Studies On Genetic Diversity Of Venturia Inaequalis Of Apple,Screening Its Resistance To Fungicides And The Mechanism Of Qinguan Resistance To Apple Scab In Northwest China

Apple scab(Venturia inaequalis)is a worldwide fungal disease of apple,which causes serious economic losses every year,especially in Europe and America.The disease damages the leaves,branches and fruits of the host,and even causes early defoliation.The disease is characterized by black scab on the leaves and fruits.The disease was originally found in Hebei Province in 1920s and later it spread to other provinces with the rapid development of apple production.In the 1980s,apple production was vigorously developed in the northwest loess plateau region,and the focus of apple production was gradually shifted from the East part of China to the West.Apple scab was once prevalent in Shaanxi,Gansu and Xinjiang in the mid and late 1990s,and latter on,the incidence of the disease obviously subsided,and it only sporadically occurred in some places of these regions.However,in recent years,with the rapid development of apple production,the occurrence and damage of apple scab in Northwest China is increasing,especially in some apple-producing areas of Shaanxi Weibei,Gansu Qinling and Liupanshan mountain areas,as well as Yili River Valley in Xinjiang,where the occurrence of apple scab is very serious,and the disease presents an increasing trend year by year.Hence,apple scab has become one of the important constraints affecting local farmers to become rich and get rid of the poverty.Therefore,In order to further clarify the genetic relationship and spread direction of Venturia inaequalis in the northwest region in recent years,to reveal its the development level of resistance to fungicides of difenoconazole and pyraclostrobin which used all year round in apple orchard,and to explore the resistance mechanism of Qinguan culitvar of apple to apple scab,we carried out all above studies and obtained results as follows:1.Method of single-spore isolation was used and 164 isolates of Venturia inaequalis of apple were isolated and purified from 457 samples of apple scab collected from Shaanxi,Gansu and Xinjiang,and the 35.89%of its samples was successfully achieved.Among them,53 isolates of Venturia inaequalis were from Shaanxi,with the rate of 32.32%;37 isolates from Gansu,with the rate of 22.56%;74 from Xinjiang,with the rate of 45.12%.2.ITS sequence analysis was used to identify all above isolated isolates of Venturia inaequalis and 11 isolates collected from the United Kingdom.The r DNA ITS region was amplified,the PCR products were detected by electrophoresis and sequenced with the universal primers ITS1 and ITS4 of eukaryotic ribosomal DNA.The sequenced results were logged into NCBI for nuclear blast comparison.The results showed that the similarity of 166isolates was over 97%and all tested isolates were identified as Venturia inaequalis(Cooke)wint.3.Using SSR molecular marker technology,the genetic diversity of 132 isolates from China and the United Kingdom was analyzed,and the genetic clustering map was constructed.The genetic distance,molecular variance and genetic structure of the isolates in different regions were analyzed.The results showed that at the level of similarity coefficient of 0.74,all the tested isolates were clustered into one group.At the level of similarity coefficient of 0.76,they were divided into two groups which the first group could be subdivided into subgroups.Some isolates from Xinjiang and a small number of isolates from UK and Gansu were classified into the first subgroup,but none of them were from Shaanxi.All isolates from Shaanxi were classified into the second subgroup,and some isolates from UK were also classified into this subgroup.Among them,the second subgroup also can be divided into several small subgroups,most of the UK isolates are in the NO.1 subgroup,including all isolates from Shaanxi apple varieties Gala,Fuji,Jiuyueqiji and Xinjiang Huadan,Huahong.The NO.2 subgroup contains all isolates from Shaanxi pink lady,Xinjiang honey crisp,and most of Xinjiang crapapple.The NO.3 subgroup only has one isolates from Shaanxi,some from Gansu and Xinjiang.The second group has only one isolate from UK and a few from Xinjiang and Gansu.The results indicated that there was a certain correlation between the genetic relationship and geographical origin of isolates in Shaanxi,but there was no significant correlation between the isolates in Xinjiang and Gansu.The results of molecular variance analysis showed that 91%of the genetic variation of 132isolates originated from intraspecific population and 9%from interspecific population which indicated that there was a relatively frequent gene exchange among the population.4.Mycelial growth rate method was adopted and 90 isolates of Venturia inaequalis selected from Shaanxi,Gansu and Xinjiang were used to determined the sensitivity or resistance to fungicides of difenoconazole and pyraclostrobin,which were used all year round in apple orchards.The results showed that the EC₅₀value of difenoconazole ranged from 0.1431 to 6.7354?g/m L,the ratio of the maximum value to the minimum value was47.07,and the average EC₅₀value was about 1.467±1.562?g/m L.The results of Shapiro Wilk test showed that the EC₅₀value did not conform to the normal distribution(w=0.9595,P<0.05),which indicated that there was some isolates emerged with low sensitivity to difenoconazole,and a relative sensitive baseline with an average inhibition rate of 0.8790±0.7589?g/m L was established.By comparing the EC₅₀values of isolates in different regions,the results showed that the sensitivity of Shaanxi isolates to difenoconazole was the highest,followed by Xinjiang and Gansu.The tested isolates of EC₅₀value range to pyraclostrobin was from 0.1369 to 2.0256?g/mL,the ratio of the maximum value to the minimum value was 15.95,and the average EC₅₀value was 0.4538±0.4362?g/m L.The EC₅₀value of Shapiro Wilk test did not conform to the normal distribution(w=0.6656,P<0.05),which indicated that there were also some isolates with low sensitivity to pyraclostrobin,and a relative sensitive baseline with an average inhibition rate of 0.2386±0.06506?g/m L was established.By comparing the EC₅₀values of isolates in different regions,the results showed that the sensitivity of Shaanxi isolates to pyraclostrobin was the highest,followed by Xinjiang and Gansu.In addition,the results also showed that there was no cross resistance of fungicides between difenoconazole and pyraclostrobin.5.The ultrastructural observation on the leaves of scab-resistant cultivar of Qinguan and scab-susceptible cultivar of Gala after inoculation with the strong pathogenisity strain Ca183 of Venturia inaequalis was carried out by electron microscope.The results of electron microscopy observation showed that the cuticle thickness of Qinguan was higher than that of Gala.The average thickness of cuticle was 1.75?m in Qinguan,and 1.06?m in Gala.By electron microscope observation,we found that after 14 days inoculation with conidia of Ca183 strain,the leaf palisade tissue of Gala was destroyed more obviously than that of Qinguan,mainly manifested as cell atrophy,loose arrangement,oval cells,chloroplast deformation,the number of the organelle reduced,serious loss of cytoplasm,almost forming empty cells;21 days after inoculation,a large number of cell necrosis appeared in Gala,while only a small amount of cell necrosis occurred in Qinguan.Therefore,it clocluded that Qinguan had the strong resistance to fungus of Venturia inaequalis infection and expansion in histocytology,and delayed the development of the disease.