| Pear chlorotic leaf spot-associated virus(PCLSaV)is newly discovered Emaravirus with five negative single stranded RNA,the polyclonal antibody against PCLSaV NP protein was prepared in our laboratory,In this study,the antibody was evaluated for the detection of PCLSaV,and the virus titer in symptomic and asymptomatic pear samples were analyzed by RNA sequencing(RNA-seq).Field investigation and transmission tests revealed that Eriophys sp.could be the vector of the virus.The study established on effective serological method for the rapid and reliable detection for PCLSaV,and provided impotant information for establishing scientific sampling and control measures for the virus.The main results are as follows:1.The polyclonal antibody prepared from PCLSaV NP protein was used to analyze the extracted virus particles by Western blot.The polyclonal antibody showed immunoreactivity with the 30.2 kDa specific expression protein NP,the polyclonal antibody of the prepared PCLSaV NP had better specificity;The cloned antibody can specifically react with the NP protein in pear leaves infected with PCLSaV,but not with the diseased leaves of pear infected with ASPV and ASGV,as well as with healthy Pyrus betulaefolia Bunge,Comice and A20;The optimal dilution concentration of the polyclonal antibody for PCLSaV detection by Dot-ELISA is 1:400 times dilution(w/v,g/mL),and 1:5 times dilution(w/v,g/mL)of pear leavesextraction,this method can effectively detect PCLSaV in the samples,Dot-ELISA and RT-nPCR were used to detect the virus in 100 pear samples.The detection rate of Dot-ELISA was 34%,and the detection rate of RT-nPCR was 47%.The detection effect of Dot-ELISA was lower than that of RT-nPCR.2.Select three pear plants showing PCLSaVdiseased and threeun-diseased pear plants in the same orchard,collect two samples of symptomsand asymptomatic leaves from each plant,and take two samples from un-diseased pear plants for RNA sequencing.The read and contig matched to the PCLSaV genome were analyzed,and it was found that the number of read matched to the PCLSaV genome chain in the symptomatic pear samples was high and the coverage rate was high,while the coverage rate of the asymptomatic samples from the same strain was very low;Among the samples of symptomatic pear plants,the number of PCLSaV readmatched by two samples on one pear plant was relatively high,and the number of PCLSaV read matched by four samples on the other two plants was very small.The sequencing results showed that the symptoms of the virus disease on leaves may be positively correlated with the virus content,and latent infection also existed in un-diseased pear plants.3.Field investigation found that the density of Eriophyid mite was high in pear orchards with severe PCLSaV occurrence.Themorphological observation of Eriophyid mite on pear leaves was carried out under stereo microscope and scanning electron microscope,and it was found that the Eriophyidmite belonged to Eriophyid mite.Typical features of(Eriophys).Eriophyid mite were collected from pear leaves showing PCLSaV symptoms,and then inoculated on the leaves of healthy Pyrus betulaefolia Bunge seedlings.RT-PCR and RT-nPCR were used to detect the difference between inoculated leaves of Pyrus betulaefolia Bunge 30 and 60 days after inoculation with Eriophyid mite,respectively.PCLSaV in young leaves,the results showed that Eriophyid mite can infect the virus,and the detection rates of PCLSaV in inoculated leaves and young leaves were 80%and 60%. |
