Etiology And Pathogenicity Mechanisms Of The Mulberry Wilt Disease In China

A new mulberry wilt disease(MWD) was observed in summer of 2006,which caused severe lost in mulberry plantations of Lin-an and Tong-lu country,Zhejiang, China.The causal agent was identified as Enterobacter cloacae complex by phenotypic and genotypic analysis including pathogenicity tests,carbon source utilization profile(Biolog),fatty acid methyl esters(FAME),analysis of 16S rDNA and rpoB gene sequences.Furthermore,representative strain of Enterobacter sp. R18-2 was labeled with green fluorescent protein(GFP),and its infection and localization profile in the plants was observed by confocal laser scanning microscopy (CLSM).In addition,the population diversity of E.cloacae complex have been determined by a serial characteristics measured such as fatty acid diversity, enterobacter repetitive intergenic consensus sequences(ERIC) and BOX element polymorphisms,heat shock proteins gene(hsp 60) and RNA polymerase beta-subunit encoding(rpoB) gene sequences analysis.The major results are listed as follows:(1) Ninety-eight MWD isolates were obtained from 33 diseased mulberry plant samples.Six representative strains(R2-2,R3-3,R6-2,R8-2 and R11-2) were selected for Koch's postulates tests.In inoculation experiments,the strains induced mulberry branch leaf wilt,discoloration,defoliation,and the shoot necrosis,caused the whole mulberry plant dark brown discolorations in vascular tissues,leaf wilt,and defoliation. The results confirmed that the testing strains were the causal agent of the MWD and were identified as Enterobacter sp.based on the carbon source metabolic and FAME profiles.The 16S rDNA and rpoB gene sequence analyses indicated the six strains belong to E.cloacae(R3-3,R8-2),E.asburiae(R2-2,R6-2) and Enterobacter sp. (R11-2,R18-2).The 3 speices of Enterobacter are belonging to the E.cloacae complex.(2) The representatives MWD isolate Enterobacter sp.R18-2 was selected and tagged with gfp and luxAB by electroporation through a pUTmini-Tn5 transposon vector containing the gfp/luxAB dual markers.A transformant designated as R18-gfp-1 exhibiting strong fluorescence under fluorescence microscope was chosen for further study.Correct insertion of the marker gene in R18-gfp-1 was confirmed by gfp-PCR amplification and the GFP expression.The cell morphology and 16S rDNA sequence similarity comparison between R18-2 and R18-gfp-1 confirmed that the transformant was derived from R18-2.GFP expression detected by flow cytometdc demonstrated that gfp gene was stable and highly expressed in R18-gfp-1.And the fluorescence intensity of GFP in R18-gfp-1 kept 100%in selective and nonselective medium after 240 h continuous transfer.The plate selective assays showed that the same colonization ability of R18-2 and R18-gfp-1 in the mulberry and tomato plants. However,the inoculation tests indicated that the wilt symptoms induced by the transformant R18-gfp-1 was delayed for 5-6 days than those of R18-2.The results indicated the pathogenicity of the transformant R18-gfp-1 was weaker to the host plant than the strain R18-2,while the colonization ability of both is consistant.The survival and colonization patterns of R18-gfp-1 in the in vitro tomato plants were speculated by confocal laser scanning microscopy(CLSM) with different parts of the plant tissues.After inoculation,a plenty of fluorescence bacteria have been observed in the root tip meristematic cell until the 3^rd day.Parts of parenchyma cell of the root and stems could detect gfp-tagged bacteria 5 days after inoculation.Then the bacteria entered the xylem vessels cell of root and stem,induced plant wilt and defoliation symptoms by blocking vessels water transportation.(3) In order to analyze the genetic structure and the phylogenetic relationships among the clusters of the phytobacterium E.cloacae complex,98 strains collected from different mulberry orchards and 9 type strains of the genus were examined with a combination of the pathogenicty on the mulberry and the fatty acid profile, ERIC-PCR and BOX-PCR fragment polymorphism.50 of 98 strains were analyzed by the two housekeeping genes hsp60 and rpoB sequence neighbor-joining tree and 10 genetic clusters were constructed according to the hsp60 sequence analyses.The robustness of the genetic clusters was confirmed by the fragment polymorphism of ERIC-PCR,BOX-PCR and rpoB sequence analyses as well as the geographical origin of the strains isolation.However,the genetic clustering presented in this study suggested the pathogenicity of E.cloacae complex is unrelated to the genetic charactcrs,such as enterobacter repetitive intergenic consensus sequences,BOX element and genes of hsp60 and rpoB.Previous studies have indicated that some strains of Enterobacter species are opportunistic pathogens of plants.Up to now,limited studies about the E.cloacae compled causing plant disease are reported.However,they have widely range of host species.It is the first report for Enterobacter as the pathogen causing plant disease in China,and mulberry as its pathogenicity host is a new record in the world.