Isolation and identification of antagonistic bacteria against tomato bacterial wilt and the mechanisms in disease prevention and plant growth promotion

Abstract:

[Objective] Isolation plant growth promoting rhizobacteria (PGPR) from disease-resistant tomato rhizosphere soil to control bacterial wilt caused by Ralstonia solanacearum and improve tomato growth, and explored the mechanisms.[Methods] PGPR bacteria with antibacterial activity was screened from tomato rhizosphere soil by inhibition zone method using a moderately pathogenic Ralstonia solanacearum HN4 as target; The highly similar strains were identified by BOX-PCR fingerprints, and the enzyme-producing activity of different genotype strains was evaluated by the plate transparent circle method; The 16S rRNA phylogenetic analysis was applied to identify the antagonistic strains with strong antibacterial ability and many kinds of enzyme production; The potential antagonistic bacteria were assessed for plant disease prevention and growth promotion in greenhouse, and the plant disease prevention and growth promotion related genes expression level were tested by real-time PCR.[Results] 29 strains were isolated and purified from tomato rhizosphere soil, of which 15 strains had antagonistic function against Ralstonia solanacearum. Moreover, four strains involved in controlling tomato bacterial wilt and promoting growth, were verified by the analysis of BOX-PCR fingerprinting and enzyme activity. In the result of 16S rRNA phylogenetic analysis, strain B2 was identified as Bacillus amyloliquefaciens. Strain B5 and B20 were identified as Bacillus subtilis, respectively. Strain B23 was identified as Bacillus velezensis. In greenhouse experiment, strain B2, B5, B20 and B23 inhibited Ralstonia solanacearum effectively, with the efficiency of 35.59%, 8.47%, 32.20% and 96.61%, respectively. Furthermore, all of them significantly increased the plant fresh weight, the total chlorophyll content and the number of root tips. The strain B2, B5 and B23 efficiently promoted plant height and root length of tomato. The strain B2, B20 and B23 efficiently promoted stem thickness of tomato. And strain B23 efficiently promoted the number of root forks. In the result of real-time PCR, strain B2, B20 and B23 upregulated the expression of PR1α and POD1 gene. Strain B2, B5 and B23 upregulated the expression of ctd1 gene. And strain B2, B5, B20 and B23 downregulated the expression of ERF2 gene.[Conclusion]] In this study, four biocontrol strains were isolated and identified to control tomato bacterial wilt and promote tomato growth, and can provide a theoretical basis for the targeted screening of plant growth-promoting and disease preventing bacteria.