Selection And Characterization Of Antagonistic Bacteria Against Tobacco Black Shank Disease

A total of 442 isolates were isolated from 18 soil samples collected from tobacco and other crop's fields in Yunnan Province, China. Among them, 44 isolates were selected based on their antagonistic activities to pathogen Phytophthora parasitica var. nicatianae on the dual culture. The pot experiments were conducted for 11 isolates from the 44 selections and other 11 isolates from the collections of Yunnan Tobacco Science Institute to test their control effect on the tobacco black shank disease. Among the tested ones, eight isolates, including B254, HJ01912, HK281, TS-4,ME2,HJ01916, 1KMB-2 and HK282, showed significant suppressive effects on the disease. Using the plate culture, this study tested the bacterial culture filtrates from 27 bacterial isolates for their antagonistic activities on P. parasitica var. nicatianae. Among them, HJ01916 cell-free culture showed obvious antagonistic activity, indicating that some antagonistic substance existed in the culture and the antagonistic mechanism of HJ01916 was antibiosis.HJ01916 isolates was selected to study on the characterization of its antagonistic substance. The cell-free culture of HJ01916 was partially thermostable and sensitive to proteinase K. The crude extract from the cell-free culture with (NH4)2SO4 precipitation had antagonistic activity, indicating that the antagonistic proteins existed in the culture. The mycelia of Phytophthora parasitica var. nicotianae incubated in the culture for 48 h showed malformation. The cultural characters and physio-biochemical tests of HJ01916 isolate were similar to Bacillus subtilis. The PCR amplified full-length 16S rDNA of the strain was 1424 bp (GenBank accession no. AY894690). The phylogentic analysis based on 16S rDNA suggested that HJ01916 isolate shared 100% homologies with B. subtilis (AY583216) and both sequences constituted a branch in phylogenetic tree. Therefore, HJ01916 was considered as an isolate of B. subtilis.The bacterial isolate HJ01912 was provided by Yunnan Tobacco Science Institute and showed suppressive effect on tobacco black shank disease. Based on the design of L27 (313) orthogonal test, this study tested the optimal prescription of the medium for fermentation culture of isolate HJ01912. Under the laboratory condition, the...