| Present population patterns of plant pathogens result from the interaction of various historical, contemporary factors of host and ecological conditions on different spatial scales. Knowledge of the genetic structure of the pathogen may offer insight into understanding the occurrence and epidemics of the disease in natural communities. Sheath blight and blast are the important rice diseases worldwide, raising the great threat to rice production and food safety. It’s necessary to acquire the population genetic structure of of Rhizoctonia solani AG-1 IA and Magnaporthe grisea, because it can reveal the origin, evolution and migration of the two pathogens in agricultural pathosystems. Based on previous sampling investigation, the genetic variation of R. solani AG-1 IA and M. grisea were investigated with SSR markers and ribosome DNA sequences, to infer the population structure and spatial distribution of genetic diversity based on phylogeographical method. The genetic patterns were discussed to assess the effects of historical, contemporary, ecological factors and pathogen biological characteristics in shaping and maintaining the geographical patterns of the two fungi. The results would provide the valuable information in elucidating the evolutionary history and potential to evolve of the pathogen populations in agricultural ecosystem, to develop effective management strategies to control the two diseases in the fields. The conclusions were as follows:1. Development of microsatellite markers of R. solani AG-1 IAMicrosatellite-enriched libraries of R. solani AG-1 IA were constructed utilizing methodologies that exploit the strong affinity between biotin and the streptavidin. The genomic DNA of R. solani AG-1 IA was digested by restriction enzyme Sau3AI, and 400-900 bp DNA fragements were captured and then ligated to artificial adaptors. The DNA fragments were annealed with the biotin-labelled oligonucleotide probes (AC) 12, (TCG)g, (GTC)g and (GCT)g, to capture the microsatellite repeat with Streptavidin Magnetic Beads. Sebsequently, the recovered fragment were amplified by PCR to transfer single strand DNA to double strand DNA. The PCR products were ligated to pMD18-T vector and transformed into DH5a competent cells, and ultimately four microsatellite enriched libraries were produced. A total of 66 sequences containing microsatellite repeat motif were selected among 500 clones randomly sampled from libraries, and the percentage of the positive clones accounted for 13.2%. Among DNA fragment containing microsatellites,8 polymorphic microsatellite loci (GenBank no. KM249342-KM249349) were proved to be polymorphic with 50 isolates of R. solani AG-1IA. The number of alleles per locus varied from 3 to 9, the observed (Ho) and expected (He) heterozygosity varied from 0.06 to 0.74 and from 0.15 to 0.72, respectively, and Shannon’s information index ranged from 0.33 to 1.58. These markers will be validated and used to estimate the the genetic diversity of R. solani AG-11 A.2. Population structure of R. solani AG-1 IA from southern China based on SSR analysisR. solani AG1-IA population containing 238 isolates collected from 8 provinces (autonomous region) in southern China were genetically assessed using 16 fluorescence-labeled SSR markers. A total of 144 polymorphic alleles were detected among the isolates of R. solani AG1-IA. The mean number of alleles (Na) and effective number of alleles (Ne) per locus were 4.768 and 2.309, respectively. Among the population of R. solani AG1-IA, Shannon’s information index (1) was 0.986, allele richness (AR) was 4.272, the observed heterozygosity (Ho) and expected beterozygosity (He) were 0.523 and 0.518, respectively. The negative value of inbreeding coefficient (FIS=-0.012) indicated excess of heterozygotes (or deficiency of homozygotes) in the total population. Nine of the ten populations significantly deviated from Hardy-Weinberg equilibrium due to heterozygote excess or deficiency, supporting the evidence for a mixed reproductive system in populations, including both asexual and sexual reproduction, while the balance between asexual and sexual reproduction was population dependent. AMOVA attributed about 89.50% of the variance to individuals within populations, indicating that the main genetic variation existed within populations. Genetic diversity among populations in the same regions were very similar. A positive correlation (r=0.418,.P=0.026) was detected between genetic and geographical distances of populations by Mantel test. Structure analysis indicated that all populations were separated into two genetically differentiated subgroups, the first included three populations (GN, CTA and CTB) located along the Pearl River was significantly distinct from the second made up of seven populations located along the Yangtze River. Extensive genetic admixture were observed in the Yangtze River populations, and high levels of gene exchange occurring within the subgroup (Nm=1.021-10.861), to prevent genetic diversity with low population differentiation (FST=0.023-0.218). These results showed that restricted long-distance migration and mixed reproductive mode of R. solani AG1-IA were a plausible explanation for the spatial structures of genetic variation of pathogen populations. This would fall into the medium-high category for pathogen evolutionary potential. Under this scenario, for long-term disease management against sheath blight including the use of fungicides and tolerant cultivars, it would be useful to minimize gene flow by reducing the spread of propagules via shared irrigation systems or contaminated machinery, and prevent pathogen transmission from infected seeds with fungicides before sowing.3. Pathogenicity differentiation of R. solani AG-1 IA from southern ChinaPathogenic variation of 238 isolates of R. solani AG-1 IA from southern China were evaluated using five rice cultivars with different resistance in seedling stage in greenhouse. The results showed that there were significant differences in pathogenicity among those tested isolates of R. solani AG-1 I A. The frequency of average disease index