The bud rot pathogens infecting cannabis (Cannabis sativa L., marijuana) inflorescences: symptomology, species identification, pathogenicity and biological control

Abstract

Bud rot pathogens cause diseases on Cannabis sativa L. (cannabis, hemp) worldwide through pre-and post-harvest infections of the inflorescence. Seven indoor or outdoor cannabis production sites and three hemp fields were sampled for bud rot and stem canker presence during 2019-2020. Among 178 isolates recovered from diseased tissues, sequences of the ITS1-5.8S-ITS2 region of rDNA, the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene and the heat shock 60 (HSP) gene identified the following: Botrytis cinerea (162 isolates), B. pseudocinerea (2), B. porri (1), Sclerotinia sclerotiorum (5), Diaporthe eres (3) and Fusarium graminearum (5). Pathogenicity studies conducted on fresh detached cannabis buds inoculated with spore suspensions or mycelial plugs showed that B. cinerea, S. sclerotiorum and F. graminearum were the most virulent, while B. pseudocinerea, B. porri and D. eres caused significantly less bud rot. Optimal growth of Botrytis species occurred at 15-25°C. In vitro antagonism tests showed that Bacillus spp., Trichoderma asperellum and Gliocladium catenulatum inhibited B. cinerea and S. sclerotiorum colony growth. When applied as a spray 48 h prior to B. cinerea inoculation, all biocontrol agents significantly (P < 0.01) reduced disease development on detached inflorescences. Prolific growth and sporulation of T. asperellum and G. catenulatum were observed on bud tissues. The pathogens B. porri, S. sclerotiorum, D. eres and F. graminearum are described for the first time as cannabis bud rot pathogens. Inoculum from neighbouring fields of diseased garlic, cabbage, blueberry and hay pasture, respectively, likely initiated infection of inflorescences. Several biological control agents show potential for disease reduction through competitive exclusion.

Exploiting Beneficial Pseudomonas spp. for Cannabis Production

“…However, these results were not validated on plants (Afzal et al, 2015;Scott et al, 2018). Ongoing research with other beneficial bacteria and fungi, like Bacillus, Streptomyces, Trichoderma, and/or Gliocladium, further supports the potential of antagonistic biocontrol agents against Botrytis on marijuana inflorescences (Punja and Ni, 2021) and hemp leaves (Balthazar et al, 2021), and against Fusarium and powdery mildew on marijuana stem cuttings and leaves, respectively (Punja, 2021;Scott and Punja, 2021). Besides, various fungal endophytes were also isolated from hemp and marijuana plants previously, but their antimicrobial activities were only tested in vitro (Gautam et al, 2013;Kusari et al, 2013;Qadri et al, 2013).…”

“…Besides, various fungal endophytes were also isolated from hemp and marijuana plants previously, but their antimicrobial activities were only tested in vitro (Gautam et al, 2013;Kusari et al, 2013;Qadri et al, 2013). Guidelines for timing of application are also important to establish as curative effects are usually harder to achieve than preventive protection (Balthazar et al, 2021;Punja and Ni, 2021).…”

“…Two main strategies are usually distinguished when selecting beneficial microorganisms for plant inoculation and microbiome engineering: the bottom-up or synthetic approach which pieces together candidates exhibiting desired traits from an extensive pool gathered from various sources, and the topdown or stepwise approach, which starts from a complex existing microbial community and identifies its keystone players (Tabacchioni et al, 2021). In cannabis, bottom-up studies seem undeniably promising given the recent results obtained with existing PGPR strains (Citterio et al, 2005;Jin et al, 2014;Pagnani et al, 2018;Comeau et al, 2021), biocontrol agents (Balthazar et al, 2020(Balthazar et al, , 2021, or commercialized bioproducts (Gonsior et al, 2004;Conant et al, 2017;Kakabouki et al, 2021a,b;Punja, 2021;Punja and Ni, 2021;Scott and Punja, 2021), thus paving the way for future work with existing Pseudomonasbased bioproducts that are already registered for cereals, fruit trees, and greenhouse vegetables (Fischer et al, 2013;Khan et al, 2016). On the other hand, several top-down studies have also started to examine the potential of harnessing cannabis microbiome residents to improve pathogen control (Gautam et al, 2013;Kusari et al, 2013;Qadri et al, 2013;Scott et al, 2018;, salinity tolerance (Afzal et al, 2015), soil phytoremediation (Liste and Prutz, 2006;Iqbal et al, 2018), cannabinoid production , and fiber retting process (Di Candilo et al, 2010;Ribeiro et al, 2015;Liu et al, 2017;Law et al, 2020).…”

“…Unfortunately, even with carefully selected beneficial microorganisms, concerns about potential carry-over of contaminants from treated plants into medical or recreational products could still limit the use of bioproducts on drug-type crops. Indeed, commercial quality control procedures and regulatory requirements usually do not distinguish between beneficial and harmful microorganisms, which could lead to unwarranted rejection of the product (McKernan et al, 2016;Boyar, 2021;Punja and Ni, 2021). Restricting treatments to plant roots and vegetative parts, and/or long before harvest time, has been suggested in order to spare the valuable inflorescences from residues while still offering beneficial effects like plant growth promotion, abiotic stress tolerance and below-ground microbiome wealth (Conant et al, 2017;Pagnani et al, 2018;Comeau et al, 2021;Kakabouki et al, 2021a,b), as well as disease prevention by direct antibiosis against soilborne pathogens (Punja, 2021), and ISR elicitation (Balthazar et al, 2020) or sporulation suppression (Balthazar et al, 2021;Scott and Punja, 2021) against aerial pathogens.…”

“…Application timing for PGPR strains that increase plant biomass should be aligned with phytochemical production goals (either maximizing inflorescence weight, or maximizing phytochemical concentration), as indicated by the distinctive effects of fertilizers when used at different cultivation stages-as explained above- (Caplan et al, 2017a,b;Saloner and Bernstein, 2021;Shiponi and Bernstein, 2021). Additionally, the use of biocontrol agents for preventive disease management should be based on forecasted risks for pathogen infestations and environmental factors (Balthazar et al, 2021;Punja and Ni, 2021). Multi-application schedules can also be advantageous for inoculants with transient effects which are preferable for their low environmental impacts (Kaminsky et al, 2019).…”