Screening And Genetic Diversity Of Antagonistic Bacteria Against Botrytis Cinerea Isolated From Tomato Rhizosphere

A total of2892isolates were obtained from11rhizosphere soil samples which werecollected from different areas of Shouguang city and Jining city. And168bacterial strainstested had some inhibitory activity against Botrytis cinerea, and34bacteria have a efficientand stable effect.The genetic diversity of antagonistic bacteria was studied with the methods ofBOXAIR-PCR,16S-RFLP,16S rDNA sequence homology and phylogenetic analysis.BOXAIR-PCR revealed that168test isolates could be divided into33BOX groups orsingle strains at87%of similarity. This includes two large groups,15and19;16small groupsand14single groups, but the result with little correlation to agrotype or geographical. TheBOX-PCR genomic fingerprints results and cluster analysis reflecting a high degree ofgenotypic among these isolates.The16S-RFLP analysis separated the168isolates into28cluster or genotypic groups at91.5%similarity level, each group presented a specific banding pattern, and clusters ofdifferent species were clearly discrepant. Moreover, groups3,4,5,10,12,14,15,16,17,18,19,21,23,25,26and28each include a single isolate. Furthermore, clusters6and13constitute two big groups and belong to the genera Bacillus and Streptomyces, respectively.For this reason, we got a conclusion that antagonistic bacteria are highly diverse at aphylogenic level.Representative isolates of each group were selected for partial16S rDNA genesequencing so as to retrieve sequence similarity and bacterial identity from NCBI on the basisof the clustering results of BOXAIR-PCR and16S-RFLP. Closest identities of representativeisolates were retrieved after16S rDNA partial sequences from76isolates. The resultsindicated a high degree of similarity at a level of no less than99﹪. A phylogenetic treedemonstrate the relatedness between isolates and type strains.76representative isolates weredivided into at least26different species from14different genera. Bacillus, Pseudomonas,Alcaligenes were the dominant genera identified, with126,10and10isolates, respectively.Species diversity was demonstrated by the genera of Pseudomonas, Bacillus and Streptomyces,including at least3,8and5species.Gram-positive isolates were more frequently encountered with a total frequency of79.2%compared to only20.8%that was Gram-negative bacteria. It was exciting to discover that20.8%of the antagonistic bacteria were strongly active against B cinerea. As we haveseen, this is the first time that Paenibacillus, Providencia, Staphylococcus, Sinorhizobium,Arthrobacter, Alcaligenes, Myroides, Brevibacillus, Acinetobacter and Tetrathiobacter arereported as antagonists of B cinerea. These antagonistic bacterial isolates demonstratepotential as future biocontrol agents.