Studies On Pathogens Of Apple Ring Rot In Shaanxi Province And Its Genetic Diversity

Apple ring rot is an important apple disease in the world, which is on trunks, branches and fruits of apple tree. And it occurred in the major apple-producing areas in China. According to 3 years investigation by our laboratory, Shaanxi Province had a high incidence on trunks and branches, and the average incidence was 60%. Thus the serious losses of the apple production in Shaanxi Province were got. So far, the species of the pathogens in Shaanxi Province were vague and the large sample analysis of genetic diversity was not found, thereby affecting the disease control and prevention research.In this study, the genetic diversity of the pathogens of apple ring rot (276 from Shaanxi Province; 70 from Shanxi, Shandong, Henan and Sichuan) was analysed by using SRAP and ISJ markers. Meanwile, 119 srains were used for direct rDNA-ITS sequencing. The main results were as follows:1. The results of rDNA-ITS sequencing showed some strains which compose 95% of the total were similiar with the sequences of B. dothidea from the GenBank database (similarity more than 99%). It indicated B. dothidea was the dominant specie of apple ring rot in Shaaxi Province.2. PCR amplified system of B. dothidea based on SRAP markers was set up and was used to analysis the genetic diversity of the tested strains. And the system was as follows: 0.5μL, dNTPs (10 mM); 2.5μL, 10×Taq Buffer; 1.0μL, Forward primer (10μM); 1.0μL, Reverse primer (10μM); 2.0μL, MgCl₂ (25 mM); 0.15μL, Taq polymerase (5 U/μL); 1.0μL, genomic DNA (200 ng/μL); 17.5μL, ddH2O. And it was used for PCR amplication with 6 pairs of SRAP primers. The electrophoretic patterns showed there were 53 polymorphic bands, (89.83% of the total amplified bands). It indicated that B. dothidea has a high SRAP polymorphism. Then a matrix was constructed and the cluster analysis was done. The results showed 272 texted strains were divided into seven groups when the similarity coefficient was 0.705. The strains of same geographical origins were scattered in two or more groups. At the same time, the strains of different regions were in the same clustering group. The results showed that there was a rich genetic diversity of B. dothidea and no significant correlation between the genetic diversity and the strains geographical origin.3. PCR amplified system of B. dothidea based on ISJ markers was set up and was also used to analysis the genetic diversity of the tested strains. And the system was as follows: 0.5μL, dNTPs (10 mM); 2.5μL, 10×Taq Buffer; 5μL, MgCl₂ (25 mM); 0.75μL, Primer (10μM); 0.13μL, Taq polymerase (5 U/μL); 1.0μL, genomic DNA (200 ng/μL); ddH2O, 16.87 μL. And it was used for PCR amplication with four primers. The electrophoretic patterns showed there were 34 polymorphic bands (82.93% of the total amplified bands). Also, the cluster analysis was done and the results showed 272 texted strains were divided into three groups when the similarity coefficient was 0.67, but the results were similar with SRAP markers.4. Based on the results above two, we can see that both SRAP markers and ISJ markers can get a stable and reliable result. Thus we got a conclusion that both of them can be used for the research of the genetic diversity of B. dothidea.5. Using ISJ primer B1 for the PCR amplication, B. dothidea and B. obtusa had the fragment of 500bp. Moreover B. obtusa had the fragment of 550bp. Therefore, the amplification results of primer B1 can be the molecular basis to divide B. dothidea and B. obtusa.