| Tobacco target spot caused by Rhizoctonia solani AG-3was one of the most economically important diseases of tobacco leaf and occurs in many countries. It was first reported at Dandong city of China in2006. There was a little report about the biological characteristics, genetic differentiation and pathogen mechanism of the pathogen. In this dissertation, the genetic diversity, quantitative detection technique and transcriptome analysis of R. solani AG-3were systematically and intensively studied, the results were as follows:1. There were an obvious physiological differentiation among R. solani AG-3from Liaoning province, and three pathotypes, which contain pathotype I, II and III with strong, medium and weak pathogenicity respectively, were determined.Some differences of ITS region of R. solani AG-3are founded. There was no correlation between the difference group and pathotypes of R. solani AG-3from tobacco.2. SRAP reaction system of R. solani AG-3from tobacco was firstly established, and genetic diversity of the R. solani AG-3from Liaoning province is analyzed by SRAP reaction system, and the relationships between SRAP groups (SGs) and the locality source of R. solani AG-3were elaborated. Using optimum reaction system and primarily screened SRAP primers which have a good polymorphism, a PCR amplication is performed, and743polymorphism DNA fragements were acquired, percentage of polymorphic loci was98.4%. R. solani AG-3strains from tobacco were divided into three SGs according to SRAP polymorphism between R. solani strains genom from tobacco. All strains with strong pathogenicity containing YC-9, YMC-2, LJT-8and DB-3strains were clusted in SG I, accouting for66.7%of the SG I; and all strains with weak pathogenicity clusted in SG II. The results showed that it is an obvious correlation between SGs and pathotype of strains but not between SGs and the locality source. Further more, there was no correlation between SGs and the anastomosis group. At present, the paper was firstly report relationship between pathotype and genetic diversity of R. solani AG-3from tobacco in the literature at home and abroad.3. Quantitative detection systems of R. solani AG-3from tobacco firstly established could be applied to early detect in tobacco tissue and determin dynamic changes in soil, and can fastly detect YC-9strain DNA post inoculation6h, and could also be used to montoring dynamic change of R. solani AG-3from tobacco in soil. Basing on ITS sequence of different anamosis group from NCBI, the specific primer Rs TqF1/R4and probe QTqFl were designed; Real-time PCR systems were respectively established to detece the pathogen from tobacco tissue and soil. The detection resulted show that DNA of R. solani YC-9strain from tobacco tissue was deteced post inoculation6h, and it was far earlier than post inoculation48h by observing the lesions. It was suggested the system was fit to early detect R. solani AG-3from tobacco. The quantitative results showed that R. solani AG-3from tobacco in soil has a dynamic change trend according to different monthly. The copies of R. solani AG-3from tobacco in soil had begun to increase since the mid to late May, and reach to the highest on June28; then it was decline until August9reach to the lowest. It reached the highest on January21, since then R. solani AG-3from tobacco began to decrease and reach to the lowest level in April.4. The firstly established transcriptome database of R. solani AG-3from tobacco could be as a foundation for analyzing its genes structure and function. Using the transcriptome sequencing and short sequence assembly, a total of630125and562259Contigs were respectively obtained from T1and T2samples, and N50is96and104nt, respectively. There were21206transcript sequences from T1with N501213bp and20400transcript sequences from T2with N501247bp, the average fragment length to836bp.21779Unigenes were received, and the average length was873nt with N50value1339nt. a comprehensive transcriptome database of R. solani AG-3from tobacco was established.5. Information of function of Unigenes is annotated and differentially expressed gene and these pathways related to pathogenicity of R. solani AG-3from tobacco were analyzed, and pathogenesis were illuminated in level of transcriptom. Through Blastx comparation, a total of14389unigenes were annotated its function. All annoated Unigenes were homology with basidiomycete’s fungi containing the pathogens T. cucumeris and R. solani. Unigenes in the category of48kinds of Gene Ontology (GO) function, the most function is metabolic process in the biological process and the catalytic activity in molecular function and cell precess and binding functional genes. Unigenes were classified in the25clusters of orthologous groups (COGs), including prediction, replication, recombination and repair function, carbohydrate transport and metabolism. Through KEGG metabolic pathway analysis, a total of3820genes were annotated in158pathways. Among them, there were more genes annotated to the chromosomes, recombinant proteins and DNA repair pathways, splicing pathway, peptides, enzymes, metabolic pathway and DNA replication proteins of ubiquitin system.Through the analysis on transcriptomes of R. solani AG-3from tobacco by the RPKM method, the results showed that827differentially expressed genes (DEGs) were acquired and730genes were annoated. The GO and COG functional entaintment and KEGG pathway enrichment analysis of the expression pattern were performed, most of differentially expressed genes were involved in protein synthesis process, including ribosomal proteins, transcription factors, translation factor and elongtation factor, etc. These genes played important roles in pathogensis process of R. solani AG-3. |
PDF Full Text Request