Study On The RT-RPA And RT-qPCR Detection And Electrotherapy Of Pear Chlorotic Leaf Spot-associated Virus

Pear chlorotic leaf spot associated virus（PCLSaV）is a new negative-sense RNA virus identified in China in recent years.The virus belongs to Emaravirus,its genome consists of at least five negative-sense RNA segments.Pear chlorotic leaf spot（PCLS）caused by PCLSaV is common in some Pyrus pyrifolia producing areas in China,which seriously affects the growth of pears.In this study,recombinase polymerase amplification（RT-RPA）technology and real-time quantitative RT-PCR（RT-qPCR）technology were established;the combination of shoot tip culture,electrotherapy treatment and chemical treatment were used to eliminate the virus and established a practical virus elimination technology system.The results provide technical support for the early diagnosis of the disease and the cultivation of virus-free pear germplasm.The main research results are as follows:1.RT-RPA detection of PCLSaV.Seven pairs of primers were designed based on the sequences of RNA3 and RNA5 of PCLSaV,the primers R3-F1/R1 with high specificity were screened by conventional RT-PCR.RT-RPA analysis of pear leaf samples with different viruses showed that the target band with the size of 243 bp was amplified only from the samples positive for PCLSaV,that is,the primers had high specificity.The minimum concentration of plasmid containing partial fragment sequences of RNA3 of PCLSaV that could be detected by RPA was 10^-7,and the sensitivity of RPA was equivalent to that of RT-PCR.38 leaf samples from field pear plant and 32 leaf samples from in vitro pear plant were tested,and 37 positive samples were detected by RT-RPA,the positive rate was 52.9%;41 positive samples were detected by RT-PCR,the positive rate was 58.6%.The consistency rate of the two methods for detecting pear leaf samples was 91.4%.The equipments required for RT-RPA are simple and the reaction speed is fast,it only needs to react at 39℃for 20 minutes,which is available for rapid detection of PCLSaV.2.RT-qPCR detection of PCLSaV.Eight pairs of primers were designed based on the sequences of RNA3 and RNA5 of PCLSaV,the primers q5-F2/R2 with high specificity were screened by conventional RT-PCR and RT-qPCR analysis,and the primer dosage and annealing temperature of RT-qPCR were optimized.The standard curve was established,the cycle value had a good linear relationship with the template concentration（R²=0.997）,and the amplification efficiency was 91.9%.The assay was highly specific for PCLSaV and had no cross reaction with other common pear viruses.The sensitivity of qPCR to detect plasmid containing partial fragment sequences of RNA5 of PCLSaV was 3.0×10³copies/μL,which was 10 times higher than that of conventional PCR.The study found that PCLSaV was unevenly distributed in pear plants,there were significant differences in the content of PCLSaV in different samples.The copy number of virus in symptomatic leaf samples was relatively high,and that in phloem of asymptomatic branches was the lowest.On the same diseased branch,the content of PCLSaV in the young leaves was higher than that in the old leaves.The virus copy in leaves with chlorotic spots and necrosis from the top was highest,and that in asymptomatic leaf from the base was lowest.32 samples from field pear plants and 46 samples from in vitro pear plants were detected,among 23 samples showing chlorotic spots symptom and 55 asymptomatic samples,23 vs 22 and 19 vs 13samples were positive for PCLSaV by RT-qPCR and RT-PCR,respectively.3.Elimination of PCLSaV from in vitro pear plants.Taking in vitro P.pyrifolia cv.’Hosui’plants as materials,the elimination effects of four methods for PCLSaV were compared.Results showed the elimination rates of PCLSaV in’Hosui’pear plants were30.0%,47.4%,44.4%and 76.5%by shoot tip culture and shoot tip culture combined with ribavirin treatment,electrotherapy treatment as well as ribavirin combined with electrotherapy treatment,respectively.Ribavirin and electrotherapy treatment combined with shoot tip culture was used to eliminate PCLSaV,ASGV and ASPV in other four pear cultivars,results showed the elimination rate of PCLSaV in‘Conference’plants was 85.7%,the average elimination rate of ASGV in‘Conference’,’Bantlett’and’Seerkefu’plants was 52.3%,the elimination rate of ASPV in‘Conference’and’Jinxiangshui’plants was 100%.In this study,electrotherapy was used to eliminate pear virus for the first time,which provided new technical support for the cultivation of pear virus-free germplasm.