential Oil Profiles and the Constituent Profiles has been considerably elaborated, and new material has been added. This edition includes 400 Essential Oil Profiles, compared to 95 previously. For each essential oil there is a full breakdown of constituents, and a clear categorization of hazards and risks, with recommended maximum doses and concentrations. All the compositional data for essential oils has been revised, expanded and referenced. There are 206 Constituent Profiles, and this section is 15 times that of the previous edition. Constituents are cross-referenced: each Constituent Profile lists the amount of that substance found in each of the 400 profiled essential oils. Third, the structure of the book has been developed. There are now separate chapters on the nervous, urinary, cardiovascular, gastrointestinal, and respiratory systems. Some sections of
text have moved from one chapter to another, and repetitive or outdated material has been deleted. We now have detailed safety advice on drug interactions, and overall there are more cautions. The new material is reflected in over 3,400 new references. A number of minor changes have also been made, such as the styling of references and the categorization of
constituents.

Not Cannabis specific
EssOilDB: A database of essential oils reflecting terpene composition and variability in the plant kingdom
Sachin Pundhir, Ganga Jeena
DOI: 10.1093/database/bau120
Database The Journal of Biological Databases and Curation · January 2014
Plant essential oils are complex mixtures of volatile organic compounds, which play indispensable roles in the environment, for the plant itself, as well as for humans. The potential biological information stored in essential oil composition data can provide an insight into the silent language of plants, and the roles of these chemical emissions in defense, communication and pollinator attraction. In order to decipher volatile profile patterns from a global perspective, we have developed the ESSential OIL DataBase
(EssOilDB), a continually updated, freely available electronic database designed to provide knowledge resource for plant essential oils, that enables one to address a multitude of queries on volatile profiles of native, invasive, normal or stressed plants, across taxonomic clades, geographical locations and several other biotic and abiotic influences.
To our knowledge, EssOilDB is the only database in the public domain providing an opportunity for context based scientific research on volatile patterns in plants. EssOilDB presently contains 123 041 essential oil records spanning a century of published reports on volatile profiles, with data from 92 plant taxonomic families, spread across diverse geographical locations all over the globe. We hope that this huge repository of VOCs will facilitate unraveling of the true significance of volatiles in plants, along with creating potential avenues for industrial applications of essential oils. We also illustrate the use of this database in terpene biology and show how EssOilDB can be used to complement data from computational genomics to gain insights into the diversity and variability of terpenoids in the plant kingdom. EssOilDB would serve as a valuable information resource, for students and researchers in plant biology, in the design and discovery of new odor profiles, as well as for entrepreneurs—the potential for generating consumer specific scents being one of the most attractive and interesting topics in the cosmetic industry.

Ethephon application stimulats cannabinoids and plastidic terpenoids production in Cannabis sativa at flowering stage
Hakimeh Mansouri, , Fatemeh Salari, Zahra Asrar
Industrial Crops and Products
doi: 10.1016/j.indcrop.2013.01.025
We studied the effect of ethephon on levels of the major cannabinoids (tetrahydrocannabinol and cannabidiol) and chlorophyll, carotenoids and ?-tocopherol in Cannabis sativa at productive stage. Results revealed that ethephon increased THCcontent of leaf in male and female plants and of male flowers. However, ethephon unable to enhancing THC content in female flowers. Treatment with etheohon increased CBD content in male and female leaf and female flowers. The treatment of male flowers with low ethephon concentration caused an increase, and those treated with high ethephon concentration resulted in a decrease in CBD content. The lowest level of ethephon (1 ?M) enhanced chlorophyll a, b and total chlorophyll in male and female plants. Both sexes treated with ethephon showed an increase in carotenoids content, but 1 ?M ethephon had the stronger effect in this regards. Male and female plants had a higher content of ?-tocopherol when treated with ethephon. These results showed ethephon is a suitable treatment for increasing cannabinoids and ?-tocopherol in productive stage of cannabis and there was not a relation between primary and secondary terpenoids.
? Ethephon treatment has considerable effects on increasing of cannabinoids in male and female cannabis. ? There is no correlation between cannabinoids and other plastidial terpenoids. ? Using of ethephon treatment in flowering stage do not adverse effect on plant growth.

ERRATUM
Evaluation of Cannabinoid and Terpenoid Content: Cannabis Flower Compared to Supercritical CO2 Concentrate
Michelle Sexton, Kyle Shelton, Pam Haley, Mike West
Planta Med, advance online publication September 19, 2017
doi:10.1055/s-0043-119361
? Table 1 was replaced by a new table.
In ? Tables 2 and 3 microgram/gram (?g/g) was modified
to mg/g.
? Fig. 2 was replaced by a new figure (in the figure microgram/
gram [?g/g] was modified to mg/gram).
The ?-caryophyllene result on page 3 in the section “Results”
was corrected, the factor is “5.1”.

Evaluation of cannabinoid and terpenoid content: Cannabis flower compared to supercritical CO2 concentrate.
Sexton M, Shelton K, Haley P, West M (2018)
Planta Med 84: 234–241
doi: 10.1055/s-0043-119361
A recent cannabis use survey revealed that 60% of cannabis users rely on smelling the flower to select their cannabis. Olfactory indicators in plants include volatile compounds, principally represented by the terpenoid fraction. Currently, medicinal- and adult-use cannabis is marketed in the United States with relatively little differentiation between products other than by a common name, association with a species type, and ?-9 tetrahydrocannabinol/cannabidiol potency. Because of this practice, how terpenoid compositions may change during an extraction process is widely overlooked. Here we report on a comparative study of terpenoid and cannabinoid potencies of flower and supercritical fluid CO2 (SC?CO2) extract from six cannabis chemovars grown in Washington State. To enable this comparison, we employed a validated high-performance liquid chromatography/diode array detector methodology for quantification of seven cannabinoids and developed an internal gas chromatography-mass spectrometry method for quantification of 42 terpenes. The relative potencies of terpenoids and cannabinoids in flower versus concentrate were significantly different. Cannabinoid potency increased by factors of 3.2 for ?-9 tetrahydrocannabinol and 4.0 for cannabidiol in concentrates compared to flower. Monoterpenes were lost in the extraction process; a ketone increased by 2.2; an ether by 2.7; monoterpene alcohols by 5.3, 7 and 9.4; and sesquiterpenes by 5.1, 4.2, 7.7, and 8.9. Our results demonstrate that the product of SC?CO2 extraction may have a significantly different chemotypic fingerprint from that of cannabis flower. These results highlight the need for more complete characterization of cannabis and associated products, beyond cannabinoid content, in order to further understand health-related consequences of inhaling or ingesting concentrated forms.

Evaluation of the terpenes β-caryophyllene, α-terpineol, and γ-terpinene in the mouse chronic constriction injury model of neuropathic pain: possible cannabinoid receptor involvement
Joshua A Bilbrey, Yuma T Ortiz, Jasmine S Felix, Lance R McMahon, Jenny L Wilkerson
Psychopharmacology . 2021 Nov 30.
doi: 10.1007/s00213-021-06031-2
Pain is one of the most common reasons to seek medical attention, and chronic pain is a worldwide epidemic. Anecdotal reports suggest cannabis may be an effective analgesic. As cannabis contains the terpenes α-terpineol, β-caryophyllene, and γ-terpinene, we hypothesized these terpenes would produce analgesia in a mouse model of neuropathic pain. We used the chronic constriction injury of the sciatic nerve mouse model, which produces mechanical allodynia, assessed via the von Frey assay, as well as thermal hyperalgesia assessed via the hotplate assay. Compounds were further assessed in tests of locomotor activity, hypothermia, and acute antinociception. Each terpene produced dose-related reversal of mechanical allodynia and thermal hyperalgesia. Thermal hyperalgesia displayed higher sensitivity to the effects of each terpene than mechanical allodynia, and the rank order potency of the terpenes was α-terpineol > β-caryophyllene > γ-terpinene. To examine the involvement of cannabinoid receptors, further tests were conducted in mice lacking either functional cannabinoid type 1 receptors (CB1R (-/-)) or cannabinoid type 2 receptors (CB2R (-/-)). Compared to wild type mice, CB1R (-/-) mice treated with α-terpineol displayed a 2.91-fold decrease in potency to reverse mechanical allodynia; in CB2R (-/-) mice, the potency of α-terpineol was decreased 11.73-fold. The potency of β-caryophyllene to reverse mechanical allodynia decreased 1.80-fold in CB2R (-/-) mice. Each terpene produced a subset of effects in tests of locomotor activity, hypothermia, and acute antinociception. These findings suggest α-terpineol, β-caryophyllene, and γ-terpinene may have differential cannabinoid receptor activity and a pharmacological profile that may yield new efficacious analgesics.
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Not Cannabis Specific
https://essentialoils.org/research
The Science Behind Essential Oils
We maintain an active role in the research of essential oils on various levels. Dr. Pappas regularly submits publications to reputable scientific journals like Journal of Essential Oil Research (JEOR) on the chemical properties of unusual essential oils as well as submitting articles relevant to aromatherapy to journals like Aromatherapy Journal (formerly Scentsitivity, published by the National Association for Holistic Aromatherapy). Dr. Pappas has also been involved with essential oil and aromatherapy education at local colleges and universities.
Some Scientific Journal Publications by Dr. Pappas related to essential oils. We hope you enjoy them

Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes
Oier Aizpurua-Olaizola†‡, Umut Soydaner†, Ekin Öztürk†, Daniele Schibano†, Yilmaz Simsir†, Patricia Navarro‡, Nestor Etxebarria‡, and Aresatz Usobiaga*‡
J. Nat. Prod.
DOI: 10.1021/acs.jnatprod.5b00949
The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.

Expression and characterization of terpene synthases from Cannabis sativa L. and Salvia sclarea L.
Nils Gu?nnewich THESIS
https://www.researchgate.net/publica...lvia_sclarea_L
Many volatile oil (or essential) oil-producing plants are particular interesting because of their high economic value. The term ‘volatile oil’ is preferred because it refers to the fact that most components of the oils, which are stored in extra cellular spaces in the epidermis or mesophyll, have low boiling points and can be recovered from the plant tissues by steam distillation. Volatile oils are quite distinct from more common triglyceride oils and fats, which are known as ‘fixed’, because of their high boiling point [1]. Vast amounts of volatile oils are terpenoids that are made up of five-carbon isoprene or isopentenoid units. Other biological molecules belong to this family, are for example; porphyrins, chlorophylls, carotenoids, steroids, gibberellins and natural rubber [2]. The production and accumulation of volatile, low molecular weight terpenes [mono-(C10), most sesqui-(C15), and some diterpenes (C20)] are not restricted to one specialized taxonomic group but occur throughout the plant kingdom. Volatile oils can be obtained from various tissues of Gymnosperms (Pinaceae: conifers; Taxaceae: the yew family; Cupressaceae: cypresses and junipers; Cycadaceae: the tree fern family). The ability to accumulate terpenes is widely distributed in Angiosperms (subphylum: Magnoliophytina), too. By temperate and tropical dicotyledons (Chenopodiaceae: beets and goosefoots; Compositae: the daisy family; Geraniaceae: the geranium family; Guttiferae/Hypericaceae: including St. Johns worts; Lamiaceae: the mint family; Cannabaceae: hemp and hop; Lauraceae: including bays; Myricacea; Myristicaceae: nutmeg and mace; Myrtaceae: myrtles, eucalypts and clove; Scrophulariaceae: olives and lilacs; Piperaceae: the pepper family; Rosaceae: the rose family; Rutaceae: the citrus family; Santalaceae: sandalwood; Apiaceae: the carrotfamily; Verbenaceae: including verbenas; Violaceae: violets and pansies) and monocotyledons (Araceae: the aroid family; Cyperaceae: the sedge family; Poaceae: the grass family; Zingiberaceae: the ginger family). This indicates that the accumulation of terpenes was a feature of the earliest seed plants [1]. In this doctoral thesis two volatile oil producing plants were investigated, due to their ability to produce economically valuable terpenoids, Cannabis sativa L. and Salvia sclarea L..

Extraction of Phenolic Compounds and Terpenes from Cannabis sativa L. By-Products: From Conventional to Intensified Processes
Emilie Isidore, Hamza Karim, Irina Ioannou
https://www.researchgate.net/publica...fied_Processes
Cannabis sativa L. is a controversial crop due to its high tetrahydrocannabinol content varieties; however, the hemp varieties get an increased interest. This paper describes (i) the main categories of phenolic compounds (flavonoids, stilbenoids and lignans) and terpenes (monoterpenes and sesquiterpenes) from C. sativa by-products and their biological activities and (ii) the main extraction techniques for their recovery. It includes not only common techniques such as conventional solvent extraction, and hydrodistillation, but also intensification and emerging techniques such as ultrasound-assisted extraction or supercritical CO2 extraction. The effect of the operating conditions on the yield and composition of these categories of phenolic compounds and terpenes was discussed. A thorough investigation of innovative extraction techniques is indeed crucial for the extraction of phenolic compounds and terpenes from cannabis toward a sustainable industrial valorization of the whole plant.

Factors influencing the yield and the quality of hemp (Cannabis sativa L.) essential oil
C. Meier, Vito Mediavilla
January 1998
https://www.internationalhempassocia.. ./jiha5107.html
https://therichardrosereport.com/dow...essential-oil/
The aim of this work was to assess the factors influencing the yield and the quality of hemp essential oil. Several strains were used to carry out field and greenhouse experiments, and as specimens for scent tests and chemical analysis of the distilled oils.
Yields of oil were highest at seeding rates of 5 kg/ha and when about a 50% of the crop had reached maturity. Pollination led to significantly lower yields, but is not easy to prevent in the field. Chemical composition showed almost no relationship to harvest dates or to scores on the scent tests. The best scent quality was always obtained from plants harvested one to three weeks before seed maturity (75% of seed matured). The interval during which both yield and quality were high was rather short and had to be assessed for each strain. The influence of other factors such as weather and harvest technique were also evaluated.

Fast, Accurate, and Precise Terpene Testing of Cannabis Samples
Lee Marotta, David Scott, Adam Floyd, Toby Astill, Cassandra (Cassie) Ereman, Ben Armstrong
PerkinElmer
https://www.perkinelmer.com/lab-solu..._014579_01.pdf
Like all botanicals and plants found in nature, cannabis also contains terpenes, which are the aromatic oils that give rise to the distinctive flavors and aromas found in cannabis varieties. There have been up to 140 different types of terpenes reported in cannabis, but multiple studies suggest that approximately 17 are the most common and can be used for examining their chemotype (chemotype: those strains that have chemical properties that differ from each other’s). Among them are monoterpenes, diterpenes, and sesquiterpenes, which are characterized by the number of repeating units of a five-carbon molecule, called isoprene, the structural hallmark of all terpenoid compounds. The diverse palate of cannabis terpenes is impressive enough, but arguably their most fascinating characteristic is their ability to interact synergistically with other compounds in the plant, like cannabinoids. In the past few decades, a significant amount of work has been performed to understand the ‘entourage effect’, which scientists refer to as synergistic interaction between terpenes and cannabinoids in the human body. This effect is believed to magnify the therapeutic benefits of the plant’s individual components — so that the medicinal impact of the whole plant is greater than the sum of its parts quantifying which terpenes are present is an important aspect of understanding the unique effects of cannabis for both medicinal and recreational users.

Fibre hemp inflorescences: From crop-residues to essential oil production
Alessandra Bertoli, Sabrina Tozzi, Luisa Pistelli, Luciana G. Angelini
Industrial Crops and Products 32 (2010) 329–337
doi: 10.1016/j.indcrop.2010.05.012
The volatile composition of ten fibre hemp (Cannabis sativa L.) varieties was investigated during two successive growing seasons under temperate climatic conditions in Central Italy.
The freshly plant inflorescences were hydrodistilled and the essential oils (EOs) were characterized by GC–MS. In addition, the composition of the aroma emitted spontaneously from the freshly plant inflorescences were analysed by SPME-GC–MS. The EO yields of eight dioecious (Carmagnola, C.S., Red Petiole, Pop 1, Pop 2, Pop 3, Pop 4, Pop 5) and two monoecious (Codimono and Felina 34) cultivars ranged from 0.11 to 0.25% (w/w) and showed a significant production of a-pinene (3–20%), b-pinene (1–8%), E-ocimene (1–10%), myrcene (8–45%) and terpinolene (0.12–22%).
The monoterpene composition was useful to distinguish the monoecious cultivars from the dioecious ones. b-Caryophyllene (7–28%), a-humulene (3–12%), and caryophyllene oxide (2–6%) were the main sesquiterpenes. Tetrahydrocannabinol (THC) was present in traces in the EOs of only two dioecious cultivars cultivated in 2005. Cannabinol (CBN) was not detected in the essential oils, while the no-hallucinogenous cannabidiol (CBD) was found as typical volatile constituent in several analysed cultivars. These findings were also confirmed by the headspace GC–MS analysis carried out on the same samples. The analysed EOs obtained from fibre hemp varieties cultivated in Central Italy were characterized by an interesting and specific terpene composition with a legal and safe cannabinoid content. They were obtained from freshly plant inflorescences, which usually represent a waste material from C. sativa L. fibre varieties. The present study strengths the hypothesis to grow hemp as a multi-use crop through a complete utilization of the plant material using inflorescences to produce essential oils as natural flavour and fragrance additives.

NOT CANNABIS SPECIFIC
Four terpene synthases contribute to the generation of chemotypes in tea tree (Melaleuca alternifolia)
Amanda Padovan, Andras Keszei, Yasmin Hassan, Sandra T. Krause, Tobias G. Köllner, Jörg Degenhardt, Jonathan Gershenzon, Carsten Külheim and William J. Foley
BMC Plant Biology (2017) 17:160
DOI 10.1186/s12870-017-1107-2
Background: Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as “chemotypes”. Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species.
Results: We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. Conclusions: Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.

Not Cannabis Specific
Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase
Narayanan Srividya, Edward M. Davis, Rodney B. Croteau1 , and B. Markus Lange
PNAS | March 17, 2015 | vol. 112 | no. 11
DOI: 10.1073/pnas.1501203112
Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (?)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.
Significance: Terpene synthases catalyze complex, chain length-specific, electrophilic cyclization reactions that constitute the first committed step in the biosynthesis of structurally diverse terpenoids. (4S)-limonene synthase [(4S)-LS] has emerged as a model enzyme for enhancing our comprehension of the reaction cycle of monoterpene (C10) synthases. While the stereochemistry of the cyclization of geranyl diphosphate to (?)-(4S)-limonene has been the subject of several mechanistic studies, the structural basis for the stabilization of carbocation intermediates and the termination of the reaction sequence have remained enigmatic. We present extensive experimental evidence that the aromatic amino acids W324 and H579 play critical roles in the stabilization of intermediate carbocations. A possible function of these residues as the terminal catalytic base is also discussed.

Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-a-pinene synthase from Cannabis sativa
Nils Gu?nnewich,Jonathan E. Page, Tobias G. Köllner, Jörg Degenhardt, Toni M. Kutchana
Natural product communications · March 2007 Natural Product Communications Vol. 0 (0) 2006
https://www.researchgate.net/profile...bis-sativa.pdf
Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-?-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 shows a Km value at 6.8 ?M and a Kcat at 8.2 x 10-2 s-1, the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 shows a Km values at 6.7 ?M and a Kcat at 8.1 x 10-2 s-1, the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperm family and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-?-pinene were detected as natural products in C. sativa trichomes.

NOT CANNABIS SPECIFIC
Genetic Control and Evolution of Sesquiterpene Biosynthesis in Lycopersicon esculentum and L. hirsutum
Rutger S. van der Hoeven, Antonio J. Monforte, David Breeden, Steven D. Tanksley, and John C. Steffens
The Plant Cell, Vol. 12, 2283–2294, November 2000,
DOI: 10.1105/tpc.12.11.2283
Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase 1 (Sst1), was identified for which the L. esculentum allele is associated with the biosynthesis of b-caryophyllene and a-humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B, and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6–associated sesquiterpenes. At a second locus, Sst2, on chromosome 8, the L. hirsutum allele specified accumulation of a-santalene, a-bergamotene, and b-bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.

Not Cannabis specific Genetic Engineering of Terpenoid Metabolism Attracts Bodyguards to Arabidopsis
Iris F. Kappers, Asaph Aharoni, Teun W. J. M. van Herpen, Ludo L. P. Luckerhoff, Marcel Dicke, Harro J. Bouwmeester
SCIENCE 23 SEPT 2005 VOL 309
DOI: 10.1126/science.1116232
Herbivore-damaged plants release complex mixtures of volatiles that attract
natural enemies of the herbivore. To study the relevance of individual components of these mixtures for predator attraction, we manipulated herbivoryinduced volatiles through genetic engineering. Metabolic engineering of terpenoids, which dominate the composition of many induced plant volatile bouquets, holds particular promise. By switching the subcellular localization of the introduced sesquiterpene synthase to the mitochondria, we obtained transgenic Arabidopsis thaliana plants emitting two new isoprenoids. These altered plants attracted carnivorous predatory mites (Phytoseiulus persimilis) that aid the plants’ defense mechanisms

*Genomic characterization of the complete terpene synthase gene family from Cannabis sativa.
Allen KD, McKernan K, Pauli C, Roe J, Torres A, Gaudino R
PLoS ONE 14 (9): e0222363. (2019)
Doi: 10.1371/journal. pone.0222363
Terpenes are responsible for most or all of the odor and flavor properties of Cannabis sativa, and may also impact effects users experience either directly or indirectly. We report the diversity of terpene profiles across samples bound for the Washington dispensary market. The remarkable degree of variation in terpene profiles ultimately results from action of a family of terpene synthase genes, only some of which have been described. Using a recently available genome assembly we describe 55 terpene synthases with genomic context, and tissue specific expression. The family is quite diverse from a protein similarity perspective, and subsets of the family are expressed in all tissues in the plant, including a set of root specific monoterpene synthases that could well have agronomic importance. Ultimately understanding and breeding for specific terpene profiles will require a good understanding of the gene family that underlies it. We intend for this work to serve as a foundation for that.

Genomic Organization of Plant Terpene Synthases and Molecular Implications
Susan C Trapp, Rodney B Croteau
Genetics 158(2):811-32July 2001
DOI: 10.1093/genetics/158.2.811
https://www.researchgate.net/publica...r_Implications
Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.

Not Cannabis specific
Geraniol Restores Antibiotic Activities against Multidrug-Resistant Isolates from Gram-Negative Species.
Lorenzi, V., Muselli, A., Bernardini, A. F., Berti, L., Pages, J.-M., Amaral, L., & Bolla, J.-M.
Antimicrobial Agents and Chemotherapy, 53(5), 2209–2211.(2009).
doi:10.1128/aac.00919-08
The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine -naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of -lactams, quinolones, and chloramphenicol.

Got Terps? Cannabis Extracts Lack the Same Compounds as Flower
Chris Roberts
https://cannabisnow.com/got-terps-ca...nds-as-flower/
It can be easy to convince yourself that concentrated cannabis is cannabis, just more of it. You can argue that concentrates have more of the good stuff and less carbon-based plant material: More terpenes, more THC, more bounce to the ounce (or bam to the gram, whatever). Just taste that surge of limonene and feel the impact on your brain and body as that wave of 80% THC crashes over your consciousness and try to say otherwise!
But contrary to what your senses might tell you about concentrate superiority, science has spoken. And science says the extraction process, as sophisticated as it may be, removes significant active ingredients from the plant-based source material — including the material that, for most consumers, determines what strain is their favourite.

Grinding and Fractionation during Distillation Alter Hemp Essential Oil Profile and Its Antimicrobial Activity.
Zheljazkov, V. D., Sikora, V., Semerdjieva, I. B., Kačániová, M., Astatkie, T., & Dincheva, I.
Molecules, 25(17), 3943.(2020).
doi:10.3390/molecules25173943
The hypothesis of this study was that we can modify the essential oil (EO) profile of hemp (Cannabis sativa L.) and obtain fractions with differential composition and antimicrobial activity. Therefore, the objective was to evaluate the effects of grinding of hemp biomass before EO extraction and fractionation during distillation on EO profile and antimicrobial activity. The study generated a several EO fractions with a diversity of chemical profile and antimicrobial activity. The highest concentrations of β-pinene and myrcene in the EO can be obtained in the 5–10 min distillation time (DT) of ground material or in the 80–120 min DT of nonground material. High δ-3-carene and limonene EO can be obtained from 0–5 min DT fraction of nonground material. High eucalyptol EO can be sampled either in the 0–5 min DT of the ground material or in the 80–120 min of nonground material. Overall, the highest concentrations of β-caryophyllene, α-(E)-bergamotene, (Z)-β-farnesene, α-humulene, caryophyllenyl alcohol, germacrene D-4-ol, spathulenol, caryophyllene oxide, humulene epoxide 2, β-bisabolol, α-bisabolol, sesquiterpenes, and cannabidiol (CBD) can be obtained when EO is sampled in the 80–120 min DT and the material is nonground. Monoterpenes in the hemp EO can be increased twofold to 85% by grinding the material prior to distillation and collecting the EO in the first 10 min. However, grinding resulted in a slight but significant decrease in the CBD concentration of the EO. CBD-rich oil can be produced by collecting at 120–180 min DT. Different EO fractions had differential antimicrobial activity. The highest antimicrobial activity of EO fraction was found against Staphylococcus aureus subsp. aureus. THC-free EO can be obtained if the EO distillation is limited to 120 min. The results can be utilized by the hemp processing industry and by companies developing new hemp EO-infused products, including perfumery, cosmetics, dietary supplements, food, and pharmaceutical industries.

Not Cannabis Specific
Handbook of Essential Oils
Science, Technology, and Applications, Second Edition
Edited ByK. Husnu Can Baser, Gerhard Buchbauer
DOI: 10.1201/b19393
DOI: 10.1201/9781420063165
Find link
The second edition of Handbook of Essential Oils: Science, Technology, and Applications provides a much-needed compilation of information related to the development, use, and marketing of essential oils. It focuses particularly on the chemistry, pharmacology, and biological activities of essential oils, with contributions from a worldwide group of

Headspace Solid Phase Microextraction (HS SPME) Gas Chromatography Mass Spectrometric Analysis of the Volatile Constituents of Cannabis sativa L. From Kashmir
Manzoor A. Rather, Bilal A. Dar, Shahnawaz N. Sofi, Tauheeda Hassan, Nasir Ali, Ashiq H. Lone, Abdul S. Shawl, Wajahat A. Shah, M. A. Qurishi and Poonam Prakash
Journal of Pharmacy Research 2011,4(8),2651-2653
https://citeseerx.ist.psu.edu/viewdo...=rep1&type=pdf
Headspace Solid-phase micro extraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) has been used to isolate and identify the volatile compounds from the leaves of Cannabis sativa growing in Kashmir. The analysis led to the identification of 17 volatile components constituting 94.8 % of the total identified components. The chemical composition of the SPME extract from the leaves of C. sativa comprised mainly of sesquiterpene hydrocarbons (64.3%), monoterpene hydrocarbons (18.4%) and alcohols (10.3%). The major components identified in the HS-SPME extract were trans-caryophyllene (36.9%), a-humulene (16.2%), a-pinene (10.7%), 3-hexen-1-ol-acetate (6.2%) and ß-pinene (4.2%). The current study is the first report involving rapid analysis of volatile components of C. sativa by HS-SPME.

Headspace SPME GC/MS Analysis of Terpenes in Cannabis
Katherine K. Stenerson
Merck
https://www.sigmaaldrich.com/technic...spme-gcms.html
A rapid method to identify cannabis terpenes for forensic and organoleptic applications
Cannabis sativa (cannabis or marijuana) contains over 100 different terpenes and terpenoids, including mono, sesqui, di and tri, as well as other miscellaneous compounds of terpenoid orgin.1 Terpenes give the plant distinct organoleptic properties and produce characteristic aromas when the buds are heated or vaporized.2 Although the terpene profile does not necessarily indicate geographic origin of a cannabis sample, it can be used in forensic applications to determine the common source of different samples.3 In addition, different cannabis strains have been developed which have distinct aromas and flavors, a result of the differing amounts of specific terpenes present

Human metabolism of α-pinene and metabolite kinetics after oral administration Lukas Schmidt & Thomas Göen Archives of Toxicology volume 91, pages677–687 (2017)
DOI 10.1007/s00204-015-1656-9
We studied the human in vivo metabolism and the elimination kinetics of α-pinene (αPN), a natural monoterpene which commonly occurs in the environment. Four volunteers were exposed to a single oral dose of 10 mg αPN. Each subject provided one pre-exposure and subsequently all post-exposure urine samples up to 24 h after administration. Additionally, blood samples were drawn hourly from two volunteers for 5 h. The analysis of the parent compound in blood was performed by a headspace GC–MS procedure, whereas the proposed αPN metabolites myrtenol (MYR) and cis- and trans-verbenol (cVER; tVER) were quantified in blood and urine using GC–PCI-MS/MS. Unknown metabolites were investigated using GC–PCI-MS full-scan analyses. The urinary concentration of the metabolites reached their maxima 1.6 h after exposure. Afterwards, they declined to the pre-exposure levels within the 24-h observation period with elimination half-lives of 1.5 h (MYR) and 1.6 h (cVER and tVER). The total eliminated amounts corresponded to 1.5 % (MYR), 5.6 % (cVER), and 4.1 % (tVER) of the orally applied dose. The GC–PCI-MS full-scan analyses identified three novel metabolites, of which one conforms to myrtenic acid (MYRA). A re-analysis of MYRA in urine showed maximum elimination 1.6 h after αPN ingestion, an elimination half-life of 1.4 h, and a share of the oral dose of 6.7 %. The study revealed that the human in vivo metabolism of αPN proceeds fast and elimination of metabolites takes places rapidly. The metabolism of αPN is dominated by extensive oxidation reactions at the methyl side-chains yielding in carboxylic acid structures as well as by allylic oxidation of the cyclohexenyl backbone, whereas predicted products of a double-bond oxidation were not detected.

