Biocontrol Activity ofBacillusspp. andPseudomonasspp. AgainstBotrytis cinereaand Other Cannabis Fungal Pathogens

Abstract

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.

Plant Growth-Promoting Activity of Pseudomonas aeruginosa FG106 and Its Ability to Act as a Biocontrol Agent against Potato, Tomato and Taro Pathogens

“…Pseudomonas spp. (Pseudomonadaceae) is one of the most abundant genera of beneficial rhizobacteria among the large and heterogeneous bacterial populations in the rhizosphere and has, therefore, attracted growing attention as a source of potential biological control agents [ 11 , 12 ]. Except for some human pathogenic strains [ 13 ], the species of P. aeruginosa is a versatile and ubiquitous bacterium that has been recognized as an active antagonist of several bacterial and fungal plant pathogens, and has potential practical applications in agricultural systems [ 14 ].…”

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).…”

“…Notably, their antagonistic activities can be used to control fungal and bacterial diseases, but also herbivorous insects (Flury et al, 2016;Khan et al, 2016), parasitic nematodes (Trivedi and Malhotra, 2013) and weeds (Trognitz et al, 2016). Accordingly, antibiotic production was suggested as the main antifungal mechanism of beneficial P. protegens and P. synxantha strains when sprayed on hemp leaves to control Botrytis gray mold, and/or inhibiting the in vitro growth of Botrytis, Sclerotinia, Fusarium, Alternaria, and Phoma (Balthazar et al, 2021). Previous studies also demonstrated that two strains of Pseudomonas koreensis and Pseudomonas taiwanensis found in the hemp rhizosphere could inhibit the growth of Aspergillus and/or Fusarium in vitro by producing siderophores, cellulases, pectinases and proteases (Afzal et al, 2015), while hemp endophytes Pseudomonas fulva and Pseudomonas orientalis inhibited the growth of Botrytis, Sclerotinia and/or Rhizoctonia in vitro by producing siderophores, cellulases, and antibiotics (Scott et al, 2018).…”

“…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).…”