A plant growth-promoting rhizobacteria (PGPR) mixture does not display synergistic effects,likely by biofilm but not growth inhibition

Combination of different PGPR strains with complementary characteristics as a mixture to reduce possible instability under fluctuating environment has been considered practical. However, PGPR mixtures do not always play synergistic roles in growth promotion or biological control as reflected in our previous findings and other publications. In this work, we accidentally discovered that a mixture containing two well compatible PGPR strains, Bacillus pumilus WP8 and Erwinia persicinus RA2, did not synergize in growth promotion or biological control of tomato bacterial wilt under field conditions. Considering the importance of PGPR biofilm formation in growth promotion and biocontrol activities, we hypothesized that this phenomenon may be related to inhibition of biofilm formation. In vitro experiments showed that biofilm-formation ability of WP8 was inhibited by both RA2 cells and filtered supernatants collected from RA2 cultures at 12 h (RA2-12) rather than 48 h (RA2-48), even at high-temperatures (within 100°C). An in vivo experiment derived from crystal violet staining yielded similar results. Using liquid chromatography-mass spectrometry (LC-MS), we compared primary and secondary metabolites of RA2 (namely RA2-12 and RA2-48) and found D-glutamine, abundant in RA2-12, as the putative inhibitory factor. Trace amounts of jasmonic acid together with viscous extracellular polysaccharides in RA2-48 likely promoted the rescue of robust biofilm formation. This work suggests that inhibition of biofilm formation should be considered in PGPR mixture development.