Human metabolism of Δ3 carene and renal elimination of Δ3 caren 10 carboxylic acid (chaminic acid) after oral administration Lukas Schmidt · Vladimir N. Belov · Thomas Göen
Arch Toxicol DOI 10.1007/s00204-014-1251-5
https://sci-hub.se/10.1007/s00204-014-1251-5
We studied the human in vivo metabolism of Δ3 -carene (CRN), a natural monoterpene which commonly occurs in the human environment. Four healthy human volunteers were orally exposed to a single dose of 10 mg CRN. Each volunteer gave one urine sample before administration and subsequently collected each urine sample within 24 h after administration. The concentration of the proposed CRN metabolites Δ3 -caren-10-ol (CRN-10-OH), Δ3 -caren-10-carboxylic acid (chaminic acid, CRN-10- COOH), and Δ3 -caren-3,4-diol (CRN-3,4-OH) were determined using a very specific and sensitive GC–MS/ MS procedure. Other CRN metabolites were investigated using GC–PCI–MS Q1 scan analyses. CRN-10-COOH was detected in each urine sample with maximum concentration (113.0–1,172.9 µg L−1 ) 2–3 h after administration, whereas CRN-10-OH and CRN-3,4-OH were not detected in any of the samples. The renal excretion kinetics of CRN-10- COOH showed an elimination half-life of about 3 h. The cumulative excretion of CRN-10-COOH within 24 h after exposure correlated with about 2 % of the applied dose. The GC–PCI–MS Q1 scan analysis indicated several additional human CRN metabolites; thereof, six spectra enabled the prediction of the corresponding chemical structure. The results of the study indicate that CRN-10-COOH is a relevant product of the human in vivo metabolism of CRN. The oxidation of its allylic methyl group proceeds until the acidic structure without interruption. Thus, the generation of the alcoholic intermediate appeared to be the rate-determining step of this metabolic route. Nevertheless, the proportion of CRN-10-COOH in the CRN metabolism is low, and other oxidative metabolites are likely. This hypothesis was confirmed by the discovery of additional human CRN metabolites, whose predicted chemical structures fit in with further oxidative products of CRN metabolism.

*Identification of a New Family of Prenylated Volatile Sulfur Compounds in Cannabis Revealed by Comprehensive TwoDimensional Gas Chromatography
Iain W. H. Oswald, Marcos A. Ojeda, Ryan J. Pobanz, Kevin A. Koby, Anthony J. Buchanan, Josh Del Rosso, Mario A. Guzman, and Thomas J. Martin
ACS OMEGA (2021)
https://pubs.acs.org/doi/pdf/10.1021/acsomega.1c04196
Cannabis sativa L. produces over 200 known secondary metabolites that contribute to its distinctive aroma. Studies on compounds traditionally associated with the scent of this plant have focused on those within the terpenoid class. These isoprene-derived compounds are ubiquitous in nature and are the major source of many plant odors. Nonetheless, there is little evidence that they provide the characteristic “skunk-like” aroma of cannabis. To uncover the chemical origins of this scent, we measured the aromatic properties of cannabis flowers and concentrated extracts using comprehensive two-dimensional gas chromatography equipped with time-of-flight mass spectrometry, flame ionization detection, and sulfur chemiluminescence. We discovered a new family of volatile sulfur compounds (VSCs) containing the prenyl (3-methylbut-2-en-1-yl) functional group that is responsible for this scent. In particular, the compound 3- methyl-2-butene-1-thiol was identified as the primary odorant. We then conducted an indoor greenhouse experiment to monitor the evolution of these compounds during the plant’s lifecycle and throughout the curing process. We found that the concentrations of these compounds increase substantially during the last weeks of the flowering stage, reach a maximum during curing, and then drop after just one week of storage. These results shed light on the chemical origins of the characteristic aroma of cannabis and how volatile sulfur compound production evolves during plant growth. Furthermore, the chemical similarity between this new family of VSCs and those found in garlic (allium sativum) suggests an opportunity to also investigate their potential health benefits.

Identification of terpenoid chemotypes among high (?)-trans-?9-tetrahydrocannabinol-producing Cannabis sativa L. cultivars.
Fischedick JT (2017)
Cannabis Cannabinoid Res 2: 34–47
DOI: 10.1089/can.2016.0040
https://www.researchgate.net/publica...va_L_Cultivars
With laws changing around the world regarding the legal status of Cannabis sativa (cannabis) it is important to develop objective classification systems that help explain the chemical variation found among various cultivars. Currently cannabis cultivars are named using obscure and inconsistent nomenclature. Terpenoids, responsible for the aroma of cannabis, are a useful group of compounds for distinguishing cannabis cultivars with similar cannabinoid content. Methods: In this study we analyzed terpenoid content of cannabis samples obtained from a single medical cannabis dispensary in California over the course of a year. Terpenoids were quantified by gas chromatography with flame ionization detection and peak identification was confirmed with gas chromatography mass spectrometry. Quantitative data from 16 major terpenoids were analyzed using hierarchical clustering analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA). Results: A total of 233 samples representing 30 cultivars were used to develop a classification scheme based on quantitative data, HCA, PCA, and OPLS-DA. Initially cultivars were divided into five major groups, which were subdivided into 13 classes based on differences in terpenoid profile. Different classification models were compared with PLS-DA and found to perform best when many representative samples of a particular class were included. Conclusion: A hierarchy of terpenoid chemotypes was observed in the data set. Some cultivars fit into distinct chemotypes, whereas others seemed to represent a continuum of chemotypes. This study has demonstrated an approach to classifying cannabis cultivars based on terpenoid profile.

Not Cannabis specific
Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases.
Greenhagen, B. T., O’Maille, P. E., Noel, J. P., & Chappell, J.
Proceedings of the National Academy of Sciences, 103(26), 9826–9831. (2006).
doi:10.1073/pnas.0601605103
Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.

Impact of four hemp (Cannabis sativa L.) varieties and stage of plant growth on yield and composition of essential oils.
Abdollahi, M., Sefidkon, F., Calagari, M., Mousavi, A., & Mahomoodally, M. F.
Industrial Crops and Products, 155, 112793. (2020). doi:10.1016/j.indcrop.2020.112793
Essential oil of Cannabis sativa L. is a valuable bio-product due to its versatility, particularly in terms of its commercial values and potential applications in medicine, cosmetics and bio-pesticide. In this study, the effect of different stages of plant growth on essential oil yield and composition of four hemp varieties, (two monoecious non-native (Fedora 17 and its progeny) and two dioecious native (Fars and Yazd) samples) were investigated. The plant materials, consist of foliage in vegetative stage, inflorescent of flower in flowering stage and in- florescent of seeds in seeding stage were subjected to hydro-distillation. The essential oils were analyzed by GC and GC/MS. The oil yields varied from 0.40 % (Fedora17) to 0.65 % (Yazd). Interaction of cultivar and growth stage showed Fed17?2 at vegetative (0.86 %) and Fed17 at flowering stage (0.20 %), had the most and least oil content, respectively. Twenty-nine compounds were identified representing 81.9%–99.5% of the essential oils. The most abundant sesquiterpenes in the oils were E-caryophyllene (16.40 %–44.70 %), ?-humulene (4.1 %–15.1 %) and Z-caryophyllene (2.4 %–10.7 %), while the major monoterpenes were (0.4–24.9 %), ?-pinene (4.6–24.3 %) and 1,8-cineole (0.8 %–9.3 %) in all growth stages and cultivars. The ratio of sesquiterpenes to monoterpenes were found to decrease during the developing plants. In conclusion, there was no significant difference between mean oil yields of native and non-native samples, but non-native samples produced the highest oil yield in vegetative stage. E-caryophyllene was found at the highest percentage in the oils of nonnative samples at vegetative stage. For all samples, the essential oils at vegetative stage contained much lower production of monoterpenes than flowering stage. In addition, to obtain the highest amount of ?-pinene and 1,8- cineole, the flowering and seeding stages of hemp are recommended.

Impact of Supercritical Fluid Extraction and Traditional Distillation on the Isolation of Aromatic Compounds from Cannabis indica and Cannabis sativa.
Naz, S., Hanif, M. A., Bhatti, H. N., & Ansari, T. M.
Journal of Essential Oil Bearing Plants, 20(1), 175–184.(2017).
doi:10.1080/0972060x.2017.1281766
The chemical composition of essential oils hydrodistilled (HD), steam distilled (SD) and supercritical fluid extracted (SCFE) from the leaves of Cannabis sativa and Cannabis indica from Pakistan were being reported. Maximum yield of essential oil for both strains was obtained at 110°C, 130°C and 45°C for hydrodistillation, steam distillation and supercritical fluid extraction respectively. Yields of essential oil using SCFE technique were more than HD and SD extraction respectively. The main compounds of C. sativa essential oil were characterized by large amounts of caryophyllene (40.6-50.0 %), humulene (9.51-16.0 %), trans-α- bergamotene (4.42-6.31 %) cis-β-farnesene (8.63-9.01 %) and δ-limonene (5.13-8.19 %) respectively. The main components of Cannabis indica were caryophyllene (21.1-25.1 %), carophyllene oxide (4.13-5.02 %), linalool (20.8-22.1 %), trans-α-bergamotene (3.23-5.16 %) and cis-β-farnesene (2.10-3.68 %), menthol (7.20-9.43 %), δ- limonene (6.13-7.19 %), eucalyptol (9.67-12.10 %), and carvone (2.11-5.13 %) respectively.

Improved Profiling of Cannabis Terpenes for Accurate Product Labelling
Laura McGregor and Elinor Hughes
The Column 6 August 2020 Vol 16 Issue 8 Pg 11-16
https://www.sepsolve.com/uploads/bro...20Ad%20Rem.pdf
There are three main sub-species of cannabis—indica, sativa, and ruderalis— but there are hundreds of commercial strains based on these sub-species and their hybrids. Profiling the terpene content in these strains is vital to provide accurate labelling of cannabis-based products, but it can be very challenging. The usual technique for this—one-dimensional gas chromatography (GC)— is not always reliable when it comes to separating the diverse classes of terpenes. This article illustrates how two-dimensional GC (GC ×GC) coupled with mass spectrometry (MS) can be used to profile cannabis terpenes with enhanced separation, resulting in the confident identification of terpenes and improved flavour interpretation.

In Defense of the “Entourage Effect”: Terpenes Found in Cannabis sativa Activate the Cannabinoid Receptor 1 In Vitro
J. LaVigne, Ryan Hecksel, J. Streicher
Published 2020 Biology The FASEB Journal
DOI:10.1096/fasebj.2020.34.s1.04020
Marijuana has been understudied for decades, primarily due to social stigma and legal restrictions. However, as legal restrictions begin to loosen among states, the potential medical benefits and pharmacological properties of marijuana are beginning to be explored. Terpenes, an expansive group of organic chemicals that impart odor and taste, are found in the Cannabis sativa plant and may work synergistically with cannabinoids, such as THC and CBD, in a term deemed the “entourage effect”. Anecdotally among the recreational and medical use community, terpenes have been reported to enhance the potency and physiological effects of marijuana. However, scientific evidence for the “entourage effect” is very limited. To evaluate this hypothesis, we obtained the C. sativa‐relevant terpenes: β‐pinene, α‐humulene, geraniol, and linalool. Utilizing Chinese hamster ovary cells (CHO) expressing the human cannabinoid receptor type 1 (CB1, CB1‐CHO) we screened these terpenes for CB1‐dependent phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2), a well‐known downstream target of CB1 activation, using Western blot. We observed that amp levels were efficaciously stimulated by all four terpenes when compared to positive control, the selective CB1 agonist WIN 55,212‐2. These results appeared to be CB1‐dependent, as pre‐treatment of these cells with a selective CB1 antagonist, rimonabant (SRI141716), blocked ERK phosphorylation by each of the terpenes. We further verified the CB1‐dependent nature of these effects by examining ERK phosphorylation by the terpenes in wild type CHO (WT CHO) cells, which do not express the CB1 receptor. In these cells, β‐pinene and α‐humulene treatment resulted in ERK phosphorylation while linalool or geraniol treatment did not. In WT CHO cells, the ERK phosphorylation induced by β‐pinene and α‐humulene was not CB1‐dependent, as rimonabant pre‐treatment did not block it. These results thus suggest that geraniol and linalool could be CB1‐selective agonists, whereas β‐pinene and α‐humulene are non‐selective and may also activate one or more receptors besides CB1. Follow‐up studies will examine other measures of CB1 activity (binding, amp signaling, GTPgS coupling) to characterize the binding and functional properties of these terpenes at the CB1 receptor, as well as identifying the other targets of β‐pinene and α‐humulene. Once we’ve characterized these terpenes individually, we aim to investigate their role in the “entourage effect”, by testing their modulation of typical cannabinoid (THC, etc.) pharmacology. Translationally, these findings could have implications in marijuana cultivar breeding and could help produce strains optimized for specific terpene profiles, which could be more efficacious for chronic pain management and other therapeutic uses.
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In silico discovery of terpenoid metabolism in Cannabis sativa
Luca Massimino
06 Feb 2017, F1000Res 6:107
doi: 10.12688/f1000research.10778.1
https://www.ncbi.nlm.nih.gov/pmc/art...ch-6-11622.pdf
Due to their efficacy, cannabis based therapies are currently being prescribed for the treatment of many different medical conditions. Interestingly, treatments based on the use of cannabis flowers or their derivatives have been shown to be very effective, while therapies based on drugs containing THC alone lack therapeutic value and lead to increased side effects, likely resulting from the absence of other pivotal entourage compounds found in the Phyto-complex. Among these compounds are terpenoids, which are not produced exclusively by cannabis plants, so other plant species must share many of the enzymes involved in their metabolism. In the present work, 23,630 transcripts from the canSat3 reference transcriptome were scanned for evolutionarily conserved protein domains and annotated in accordance with their predicted molecular functions. A total of 215 evolutionarily conserved genes encoding enzymes presumably involved in terpenoid metabolism are described, together with their expression profiles in different cannabis plant tissues at different developmental stages. The resource presented here will aid future investigations on terpenoid metabolism in Cannabis sativa.

Not Cannabis specific:
Large-Scale Evolutionary Analysis of Genes and Supergene Clusters from Terpenoid Modular Pathways Provides Insights into Metabolic Diversification in Flowering Plants
Johannes A. Hofberger, Aldana M. Ramirez, Erik van den Bergh, Xinguang Zhu, Harro J. Bouwmeester, Robert C. Schuurink, M. Eric Schranz
doi:10.1371/journal.pone.0128808
An important component of plant evolution is the plethora of pathways producing more than 200,000 biochemically diverse specialized metabolites with pharmacological, nutritional and ecological significance. To unravel dynamics underlying metabolic diversification, it is critical to determine lineage-specific gene family expansion in a phylogenomics framework. However, robust functional annotation is often only available for core enzymes catalyzing committed reaction steps within few model systems. In a genome informatics approach, we extracted information from early-draft gene-space assemblies and non-redundant transcriptomes to identify protein families involved in isoprenoid biosynthesis. Isoprenoids comprise terpenoids with various roles in plant-environment interaction, such as pollinator attraction or pathogen defense. Combining lines of evidence provided by synteny, sequence homology and Hidden-Markov-Modelling, we screened 17 genomes including 12 major crops and found evidence for 1,904 proteins associated with terpenoid biosynthesis. Our terpenoid genes set contains evidence for 840 core terpene-synthases and 338 triterpene-specific synthases. We further identified 190 prenyltransferases, 39 isopentenyl-diphosphate isomerases as well as 278 and 219 proteins involved in mevalonate and methylerithrol pathways, respectively. Assessing the impact of gene and genome duplication to lineage-specific terpenoid pathway expansion, we illustrated key events underlying terpenoid metabolic diversification within 250 million years of flowering plant radiation. By quantifying Angiosperm-wide versatility and phylogenetic relationships of pleiotropic gene families in terpenoid modular pathways, our analysis offers significant insight into evolutionary dynamics underlying diversification of plant secondary metabolism. Furthermore, our data provide a blueprint for future efforts to identify and more rapidly clone terpenoid biosynthetic genes from any plant species.

Leaf enclosure measurements for determining volatile organic compound emission capacity from Cannabis spp.
Chi-Tsan Wang, Christine Wiedinmyer, Kirsti Ashworth, William Vizuete Project: Potential Air Quality Impacts of Marijuana Cultivation Facilities in Denver, Colorado October 2018
DOI: 10.1016/j.atmosenv.2018.10.049
The legal commercialization of Cannabis for recreational and medical use in certain US states has effectively created a new and nearly unregulated cultivation industry. Within the city limits of Denver, Colorado, there are now more than 600 registered Cannabis spp. cultivation facilities (CCFs) for recreational and medical uses, each containing thousands of plants. Ambient measurements collected inside growing operations pre-legalization have found concentrations as high as 50–100 ppbv of terpenes; a group of highly reactive biogenic volatile organic compounds (BVOCs) and known precursors for the formation of ozone and particulate matter (PM). Due to its illicit nature there has been insufficient experimental data produced to determine Cannabis spp. emission rates. This study used, for the first time, an enclosure chamber and live Cannabis spp. plants during a 90-day growing period consisting of four different strains of Cannabis spp.: Critical Mass, Lemon Wheel, Elephant Purple, and Rockstar Kush. These measurements enabled characterization of terpenes and estimates of emission capacity (EC, ?gC g?¹ hr?¹) at standard conditions. During peak growth, the percentages of individual BVOC emissions were dominated by ?-myrcene (18–60%), eucalyptol (17–38%), and d-limonene (3–10%) for all strains. Our results showed large variability in the rate and composition of terpene emissions across different strains. For the Critical Mass and Lemon Wheel, the dominant terpenoid was eucalyptol (32% and 38%), and it was ?-myrcene (60% and 45%) for the Elephant Purple and Rockstar Kush. Critical Mass produced the highest terpene emission capacity (8.7 ?gC g?¹ hr?¹) and Rockstar Kush the lowest (4.9 ?gC g?¹ hr?¹). With 600 CCFs in Denver, and assuming 10,000 plants per CCF, an emission capacity of 8.7 ?gC g?¹ hr?¹ would more than double the existing rate of BVOC emissions to 520 metric ton year?¹. Using Maximum Incremental Reactivity (MIR) values the total ozone formation potential from all these emitted species could produce 2100 metric tons year?¹ of ozone, and based on published secondary organic aerosols yields 131 metric tons year?¹ of PM. It is likely that the ECs calculated here are lower than those achieved in CCFs where growing conditions are optimized for rapid growth and higher biomass yields. Further studies including a greater number of the 620 available Cannabis spp. strains and a wider range of treatments are needed to generate a representative dataset. Such a dataset could then better enable assessments of the potential impacts of this new industry on indoor and regional air quality.

Medicinal properties of terpenes found in Cannabis sativa and Humulus lupulus
Tarmo Nuutinen
European Journal of Medicinal Chemistry August 2018
DOI: 10.1016/j.ejmech.2018.07.076
Cannabaceae plants Cannabis sativa L. and Humulus lupulus L. are rich in terpenes-both are typically comprised of terpenes as up to 3-5% of the dry-mass of the female inflorescence. Terpenes of cannabis and hops are typically simple mono-and sesquiterpenes derived from two and three isoprene units, respectively. Some terpenes are relatively well known for their potential in biomedicine and have been used in traditional medicine for centuries, while others are yet to be studied in detail. The current, comprehensive review presents terpenes found in cannabis and hops. Terpenes' medicinal properties are supported by numerous in vitro, animal and clinical trials and show anti-inflammatory, antioxidant, analgesic, anticonvulsive, antidepressant, anxiolytic, anticancer, antitumor, neuroprotective, anti-mutagenic, anti-allergic, antibiotic and anti-diabetic attributes, among others. Because of the very low toxicity, these terpenes are already widely used as food additives and in cosmetic products. Thus, they have been proven safe and well-tolerated.

Metabolic Products of Linalool and Modulation of GABAA Receptors
Sinem Milanos, Shaimaa A. Elsharif, Dieter Janzen, Andrea Buettner and Carmen Villmann
Front. Chem., 21 June 2017
DOI: 10.3389/fchem.2017.00046
https://www.frontiersin.org/articles...017.00046/full
Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABAA receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC10−30 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.

Metabolism of β-myrcene in vivo and in vitro: its effects on rat-liver microsomal enzymes
K. Madhava Madyastha & V. Srivatsan
XENOBIOTICA, 1987, VOL. 17, NO. 5, 539-549 DOI: 10.3109/00498258709043961
1. Metabolites isolated from the urine of rats after oral administration of /I-myrcene (I) were: 10-hydroxylinalool (11), 7-methyl-3-methylene-oct-6-ene-1,2-diol (IV), 1 - hydroxymethyl-4-isopropenyl cyclohexanol (VI), 10-carboxylinalool (I 11) and 2-hpdroxy7-methyl-3-methylene-oct-6-enoic acid (V).
2. Liver microsomes prepared from phenobarbital-treated rats convert β-myrcene (I) to 10-hydroxylinalool (II) in the presence of NADPH and oxygen. NADH neither supported this reaction nor did it show any synergistic effect. The rate of conversion was significantly greater in microsomes prepared from phenobarbital-treated rats than from 3-methylcholanthrene-treated or control microsomal preparations. The formation of 10-hydroxylinalool (II) was inhibited by metyrapone, carbon monoxide, SKF-525A, p-chloromercuric benzoate (p-CMB) and cytochrome c.
3. Titration of phenobarbital-induced liver microsomes with β-myrcene (I) produced a series of type I difference spectra with peaks around 387–390 nm and troughs around 421–425 nm. The Ks for β-myrcene was 10.6 μM.
4. Administration (four days) of β-myrcene (I) to rats did not result in any significant effect on the hepatic drug-metabolizing enzymes.

Metabolomics and bioanalysis of terpenoid derived secondary metabolites: Analysis of Cannabis sativa L. metabolite production and prenylases for cannabinoid production.
Muntendam, R. (2015). PHD Thesis
https://www.rug.nl/research/portal/f...ete_thesis.pdf
The research described within this thesis is focused on the analysis of cannabinoid production patterns exhibited by selected strains from the medicinal Cannabis cultivator Bedrocan (Bedrocan B.V., Veendam). Furthermore, the enzymatic production of cannabigerolic acid is explored by analyzing the catalytic mechanism of a prenyltransferase. At the end the putative terpene based production host Xanthophyllomyces dendrorhous is tested for metabolic engineering purposes by the introduction of a terpenoid cyclase to serve as proof of principle and to obtain molecular strategies to genetically alter the host for non-native compound production, such as cannabinoids

Modelling of a limonene/pinene synthase and product specifity of the two monoterpene synthases of Cannabis sativa L
Brandt W, Weber R, Gu?nnewich N, Bräuer L, Schulze D, Rausch F, Page JE,
Schmidt J, Kutchan TM, Wessjohann LA. (2008)
Find DOI or Link I asked Dr Gu?nnewich where to find it.

MONO- AND SESQUI-TERPENE HYDROCARBONS OF THE ESSENTIAL OIL OF CANNABIS SATIVA
HENK HENDRIKS, THEO M. MALINGR, SIEB BATTERMAN, REIN BOS
Phytochemistry, 1975, Vol. 14. Pp 814-815
FIND DOI or Link
The presence of cannabinoids in the essential oil of Cannabis sativu L. has been reported previously In this communication the results of the investigation into the mono- and sesqui-terpenc hydrocarbon fraction are presented. together with previous work (Table I).

Monocyclic and bicyclic monoterpenes in air of German daycare centers and human biomonitoring in visiting children, the LUPE 3 study Lukas Schmidt, Thomas Lahrz, Martin Kraft, Thomas Göen, Hermann Fromme
Environment International 83 (2015) 86–9
DOI: 10.1016/j.envint.2015.06.004
To investigate the assumed association between indoor air pollution with monoterpenes (MTps) and the internal MTp exposure of occupants, a comparative study was performed in daycare centers in two federal states of Germany. Three well-known monoterpenoid air pollutants, viz. α-pinene (αPN), Δ3 -carene (CRN), and R-limonene (LMN), were measured in indoor air in 45 daycare centers. Additionally, urine samples of 222 children visiting these facilities were collected in the evening after a full-day stay. Altogether 11 MTp metabolites were analyzed in the urine samples using a novel highly sensitive and selective gas chromatographic–tandemmass spectrometric procedure. The medians (95th percentiles) of the MTp levels in indoor air were 9.1 μg m−3 (94 μg m−3 ) for LMN, 2.6 μg m−3 (13 μg m−3 ) for αPN, and b1.0 μg m−3 (3.2 μg m−3 ) for CRN. None of the day care centers exceeded the German health precaution or hazard guide value. In spite of the low MTp air exposure, the urine analyses revealed an exposure to the three monoterpenes in almost all children. The median levels of MTp metabolites in urine were 0.11 mg L−1 for LMN-8,9-OH, 0.10 mg L−1 for LMN-1,2- OH, 49 μg L−1 for PA, 2.9 μg L−1 for POH, 5.2 μg L−1 for tCAR, and 4.1 μg L−1 for cCAR (LMN metabolites), 7.2 μg L−1 for MYR, 19 μg L−1 for tVER, and 19 μg L−1 for cVER (αPN metabolites), as well as 8.2 μg L−1 for CRN-10-COOH (CRN metabolite). Statistically significant and strong correlations among the urinary metabolites of each MTp were found. Moreover, statistical associations between LMN metabolites and the LMN indoor air levels were revealed. However, the weakness of the associations indicates a considerable impact of other MTp sources, e.g. diet and consumer products, on the internal exposure

Not Cannabis Specific
Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.
Degenhardt, J., Köllner, T. G., & Gershenzon,
J. Phytochemistry, 70(15-16), 1621–1637. (2009).
doi:10.1016/j.phytochem.2009.07.030
The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation

Not Cannabis specific
More is better: the diversity of terpene metabolism in plants.
Zhou, F., & Pichersky, E.
Current Opinion in Plant Biology, 55, 1–10.(2020).
doi:10.1016/j.pbi.2020.01.005
All plants synthesize a diverse array of terpenoid metabolites. Some are common to all, but many are synthesized only in specific taxa and presumably evolved as adaptations to specific ecological conditions. While the basic terpenoid biosynthetic pathways are common in all plants, recent discoveries have revealed many variations in the way plants synthesized specific terpenes. A major theme is the much greater number of substrates that can be used by enzymes belonging to the terpene synthase (TPS) family. Other recent discoveries include non-TPS enzymes that catalyze the formation of terpenes, and novel transport mechanisms.

[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]More Than Just Taste and Smell: Examining the Complexity and Diversity of Terpene Profiles from Popular Cannabis Strains
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]Andrew Defries [/FONT]
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]https://thecannabinoidchronicles.com/author/andrew/[/FONT]
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]https://terpenecharts.com/author/andrew/[/FONT]
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]https://cannabinoidcharts.com/author/andrew/[/FONT][/FONT]
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]Poster · April 2018
[FONT=PÊˇø◊îúY¿¥*†°∂‡XËÊˇø0IπY¥ü]https://www.researchgate.net/publication/325270252_More_Than_Just_Taste _and_Smell_Examining_the_Compl exity_and_Diversity_of_Terpene _Profiles_from_Popular_Cannabi s_Strains?enrichId=rgreq-39ad91745ca344fd0905646f59c829 f3-XXX&enrichSource=Y292ZXJQYWdlO zMyNTI3MDI1MjtBUzo2OTA5OTMxNzk 0MDIyNDdAMTU0MTc1NzAyMDk1Mw%3D %3D&el=1_x_3&_esc=publicationC overPdf[/FONT] [/FONT]
Cannabis synthesizes a diversity of secondary metabolites. Selection and breeding has resulted in strains with enhanced cannabinoid potency and unique flavor profiles called chemotypes. Volatile organic compounds known as terpenes responsible for the smell and taste of cannabis are becoming recognized as medicinal molecules in their own right. Our goal is to create a botanical classification system to delineate chemotype groups and subgroups in popular strains using terpene profiles. When used in conjunction with genetic data we aim to determine the lineage of unknown strains and to guide future breeding projects to develop specific and unique chemotypes.



Multidimensional analysis of cannabis volatile constituents: Identification of 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane as a volatile marker of hashish, the resin of Cannabis sativa L.
Marie Marchinia, Céline Charvozb, Laurence Dujourdyb, Nicolas Baldovinia, Jean-Jacques Filippia, ,
Journal of Chromatography Volume 1370, 28 November 2014, Pages 200–215
Doi: 10.1016/j.chroma.2014.10.045
•Analysis of cannabis herb and hashish volatile constituents by HS-SPME-GC × GC–MS.
•Identification of a new volatile marker of hashish.
•Photolytic rearrangement of ?-myrcene into hashishene.
•Formation of photo-oxidation products during hashish manufacture.
The volatile constituents of drug samples derived from Cannabis sativa L. were investigated by means of headspace solid phase microextraction (HS-SPME) and gas chromatography techniques (GC–MS, GC × GC–MS). Samples of cannabis herb andhashish showed clear differences in their volatile chemical profiles, mostly resulting from photo-oxidation processes occurring during the transformation of fresh cannabis herb into hashish. Most unexpectedly, we could demonstrate hashish samples as containing remarkable amounts of a rare and unusual monoterpene – 5,5-dimethyl-1-vinylbicyclo[2.1.1]hexane – among the volatile compounds detected in their headspaces. We gave evidence for the formation of this compound from the light induced rearrangement of ?-myrcene during the manufacture of hashish. In view of its high abundance among volatile constituents of cannabis resin and its scarce occurrence in other natural volatile extracts, we propose to rename this specific monoterpene hashishene.

Natural Borneol, a Monoterpenoid Compound, Potentiates Selenocystine-Induced Apoptosis in Human Hepatocellular Carcinoma Cells by Enhancement of Cellular Uptake and Activation of ROS-Mediated DNA Damage.
Su, J., Lai, H., Chen, J., Li, L., Wong, Y.-S., Chen, T., & Li, X. (2013).
PLoS ONE, 8(5), e63502.
doi:10.1371/journal.pone.0063502
Selenocystine (SeC) has been identified as a novel compound with broad-spectrum anticancer activities. Natural borneol (NB) is a monoterpenoid compound that has been used as a promoter of drug absorption. In the present study, we demonstrated that NB significantly enhanced the cellular uptake of SeC and potentiated its antiproliferative activity on HepG2 cells by induction of apoptosis. NB effectively synergized with SeC to reduce cancer cell growth through the triggering apoptotic cell death. Further mechanistic studies by Western blotting showed that treatment of the cells with NB and SeC activated the intrinsic apoptotic pathway by regulation of pro-survival and pro-apoptotic Bcl-2 family proteins. Treatment of the cells with NB and SeC induced the activation of p38MAPK and inactivation of Akt and ERK. NB also potentiated SeC to trigger intracellular ROS generation and DNA strand breaks as examined by Comet assay. Moreover, the thiol-reducing antioxidants effectively blocked the occurrence of cell apoptosis, which confirmed the important role of ROS in cell apoptosis. Taken together, these results reveal that NB strongly potentiates SeC-induced apoptosis in cancer cells by enhancement of cellular uptake and activation of ROS-mediated DNA damage. NB could be further developed as a chemosensitizer of SeC in treatment of human cancers.

