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We established transformation technologies using Agrobacterium tumefaciens (Smith & Townsend) Conn to insert foreign genes into high THC-containing cannabis (Cannabis sativa L.). The Arabidopsis non-expressor of pathogenesis-related protein 1 (AtNPR1) gene was selected as a potentially useful agronomic gene, which was linked to the bar gene from Streptomyces that encodes herbicide resistance. We investigated how Agrobacterium strains (EHA105 and GV3101), glufosinate concentrations, explant source, and light intensity affected transformation frequency (TF). Transformation was confirmed by RT-PCR with primers for the NPR1 or bar genes. Glufosinate at 0.5-1 mg/L inhibited growth of non-transformed calli within 8 weeks following A. tumefaciens infection. Strain EHA105 yielded a higher TF compared to strain GV3101. Whole leaflets yielded a higher TF compared to sectioned leaf explants with strain GV3101. However, this effect was not seen with EHA105. Petiole segments showed a higher TF than leaf sections with strain EHA105. Placing explants under light or dark conditions did not affect TF, which ranged from 5% to 95% in different experiments. This is the first report of successful transformation of two high THC-containing C. sativa genotypes with two foreign genes simultaneously--AtNPR1 and bar. The recovery of plantlets from transgenic calli was not attempted and awaits further research. Key words: marijuana, genetic engineering, biotechnology, transgenic plants, Introduction Agrobacterium-mediated transformation is a widely used technique for gene insertion into plant genomes (Gelvin 2000, 2003; Anjanappa and Gruissem 2021). The Agrobacterium tumefaciens (Smith & Townsend) Conn (syn. Agrobacterium [...]

We investigated the diversity of fungal species present on inflorescences as epiphytes and in stem tissues as endophytes in flowering plants of cannabis grown organically in British Columbia during 2019-2021. Fresh and dried inflorescence samples were obtained at various times during production while stems were obtained at harvest. Fungal species in the air were assessed by exposing Petri dishes containing potato dextrose agar + streptomycin sulfate for 1 h in the growing environment while soil samples were dilution-plated to assess soil fungal diversity. Colonies were identified from PCR-derived sequences of the ITS1-5.8S-ITS2 region of ribosomal deoxyribonucleic acid. Twenty-nine species in 26 genera were recovered from inflorescences and 17 species in 11 genera originated from stem tissues. Approximately 96% of species found on inflorescences were present in air and 45% were present in organic soil. The fungi comprised plant pathogens, saprophytes, and opportunistic human pathogens. A large proportion of the species found in air and soil in organic facilities are present on cannabis inflorescences, where they may increase total colony forming units and negatively affect product quality. Some species could contribute to allergies or secondary infections in humans. The potential benefits of endophytes within organically grown cannabis plants remain unexplored. Key words: Cannabis sativa, post-harvest quality, soil fungi, organic production, plant pathogens, endophytes, Introduction Large-scale production of THC-containing Cannabis sativa L. (referred to here as cannabis) for medicinal and recreational use is currently permitted in Canada following its legalization in 2018. Licensed producers [...]

Botrytis cinerea is a widespread necrotrophic plant pathogen that causes diseases on >1000 plant species, including vegetables and ornamental greenhouse crops. On cannabis (Cannabis sativa L.), the pathogen is responsible for causing 'bud rot', a major disease affecting the inflorescences (compound flowers), as well as seedling damping-off and leaf blight under certain conditions. During greenhouse cultivation, Botrytis cinerea can destroy cannabis inflorescences rapidly under optimal relative humidity conditions (>70%) and moderate temperatures (17-24 [degrees]C). Little is currently known about the host-pathogen interactions of Botrytis cinerea on cannabis. Information gleaned from other hosts can provide valuable insights for comparative purposes to understand disease development, epidemiology, and pathogenicity of Botrytis cinerea on cannabis crops. This review describes the pathogenesis and host responses to Botrytis infection and assesses potential mechanisms involved in disease resistance. The effects of microclimatic and other environmental conditions on disease development, strategies for early disease detection using prediction models, and the application of biological control agents that can prevent Botrytis cinerea development on cannabis are discussed. Other potential disease management approaches to reduce the impact of Botrytis bud rot are also reviewed. Numerous opportunities for conducting additional research to better understand the cannabis-Botrytis cinerea interaction are identified. Key words: cannabis, epidemiology, molecular detection, disease prediction, biological control, integrated disease management, Introduction Agricultural plants are continuously confronted with biotic stressors that negatively affect their development, yield, and quality. Significant crop losses worldwide are caused by pathogens that result in economic losses [...]

