Evolution And Molecular Mechanism Of Ralstonia Solanacearum In Response To Acid And Low Temperature Stress

Ralstonia solanacearum,one of the top ten plant pathogenic bacteria in the world,is considered as a variable compound species due to large host range,wide distribution and rich genetic diversity.As one of the hosts,tobacco suffers severe bacterial wilt.In China,tobacco planting region distributed widely,containing several geographic areas with different climate and environmental factors.In recent years,due to the global climate warming,and changes in the cropping systems,it is reported that the disease has frequently outbroken in cool highlands.R.solanacearum is phenotypic polymorphisms.An important reason for its polymorphism is the variation of genome,which affected by many factors such as temperature,humidity,soil factors,host or non-host plants,and human activities.The evolution and adaptation of R.solanacearum influenced by these factors need further exploration.Based on this background,this thesis for degree concentrated on evolution and molecular mechanism of R.solanacearum in response to acid and low temperature stress.From one aspect,the thesis analyzed the genetic diversity and evolution of wild strains from 14 tobacco planting regions.From another aspect,it studied the process and molecular mechanism of R.solanacearum adapting to abiotic pH and temperature by experimental evolution with genome resequencing technology.From these two aspects,the diversity and evolutionary trends of R.solanacearum were expounded.The main findings of this research are as follows:1 The genetic diversity and phylogenetic relationship of R.solanacearum strains collected from tobacco in China were evaluated:there were 7 clusters among all tested strains,and the prevalence and distribution of sequevars were found to be related to factors such as altitude.The diversity of 107 R.solanacearum isolates collected from tobacco were analyzed by the egl phylogenetic tree.The result showed that all isolates from tobacco were sequevar diversity,which could be divided into sequevar 13,14,15,17,34,44,54,and 55.Among them,sequevar 55was a newly classified group.The distribution of sequevar had relationship with altitude.Specifically,sequevar 15 and 34 were mainly prevalent in low altitude regions;sequevar 54 was prevalent in higher altitude areas.According to the statistical analysis,the sequevar distribution is significantly different in different eco-zone?E1 and E2?with different altitude,temperature,and soil pH,which suggested that altitude,temperature,and soil pH were important factors affecting the sequevar diversity of R.solanacearum.A phylogenetic tree and STRUCTURE analysis were performed based on the SNPs of all genomes of R.solanacearum isolates.The results showed that these isolates may have 7 ancestor populations,and some isolates were heterozygous.Most of the sequevars based on the egl gene can be clustered into the same branch in the phylogenetic tree of genomes.The strains from the same tobacco-planting eco-zone were distributed in different genetic structural groups,indicating that strains were cross-spreading between eco-zones across the country.Positive selection analysis was performed on all gene clusters in these isolates.A total of 628 gene clusters were calculated under positive selections.Among the top 50 genes with the most selected sites,pathogenic-related genes such as T3Es and motility-related genes were more strongly positively selected.2 With long-term stress,experimentally evolved strains of R.solanacearum adapted to acid and low-temperature environments were obtained,and the phenotypic changes of each experimental strain were identifiedThe experimental evolution was performed on R.solanacearum strain CQPS-1 under acid and low temperature stress.After long-term culturing,experimental strains adapted to acid and low-temperature environments were obtained.After 1500 generations of serial passage experiment in the acid environment?minimal medium with pH 4.9?,the growth of the acid-adapted experimental strains in the acid environment was significantly better than that of the strains cultured in the control environment.The growth rate of acid-adapted experimental strains was1.67-fold higher than that of the control experimental strains.About the experiment in different temperature environment,the Tm20-series and Tm30-series strains were serial passage cultured for400 d.Tm20-series strains showed significantly enhanced growth ability compared with the original strain CQPS-1 under the shaking condition at 20?.However,compared with the control Tm30-series strains,Tm20-series strains did not show significant difference.The phenotypic characteristics of all experimental strains were evaluated,including the colony morphology,culture phenotype,motility,and carbon source metabolism.After long-term serial passage experiment,the colony morphology of all experimental strains changed from pink center and strong fluidity?the colony of typical virulence strain?to smaller,red,and poor fluidity?the colony of avirulence strain?.In addition,two Tm20 strains?Tm20-2 and Tm20-5?appeared brown when grew on solid plates.The motility of the acid-adapted experimental strains was significantly increased,almost 1.68-fold larger than the original strain CQPS-1,while the motility of all other experimental strains was significantly reduced.Three experimental Tm20 strains?Tm20-2,Tm20-4,and Tm20-5?were chosen for evaluating carbon source metabolism of experimental strains adapted to low temperature stress.The results showed that the capacity of carbon source metabolism of three experimental Tm20 strains had improved compared with the original strain CQPS-1.Specifically,there were 29 carbon source materials were utilized better by experimental Tm20strains than orther carbon source materials.3 The adaptive molecular mechanism of acid-adapted and low temperature-adapted strains by using the method of genome resequencing,and studying the function of related genesGenome resequencing was performed on the acid-adapted experimental strains?5 C49 strains serial passage culturing in acid environment and 5 C65 strains culturing in control environment?and the low temperature-adapted experimental strains?5 