followed a normal school distribution. Based on their disease index to five cultivars, the isolates were classified into 3 distinct groups by dynamic clustering, weak, moderate and strong pathotypes, accounting for 24.37%,50.0% and 25.63%, respectively. The moderate pathotype was dominant. The discriminant functions of three pathotypes were calculated based on the Bayes method. The accurate rate for discrimination was up to 96.00%. The results indicated that the dynamic cluster and discriminant analysis can be used to evalute the pathogenicity differentiation of R. solani AG-1 I A. Random distribution of pathotypes implied that R. solani AG-1 IA population were a mixture of pathotypes naturally, and the pathogenic variation was not obviously correlated with the collective location and year of samples.4. Population structure of R. solani AG-1 IA from southern China based on ITS sequencingTo assess the genetic variation of R. solani AG1-IA population,173 isolates collected from 7 provinces (autonomous region) in southern China were studies based on ITS-5.8S rDNA partial sequences. Fifty-two haplotypes were defined based on nucleotide variation of ITS sequencing, and haplotypes H4 and H7 were appeared on all 7 populations with the frequency of 57.80%. The shared haplotypes supplied the molecular evidence for gene flow among populations. A high genetic diversity was observed among populations with 0.804 for haplotype diversity and 0.402% for nucleotide diversity. The value of genetic differentiation (Fst) and gene flow (Nm) among populations were 0.026 to 0.433 and 0.327 to 9.329, respectively. AMOVA analysis showed that only 10.46% of genetic variation occurred among populations whereas 89.57% existed within populations. Phylogenetic tree of haplotypes exhibited highly mixed branches among different areas. Neutral test and haplotypes network analysis indicated that the population of R. solani AG-1 IA had experienced historical expansion during its evolution. UPGMA dendrogram indicated that all populations were separated into two genetically differentiated subgroups, the populations located along the Pearl River were significantly distinct from the populations located along the Yangtze River, which was consistent with the results of SSR analysis.5. Population structure of M. grisea from southern China based on SSR analysisThe genetic diversity of 250 isolates of M. grisea collected from 7 provinces (autonomous region) in southern China, were analyzed using 8 fluorescence-labeled SSR markers. A total of 114 polymorphic alleles were detected among the isolates of M. grisea. The mean number of alleles (Na) and effective number of alleles (Ne) per locus were 4.96 and 2.57, respectively. Among the population of M. grisea, Shannon’s information index (I) was 0.99, allele richness (AR) was 4.265. The observed heterozygosity (Ho=0.39) was lower than the expected heterozygosity (He=0.51), suggesting that there was heterozygosity deficiency within population due to inbreeding. Eight of the ten populations significantly deviated from Hardy-Weinberg equilibrium due to heterozygote deficiency, supporting the evidence for an obvious sexual reproduction existed in the populations of M. grisea. Analysis of molecular variance (AMOVA) showed that 75.98% of the total genetic variation was attributed to differences among isolates within population, while 24.02% of genetic variation occurred among populations. There were different levels of gene flow (Nm=0.181-2.529) and moderate to high differentiation (FST=0.090-0.578) between populations. Mantel test revealed that there was positive correlation (r=0.396, P=0.041) between geographical and genetic distances of populations. Low genetic differentiation (Fst<0.05) was obtained among populations from different years in the same region. Although there was a little change, the population genetic structure of M. grisea from the same region were stability. Structure analysis based on the Bayesian clustering method, showed that different levels of gene flow (Nm=0.182-2.529) existed among populations. Gene flow among some populations were relatively weak, which might have caused the genetic differentiation among the populations. There was natural migration capacities of M. grisea among different agro-ecological areas, which should lead to increased vigilance on the risk of introductions of new genotypes of the pathogen through the exchanges of rice seeds.6. Pathogenicity variation of M. grisea from southern ChinaSeventy-two isolates of M. grisea collected from southern China were identified with 7 Chinese differentials. Six groups (ZA, ZB, ZC, ZD, ZE and ZG) with 38 physiological races were distributed among different populations. ZA and ZB were the predominant groups with the average frequencies of 45.8% and 37.5%, respectively, and ZA1, ZB15 and ZB17 were the dominant races. The results suggest that M, grisea have evolved as a major pathogen on cultivated rice through a host-tracking process, leading to adapting independently on indica rice varieties in southern China. The existance of seven avirulence genes (ACE1, Avr-CO39, Avr-Pib, Avr-Pik, Avr-Pita, Avr-Piz-t and PWL2) of 201 isolates of M. grisea were tested. The frequency of PWL2 gene was highest with 100%, the frequency of Avr-Pib gene was 98.51%, and Avr-Pita gene showed the lowest frequency, that was 42.79%. Seven avirulence genes can be detected in the isolates from different regions, and there was significant difference of frequencies of avirulence genes in different geographical origin. Distincitve patterns of pathogenicity suggested the adaptive divergence of host-specific forms of M. grisea populations in southern China linked to local environment and grown cultivars, driven by the selection pressure of host resistance genes. These epidemiological significance needs to be carefully considered, M. grisea is capable of generating considerable genetic variation even in the absence of sexual reproduction with the potential for coevolutionary interaction between host resistance and pathogen virulence. |
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