Not Cannabis specific
Natural Terpenes Prevent Mitochondrial Dysfunction, Oxidative Stress and Release of Apoptotic Proteins during Nimesulide-Hepatotoxicity in Rats
Brijesh Kumar Singh1, Madhulika Tripathi, Bhushan P. Chaudhari, Pramod K. Pandey, Poonam Kakkar
PLoS ONE 7(4): e34200.
doi:10.1371/journal.pone.0034200
Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P,0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P,0.05). A combination of camphene and geraniol (CG; 1:1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a combination of natural terpenes prevented nimesulide induced cellular damage and ensuing hepatotoxicity

Odor Scrubbing in a Growhouse: Ways to Remove Odor from Cannabis Terpenes
Antonio DeRose
Terpenes And Testing Magazine
https://terpenesandtesting.com/odor-...abis-terpenes/
The smell of cannabis comes from its terpene profile. Terpenes have very distinct and often strong odors, and the collective odor of a given plant is one way many consumers choose which types of flower they want to consume. The strong aromas from terpenes may be well-liked by many consumers, but odor is an ongoing concern for cannabis grow facilities that must follow local city and state regulations regarding odor output. For this reason, many facilities utilize different odor scrubbing techniques to reduce and eliminate the strong odors of the cannabis being cultivated.

Opioids Out, Cannabis In Negotiating the Unknowns in Patient Care for Chronic Pain
JAMA November 1, 2016 Volume 316, Number 17 Pg 1763-4
DOI: 10.1001/jama.2016.13677
With thecurrent nationwide epidemicof opioidabuse, dependence, and fatalities, clinicians are being asked by federal agencies and professional societies to control their prescribing of narcotic medications for pain. Federal guidelines emphasize tapering, discontinuing, and limiting initiation of these drugs except in provision of end of-life care.1 Reducing reliance on opioids, however, is a
massive task. According to one estimate, more than 650 000 opioid prescriptions are dispensed each day in the United States. Unless the nation develops an increased tolerance to chronic pain, reduction in opioid prescribing leaves a vacuum that will be filled with other therapies

para-Menthane as a Stable Terpene Derived from Orange By-Products as a Novel Solvent for Green Extraction and Solubilization of Natural Substances.
Madji, Hilali, Fabiano-Tixier, Tenon, Bily, Laguerre, & Chemat.
Molecules, 24(11), 2170.(2019).
doi:10.3390/molecules24112170
This study aims at investigating p-menthane, a novel bio-based solvent resulting from the hydrogenation of d-limonene, as a green alternative to n-hexane or toluene for the extraction and solubilization of natural substances. First, conductor-like combination of quantum chemistry (COSMO) coupled with statistical thermodynamics (RS) calculations show a comparable solubilization profile of p-menthane and n-hexane for carotene, volatile monoterpenes such as carvone and limonene, and model triglycerides. Other data obtained experimentally in solid/liquid extraction conditions further indicate that p-menthane showed similar performances to n-hexane for extracting carotenes from carrots, aromas from caraway seeds, and oils from rapeseeds, as these products showed a comparable composition. p-Menthane was also tested using common analytical extraction procedures such as Soxhlet for determination of oil content via multiple extraction stages, and Dean–Stark for determination of water content via azeotropic distillation. For both systems, yields were comparable, but for Dean–Stark, the distillation curve slope was higher when using p-menthane, and the time needed to attain 100% water recovery was 55% shorter than for toluene. Taken together, these results reveal the potential of p-menthane as a green replacer for petroleum-based solvents such as n-hexane or toluene.

Phytol, not propylene glycol, causes severe pulmonary injury after inhalation dosing in Sprague-Dawley rats
Daniela Schwotzer, Andrew Gigliotti, Hammad Irshad, Wendy Dye & Jacob McDonald
Inhalation Toxicology 2021, VOL. 33, NO. 1, 33–40
DOI: 10.1080/08958378.2020.1867260
Introduction: The use of vaping pens for inhalation of cannabinoid derived products is rising and has become a popular alternative to smoking combustible products. For efficient product delivery, additives are sometimes added and vaping pens often may include compounds like Phytol or Propylene
Glycol as thinning agents. This study aimed at comparing Phytol and Propylene Glycol with respect to potential toxicity and safe use in vaping products.
Methods: Male and female Sprague Dawley rats were exposed to 5mg/L of Phytol or Propylene Glycol for up to 6 hours over up to 14 days and monitored for clinical signs and changes in body weight. Gross necropsy and histopathology of respiratory tissue was performed to assess potential adverse effects.
Results: Phytol exposed animals expressed severe clinical signs, body weight loss and mortality after one or two exposure days, leading to termination of all dose groups for this compound. Lung weights were increased and respiratory tissue was severely affected, demonstrating dose-responsive tissue degeneration, necrosis, edema, hemorrhage and inflammation. Propylene Glycol exposed animals did not show any adverse reactions after 14 days of high dose exposure.
Conclusions: For Phytol, a low observed adverse effect level (LOAEL) was determined at _109.0/ 10.9 mg/kg/day presented/deposited dose and therefore its use as excipient in vaping product is not recommend; a safe exposure range was not established for Phytol. Propylene Glycol, in contrast, is considered safe with a no observed adverse effect level (NOAEL) at 1151.7/115.2mg/kg/day presented/ deposited dose in rats.

Not Cannabis specific
Plant terpenoid synthases: Molecular biology and phylogenetic analysis
JO¨RG BOHLMANN, GILBERT MEYER-GAUEN AND RODNEY CROTEAU
Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4126–4133, April 1998
doi: 10.1073/pnas.95.8.4126
https://www.pnas.org/content/pnas/95/8/4126.full.pdf
This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C10, C15, and C20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the Tps gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes.

Plucking Terpenes from Plants
Jason S. Lupoi
https://extractionmagazine.com/2020/...zHZA8S0Yq2A_wQ
Three ways for extracting fragrant botanical essences
Steam Distillation
Subcritical CO2
Hexane

Potentiating effect of ?-caryophyllene on anticancer activity of ?-humulene, isocaryophyllene and paclitaxel.
Legault, J., & Pichette, A.
Journal of Pharmacy and Pharmacology, 59(12), 1643–1647 (2007)
doi:10.1211/jpp.59.12.0005
b-caryophyllene is a sesquiterpene widely distributed in essential oils of various plants. Several biological activities are attributed to b-caryophyllene, such as anti-inflammatory, antibiotic, antioxidant, anticarcinogenic and local anaesthetic activities. In this work, the potentiating effect of bcaryophyllene on the anticancer activity of a-humulene, isocaryophyllene and paclitaxel against MCF-7, DLD-1 and L-929 human tumour cell lines was evaluated. A non-cytotoxic concentration of bcaryophyllene significantly increased the anticancer activity of a-humulene and isocaryophyllene on MCF-7 cells: a-humulene or isocaryophyllene alone (32 mg mL?1) inhibited cell growth by about 50% and 69%, respectively, compared with 75% and 90% when combined with 10 mg mL?1 b-caryophyllene. Moreover, b-caryophyllene potentiated the anticancer activity of paclitaxel on MCF-7, DLD-1 and L-929 cell lines. The highest potentiating effect was obtained in DLD-1 cells treated with paclitaxel combined with 10 mg mL?1 b-caryophyllene, which increased the paclitaxel activity about 10-fold. The intracellular accumulation of paclitaxel-oregon green was evaluated in combination with concentrations of b-caryophyllene ranging from 2.5 to 40 mg mL?1. b-Caryophyllene (10 mg mL?1) significantly increased the intracellular accumulation of paclitaxel-oregon green (about 64% over controls). Moreover, b-caryophyllene induced intracellular accumulation of calcein but not verapamil, an inhibitor of P-glycoprotein and multidrug resistance related protein transporters, suggesting that b-caryophyllene promotes drug accumulation by a different mechanism of action. These results
suggest that b-caryophyllene facilitates the passage of paclitaxel through the membrane and thus potentiates its anticancer activity.

Predicting chemovar cluster and variety verification in vegetative cannabis accessions using targeted single nucleotide polymorphisms
Philippe Henry, Aaron Hilyard , Steve Johnson , Cindy Orser
PeerJ Preprints (2018)
DOI: 10.7287/peerj.preprints.27442v1
The cannabis industry has gained momentum and global acceptance recently, culminating in the legalization of adult use at the federal level in Canada, a first among G20 countries. Inherent to legalization, a highly regulated regime has emerged, mostly centered on end user safety, restriction of access to youth, and diversion of market shares away from the black market and organized crime. The lack of authentication of cannabis varieties remains as an issue often unaddressed by the regulators, although this has the potential to seriously hamper research and the medical application of cannabis derived products. Here, we extend upon previous work that aims to classify cannabis accessions based on their dominant terpene profiles, focusing on four main informative terpenes, betamyrcene, terpinolene, limonene and beta-caryophyllene. We identify three major terpene groups and present a simple genetic-based tool to bridge the variety identification gap and to enable the prediction of terpenoid expression in vegetative cannabis. This genetic tool offers promise to sorting out the strain name game that has been ongoing, thus providing greater transparency in the industry and contributing to an enhanced understanding of cannabis medicine for the end user

Not Cannabis related
Prevalence of cilantro (Coriandrum sativum) disliking among different ethnocultural groups.
Mauer, L., & El-Sohemy, A.
Flavour, 1(1), 8. (2012).
doi:10.1186/2044-7248-1-8
Background: Cilantro, the leaf of the Coriandrum sativum plant, is an herb that is widely consumed globally and has purported health benefits ranging from antibacterial to anticancer activities. Some individuals report an extreme dislike for cilantro, and this may explain the different cilantro consumption habits between populations. However, the prevalence of cilantro dislike has not previously been reported in any population. The objective of this study was to determine the prevalence of cilantro dislike among different ethnocultural groups from a population of young adults living in Canada. Subjects (n = 1,639) between the ages of 20 and 29 years were participants of the Toronto Nutrigenomics and Health Study. Individuals rated their preference for cilantro on a 9-point scale from ‘dislike extremely’ to ‘like extremely’. Subjects also had the option to select ‘have not tried’ or ‘would not try’. Subjects who selected 1 to 4 were classified as disliking cilantro. Results: The prevalence of dislike ranged from 3 to 21%. The proportion of subjects classified as disliking cilantro was 21% for East Asians, 17% for Caucasians, 14% for those of African descent, 7% for South Asians, 4% for Hispanics, and 3% for Middle Eastern subjects. Conclusions: These findings show that the prevalence of cilantro dislike differs widely between various ethnocultural groups.

Quantitative Determination of Terpenes in Cannabis Using Headspace Solid Phase Microextraction and GC/MS
Michael Halpenny, Katherine K. Stenerson
GERSTEL Application Note No. 189 , 2017
https://www.gerstel.com/pdf/AppNote-189.pdf
Well known for their characteristic fl avor and fragrance characteristics, terpenes are contained in the derived essential oils of cannabis. Analysis of cannabis for terpene concentrations can be applied to strain identifi cation, referred to as fi ngerprinting, and for concentration accuracy when applied to medicinal treatments. Terpenes have high vapor pressures, are extremely volatile and thus are an excellent candidate for static headspace GC analysis. In this work, headspace SPME (HS-SPME) was combined with GC/ MS for the quantitative analysis of several selected terpenes in cannabis. The conventional approach for terpene analysis in cannabis involves a solvent extraction followed by GC/FID analysis. HS-SPME offers several advantages over the solvent extraction method in that it is non-destructive to the sample, requires a very small sample size, produces minimal interference from co-extracted matrix, protects the GC instrument from contamination, and can be easily automated

Qualitative terpene profiling of Cannabis varieties cultivated for medical purposes
Ernesto D Rocha, Vitória EA Silva, Fernanda CS Pereira, Valery M Jean, Fabio L Costa Souza, Leopoldo Clemente Baratto, Ana Vieira, Virginia M Carvalho
Rodriguesia 71January 2020
DOI: 10.1590/2175-7860202071040
https://www.researchgate.net/publica...dical_purposes
https://www.scielo.br/j/rod/a/k69ddv...BGqmS/?lang=en
With the upcoming medical Cannabis regulation, quality control methods on raw material will be required. Besides testing for contaminants and potency, there are also pharmaceutical and forensic interests in the determination of the terpene profile in different strains of Cannabis as complementary identification methods. A simple non-destructive HS-SPME GC-MS method was used to identify the terpene content in twelve Cannabis samples, four of them were of the hemp type (Harle-tsu), seven from various marihuana types and one of the intermediate type. They all were previously analyzed by HPLC to determine the potency (THC and CBD content). Spectral library matching was used to identify the terpenes compounds. Thirty terpenes compounds were detected, nine of them were present in all Cannabis samples and used to find their terpene profile: α-pinene, β-pinene, β-myrcene, D-limonene, terpinolene, linalool, caryophyllene, α-bergamotene and humulene. Three of them, caryophyllene, α-pinene and β-myrcene were found as larger components in most of samples. A principal components analyses (PCA) was performed. The four hemp type samples showed two different profiles, two samples showed caryophyllene as main component and the others two with β-myrcene as such. The marihuana type samples showed wider profiles with no clear patterns at all, which is not surprising because of the low number of samples. The simple methodology shows viable to set the terpenes profile for analyses of raw Cannabis material. Suitability for differentiation between different sorts of types needs more studies, with increasing numbers of samples.

Quantitation of Select Terpenes/Terpenoids and Nicotine Using Gas Chromatography−Mass Spectrometry with High-Temperature Headspace Sampling
Trinh-Don Nguyen, Seamus Riordan-Short, Thu-Thuy T. Dang, Rob O’Brien, and Matthew Noestheden
ACS Omega 2020, 5, 5565−5573
DOI:10.1021/acsomega.0c00384
https://www.ncbi.nlm.nih.gov/pmc/art.../ao0c00384.pdf
Plants are the main sources of many high-value bioactive terpenoids used in the medical, fragrance, and food industries. Increasing demand for these bioactive plants and their derivative products (e.g., cannabis and extracts thereof) requires robust approaches to verify feedstock, identify product adulteration, and ensure product safety. Reported here are singlelaboratory validation details for a robust testing method to quantitate select terpenes and terpenoids in dry plant materials and terpenoid-containing vaping liquids (e.g., a derivative product) using high-temperature headspace gas chromatography−mass spectrometry, with glycerol used as a headspace solvent. Validated method recoveries were 75−103%, with excellent repeatability (relative standard deviation (RSD) < 5%) and intermediate precision (RSD < 12%). The use of high-temperature headspace (180 °C) permitted terpene and terpenoid profiles to be monitored at temperatures consistent with vaping conditions.

Quantitation of Terpenes in Cannabis Products Using APCI LC-MS/MS
Katherine C Hyland, C Borton, P Winkler, Matthew Noestheden
Planta Medica 82(05) March 2016
DOI: 10.1055/s-0036-1579770
With the recent legalization of cannabis in several states, there is a growing need for robust, cost-effective analytical methods to facilitate routine testing. While testing of potency (and for pesticide and herbicide residues) is important, manufacturers of cannabis products also need fit-to-purpose analytical methods that provide information on the sensory profile of their products to ensure lot-to-lot consistency. Here, we present an LCMS/MS method that uses atmospheric pressure chemical ionization (APCI) for the analysis of terpenes (major determinant of aroma) in cannabis products. Examples of accurate quantitation are shown for a variety of cannabis products.

R Limonene metabolism in humans and metabolite kinetics after oral administration
Lukas Schmidt · Thomas Göen
Arch Toxicol
DOI 10.1007/s00204-016-1751-6
We studied the R-limonene (LMN) metabolism and elimination kinetics in a human in vivo study. Four volunteers were orally exposed to a single LMN dose of 100–130 µg kg−1 bw. In each case, one pre-exposure and subsequently all 24 h post-exposure urine samples were collected. From two subjects, blood samples were drawn up to 5 h after exposure. The parent compound was analysed in blood using headspace GC–MS. The metabolites cis- and trans-carveol (cCAR), perillyl alcohol (POH), perillic acid (PA), limonene-1,2-diol (LMN-1,2-OH), and limonene-8,9-diol (LMN-8,9-OH) were quantified in both blood and urine using GC-PCI-MS/MS. Moreover, GC-PCI-MS full-scan experiments were applied for identification of unknown metabolites in urine. In both matrices, metabolites reached maximum concentrations 1–2 h post-exposure followed by rapid elimination with half-lives of 0.7–2.5 h. In relation to the other metabolites, LMN-1,2-OH was eliminated slowest. Nonetheless, overall renal metabolite elimination was completed within the 24-h observation period. The metabolite amounts excreted via urine corresponded to 0.2 % (cCAR), 0.2 % (tCAR), <0.1 % (POH), 2.0 % (PA), 4.3 % (LMN-1,2-OH), and 32 % (LMN-8,9-OH) of the orally administered dose. GC-PCI-MS full-scan analyses revealed dihydroperillic acid (DHPA) as an additional LMN metabolite. DHPA was estimated to account for 5 % of the orally administered dose. The study revealed that human LMN metabolism proceeds fast and is characterised by oxidation mainly of the

exo-cyclic double bond but also of the endo-cyclic double bond and of the methyl side chain. The study results may support the prediction of the metabolism of other terpenes or comparable chemical structures.

Removal of floral microbiota reduces floral terpene emissions
Josep Penuelas, Gerard Farre-Armengol, Joan Llusia, Albert Gargallo-Garriga, Laura Rico, Jordi Sardans, Jaume Terradas & Iolanda Filella.
DOI: 10.1038/srep06727
The emission of floral terpenes plays a key role in pollination in many plant species. We hypothesized that the floral phyllospheric microbiota could significantly influence these floral terpene emissions because microorganisms also produce and emit terpenes. We tested this hypothesis by analyzing the effect of removing the microbiota from flowers. We fumigated Sambucus nigra L. plants, including their flowers, with a combination of three broad-spectrum antibiotics and measured the floral emissions and tissular concentrations in both antibiotic-fumigated and non-fumigated plants. Floral terpene emissions decreased by ca. two thirds after fumigation. The concentration of terpenes in floral tissues did not decrease, and floral respiration rates did not change, indicating an absence of damage to the floral tissues. The suppression of the phyllospheric microbial communities also changed the composition and proportion of terpenes in the volatile blend. One week after fumigation, the flowers were not emitting b-ocimene, linalool, epoxylinalool, and linalool oxide. These results show a key role of the floral phyllospheric microbiota in the quantity and quality of floral terpene emissions and therefore a possible key role in pollination.

Selective recovery of terpenes, polyphenols and cannabinoids from Cannabis sativa L. inflorescences under microwaves Veronika Gunjević, Giorgio Grillo, Diego Carnaroglio, Arianna Binello, Alessandro Barge, Giancarlo Cravotto
Industrial Crops and Products Volume 162, April 2021 DOI: 10.1016/j.indcrop.2021.113247
In recent years, hemps health and nutritional properties recognition has led to an impressive growth of Cannabis research, industrial processing, and the related market. Moreover, the demand for natural Cannabis-derived compounds (i.e. terpenes, polyphenols, and cannabinoids) is constantly growing. In spite of the strict regulation of some countries, the global market needs suitable technologies for the smart recovery of bioactive Cannabis metabolites. Conventional extraction procedures can show drawbacks, in terms of environmental impact and their high energy consumption. Microwaves (MW), a mature technique for extraction-process intensification, is attracting great amounts of attention in academic-research and industrial-application fields for its technological advantages. This work aims to design a fast and cost-efficient MW-assisted cascade protocol for bioactive Cannabis compounds recovery in a pilot-scale reactor. Microwave-assisted hydrodistillation (MAHD) can provide a volatile hydrodistillate that is rich in monoterpenes, sesquiterpenes, and a small amount of phytocannabinoids. Using non-canonical protocol of hydrodistillation, the definition of “volatile fraction” is generally considered more appropriate than “essential oil”. The health-promoting activity of this combination has been proposed in literature, and can constitute matter of further investigations. The optimized MAHD procedure yielded 0.35 ± 0.02 % w/w of hydrodistillate, while conventional hydrodistillation gave only 0.12 ± 0.01 %, w/w (in relation to dry inflorescence mass). The water resulting in the vessel after MAHD showed a high total polyphenolic content (5.35 ± 0.23 %, w/w). Two flavones known for their beneficial effects to health, namely luteolin-7-O-glucoside and apigenin-7-O-glucoside, were detected and quantified. An attempt to recover phytocannabinoid using the MW-assisted hydrodiffusion and gravity method (MAHG) was also carried out. Cannabinoids (CBD and THC) content was determined in fresh Cannabis and in production streams. During MAHD, phytocannabinoid decarboxylation inside the residual matrix was around 70 % (69.01 ± 0.98 % and 74.32 ± 1.02 % for THC and CBD respectively). Furthermore, the overall content of these metabolites was not affected by the hydrodistillation, preserving the processed plant material for subsequent ethanolic extraction.

Separation of aroma compounds from industrial hemp inflorescences (Cannabis sativa L.) by supercritical CO2 extraction and on-line fractionation
Carla Da Porto, Deborha Decorti, Andrea Natolino
Industrial Crops and Products 58 (2014) 99–103
doi.org/10.1016/j.indcrop.2014.03.042
The use of supercritical carbon dioxide (Sc-CO2) extraction at 10 and 14 MPa and 40?C and on-line frac-tionation using two separators (Sep 1: 7 MPa/25?C; Sep2: 5 MPa/15?C) to recovery volatile compoundsfrom the inflorescences of fiber type Cannabis sativa L. was investigated by HS-SPME/GC–MS and direct GC–MS and compared with hydrodistillation. The best results were obtained by Sc-CO2extraction car-ried out at 10 MPa and 40?C. Under these operating conditions, cuticular waxes covering the surface offlowers were collected in the first separator and volatile compounds (100%) in the second. The superiorquality of this last extract was proved by the perfect overlapping of its HS-SPME/GC–MS volatile profileto that of inflorescences. The recovery of fractions with different composition and biological properties,made the inflorescences of fiber type Cannabis sativa L suitable for cosmetic and/or food industry.

Simplified Cannabis Terpene Profiling by GCMS
SHIMADZU
First Edition: September 2016
https://www.shimadzu.eu.com/sites/sh...ngcannabis.pdf
Terpene and terpenoid compounds are naturally occurring aromatic compounds that give cannabis its unique flavor and fragrance. Aside from their aromatic properties, terpenes have advantageous health benefits. They also have a synergistic relationship with cannabinoids, which further enhance the therapeutic effect of THC. Cannabis has over 140 terpene components, many of which are of medicinal interest. 1 Predominant terpenes in cannabis include β-myrcene, which has antibiotic properties and enhances the THC muscle relaxant effect; α-pinene, which has antiinflammatory properties and enhances the THC bronchodilator effect; and β-caryophyllene, which also has anti-inflammatory properties and enhances the THC gastric cytoprotective effect amongst other health benefits. 2, 3 The concentration of individual terpenes varies by strain, can be anywhere from 0.1 to 1.5% of its total dry weight, and can vary depending on harvest time, drying and storage conditions. 1, 4 Terpene levels can decrease over time, and after three months of storage, can reduce terpene levels by more than half.4 The decrease in terpene amount over time varies for different terpenes.

Susan C, Trapp
http://susanctrapp.com/
Terpene expert

Not Cannabis specific
Stability of Essential Oils: A Review.
Turek, C., & Stintzing, F. C.
Comprehensive Reviews in Food Science and Food Safety, 12(1), 40–53.(2013).
doi:10.1111/1541-4337.12006
In recent years, consumers have developed an ever-increasing interest in natural products as alternatives for artificial additives or pharmacologically relevant agents. Among them, essential oils have gained great popularity in the food, cosmetic, as well as the pharmaceutical industries. Constituting an array of many lipophilic and highly volatile components derived from a great range of different chemical classes, essential oils are known to be susceptible to conversion and degradation reactions. Oxidative and polymerization processes may result in a loss of quality and pharmacological properties. Despite their relevance for consumers, there is a paucity of information available addressing this issue. Therefore, the present review provides a comprehensive summary on possible changes in essential oils and factors affecting their stability. Focusing on individual essential oils, the various paths of degradation upon exposure to extrinsic parameters are outlined. Especially temperature, light, and oxygen availability are recognized to have a crucial impact on essential oil integrity. Finally, analytical methods to assess both genuine as well as altered essential oil profiles are evaluated with respect to their suitability to track chemical alterations. It is believed that only a careful inspection of essential oils by a set of convenient methods allows profound quality assessment that is relevant to producers and consumers alike.

Not Cannabis specific
Structure of limonene synthase, a simple model for terpenoid cyclase catalysis
David C. Hyatt, Buhyun Youn, Yuxin Zhao, Bindu Santhamma, Robert M. Coates, Rodney B. Croteau and ChulHee Kang
PNAS March 27, 2007 _ vol. 104 _ no. 13
DOI: 10.1073_pnas.0700915104
https://www.pnas.org/content/pnas/104/13/5360.full.pdf
The crystal structure of (4S)-limonene synthase from Mentha spicata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-Å resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2- fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate–metal ion coordination and substrate binding conformation in the course of the multistep reaction.

Systems and methodologies for rapid and robust Cannabis Terpene Analysis 5 minute analysis time = more productivity
Ron Honnold, Agilent
Conference: 2017 Cannabis Science Conference At: Portland, OR August 2017
DOI: 10.13140/RG.2.2.34481.89441
Headspace-Gas Chromatography Flame Ionization Detector with Mass Spectrometer methodology enhancements allow full scan acquisition with a 5 minute run time with separation using both FID and MS for quantitation.

Taming THC: potential cannabis synergy and phytocannabinoid terpenoid entourage effects
Ethan B Russo
British Journal of Pharmacology (2011) 163 1344–1364
doi: 10.1111/j.1476-5381.2011.01238.x
Tetrahydrocannabinol (THC) has been the primary focus of cannabis research since 1964, when Raphael Mechoulam isolated and synthesized it. More recently, the synergistic contributions of cannabidiol to cannabis pharmacology and analgesia have been scientifically demonstrated. Other phytocannabinoids, including tetrahydrocannabivarin, cannabigerol and cannabichromene, exert additional effects of therapeutic interest. Innovative conventional plant breeding has yielded cannabis chemotypes expressing high titres of each component for future study. This review will explore another echelon of phytotherapeutic agents, the cannabis terpenoids: limonene, myrcene, ??pinene, linalool, ??caryophyllene, caryophyllene oxide, nerolidol and phytol. Terpenoids share a precursor with phytocannabinoids, and are all flavour and fragrance components common to human diets that have been designated Generally Recognized as Safe by the US Food and Drug Administration and other regulatory agencies. Terpenoids are quite potent, and affect animal and even human behaviour when inhaled from ambient air at serum levels in the single digits ng·mL?1. They display unique therapeutic effects that may contribute meaningfully to the entourage effects of cannabis?based medicinal extracts. Particular focus will be placed on phytocannabinoid?terpenoid interactions that could produce synergy with respect to treatment of pain, inflammation, depression, anxiety, addiction, epilepsy, cancer, fungal and bacterial infections (including methicillin?resistant Staphylococcus aureus). Scientific evidence is presented for non?cannabinoid plant components as putative antidotes to intoxicating effects of THC that could increase its therapeutic index. Methods for investigating entourage effects in future experiments will be proposed. Phytocannabinoid?terpenoid synergy, if proven, increases the likelihood that an extensive pipeline of new therapeutic products is possible from this venerable plant.

Tandem mass spectrometric quantification of 93 terpenoids in Cannabis using static headspace (SHS) injections.
Shapira, A., Berman, P., Futoran, K., Guberman, O., & Meiri, D.
Analytical Chemistry. (2019).
doi:10.1021/acs.analchem.9b02844
The therapeutic effect of Cannabis largely depends on the content of its pharmacologically active secondary metabolites, mainly phytocannabinoids, flavonoids, and terpenoids. Recent studies suggest there are therapeutic effects of specific terpenoids as well as synergistic effects with other active compounds in the plant. Although Cannabis contains an overwhelming milieu of terpenoids, only a limited number are currently reported and used for metabolic analysis of Cannabis chemovars. In this study, we report the development and validation of a method for simultaneous quantification of 93 terpenoids in Cannabis air-dried inflorescences and extracts. This method employs the full evaporation technique via a static headspace sampler, followed by gas chromatography?mass spectrometry (SHS-GC/MS/MS). In the validation process, spiked terpenoids were quantified with acceptable repeatability, reproducibility, sensitivity, and accuracy. Three medical Cannabis chemovars were used to study the effect of sample preparation and extraction methods on terpenoid profiles. This method was further applied for studying the terpenoid profiles of 16 different chemovars acquired at different dates. Our results demonstrate that sample preparation methods may significantly impact the chemical fingerprint compared to the nontreated Cannabis. This emphasizes the importance of performing SHS extraction in order to study the natural terpenoid contents of chemovars. We also concluded that most inflorescences expressed relatively unique terpenoid profiles for the most pronounced terpenoids, even when sampled at different dates, although absolute concentrations may vary due to aging. The suggested method offers an ideal tool for terpenoid profiling of Cannabis and sets the scene for more comprehensive works in the future.

Not Cannabis specific
Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis.
Köksal, M., Jin, Y., Coates, R. M., Croteau, R., & Christianson, D. W.
Nature, 469(7328), 116–120.(2010).
doi:10.1038/nature09628
With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this familyis the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization1 and shows remarkable efficacy in cancer chemotherapy2 . The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) 3 is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene4 , which is catalysed by taxadiene synthase5 . The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids6 . We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 A? resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 A? resolution). The TXS structure reveals a modular assembly of three ahelical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third ‘insertion’ domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis

TERPEDIA The Terpene Encyclopedia
https://terpedia.com/

Not Specific to Cannabis
Terpene biosynthesis in glandular trichomes of hop.
Wang G, Tian L, Aziz N, Broun P, Dai X, He J, King A, Zhao PX, Dixon RA.
Plant Physiol. 2008 Nov;148(3):1254-66.
doi: 10.1104/pp.108.125187.
Hop (Humulus lupulus L. Cannabaceae) is an economically important crop for the brewing industry, where it is used to impart flavor and aroma to beer, and has also drawn attention in recent years due to its potential pharmaceutical applications. Essential oils (mono- and sesquiterpenes), bitter acids (prenylated polyketides), and prenylflavonoids are the primary phytochemical components that account for these traits, and all accumulate at high concentrations in glandular trichomes of hop cones. To understand the molecular basis for terpene accumulation in hop trichomes, a trichome cDNA library was constructed and 9,816 cleansed expressed sequence tag (EST) sequences were obtained from random sequencing of 16,152 cDNA clones. The ESTs were assembled into 3,619 unigenes (1,101 contigs and 2,518 singletons). Putative functions were assigned to the unigenes based on their homology to annotated sequences in the GenBank database. Two mono- and two sesquiterpene synthases identified from the EST collection were expressed in Escherichia coli. Hop MONOTERPENE SYNTHASE2 formed the linear monterpene myrcene from geranyl pyrophosphate, whereas hop SESQUITERPENE SYNTHASE1 (HlSTS1) formed both caryophyllene and humulene from farnesyl pyrophosphate. Together, these enzymes account for the production of the major terpene constituents of the hop trichomes. HlSTS2 formed the minor sesquiterpene constituent germacrene A, which was converted to beta-elemene on chromatography at elevated temperature. We discuss potential functions for other genes expressed at high levels in developing hop trichomes.

