Identification And Detection Of Viruses Infecting Kiwifruit In Fengxin County

Fengxin is the largest kiwifruit-growing county in Jiangxi Province and a characteristic county.Its kiwifruit production plays an important role in local economic development and guaranteeing market supply.In recent years,with the continuous expansion of kiwifruit planting scale and the frequent transfer of seedlings,scions and other reproductive materials,the occurrence of kiwifruit virus diseases and other pests has become increasingly serious.In order to clarify the current species and occurrence status of Fengxin kiwifruit virus disease,and to carry out rapid,efficient and simple detection of kiwifruit virus,this paper carried out small RNA deep sequencing identification and RT-PCR,multiplex PCR,and LAMP detection on Fengxin kiwifruit virus disease.The following experimental results were obtained:23 samples of suspected viral diseases were collected in the agricultural cooperatives,Shankou,Xinxilan and other bases in Fengxin County.After the samples were mixed,small RNA deep sequencing was performed.After sequence preprocessing,splicing and comparative analysis,a total of 4 viruses were detected,namely Actinidia virus A（Ac VA）,Actinidia virus B（Ac VB）,Actinidia chlorotic ringspots associated virus（Ac CRa V）and Citrus leaf blotch virus（CLBV）.100 leaf samples with suspected viral diseases were collected from various bases such as Shankou,Nonghe and Xinxilan,and 4 viruses of Ac VA,Ac VB,Ac CRa V,and CLBV were detected on each sample by RT-PCR technology.The results showed that the recovery rates were 70%,67%,70%and 73%,respectively,in which 94%of the samples were infected by 2-4 kinds of viruses,while the number of samples infected by a single virus only accounted for 6%.Ac VA,Ac VB and Ac CRa V were the dominant pathogens of kiwifruit virus disease in Fengxin County,and the combined infection of kiwifruit virus disease was common.The corresponding symptoms of the four viruses were preliminarily summarized:Ac VA and Ac VB often co-infect kiwifruit leaves,mainly causing bright veins,chlorosis,and slight shrinkage of leaves;Ac CRa V mainly caused irregular mottled leaves and slight chlorosis;CLBV mainly causes yellowing of large areas of leaves.The symptoms of diseased leaves infected by multiple viruses will be superimposed,and the symptoms will be more serious and complex,which is easy to be detected in time.The symptoms of diseased leaves infected by one virus will be mild,or even have no obvious symptoms.A multiplex PCR detection system was established and optimized to improve the detection efficiency of the four viruses Ac VA,Ac VB,Ac CRa V,and CLBV,and to achieve the purpose of saving detection cost and time.Using diseased leaves containing four viruses as samples,specific primers were designed for the conserved sequence intervals of each virus,and the annealing temperature and primer concentration ratio in the detection system were optimized.The results showed that the specificity of the primers was good,and the optimal annealing temperature for the reaction was 55℃.The optimal primer concentration ratio for the reaction was 6:2:3:2.Eight groups of 10-fold concentration gradient dilutions were performed on the c DNA to verify the sensitivity of the reaction system.The results showed that the sensitivity of the multiple PCR reaction system was 1μg.μL^-1,which was high but 10-100 times lower than the sensitivity of single PCR.A RT-LAMP reaction system was established and optimized for CLBV to solve the problems of lack of experimental equipment and relatively simple experimental conditions in grassroots units.Using the diseased leaves containing CLBV as samples,five specific LAMP primers were designed,and a group with the best effect was screened out.The three reaction conditions of annealing time,annealing temperature,and inner and outer primer concentration ratio were then optimized.The results showed that Primer No.1 was the most suitable primer,and the optimal reaction system was:reaction at 61℃for 60 minute under the condition that the concentration ratio of inner and outer primers was 8:1.The sensitivity of the reaction system was then verified by 8 groups of 10-fold serial dilutions of c DNA.The results showed that the sensitivity of the RT-LAMP reaction system was 100pg.μL^-1,which was high and 10 times higher than that of the ordinary RT-PCR detection method.This RT-LAMP reaction system has good specificity and can be applied to the detection of CLBV in actual production.