Phenazine-1-carboxylic acid is a more important contributor to biocontrol Fusarium oxysporum than pyrrolnitrin in Pseudomonas fluorescens strain Psd

Abstract

Phenazines and pyrrolnitrin (Prn) are broad spectrum antibiotics, produced by bacteria, more so by the biocontrol strains to kill the phytopathogens in soil. We have studied a rhizospheric soil isolate of Pseudomonas fluorescens strain Psd producing both phenazine-1-carboxylic acid (PCA) and Prn. In order to study the contribution of these antibiotics, the phzD and prnC genes involved in PCA and Prn biosynthesis, were disrupted in a site-specific manner using a group II intron-based Targetron gene-knockout system, and gene disruption followed by allelic exchange through homologous recombination, respectively. The resulting knockout strains Psdphz122s-34 and PsdprnC::gen did not produce PCA and Prn, respectively. In fact, by combining these two strategies, a Psdphz122s-34prnC::gen double mutant could also be generated. Identification and lack of PCA production was corroborated by HPLC/APCI-MS analysis, and TLC detection for both the antibiotics in these mutants. Loss of antifungal activity against the phytopathogenic fungus Fusarium oxysporum was observed using in vitro growth assays on plates or growth chamber experiments with tomato seedling on an artificial substrate. Based on the characterization of these gene knockout mutants, we propose that PCA and Prn have a major role in antifungal activity of strain Psd.