Terpene Comparison chart
Yewande Okuleye
DOI: 10.13140/RG.2.2.36575.87203
Conference: Psychoactive Supper At: London May 2016
Affiliation: University of Leicester
This visual/odour profile card was designed to communicate ideas about the ubiquity of terpenes in plants. The comparison between food substances and cannabis asked question about the cultural meanings which cannabis has engendered due to the international drug regime

Terpene Contents Differ in Flowers and Supercritical Co2 Extract
Cara Wietstock
https://terpenesandtesting.com/terpe...l-co2-extract/
From how to best extract them to their best use medicinally; terpenes have been the topic of conversation for everyone from the consumer to the research analyst. This fall, Dr. Michelle Sexton conducted a comparative study [1] of the cannabinoid and terpene content in both cannabinoid flowers and supercritical CO2 extract. Their findings support the idea that the current nomenclature and consumer labeling habits do not help the consumer find the product that they’re looking for.The quantification of seven cannabinoids analysts in the project used a validatedhigh-performance liquid chromatography/diode array detector methodology. To identify which of 42 terpenes were in each sample they utilized internal gas chromatography-mass spectrometry method. What they found was shocking. Between the flower and the extract, the terpenoid and cannabinoid contents were significantly different.Cannabinoid potency in extracts increased by factors of 4.0 for cannabidiol and 3.2 for ?-9 tetrahydrocannabinol when compared to the flower used for start materials. The study also found that monoterpenes were lost in the extraction process. Ketone, monoterpene alcohols, sesquiterpenes all showed an increase in the supercritical CO2 extract as compared to the results from the original dried, cured cannabis flowers.

Terpene Exhaust Emissions and Impact Ozone Modeling from Cannabis Plants at Commercial Indoor Cultivation Facilities in Colorado
Kaitlin Urso,Alicia Frazier, Sara Heald & Andrey Khlystov
DOI: 10.1080/10962247.2022.2046206
https://www.tandfonline.com/doi/full...eedAccess=true
In 2019, an air emissions field sampling study was conducted by the Colorado Department of Public Health and Environment (CDPHE) Air Pollution Control Division (APCD) at three commercial cannabis cultivation facilities. The goal of the study was to quantify biogenic-terpene volatile organic compound (VOC) emissions from growing cannabis at cultivation facility exhaust points to estimate a VOC emission rate by a top-down approach. The resulting VOC emission rates were then used in combination with 2019 commercial cannabis cultivation facility biomass production volumes (harvest weight) and cultivation locations from the Colorado Department of Revenue’s Marijuana Enforcement Division (MED) to model the potential ozone and PM
2.5 formation impacts of the cannabis industry in the Denver Metro North Front Range (DM/NFR) Ozone Nonattainment Area (NAA).

Despite cannabis cultivation facilities’ high nuisance odors, this study found the biogenic VOC emissions rate from the sampled indoor facilities to be low (2.13 lbs to 11.12 lbs of VOC/ton of cannabis harvested), even at large production facilities. The dominant terpenes from this sampling study present in most samples were β-caryophyllene, D-limonene, terpinolene, α-pinene, β-pinene and β-myrcene respectively by concentration. Interestingly, the cannabis emissions exhaust profile lacked isoprene, a terpene commonly emitted from other plants that is highly reactive and has great potential to contribute to ozone formation [Sharkey et al 2007, Wang et al. 2019]. The low biogenic VOC emissions rate and the lack of isoprene from the cannabis cultivation facilities sampled resulted in a very low to negligible impact on both ozone formation (0.005% - 0.009% increase in ozone from cannabis cultivation) and PM2.5 formation (largest maximum 24-hour PM2.5 difference of 0.009 µg/m3) in the DM/NFR NAA.
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Terpene Extraction by Ultrasonics
Derek Johnson
Extraction Magazine
https://extractionmagazine.com/2021/...y-ultrasonics/
There are many techniques for extracting terpenes, each with its own advantages. Of them all, ultrasonic extraction (ultrasonics) offers many benefits, which is why it’s quickly becoming a preferred extraction method for botanicals including cannabis and hops.
The very nature of ultrasonic extraction makes it an efficient and eco-friendly method of production. A solvent, water, or oil is used as a medium to hold the material meant to undergo extraction. Ultrasound extraction works with any solvent, including pressurized solvents like supercritical carbon dioxide and hydrocarbons like butane.
Ultrasonic extraction uses sound energy to create waves and vibrations greater than 20 kilohertz. These waves and vibrations are capable of agitating materials intended for extraction and can lead to superior terpene and cannabinoid extract yields. The method helps catalyze an extraction by deagglomerating biomass particles and reducing their size.
During the extraction, a process called cavitation takes place, which leads to the production of innumerable tiny bubbles that expand and implode rapidly and with great force. The energy generated is what destroys the cell walls of the material undergoing extraction, causing the desired compounds to flow more freely from the cells into the liquid or gel holding the source material.

Terpene Extractors Milestone
https://www.milestonesrl.com/product...ave-extraction
Get links for all
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Isolation Of Strain Specific HIGH QUALITY TERPENES Through Microwave Assisted Extraction
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Terpene Isolation Could Be The Future Of Cannabis
Drake Dorm
https://www.medicaljane.com/2013/10/...e-of-cannabis/
Terpenes are aromatic compounds that are largely responsible for the smell and taste of cannabis. Terpenes are believed to interact synergistically with cannabinoids to improve the efficacy of medical marijuana.
With that said, relatively little is known about terpenes as far as cannabis is concerned. Most often, the cannabis community thinks of terpenes solely as a source of aroma and flavor in their favorite strain of cannabis. However, there are a number of possible applications for terpenes that are unknown to many people who consider themselves a cannabis connoisseur.
For instance, many third-party testing facilities lab-test various cannabis strains and infused products. They are then able to use their database to determine what the average terpene profile for each strain is, and compare future test samples with these standards. The same process could potentially help determine the genetic lineage of a particular strain in a case where it is unknown.
Even further, terpenes are of interest to a number of investigators in their own right. A great deal of focus in the cannabis industry has turned towards the concept of isolating terpenes and the potential application of such a process. In fact, there are rumors about a number of pure terpene products expected to be on the market in the very near future – most notably from The Werc Shop and Cannabis Biotech.

Terpene Synthases and Terpene Variation in Cannabis sativa.
Booth, J. K., Yuen, M. M. S., Jancsik, S., Madilao, L., Page, J., & Bohlmann, J.
Plant Physiology, pp.00593.2020. (2020).
doi:10.1104/pp.20.00593
Cannabis (Cannabis sativa) resin is the foundation of a multi-billion dollar medicinal and recreational plant bioproducts industry. Major components of the cannabis resin are the cannabinoids and terpenes. Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences. A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling. We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar. We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling and trichome-specific transcriptome sequencing. Sequence analysis of these five cultivars and the reference genome of the Purple Kush (PK) cultivar revealed a total of 33 different cannabis terpene synthase (CsTPS) genes as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars. Our annotation of the PK reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes. An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases. The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and collectively explain a diverse range of cannabis terpenes.

Terpene synthases from Cannabis sativa
Judith K. Booth, Jonathan E. Page, Jorg Bohlmann
PLoS ONE 12(3): e0173911 2017
Doi: 10.1371/journal.pone.0173911
Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different
monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as ?-myrcene, (E)-?-ocimene, (-)-limonene, (+)-?-pinene, ?-caryophyllene, and ?-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

NOT CANNABIS SPECIFIC
TERPENES
Eberhard Breitmaier
https://epdf.pub/terpenes1f1560e0e08...7a851619 2.html

Terpenes and derivatives as a new perspective for pain treatment: a patent review
Adriana G Guimaraes, Mairim R Serafini & Lucindo J Quintans-Junior
Expert Opin. Ther. Patents (2013) 24(3)
DOI: 10.1517/13543776.2014.870154
Introduction: Terpenes are natural compounds found in several organisms belonging to the animal and plant kingdoms. They constitute the largest class of natural products with > 55,000 known compounds structurally diversified. Several studies have attributed to this big family of compounds a range of pharmacological properties, such as anticancer, antimicrobial, antifungal, antiviral, antihyperglycemic, analgesic, anti-inflammatory and antiparasitic.
Areas covered: In this review, the authors summarize therapeutic patent applications concerning the employment of terpenes for pain relief, focusing on the perspective for these compounds to become candidates for new drugs intended to control painful syndromes.
Expert opinion: Over years of tremendous academic and industrial investment in the characterization of the analgesic action of terpenes, there was the development of a successful product that has been well-accepted clinically. Furthermore, there is still hope that new therapeutic options for the control of painful syndromes will be developed from terpenes, which have been shown to be great candidates for this purpose because of the range of pharmacological mechanisms in important target sites

NOT CANNABIS SPECIFIC
Terpenes and Terpenoids
Shagufta Perveen, Areej Al-Taweel
https://www.researchgate.net/publica...and_Terpenoids
Natural products are the compounds which isolate from different natural sources such as plants, animals, microbes, insects, plant pathogens, and endophytes and marine. These are known as secondary metabolites since they are formed due to the enzymatic resections of primary metabolites (amino acids, sugars, vitamins, etc.). Terpenes belong to the biggest class of secondary metabolites and basically consist of five carbon isoprene units which are assembled to each other (many isoprene units) by thousands of ways. Terpenes are simple hydrocarbons, while terpenoids are modified class of terpenes with different functional groups and oxidized methyl group moved or removed at various positions. Terpenoids are divided into monoterpenes, sesquiterpenes, diterpenes, sesterpenes, and triterpenes depending on its carbon units (Figure 1). Most of the terpenoids with the variation in their structures are biologically active and are used worldwide for the treatment of many diseases. Many terpenoids inhibited different human cancer cells and are used as anticancer drugs such as Taxol and its derivatives. Many flavorings and nice fragrances are consisting on terpenes because of its nice aroma. Terpenes and its derivatives are used as antimalarial drugs such as artemisinin and related compounds. Meanwhile, terpenoids play a diverse role in the field of foods, drugs, cosmetics, hormones, vitamins, and so on. This chapter provides introduction and information on the bioactive terpenes isolated currently from different natural sources

Terpenoids From Cannabis Do Not Mediate an Entourage Effect by Acting at Cannabinoid Receptors.
Finlay, D. B., Sircombe, K. J., Nimick, M., Jones, C., & Glass, M.
Frontiers in Pharmacology, 11.(2020).
doi:10.3389/fphar.2020.00359
The entourage effect was a proposed explanation for biological observations that endocannabinoid ligand activities can be modified by other lipids released from cells at the same time. An increasing volume of anecdotal reports and interest in the plant have provoked research into the activity of minor chemical constituents of the plant—including volatile terpenoids such as myrcene, a- and b- pinene, b-caryophyllene, and limonene. However, to date, no clear interaction has been identified. The current study was designed to determine whether terpenes in the cannabis plant have detectable receptor-mediated activity, or modify the activity of D9 -tetrahydrocannabinol, cannabidiol, or the endocannabinoid 2-arachidonylglycerol at the cannabinoid receptors. In addition, we have utilized a standard radioligand binding paradigm with ability to detect orthosteric and allosteric interactions of test compounds. With the possible exception of a weak interaction of b-caryophyllene with CB2, no data were produced to support the hypothesis that any of the five terpenes tested (either alone or in mixtures) have direct interactions with CB1 or CB2, as the binding of radioligand ([3 H]-CP55,940), D9 - tetrahydrocannabinol, and cannabidiol were unaltered by the presence of terpenes. Similarly, terpene functional effects were also not detected, either alone or in combination with D9 -tetrahydrocannabinol, cannabidiol, or 2-arachidonoylglycerol. This study adds to the evidence that the putative entourage effect cannot be explained by direct effects at CB1 or CB2.

Terpenes in Cannabis sativa – from plant genome to humans.
Booth JK, Bohlmann J (2019)
Plant Sci 284: 67-72
Doi: 10.1016/j.plantsci.2019.03.022
Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties. Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions that have prevented many researchers from studying cannabis, its products, and their effects in humans. Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid composition of different cannabis ‘strains’. Likewise, claims of some of the medicinal properties attributed to cannabis metabolites would benefit from thorough scientific validation.

Terpenes in Cannabis: Solving the Puzzle of How to Predict Taste and Smell.
Roell, M.-S.
Plant Physiology, 184(1), 8–9. (2020).
doi:10.1104/pp.20.00919
In this issue of Plant Physiology, Booth et al. (2020) provide the framework for future breeding efforts to produce cannabis fragrance and flavor features demanded by consumers. Specifically, they analyzed terpene profiles of eight cannabis cultivars and characterized 13 new cannabis terpene synthases. In plants, terpenes form a diverse group of hydrocarbon-based metabolites estimated to encompass thousands of different molecules (Pichersky and Raguso, 2018). Terpenes have diverse roles. They function as primary cellular components, e.g. as hormones or antioxidants, and they are indispensable for ecological interactions, e.g. signaling and defense against herbivores (Pichersky and Raguso, 2018). In cannabis, more than 100 different terpenes have been identified that define odor and flavor of different cultivars (Rothschild et al., 2005; Andre et al., 2016). Terpene- and cannabinoid-biosynthesis depend on the five carbon building block isopentenyl pyrophosphate (IPP; Fig. 1). IPP is produced by the plastidial methylerythritol phosphate pathway and the cytosolic mevalonate pathway. Metabolic fluxes within both pathways contribute to the substrate pools available for terpene synthases (TPSs). TPSs produce the diversity of cyclic and acyclic terpene core structures, using geranyl diphosphate or farnesyl diphosphate for monoterpene or sesquiterpene synthesis, respectively (Fig. 1). The accurate predicting and design of cannabis terpene profiles requires understanding of TPS, the key enzyme in terpene biosynthesis (Fig. 1). Booth et al. (2020) identified 19 TPS gene models in the ‘Purple Kush’ cannabis reference genome. TPS genes show multicopy gene clustering, a common phenomenon previously observed for genes of the IPP and cannabinoid biosynthetic pathways (Taura et al., 2009). Foliar terpene profiling of eight cannabis cultivars revealed a total of 48 different terpenes with three monoterpenes (myrcene, a?pinene, and limonene) and two sesquiterpenes (b?caryophyllene and a?humulene) present in all cultivars. In six selected cultivars, monoterpenes accumulated during the life cycle, in tissues including leaves, juvenile flowers, and adult flowers. Using trichome transcriptome profiling, Booth et al. (2020) identified 33 TPS genes among the selected six cultivars. Further, 13 new TPSs were biochemically characterized regarding product formation using both geranyl diphosphate and farnesyl diphosphate as substrates. Overall TPS specificity varied between the production of a single mono- or sesquiterpene to as many as 13 different sesquiterpenes produced by a single TPS enzyme. With these results, simple assumptions regarding TPS transcript abundance and terpene profiles can be made. However, spatiotemporal profiling of TPS transcript levels and terpene quantities will be necessary for more accurate predictions. Integrating the 13 TPSs characterized in this study with previously characterized TPS brings the total number to 30 known TPSs across 14 different cannabis cultivars (Booth et al., 2017; Zager et al., 2019; Livingston et al., 2020). Harmonizing trichome transcriptomics tools, knowledge of TPS function, and terpene profiling sets the framework for cannabis breeders to predictively shape and design terpene composition on demand

LIST: Terpenes in vape
EPA
https://comptox.epa.gov/dashboard/ch...s/VAPETERPENES
Description: Terpenes are organic compounds found in the marijuana plant that give strains their distinct aromatic and flavor profiles. They are now being isolated and concentrated into oils for individual vaping.
Number of Chemicals: 37

Terpenes May Improve Effectiveness of Medical Marijuana
Drake Dorm
https://www.medicaljane.com/2013/09/...cal-marijuana/
Terpenes Influence the Synergy Effect of Cannabis
As we know, science has identified and characterized the molecular structure of around 20,000 terpenes, which makes it the largest category of plant chemicals. These aromatic compounds are found in the essential oils of plants and flowers, and plenty of studies have been done on their effects. Of the 20,000 identified terpenes, about 140 of these have been found in cannabis. Only a few of them appear in high concentrations, but they have been found to have a number of benefits. Terpenes play very important individual roles, but recent research also suggests an “entourage effect” may exist. In his 2011 study, Ethan Russo (GW Pharmaceuticals), discussed the interaction between terpenes and cannabinoids. In fact, Russo points out that terpenes likely played a role in a number of ancient antidotes for the less desirable effects of THC. For instance, citrus fruits (high in limonene) were used as a “cannabis antidote” in 10th century Persia. Other ancient antidotes include calamus plant roots and pine nuts (high in pinene), as well as black pepper (high in caryophyllene and myrcene).

Not directly Cannabis related
Terpenes solubility in water and their environmental distribution.
Martins, M. A. R., Silva, L. P., Ferreira, O., Schröder, B., Coutinho, J. A. P., & Pinho, S. P.
Journal of Molecular Liquids, 241, 996–1002.(2017).
doi:10.1016/j.molliq.2017.06.099
Terpenes and terpenoids belong to the largest and most diverse class of natural products. Due to the increasing importance of their applications and the emerging perception of their impact on the environment, the available physico-chemical characterization is insufficient. In this work the water solubility of geraniol, linalool, DL-citronellol, thymol, eugenol, carvacrol and pcymene, in the temperature range from (298.15 to 323.15) K, and at atmospheric pressure, is studied. Due to the low solubility of these compounds a novel technique was adopted for their measurements and validated using the aqueous solubility data for sparingly soluble aromatic compounds. The thermodynamic properties of solution were derived from the experimental data at infinite dilution. It is shown that the solubility of terpenes in water is an endothermic process confirming the existence of UCST phase diagrams, and only for carvacrol and eugenol is entropically driven. The experimental information is shown in a two-dimensional chemical space diagram providing indications to their probable distribution in the environment once released.

Terpenes/Terpenoids in Cannabis: Are They Important?
Lumír Ond?ej, Hanuš, Yotam Hod
Med Cannabis Cannabinoids, 1-36 (2020)
DOI: 10.1159/000509733
Cannabis sativa plant has not only cannabinoids as crucial compounds but also the other compounds that play important role as synergistic and/or entourage compound. Cannabis/hemp plant materials and essential oils were analyzed with the help of gas chromatography/mass spectrometry detector for the content of terpenes and terpenoids. The main terpenes/terpenoids and their abundance in the samples were evaluated. Results of this study will be helpful in the next evaluation of these compound in mixture with cannabinoids and their importance in medical treatment.

Terpenoid biosynthesis in trichomes—current status and future opportunities
B. Markus Lange and Glenn W. Turner
Plant Biotechnology Journal (2012), pp. 1–21
doi: 10.1111/j.1467-7652.2012.00737.x
Glandular trichomes are anatomical structures specialized for the synthesis of secreted natural products. In this review we focus on the description of glands that accumulate terpenoid essential oils and oleoresins. We also provide an in-depth account of the current knowledge about the biosynthesis of terpenoids and secretion mechanisms in the highly specialized secretory cells of glandular trichomes, and highlight the implications for metabolic engineering
efforts.

Terpenoid chemoprofiles distinguish drug-type Cannabis sativa L. cultivars in Nevada.
Orser C, Johnson S, Speck M, Hilyard A, Afia I (2018)
Nat Prod Chem Res 6: 304
doi:10.4172/2475-7675.1000304
https://www.researchgate.net/publica...vars_in_Nevada
An unintended consequence of state-mandated cannabis testing regulations has been the resulting database from the analysis of thousands of individual cannabis flower samples from artificially restricted geographical regions. The resulting detailed chemical database can serve as the basis for the development of a chemotaxonomic classification scheme outside of conjectural cultivar naming by strain. Chemotaxonomic classification schemes for cannabis cultivars have previously been reported by others based largely on cannabis strains grown in California under an unregulated testing environment or in Europe from strains grown by a single cultivator. In this study 2,237 individual cannabis flower samples, representing 204 individual strains across 27 cultivators in a tightly regulated Nevada cannabis testing market, were analyzed across 11 cannabinoids and 19 terpenoids. Even though 98.3% of the samples were from Type I cannabis strains by cannabidiolic acid/tetrahydrocannabinolic acid (THCA) ratio of <0.5 CBDA, principal component analysis (PCA) of the combined dataset resulted in three distinct clusters that were distinguishable by terpene profiles alone. Further dissection of individual strains by cultivators within clusters revealed striking fidelity of terpenoid profiles and also revealed a few outliers. We propose that three terpenoid cluster assignments account for the diversity of drug type cannabis strains currently being grown in Nevada.

Terpenoid Chemoprofiles Distinguish Drug-type Cannabis sativa L. Cultivars in Nevada
Philippe Henry, Cindy Orser, Steve Johnson, Aaron Hilyard, S Thoslon, A Everett, Mark D Speck
Conference: The Emerald Conference February 2018 (Poster)
DOI: 10.13140/RG.2.2.20022.40006
https://www.researchgate.net/publica...vars_in_Nevada
One consequence of state-mandated cannabis testing regulations has been the resulting database from the analysis of thousands of individual cannabis flower samples from artificially restricted geographical regions. The resulting detailed chemical database can serve as the basis for the development of a chemotaxonomic classification scheme outside of conjectural cultivar naming by strain. Chemotaxonomic classification schemes for cannabis cultivars have previously been reported by others based largely on cannabis strains grown in unregulated testing environments or in Europe from strains grown by a single cultivator. In this study 2,237 individual cannabis flower samples, representing 204 individual strains across 27 cultivators in a tightly regulated Nevada cannabis testing market, were analyzed across 11 cannabinoids and 19 terpenoids. Even though 98.3% of the samples were from Type I cannabis strains based on CBDA/THCA cannabinoid ratio, principal component analysis (PCA) of the combined dataset resulted in three distinct clusters that were distinguishable by terpene profiles alone. Further dissection of individual strains by cultivators within clusters revealed striking fidelity of terpenoid profiles but also revealed a few outliers. We propose that three terpenoid cluster assignments, and as few as three terpenoids, account for the diversity of drug-type cannabis strains currently being grown in Nevada.

Not Cannabis specific
Terpenoid synthase structures: a so far incomplete view of complex catalysis.
Gao, Y., Honzatko, R. B., & Peters, R. J.
Natural Product Reports, 29(10), 1153. (2012).
doi:10.1039/c2np20059g
The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the initial carbocation is generated does vary. High-resolution crystal structures have been obtained for at least representative examples from each of the various types of enzymes involved in producing terpenoid hydrocarbon backbones. However, while this has certainly led to some insights into the enzymatic structure– function relationships underlying the elongation and simpler cyclization reactions, our understanding of the more complex cyclization and/or rearrangement reactions remains limited. Accordingly, selected examples are discussed here to demonstrate our current understanding, its limits, and potential ways forward.

Terpenoids and Phytocannabinoids Co-Produced in Cannabis Sativa Strains Show Specific Interaction for Cell Cytotoxic Activity.
Namdar, D., Voet, H., Ajjampura, V., Nadarajan, S., Mayzlish-Gati, E., Mazuz, M., … Koltai, H.
Molecules, 24(17), 3031. (2019).
doi:10.3390/molecules24173031
: Mixtures of different Cannabis sativa phytocannabinoids are more active biologically than single phytocannabinoids. However, cannabis terpenoids as potential instigators of phytocannabinoid activity have not yet been explored in detail. Terpenoid groups were statistically co-related to certain cannabis strains rich in ? 9 -tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), and their ability to enhance the activity of decarboxylase phytocannabinoids (i.e., THC or CBD) was determined. Analytical HPLC and GC/MS were used to identify and quantify the secondary metabolites in 17 strains of C. sativa, and correlations between cannabinoids and terpenoids in each strain were determined. Column separation was used to separate and collect the compounds, and cell viability assay was used to assess biological activity. We found that in “high THC” or “high CBD” strains, phytocannabinoids are produced alongside certain sets of terpenoids. Only co-related terpenoids enhanced the cytotoxic activity of phytocannabinoids on MDA-MB-231 and HCT-116 cell lines. This was found to be most effective in natural ratios found in extracts of cannabis inflorescence. The correlation in a particular strain between THCA or CBDA and a certain set of terpenoids, and the partial specificity in interaction may have influenced the cultivation of cannabis and may have implications for therapeutic treatments

Terpenoids Biotransformation in Mammals III: Biotransformation of a-Pinene, ,8-Pinene, Pinane, 3-Carene, Carane, Myrcene, and p-Cymene in Rabbits
T. ISHIDA, Y. ASAKAWA, T. TAKEMOTO, and T. ARATANT
Journal of Pharmeceutlcal Sciences Vol. 70, No. 4, April 1981
DOI: 10.1002/JPS.2600700417
The biotransformation of (+)-, (-)-, and (*)-a-pinenes, (-)-/?-pinene (nopinene), (-)-cis-pinane, (+)-3-carene, (-)-cis-carane, myrcene, and p-cymene in rabbits was investigated. The major metabolites were as follows: (-)-trans-verbenol from (+)-, (-)-, and (&Ia-pinenes; (-)-10-pinanol and (-)-l-p-menthene-7,8-diol from (-)- /?-pinene; (-)-a-terpineol and (-)-trans-sobrerol from (-)Aspinane; (-)-m-rnentha-4,6-dien-8-01, 3-caren-9-01, (-)-3-carene-9-carboxylic acid, and 3-carene-9,10-dicarboxylic acid from (+)-3-carene; carane9,lO-dicarboxylic acid from (-)-cis-carane; and myrcene-3(10)-glycol, myrcene-1,2-glycol, uroterpenol, and p-cymene-9-carboxylic acid from p-cymene. These metabolisms include allylic oxidation, epoxidation, stereoselective gem-dimethyl hydroxylation and its oxidation, cleavage of a conjugated double bond by epoxidation, and regioselective oxidation, some of which are not found usually in chemical reactions, and due to which various new compounds were determined. This biotransformation of the monoterpene hydrocarbons gave some insect pheromones in high yield.

Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels.
Heblinski, M., Santiago, M., Fletcher, C., Stuart, J., Connor, M., McGregor, I. S., & Arnold, J. C.
Cannabis and Cannabinoid Research. (2020).
doi:10.1089/can.2019.0099
Introduction: Cannabis sativa produces hundreds of bioactive compounds, including cannabinoids and terpenoids. It has been proposed that cannabinoids act in synergy with terpenoids to produce the entourage effect, a concept used to explain the therapeutic benefits of medicinal cannabis. One molecular explanation for the entourage effect is that the terpenoids augment the actions of cannabinoids at their molecular drug targets in cells. We recently reported that terpenoids commonly found in cannabis do not influence the functional effects of D9-tetrahydrocannabinol (D9-THC) on cannabinoid 1 and cannabinoid 2 receptors. The present study aimed to extend on this research by examining whether terpenoids influence the effects of phytocannabinoids and endocannabinoids on human transient receptor potential ankyrin 1 (hTRPA1) and human transient receptor potential vanilloid 1 (hTRPV1) channels heterologously expressed in mammalian cells.
Materials and Methods: The activity of terpenoids, phytocannabinoids, and endocannabinoids was assessed in inducible HEK Flp-In T-Rex cells transfected with hTRPA1 and hTRPV1 channels, respectively. Real-time changes in intracellular calcium ([Ca]i) were measured using the Calcium 5 dye and a FlexStation 3 plate reader.
Results: a-pinene, b-pinene, b-caryophyllene, linalool, limonene, b-myrcene or a-humulene did not affect [Ca]i in hTRPA1 and hTRPV1 overexpressing cells. Cinnamaldehyde (CA), D9-THC, and 2-arachidonoylglycerol (2-AG) activated TRPA1 receptors with high efficacy and similar potency (EC50s of*10 lM). Capsaicin and anandamide (AEA) activated TRPV1 receptors with an EC50 of 61nM and 4.3 lM, respectively, but TRPV1 showed no response to D9-THC, cannabidiol, and other minor cannabinoids. Terpenoids did not significantly affect the responses of TRPA1 and TRPV1 receptors to submaximal and maximal concentrations of CA and D9-THC or the endocannabinoids AEA and 2-AG.
Discussion: We could not find any evidence that the terpenoids tested here activate TRPA1 and TRPV1 channels or modulate their activation by D9-THC and other agonists, including endocannabinoids

Terpenoids From Cannabis Do Not Mediate an Entourage Effect by Acting at Cannabinoid Receptors.
Finlay, D. B., Sircombe, K. J., Nimick, M., Jones, C., & Glass, M.
Frontiers in Pharmacology, 11.(2020).
doi:10.3389/fphar.2020.00359
The entourage effect was a proposed explanation for biological observations that endocannabinoid ligand activities can be modified by other lipids released from cells at the same time. An increasing volume of anecdotal reports and interest in the plant have provoked research into the activity of minor chemical constituents of the plant—including volatile terpenoids such as myrcene, a- and b- pinene, b-caryophyllene, and limonene. However, to date, no clear interaction has been identified. The current study was designed to determine whether terpenes in the cannabis plant have detectable receptor-mediated activity, or modify the activity of D9 -tetrahydrocannabinol, cannabidiol, or the endocannabinoid 2-arachidonylglycerol at the cannabinoid receptors. In addition, we have utilized a standard radioligand binding paradigm with ability to detect orthosteric and allosteric interactions of test compounds. With the possible exception of a weak interaction of b-caryophyllene with CB2, no data were produced to support the hypothesis that any of the five terpenes tested (either alone or in mixtures) have direct interactions with CB1 or CB2, as the binding of radioligand ([3 H]-CP55,940), D9 - tetrahydrocannabinol, and cannabidiol were unaltered by the presence of terpenes. Similarly, terpene functional effects were also not detected, either alone or in combination with D9 -tetrahydrocannabinol, cannabidiol, or 2-arachidonoylglycerol. This study adds to the evidence that the putative entourage effect cannot be explained by direct effects at CB1 or CB2.

Terpinolene, a component of herbal sage, downregulates AKT1 expression in K562 cells
DOI: 10.3892/ol.2011.491
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362481/
Protein kinase AKT mediates cell proliferation and survival signals, and also contributes to cancer progression. Increased expression and/or activation of AKT is involved in a variety of human cancers. In cells treated with sage or rosemary extract, mRNA and protein expression levels of AKT1 were reduced compared with those of the control cells 48 h after the herbal treatments. We found that terpinolene, a common component of sage and rosemary, markedly reduced the protein expression of AKT1 in K562 cells and inhibited cell proliferation.

Terpli: A Future Driven by Terpenes Versus Anything Else
Jason S. Lupoi
Terpenes and Testing magazine
https://terpenesandtesting.com/terpl...anything-else/
A talk with Terpli CEO Peter Kasper,
Peter: Terpli interprets batch-specific certificates of analysis to analyze the actual chemistry behind each product via the full cannabinoid and terpene profile. We use an algorithm to analyze the sample, submitted reviews, and research information to interpret what a user might expect from a particular product in their hand.
Terpli has designed a unique and personalized mobile app that offers users specific insights into effects they can anticipate from a cannabis variety based on the terpene profile and what other users have experienced from the plant.
The app allows users to look up a particular product from a represented brand via its product name, orby typing in the batch ID from the packaging label, which is run through an algorithm that curates specific effects and outcomes that a user might experience based on the cannabinoid and terpene profile. We work with brands and retailers to provide discounts that act as incentives for our users to write genuine reviews of the products on the platform and help with the high cost of cannabis goods.

