RPA-LFD Detection And Exogenous Salicylic Acid Eradication Of Apple Virus

Virus disease is one of the main factors that restrict the high-quality,efficient and sustainable production of apple.At present,there is no effective method to cure viral diseases once the tree get infected,therefore,the use of virus-free propagules is still served as the most important strategy to combat viral diseases in apple.The establishment of simple and efficient method for the detection and eradication of apple viruses is prerequisite for the obtaining and cultivation of virus-free trees.The purpose of this study was therefore(1)to evaluate a multiplex RT-PCR detection technique for apple latent viruses;(2)to establish a Reverse Transcription Recombinase Polymerase Amplification(RT-RPA)and Lateral Flow Dipstick(LFD)technology for the detection of Apple Stem Grooving Virus(ASGV);(3)to test the effects of exogenous salicylic acid(SA)treatment on the eradication of apple ASGV and Apple Chlorotic Leaf Spot Virus(ACLSV)and to investigate the mechanism behind virus eradication.The results obtained from this study is presented as follows:(1)The multiplex RT-PCR detection system was established and optimized.In this study,apple cultivar ‘Yanfu-8’ was infected with ASPV(Apple stem pitting virus),ASGV and ACLSV,and c DNA was used as the template for multiplex RT-PCR with a reaction volume of 25 μL.Firstly,the reaction conditions were optimized,and the optimal reaction conditions were determined as follows : 1 μL 10 μM F and R primers,2 μL c DNA,35 cycles at Tm 57°C.Secondly,the sensitivity comparison and specificity verification were carried out,and the results showed that the single RT-PCR was 10 times more sensitive than the multiple RT-PCR,and multiple RT-PCR was specific.Finally,the reliability of multiple RT-PCR detection was verified by field samples.The results showed that the virus detection results of multiple RT-PCR for test field samples were almost consistent with those of single RT-PCR.When the ASGV virus concentration was low,multiple RT-PCR could not be detected.(2)An efficient ASGV detection technique based on recombinant polymerase amplification(RPA)was developed.In this study,‘Gala’ single-infected with ASGV was used as test material RNA was used as the template,M-MLV reverse transcriptase were added into the RPA reaction for the reverse transcription of RNA and RPA-specific amplification,so that the reverse transcription and amplification were synchronized..Firstly,the reaction temperature and time were optimized.Results showed that the amplification(3)effect was the best when incubated at 40 °C for 30 min..Secondly,the sensitivity and specificity of the optimized RPA reaction system were tested,the 500 ng/μL RNA was diluted 10 times in gradient.And the results showed that the RT-RPA detection method was100 times more sensitive than RT-PCR,specificity test showed that only samples infected with ASGV showed positive response.Finally,a simple,cheap and fast crude RNA extraction method was applied using apple leaves for ASGV detection by one-step RT-RPA.This method was applied to test tube seedlings and field samples,the results were consistent with those of RT-PCR,indicating that the one-step RT-RPA method can be used for the detection of apple ASGV.(4)Exogenous supply of SA was combined with thermotherapy followed by shoot tip culture or cryotherapy for the eradication of apple viruses,and the role of SA was studied.Apple cultivar ’Yanfu 8’ infected with ASGV and ACLSV.Firstly,the optimal SA concentration was screened at 0,1,10,50 and 100 μM,results showed that the SA treatment at 10 μM significantly reduce the virus concentration in plants without affecting the vegetative growth.Then,thermotherapy was applied to in vitro stock shoots cultured on proliferation medium with or without 10 μM SA for 2 weeks.The RT-q PCR test showed that as compared with shoots only treated by thermotherapy,a significant decrease in the concentration of viruses was found in shoots treated by SA and thermotherapy.Virus detection showed that 100% virus-free rates were obtained from SA treatment and the control group after thermotherapy.Noticeably,the higher level of shoot tip regeneration(89%)was obtained from the SA treatment group,higher than the 75% obtained from the control group.In combining SA with thermotherapy followed by shoot tip cryotherapy,a shoot tip regrowth level of 13% was obtained,higher than the 7% of shoot tip regrowth of the control group,and all the plants regenerated after cryotherapy were virus-free.Finally,by applying thermotherapy,the treatment of 10 μM SA was compared with 25 μM of ribavirin(R)or the 30 μM sodium nitroprusside(SNP)for virus eradication.The results showed all the treatments produced 100 % of virus eradication efficacies,but the higher level of shoot tip regeneration was produced from the SA treatment than those obtained from the R and SNP treatments.These results indicated that SA could reduce the virus titers in apple plants,and increase the regrowth of shoot tip culture and cryotherapy without lowering the virus eradication efficiencies.