High‐throughput analysis of genes involved in biocontrol performance ofPseudomonas fluorescensNBC275 against Gray mold

Abstract

Aims: Many physiological and microbial characteristics influence the biocontrol performance of the biological control agents (BCAs) in agricultural fields. To implement effective biocontrol, the contribution of specific genes, mechanisms and traits to the biocontrol performance of BCAs need to be characterized and explored in greater detail. Methods and results: In this study, a transposon (Tn) mutant library using the BCA Pseudomonas fluorescens NBC275 (Pf275) was generated to explore genes and bacterial characteristics involved in antifungal activity and biocontrol performance. Among the Tn mutants, 205 strains showing variations in antifungal activity compared to wild-type (WT) were selected and further analysed for biocontrol efficacy against gray mold in pepper fruits. The genes involved in pyoverdine biosynthesis (pvdI and pvdD) and chitin-binding protein (gbpA) played essential roles in the antifungal activity and biocontrol capacity of Pf275. In addition, a mutation in phlD completely abolished the antifungal activity and significantly suppressed the biocontrol ability of the strain. Genes affecting antifungal activity of Pf275 significantly influenced swimming motility, which was identified as an important trait for the biocontrol ability of the bacterial strain. Conclusions: Overall, our results suggest that antifungal compound production, siderophore biosynthesis and swimming motility synergistically contribute to Pf275 biocontrol performance. The utility of this library was demonstrated by identifying genes for antagonism and biocontrol ability in this BCA strain. The functional roles of many genes identified as contributing to antagonism and in vivo biocontrol activity require further study. Significance and Impact of this Study: Genes contributing to antifungal activity and biocontrol performance of P. fluorescens were identified and highlighted by Tn mutagenesis, which will give insight to improve the biocontrol performance of this BCA.

Assessment of the Contribution of Antagonistic Secondary Metabolites to the Antifungal and Biocontrol Activities of Pseudomonas fluorescens NBC275

“…We previously confirmed the role of pyoverdine in antagonism and biocontrol efficacy of Pf275 with mutants in genes pvdI and pvdD ( Dutta et al, 2019a ). In this study, antiSMASH identified two BGCs for pyoverdine (clusters 3 and 11) in Pf275 genome with conserved chromophore biosynthetic gene pvdL ( Gross and Loper, 2009 ) in both clusters.…”

“…In our previous studies, P. fluorescens NBC275 (Pf275) showed strong antagonism against several plant fungal pathogens ( Dutta et al, 2019b ) and biocontrol activity against gray mold in pepper fruit. In addition, overall analysis of Tn-mutant library of Pf275 revealed that production of antifungal SMs contributes to the strain’s biocontrol performance ( Dutta et al, 2019a ). However, the functional roles of the genes and SMs towards antagonism and biocontrol performance remain unclear.…”

“…Tn insertion mutant analysis identified 13 mutants that resulted in the suppression of antifungal activity; the genes belonged to 6 BGCs of the 14 BGCs predicted by antiSMASH ( Supplementary Table 3 ). In this study, to evaluate the contribution of each BGC to the overall biocontrol performance of Pf275, 13 mutants were assessed for the biocontrol efficacy against gray mold following Dutta et al (2019a) . The average disease area per pepper fruit was recorded from 3 replications (10 fruits per replication) ( Fig.…”

“…Four mutants had mutation in the genes for 2,4-DAPG production, including two core biosynthetic genes phlD (Tn57-74) and phlC (Tn25-17), a gene mexB_2 (Tn45-28) encoding a multidrug efflux RND transporter permease subunit, and a gene citN_1 (Tn03-62) for a citrate transporter. The mutants impaired in phlD and phlC completely lost the antifungal activity and their biocontrol ability was also drastically reduced ( Dutta et al, 2019a ). The BGC for 2,4-DAPG production is composed of six structural ( phlA, phlC, phlB, phlD, phlE, and phlI ) and three regulatory ( phlF, phlG, and phlH ) genes (Mandryk-Litvinkovich et al, 2017) ( Supplementary Figs.…”

“…AntiSMASH identified three clusters of bacteriocin in the Pf275 genome. In our previous study a mutant Tn03-39, impaired in ydaP , which predicted as an additional biosynthetic gene in bacteriocin BGC (Cluster 6) by antiSMASH, showed reduced antifungal activity ( Dutta et al, 2019a ). Bacteriocin are predominantly antibacterial compounds ( Mataragas et al, 2002 ), but bacteriocins from the Lactobacillus spp.…”