Genetic Diversity Of Xanthomonas Citri Subsp. Citri Isolates From China On The Basis Of Simple Sequence Repeat (SSR)

Citrus Bacterial Canker Disease（CBCD）, caused by Xanthomonas axonopodispv.citri, is one of the most destructive disease. The pathogen Xanthomonas axonopodispv.citri infects citru species of family Rutaceae. The citrus production is the mostimportant products of China’s southern provinces, but citrus canker is found in most ofprovinces,causing serious harm to the yield and quality of citrus, a serious impact onthe local citrus industry and economic development. Before1990s, based on itsdistribution and the pathogenicity, Xanthomonas axonopodis pv.citri could be dividedinto five strains: A/B/C/D/E. In recent years, according to the classification of the16SrRNA gene and multi-gene classification, the original citrus canker A, B/C/D and Estrains are divided into three different bacterial subspecies: Xanthomnas citri subsp.citri,X.fuscans subsp. aurantifolii, X. alfalfae subsp. citrumelonis. Some Chinese researchershave studied isolates collected from some provinces in China on the level ofphysiological and biochemical, and most of them suggested that citrus canker bacteriawhich was found in China belong to the A strain, that is the Xanthomnas citri subsp.citriin the new classification system. Citrus is one of the most important fruit and more thanten provinces have the citrus tree planting. In this thesis, we collected hundreds citruscanker infected sample from ten province of China, isolated the pathogenic strains,analysed the genetic variation and population composition of citrus bacterial cankerdisease pathogen Xanthomonas citri subsp. citri （Xcc） by Simple Sequence Repeat（SSR）. Our study will helpful for the further study of citrus canker disease origin,epidemiology in China. Belows are some of our results from research:①We collected the hundreds of bacterial pathogens of infected leave, stem andfruit of citrus tissue from ten citrus cultivation provinces. After isolated, purified andconfirmed the pure cultures by pathogenesis assay and PCR test, we extracted thegenomic DNA of pathogen isolates as a template for SSR analysis. The isolates wereidentified at the morphological and molecular level, and inoculated on detached leavesfor Koch’s certification. We final got51citrus canker bacteria isolates.②We searched for the suitable tandem repeat sequences using tandem repeatsfinder software. Lastly, we selected five highly polymorphic simple sequence repeatmolecular markers for the follow-up study.③SSR was amplified use52isolates （Fifty-one from collected and one from America as model strain） as templets. The results showed that44alleles were detectedwith5pairs of selected primers of SSR maker from52Xanthomonas citri subsp. citriisolates. The number of detected alleles varied from5to12, with an average of8.8alleles per locus. The estimated gene diversity were0.83、0.64、0.86、0.86、0.54acrossthe5SSR loci, with an average of0.75. The clustering analysis results of the52Xanthomonas citri subsp. citri isolates using the unweighted paired-group method usingarithmetic averages cluster analysis of data from5simple sequence repeat （SSR） loci（01,02,03,04,05） were classified into6groups.④The genotype （AACAGCC）₁₈ at02locus and the genotype （TGGGGAT）₆at05locus were more than50%, so the isolates which had one of the two genotypes weredominant strains. The ratio of these two genotypes in the same strain accounted for30.8%and were higher than the other genotypes.⑤Dendrogram analysis showed that the clusters were mostly consistent with thegeographical origins of the isolates, but there is little relationships between varietieorigens. The isolates from Jiangxi, Fujian, Zhejiang had relatively close geneticrelationship, the three provinces were adjacented to each other in the geography.Conclusion: A higher diversity in molecular level of genetic variability wasobserved among the tested52Xcc isolates. In the region, genetic diversity was abundant（H>0.5）. Dendrogram analysis showed that the clusters were mostly consistent with thegeographical origins of the isolates. The SSR markers developed were useful forpopulation genetics studies of Xanthomonas citri subsp. citri isolates.