| Tobacco bacterial wilt(TBW)is a soil-borne bacterial disease caused by Ralstonia solanacearum with a large of host plants,and it occurs in areas with high temperature and high humidity in the whole year which seriously affects the yield and quality of tobacco leaves,eventually causing huge economic losses.It is an effective method to control the disease by breeding excellent disease-resistant varieties,and using the next-generation sequencing technology RNA-seq mining of candidate genes related to disease resistance can accelerate the process of disease resistance breeding working.In recent years,although the transcriptome of roots and stems which were before and after inoculated with Ralstonia solanacearum for tobacco seedling cultivated in soil has been reported to explore the genes respond to Ralstonia solanacearum infestation,no studies have been reported on the use of root system as an object to analyze the expression profiles of several tobacco materials contrasting in disease resistance,given that the root system is the main channel for plant exposure or resistance to Ralstonia solanacearum infestation.Accordingly,four tobacco varieties with significant and different resistance to TBW in this study were selected as materials to establish a method for the identification of tobacco seedling resistance to Ralstonia solanacearum in hydroponics,and RNA-seq was used to analyse the transcriptomes of the roots before and after exposure to the Ralstonia solanacearum inoculation so as to explore the changes in the expression profiles of different tobacco varieties under hydroponic conditions,contributing to identifying candidate genes associated with anti-bacterial wilt in tobacco.The main results are presented as follows:1.Ralstonia solanacearum has been screened for tobacco inoculation trials.A typical TBW in tobacco plant from the tobacco growing area of Nanxiong City,Guangdong province was collected and a strain(GDNX-1)belongs to Ralstonia solanacearum was isolated and screened by streaking plate method.The morphological observations revealed that the strain was single-colony and gram-negative in medium,meanwhile,PCR amplification using three pairs of primers specific for Ralstonia solanacearum indicated that the strain was able to amplify the corresponding specific bands.Moreover,the results of phylotype,biovar and race identification manifested that the pathogen isolated from tobacco was Ralstonia solanacearum,belonging to phylotype I,biovar III and race I.The isolated Ralstonia solanacearum strains were further used for the identification and evaluation of disease resistance in different tobacco varieties studied.2.A rapid and effective hydroponic inoculation method for the identification of tobacco seedling to TBW was established.Four tobacco varieties of Honghuadajinyuan,Qinggeng,Yanyan 97 and D101 were used as test materials to identify and evaluate the resistance among them that responding to Ralstonia solanacearum by inoculation with root-damaged and rootundamaged in hydroponics,the results showed that D101 was moderately resistant and the remaining three varieties were highly susceptible or susceptible,which was in general agreement with the evaluation of the traditional method by injurying the root infected with Ralstonia solanacearum for tobacco seedling cultivated in soil.The inoculation of Ralstonia solanacearum in hydroponics could determine the resistance of each variety in 7-10 days,whereas,it takes 3 ～ 4 weeks for inoculation with root-wounding and irrigation method in soil,pointing out that the hydroponic inoculation method established in this study is a rapid,efficient and accurate method for identification and evaluation of tobacco seedling resistance to BW.The hydroponic inoculation method in this study was further used to perform the RNAsequencing for different resistant tobacco varieties in root inoculated with Ralstonia solanacearum.3.Transcriptome sequencing data were obtained for tobacco materials with significant differences in tobacco bacterial wilt disease resistance.Using RNA-seq technique,transcriptome data of two resistant tobacco varieties(D101,Yanyan 97)and two susceptible tobacco varieties(Honghuadajinyuan,Qinggeng)were obtained at five time points including0 h,6 h,1 d,3 d and 7 d of inoculation with Ralstonia solanacearum,and a total of 81,534 genes were obtained.Furthermore,all the obtained gene sequences were compared to NR,Swiss Prot and GO databases for functional annotation,and the results showed that 75928(93.12 %),46503(57.04 %)and 22915(28.10 %)genes could be annotated,respectively.4.Differentially expressed genes were found in tobacco accessions contrasting in disease resistance,and the possible molecular mechanisms of tobacco disease resistance were hypothesized.Comparing the differentially expressed genes of four tobacco varieties at multiple time points after inoculation with Ralstonia solanacearum,a total of 39 differentially expressed genes were found in the four tobacco materials at four time points,of which 34 were upregulated and 5 were down-regulated,and these genes were mainly involved in glutathione metabolism,plant rhythm and so on,which together constitute the mechanism of tobacco materials responding to Ralstonia solanacearum infestation.There were 128(92 up-regulated and 36 down-regulated)differentially expressed genes only in the two disease resistant tobacco varieties,which may play a more important role in tobacco disease resistance and belong to the candidate genes related to disease resistance.The differentially expressed genes that were shared in only two disease-resistant tobacco materials were mainly associated with MAPK signaling pathway,phenylpropanoid biosynthesis,phytopathogen interactions,phytohormone signaling,which may constituted the unique disease resistance mechanism of the diseaseresistant tobacco materials after inoculation of Ralstonia solanacearum. |
