Detection Of Pathotype Of Xanthomans Oryzae Pv. Oryzicola Population And Their Genetic Structure Analysis From Anhui Province

Rice bacterial leaf streak disease is caused by Xanthomonas oryzae pv. oryzicola, it become a important bacterial diseases and pest affecting rice production in China. Bacterial leaf streak occurs mainly affected by three main factors:pathogens, resistant varieties and climatic conditions. Recently, this disease had harm gradually expanded in Anhui province and has risen to major diseases in Anhui province. Breeding resistant varieties is the most effective prevention measures, but the premise is proved pathogenic type and genetic background, so in 2009-2011 we conducted and separation diseased leaves of rice from 11 different areas in Anhui province, and analyzed the populations pathogenic type and genetic structure, the results were as follows:1. Isolation and molecular identification Xanthomonas oryzae pv. oryzicola.In this study, using the plate dilution methods and BPA medium recovered from diseased leaves of rice plants associated with bacterial leaf streak located in 11 different regions in Anhui Province, in total receive 94 representative strains by random.collected from 11 different regions of Anhui rice diseased leaves carry bacteria isolated were randomized to receive 94 representative strains. These strains showed a consistent trait on BPA culture, strains were yellow, round, smooth surface. The obtained isolates were inoculated by acupuncture in the booting stage of the susceptible rice "Jingang 30" to pathogenicity determination, the results showed that 94 isolated strains were make the susceptible rice " Jingang 30" onset. Further to 94 strains isolated identified by PCR detection and found that 94 strains after PCR amplification achieve the amplification signal, it can be determined that the 94 strains isolated were Xanthomonas oryzae pv. oryzicola.2. Analysis pathogenic type of Xanthomonas oryzae pv. oryzicola populations.In this experiment,using IRBB5, IRBB14, IR24, IRBB4, IRBB21 and Jingang 30 rice varieties to pathogenic type analysis for 72 strains by identification tested, the results showed that:the 72 tested strains of Xanthomonas oryzae pv. oryzicola had clear pathotypes differentiation and can be divided into Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ six pathogenic type. According to the frequency of low pathogenic type of share, respectively:pathogenic type Ⅰ> pathogenic type Ⅱ> pathogenic type Ⅲ> Pathotypes Ⅵ> pathogenic type Ⅳ> pathogenic type Ⅴ. Which type Ⅰ was the dominant causative pathogenic type, accounting for 27.8% of the test strains. As can be seen from the strains tested in the identification of host pathogenic response, from the Huaiyuan and Fengyang strains have highly toxic, weaker toxic from Mingguang strains, the results show that the pathogenic type of Xanthomonas oryzae pv. oryzicola and geographical distribution has some relevance.3. Analysis genetic differentiation and genetic diversity of Xanthomonas oryzae pv. oryzicola.Using CTAB method extracte bacteria genomic DNA from 11 different regions of Anhui Province and using the BOX, REP and ERIC three sets of primers to PCR amplification for their genomic DNA, and then analyze their genetic diversity situations. The results showed that three sets of primers were amplified 49 fingerprint bands, and the amplified DNA bands were polymorphic, the percentage of polymorphic bands was 100%, the molecular size of the amplified fragments were between 100-2000 bp. By comparison, the REP primers had highest resolution, can be generated from 2 to 13 primary band, follow is BOX primers, can be generated from 2 to 10 primary band, ERIC primers having the lowest resolution, generating 1 to 8 main fingerprint.In order to system understand genetic diversity and genetic differentiation of Xanthomonas oryzae pv. oryzicola population from Anhui province, based on geographic area characteristics of the tested strains, the test will classify 11 different populations from Anhui province to southwest region of Anhui, northeast region of Anhui, northwest region of Anhui, central region of Anhui and southeast region of Anhui five groups. Use POPGENE version 1.3.1 software analysis genetic diversity of Xanthomonas oryzae pv. oryzicola can be drawn, the five geographic regions population of Xanthomonas oryzae pv. oryzicola from Anhui province, the average level of genetic parameters as follws:Nei’s genetic diversity index (H) 0.32, Shannon’s information index (I) was 0.49, genetic diversity (Ht) 0.3232, genetic diversity within populations (Hs) and genetic diversity among populations (Dst) values were 0.3232,0.2261 and 0.0971. Diversity within populations greater diversity among populations. Gst mean genetic differentiation coefficient 0.3006. The results show that Xanthomonas oryzae pv. oryzicola from Anhui province have some genetic differentiation among different geographical regions, genetic variation mainly comes from internal groups.In order to understand the different areas of Xanthomonas oryzae pv. oryzicola from Anhui province genetic background differences, in this study, cluster analysis by use NTSYS-pc2.1 software after amplification by BOX, ERIC and REP primers, the results showed that in a similarity coefficient 0.73 to be threshold, ERIC primers can be divided 94 strains into two categories, BOX and REP primers can be divided 94 strains into three categories. By comparison, three kinds of primers dominant groups has relatively higher cross rate strains, both the three dominant groups contain Huoqiu, Mingguang, Fengyang and Wuwei strains. In order to reduce the accidental errors amplification by single primer, making research results more objective, comprehensive three primers amplification results for 11 different populations from Anhui to UPGMA cluster analysis, the results showed that similarity coefficient 0.70 is the threshold can be divided 11 populations into three groups, from Qianshan in southwest region of Anhui is a taxon, the northeast region of Anhui Chuzhou and cenral region of Anhui Feidong as one group, other eight regions populations is another taxon. As can be seen from the clustering results, strains from the same geographic area population genetic similarity higher, clustered together, such as the northeast region of Anhui Huaiyuan and Fengyang. In addition, the study also found that among the different geographic regions also showed a high degree of genetic similarity, clustered together, such as southeast region of Anhui Wuwei and northeast region of Mingguang srains. The clustering results are basically consistent with the results of a single cluster by three primers.