Copyright of Canadian Journal of Plant Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The incidence of Hop latent viroid (HLVd) affecting cannabis plants in licenced production facilities in Canada during 2020–2023 was determined by RT-PCR analyses of 15 947 samples from nine provinces. Positive detection ranged from 5.3% to 92% of samples submitted, depending on province and year of sampling. The average country-wide HLVd infection incidence was 25.6%. Symptoms on affected plants varied with plant growth stage, and ranged from asymptomatic to mild leaf curl and mottling on stock (mother) plants and on vegetative plants, to extreme stunting and reduced inflorescence development on flowering plants. All symptomatic and some asymptomatic plants contained a 256 nucleotide RNA fragment with 100% sequence homology to HLVd from GenBank. The viroid was detected in various tissues of stock, vegetative, and flowering plants, including the uppermost leaves, at middle and lower positions in the canopy, and in the roots. The incidence of infection varied with the cannabis genotype. Flowering plants displayed yellowing or darkening of inflorescence leaves surrounding the pistillate flowers. Reductions of 12–42% in inflorescence stem lengths, fresh weights, and plant heights were observed in infected plants compared to noninfected plants, depending on the genotype. Levels of tetrahydrocannabinol (THC) and terpenes in diseased inflorescences were also significantly lower. The development of glandular trichomes which produce and store cannabinoids and terpenes was greatly reduced by HLVd infection. The affected trichomes had shorter stalk lengths and smaller glandular head sizes, and appeared shrivelled upon drying of the inflorescences. Hop latent viroid poses a significant economic threat to the cannabis industry. [ABSTRACT FROM AUTHOR]

The increased cultivation of high THC-containing Cannabis sativa L. (cannabis), particularly in greenhouses, has resulted in a greater incidence of diseases and molds that can negatively affect the growth and quality of the crop. Among them, the most important diseases are root rots (Fusarium and Pythium spp.), bud rot (Botrytis cinerea), powdery mildew (Golovinomyces ambrosiae), cannabis stunt disease (caused by hop latent viroid), and a range of microbes that reduce post-harvest quality. An integrated management approach to reduce the impact of these diseases/microbes requires combining different approaches that target the reproduction, spread, and survival of the associated pathogens, many of which can occur on the same plant simultaneously. These approaches will be discussed in the context of developing an integrated plan to manage the important pathogens of greenhouse-grown cannabis at different stages of plant development. These stages include the maintenance of stock plants, propagation through cuttings, vegetative growth of plants, and flowering. The cultivation of cannabis genotypes with tolerance or resistance to various pathogens is a very important approach, as well as the maintenance of pathogen-free stock plants. When combined with cultural approaches (sanitation, management of irrigation, and monitoring for diseases) and environmental approaches (greenhouse climate modification), a significant reduction in pathogen development and spread can be achieved. The use of preventive applications of microbial biological control agents and reduced-risk biorational products can also reduce disease development at all stages of production in jurisdictions where they are registered for use. The combined use of promising strategies for integrated disease management in cannabis plants during greenhouse production will be reviewed. Future areas for research are identified. [ABSTRACT FROM AUTHOR]

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ9-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures. [ABSTRACT FROM AUTHOR]

Copyright of Canadian Journal of Plant Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Total yeast and mold (TYM) levels in inflorescences of high THC-containing Cannabis sativa (cannabis) are regulated to ensure that medicinal and recreational users, especially those with immunocompromised systems, are not exposed to potentially harmful levels. In North America, the limits imposed range from 1,000-10,000 cfu/g of dried product to 50,000-100,000 cfu/g, depending on the jurisdiction. Factors affecting a build-up of TYM in cannabis inflorescences have not been previously researched. In this study, >2,000 fresh and dried samples were assayed for TYM over a 3-year period (2019-2022) to identify specific factors which can contribute to TYM levels. Greenhouse-grown inflorescences were sampled before and after commercial harvest, homogenized for 30 s, and plated onto potato dextrose agar (PDA) with 140 mg/L streptomycin sulfate. Colonyforming-units (cfu) were rated after 5 days of incubation at 23°C under 10-14 h light. PDA provided more consistent counts of cfu compared to Sabouraud dextrose and tryptic soy agars. The predominant fungal genera identified by PCR of the ITS1-5.8S-ITS2 region of rDNA were Penicillium, Aspergillus, Cladosporium, and Fusarium. In addition, four yeast genera were recovered. In total, 21 species of fungi and yeasts constituted the total cfu present in the inflorescences. The variables that significantly (p < 0.05) increased these TYM levels in inflorescences were: the genotype (strain) grown, presence of leaf litter in the greenhouse, harvesting activity by workers, genotypes with a higher abundance of stigmatic tissues and inflorescence leaves, higher temperature and relative humidity within the inflorescence microclimate, time of year (May-October), method of drying buds after harvest, and inadequate drying of buds. The variables which significantly (p < 0.05) decreased TYM in samples were: genotypes with lower numbers of inflorescence leaves, air circulation achieved by fans during inflorescence maturation, harvesting during November-April, hang-drying of entire inflorescence stems, and drying to a moisture content of 12-14% (water activity of 0.65-0.7) or lower which was inversely correlated with cfu levels. Under these conditions, the majority of dried commercial cannabis samples contained <1,000-5,000 cfu/g. Our findings indicate that TYM in cannabis inflorescences are the result of a dynamic interaction between genotype, environment, and post-harvest handling methods. Some of these factors may be altered by cannabis producers to reduce the potential build-up of these microbes. Among the 21 fungal and yeast species recovered from greenhouse-grown cannabis inflorescences, a few could pose a potential threat to human health, while many do not and they could provide beneficial interactions within the cannabis plant. The currently recommended plating methods onto agar media and enumeration of total cfu are unable to distinguish between these two groups. [ABSTRACT FROM AUTHOR]