Characterization Of Molecular Variation Of Ralstonia Solanacearum Causing Tobacco Bacterial Wilt And Technique Exploration For The Disease Control

Tobacco bacterial wilt(TBW),caused by Ralstonia solanacearum,is one of the most serious soil-borne disease of tobacco(Nicotiana tabacum).In this study,the molecular variation and pathogenicity of R.solanacearum causing TBW from 14 provinces in China were analyzed;based on the response of different tobacco cultivars to bacterial wilt in field,the effects of grafting on controlling TBW were evaluated by using resistant cultivar as the stocks;the differential expressed genes(DEGs)and relative resistant pathways of tobacco resistant cultivar(Fandi 3)and susceptive cultivar(Yunyan87)in response to R.solanacearum were analyzed by using the Digital Gene Expression(DGE)profiling;the effects and mechanism of tobacco-marigold intercropping on controlling TBW were explored.The main results of the study are described as follows:1.In China,TBW is distributed in southeastern China,southwestern China,the mid-basin region of the Yangtze River and the lower Yellow/Huai River region.89 R.solanacearum strains infecting tobacco from the four regions were characterized as bv 3and 4.And bv 3 was dominated,with proportion of 89.89%.Based on polymorphisms in the egl gene,the strains were all identified as phylotype I and further divided into seven sequevars(13,14,15,17,34,44 and 54).Among the seven sequevars,sequevar 15 constituted the largest group and was widely distributed in all four regions in China,with proportion of 33.71%.Sequevar 13 and 14 which are reported here on tobacco,were previously found only on other plants.And a new sequevar 54 was also found in this study.All the seven sequevars were found in southeastern and southwestern China,sequevars 15,17 and 54 were present in the midbasin region of the Yangtze River,and all strains from the lower Yellow/Huai River region were only identified as sequevar 15,indicating the farther north the distribution,the fewer the number of sequevars.Pathogenicity of 27 representative strains was assessed by inoculation onto three tobacco cultivars with different resistance levels.Through cluster analysis of AUDPC values,the 27 strains were classified into three pathotypes with high,medium and low virulence.The strains from the lower Yellow/Huai River region have lower pathogenicity than the strains from southeastern China,southwestern China and the mid-basin region ofthe Yangtze River,indicating an association between pathotype and geographic distribution for R.solanacearum.However,no obvious associations were found between sequevars and pathotype.2.The resistance of 20 tobacco cultivars against TBW was evaluated in field.By using Yanyan97,Fandi3 and Coker176 as the stocks and Yunyan87 as the scion,the effects of the three stions on controlling TBW,yield and quality of tobacco were analyzed.The results indicated that there are strong affinities in Yunyan87/Yanyan97,Yunyan87/Fandi3 and Yunyan87/Coker176.The survival rates of the three stions were all higher than 75%.No significant difference was found in agronomic traits of plant and chemical composition contents between grafted plants and own-root plants.The incidence and disease index of TBW of grafted plants were significantly lower than that of own-root plants.The control efficiency of stions on TBW varied from 72.07% to 77.16%.The yield per hectare,economic income per hectare and unit price of grafted plants were significantly higher than that of own-root plants,which increased by 15.23%-23.92%,19.45%-37.54% and 3.66%-10.99%,respectively.Among the three stions,the grafting plant of Yunyan87/Yanyan97 showed better comprehensive characterizations.3.The stem samples of tobacco resistant cultivar(Fandi 3)and susceptible cultivar(Yunyan87)at 1 d,3 d and 7 d post-inoculated with or without R.solanacearum were sequenced by Illumina Hiseq 2500 platform using DGE profiling.The results of gene expression profile indicated that at 3 dpi and 7 dpi,the number of up-regulated differential expressed genes in resistant cultivar increased significantly.WRKY6 and WRKY11 family genes in WRKY transcription factors,ERF5 and ERF15 family genes in ERFs transcription factors and genes encoding PR5 were significantly up-regulated in resistant cultivar in response to R.solanacearum infection,and WRKY11 and ERF15 were firstly found to might be involved in the disease resistance.The analysis of KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway on differential expressed genes demonstrated that cysteine and methionine metabolism,glutathione metabolism and phenylpropane pathways were the main resistance pathways to R.solanacearum infection.In resistant cultivar,the differential expressed genes encoding CYP450,TCM,CCo AOMT,4CL,PAL,CCR,CSE and CADH enriched in phenylpropane pathway were up-regulated at 3 dpi and 7 dpi,which are involved in the synthesis of plant antitoxinssuch as flavonoid,stilbenoid and lignin.Subsequent q-PCR analysis showed that 9candidate differential expressed genes had higher expression levels in resistant cultivar than that in susceptible cultivar,indicating the reliability of DGE profiling.In pot experiment,the control efficiency of flavonoid with 1-4 mmol/L on TBW varied from56.10% to 84.15%,which verified that flavonoid showed good control effect on bacterial wilt.4.The disease index of TBW,soil chemical properties,soil microbial community diversity and structure were measured and analyzed in the tobacco-marigold intercropping field and tobacco monocropping field.Compared with tobacco monocropping field,the incidence and disease index of TBW in the intercropping field was significantly lower with control efficiency of 58.25%.Soil p H,exchangeable calcium and organic matter content in soil from the intercropping field were significantly higher than those from tobacco monocropping field.Soil bacterial microbial community in the intercropping field exhibited higher diversity and richness,especially at 50 d post-transplanted,than that in the tobacco monocropping field.The relative abundance of soil beneficial microorganisms,such as Lysobacter,Burkholderia,Trichoderma,Mortierella,Chaetomium and Penicillium increased in the intercropping field.And the relative abundance of R.solanacearum in the intercropping field was lower by6.81%-10.10%.The results confirmed that the suppression of TBW in the tobacco-marigold intercropping field is due to the construction of disease suppressive soil.In this study,the molecular variation and pathogenicity of tobacco R.solanacearum in China were clarified,the genes related with the resistant to R.solanacearum in tobacco were discovered,the effects of tobacco grafting and tobacco-marigold intercropping on controlling TBW were ascertained.The results may provide the methods for the green control of TBW.