High-throughput identification of genes influencing the competitive ability to obtain nutrients and performance of biocontrol in Pseudomonas putida JBC17

Abstract

Elucidating underlying mechanisms of biocontrol agents (BCAs) could aid in selecting potent BCAs and increasing their biocontrol efficacy. Nutrient competition is an important biocontrol mechanism; however, essential nutrient sources, and contributing genes for nutrient competition still remain to be explored. Pseudomonas putida JBC17 (JBC17WT) suppressed green mold in satsuma mandarins by inhibiting conidial germination of Penicillium digitatum via nutrient competition. To analyze genes essential for biocontrol performance of JBC17WT, we generated a transposon (Tn)-mediated mutant library and selected mutants with the ability to suppress conidial germination. Several mutants in the genes of flagella-formation, including fliR, fliH, and flgG, increased biocontrol performance and enhanced inhibition of conidial germination. They lost swimming motility, exhibited increased growth and rapid carbon and nitrogen utilization than the wild type under nutrient-poor conditions. The nutrient competition assay using polytetrafluoroethylene cylinders revealed that conidial germination was inhibited by nutrient absorption under nutrient-poor conditions. In addition, genes, including amidohydrolase (ytcJ), tonB-dependent receptor (cirA), argininosuccinate synthase (argG), D-3-phosphoglycerate dehydrogenase (serA), and chaperone protein (dnaJ), were involved in the inhibition of conidial germination. The results of this study indicate that rapid and continuous absorption of nutrients by JBC17WT restrict nutrient availability for conidial germination on nutrient-limited fruit surfaces, thereby decreasing the chances of fungal spores infecting fruits. The high-throughput analysis of Tn mutants of this study highlighted the importance of nutrient competition and the genes that influence biocontrol ability, which contributes to the development of biocontrol applications.