Isolation Of Antagonistic Strains Against Tomato Bacterial Wilt And Identification Of Their Biological Control Mechanisms

There are no effective chemical methods to control tomato bacterial wilt(TBW), which caused by Ralstonia solanacearum(RS). Biological control has great potential. In this paper, three effective antagonistic strains JK6, F7 and Y-2 were isolated from tomato rhizosphere soil through plate inhibition tests. Firstly, pot experiments were carried out to evaluate their biological control efficacies. The antibacterial metabolites in their culture filtrate were identified by MS and HPLC analysis. Real-Time PCR analysis was carried out to estimate the quantification of functional genes in the rhizosphere soil after treated with antagonistic strains. The main results were as follows:(1) The inhibition diameters of JK6, F7 and Y-2 against RS were up to 34.7, 20.3 and 21.3 mm, respectively. Furthermore, they can significantly suppress the growth of F. oxysporum f.sp. cubense, F. oxysporum f.sp. cucumerinum, and Colletotrichum gloeosporioides, ranging from 34.5 to 45.0 mm. Additional, JK6 was identified as B. amyloliquefaciens, F7 and Y-2 were identified as B. methylotrophicus through their 16 S r RNA gene sequences, physiological and biochemical characteristics.(2) The biocontrol efficacies(BEs) of JK6 suppressing TBW were up to 58.6% in pot experiments and it could significantly promote plant growth. Additional, F7 and Y-2 also could significantly suppress TBW and their BEs were up 46.0 % and 55.5%.(3) Our study demonstrated the presence of lipopeptides surfactin in the fermentation of JK6, F7 and Y-2 strains with C18 column filtering and methanol extracting. Their concentrations were 64.24, 53.09 and 31.34 mg.L-1 through MS and HPLC analysis. To further verify the inhibitory effect of methanol extract of fermented liquid against RS, the inhibition zones were up to 23, 18 and 19 mm, respectively. It proved that the surfactin was the important evidence for the biocontrol potential of the antagonistic strains.(4) The results of functional gene analysis showed that: all of the antagonistic strains contained srf AB?fen D?itu A?itu C?itu D and ynd J genes. Furthermore, Real-Time PCR analysis showed that the ynd J, fen D and srf AB gene contents(copy number per 100 ng of total soil DNA) in the antagonistic strains JK6, F7 and Y-2 treated soil were much higher than those of CK treatments.In a word, the antagonistic strains B. amyloliquefaciens JK6, B. methylotrophicus F7 and Y-2, were proved to be excellent biocontrol strains against RS and other soil-borne diseases. They could significantly suppress TBW. Lipepoptides surfactin has been identified to be the main antibacterial metabolites. Furthermore, the srf AB, fen D, and ynd J DNA content in their treated soil were significantly higher than those of control. These results provided preliminary reference and method for illustrating antagonistic activity components and biocontrol molecular mechanisms in field soil. We also found the biocontrol potential and mechanisms of novel B. methylotrophicus strains, which provide new idea for antagonistic microbial resources. They showed abroad application prospects in microbial fertilizer.