Screening Of Bacillus Strains Against Grapevine Downy Mildew Disease And Study On The Biocontrol Mechanisms

Grapevine downy mildew disease is a world spread disease which caused by the biotrophic Plasmopara viticola.The aim of this study was to isolate and screen bacterial endoph^es with potential applications for biocontrol of grapevine downy mildew,and research on their biocontrol mechanisms.Our findings will provide high-efficient strains and practical guidance for industrialization of biofungicides.1.Isolation,screening and identification of bacterial endophytesA total of 239 bacterial endophytes were isolated from grapevine leaves and screened by leaf disk assays.Two strains,GLB191 and GLB197,which exhibited the most robust preventive effects against P.viticola in field conditions.The 16S rDNA and the gyrA/gyrB gene analysis identified GLB191 and GLB197 as Bacillus subtilis and B.pumilus,respectively.2.Research on the colonization mechanism of B.subtilis GLB191In order to research the effect of biofilm formation and chemotaxis on GLB191 colonization ability and the relationship between colonization and bicontrol efficiency,we knocked out colonization related genes srfAC,hag and the chemotaxis related gene cheA.Results from the detection of colonization abilities showed that the colonization quantity of AsrfAC and Ahag mutations significantly lower than wild type strains,in both leaf surface and internal.However,?cheA exhibited no effect on colonization quantity.The control efficiency of ?srfAC,Ahag and AcheA mutations was also ditected by leaf disk assays.Results demonstrated that AsrfAC and Ahag lost part ability for grapevine downy mildew controlling while AcheA showed no effect.3.Research on the metabolite of B.subtilis GLB 191In the present research,we found that the metabolites of GLB 191 demonstrate the same effect on sporangial germination and downy mildew disease controlling as fermentation broth.To find the active compound in GLB191 metabolites in a genomic level,the whole genome sequence of GLB191 were obtained.Genome sequence analysis revealed the presence of 13 secondary metabolism gene clusters,and the gene cluster responsible for lactobin A synthesize is specific in GLB 191.Then we knocked out the key synthetic genes of these 13 clusters respectively,and detected their control efficiency on downy mildew disease.Four of these mutants lost partial bioactivity,which suggested that plipastatin,surfactin,bacillibactin and diketopiperazine act in an active manner.4.Research on the metabolite of B.pumilus GLB 197The metabolite of GLB 197 plays the main role of bicontrol efficiency,as it directly inhibit the sporangial germination as well as downy mildew disease development.To fully research the active compounds of GLB 197 in genomic level,the whole genome sequence of B.pumilus GLB 197 were obtained.Analysis of the related secondary metabolism genes revealed the presence of 6 secondary metabolism gene clusters in the genome.The function and bioactivity of these clusters are needed to be further studied.