AUTHOR=Balthazar Carole , St-Onge Renée , Léger Geneviève , Lamarre Simon G. , Joly David L. , Filion Martin 

TITLE=Pyoluteorin and 2,4-diacetylphloroglucinol are major contributors to Pseudomonas protegens Pf-5 biocontrol against Botrytis cinerea in cannabis

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.945498

DOI=10.3389/fmicb.2022.945498

ISSN=1664-302X

ABSTRACT=<p><italic>Pseudomonas protegens</italic> Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen <italic>Botrytis cinerea</italic> causing gray mold disease in <italic>Cannabis sativa</italic> crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against <italic>B. cinerea</italic> in the phyllosphere of <italic>C. sativa</italic>, two knockout Pf-5 mutants were generated by in-frame deletion of genes <italic>pltD</italic> or <italic>phlA</italic>, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. <italic>In vitro</italic> confrontation assays revealed that deletion mutant ∆<italic>pltD</italic> inhibited <italic>B. cinerea</italic> growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆<italic>phlA</italic> inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered <italic>in vitro</italic> inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆<italic>pltD</italic> and ∆<italic>phlA</italic> significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities <italic>in planta</italic> similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of <italic>P. protegens</italic> Pf-5 against <italic>B. cinerea</italic> in the cannabis phyllosphere.</p>