That skunky smell? Blame ‘321 MBT’
Hemp Today April 2021
https://hemptoday.net/that-skunky-smell-blame-321-mbt/
American researchers say they have determined what gives cannabis its skunky smell. Scientists from Indiana-based Byers Scientific and Iowa State University say the compound 3-methyl-2-butene-1-thiol (“321 MBT”) is the main source of the odor, and not cannabis plant terpenes, which have generally been considered to cause the smell. The research team used analytical chemistry, leaf enclosure study and field observation to isolate and identify the aroma’s source. 321 MBT is the same chemical that causes the skunky-like aroma in spoiled beer, the scientists said. Hops, the main ingredient in beer, is also of the Cannabaceae plant family. The research is part of efforts to devise ways to eliminate the smell.

The Cannabis sativa Versus Cannabis indica Debate: An Interview with Ethan Russo, MD
Daniele Piomelli and Ethan B. Russo
Cannabis and Cannabinoid Research Jan 2016
DOI: 10.1089/can.2015.29003.ebr
Dr. Ethan Russo, MD, is a board-certified neurologist, psychopharmacology researcher, and Medical Director of PHYTECS, a biotechnology company researching and developing innovative approaches targeting the human endocannabinoid system. Previously, from 2003 to 2014, he served as Senior Medical Advisor and study physician to GW Pharmaceuticals for three Phase III clinical trials of Sativex for alleviation of cancer pain unresponsive to optimized opioid treatment and studies of Epidiolex for intractable epilepsy. He has held faculty appointments in Pharmaceutical Sciences at the University of Montana, in Medicine at the University of Washington, and as visiting Professor, Chinese Academy of Sciences. He is a past President of the International Cannabinoid Research
Society and former Chairman of the International Association for Cannabinoid Medicines. He serves on the Scientific Advisory Board for the American Botanical Council. He is the author of numerous books, book chapters, and articles on Cannabis, ethnobotany, and herbal medicine. His research interests have included correlations of historical uses of Cannabis with modern pharmacological mechanisms, phytopharmaceutical treatment of migraine and chronic pain, and phytocannabinoid/terpenoid/serotonergic/vanilloid interactions.

The Cannabis Terpenes
Sarana Rose Sommano , Chuda Chittasupho , Warintorn Ruksiriwanich and Pensak Jantrawut
Molecules 2020, 25, 5792;
doi:10.3390/molecules25245792
Terpenes are the primary constituents of essential oils and are responsible for the aroma characteristics of cannabis. Together with the cannabinoids, terpenes illustrate synergic and/or entourage effect and their interactions have only been speculated in for the last few decades.
Hundreds of terpenes are identified that allude to cannabis sensory attributes, contributing largely to the consumer’s experiences and market price. They also enhance many therapeutic benefits, especially as aromatherapy. To shed light on the importance of terpenes in the cannabis industry, the purpose of this review is to morphologically describe sources of cannabis terpenes and to explain the biosynthesis and diversity of terpene profiles in different cannabis chemovars.

The effect of five day dosing with THCV on THC-induced cognitive, psychological and physiological effects in healthy male human volunteers: a placebo-controlled, double-blind, crossover pilot trial.
Amir Englund, Zerrin Atakan, Aleksandra Kralj, Nigel Tunstall, Robin Murray and Paul Morrison
DOI: 10.1177/0269881115615104
In this small pilot study with healthy infrequent cannabis users, results indicate that the dose of 10mg oral THCV is well tolerated with no serious adverse reactions, and was subjectively not distinguishable from placebo. Furthermore, the lower dose of 1mg iv THC did not produce any significant short-term memory impairment, or psychotic or paranoid reactions. THCV significantly inhibited THC-induced impairment to delayed recall as well as THC-induced increase of heart rate. THCV on its own showed signs towards improved performance on the harder working-memory task, while also producing a slight increase in
anxiety. However, these effects were small and need to be further studied in a larger sample.
RATIONALE:
Cannabis is mostly grown under illegal and unregulated circumstances, which seems to favour a product increasingly high in its main cannabinoid ?-9-tetrahydrocannabinol (THC). ?-9-tetrahydrocannabivarin (THCV) is a relatively untestedcannabinoid which is said to be a cannabinoid receptor neutral antagonist, and may inhibit the effects of THC.
OBJECTIVES:
To explore the safety and tolerability of repeated THCV administration and its effects on symptoms normally induced by THC in a sample of healthy volunteers.
METHODS:
Ten male cannabis users (<25 use occasions) were recruited for this within-subjects, placebo-controlled, double-blind, cross-over pilot study. 10mg oral pure THCV or placebo were administered daily for five days, followed by 1mg intravenous THCon the fifth day.
RESULTS:
THCV was well tolerated and subjectively indistinguishable from placebo. THC did not significantly increase psychotic symptoms, paranoia or impair short-term memory, while still producing significant intoxicating effects. Delayed verbal recall was impaired by THC and only occurred under placebo condition (Z=-2.201, p=0.028), suggesting a protective effect of THCV. THCV also inhibited THC-induced increased heart rate (Z=-2.193, p=0.028). Nine out of ten participants reported THC underTHCV condition (compared to placebo) to be subjectively weaker or less intense (?2=6.4, p=0.011). THCV in combination with THC significantly increased memory intrusions (Z=-2.155, p=0.031).
CONCLUSION:
In this first study of THC and THCV, THCV inhibited some of the well-known effects of THC, while potentiating others. These findings need to be interpreted with caution due to a small sample size and lack of THC-induced psychotomimetic and memory-impairing effect, probably owing to the choice of dose.

FYI, I did THCV/THC trials 15 years ago, unpublished, but we found much the same, THCV is not for recreational users unless they like their THC tuned down. THCV delays THC onset, reduces peak experiences, and maybe lengthens the reduced effects.
-SamS

The essential oil of Cannabis sativa.
Malingre T, Hendriks H, Batterman S, Bos R, Visser J (1975)
Planta Med 28: 56–61
DOI: 10.1055/s-0028-1097829
In previous reports the presence of cannabinoids in the distilled essential oil of Cannabis sativa L. was proved, besides the presence of mono- and sesquiterpene hydrocarbons. In this paper the localization of the cannabinoids in the hairs of the leaves and with that the possible biogenetic relation with the components of the essential oil are demonstrated by microscopic examination after colouring tests and gaschromatographic analysis of the isolated contents of individual glandular hairs. Quantitative data about the relation between essential oil and cannabinoids are obtained by comparing the extracts without and after preceding steam distillation. On acount of the origin of the seed (birdseed), special attention was paid to the botanical description of the plant material and to the counting of chromosomes.

NOT CANNABIS SPECIFIC
The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom.
Chen F, Tholl D, Bohlmann J, Pichersky E.
Plant J. 2011 Apr;66(1):212-29.
doi: 10.1111/j.1365-313X.2011.04520.x.
Some plant terpenes such as sterols and carotenes are part of primary metabolism and found essentially in all plants. However, the majority of the terpenes found in plants are classified as 'secondary' compounds, those chemicals whose synthesis has evolved in plants as a result of selection for increased fitness via better adaptation to the local ecological niche of each species. Thousands of such terpenes have been found in the plant kingdom, but each species is capable of synthesizing only a small fraction of this total. In plants, a family of terpene synthases (TPSs) is responsible for the synthesis of the various terpene molecules from two isomeric 5-carbon precursor 'building blocks', leading to 5-carbon isoprene, 10-carbon monoterpenes, 15-carbon sesquiterpenes and 20-carbon diterpenes. The bryophyte Physcomitrella patens has a single TPS gene, copalyl synthase/kaurene synthase (CPS/KS), encoding a bifunctional enzyme producing ent-kaurene, which is a precursor of gibberellins. The genome of the lycophyte Selaginella moellendorffii contains 18 TPS genes, and the genomes of some model angiosperms and gymnosperms contain 40-152 TPS genes, not all of them functional and most of the functional ones having lost activity in either the CPS- or KS-type domains. TPS genes are generally divided into seven clades, with some plant lineages having a majority of their TPS genes in one or two clades, indicating lineage-specific expansion of specific types of genes. Evolutionary plasticity is evident in the TPS family, with closely related enzymes differing in their product profiles, subcellular localization, or the in planta substrates they use.

THE INFLUENCE OF TERPENES ON THE RELEASE OF VOLATILE ORGANIC COMPOUNDS AND ACTIVE INGREDIENTS TO CANNABIS VAPING AEROSOLS
Jiries Meehan-Atrash, Wentai Luo, Kevin J. McWhirter David G. Dennis, David Sarlah, Robert P. Jensen, Isaac Afreh, Jia Jiang, Kelley Barsanti, Alisha Ortiz, Robert M. Strongin
RSC Adv., 2021, 11,11714-11723
DOI: 10.1039/D1RA00934F
Dabbing and vaping cannabis extracts have gained large popularity in the United States as alternatives to cannabis smoking, but diversity in both available products and consumption habits make it difficult to assess consumer exposure to psychoactive ingredients and potentially harmful components. This work studies the how relative ratios of the two primary components of cannabis extracts, D9-tetrahydrocannabinol (THC) and terpenes, affect dosage of these and exposure to harmful or potentially harmful components (HPHCs). THC contains a monoterpene moiety and has been previously shown to emit similar volatile degradation products to terpenes when vaporized. Herein, the major thermal degradation mechanisms for THC and b-myrcene are elucidated via analysis of their aerosol gas phase
products using automated thermal desorption-gas chromatography-mass spectrometry with the aid of isotopic labelling and chemical mechanism modelling. Four abundant products – isoprene, 2-methyl-2-butene, 3-methylcrotonaldehyde, and 3-methyl-1-butene – are shown to derive from a common radical intermediate for both THC and b-myrcene and these products comprise 18–30% of the aerosol gas phase. The relative levels of these four products are highly correlated with applied power to the ecigarette, which indicates formation of these products is temperature dependent. Vaping THC–bmyrcene mixtures with increasing % mass of b-myrcene is correlated with less degradation of the starting material and a product distribution suggestive of a lower aerosolization temperature. By contrast, dabbing THC–b-myrcene mixtures with increasing % mass of b-myrcene is associated with higher levels of HPHCs, and isotopic labelling showed this is due to increased reactivity of b-myrcene relative to THC.

Not Cannabis specific
The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers
Natalia Dudareva, Susanna Andersson, Irina Orlova, Nathalie Gatto, Michael Reichelt, David Rhodes, Wilhelm Boland, and Jonathan Gershenzon
Proc Natl Acad Sci U S A. Jan 18, 2005; 102(3): 933–938.
doi: 10.1073/pnas.0407360102
Terpenoids, the largest class of plant secondary metabolites, play essential roles in both plant and human life. In higher plants, the five-carbon building blocks of all terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate, are derived from two independent pathways localized in different cellular compartments. The methylerythritol phosphate (MEP or nonmevalonate) pathway, localized in the plastids, is thought to provide IPP and dimethylallyl diphosphate for hemiterpene, monoterpene, and diterpene biosynthesis, whereas the cytosol-localized mevalonate pathway provides C5 units for sesquiterpene biosynthesis. Stable isotope-labeled, pathway-specific precursors (1-deoxy-[5,5-2H2]-d-xylulose and [2,2-2H2]-mevalolactone) were supplied to cut snapdragon flowers, which emit both monoterpenes and the sesquiterpene, nerolidol. We show that only one of the two pathways, the plastid-localized MEP pathway, is active in the formation of volatile terpenes. The MEP pathway provides IPP precursors for both plastidial monoterpene and cytosolic sesquiterpene biosynthesis in the epidermis of snapdragon petals. The trafficking of IPP occurs unidirectionally from the plastids to cytosol. The MEP pathway operates in a rhythmic manner controlled by the circadian clock, which determines the rhythmicity of terpenoid emission

The Monoterpene Ocimene: Characteristics, Sources, and Benefits
Derek Johnson
Terpenes and Testing magazine
https://terpenesandtesting.com/the-m...-and-benefits/
Ocimene is an isomeric hydrocarbon as well as a monoterpene. When exposed to air, it becomes extremely volatile, which helps explain its highly aromatic characteristics. Ocimene occurs as three different isomers: α-ocimene, cis-β-ocimene, and trans-β-ocimene.
Ocimene is highly valued for its aromatic profile. It contributes to the strong and pleasant aromas of many fruits, herbs, and spices, including tarragon, pepper, basil, mango, mint, and parsley. The odor has been described as herbaceous, citrusy, tropical, and woody.
Ocimene is also present in cannabis. One popular dispensary cites cultivars Jack Herer and Mandarin Dream. Another adds Sour Diesel, Clementine, and Snowcone to the list. It is also a key terpene in the medical cultivar Bedrocan.
Ocimene is associated with anti-fungal, anticonvulsant, and anti-tumor activities. It has also been reported to play an important role as a pheromone for the social regulation of honeybees.
One study focused on essential oil from the Angelica plant, which contains principally α-pinene and cis-β-ocimene. The oil and the two isolated terpenes proved effective against Candida albicans, Cryptococcus neoformans, and dermatophytes (skin fungus).

The next evolution in Horticulture lighting –Impact of Using Supplemental UV
Peter Barber
https://issuu.com/amsterdamrai/docs/peter_barber.pptx
Shows that a very narrow band of UVB can help increase Terpenes and Cannabinoids as well as help control PM and Botrytris on Cannabis crops

The preservation and augmentation of volatile terpenes in cannabis inflorescence
Justin Bueno, Emily Leuer, Michael Kearney Jr, Edward H. Green and Eric A. Greenbaum
Journal of Cannabis Research (2020) 2:27
DOI: 10.1186/s42238-020-00035-z
Terpenes contribute to the pharmacology, efficacy, aroma, and flavor of cannabis inflorescence, improving the experience for medical and recreational users. Terpenes are inherently volatile, resulting in the loss of terpene content as inflorescence ages. A method to establish and/or maintain a desired terpene content of cannabis inflorescence is needed. A novel packaging method was investigated for the preservation of native terpenes and the replenishment of terpenes to depleted inflorescence over various storage durations. Methods: Inflorescence samples from two different chemotypes (DJ’s Gold, Cream Caramel) were obtained from a state licensed medical cannabis organization. Samples from the DJ’s Gold chemotype were depleted of terpenes whereas samples from the Cream Caramel chemotype had a terpene content representative of inflorescence available for medicinal or recreational purposes. Inflorescence samples were stored using the novel packaging approach, in airtight containers in the presence of external terpenes. Control samples were similarly stored without external terpenes. Terpene content of the inflorescence samples were quantitively determined by headspace gas chromatography mass spectrometry (HS GC-MS) after various storage durations. Main effects analysis was used to determine the impact of various parameters on the effectiveness of the system. Results: All samples stored using the novel packaging approach had a higher terpene content than their corresponding control. 1.18% (w/w) of external terpene, relative to inflorescence weight, was the minimum amount required to maintain the initial terpene content of the inflorescence after 6 weeks of storage. Main effects analysis showed that augmentation of inflorescence terpene content was dependent upon the amount and type of external volatile utilized. The terpene profile of inflorescence samples from two separate harvests were selectively adjusted, reducing the percent difference of the two sample’s terpene profiles by 39.5%. Conclusions: A successful proof of concept was achieved for preservation, augmentation, and replenishment of terpenes to cannabis inflorescence over various storage durations. Inflorescence stored using the novel packaging approach is a significant step towards providing patients with cannabis inflorescence of reproducible and reliable terpene content, an important component of inflorescence efficacy. The novel approach for replenishment of terpenes to depleted inflorescence represents an exciting development for patients and manufacturers.

The Problem of Concentrate Consistency: Terpene Loss in Extraction
Steep Hill
https://static1.squarespace.com/stat...Terpenes_2.pdf
Cannabis Extraction Methods Alter Terpene Concentrations
The modern cannabis consumer doesn’t rely solely on whole-flower methods of consumption. Many modern cannabis consumers—whether medical cannabis patients or recreational consumers—have embraced the convenience, discretion, and rapid relief offered by personal vaping pens or concentrate rigs, which offer consistent, convenient ways to dose discreetly. The question, then, is whether consumers who prefer to use cannabis extracts as opposed to whole-flower use are getting the consistency of experience that they need, as well as the certainty that the product they’re purchasing lives up to the chemovar profile of the strain used in its labeling and marketing.

THE PRODUCTION OF ESSENTIAL HEMP OIL IN SWITZERLAND
Vito Mediavilla
https://www.researchgate.net/publica...IN_SWITZERLAND
Essential hemp oil is an interesting product, which may be obtained from hemp. This concentrate of the typical hemp aroma is utilised in cosmetics, additives to food, aroma therapy and perfume. Since 1995 a couple of Swiss farmers produced with very high labour input high quality oils. Their sale
potential is not clear yet and at present it is a niche product. Hemp regulations in some countries (e.g. USA) limit its import. We assessed the factors influencing yield and quality in some experiments. Recommendations about variety choice, harvest time, weather influence, seed density, harvest
techniques and prevention of pollination in function to yield and smell scent are discussed. For a broader propagation of essential hemp oil additional information about medical (pharmaceutical and dermatological) use is needed.

Not cannabis specific
The Revaluation of Plant-Derived Terpenes to Fight Antibiotic-Resistant Infections.
Cappiello, F., Loffredo, M. R., Del Plato, C., Cammarone, S., Casciaro, B., Quaglio, D., … Ghirga, F.
Antibiotics, 9(6), 325.(2020).
doi:10.3390/antibiotics9060325
he discovery of antibiotics has revolutionized the medicine and treatment of microbial infections. However, the current scenario has highlighted the difficulties in marketing new antibiotics and an exponential increase in the appearance of resistant strains. On the other hand, research in the field of drug-discovery has revaluated the potential of natural products as a unique source for new biologically active molecules and scaffolds for the medicinal chemistry. In this review, we first contextualized the worldwide problem of antibiotic resistance and the importance that natural products of plant origin acquire as a source of new lead compounds. We then focused on terpenes and their potential development as antimicrobials, highlighting those studies that showed an activity against conventional antibiotic-resistant strains

Not Cannabis specific
The role of structure and molecular properties of terpenoids in determining their antimicrobial activity.
Griffin, S. G., Wyllie, S. G., Markham, J. L., & Leach, D. N.
Flavour and Fragrance Journal, 14(5), 322–332.(1999).
doi:10.1002/(sici)1099-1026(199909/10)14:5<322::aid-ffj837>3.0.co;2-4
The minimum inhibitory concentrations (MIC) of 60 terpenoids against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans have been determined. Hierarchical cluster analysis was used to group the compounds into ®ve groups according to their activity patterns against the four microorganisms. K-Means cluster analysis was then used to con®rm these groupings and to show the di€erences in the activity patterns of the groups. Ten molecular properties of the terpenoids, either calculated via molecular modelling or determined by direct measurement, were then used as variables in a forward stepwise discriminant analysis to identify which variables discriminated between groups. Low water solubility of Group IV compounds, mainly hydrocarbons and acetates, was found to be associated with their relative inactivity. The remaining groups, all containing oxygenated terpenoids, showed characteristic but distinct activity patterns towards the four test organisms. Hydrogen bonding parameters were found to be associated with antimicrobial activity in all cases. Activity against Gram-negative E. coli and P. aeruginosa was associated with a combination of a hydrogen bonding and size parameters. This was not found to be the case for the Gram-positive S. aureus or the yeast C. albicans.

The sedative effect of inhaled terpinolene in mice and its structure–activity relationships.
Ken Ito, Michiho Ito
Journal of Natural Medicines 67(4) January 2013
DOI: 10.1007/s11418-012-0732-1
Terpinolene is a cyclic monoterpene compound found in some Labiatae herbs. In our previous study, we evaluated the sedative effect of inhaled essential oils of Microtoena patchoulii leaves in mice and isolated terpinolene as an active ingredient. We investigated the structure-activity relationships of terpinolene to identify the structural part essential to its sedative effect. Comparison of terpinolene analog activities showed that a double bond in the side-chain or pi bonds in the six-membered ring play important roles in the sedative effect. In another experiment using olfactory impaired mice, we further revealed that inhaled terpinolene exerted the effect after nasal absorption into the body.

The Terroir of Cannabis: Terpene Metabolomics as a Tool to Understand Cannabis sativa Selections.
Mudge, E., Brown, P., & Murch, S. (2019).
Planta Medica.
doi:10.1055/a-0915-2550
The phytochemical diversity of Cannabis chemovars is not well understood, and many chemovars were created in informal breeding programs without records of parentage or the criteria for selection. Key criteria for selection sometimes included aroma notes and visual cues, which some breeders associated with pharmacological activity. We hypothesized that the process of selection for scents believed to be related to specific tetrahydrocannabinol levels has resulted in modified terpene biosynthesis in these chemovars. Thirty-two cannabinoids, 29 monoterpenes and 38 sesquiterpenes were measured in 33 chemovars from 5 licensed producers. A classification system based on cannabinoid content was used with targeted metabolomic tools to determine relationships in the phytochemistry. Three monoterpenes, limonene, ?-myrcene, and ?-pinene, and two sesquiterpenes, caryophyllene and humulene, were abundant in the majority of chemovars. Nine terpenes were present in tetrahydrocannabinol-dominant chemovars. Three monoterpenes and four sesquiterpenes were predominantly found in cannabidiol-containing chemovars. Low abundance terpenes may have been the aromatic cues identified by breeders. The medicinal activity of some of the terpenes is likely to contribute to the pharmacological effect of specific chemovars. Together, these data demonstrate the synergy of compounds in Cannabis chemovars and point to the need for additional research to understand the phytochemical complexity.

The Volatile Oil Composition of Fresh and Air-Dried Buds of Cannabis sativa
Samir A. Rossi and Mahmoud A. ElSohly
J. Nut. Prod. 1996, 59, 49-51
DOI: 10.1021/np960004a
The composition of the steam-distilled volatile oil of fresh and air-dried, indoor-grown marijuana was studied by GC/FID and GC/MS. In all, 68 components were detected of which 57 were fully identified. Drying of the plant material had no effect on the qualitative composition of the oil and did not affect the ability of individuals familiar with marijuana smell to recognize the odor

Therapeutic and Medicinal Uses of Terpenes
Destinney Cox-Georgian, Niveditha Ramadoss, Chathu Dona, and Chhandak Basu
Book, Medicinal Plants, N. Joshee et al. (eds.), Chapter 15
DOI: 10.1007/978-3-030-31269-5
DOI: 10.1007%2F978-3-030-31269-5_15
Terpenes, also known as terpenoids are the largest and most diverse group of naturally occurring compounds. Based on the number of isoprene units they have, they are classified as mono, di, tri, tetra, and sesquiterpenes. They are mostly found in plants and form the major constituent of essential oils from plants. Among the natural products that provide medical benefits for an organism, terpenes play a major and variety of roles. The common plant sources of terpenes are tea, thyme, cannabis, Spanish sage, and citrus fruits (e.g., lemon, orange, mandarin). Terpenes have a wide range of medicinal uses among which antiplasmodial activity is notable as its mechanism of action is similar to the popular antimalarial drug in use—chloroquine. Monoterpenes specifically are widely studied for their antiviral property. With growing incidents of cancer and diabetes in modern world, terpenes also have the potential to serve as anticancer and antidiabetic reagents. Along with these properties, terpenes also allow for flexibility in route of administration and suppression of side effects. Certain terpenes were widely used in natural folk medicine. One such terpene is curcumin which holds anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic, and many other properties. Curcumin has also become a recent trend in healthy foods and open doors for several medical researches. This chapter summarizes the various terpenes, their sources, medicinal properties, mechanism of action, and the recent studies that are underway for designing terpenes as a lead molecule in the modern medicine

Thermal Degradation of Terpenes: Camphene, A3-Carene, Limonene, and ot=Terpinene
GERALD W. MCGRAW, RICHARD W. HEMINGWAY, LEONARD L. INGRAM, JR., CATHERINE S. CANADY, AND WILLIAM B. MCGRAW
VOL. 33, NO. 22,1999 I ENVIRONMENTAL SCIENCE & TECHNOLOGY
https://www.srs.fs.fed.us/pubs/ja/ja_mcgraw001.pdf
Emissions from wood dryers have been of some concern for a number of years, and recent policy changes by the Environmental Protection Agency have placed emphasis upon the gaseous emissions that lead to the formation of particulate matter as small as 2.5 pm diameter. In this qualitative study, camphene, AQarene, limonene, and a-terpinene were thermally degraded in the presence of airto determine the number and kind of oxidative degradation products that might be expected under drying conditions used in processing wood productsvarious chromatographic methods were used to isolate the products for proof of structure by NMR and/or GC-MS.The degradation products resulted from dehydrogenations, epoxidations, double bond cleavages, allylic oxidations, and rearrangements. A number of compounds not previously associated with the thermal degradation of these terpenes were identified

Tingenone, a pentacyclic triterpene, induces peripheral antinociception due to cannabinoid receptors activation in mice
C. C. Veloso, R. C. M. Ferreira, V. G. Rodrigues, L. P. Duarte, A. Klein
I. D. Duarte, T. R. L. Romero, A. C. Perez
Inflammopharmacol
DOI 10.1007/s10787-017-0391-7
Several works have shown that triterpenes induce peripheral antinociception by activation of cannabinoid receptors and endocannabinoids; besides, several research groups have reported activation of cannabinoid receptors in peripheral antinociception. The aim of this study was to assess the involvement of the cannabinoid system in the antinociceptive effect induced
by tingenone against hyperalgesia evoked by prostaglandin E2 (PGE2 ) at peripheral level. The paw pressure test was used and the hyperalgesia was induced by intraplantar injection of PGE2 (2 l g/paw). All drugs were injected subcutaneously in the hind paws of male Swiss mice. Tingenone (200 l g/paw) administered into the right hind paw induced a local antinociceptive effect, that was antagonized by AM630, a selective antagonist to CB2 cannabinoid receptor. AM251, a selective antagonist to CB1 cannabinoid receptor, did not alter the peripheral antinociceptive effect of tingenone. MAFP, a fatty acid amide hydrolase (FAAH) inhibitor; VDM11, an anandamide reuptake inhibitor; and JZL184, monoacylglycerol lipase (MAGL) inhibitor did not potentiate the peripheral antinociceptive effect of the lower dose of tingenone (50 l g/paw). The results suggest that tingenone induced a peripheral antinociceptive effect via cannabinoid receptor activation. Therefore, this study suggests a pharmacological potential for a new analgesic drug.

Toxicant Formation in Dabbing: The Terpene Story
Jiries Meehan-Atrash, Wentai Luo, Robert M Strongin
Project: Cannabis vaporizing September 2017 DOI: 10.1021/acsomega.7b01130
Inhalable, noncombustible cannabis products are playing a central role in the expansion of the medical and recreational use of cannabis. In particular, the practice of “dabbing” with butane hash oil has emerged with great popularity in states that have legalized cannabis. Despite their growing popularity, the degradation product profiles of these new products have not been extensively investigated. The study herein focuses on the chemistry of myrcene and other common terpenes found in cannabis extracts. Methacrolein, benzene, and several other products of concern to human health were formed under the conditions that simulated real-world dabbing. The terpene degradation products observed are consistent with those reported in the atmospheric chemistry literature.

Not Cannabis specific
Traversing the fungal terpenome
Maureen B. Quin, Christopher M. Flynn and Claudia Schmidt-Dannert
Nat. Prod. Rep., 2014, 31, 1449
DOI: 10.1039/c4np00075g
Fungi (Ascomycota and Basidiomycota) are prolific producers of structurally diverse terpenoid compounds. Classes of terpenoids identified in fungi include the sesqui-, di- and triterpenoids. Biosynthetic pathways and enzymes to terpenoids from each of these classes have been described. These typically involve the scaffold generating terpene synthases and cyclases, and scaffold tailoring enzymes such as e.g. cytochrome P450 monoxygenases, NAD(P)+ and flavin dependent oxidoreductases, and various group transferases that generate the final bioactive structures. The biosynthesis of several sesquiterpenoid mycotoxins and bioactive diterpenoids has been well-studied in Ascomycota (e.g. filamentous fungi). Little is known about the terpenoid biosynthetic pathways in Basidiomycota (e.g. mushroom forming fungi), although they produce a huge diversity of terpenoid natural products. Specifically, many trans-humulyl cation derived sesquiterpenoid natural products with potent bioactivities have been isolated. Biosynthetic gene clusters responsible for the production of transhumulylcation derived protoilludanes, and other sesquiterpenoids, can be rapidly identified by genome sequencing and bioinformatic methods. Genome mining combined with heterologous biosynthetic pathway refactoring has the potential to facilitate discovery and production of pharmaceutically relevant fungal terpenoids.

Ultrasound-assisted extraction of volatile compounds from industrial Cannabis sativa L. inflorescences
Da Porto C, Decorti D, Natolino A
International Journal of Applied Research in Natural Products
2014 Vol. 7 (1), pp. 8-14
doi: 10.1111/1750-3841.14075
This study investigated the use of ultrasound-assisted extraction (UAE) to recovery volatile compounds from the inflorescences of a fiber type Cannabis sativa L. cultivar. The results show that ultrasonic treatment not longer than 5 min allows to obtain an enhanced concentration of terpenes in comparison with maceration. Instead, an ultrasonic treatment longer than 5 min increased the concentration of ?-9-tetraidrocannabinol (THC). A preliminary screening of cannabis inflorescences scent was performed by headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS) avoiding the chemical modification and artifact formation that can occur in conventional methods.

Use of ABA/Methyl Jasmonate/Sugar to increase terpene and cannabinoid production in cannabis sativa
https://www.patentguru.com/US10874102B2
https://www.patentguru.com/US2018279611A1
https://patents.google.com/patent/US20180279611A1
The invention involves the use of a combination of s-abscisic acid (S-ABA), methyl jasmonate (MJ), glucose, sucrose and fructose to raise the level of cannabinoids and terpenes (flavor and scent molecules) in the Cannabis Sativa plant.

