| Verticillium wilt is a very harmful vascular disease,which seriously affects the yield and quality of the eggplant.Many studies showed that genomes of wild eggplant relatives harbour a large number of genes that confer disease and insect resistance,and that there were abundant wild relatives of the eggplant in Yunnan.Due to the lack of research on the resistance mechanism of wild eggplant relatives to Verticillium wilt,the utilization of their genetic resources for breeding resistant varieties is scarce.In this thesis,the wild eggplant relative accessions collected in a previous project were used as the materials.Artificial pathogen inoculation was performed to investigate the resistance that these wile eggplant relatives exhibited to Verticillium wilt,and some accessions with strong resistance to Verticillium wilt were screened out.I also observed how the tissue structures of the wild eggplant relatives changed after being infected by Verticillium dahliae,and related physiological and biochemical indexes over the pathogenesis were also recorded.To gain insights of the molecular mechanism,gene expression changes were also analysed by using transcriptomics and q RT-PCR.I aimed to illustrate the microstructural,physiological and molecular responses of wild eggplant relatives to Verticillium wilt.The results and main conclusions of this study are as follows:1.Through procedures of isolation,identification and pathogenicity analysis,a hyper-virulent V.dahlia strain QZ-S was screened out,and it was used to inoculate 45 wild eggplant relative accessions collected in Yunnan.In total,ten resistant accessions were identified,and two of them exhibited high resistance—Solanum sisymbriifolium（197）and S.aculatissimum Jacqu;two showed slight disease resistance—S.Torvum Swartz.and S.chrysotrichum Schltdl.;and six showed moderate resistance,including one S.violaceum Ort accession and five S.integrifolium Poir accessions.One highly susceptible accession—Daliyeqie（239）,with a disease index of 88.0—was also obtained.2.S.sisymbriifolium（197）with high resistance and Daliyeqie（239）with high susceptibility were used as the materials in this study.Through analyses of morphological and physiological index changes in roots after V.dahliae inoculation,I found that before the pathogen infection,compared with 239,the root growth of 197 was better and the enzyme activities were higher,which might be beneficial to resist the pathogen invasion.The root morphological and physiological characteristics of 197 and 239 showed similar change trends after the pathogen infection,so it was speculated that the changes in enzyme activities caused by pathogen infection might be part of the plant stress response reaction.Compared with 239,roots morphology,enzyme activities and osmoregulation substances of 197 changed more rapidly,indicating that the response of 197 to pathogen was more rapid and that there exists a faster regulatory mechanism.After comprehensive analyses,I chose 12 h and 48 h as the key time points for studying the short-term plant-pathogen interaction,14 d as the key time point for the long-term interaction.Contents of SOD,POD,PAL,MDA and soluble sugar could be used as the main indices of the physiological and biochemical changes during the eggplant’s responses to Verticillium wilt.3.By using an improved paraffin sectioning method,the changes in main microstructural indexes of the stem bases of 197 and 239 were observed after V.dahliae infection.Before the infection,197 stem bases,in comparison with 239 ones,contained closely arranged epidermal and parenchyma cells with fewer layers,showed a higher degree of lignification,and had larger xylem vessels and wider pith.These observations were indicative for the selection of eggplant accessions resistant to Verticillium wilt.After the pathogen infection,both 197 and 239 plants grew slowly,and their stem bases contained increased xylem area,increased number of vessels,and showed the phenomenon of spore tissue diffusion.Compared with 197,more marked slow growth,earlier invasion time of spores,and more severe vessel blockages were observed with239 plants.Xylem area,number of xylem vessels and pith width could be used as the main tissue structure indices for selecting eggplant accessions against Verticillium wilt.4.Global genes expression profiles of 197 and 239 after V.dahliae inoculation（0h,12 h and 48 h）were investigated by using transcriptome sequencing.More differentially expressed genes,more KEGG enrichment pathways of the differentially expressed genes and more important pathways related to disease resistance were found for 197 plants than for 239 plants,but there were many primary metabolism related KEGG enrichment pathways were obtained for 239 plants.Through the analyses of three important pathways（PHST,PPI and PB）,which are related to Verticillium wilt resistance,I found that many important genes,such as some key genes of BR and ET syntheses in the PHST pathway,some upstream genes（Pti1,RIN4,RPM1 and RPS2）activating hypersensitivity reaction in the PPI pathway,and genes encoding COMT,CCo AOMT and UGT72 E enzymes in the PB pathway.The results also showed that Ve1,PR10,WRKY22 and WRKY33 might play key roles in the interaction between the eggplant and V.dahliae.5.Three Ve homologous genes Sm Ve2,Ss Ve1 and Ss Ve2 were identified in the genomes of 197 and 239.On the basis of sequence comparison,homology analysis,subcellular localization and protein secondary and tertiary structures prediction,the gene expression profiles of Ve at different time points（before and after inoculation）and different tissue parts（root,stem and leaf）in 239 and 197 were analysed by q RT-PCR.The correlation between the relative expressions of the Ve homologous genes and the disease index of Verticillium wilt in the wild eggplant relatives were analysed.I found that the three Ve homologous genes had complete structures,complete ORF frames;they are highly homologous to other reported Ve genes of Solanaceae plants,and the predicted secondary and tertiary structures of the encoded proteins of these proteins from eggplant relatives and Solanaceae plants were also highly similar.Their expressions were all detectable in roots,stems and leaves,but the expression levels in roots and stems were higher than that in leaves.The expression of Ve1 varied significantly in 31 wild eggplant relative accessions,and in many accessions,no expression was detectable;however,Ve2 was expressed in all the tested accessions.The relative gene expression of Ve1 was negatively correlated with the disease index;the gene expression of Ve2 was not significantly correlated with the disease index,while the relative expression of Ve1 + Ve2 was negatively correlated with the disease index.Different aspects of responses of wild eggplant relatives to V.dahlia,including tissue structure,physiology,biochemistry and gene expressions,were preliminarily studies,and some candidate genes that may play key roles in the interaction between wild eggplant relatives and V.dahliae were identified in this thesis.The results laid a foundation for the exploitation and utilization of genes conferring resistance to Verticillium wilt in wild eggplant relatives,and provided knowledge for people to study the resistance to Verticillium wilt in other crops,as well as for understanding the disease resistance mechanism. |
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