Tm20 series experimental strains serial passage culturing in 20?,and 5 Tm30 series experimental strains culturing in 30??.A total of23.96 Gbp Clean Data was obtained for the acid-adapted experimental strains,and a total of 23.80Gbp Clean Data for the low temperature-adapted experimental strains.The variation difference between C49 and C65 series strains was compared.Venn diagram results showed that there were 127 common mutation genes in the two series strains.There were 10unique mutation genes in C49 series strains,while 7 unique mutation genes in C65 series strains.Based on the mutation site of each strain,three genes,phcA?gene ID BC350₁0400?,pehR?gene ID BC350₁0115?,and spp?gene ID BC350₁1470?,may related to that strains adapted to acid environment and changed motility.The gene knockout method was used to verify the function of two genes?spp and pehR?.The results showed that the signal peptide protein gene?spp?had no effect on the colony morphology,normal growth process,motility,and adaptation to the acid environment of the strain.However,it played important role in the pathogenicity of R.solanacearum.The pehR gene also had no effect on the colony morphology and growth of the strain,but deletion of pehR in R.solanacearum CQPS-1 led to decreased swimming motility,and the virulence of the pehR deletion mutant on susceptible tobacco plants was significantly attenuated compared to the wild-type strain.Combined with references,phcA and pehR played an important role in R.solanacearum strain adapting to the acid environment and changing motility.The variation difference between Tm20 series and Tm30 series strains was compared.Venn diagram results showed that there were 83 common mutation genes in the two series strains.There were 8 unique mutation genes in Tm20 series strains,while 5 unique mutation genes in Tm30 series strains.The comparison of mutation sites showed that there were no common mutation sites in the5 Tm20 strains or the 5 Tm30 strains,which indicated that the mutations in all low temperature-adapted strains were random mutations.There were no mutation genes related to low temperature adaptation.In addition,the molecular mechanism of the phenotypic change that two Tm20 strains appeared brown when grew on solid plates was analyzed by comparing the mutation sites.Two genes may relate to the phenotypic change,one was trmD?gene ID:BC350₀1540?,another was wecA?gene ID:BC350₀3300?.The annotation of trmD was tRNA?guanine-N1?-methyltransferase,and the wecA was glycosyl transferase.4 The experimental strains had better environmental adaptability after adapting to acid/low temperature:temperature adaptability of acid-adapted strains was improved,and drug tolerance of acid/low temperature-adapted strains was enhancedThe tolerance of all experimental strains to other stresses was evaluated,including the tolerance of acid-adapted strains on low temperature/high temperature,the tolerance of low temperature-adapted strains on poor nutrition/acid environment,and the tolerance of all experimental strains on streptomycin.The OD_600nm of C49 series strains?acid-adapted experimental strains?in relatively low temperature?20??was 1.54-fold larger than that of C65 series strains?control series strains?,and the OD_600nm of C49 series strains was 1.83-fold larger than that of C65series strains in relatively high temperature?38??environment,which suggested that temperature adaptability of strains increased after adapting to the acid environment.The growth of low temperature-adapted experimental strains?Tm20 series strains?did not show significant difference in the poor nutrition or acid environment compared with the control experimental strains?Tm30series strains?.The growths of Tm20-2 and Tm20-5 that showed brown on solid plates were significantly lower in the poor nutrition or acid environment than that of other low temperature experimental strains and original strains,indicating that the phenotypic change will affect the environmental adaptability of the strains.In addition,the sensitivity of the experimental strains to streptomycin was analyzed by evaluating the MIC of streptomycin.The MICs of acid-adapted experimental strains?C49 series?,the C65 series atrains,and the original strain CQPS-1 were 5mg/L,3.5 mg/L,and 2.5mg/L,respectively.The MIC value of C49 strains was significantly increased,which was 2 times higher than the original strain,and 1.43 times higher than the C65series atrains.Among the Tm20 series strains,the MIC of 2 strains?Tm20-3 and Tm20-4?increased to 5 mg/L.The average MIC value of the Tm20 series strains and the control Tm30 series strains were 3.5 mg/L and 1.9 mg/L,respectively.Compared with the original strain,the MIC of the Tm20 series strains was increased by 1.43 times;compared with the control Tm30 series strains,it was increased by 1.84 times.The results showed that R.solanacearum can improve its drug resistance after adapting to acid/low temperature environment.In summary,this dissertation took R.solanacearum as the research object to analyze the genetic evolution of wild-type strains from natural environmental and the adaptation mechanism of experimental strains affected by single abiotic factor?acid and low temperature?.First,the genetic diversity and evolution of wild-type strains were clarified.Second,experimental strains adapted to acid or low temperature environments by experimental evolution were obtained.It was found that R.solanacearum could quickly adapt to stress environments and change the phenotype.The possible molecular mechanism were analyzed by genome resequencing,and the functions of two genes were verified by gene knockout method.Finally,the results of the tolerance of experimental strains to other stresses suggested that the strains adapted to acid/low temperature would improve their tolerance to other stress environments.The results of this study laid a theoretical foundation for fully understanding the genetic evolution of R.solanacearum collected from single host.Moreover,this study provided evidence to understand the adaptation mechanism of R.solanacearum adapting to the long-term acidified soil or cold environment,which was of great significance for future control of this disease in the soil acidification and cold regions.