*UVB lights to increase terpenes and secondary metabolites
Peter Barber
GreenTech Amsterdam Rai 2018
https://issuu.com/amsterdamrai/docs/peter_barber.pptx

Variation in the compositions of cannabinoid and terpenoids in Cannabis sativa derived from inflorescence position along the stem and extraction methods.
Namdar, D., Mazuz, M., Ion, A., & Koltai, H.
Industrial Crops and Products, 113, 376–382.(2018).
doi:10.1016/j.indcrop.2018.01.060
In the last decade, recognition of the therapeutic abilities of Cannabis sativa has risen, along with the need to standardize its products. Standardization requires grading the methods for growing the plant and extracting the active compounds accumulated in its inflorescence. We explored the results of different methods used today and their effect on the levels of compounds extracted from inflorescences positioned along the C. sativa flowering stem. The polarity of the solvent used for the extraction, drying processes and separation methods influenced the chemical composition of the extract. However, regardless of extraction and analytical methods applied, the amounts of cannabinoids and terpenoids in the inflorescences decreased with the position of the sampled in- florescence from top to bottom of the flowering stem. These results have significant implications for the development of growth protocols for C. sativa cultivation and flower extraction methods to standardize cannabisbased products

Variations in Terpene Profiles of Different Strains of Cannabis sativa L
Salvatore Casano, Gianpaolo Grassi, V Martini, Marco Michelozzi
December 2011 Acta horticulturae 925(925):115-121
DOI: 10.17660/ActaHortic.2011.925.15
Secondary compounds of the plant are indispensable to cope with its often hostile environment and the great chemical diversity and variability of intraspecific and interspecific secondary metabolism is the result of natural selection. Recognition of the biological properties of secondary compounds have increased their great utility for human uses; numerous compounds now are receiving particular attention from the pharmaceutical industry and are important sources of a wide variety of commercially useful base products. Medical and other effects of Cannabis sativa L. are due to concentration and balance of various active secondary metabolites, particularly the cannabinoids, but including also a wide range of terpenoids and flavonoids. A wide qualitative and quantitative variability in cannabinoids, terpenoids, and flavonoids contents in Cannabis species are apparent from reports in the literature. Terpenes are strongly inherited and little influenced by environmental factors and, therefore, have been widely used as biochemical marker in chemosystematic studies to characterize plant species, provenances, clones, and hybrids. This study investigated the variability of terpene profiles in C. sativa. The terpene composition in inflorescences of samples collected from progenies of 16 plants derived from different strains was analysed by GC/FID. The amount of each terpene (in sufficient quantities to be considered in statistical analysis) was expressed as a percentage of total terpenes. Results showed a large variation between different strains in the relative contents for several monoterpenes (?-pinene, camphene, ?-pinene, sabinene, ?-3-carene, ?-phellandrene, ?-myrcene, ?-terpinene, limonene, 1.8-cineole, ?-terpinene, cis-?-ocimene, trans-?- ocimene, ?-terpinolene) and one sesquiterpene, ?-caryophyllene. This variability in terpene composition can provide a potential tool for the characterization of Cannabis biotypes and warrant further research to evaluate the drug’s medical value and, at the same time, to select less susceptible chemotypes to the attack of herbivores and diseases.

Part 2, Which Terpenes Are in ‘Haze’ Strains?
WILL HYDE
https://www.leafly.ca/news/strains-p...-strain-family
In this series, Leafly explores what makes each family of strains unique based on their terpene profiles. A strain “family” refers to a line of hybrids branching from one genetic matriarch that expresses unique and desirable characteristics that breeders seek to build upon. This introductory primer will help you learn a little more about cannabis breeding and strain variability

Not Cannabis specific
The role of structure and molecular properties of terpenoids in determining their antimicrobial activity.
Griffin, S. G., Wyllie, S. G., Markham, J. L., & Leach, D. N.
Flavour and Fragrance Journal, 14(5), 322–332.(1999).
doi:10.1002/(sici)1099-1026(199909/10)14:5<322::aid-ffj837>3.0.co;2-4
The minimum inhibitory concentrations (MIC) of 60 terpenoids against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans have been determined. Hierarchical cluster analysis was used to group the compounds into ®ve groups according to their activity patterns against the four microorganisms. K-Means cluster analysis was then used to con®rm these groupings and to show the di€erences in the activity patterns of the groups. Ten molecular properties of the terpenoids, either calculated via molecular modelling or determined by direct measurement, were then used as variables in a forward stepwise discriminant analysis to identify which variables discriminated between groups. Low water solubility of Group IV compounds, mainly hydrocarbons and acetates, was found to be associated with their relative inactivity. The remaining groups, all containing oxygenated terpenoids, showed characteristic but distinct activity patterns towards the four test organisms. Hydrogen bonding parameters were found to be associated with antimicrobial activity in all cases. Activity against Gram-negative E. coli and P. aeruginosa was associated with a combination of a hydrogen bonding and size parameters. This was not found to be the case for the Gram-positive S. aureus or the yeast C. albicans.

The sedative effect of inhaled terpinolene in mice and its structure–activity relationships.
Ken Ito, Michiho Ito
Journal of Natural Medicines 67(4) January 2013
DOI: 10.1007/s11418-012-0732-1
Terpinolene is a cyclic monoterpene compound found in some Labiatae herbs. In our previous study, we evaluated the sedative effect of inhaled essential oils of Microtoena patchoulii leaves in mice and isolated terpinolene as an active ingredient. We investigated the structure-activity relationships of terpinolene to identify the structural part essential to its sedative effect. Comparison of terpinolene analog activities showed that a double bond in the side-chain or pi bonds in the six-membered ring play important roles in the sedative effect. In another experiment using olfactory impaired mice, we further revealed that inhaled terpinolene exerted the effect after nasal absorption into the body.

Valorizing industrial hemp (Cannabis sativa L.) by-products: Cannabidiol enrichment in the inflorescence essential oil optimizing sample pre-treatment prior to distillation.
Fiorini, Dennis; Molle, Arnaud; Nabissi, Massimo; Santini, Giuseppe; Benelli, Giovanni; Maggi, Filippo
Industrial Crops and Products, 128(), 581–589. (2019).
doi:10.1016/j.indcrop.2018.10.045
By products of industrial hemp (Cannabis sativa L.), including inflorescences, represent an exploitable material to produce niche products for the pharmaceutical, nutraceutical, cosmetic and pesticide industry. One of them is the essential oil, whose composition can be properly modulated on an industrial level by optimizing the extractive conditions and sample pretreatment. This allows to achieve high concentrations of bioactive compounds, such as cannabidiol (CBD) and sesquiterpenes [e.g. (E)-caryophyllene]. In the present work, we evaluated the effects of type of distillation apparatus, status of conservation of the plant material, grinding and sample-pretreatment with microwave and heat, on the hemp essential oil chemical profile obtained from the monoecious cultivar Felina 32. Seven marker compounds, including the monoterpenes α-pinene, myrcene and terpinolene, the sesquiterpenes (E)-caryophyllene, α-humulene and caryophyllene oxide, and the cannabinoid CBD were quantified in the different hemp essential oil samples by gas chromatography-flame ionization detection (GC-FID) analysis, whereas the overall chemical profiles were achieved by gas chromatography-mass spectrometry (GC–MS) analysis. Results showed that hydrodistillation (HD) in comparison with steam distillation (SD) gave a higher content of cannabinoids. Drying was fundamental to induce decarboxylation of cannabinoid acids to the relative alcoholic forms, coupled with an increase of the sesquiterpene fraction. The optimization of sample pretreatments pointed out that the exposure of dry inflorescences to microwave heating at 900 W power for 1 min was the best method to increase the abundance of bioactive compounds in the essential oil, with special reference to CBD, (E)-caryophyllene and caryophyllene oxide. Overall, these results give new insights into the exploitation of hemp byproducts in different fields such as pharmaceuticals, nutraceuticals and eco-friendly insecticides.

Vapor Phase Terpenes Mitigate Oxidative Degradation of Cannabis sativa Inflorescence Cannabinoid Content in an Accelerated Stability Study
Justin Bueno, Solmaz Alborzi, and Eric A. Greenbaum Cannabis and Cannabinoid Research Volume X, Number X, 2022
DOI: 10.1089/can.2021.0207
Introduction: As Cannabis sativa L. (Cannabaceae) ages, inflorescence phytochemicals are susceptible to oxidative degradation. Reduction of D9-tetrahydrocannabinol (D9-THC) content has the potential to impact the reliability and accuracy of dosing. Advances that improve cannabinoid stability during storage would have an important impact in medical cannabis markets. Reported here is the use of C. sativa terpenes with antioxidant properties that improve inflorescence cannabinoid stability.
Materials and Methods: Killer Kush inflorescence samples were stored in a temperature-controlled environment, in opaque jars. To accelerate the rate of oxidate degradation, samples were stored with the oxidizing agent hydrogen peroxide. Vapor phase terpenes were added to inflorescence packaging. Two terpene blends and three different dosage amounts were evaluated. Inflorescence stability samples were prepared in triplicate for each sample type. Cannabinoid content was quantitatively assessed after 24, 81, and 127 days of storage using high-performance liquid chromatography. Terpene content was assessed using headspace gas chromatography mass spectrometry. Results from inflorescence stored with and without external terpenes were compared by analysis of variance (ANOVA) data processing.
Results: After 127 days of storage, inflorescence in the accelerated study experienced a loss of 18.0% and 34.3% total D9-THC content for samples stored with and without external terpenes, respectively. The differences in cannabinoid content were found to be statistically significant at all time points using ANOVA processing. In the non accelerated study, only one of the six sample types investigated had a statistically significant greater total D9-THC content than control at all time points. Nevertheless, a dose-dependent relationship between the amount of external terpenes added to inflorescence and the preservation of total D9-THC content was observed.
Discussion: In the accelerated study, exogenous terpenes reduced the degradation of inflorescence cannabinoid content by 47.4%. This represents the first reported addition of terpene antioxidants to inflorescence packaging for cannabinoid preservation. Of note, the antioxidants used in this system can be obtained from C. sativa. This is advantageous from a toxicological perspective as inhaling synthetic antioxidants presents unknown and unpredictable risks. When fully developed, the novel system has applications for inflorescence packaged for individual sale, as well as long-term storage of bulk biomass.
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bsgospel

Bat Macumba
Veteran
https://www.nature.com/articles/s41593-019-0416-1

Genome-wide association study implicates CHRNA2 in cannabis use disorder

"Cannabis is the most frequently used illicit psychoactive substance worldwide; around one in ten users become dependent. The risk for cannabis use disorder (CUD) has a strong genetic component, with twin heritability estimates ranging from 51 to 70%."



I will add it-SamS
https://sci-hub.tw/10.1038/s41593-019-0416-1

Genome-wide association study implicates CHRNA2 in cannabis use disorder
Nat Neurosci. 2019 Jun 17. doi: 10.1038/s41593-019-0416-1. [Epub ahead of print]
Ditte Demontis , Veera Manikandan Rajagopal , Thorgeir E. Thorgeirsson , Thomas D. Als , Jakob Grove, Kalle Leppälä, Daniel F. Gudbjartsson , Jonatan Pallesen, Carsten Hjorthøj , Gunnar W. Reginsson, Thorarinn Tyrfingsson, Valgerdur Runarsdottir, Per Qvist , Jane Hvarregaard Christensen, Jonas Bybjerg-Grauholm , Marie Bækvad-Hansen, Laura M. Huckins , Eli A. Stahl , Allan Timmermann, Esben Agerbo, David M. Hougaard , Thomas Werge, Ole Mors, Preben Bo Mortensen, Merete Nordentoft, Mark J. Daly, Hreinn Stefansson , Kari Stefansson , Mette Nyegaard, and Anders D. Børglum 
Cannabis is the most frequently used illicit psychoactive substance worldwide; around one in ten users become dependent. The risk for cannabis use disorder (CUD) has a strong genetic component, with twin heritability estimates ranging from 51 to 70%. Here we performed a genome-wide association study of CUD in 2,387 cases and 48,985 controls, followed by replication in 5,501 cases and 301,041 controls. We report a genome-wide significant risk locus for CUD (P= 9.31 × 10−12) that replicates in an independent population (Preplication= 3.27 × 10−3, Pmeta-analysis= 9.09 × 10−12). The index variant (rs56372821) is a strong expression quantitative trait locus for cholinergic receptor nicotinic α2 subunit (CHRNA2); analyses of the genetically regulated gene expression identified a significant association of CHRNA2 expression with CUD in brain tissue. At the polygenic level, analyses revealed a significant decrease in the risk of CUD with increased load of variants associated with cognitive performance. The results provide biological insights and inform on the genetic architecture of CUD.
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
IC Trichomes


Accumulation of Cannabinoids in Glandular Trichomes of Cannabis(Cannabaceae).
Mahlberg, P. G., & Kim, E. S.
Journal of Industrial Hemp, 9(1), 15–36. (2004).
doi:10.1300/j237v09n01_04
Sessile- and capitate-stalked secretory glands are sites of cannabinoid accumulation in Cannabis (Cannabaceae). Analyses show cannabinoids to be abundant in glands isolated from bracts or leaves of pistillate plants. Cannabinoids are concentrated in the secretory cavity formed as an intrawall cavity in the outer wall of the disc cells. Specialized plastids, lipoplasts, in the disc cells synthesize lipophilic substances, such as terpenes, that migrate through the plasma membrane and into the cell wall adjacent to the secretory cavity. These substances enter the cavity as secretory vesicles. An antibody probe for THC shows it to be most abundant along the surface of vesicles, associated with fibrillar material in the cavity, in the cell wall and in the cuticle; little THC was detected in the cytoplasm of disc or other cells. The phenol, phloroglucinol, is abundant in both gland types. A working hypothesis for the site of cannabinoid synthesis is proposed, and must be examined further. Knowledge of the mechanism of cannabinoid synthesis and localization can contribute to efforts to further reduce the THC content in hemp strains for potential agricultural use in the United States and elsewhere.

An Investigation of Potential Staining Reagents for the Glandular Trichomes of Cannabis sativa.
Corrigan, D., & Lynch, J.
Planta Medica, 40(S 1), 163–169. (1980).
doi:10.1055/s-2008-1075020
Various chromogenic chemical tests for Cannabis were evaluated as stains for the microscopical identification of the glandular trichomes of Cannabis sativa. Only vanillin in ethanolic sulphuric acid gave results comparable with the staining obtained with Fast Blue B. The specificity of these two stains was examined using 222 nonCannabis species from 21 selected plant families. None of the species possessed glandular trichomes which stained with Fast Blue B. Two Mentha and three Callicarpa species had stained glandular trichomes with the vanillin reagent. It is concluded that Fast Blue B is a very specific stain for the cannabinoids in the glandular trichomes of C. sativa L.

Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR
Nizar Happyanaa, b, Sara Agnoletc, Remco Muntendamd, Annie Van Dame, Bernd Schneiderc, Oliver Kaysera, Emil-Figge
Phytochemistry
Doi: 10.1016/j.phytochem.2012.11.001
Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8 weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25–143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. ?9-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, ?9-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.

not Cannabis specific
Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin.
Teoh KH, Polichuk DR, Reed DW, Nowak G, Covello PS.
FEBS Lett. 2006 Feb 20;580(5):1411-6. Epub 2006 Jan 30.
DOI: 10.1016/j.febslet.2006.01.065
Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.

Not Cannabis specific
Assessing Flux Distribution Associated with Metabolic Specialization of Glandular Trichomes.
Zager, J. J., & Lange, B. M.
Trends in Plant Science, 23(7), 638–647.(2018).
doi:10.1016/j.tplants.2018.04.003
Many aromatic plants accumulate mixtures of secondary (or specialized) metabolites in anatomical structures called glandular trichomes (GTs). Different GT types may also synthesize different mixtures of secreted metabolites, and this contributes to the enormous chemical diversity reported to occur across species. Over the past two decades, significant progress has been made in characterizing the genes and enzymes that are responsible for the unique metabolic capabilities of GTs in different lineages of flowering plants. Less is known about the processes that regulate flux distribution through precursor pathways toward metabolic end-products. We discuss here the results from a meta-analysis of genome-scale models that were developed to capture the unique metabolic capabilities of different GT types.

A Temporal Study of Cannabinoid Composition in Continual Clones of Cannabis sativa L. (Cannabaceae).
Turner, J. C., Mahlberg, P. G., Lanyon, V. S., & Pleszczynska, J.
Botanical Gazette, 146(1), 32–38. (1985).
doi:10.1086/337497
Genetically and developmentally defined vegetative samples of three clones of Cannabis sativa L. were grown in a common environment and analyzed for cannabinoid production. Significant variations occurred in cannabinoid levels in each clonal population. Throughout the 2-yr study, the cannabinoid fluctuations were random rather than cyclic. Although within each clone all cannabinoids increased or decreased simultaneously, fluctuations in cannabinoid levels occurred independently from clone to clone. In addition, each clone retained its distinctive morphology and cannabinoid profile throughout the study.

Cannabinoid composition and gland distribution in clone of Cannabis sativa L. (Cannabaceae)
Jocelyn C. TURNER,, John K. HEMPHILL, Paul G. MAHLBERG
Bulletin on Narcotics, Vol. XXX, No. 1
https://www.unodc.org/unodc/en/data-...1_page007.html
https://pdfs.semanticscholar.org/d76...7b733bdba3.pdf
Gladnular trichomes covering the plant surface have been implicated as the source at cannabinoids in Cannabis sativa L. (DePasquale, 1974: Malingre et al., 1975). Harmmond and Mahlberg (1973,1977), in their scanning electron microscope study of these trichomes, described three gland types: the bulbous gland which consists of a large multi-cellular head, and the capitate-stalked gland which consists of a large multi-cellular head, that terminates a stalk of variable height. Non-glandular trichomes are also present in abundance on the plant epiderrmis (Ledbetter and Krikorian, 1975). Fairbairn (1972) reported the presence of cannabionds in both capitate-sessile and capitate-stalked glands and indicated that capitate-stalked glands were the major cannabinoid containing glands. DePasquale (1974), in an ultrastructural-study of be a capitate-stalked glands, interpreted the secretory part of the gland to be combination of the gland head and perhaps apical stalk cells. Malingre et al. (1975) concluded that cannabinoids were present mainly in the epidermal glands and not in mesophyll cells or non-glandular trichomes, although there was some indication of cannabinoid content in the least mid-rib.
The purpose of this investigation is to determine whether a correlation exists between glandular trichomes and cannabinoid content in cannabis. If specific glands are associated with cannabinoid content, a correlation should exist between the gland number present on a particular plant and the cannabinoid content of that part. Futhermore, it should also be possible to establish a correlation between the number of each gland type and cannabinoid content.

Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes.
Rodziewicz, P., Loroch, S., Marczak, ?., Sickmann, A., & Kayser, O.
Plant Science. Volume 284, July 2019, Pages 108-116 (2019)
doi:10.1016/j.plantsci.2019.04.008
Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ~65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes.

Cannabis glandular trichomes alter morphology and metabolite content during flower maturation.
Livingston, S. J., Quilichini, T. D., Booth, J. K., Wong, D. C. J., Rensing, K. H., Laflamme?Yonkman, J., … Samuels, A. L.
The Plant Journal. (2019).
doi:10.1111/tpj.14516
The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile-like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two-photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12–16 secretory disc cells and strongly monoterpene-dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed redshifted autofluorescence, eight secretory disc cells and less monoterpene-dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co-expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products.

Cannabis trichomes – a cellular metabolite factory
Cailun A. Tanney, Rachel G. Backer, Anja Geitmann, Donald L. Smith
Front. Plant Sci. (2021)
doi: 10.3389/fpls.2021.721986
https://www.frontiersin.org/articles...21986/abstract
Cannabis has been legalized for recreational use in several countries and medical use is authorized in an expanding list of countries; markets are growing internationally, causing an increase in demand for high quality products with well-defined properties. The key compounds of Cannabis plants are cannabinoids, which are produced by stalked glandular trichomes located on female flowers. These trichomes produce resin that contains cannabinoids such as tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), and an array of other secondary metabolites of varying degrees of commercial interest. While growing tend to focus on improving whole flower yields, our understanding of the “goldmines” of the plant – the trichomes – is limited despite their being the true source of revenue for a multi-billion-dollar industry. This review aims to provide an overview of our current understanding of cannabis glandular trichomes and their metabolite products in order to identify current gaps in knowledge and to outline future research directions.
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Characterization of the Cannabis sativa glandular trichome proteome
Lee James ConneelyID, Ramil Mauleon, Jos Mieog, Bronwyn J. Barkla, Tobias Kretzschmar
PLoS ONE 16(4): e0242633.
DOI: 10.1371/journal.pone.0242633
Cannabis sativa has been cultivated since antiquity as a source of fibre, food and medicine. The recent resurgence of C. sativa as a cash crop is mainly driven by the medicinal and therapeutic
properties of its resin, which contains compounds that interact with the human endocannabinoid system. Compared to other medicinal crops of similar value, however, little is known about the biology of C. sativa. Glandular trichomes are small hair-like projections made up of stalk and head tissue and are responsible for the production of the resin in C. sativa. Trichome productivity, as determined by C. sativa resin yield and composition, is only beginning to be understood at the molecular level. In this study the proteomes of glandular trichome stalks and heads, were investigated and compared to the proteome of the whole flower tissue, to help further elucidate C. sativa glandular trichome biochemistry. The data suggested that the floral tissue acts as a major source of carbon and energy to the glandular trichome head sink tissue, supplying sugars which drive secondary metabolite biosynthesis. The trichome stalk seems to play only a limited role in secondary metabolism and acts as both source and sink.

Chemical fingerprinting of single glandular trichomes of Cannabis sativa by Coherent anti-Stokes Raman scattering (CARS) microscopy
Paul Ebersbach, Felix Stehle, Oliver Kayser and Erik Freier
BMC Plant Biology (2018) 18:275
Doi: 10.1186/s12870-018-1481-4
Backround: Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type.
Results: Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise ?9-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures.
Conclusion: Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis-related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.

CUTICLE DEVELOPMENT ON GLANDULAR TRICHOMES OF CANNABIS SATIVA (CANNABACEAE).
Mahlberg, P. G., & Kim, E.-S.
American Journal of Botany, 78(8), 1113–1122. (1991)
doi:10.1002/j.1537-2197.1991.tb14518.x
DOI: 10.2307/2444899
The dermal sheath of glandular trichomes of Cannabis sativa L., consisting of cuticle and a subcuticular wall, was examined by transmission electron microscopy. Cuticle thickened selectively on the outer wall of disc cells of each trichome prior to formation of the secretory cavity, where as thickening was less evident on the dermal cells of the bract. Membraned secretory vesicles that differ in size and appearance in the secretory cavity were the source of precursors for synthesis of cuticle. Vesicle contents, released following the degradation of the vesicle membrane upon contact with the subcuticular wall, contributed to both structured and amorphous phases of cuticle development. The structured phase was represented by deposition and thickening of cuticle at the subcuticular wall-cuticle interface to form a thickened cuticle. In the amorphous phase precursors permeated the cuticle in a liquid state, as shown by fusion of cuticles and wax layers between contiguous glands, and may have contributed to growth in surface area of the expanding sheath. Disc cells are interpreted to control growth of secretory cavity by secretion ofmembraned vesicles into the cavity. The thickened cuticle, which increased eightfold in thickness during enlargement of the gland, provided structural strength for the extensive surface area of the dermal sheath. The gland of Cannabis in which vesicle contents contribute to the growth in thickness and surface area ofthe cuticle of the sheath is interpreted to represent a phylogenetically derived state as contrasted to secretory glands possessing only cuticle and lacking a complement of secretory vesicles.

Cytochemical localization of cellulase in glandular trichomes of cannabis (cannabaceae).
Kim, E.-S., & Mahlberg, P. G.
Journal of Plant Biology, 40(1), 61–66. (1997).
doi:10.1007/bf03030322
Cellulase reaction product was localized cytochemically at the ultrastructural level in the cell wall of disc cells, the secretory cavity and in the subcuticular wall of glands in Cannabis. Cellulase reaction product was evident in the less dense region of the disc cell wall prior to secretory cavity formation. Reactivity in this region was associated with separation of an outer zone, forming the subcuticular wall, from the inner wall zone adjacent to the plasma membrane of the disc cells. Reaction product was associated with the disc cell wall and fibrillar matrix extending from it into the secretory cavity. Reactivity remained evident over the subcuticular wall throughout enlargement of the secretory cavity. Reaction product also was present over fibrillar matrix in the secretory cavity associated with both the inner wall and the subcuticular wall. The distribution of cellulase reaction product supports an interpretation that cellulase is involved in formation of the secretory cavity and subsequent redistribution of wall products to form the subcuticular wall during development of the secretory cavity.

not Cannabis specific
Ecophysiology of leaf trichomes
Christopher P. Bickford
Functional Plant Biology, 2016, 43, 807–814 Review
doi: /10.1071/FP16095
This review examines how leaf trichomes influence leaf physiological responses to abiotic environmental drivers. Leaf trichomes are known to modulate leaf traits, particularly radiation absorptance, but studies in recent decades have demonstrated that trichomes have a more expansive role in the plant–environment interaction. Although best known as light reflectors, dense trichome canopies modulate leaf heat balance and photon interception, and consequently affect gas exchange traits. Analysis of published studies shows that dense pubescence generally increases reflectance of visible light and near-infrared and infrared radiation. Reflective trichomes are also protective, reducing photoinhibition and UV-B related damage to leaf photochemistry. Little support exists for a strong trichome effect on leaf boundary layer
resistance and transpiration, but recent studies indicate they may play a substantive role in leaf water relations affecting leaf wettability, droplet retention and leaf water uptake. Different lines of evidence indicate that adaxial and abaxial trichomes may function quite differently, even within the same leaf. Overall, this review synthesises and re-examines the diverse
array of relevant studies from the past 40 years, illustrating our current understanding of how trichomes influence the energy, carbon and water balance of plants, and highlighting promising areas for future research.

Not Cannabis specific
EST Analysis of Hop Glandular Trichomes Identifies an O-Methyltransferase That Catalyzes the Biosynthesis of Xanthohumol.
Nagel, J., Culley, L. K., Lu, Y., Liu, E., Matthews, P. D., Stevens, J. F., & Page, J. E.
THE PLANT CELL ONLINE, 20(1), 186–200.(2008).
doi:10.1105/tpc.107.055178
The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-L-methionine–dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes.

Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-a-pinene synthase from Cannabis sativa
Nils Gu?nnewich,Jonathan E. Page, Tobias G. Köllner, Jörg Degenhardt, Toni M. Kutchana
Natural product communications · March 2007 Natural Product Communications Vol. 0 (0) 2006
https://www.researchgate.net/profile...bis-sativa.pdf
Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-?-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 shows a Km value at 6.8 ?M and a Kcat at 8.2 x 10-2 s-1, the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 shows a Km values at 6.7 ?M and a Kcat at 8.1 x 10-2 s-1, the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperm family and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-?-pinene were detected as natural products in C. sativa trichomes.

Gene Networks Underlying Cannabinoid and Terpenoid Accumulation in Cannabis.
Zager, J. J., Lange, I., Srividya, N., Smith, A., & Lange, B. M.
Plant Physiology, pp.01506.2018. (2019).
doi:10.1104/pp.18.01506
Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant–environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell and genetic origin. Glandular trichomes were isolated from each of these strains and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from imported precursors. Terpene synthase genes involved in the biosynthesis of the major mono- and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT ((-)-limonene synthase) and CsTPS15CT (?-myrcene synthase) we functionally evaluated genes that encode enzymes with activities not previously described in cannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases); CsTPS16CC (germacrene B synthase); and CsTPS20CT (hedycaryol synthase). This study lays the groundwork or developing a better understanding of the complex chemistry and biochemistry underlying resin accumulation across commercial cannabis strains.

Gland Distribution and Cannabinoid Content in Clones of Cannabis sativa L.
Turner, J. C., Hemphill, J. K., & Mahlberg, P. G.
American Journal of Botany, 64(6), 687.doi:10.2307/2441721 (1977).
The relationship between glandular trichomes and cannabinoid content in Cannabis sativa L. was investigated. Three strains of Cannabis, which are annuals, were selected for either a drug, a non-drug, or a fiber trait and then cloned to provide genetically uniform material for analyses over several years. The distribution of the number and type of glands was determined for several organs of different ages including the bract and its subtending monoleaflet leaf and the compound leaf on pistillate plants. Quantitation of glands on these structures was integrated with gas chromatographic analyses of organ cannabinoid profiles. A negative correlation was found between cannabinoid content and gland number for each of the three clones. Isolated heads of the capitate-stalked glands also were analyzed for cannabinoid content and found to vary in relation to clone and gland age. These studies indicate that cannabinoids may occur in plant cells other than glandular trichomes. The results of these studies emphasize the need for stringent sampling procedures in micromorphological studies on trichome distribution and analytical determinations ofcannabinoid content in Cannabis.

GLANDULAR CUTICLE FORMATION IN CANNABIS (CANNABACEAE).
Kim, E.-S., & Mahlberg, P. G. (1995).
American Journal of Botany, 82(10), 1207–1214.
doi:10.1002/j.1537-2197.1995.tb12653.x
Formation of the cuticle from components of the secretory cavity and subcuticular wall was studied by transmission electron microscopy of glandular trichomes of Cannabis prepared by high pressure cryofixation-cryosubstitution. Secretory vesicles in the secretory cavity resembled those localized in the subcuticular wall as well as the vesicle-related material associated with the irregular inner surface of the cuticle and appeared to provide precursors for thickening of the cuticle. Some contiguous vesicles in the secretory cavity and subcuticular wall lacked a surface feature at their point of contact, supporting an interpretation of vesicle fusion. Fibrillar matrix from the secretory cavity contributed fibrillar matrix to the subcuticular wall, and persisted as residual fibrillar matrix associated with secretory materials coalesced to the thickened inner surface of the cuticle. Elongated fibrils arranged in uniformly spaced parallel pairs contributed to the organization of fibrillar matrix in the subcuticular wall. Striae were evident in the outer portion of the cuticle, and appeared to represent sites of degraded residual fibrillar matrix associated with secretory materials coalesced to the inner cuticular surface. This study supports an interpretation that contents of secretory vesicles from the secretory cavity contribute to formation of glandular cuticle.

Glandular trichomes: what comes after expressed sequence tags?
Alain Tissier
The Plant Journal (2012) 70, 51–68
doi: 10.1111/j.1365-313X.2012.04913.x
Glandular trichomes cover the surface of many plant species. They exhibit tremendous diversity, be it in their shape or the compounds they secrete. This diversity is expressed between species but also within species or even individual plants. The industrial uses of some trichome secretions and their potential as a defense barrier, for example against arthropod pests, has spurred research into the biosynthesis pathways that lead to these specialized metabolites. Because complete biosynthesis pathways take place in the secretory cells, the establishment of trichome-specific expressed sequence tag libraries has greatly accelerated their elucidation. Glandular trichomes also have an important metabolic capacity and may be considered as true cell factories. To fully exploit the potential of glandular trichomes as breeding or engineering objects, several research areas will have to be further investigated, such as development, patterning, metabolic fluxes and transcription regulation. The purpose of this review is to provide an update on the methods and technologies which have been used to investigate glandular trichomes and to propose new avenues of research to deepen our understanding of these specialized structures.

