Development Of RT-PCR Techniques For The Detection Of Pear Chlorotic Leaf Spot-associated Virus And Evaluation Of The Effects Of Virus Infection On Rooting Of In Vitro-cultured Pear Plants

Apple stem grooving virus,Apple stem pitting virus and Apple chlorotic leaf spot virus are common viruses infecting pear trees.The mixed infection of these viruses often affects the growth and product quality of pear.Previously,a novel virus in the genus Emaravirus infecting pear trees was found by deep sequencing in our laboratory.The affected pear trees showed severe symptoms of semi-transparent chlorotic spots.Then,the virus was tentatively named pear chlorotic leaf spot associated virus(PCLSaV).The genome of the virus consists of five negative single-stranded RNAs(RNAs 1-5).In this study,primers were designed for the detection of PCLSaV by RT-PCR,and nested RT-PCR(nRT-PCR)assays were developed and used for the detection of PCLSaV in in vitro-cultured pear plants.Meanwhile,the rooting efficiency and transplanting survival rate of virus-infected and virus-free in-vitro plants of three pear cultivars were comparatively evaluated.The main results are as follows:1.Two sets of nested RT-PCR(nRT-PCR)primers,Out-F1/R1 and Inner-F1/R1,and Out-F2/R2 and Inner-F2/R2,were designed based on the obtained RNA3 sequence of PCLSaV genome.Leaf samples of 22 pear plants known to be infected with PCLSaV were collected and their RNAs were individually subjected to reverse transcription by using random primers and a primers 3C complementary to the 3' terminal sequence of the genomic RNAs of emaraviruses,respectively.Then,the PCLSaV detection efficiency of RT-PCR and nRT-PCR assays using primers designed in this study and those designed previously in our laboratory based on the sequence of RNA 3 and RNA 5 of the virus were evaluated.When the cDNAs obtained from random-primer reverse transcription were used as templets,the rates of PCLSaV-positive samples were 90.9% and 42.9% in RT-PCR tests using primer sets RNA3-F/R and RNA5-F/R,respectively.Whilst,using the cDNAs obtained from primer 3C reverse transcription as templets,the PCLSaV-positive rates were 77.3% and 13.6% in RT-PCR tests using the two primer sets,respectively.Moreover,by using the cDNAs obtained from random-primer and 3C reverse transcription as templets,the rates of PCLSaV-positive samples were 100% vs66.7% and 100% vs 62.5% in nested PCR assays using two primer sets Out-F1/R1 combined with Inner-F1/R1 and Out-F2/R2 combined with Inner-F2/R2,respectively.Using the cDNAs obtained from primer 3C reverse transcription as templets and productsobtained by using 3C/5H as outer primers,the rates of PCLSaV-positive samples were77.3%?72.75%,0% and 95.4% in nested PCR assays using RNA3-F/R,RNA5-F/R,Out-F1/R1 and Out-F2/R2 as inner primers,respectively.The results indicated that nRT-PCR assays by using random primers for reverse transcription and the two sets of nRT-PCR primers could efficiently detect PCLSaV in pear leaf samples.2.In-vitro plants of 13 pear cultivars were tested for the presence of three common viruses ASGV,ACLSV and ASPV and the novel virus PCLSaV.The nRT-PCR assays using random primers for reverse transcription and the primer set Out-F1/R1 combined with Inner-F1/R1 showed that 6 out of 12 plants of Cuiguan without virus-elimination treatment,2 out of 8 plants of Cuiguan and Jinshui 2 previously subjected to virus-elimination treatments were PCLSaV positive,respectively.In-vitro plants of other10 pear cultivars were PCLSaV free.Meanwhile,nested multiple RT-PCR showed that plants of all tested cultivars without virus-elimination treatment were positive for one or more viruses of ASGV,ACLSV and ASPV,but all tested plants w previously subjected to virus-elimination treatments were free for the three viruses.3.Using virus-free and virus-infected in-vitro plants of pear cultivars Seerkefu,Red Bastlett and Bantlett as materials,the effects of virus infections on their rooting and transplantation were evaluated.The results showed that the effect of virus on rooting of in vitro pear plants varied among cultivars.The root number and length of virus-free plants of Seerkefu and Red Bastlett were significantly higher than those of their ASGV-infected plants.Whilst,there were no significant differences in rooting rate,root numbers,root length and callus size between virus-infected and virus-free plants of Bantlett.When the rooted plants were transplanted,the survival rates of ASGV-infected verse virus free plants of Seerkefu and Red Bastlett were 24.6% vs 66.6% and 33.2% vs 66.7%,respectively.The survival rates of ASGV-infected,ASGV and ASGV co-infected and virus free Bantlett plants were 74% vs 59% vs 70%,respectively.At 20 d and 40 d post transplantation,the plant height and new leaf number showed no significant difference among ASGV-infected and virus-free Red Bastlett plants.However,plant height and new leaf number of virus-free Seerkefu and Bantlett were higher than thoses of virus-infected Seerkefu and Bantlett plants.