The inhibitory mechanisms by mixtures of two endophytic bacterial strains isolated from Ginkgo biloba against pepper phytophthora blight

Two endophytic bacterial strains, Fy11 and Zy44 (Bacillus amyloliquefaciens), isolated from Ginkgo biloba leaves, were tested as a single application and in combination for their abilities to suppress pepper phytophthora blight under greenhouse conditions. When both strains were applied in a mixture, the disease severity was more reduced. The objective of this study was to find out the inhibitory mechanisms of synergistic activity against pepper phytophthora blight through a mixture of Fy11+Zy44. Firstly, the colonization ability of the two endophytic bacterial strains was tested in pepper tissues. The result indicated that both endophytic strains were efficient colonizers of pepper, and could vertically transfer from roots to shoots and leaves. After inoculation for 7days, the population densities of Fy11-gfp and Zy44-gfp were 24.7×103 CFU/g FW and 13.0×103CFU/g FW in pepper roots, respectively. The cell counts of co-inoculated Fy11+Zy44 were not significant different comparing to a single strain inter pepper tissues. Secondly, the direct antagonism of antifungal compounds produced by the endophytic bacteria were investigated against pepper phytophthora blight. The crude lipopeptides produced by strain Zy44 highly effectively reduced disease index, and the control efficacy reached 84.8% and 67.2% in 7d and 15d after inoculation with the pathogen, respectively. The control effect of the secondary metabolites produced by strain Fy11 was not obvious. In the end, the induced systemic resistance (ISR) mediated by strains Fy11 and Zy44 on pepper were further analysed using priming effect of the defense-related genes after pathogen challenge. The expressions of CaPR4 (Capsicum annuum pathogenesis-protein 4) and CaBGLU (C. annuum β-1,3-glucanase) were strongly induced in the plants treated with strain Fy11 in 24h after pathogen challenge, but CaPR4 and CaBGLU transcripts were not increased in the plants treated with strain Zy44. These results indicated that the mechanisms of additive effect through combined strains Zy44 and Fy11 might be due to direct antagonism of antifungal lipopeptides produced by strain Zy44, as well as indirect inhibition through ISR induced by strain Fy11. Therefore, the synergistic interactions were due to the summation of biocontrol mechanisms of both biocontrol agents. This work provides a theoretical explanation for our findings of reduced disease severity with a mixture of endophytic bacteria.