Histochemical Analyses of Laticifers and Glandular Trichomes in Cannabis sativa.
Furr, M., & Mahlberg, P. G.
Journal of Natural Products, 44(2), 153–159. (1981).
doi:10.1021/np50014a002
The unbranched nonarticulated laticifer, including its latex, and capitate glandular trichomes from Cannabis satirma L. were analyzed in fresh and cryostat preparations with histochemical procedures for the presence of cannabinoids, alkaloids, and other selected cellular components. A positive response to cannabinoid indicators, Duquenois-Segm, fast blue salt B, Gibb, and Beam reagents occurred in laticifers, as well as exuded latex and in disc cells of epidermal capitate glandular trichomes. No response or only an apparent background response to these reagents was detected in other cells. Alkaloids were detected histochemically in laticifers and exuded latex a-ith Wagner, Dittmar, Ellram, chromic acid, Hager, and Dragendorff reagents. Alkaloid indicators also reacted with capitate glandular trichomes but did not show- a positive response in other cells of the plant. Laticifers also contained other specialized contents including the enzymes, cytochrome oxidase, and lipase. Free lipids and storage proteins n-ere not detected in laticifers. Qualitative responses to these histochemical procedures were similar in laticifers and capitate glandular trichomes from various regions and organs of the plant axis. Histochemical indicators can be useful for preliminary surveying of specialized cell types and tissues for the presence of specialized substances.

H NMR-Based Metabolomics and Cannabinoids Analysis of Medicinal Cannabis Trichomes during Flowering Period
Nizar Happyana, Oliver Kayser

August 2014
Conference: The 29th International Horticultural CongressAt: Brisbane, AustraliaVolume: Abstract Book
Project: Studies on Cannabis sativa and Cannabinoids
Cannabis sativa L. trichomes are known as the main site of cannabinoids production, which are the responsible compounds for most biological activities of the plant. This study reports 1 H NMR based-metabolomics and cannabinoids analysis of trichomes of four medicinal Cannabis varieties, Bediol, Bedica, Bedrobinol, and Bedrocan, in order to investigate cannabinoids production and metabolites profiles of the trichomes during the last four weeks of flowering period. Analysis of 1 H NMR spectra (Figure 1) revealed totally six identified cannabinoids in the chloroform extracts, ? 9 -tetrahydrocannabinolic acid [THCA], cannabidiolic acid [CBDA], cannabichromenic acid [CBCA], cannabigerolic acid [CBGA], ? 9 -tetrahydrocannabinol [THC] and cannabidiol [CBD], and 20 compounds in the water extracts including sugars, amino acids, and other acidic compounds.

Immunochemical localization of tetrahydrocannabinol (THC) in chemically fixed glandular trichomes of Cannabis (Cannabaceae).
Kim, E. S., & Mahlberg, P. G.
Korean Journal of Biological Sciences, 3(2), 215–219.(1999).
doi:10.1080/12265071.1999.9647488
Monoclonal antibody for delta-9-tetrahydrocannabiol (THC Ab), conjugated with protein A-gold, was employed as a probe to detect THC localization in the gland and subjacent cells of chemically fixed bracts of Cannabis. THC was detected in the outer wall of the disc cells, fibrillar matrix, the surface feature of secretory vesicles, and sheath throughout development of the secretory cavity. The probe was absent from vesicles. Label was also present in anticlinal walls of disc cells and walls of dermal and mesophyll cells. Little or no THC Ab was present in disc cells and none were detected in control tissues. This distribution pattern of THC Ab was similar to that in tissues prepared by high pressure cryofixation-cryosubstitution. Consistent association of THC with wall and wall-derived materials suggests that cannnabinoids are synthesized outside the plasma membrane and bound to a wall component, where-upon they are transported to the cavity with wall materials released from the disc cell wall during development of the secretory cavity.

Immunochemical localization of Tetrahydrocannabinol (THC) in Cryofixed Glandular Trichomes of Cannabis (Cannabaceae).
Kim, E.-S., & Mahlberg, P. G.
American Journal of Botany, 84(3), 336–342. (1997).
doi:10.2307/2446007
Delta 9-tetrahydrocannabinol (THC) localization in glandular trichomes and bracteal tissues of Cannabis, prepared by high pressure cryofixation-cryosubstitution, was examined with a monoclonal antibody-colloidal gold probe by electron microscopy (EM). The antibody detected THC in the outer wall of disc cells during the presecretory cavity phase of gland development. Upon formation of the secretory cavity, the immunolabel detected THC in the disc cell wall facing the cavity as well as the subcuticular wall and cuticle throughout development of the secretory cavity. THC was detected in the fibrillar matrix associated with the disc cell and with this matrix in the secretory cavity. The antibody identified THC on the surface of secretory vesicles, but not in the secretory vesicles. Gold label also was localized in the anticlinal walls between adjacent disc cells and in the wall of dermal and mesophyll cells of the bract. Grains were absent or detected only occasionally in the cytoplasm of disc or other cells of the bract. No THC was detected in controls. These results indicate THC to be a natural product secreted particularly from disc cells and accumulated in the cell wall, the fibrillar matrix and surface feature of vesicles in the secretory cavity, the subcuticular wall, and the cuticle of glandular trichomes. THC, among other chemicals, accumulated in the cuticle may serve as a plant recognition signal to other organisms in the environment.

Interrelationships of glandular trichomes and cannabinoid content. I : Developing pistillate bracts of Cannabis sativa L. (Cannabaceae)
J.C. TURNER, J. K. HEMPHILL and P. G. MAHLBERG
Bulletin on Narcotics, Vol. XXXIII, No. 2
https://www.realhemp.com/wp-content/...annabaceae.pdf
Gland number and cannabinoid content for three clones of Cannabis were compared. Cannabinoid levels and mature glands were quantified throughout progressive stages of pistillate bract development in order to define possible interrelationships between glands and cannabinoids. Similar trends occurred among the clones for each gland type while individual cannabinoids displayed a different pattern in each clone. Gland initiation and development as well as cannabinoid synthesis were found to occur during bract ontogeny for all three clones. A positive correlation existed for the total number of capitate glands per bract compared to the total cannabinoid content of the bract. The study also suggested that the glands may contain the majority of the cannabinoidspresent in the bract.

Interrelationships of glandular trichomes and cannabinoid content II. Developing vegetative leaves of Cannabis sativa L. (Cannabaceae)
J.C. TURNER, J. K. HEMPHILL and P. G. MAHLBERG
Bulletin on narcotics 33(2):59-69
https://pdfs.semanticscholar.org/ceb...5dfd7383b9.pdf
Gland number and cannabinoid content were quantified during ontogeny of vegetative leaves from three clones of Cannabis. Initiation of capitatesessile and bulbous glands was found to occur uniformly during leaf development. Cannabinoids were synthesized throughout leaf development as well, but at a decreasing rate. A positive correlation was found for total capitate-sessile glands per leaf as compared with total cannabinoid content of the leaf. The data also indicated that other leaf tissues in addition to the glands may contain cannabinoids.

NOT CANNABIS SPECIFIC but applicable to CANNABIS
Metabolic, Genomic, and Biochemical Analyses of Glandular Trichomes from the Wild Tomato Species Lycopersicon hirsutum Identify a Key Enzyme in the Biosynthesis of Methylketones
Eyal Fridman, Jihong Wang, Yoko Iijima, John E. Froehlich, David R. Gang,
John Ohlrogge, and Eran Pichersky,
The Plant Cell, Vol. 17, 1252–1267, April 2005,
Medium-length methylketones (C7-C15) are highly effective in protecting plants from numerous pests. We used a biochemical genomics approach to elucidate the pathway leading to synthesis of methylketones in the glandular trichomes of the wild tomato Lycopersicon hirsutum f glabratum (accession PI126449). A comparison of gland EST databases from accession PI126449 and a second L. hirsutum accession, LA1777, whose glands do not contain methylketones, showed that the expression of genes for fatty acid biosynthesis is elevated in PI126449 glands, suggesting de novo biosynthesis of methylketones. A cDNA abundant in the PI126449 gland EST database but rare in the LA1777 database was similar in sequence to plant esterases. This cDNA, designated Methylketone Synthase 1 (MKS1), was expressed in Escherichia coli and the purified protein used to catalyze in vitro reactions in which C12, C14, and C16 b-ketoacyl–acyl-carrier-proteins (intermediates in fatty acid biosynthesis) were hydrolyzed and decarboxylated to give C11, C13, and C15 methylketones, respectively. Although MKS1 does not contain a classical transit peptide, in vitro import assays showed that it was targeted to the stroma of plastids, where fatty acid biosynthesis occurs. Levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.

Metabolic profiling and metabolome analysis of laser dissected trichomes from Cannabis sativa L.
Oliver Kayser, Nizar Happyana, Sara Agnolet, Remco Muntendam, Bernd Schneider
https://www.researchgate.net/publica...nabis_sativa_L
Cannabis sativa (Cannabaceae), has been used as a medicine by humankind before Christian era. In this plant, cannabinoids are the most studied compounds as consequences of their interesting biological activities. Cannabinoids are produced mainly in the trichomes, especially capitate-stalked and capitate-sessile types. To study cannabinoids production in the specific trichomes and metabolome patterns over the complete development periode comprehensively, capitate-stalked trichomes were dissected and departed two parts, the gland (head) and the stem by laser microdissection (LMD), while intact capitate-sessile trichomes were dissected as well. LCMS analysis was conducted on 25-143 collected cells, and cannabinoid profiles in the dissected trichomes were analysed. Cryogenic NMR was used to identify and determine the structures of cannabinoids in the dissected trichomes. Based on LCMS analysis, cannabinoids profiles among tested samples did not differ significantly. THCA, CBDA and CBGA were identified as major compounds, while THC, CBD, and CBG were detected as minor compounds in all samples. CBC as other minor compound along with CBN as a side product of THC were also identified on the intact of capitate-stalked and head of capitate-stalked old plants (8 weeks). The presence of THCA and CBDA in the dissected trichomes was confirmed clearly by 1H NMR spectra that resulted from cryogenic NMR measurements. Moreover, CBCA which could not be analyzed by LCMS as its reference compound was unavailable, could be detected by cryogenic NMR in the dissected capitate-stalked trichomes. It is first report about the presence of cannabinoids on the stem of capitate-stalked. Furthermore, the discovery of metabolites in the stem of capitate-stalked confirmed the appearance of other spots for cannabinoid production besides the head of capitate-stalked. In addition, this presentation shows a promising method that combines LMD, LCMS and cryogenic NMR for cannabinoid analysis comprehensively on the specific trichomes of Cannabis. Besides that, this method is capable to be applied for analysing compounds on specific cells or tissues of other plants comprehensively.

Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR.
Happyana, N., & Kayser, O.
Planta Medica, 82(13), 1217–1223. (2016).
doi:10.1055/s-0042-108058
Cannabis sativa trichomes are glandular structures predominantly responsible for the biosynthesis of cannabinoids, the biologically active compounds unique to this plant. To the best of our knowledge, most metabolomic works on C. sativa that have been reported previously focused their investigations on the flowers and leaves of this plant. In this study, 1H NMR-based metabolomics and real-time PCR analysis were applied for monitoring the metabolite profiles of C. sativa trichomes, variety Bediol, during the last 4 weeks of the flowering period. Partial least squares discriminant analysis models successfully classified metabolites of the trichomes based on the harvest time. ?9-Tetrahydrocannabinolic acid and cannabidiolic acid constituted the vital differential components of the organic preparations, while asparagine, glutamine, fructose, and glucose proved to be their water-extracted counterparts. According to RT?PCR analysis, gene expression levels of olivetol synthase and olivetolic acid cyclase influenced the accumulation of cannabinoids in the Cannabis trichomes during the monitoring time. Moreover, quantitative 1H NMR and RT?PCR analysis of the Cannabis trichomes suggested that the gene regulation of cannabinoid biosynthesis in the C. sativa variety Bediol is unique when compared with other C. sativa varieties.

Monitoring metabolites production and cannabinoids analysis in medicinal Cannabis trichomes during flowering period by 1H NMR-based metabolomics
Oliver Kayser
Conference: International Congress on Natural Products Research (ICNPR 2012), the 8th Joint Meeting of AFERP, ASP, GA, PSE and SIF, New York, USA; 07/2012At: New York
Volume: Planta Med 2013; 79 - SL44
Project: Heterologous Biosynthesis of plant secondary natural Products
FIND DOI or LINK
Cannabis trichomes are known as the main site of cannabinoids production, the responsible compounds for most biological activities of the plant. This study reports 1H NMR based-metabolomics and cannabinoids analysis of trichomes of 4 medicinal Cannabis varieties, Bediol, Bedica, Bedrobinol, and Bedrocan, in order to investigate cannabinoids production and metabolites profiles of the trichomes during the last 4 weeks of flowering period. Analysis of 1H NMR spectra revealed totally 6 identified cannabinoids in the chloroform extracts, THCA (1), CBDA (2), CBCA (3), CBGA (4), THC (5) and CBD (6), and 20 compounds in the water extracts including sugars, amino acids, and other acidic compounds. Quantification analysis using 1H NMR method showed that production of total cannabinoids of Bedrocan and Bedica trichomes increased from week 5 (Bedrocan: 63.01 mg/g) till week 7 (Bedrocan: 111.04 mg/g) and then decreased at week 8 (Bedrocan: 79.76 mg/g). Meanwhile cannabinoids production of Bedrobinol and Bediol trichomes increased during the monitoring time. Different metabolites profiles within trichomes varieties were revealed by PLSDA models of metabolomics. Important differential metabolites in this discrimination were THCA (1) and CBDA (2) on the chloroform extracts, and were asparagine, choline, fructose and glucose on the water extracts. Furthermore PLSDA classified the trichomes of every variety based on their harvested weeks. THCA (1) was found as an important discriminant compound in the chloroform extracts of every variety. Meanwhile, threonine, asparagine and fructose were detected as differential metabolites in the water extracts of every variety. This study indicated that Cannabis tricomes during flowering period produced metabolites, particularly cannabinoids in different amounts depending on the time and the plant variety. Furthermore it is the first report for monitoring metabolites production in plant trichomes using 1H NMR-based metabolomics.

Morphogenesis of Capitate Glandular Hairs of Cannabis sativa (Cannabaceae).
Hammond, C. T., & Mahlberg, P. G.
American Journal of Botany, 64(8), 1023.(1977).
doi:10.2307/2442258
The glandular secretory system in Cannabis sativa L. (marihuana) consists of three types of capitate glandular hairs (termed bulbous, capitate-sessile, and capitate-stalked) distinguishable by their morphology, development, and physiology. These gland types occur together in greatest abundance and developmental complexity on the abaxial surface of bracts which ensheath the developing ovary. Bulbous and capitate-sessile glands are initiated on very young bract primordia and attain maturity during early stages of bract growth. Capitate-stalked glands are initiated later in bract growth and undergo development and maturation on medium, to full sized bracts. Glands are epidermal in origin and derived, with one exception, from a single epidermal initial. The capitate-stalked gland is the exception and is of special interest because it possesses a multicellular stalk secondarily derived from surrounding epidermal and subepidermal cells. Glands differentiate early in development into an upper secretory portion and a subtending auxiliary portion. The secretory portion, depending on gland type, may range from a few cells to a large, flattened multicellular disc of secretory cells. The secretory portion produces a membrane-bound resinous product which caps the secretory cells. Capitatestalked glands are considered to be of particular evolutionary significance because they may represent a gland type secondarily derived from existing capitate-sessile glands

PKS Activities and Biosynthesis of Cannabinoids and Flavonoids in Cannabis sativa L. Plants
Isvett Josefina Flores-Sanchez, Robert Verpoorte
Plant and Cell Physiology, Volume 49, Issue 12, December 2008, Pages 1767–1782,https://doi.org/10.1093/pcp/pcn150
Polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Chalcone synthase (CHS, EC 2.3.1.74), stilbene synthase (STS, EC 2.3.1.95), phlorisovalerophenone synthase (VPS, EC 2.3.1.156), isobutyrophenone synthase (BUS) and olivetol synthase activities were detected during the development and growth of glandular trichomes on bracts. Cannabinoid biosynthesis and accumulation take place in these glandular trichomes. In the biosynthesis of the first precursor of cannabinoids, olivetolic acid, a PKS could be involved; however, no activity for an olivetolic acid-forming PKS was detected. Content analyses of cannabinoids and flavonoids, two secondary metabolites present in this plant, from plant tissues revealed differences in their distribution, suggesting a diverse regulatory control for these biosynthetic fluxes in the plant.

Plastid Development in Disc Cells of Glandular Trichomes of Cannabis (Cannabaceae)
Paul Mahlberg, Eun-Soo Kim
July 1997
Molecules and Cells 7(3):352-9
https://pdfs.semanticscholar.org/cb3...29271f5b5d.pdf
Plastids in lipophilic glandular trichomes of chemically fixed (CF) and high pressure cryofixed-cryosubstituted (HPC-CS) bracteal tissues of Cannabis were examined by transmission electron microscopy. In CF preparations, plastids in disc cells prior to secretory cavity formation possessed several lobed and dilated thylakoid-like features. In glands with secretory cavities, thylakoid-like features aggregated to form reticulate bodies that distended regions of the elongated plastids. Electron-gray inclusions evident on the plastid surface appeared continuous with the reticulate body. Inclusions of similar electron density also appeared in the cell cytoplasm, along the plasma membrane, between the plasma membrane and cell wall facing the cavity, and in the secretory cavity in both CF and HPC-CS preparations. The bilayer structure of membranes of the plastid envelope was evident in HPC-CS but not in CF preparations. In HPC-CS preparations, secretions were evident on the plastid surface and were continuous with those in the plastid through pores in the envelope. This study supports an interpretation that these specialized plastids, lipoplasts, synthesize secretions that are transported through the plasma membrane and cell wall to subsequently accumulate in the secretory cavity.

Quantitative Analysis of Cannabinoids in the Secretory Product from Capitate-Stalked Glands of Cannabis sativa L. (Cannabaceae)
Vicki S. Lanyon, Jocelyn C. Turner and Paul G. Mahlberg
Botanical Gazette Vol. 142, No. 3 (Sep., 1981), pp. 316-319
DOI: 10.1086/337229
Capitate-stalked glandular trichomes from pistillate bracts of Cannabis sativa L. were analyzed to determine the cannabinoid composition of secretory products within the secretory sac. Analyses were performed on cloned materials of a strain characteristically high in cannabidiol. The secretory product is accumulated in a single large secretory sac, which develops above a multicellular disk of secretory cells during gland ontogeny. The content of the secretory sac was removed with micropipets, using a micromanipulator without damaging or removing disk cells or their contents, and analyzed by gas-liquid chromatography. The cannabinoid content of the secretory sac was compared with previously quantitated cannabinoids of whole capitate-stalked glands. Results indicated that nearly all of the cannabinoid content of capitate-stalked glands was present in the secretory sac.

QUANTITATIVE DETERMINATION OF CANNABINOIDS IN INDIVIDUAL GLANDULAR TRICHOMES OF CANNABIS SATIVA L. (CANNABACEAE).
Turner, J. C., Hemphill, J. K., & Mahlberg, P. G.
American Journal of Botany, 65(10), 1103–1106. (1978).
doi:10.1002/j.1537-2197.1978.tb06177.x
Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis saliva L., which included both drug and fiber phenotypes. were investigated by gas-liquid
chromatographic analyses. Capitate-stalked glands were selectively harvestedfrom vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspicuously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis representa dynamic system within the cannabinoid synthesizing activities of this plant.

Secretory Cavity Development in Glandular Trichomes of Cannabis sativa L. (Cannabaceae).
Kim, E.-S., & Mahlberg, P. G.
American Journal of Botany, 78(2), 220. (1991).
doi:10.2307/2445245
Formation of secretory vesicles in the noncellular secretory cavity of glandular trichomes of Cannabis sativa L. was examined by transmission electron microscopy. Two patterns of vesicle formation occurred during gland morphogenesis. During initial phases of cavity formation small hyaline areas arose in the wall near the plasma membrane of the disc cell. Hyaline areas of elongated shape and different sizes were distributed throughout the wall and adjacent to the secretory cavity. Hyaline areas increased in size, some possibly fusing with others. These hyaline areas, possessing a membrane, moved into the cavity where they formed vesicles. As membraned vesicles they developed a more or less round shape and their contents became electron-dense. During development of the secretory cavity and when abundant secretions were present in the disc cells, these secretions passed through the wall to accumulate as membraned vesicles of different sizes in the cavity. As secretions emerged from the wall, a membrane of wall origin delimited the secretory material from cavity contents. Vesicles released from the wall migrated in the secretory cavity and contacted the sheath where their contents permeated into the subcuticular wall as large or diffused quantities of secretions. In the subcuticular wall these secretions migrated to the wall-cuticle interface where they contributed to structural thickening of the cuticle. This study demonstrates that the secretory process in glands of Cannabis involves not only secretion of materials from the disc cell, but that the disc cell somehow packages these secretions into membraned vesicles outside the cell wall prior to deposition into the secretory cavity for subsequent structural development of the sheath.

Secretory Vesicle Formation in Glandular Trichomes of Cannabis sativa (Cannabaceae).
Mahlberg, P. G., & Kim, E.-S.
American Journal of Botany, 79(2), 166. (1992).
doi:10.2307/2445104
Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall-cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacento the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane-bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron-dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerousecretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased fivefold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperm

Secretory Vesicle Formation in the Secretory Cavity of Glandular
Trichomes of Cannabis sativa L. (Cannabaceae)
Eun Soo Kim and Paul G. Mahlberg
Mol. Cells, Vol. 15, No. 3, pp. 387-395
https://www.molcells.org/journal/lis...s_n=&x=42&y=11
The disc cell wall facing the secretory cavity in lipophilic glands of Cannabis was studied for origin and distribution of hyaline areas, secretory vesicles, fibrillar matrix and particulate material. Secretions evident as light areas in the disc cell cytoplasm pass through modified regions in the plasma membrane and appear as hyaline areas in the cell wall. Hyaline areas, surrounded with a filamentous outline, accumulate near the wall surface facing the secretory cavity where they fuse to form enlarged hyaline areas. Fibrillar matrix is related to and may originate from the dense outer layer of the plasma membrane. This matrix becomes distributed throughout the wall material and contributes in part to the composition of the surface feature of secretory vesicles. Thickening of the cell wall is associated with secretions from the disc cells that facilitates movement of hyaline areas, fibrillar matrix and other possible secretions through the wall to form secretory vesicles and intervesicular materials in the secretory cavity. The outer wall of disc cells in aggregate forms the basilar wall surface of the secretory cavity which facilitates the organization of secretory vesicles that fill the secretory cavity.

SECRETORY VESICLE FORMATION IN GLANDULAR TRICHOMES OF CANNABIS SATIVA (CANNABACEAE)
Paul G. Mahlberg, Eun-Soo Kim
American Journal of Botany 79(2):166-173 February 1992
DOI: 10.1002/j.1537-2197.1992.tb13634.x
Formation of secretory vesicles in the noncellular secretory cavity of glandular trichomes of Cannabis saliva L. was examined by transmission electron microscopy. Two patterns of vesicle formation occurred during gland morphogenesis. 1) During initial phases of cavity formation small hyaline areas arose in the wall near the plasma membrane of the disc cell. Hyaline areas of elongated shape and different sizes were distributed throughout the wall and adjacent to the secretory cavity. Hyaline areas increased in size, some possibly fusing with others. These hyaline areas, possessing a membrane, moved into the cavity where they formed vesicles. As membraned vesicles they developed a more or less round shape and their contents became electron-dense. 2) During development of the secretory cavity and when abundant secretions were present in the disc cells, these secretions passed through the wall to accumulate as membraned vesicles of different sizes in the cavity. As secretions emerged from the wall, a membrane of wall origin delimited the secretory material from cavity contents. Vesicles released from the wall migrated in the secretory cavity and contacted the sheath where their contents permeated into the subcuticular wall as large or diffused quantities of secretions. In the subcuticular wall these secretions migrated to the wall–cuticle interface where they contributed to structural thickening of the cuticle. This study demonstrates that the secretory process in glands of Cannabis involves not only secretion of materials from the disc cell, but that the disc cell somehow packages these secretions into membraned vesicles outside the cell wall prior to deposition into the secretory cavity for subsequent structural development of the sheath.

Size matters: evolution of large drug-secreting resin glands in elite pharmaceutical strains of Cannabis sativa (marijuana)
Ernest Small . Steve G. U. Naraine
Genet Resour Crop Evol
DOI: 10.1007/s10722-015-0254-2
Most tetrahydrocannabinol (THC) of Cannabis sativa is located in the resin heads of capitate-stalked glandular trichomes. We found that after harvest the resin heads shrink in diameter in exponential decay fashion under ambient room conditions, losing about 15 % in the first month, rising to 24 % over the first year, 32 % by 50 years, and 34 % after a century. An equation accounting for the asymptotic curve descriptive of the progression of shrinkage was determined [original gland head diameter in microns = observed diameter divided by (0.5255 + 0.4745 multiplied by time in days to the power ?0.1185)], so that if the age of a specimen is known, the original diameter of the gland heads in the fresh state can be extrapolated. This equation was employed to compare gland head size in samples of different ages. A sample of high-THC medical marijuana strains marketed under license possessed resin head diameters averaging 129 ?m, while a sample of low-THC industrial hemp cultivars possessed gland head diameters averaging 80 ?m. The mean volume of the resin heads of the narcotic strains was more than four times larger than that of the industrial hemp strains. This is the first documented report of a consistent morphological separator of elite narcotic strains and non-narcotic plants. Most recognized strains of marijuana were bred clandestinely and illicitly during the last half century. The occurrence of large resin gland heads in a sample of officially marketed pharmaceutical strains is an obvious correlate of selection for higher quantity of resin production.

NOT DIRECTLY CANNABIS RELATED
Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa: identification of a trichome-specific isoform.
Clark IM, Forde BG, Hallahan DL.
Plant Mol Biol. 1997 Mar;33(5):875-85.
DOI: 10.1023/A:1005706609510
Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.

Not Cannabis specific
Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics.
Schilmiller, A. L., Miner, D. P., Larson, M., McDowell, E., Gang, D. R., Wilkerson, C., & Last, R. L.
PLANT PHYSIOLOGY, 153(3), 1212–1223. (2010).
doi:10.1104/pp.110.157214
Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces b-caryophyllene and a-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes.
Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S, Shoyama Y (2005)
Plant Cell Physiol 46: 1578–1582
DOI: 10.1093/pcp/pci166
Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acylactivating enzyme in Cannabis sativa trichomes
Jake M Stout, Zakia Boubakir, Stephen J Ambrose, Jonathan E Page
February 2012 The Plant Journal 71(3):353-65
DOI: 10.1111/j.1365-313X.2012.04949.x
The psychoactive and analgesic cannabinoids (e.g. ?(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.

The identification of tetrahydrocannabinolic acid synthase in the non-aqueous secretions of the storage cavities from Cannabis sativa glandular trichomes
P Rodziewicz, S Loroch, I Feldmann, C Schumbrutzki, O Kayser
Planta Medica International Open 4 2017
DOI: 10.1055/s-0037-1608363
http://www.tb.bci.tu-dortmund.de/Pos...Rodziewicz.pdf
Cannabis sativa is an important herbaceous species cultivated since ancient times due to its unique medical and recreational properties, but also as a source of valuable seed oil and high quality fibre. Among numerous phytochemicals synthesized in this herb, cannabinoids represent the unique class of secondary metabolites, which largely contribute to the pharmacological properties of this species. More than 100 cannabinoids have been described in the literature, but Δ 9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) are present in the largest quantities in this plant. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers and their main reservoirs are storage cavities of these hair-like structures.
Tetrahydrocannabinolic acid synthase catalyzes the unique oxidative cyclization of cannabigerolic acid (CBGA) into THCA, which is a direct precursor of the mind-affecting compound – Δ 9- tetrahydrocannabinol (THC). Thus, it is considered to be a key enzyme controlling the psychoactive properties of C. sativa. In previous research it was suggested that THCA synthase is secreted into the storage cavities of glandular trichomes, where it might also synthesize the final product – THCA [2]. However, no direct evidence on protein level was presented.
To examine the putative extracellular location of THCA synthase, the storage cavity secretions from C. sativa trichomes were obtained and submitted for proteomic analysis. By using mass spectrometry we were able to identify the THCA synthase in the non-aqueous content of the storage cavities from C. sativa glandular trichomes.

The trichomes and glands of Cannabis sativa L.
J. W. r FAIRBAIRN (1972)
https://www.unodc.org/unodc/en/data-...4_page005.html
The main object of this paper is to describe a new type of sessile gland in cannabis and to report gas chromatographic results on the presence of cannabinoids in the glands, but the opportunity is taken to include descriptions, with scanning electron micrographs, of all the trichomes.

Time course of cannabinoid accumulation and chemotype development during the growth of Cannabis sativa L.
Pacifico, D., Miselli, F., Carboni, A., Moschella, A., & Mandolino, G.
Euphytica, 160(2), 231–240. (2007).
doi:10.1007/s10681-007-9543-y
The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gaschromatographic analysis in greenhouse-grown plants. The total amounts and the concentration ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R £ 15R) £ NL, and plants from a seized seed lot) were found chemotypically uniform, with all plants belonging to chemotpe II (mixed) and I (high THC) respectively. The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype III. The CBD/THC and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103 days after sowing, and consistent with the ratios determined on mature inXorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves around 80 days from sowing, and were shown to be simultaneous during the growth period, irrespective of the chemotypes. Callus cultures were obtained from all the five different chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently of the type and amount of
cannabinoids in the mother plants, it was confirmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.

TRICHOMES AND CANNABINOID CONTENT OF DEVELOPING LEAVES AND BRACTS OF CANNABIS SATIVA L. (CANNABACEAE)
Jocelyn C. Turner , John K. Hemphill , Paul G. Mahlberg
American Journal of Botany, 67(10), 1397–1406
DOI: 10.1002/j.1537-2197.1980.tb07774.x
Trichome density and type and cannabinoid content of leaves and bracts were quantitated during organ ontogeny for three clones of Cannabis sativa L. Trichome initiation and development were found to occur throughout leaf and bract ontogeny. On leaves, bulbous glands were more abundant than capitate?sessile glands for all clones, although differences in density for each gland type were evident between clones. On pistillate bracts, capitate?sessile glands were more abundant than the bulbous form on all clones, and both types decreased in relative density during bract ontogeny for each clone. The capitate?stalked gland, present on bracts but absent from vegetative leaves, increased in density during bract ontogeny. The capitate?stalked gland appeared to be initiated later than bulbous or capitate?sessile glands during bract development and on one clone it was first found midway in bract ontogeny. Nonglandular trichomes decreased in density during organ ontogeny, but the densities differed between leaves and bracts and also between clones. Specific regulatory mechanisms appear to exist to control the development of each trichome type independently. In addition, control of trichome density seems to be related to the plant organ and clone on which the gland type is located. Cannabinoid synthesis occurs throughout organ development and is selectively regulated in each organ. Typically, cannabinoid synthesis occurred at an increasing rate during bract development, whereas in developing leaves synthesis occurred at a decreasing rate. Cannabinoid content on a dry weight basis was generally greater for bracts than leaves. Analyses of leaves indicate that other tissues in addition to glands may contain cannabinoids, while for bracts the gland population can accommodate the cannabinoid content for this organ. The functional significance of trichomes and cannabinoids in relation to evolution is discussed.

