Isolation, Identification And The Establishment Of The Molecular Detection Methods Of The Pathogen Of Mulberry Bacterial Wilt Disease

In recent years, a new kind of mulberry bacterial wilt disease which was similar toRalstonia solanacearum were found in the main silkworm planting area Zhejiang andGuangdong province. The new disease caused serious damage to silkworm production. Weisolated, identified and detected the pathogen which caused mulberry bacterial wilt diseaseand discussed it in this study. This provided another evidence for Enterubacterium causedmulberry bacterial wilt disease in China. Koch’s postulates, biochemical and molecularidentification were performed on the pathogen isolated from xylem of the diseased plant,and the pathogen proved to be Enterubacterium. At the same time, we designed specificprimers according to the rpoB gene of the four typical isolated strains, and tested thepathogen. The main achievements were listed as follows:1) Forty mulberry bacterial wilt disease isolates were obtained from xylem of the newinfected mulberry trees in Guangzhou, Guangdong province, P. R. China. Four inoculationmethods to the four typical representative pathogenic strains showed that the most effectiveone was weal inoculation method, and this method was applied for Koch’s postulates. Theresults showed that the four typical tested strains caused mulberry seedling discoloration,leaf wilt and top necrosis. Also they can induce whole plant defoliation and dark browndiscoloration of vascular tissue. The same pathogens can be separated out and the samesymptoms emerged after inoculation. To ascertain the real pathogen of mulberry wilt diseasein Guangzhou, four typical representative pathogenic strains were studied. Conventionalbacteriology, along with light microscope and electron microscope observation,morphological, biochemical and physiological characterization and molecular identificationwere performed on these4strains. The results showed that the four strains were gramnegative bacteria, short rods. It was confirmed that “mulberry wilt” disease was caused byEnterubacterium. It was another new evidence that Enterobacter sp. caused novel diseaseon plant and this disease was only reported by Zhejiang province. So far, it was consideredthat vascular bundles pathogen in Guangzhou was mulberry Ralstonia solanacearum. Andthis study had an important directing meaning in recognizing mulberry diease in this area.2) Two primers of the pathogen of mulberry bacterial wilt disease were designed withinregions of the rpoB gene. A semi-nested PCR method was established to detect4strains ofmulberry bacterial wilt disease,1Pseudomonas syringae pv. Mori strain,1Ralstoniasolanacearum strain,12unidentified saprophytic(epiphytic) bacterial isolates from mulberry trees and4standard Enterubacterium, strong specificity was performed and met theexpected requirements. Samples can be directly detected whether it has obvious symptomsor not. Both the purified DNA of the strain and samples can amplify167bp fragments of therpoB gene. Thus it was not necessary to purify strains, and sample bacterial suspension canbe directly applied to PCR detection. Meanwhile, sensitivity was tested to mulberry samples.We could detect10-3DNA dilution and10-4strain dilution, while10-5,10-6dilutions couldnot.