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

Abstract

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

Structures and Biosynthesis of Caryoynencins, Unstable Bacterial Polyynes from Pseudomonas protegens Recombinant Expressing the cayG Gene

“…protegens Cab57, was not pathogenic on plants, such as B. caryophylli strains. − We expect that the usefulness of P. protegens Cab57 and its transformants can be further demonstrated by introducing other modifying enzymes for bacterial polyynes.…”

“…Pseudomonas protegens strains are well-known as biocontrol agents that protect crops from pathogens (e.g., fungi, oomycetes, and nematodes). − We recently identified new bacterial polyynes, protegenins A–D, from P. protegens Cab57 by genome mining (Figure A) .…”

Biocontrol potential of Pseudomonas protegens ML15 against Botrytis cinerea causing gray mold on postharvest tomato (Solanum lycopersicum var. cerasiforme)

“…It has been recently shown that pyoluteorin and 2,4-DAPG play a crucial role in the P. protegens Pf-5 biocontrol against B. cinerea . Moreover, pyrrolnitrin is known for its broad-spectrum antimicrobial activity and low phototoxicity which has made it an interesting target for biotechnological investigations ( Pawar et al., 2019 ; Balthazar et al., 2022b ). Cluster Blast analysis showed that all five of the highly similar BGCs are conserved within the Pseudomonas genus, and most of them are specific to the P. protegens species.…”

Microbial Biopesticides: Diversity, Scope, and Mechanisms Involved in Plant Disease Control

“…Antibiotic concentrations on plant surfaces or in the soil, however, are considerable if not exponentially lower than those obtained in culture media [144]. Despite this difference, the role of antibiotics produced by biocontrol agents in the suppression of many plant diseases in planta has been confirmed through the use of mutant strains that are unable to produce antibiotics and do not suppress disease symptoms [109].…”