| Tobacco bacterial wilt is a destructive soil borne bacteria disease that is caused by ralstonia solanacearum.This disease has brought great harm to the yield and quality of tobacco as well as the comprehensive use of the land.It has become one of the main factors restricting the development of China’s tobacco industry.Cultivation of disease-resistant varieties has proved to be the most effective method to control this disease.This research firstly studied the differentiation of tobacco ralstonia solanacearum in Taining,Fujian Province and Xuancheng,Anhui Province.Secondly,it built an indoor accurate identif ication system to test the tobacco bacterial wilt resistance.Then,the resistance identification of 14 mutants and three control varieties was carried out,and the genetic regularity of their resistance was preliminarily analyzed.Finally,by RNA-Sequencing technology,the study revealed the difference in gene expression during the process of tobacco-pathogen interaction.At the same time,the differential genes involved in the resistance to bacterial wilt were analyzed with qRT-PCR technology.Therefore,such efforts will provide genetic resources and theoretical basis for studies on resistance mechanism and the prevention and control of tobacco bacterial wilt.The main research results are as follows.1.The bacterial strains in Taining,Fujian Province and Xuancheng,Anhui Province were respectively inoculated with three kinds of disaccharide(lactose,maltose,and cellobiose)and three kinds of hexanol(mannitol,sorbitol,and dulcito).After fifteen days,the results showed that the strains were able to grow normally in nutrient mediums,and the newly emerging bacterial colony were orange.It proved that the strains had the ability to make use of three kinds of disaccharide and three kinds of hexanol.Therefore,according to the related literature,it could be concluded that the tested strains were biochemical type III.This result was consistent with the report that the ralstonia solanacearum which infected Chinese tobacco were mainly biochemical type III..2.Three control varieties,susceptible var ieties"Honghuadajinyuan"and"Cuibi No.1"and disease-resistant variety"Yanyan 97",were selected as subjects to explore indoor accurate identif ication system.According to the root soaking method,the tested strains were soaked with 1 x 10~8 cfu/ml concentration of ralstonia solanacearum suspension.On the twelfth day,the results showed that the indoor disease index of the tested materials was in accordance with the true index accounted in the literature.It can be seen that the root soaking method under indoor controllable conditions can truly reflect the resistance of the materials.This method is fast,economical and reliable,and it can be used as an important way to identify the resistance of tobacco bacterial wilt..3.Fourteen bacterial wilt resistant mutants obtained by EMS mutagenesis and three control varieties,"Honghuadajinyuan","Cuibi No.1"and"Yanyan 97"were selected as materials for indoor inoculation and identification.The results showed that the rate of disease resistance of the 17 indoor tested materials was very close to that of natural strains in the field.There was a very signif icant positive correlation,and the correlation coefficient was 0.964.Reciprocal crosses were perf ormed respectively between susceptible varieties"Honghuadajinyuan"and"Cuibi No.1"and disease-resistant varieties 359-k,633-k,N1561-1 and 1412-k1.Both F1 and RF1 in all cross combinations were affected,and all indexes reflecting the state of illness were above 90%.The results showed that the inheritance of resistance of bacterial wilt resistant mutants was controlled by recessive nuclear gene.4.The transcriptome sequencing was performed after the inoculation of virulent strains of ralstonia solanacearum into bacterial wilt resistant mutants and the susceptible variety"Cuibi No.1".The results showed that a large number of differentially expressed genes were screened out by RNA-Sequencing technology,and the genes were mainly up-regulated.The infection of strains could affect multiple metabolic pathways(the signal transduction pathway of plant hormone,the plant-pathogen interaction,oxidative phosphorylation,pyrimidine metabolism,etc.)It could also induce the gene expression of related plants(signal transduction mechanism,catalytic activity,organic metabolism,biosynthesis,etc.)This also indicated that the tobacco-pathogen interaction was a very complex process.5.Eight differentially expressed genes that might be associated with disease resistance were selected for qRT-PRC verification in the wild type and mutant 633-k and 1412-k1 to determine whether they were involved in the tobacco bacterial wilt reaction.The results revealed that the expression intensities of Ntab0876820 and Ntab0833810 were different after the inoculation into infection-resistant materials.The expression intensities of the mutant 633-k and 1412-k1 increased with the increase of the number of days,while that of the wild type was not changed signif icantly.T he structure domain of Ntab0876820 contained transcription factor WRKY.Ntab0833810 participated in the oxidation-reduction reaction of the cell. |
PDF Full Text Request