Pyoluteorin and 2,4-diacetylphloroglucinol are major contributors to Pf-5 biocontrol against in cannabis.

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