Population Diversity Of “Candidatus Liberibacter Asiaticus” In Southern China Based On Different Gene Loci

Citrus Huanglongbing(HLB)is one of the most devastating diseases in citrus production,which causes big economic losses in citrus industry worldwide.This disease is known to be associated with a fastidious,phloem-limited α-Proteobacteriacea Gram-negative,uncultured bacterium as “Candidatus Liberibacter”.At present,there are three species: “Ca.L.asiaticus”,“Ca.L.africanus”and “Ca.L.americaus”.We identified only “Ca.L.asiaticus”(CLas)in China.Huanglongbing is a centry-old history in China.The genetic diversity is abundant for the Chinese “Ca.L.asiaticus” strains.However,the study of the intraspecific genetic structure of “Ca.L.asiaticus” is usually based on single gene or locus.The objective of this study is to explore the intraspecific genetic structure and genetic diversity of“Candidatus Liberibacter asiaticus”(CLas)in nine provinces of China,and to evaluate the feasibility of studying genetic diversity of specific species using multiple loci.Three kinds of polymorphic gene loci including three phage regions(SC1＿gp045,SC2＿gp035 and PJXGC),transposon site(CLIBASIA＿05620 ～ CLIBASIA＿05625)and short tandem repeat(STR)genes(CLIBASIA＿03080 and CLIBASIA＿01215)were used to evaluate genetic diversity of 669 CLas samples from 9 province in southern China.The main results were as follows:1.For the first time,three different polymorphic loci including prophage region,transposon site and short tandem repeat sequence were combined to analyze the population diversity of pathogens of citrus HLB from different geographical regions in China.The diversity between different loci varies greatly.The results of this study indicate that the diversity analysis using multiple loci are are not the same as using a single locus.2.The CLas populations in the southern provinces of China have different dominant groups based on different loci.Based on the prophage types,the CLas isolates were classified into 8 groups,among which the Type 2 prophage was predominate(67.41%).Four types of isolates were identified based on transposon sites,and the type containing the B350 fragment(non-MCLas-A type)transposon was the dominant species(70.40%).Based on the two short tandem repeats loci,ten band types and 30 band types were identified,respectively,and the isolates with the band type "2"(TRN=3)at the STR1 locus was the dominant species(57.55%),and there were no dominant strains at the STR12 locus.the strains were diverse.However,the matching of the three dominant populations was as high as 74.72%.3.Cluster analysis showed that the CLas population in southern China can be divided into two groups,namely,Group I(strains from Guangdong,Guangxi,Hunan,Fujian,Jiangxi,Hainan,and Zhejiang)and Group II(strains from Yunnan and Guizhou).The clustering results based on different loci were slightly different,but the strains in the Guangdong and Guangxi provinces always clustered together,while the higher altitude Yungui area and other provinces had farther genetic distances.4.Three kinds of polymorphic gene loci were used to evaluate genetic diversity of 178 CLas samples from 8 cities in Guangdong Province.Based on the prophage types,the CLas isolates were classified into 6 groups,among which the Type 2 prophage was predominate(86.52%).Four types of isolates were identified based on transposon sites,and the type containing the B350 fragment(non-MCLas-A type)transposon was the dominant species(73.60%).Based on the two short tandem repeats loci,nine band types and 17 band types were identified,respectively,and the isolates with the band type "2"(TRN=3)at the STR1 locus was the dominant species(59.60%).The STR12 site had a variety of strains.Cluster analysis showed that the genetic distance between the strains in Maoming and those in other regions was far,while the CLas in Qingyuan and Meizhou were similar based on these genes.5.The total clustering results of the strains in Guangdong Province based on all four loci were different from those based on each locus,indicating that the genetic diversity at the gene level is more complicated.In summary,the CLas samples were analyzed simultaneously at multiple loci to assess the population diversity of the pathogens of citrus Huanglongbing in China,providing information to determine the origin and epidemiology of CLas dominant species in China,and suggesting a certain molecular basis for the epidemic trend of Huanglongbing.