TRICHOMES OF CANNABIS SATIVA L. (CANNABACEAE)
Amer. J. Bot. 63(5): 578-591. 1976
P Dayanandan, Pernn B Klunvrln
https://www.academia.edu/22556023/TR...?auto=download
The diversity of non-glandular and glandular hairs of Cannabis sativa I:. (marihuana) aredescribed by scanning electron microscopy. The non-glandular hairs are of two major types,as distinguished by size differences and locations, and all of them are highly silicified. Thepresence of silica as well as cystoliths of calcium carbonate help in the identification of mari-huana even in its ash residues. X-ray microanalyses of Cannabis hairs are compared withthose of Humulus lupulus and Lantana camera, whose hairs have been considered to resemblethose of marihuana. Glandular hairs are found to be of two major categories, One group con-sists of glands whose heads are generally made up of eight cells and the other group whoseheads are generally made up of two cells but never more than four cells. All glands of bothcategories are stalked. Some glands of the first category are massively stalked and these arerestricted solely to anthers and bracts of staminate and pistillate plants. The massive stalk isconsidered to be made up of epidermal and hypodermal cells that have grown in response tosome stimulation during anthesis. Fine details of the shoot system of Cannabis, such as cuticu-lar ridges on epidermal cells, warty protuberances on non-glandular hairs, and surface viewsof glands in developing stages are also reported. Glandular hairs on the bracts of HumulusIupulus resemble hose of Cannabis.

Not Cannabis specific
Trichomes: One Way to Understand the Beautiful Bricks of Nature’s Great Structures
Jacob Haas
https://cannabisgenomics.org/blog/20...eat-structures
If you start with the right building blocks, you can build magnificent cathedrals. The building blocks can come from humble sources: even in the Cathedral at Cologne, some bricks are made of slimy mud and pretty stained glass windows from tiny, useless, sand.
Natural things, too, have to be built by smaller things. From the bodies of many plants and animals just tiny compounds called terpenes. They are produced by a variety of plants, particularly evergreen trees, but also by termites and butterflies. They look sappy, sticky, meager and probably useless, but they glisten, and this sparkly hints at their enormous value. It turns out that they are a versatile, valuable and nearly ubiquitous building block for all kinds of magnificent things. With terpenes are built the rubber in some tires, many of the fragrances you encounter -- including the smell of a hoppy beer. Terpenes even make some of the steroids inside your favorite athletes. Yes, Sammy Sosa may have built his career on terpenes. The terpene farnesene, which can be found in many plants and insects, has great potential as a renewable carbon building block, and is currently being mass-produced by a genetically modified yeast, which instead of producing alcohol produces this compound (Schofer et al. 2014). Terpenes can also serve as signaling chemicals for the plant cell, and as antimicrobials, and are oftentimes found in soaps and hygienic products.
If we want to do a more competent job of building tires, brewing beer, and making soaps out of terpenes, it might help to know something more about what the terpene is. Terpenes and terpenoids are the most common group of chemicals produced by plants, constructed from a common building block called isoprene, which is a simple five carbon molecule (Zwenger and Basu 2008)(figure3). Terpenoids can be thought of as modified terpenes, usually with added or removed methyl groups (CH3), accompanied by the presence of oxygen atoms in the molecule (Zwenger and Basu 2008). Limonene, in the top panel of figure 2, is a terpene, while the others are considered terpenoids

ULTRASTRUCTURAL DEVELOPMENT OF CAPITATE GLANDULAR HAIRS OF CANNABIS SATIVA L. (CANNABACEAE).
Hammond, C. T., & Mahlberg, P. G. (1978).
American Journal of Botany, 65(2), 140–151.
doi:10.1002/j.1537-2197.1978.tb06051.x
The capitate-sessile and capitate-stalked glands of the glandular secretory system in Canna his , which are interpreted as lipophilic type glandular hairs, were studied from floral bracts of pistillate plants. These glands develop a flattened multicellular disc of secretory cells, which with the extruded secretory product forms the gland head and the auxiliary cells which support the gland head. The secretory product accumulates beneath a sheath derived from separation of the outer wall surface of the cellular disc. The ultrastructure of secretory cells in pre-secretory stages is characterized by a dense ground plasm, transitory lipid bodies and fibrillar material, and well developed endoplasmic reticulum. Dictyosomes and dictyosome-derived secretory vesicles are present, but never abundant. Secretory stages of gland development are characterized by abundant mitochondria and leucoplasts and by a large vacuolar system. Production of the secretory product is associated with plastids which increase in number and structural complexity. The plastids develop a paracrystalline body which nearly fills the mature plastid. Material interpreted as a secretion appears at the surface of plastids, migrates, and accumulates along the cell surface adjoining the secretory cavity. Extrusion of the material into the secretory cavity occurs directly through the plasma membrane-cell wall barrier.
 
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CannaRed

Cannabinerd
Sam, I tried to read this one, but I believe it links to a separate study by different scientists.


Size matters: evolution of large drug-secreting resin glands
in elite pharmaceutical strains of Cannabis sativa (marijuana)
Ernest Small . Steve G. U. Naraine
Genet Resour Crop Evol
DOI: 10.1007/s10722-015-0254-2

FIXED Thanks for the help ANTONE ELSE FIND ANY LINKS WITH PROBLEMS?-SamS
 
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bsgospel

Bat Macumba
Veteran
Vetting Archaeology/History:

A 2014 update on the Ice Maiden.

https://siberiantimes.com/science/c...d-from-breast-cancer-reveals-unique-mri-scan/
I CAN ADD THIS

The original National Geographic article is available in a back-issue as listed but you can also get the story from the team in a NOVA transcript circa 1998

https://www.pbs.org/wgbh/nova/transcripts/2517siberian.html
THIS LINK DOES NOT HAVE THE NAT GEO ARTICLE IT IS A NOVA SHOW TRANSCRIPT ABOUT THE ICE MAIDEN I CAN ADD IT

The DOI to list for the Rudenko paper is doi:10.1525/aa.1973.75.4.02a01350 sci-hub link checks out
IT IS A 3 PAGE REVIEW OF THE BOOK THE OTHER LINKS I GAVE ARE FURTHER INFO BUT I STILL AM LOOKING FOR THE DOI OR A LINK TO THE WHOLE BOOK, I HAVE THE BOOK AT HOME.

Here is an abstract/introduction for the Hui-Lin Li paper if you'd like to include it. I ADDED IT

"From a historical vantage, Cannabis has been found in China since Neolithic times, about 6,000 years ago, with a continuous record of cultivation down to the present. This record stands unique in comparison to those of other regions in Asia, and it strongly indicates the plant to be indigenous. New archeological finds in recent years considerably substantiate and extend its early history. The very scattered references in historical literature are in need of organization and analysis. These records are assembled here, followed by some notes on the possible routes of early diffusion of the plant in relation to its usage."

Did a thread search and I didn't find anything for this new entry (still under archaeology and/or DNA)

https://sci-hub.tw/10.1007/s10722-008-9343-9

Results of molecular analysis of an archaeological hemp(Cannabis sativa L.) DNA sample from North West China
Ashutosh Mukherjee, Satyesh Chandra Roy, S. De Bera, Hong-En Jiang, Xiao Li, Cheng-Sen Li, Subir Bera.
Genetic Resources and Crop Evolution 55(4):481-485 · June 2008
DOI: 10.1007/s10722-008-9343-9
ADDED TO THREAD

"Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C.sativa sample showed that C.sativa races might have been introduced into China from the European–Siberian center of diversity."

File under Medical Cannabis: I ADDED BOTH.

Personal Account of Medical Use of Cannabis
Hodges, Clare. (2002). Personal Account of Medical Use of Cannabis. Journal of Cannabis Therapeutics. 2(3-4).
DOI: 10.1300/J175v02n03_11.
The author provides a personal account of her sojourn with multiple sclerosis and its treatment with smoked and oral preparations of cannabis. Additional information is provided as to the effects, dosing and delivery of cannabis employed by 250 members of the Alliance for Cannabis Therapeutics.

Effect of baseline cannabis use and working-memory network function on changes in cannabis use in heavy cannabis users: a prospective fMRI study
Cousijn, J.; Wiers, R.W.; Ridderinkhof, K.Richard.; van den Brink, W.; Veltman, D.J.; Goudriaan, A.E.
Human Brain Mapping 35(5): 2470-2482. 2014.
"Theoretical models of addiction suggest that a substance use disorder represents an imbalance between hypersensitive motivational processes and deficient regulatory executive functions. Working-memory (a central executive function) may be a powerful predictor of the course of drug use and drug-related problems. Goal of the current functional magnetic resonance imaging study was to assess the predictive power of working-memory network function for future cannabis use and cannabis-related problem severity in heavy cannabis users. Tensor independent component analysis was used to investigate differences in working-memory network function between 32 heavy cannabis users and 41 nonusing controls during an N-back working-memory task. In addition, associations were examined between working-memory network function and cannabis use and problem severity at baseline and at 6-month follow-up. Behavioral performance and working-memory network function did not significantly differ between heavy cannabis users and controls. However, among heavy cannabis users, individual differences in working-memory network response had an independent effect on change in weekly cannabis use 6 months later (ΔR(2) = 0.11, P = 0.006, f(2) = 0.37) beyond baseline cannabis use (ΔR(2) = 0.41) and a behavioral measure of approach bias (ΔR(2) = 0.18): a stronger network response during the N-back task was related to an increase in weekly cannabis use. These findings imply that heavy cannabis users requiring greater effort to accurately complete an N-back working-memory task have a higher probability of escalating cannabis use. Working-memory network function may be a biomarker for the prediction of course and treatment outcome in cannabis users. "

THANKS FOR THE HELP,
-SAMS
 
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bsgospel

Bat Macumba
Veteran
Ancient Cannabis Burial Shroud in a Central Eurasian Cemetery
HONGEN JIANG, LONG WANG, MARK D. MERLIN, ROBERT C. CLARKE,
YAN PAN, YONG ZHANG, GUOQIANG XIAO, AND XIAOLIAN DING
Economic Botany, Vol 70(X), 2016, 9 pp.
DOI: 10.1007/s12231-016-9351-1
We have recently the reported the isolation, structure elucidation and partial synthesis of (-)- A’-tetrahydrocannabinol the major active principle in hashish, as well as a total
reported synthesis of the racemate of I. We wish now to present data showing that the absolute configuration of the naturally occuring (-)-I is D. relative to D-(+)-glyceraldehyde.
Adams has reported that tetrahydrocannabidiol (III) obtained by reduction of cannabidiol which has since been shown to possess structure II, can be oxidized to menthane carboxylic acid (IVa). The anilide of IVa, thus obtained did not depress the melting point of the anilide of IVa prepared from menthol (V) through the menthyl chloride (VI) followed by carbonation of the Grignard derivative of VI. Unfortunately the rotation of the anilide of IVa prepared by the degradation of the natural product was not reported.


I FIXED IT THANKS A LOT, AFTER DOING THIS FOR HOURS I BEGIN TO SPIN I CAN USE THE HELP IF ANYONE SEES ANYTHING SCREWED UP OR LINKS NOT WORKING PLEASE TELL ME SO I CAN FIX IT. Also if anyone has an idea where a post could be additional listed under another SUBJECT please let me know so I can do it.
-SAMS

This abstract should actually read: An extraordinary cache of ancient, well-preserved Cannabis plant remains was recently discovered in a tomb in the Jiayi cemetery of Turpan, NW China. Radiometric dating of this tomb and the archeobotanical remains it contained indicate that they are approximately 2800–2400 years old. Both morphological and anatomical features support the identification of the plant remains as Cannabis. Research discussed in this paper describes 13 nearly whole plants of Cannabis that appear to have been locally produced and purposefully arranged and used as a burial shroud which was placed upon a male corpse. This unique discovery provides new insight into the ritualistic use of Cannabis in prehistoric Central Eurasia. Furthermore, the fragmented infructescences of Cannabis discovered in other tombs of the Jiayi cemetery, together with similar Cannabis remains recovered from coeval tombs in the ancient Turpan cemetery along with those found in the Altai Mountains region, reveal that Cannabis was used by the local Central Eurasian people for ritual and/or medicinal purposes in the first millennium before the Christian era.

I'll try to find where that other abstract actually belongs if it hasn't been attached to it already. *Edit: the paper it belongs to has the correct abstract.
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
BUMP, I ADDED ANOTHER 25 PAPERS TODAY, LOOK IN
MEDICAL CANNABIS/ENDOCANNBINOIDS

19 ARE FROM THE SAME JOURNAL British Journal of Pharmacology Volume 163, Issue 7
Special Issue:Cannabinoids in Biology and Medicine, Part I. Guest Editors: Itai Bab and Steve Alexander

-SAMS
 
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Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
BUMP I just added another 35 papers today look in
MEDICAL CANNABIS/ENDOCANNBINOIDS

THEY ARE ALL FROM THE SAME JOURNAL
British Journal of Pharmacology Volume 165, Issue 8
Special Issue:Themed Section: Cannabinoids in Biology and Medicine, Part II.
Guest Editors: Itai Bab and Steve Alexander

-SamS
 

Sam_Skunkman

"RESIN BREEDER"
Moderator
Veteran
Bump, Added a bunch of DNA, Cannabinoids, and In Vitro papers

Also paper by paper I am adding all their abstracts to help users find papers in the Bib they want to download and read.
-SamS
 
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mexcurandero420

See the world through a puff of smoke
Veteran
Biological and Chemical Evaluation of a 43-Year-Old Sample of Cannabis Fluidextract.
Kubena RK, et al. J Pharm Sci. 1972.

Stability of cannabinoids in dried samples of cannabis dating from around 1896–1905.
https://doi.org/10.1016/0378-8741(90)90068-5


DONE thanks for the post.
-SamS
 
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bsgospel

Bat Macumba
Veteran
I'm thinking Breeding or Cannabis Analysis:

Variations of 9-THC content in single plants of hemp varieties
Klemens Mechtler∗, Josef Bailer, Karl de Hueber
Industrial Crops and Products. 19(1), 19-24
DOI: 10.1016/S0926-6690(03)00077-3

ADDED-SamS

Within a given plant population, the concentration of any constituent is expected to vary within a certain bandwidth. To test the distribution of 9-tetrahydrocannabinol (THC) in hemp populations, a number of single plants were taken from populations of five well-known hemp accessions (Fasamo, Beniko, Bialobrzeskie, Félina 34, Kompolti) and a Hungarian provenance. The quantitative analysis of single plants delivered a set of 30–61 THC and Cannabidiol (CBD) values for each of the six hemp accessions under consideration. The distribution of THC within a number of hemp plants often shows no Gaussian distribution, the different varieties have quite characteristic distributions of THC. Most single-plant values are close together, the variation, however, differing from variety to variety. In addition, single plants are found with THC values far outside this bandwidth.
 
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bsgospel

Bat Macumba
Veteran
SamS said:
"We therefore consider CBDAS as the ancestral type of these synthases."
-----------
Does this mean THC synthase evolved or mutated from CBD synthase? Maybe man helped select the few that were THC synthase however they came to be, and spread them worldwide? I would not be surprised......
Man selects for THC and against CBD, in the case of Ganja, he selects for high resin contents regardless of Cannabinoid for Hashish, for hemp until recently there was little selections for Cannabinoids until after Cannabis became illegal less then 100 years ago, and very recently for CBD for mediacal varieties.

I think you have a point: "Another feature common to THCA- and CBDA-synthases is the
presence of a domain showing high homology with the berberine-bridge enzyme involved in the alkaloid biosynthesis of Eschscholtzia californica."

If there is high conservation SNP across the species & others in the domain, the SNP's found in each pathway may be crucial.

In the Keith Allen talk, we might combine the findings in this paper with his demonstration about 3/4 of the way through the labtube video. When he speaks about the production of other smaller quantity random components...That may play a factor regardless of transcription or gene operation.
 

bsgospel

Bat Macumba
Veteran
Under cannabinoids

Cannabinoid Ester Constituents from High-Potency Cannabis sativa
Safwat A Ahmed, Samir A Ross, Desmond Slade, Mahmoud A Elsohly
July 2008 Journal of Natural Products 71(6):1119
DOI: 10.1021/np800261x

Doi is actually 10.1021/np070454a
I FIXED IT THANKS, -SamS
 
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ahortator

Well-known member
Veteran
IC Archaeology/History

Cultivo y manufactura de lino y cáñamo en Nueva España, 1777-1800
Escrito por Ramón María Serrera Contreras. Sevilla 1974.


https://books.google.es/books?id=p9...&pg=PA37#v=onepage&q=lino cañamo grua&f=false

Instruccion para sembrar, cultivar y beneficiar el lino y cañamo en Nueva España
Escrito por Miguel de la Grua Talamanca y Branciforte. México 1796.


https://books.google.es/books?id=98...s&pg=PP5#v=onepage&q=lino cañamo grua&f=false

I DID ADD THESE BUT I DO NOT KNOW WHAT TO SAY, SPANISH AND NO PLACE TO DOWNLOAD THE ARTICLE, I CAN READ THEM ONLINE WITH THE LINKS YOU HAVE, BUT NO PDF-SamS
 
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ahortator

Well-known member
Veteran
Hi Sam

The first link is from a book which have author rights so it is uncomplete and I have found no way to download it. However it is very interesting about a late introduction of hemp growing from Spain in the last quarter of the 18th C. They carried to Mexico Spanish seeds and growers from Granada. However hemp had been already introduced as early as the 16th C. by Pedro Cuadrado.

The second link is from the oldest Cannabis growing guide I know (the first part is about flax growing), but only for fiber purposes.
If you click the gear in the upper right of the page you can download the PDF. This is the PDF link, but I don't know if it works:

https://books.googleusercontent.com...UOtxS53eBntq9r4PECD0n4FO7sDlJkwoVwPXfZZ9u0ZAA

Sadly the two books are only in Spanish.

There is too this work written by José Antonio Alzate y Ramírez in 1772: Memory on the use that the Indians make of the pipiltzintzintlis.

Here you have a link to an uncomplete book, in Spanish. But again I have found no way to download it.

https://books.google.es/books?id=Hb...age&q=ramirez alzate pipiltzintzintli&f=false

But the same text is in this web:

https://circulodepoesia.com/2015/11...ue-hacen-los-indios-de-los-pipiltzintzintlis/

This is the text translated by Google translator. I hope it will be useful.

In this day, in which this text of 1772 has finally been understood. The Supreme Court of Justice of the Nation has granted an writ of protection for the recreational use of hemp or marijuana. It is the text: Memory on the use made by the Indians of the pipiltzintzintlis written by José Antonio Alzate y Ramírez (1737-1799). Naturalist, cartographer and botanist of New Spain, nephew of our poet Sor Juana Ines de la Cruz.



















Memory on the use that the Indians make of the pipiltzintzintlis

In the moral history of the world the description of the virtues and vices of its inhabitants does not occupy the smallest place. What important service would do to literature who was dedicated to give an idea of ​​the passions, usages and inclinations of the Indians? This part is less in all its historians. They have scarcely given us superficial ideas, the ones most unconnected with the truth; Who should not admire in them the lack, usually [1], of greed and revenge; passions that cause so much damage to humanity? Miserable in whom the pain of our first parents to request sustenance with anxiety and fatigue, is verified in its greatest extent; objects worthy of compassion, have achieved the pardons, privileges and favors that our kings have taken pains to grant them; Those who, in a tone of contempt, treat them as idolaters, make a notable grievance to the prelates and pastors who have carefully sought to uproot this effect from our malice: what nation in its origin has not been an idolater? The Hebrews, people chosen by God for their worship, and who felt at every step the wonders of Omnipotence, did not let go the reins of their malice to worship the works of their hands? Well, we will not reproach them with an epithet that equally understands us all, with only the difference of time.

If we notice in them some relics of paganism, we must consider that only a little more than two and a half centuries has the precise light of the gospel struck them; time that is not enough to erase those traditions proceeded from the depraved human heart. What centuries has the gospel preached in Italy, England and other kingdoms? For their authors describe the superstitions and abuses of the small town. A famous English author attributes many of them, not only to the English commoners, but to the people of some sphere.

The custom that is practiced in Italy in the gift of beans the day of the dead, recognizes a pagan origin; This was proved not so long ago by an Italian sage; the talismans, amulets, etc., have no destination in Spain; France and other kingdoms are those that look with appreciation for those who make up the common people. ·

Let's read in the beautiful book of the world, reflect on preventative nudes and give thanks to God to see so many people, so many tribe, in thousands of earth converted to true religion in the space of a few years. Let us rejoice to see ancient Spain execute with its eminent prelates, zeal for our kings and fervor of the ministers of the gospel a company that does not count equally another nation, and so notorious that the most desperate Pyrrhonist can not have breath to refute it.

The abuse of the pipiltzintzintlis [2] is one of those relics of gentility that are preserved among some of the Indians; this is expressed in the edicts published by the prelates of this kingdom, and lately in the year of 1769, in which the parish priests are instructed to use all their desire to eradicate this superstition in which the spiritual health of the Indians goes the temporary one can also be added [3]. Some observations and discoveries that have entered my eyes provide me with an issue for the present memory, because of the great utility that can result. The superstition of the Indians, in the use of pipiltzintzintlis, is reduced to taking certain seeds, believing that by their means they divine and have a thousand fits, in which the most recondite things are manifested to them, with other peculiarities proceeded according to their own ignorance and malice. The effects produced in them are frightful: some manifest a ridiculous joy, others remain for some time stupid, others, and this is the most common, vividly represent a furious; and all these effects are believed by many of them as having happened through the mediation of the devil.

What are pipiltzintzintilis? Is its effect natural or preternatural? To the first I satisfy with the experience: there will be like ten years that the chance gave me the occasion of the disappointment; get a small amount of said pipiltzintzintlis, which was composed of a mixture of seeds and dried herbs; at first sight I then recognized they were nothing but the leaves and seeds of hemp; warning that I had to the point, to have seen before in a garden the hemp plant. Despite this, which for me was a demonstration, on the first occasion and to be completely convinced, I sowed those seeds with all possible precaution and achieved some hemp plants, as well as that of Europe, which the Indians recognized as pipiltzintzintlis, it was necessary to tear off the plants after the seeds began to mature because they were trying to catch as much as they could.

This is still verified with another fact that must banish all doubt: at the time when the expedition of Sonora was meditated, and when that navigation of the southern sea was raised, a subject charged with preventing some of the things necessary for a new establishment , he warned very well would be conducive to plant hemp in some immediate vicinity of those coasts to manufacture cables, sails and others in which hemp is necessary for the handling of the ships, trying to avoid the excessive expenses that are incurred when driving from Veracruz even those provinces. His idea was applauded, and there was no more difficulty than getting the seed; He appealed to me and I warned him, already well informed of what had happened to me, it was very easy business, because among the herbalists [4] of this city, he would find some portion: my conjecture was very successful, because they found quite a number of bushels, and not at the highest price. Somebody will ask me: where do the Indians get the pipiltzintzintli or hemp, when it is well known that in the kingdom there is no cultivation destined, except for this and that plant, that one or the other particular sows out of curiosity? The answer is short, it is wild in the hot lands and I have also reported that the Indians tend to sow some, either for the purpose I have expressed, or to apply it externally in some of their diseases [5].

Demonstrated that the pipiltzintzintlis are nothing but hemp, I still have to satisfy the second question, what I will execute, warning, first, not only the Indians of New Spain who practice the inner use of the seed and leaves of hemp for their extravagant visions. The second is that the effects observed in those who use hemp inside or pipiltzintzintli are usually natural. For the former, what is said by Monsieur Petit, in his dissertation on the nepenthes of Homer [6], printed in 1689, is very useful; it is explained like this, speaking of the herbs and other productions of nature that disrupt the brain. "Among the drugs (he says) that have this use the Egyptians also use another composition, which they call asís; these are powders composed of hemp leaves, which are mixed by mixing water and forming pills when they want to forget about their melancholies, their care, and seek happiness; they swallow five or six of these pills, which are the size of a chestnut; this drug that intoxicates them to the point, makes them shortly afterwards to pass to a kind of abduction or static dream, during which they see the most pleasant things in the world: forests, fountains, meadows or gardens, adorned with the most beautiful flowers; the enchanted places where ... [7] in a word, the true Fortunatas Islands [8], or to speak more properly, a true paradise of Muhammad. "

The same news is presented to us by the famous Valmont de Bomare in his Universal Dictionary of natural history, et cetera, printed in Paris in 1767, in the word chambre hemp, it is explained in this way: "The leaves of hemp seem to contain a virtue that intoxicates, and numb. Koempser [9] tells how in some places of the Indies (Eastern) a drink is prepared that intoxicates, which is of use of this country. Some mix the hemp seed with food ... but this fills their heads with smoke and if eaten in abundance excites delirium, according to and like the coriander. "What shall we say of the daily and general use throughout the kingdom of coriander? For now I can not elaborate more on the subject.

The testimony of Monsieur Valmont is very important. Who else to a naturalist must believe about the virtues that contain the productions of nature? According to what he refers to, hemp is narcotic [10], and therefore, its effects are natural, with which it is not much that the Indians who take it suffer from a brain disorder, by a very natural effect.

Recklessness would be to affirm that in some occasions the effects of hemp on the Indians are not helped by the spirit of darkness, by that implicit or explicit agreement that some of them may contract with it; but usually, we must confess that in the most, the effects and visions are purely natural. Piety, reason and criticism dictate that we should not regard as preternatural everything that does not extend beyond the limits of nature.

What important service would it do to the spiritual health of these unfortunates who taught them that in the use of the pipiltzintzintlis the devil has no more part than what he is wanted to give? I think it would be the shortest way to destroy this bunch of superstition. The prohibition of its use is very necessary interim perseveren Indians in believing its diabolic effects; But is it not constant that the prohibition incites more and more the desire to execute the forbidden thing, for that malice to which we are so prone? A practical case seems to me to prove with evidence the precision of instructing the Indians in the natural effect caused by the pipiltzintzintlis. The seed of hemp has many uses in medicine, according to Bomare, cited above: it is emulsive and, boiled in milk, it is useful to cure cough and jaundice. Some other authors regard it as a specific against gonorrhea. Lemery in his treatise on drugs, describes its virtues in this way: "Hemp contains much oil and little salt, it is very good for the burned and for the ringing of the ears; the seed is reputed to be very good at combating lasciviousness; taken for many days pacifies the cough. "The dosage is from a scruple to a dram: in the Economic Dictionary these same virtues are referred; in more number and treated with extension in the Compendium of the usual plants by Monsieur Chomel, doctor regent of the medical faculty of Paris.

So if a doctor ordered the use of hemp seed by virtue of its usefulness in medical uses, ignoring, as it is credible ignore, the abuses that the Indians have in this seed, which they know by pipiltzintzintli, there was not a very serious damage because a new incentive to remain in those sinful creatures was ministered to their ignorance? What portions of the aforementioned seed will have been consumed in the Royal Hospital of this court ordered by the doctors in charge of the healing of the multitude of Indians who annually tend in this hospital with very healthy intention, regarding the doctors, and spiritual damage of the Indians? I am persuaded that some of them, who will have been administered in their ailments, regained their temporary health, will have been deeply rooted in the abuse of the pipiltzintzintlis. It seems I have demonstrated the virtue of the decanted pipiltzintzintlis, so we will have to say with the language of theologians, that they are bad because they are forbidden, not forbidden because they are bad.











Notes



[1] It should be understood I speak of the Indians recognized as such, because the mixture with other castes and the different education changes their character.

[2] Pipiltzintzintli is equivalent in our Spanish to little boy, little, little son.

[3] There is no doubt that your temporary health is involved. The violent effect of narcotics proves it a lot; it has not been many months that a person to whom they administered him, I do not know for what purpose, the pipiltzintzintlis, perhaps in too much quantity, lost the judgment.

[4] We call herbalists to the indian women that in the market are engaged in selling herbs or other medicinal things; they do in part what the drugstore keepers in Europe.

[5] In the outer use of them they still have their abuses, except that those expressions with which they are explained, saying that it is not convenient to fry or heat the pipiltzintzintlis, this is the little children, when they have to apply to some destination, because they die , understand it figuratively; this only a deep knowledge of the language and a handling with them executed with discretion, can solve the doubt.

[6] Nepenthes of Homer; This famous poet describes it as very propitious to banish sadness; its commentators have tired themselves uselessly to find out what it is.

[7] I have truncated part of what Petit says for being obscene; our language, and much more my state, requires me to pass in silence everything that faces modesty. The famous Desfontaines was reprimanded over his ignorance in anatomy, to which he responded with that customary and wise discretion: I possess a wise ignorance of anatomy, but that which suits an ecclesiastic; his dissertation or memory in which he gives the reason why obscene things are treated with more relief in Latin and not in vulgar language, is the best that is printed; the reason given is because Latin is a Gentile language, the vulgar are Christianized languages, by when they began to be used after the preaching of the Gospel.

[8] The Canary Islands.

[9] The author who has given us the best history of Japan.

[10] The way of working of the narcotic refers to the medical authors who have written about the virtue of the medicines. To give a slight idea, I will expose the narcotics article from the famous Trévoux Dictionary; which will give a slight idea of ​​what the best doctors have written ... "Narcotics, a term of medicine that is said of the remedies that seek numbness; the narcotics act according to how they calm and diminish the movement of the spirits and prevent them for some time from moving with all the vitality that is necessary. The poppy, the opium, the mandrake, the narcissus, the henbane and so on, are narcotics; the ancients, attribute it to the coldness they have of their nature: Etmullero after Willis thinks that animal spirits are composed of a volatile fluid salt, and that by the mixture of sulfur or oils in which narcotics abound, they dissolve . The opinion of Monsieur Andry, who warns Monsieur Fayon, is that the salt of the narcotics is dissolved by a liquor, whatever it may be, and that its branched particles that remain free of the salts, get entangled with each other and they stop the course of the blood and the spirits. Finally, others judge that narcotics shut the orifice of the nerves at their origin. Linder, in his Treatise on Poisons, says that the action of narcotics is not the same in all, when there are so many different causes that can cause sleep and numbness, as the deficit of abundance in the brain, which dilates the arteries and veins, it compresses the origin of the nerves: the coagulation and thickness of the blood, a cluster of phlegm or blood lost in the brain and a thousand other things. "
 
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