Research On Genetic Diversity And Molecular Rapid Detection Of Apple Scab Fungus Venturia Inaequlis

Apple scab is one of the main diseases of apples. It is characterized by fast epidemic, serious damage and difficalf to control. It is one of the most important apple diseases in Europe and America and often causes yield and economic losses. In China, apple scab distributes mainly in Liaoning province, Heilongjiang province, Gansu province, and same areas of Shaanxi province, Xinjiang province and Shandong province. It has caused serious effect on industry of apple because of large-scale epidemic. In this study, we performed research on genetic diversity and molecular rapid detection of Venturia inaequalis and obtained the results as follows:1. Eighteen pairs of primers were selected for study on genetic diversity of 90 isolates of V. inaequalis from China, India and United kingdom by using simple sequence repeats (SSR) technique. Genetic clustering map was constructed based on these results. The results indicated that, all the 90 isolates were clustered together at the 0.76 genetic similarity level. At the 0.80 genetic similarity level, all the isolates (18) from United kingdom were clustered in groupⅠand isolates from China and India (72) were clustered into groupⅡ. At the 0.83 genetic similarity level, isolates from groupⅡwere divided into 3 subgroup:all the Indian isolates belonged to subgroup A, all the isolates from Xingping Fuji, Starking Delicious, Qinguan and 11% isolates from Xingping Gala had clustered into subgroup B and all the isolates from Xunyi Gala, Xunyi fuji and 89% isolates from Xingping Gala were clustered into subgroup C. We can conclude that the isolates of V. inaequalis from three countries were relative relationship based on the regional source. The isolates from China were relatively close to that from India and far from the UK. All the isolates from China were not relatively close based on the source but had a relative relationship on cultivars. Five pairs of primers were used in PCR amplification of 20 isolates of V. inaequalis, which belong to 7 physiological races, through SSR technique. Fourty three alleles were detected with an average of 8.6 alleles per SSR primer pair.A genetic clustering map was constructed and genetic similarity for isolates of V. inaequalis was from 0.72 to 1.0 with an average of 0.85. At the genetic distance of 0.81,20 isolates were divided into 6 groups. Except group 1 and group 4, isolates in the same group belonged to different races, which indicated that genetic diversity of 20 isolates of V. inaequalis had minor relevance with their races.2. Using amplified fragment length polymorphism (AFLP) technique, we obtained 25 pairs of primers for study on genetic diversity of 46 isolates of V. inaequalis from Xingping and Xunyi in shaanxi province. Genetic clustering map was constructed based on these results. The results indicated that, all the 46 isolates were clustered into two groups at the 0.68 genetic similarity level. Group I contained all the isolates from Xunyi fuji, Xunyi Gala and Xingping Gala. Group II contained all the isolates from Xunyi Starking Delicious, Xunyi Qinguan, Xingping Starking Delicious and Xingping Qinguan. These results showed that the isolates of Venturia inaequalis from Xingping and Xunyi didn't have a relative relationship based on the regional source but had a relative relationship on the host cultivars.3. Universal primers ITS1 and ITS4 were used for PCR amplification of 26 isolates from V. inaequalis, Alternaria mali, Marssonina cororlar and Podospharea leucotricha. Twenty-six ITS sequences were obtained. Based on sequence alignment result, one specific primer pair was obtained and the size of PCR product was 320 bp. The primer pair 320A/320B was specific to V. inaequalis and the pathogen could be detected in apple leaves without symptom with the primer pair. The sensitivity of this primer pair for DNA of V. inaequalis was 100 fp/μl.