Study On Seed Transmission And Molecular Characterization Of Three Viruses In Pyrus Betuleafolia And Pyrus Calleryana

Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV) are naturally spreaded by propagation materials and plants. It has been reported that ASGV can be trasmitted through the seeds of lily, Chenopodium quinoa and apple. Recently, it was found that seedlings of Pyrus betulaefolia were positive for the three viruses in our laboratory as detection by RT-PCR. To understand whether these viruses could be trasmitted through seeds of P. betulaefolia and Pyrus calleryana^ in this study, we analyzed the efficiency of seed transmission of the three viruses by using nested-multiplex RT-PCR (nmRT-PCR), and the molecular characteristics of the three viruses from these samples.The obtained results are listed as followings.1. Samples of 31 fruiting trees of P. betulaefolia from Shandong, Shanxi, Gansu and Hubei provinces and three fruiting trees of P. calleryana were collected, and tested for the presence of the three viruses. Results showed that plants positive for ASGV, ACLSV and ASPV accounted of 79.4%,8.8% and 32.4%, respectively. The three viruses were detected in different tissues, including leaves, phloem, flower, petal and pollen. However, the virus detection efficiency differend dependeing the tissues used.2. The seeds of eight P. betulaefolia and two P. calleryana fruiting tree that infected by the three viruses can be detected viruses use nested-multiplex RT-PCR. In the different sources of seeds, the seed-borne rate for ASGV is 3.3% to 73.9%, the rate for ACLSV is 2.2% to 40.4%, the rate for ASPV is 2.2% to 40.4%. Seedlings from two P. betulaefolia and two P. calleryana in the 6-8 leaf stage were detected for viruses by nmRT-PCR, the result showed that, three viruses can spread by seed. The seed-transmission rate of ASGV from different fruiting tree has a big different is 2.2% to 19.7%, seed-transmission rate of ACLSV is 15.6%, seed-transmission rate of ASPV is 2.2% to 3.0%. So, seed infection rate is higher than the rate of seed transmission.3. Samples of 821 P.betuleafolia from Shandong and Hubei province and nine Pyrus pashia from Yunnan province were detected for viruses by nested-multiplex RT-PCR. Results showed that, the plants positive for ASGV, ACLSV and ASPV is 10.1%,5.3% and 3.5%, respectively. The plants positive for ASGV and ACLSV were close to or less than seed-transmission from plants were infected by viruses, except ASPV.4. The CP and MP genes of 24 and 11 ASGV isolates from P. betuleafolia and P. calleryana were sequenced, respectively. The CP gene of those isolates showed 87.3% to 100% and 94.1% to 100% sequence identities at nucleotide and amino acid levels, respectively. Identity of nucleotides and amino acid of MP gene of those isolates are 84% to 99.9% and 94.4% to 100%, respectively. The phylogenetic analysis based on aa sequences of CP and MP genes indicated that the phylogenetic positions of ASGV isolates from P. betuleafolia, P. calleryana and other hosts were no relation to their host origins. Some ASGV isolates showed intro-isolate variation, and molecular variants from the same source plant grouped into different clusters.5. The CP gene of 11 isolates from P. betuleafolia and P. calleryana showed 88.8% to 100% and 93.8% to 100% sequence identities at nucleotide and amino acid levels, respectively. The phylogenetic tree was generated base on aa sequence of CP gene showed that isolates from this study were clustered into a cluster. Isolates from P. betuleafolia and P. calleryana were presence conserved amino acid sequence (S73-S79-D82-G98).6. The CP gene of ASPV nine isolates from P. betuleafolia and P. calleryana is highly variable, those isolates showed 65.3% to 99.8% and 70.2% to 99.7% sequence identities at nucleotide and amino acid levels, respectively. The nucleotide sequence similarity of MP gene seven isolates from P. betuleafolia, and amino acid sequence similarity of MP gene isolates are 86.5% to 100%,72.5% to 100% and 67.1% to 100%, respectively. The phylogenetic analysis based on aa sequences of CP and MP genes suggested that the phylogenetic positions of ASPV isolates from P. betuleafolia and P. calleryana were no relation to their host origins, but CP gene of ASPV of isolates from other host was correlated to their host origins. Some CP gene of isolates from the same source displayed variable, were grouped into different clusters.