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

Abstract

<i>Pseudomonas protegens</i> Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen <i>Botrytis cinerea</i> causing gray mold disease in <i>Cannabis sativa</i> 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 <i>B. cinerea</i> in the phyllosphere of <i>C. sativa</i>, two knockout Pf-5 mutants were generated by in-frame deletion of genes <i>pltD</i> or <i>phlA</i>, 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. <i>In vitro</i> confrontation assays revealed that deletion mutant ∆<i>pltD</i> inhibited <i>B. cinerea</i> growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆<i>phlA</i> 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 <i>in vitro</i> 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 ∆<i>pltD</i> and ∆<i>phlA</i> 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 <i>in planta</i> 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 <i>P. protegens</i> Pf-5 against <i>B. cinerea</i> in the cannabis phyllosphere.