RESUMEN
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. Colony-forming-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.
RESUMEN
Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 µM) and naphthaleneacetic acid (0.5 µM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.
RESUMEN
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 PCR-based 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.