Research On Fast Detection And Elimination Of Major Viruses In Pyrus Pyrifolia

Virus diseases are causing serious losses in pears in north China. The cultivation of virus-free pears is the most effective way to reduce the losses caused by viruses. The viruses' detection is the critical step in the cultivation of virus-free varieties. This thesis conducted some research on the rapid detection and elimination of three major virusesACLSV, ASGV and ASPV in Pyrus pyrifolia. The results are described as following:Eight, two and one sample out often were found to have ACLSV, ASGV and ASPV respectively with the indexing of herbaceous indicators in the green house. The equivalent numbers for indexing of woody indicators were five three and seven respectively. The results indicate that indexing of herbaceous indicators for ACLSV and woody indicators for ASPV and ASGV are more accurate.An improved PAS-ELISA, PAS-ELISA and plate trapped antigen indirect ELISA (PTA-ELISA) methods were applied to detect isolates of ACLSV-C from an apple and ASGV from pears. The results showed that nonspecific reaction could be reduced efficiently and the absorbance ratio of positive and negative samples was enhanced by adding the second antibody and protein A enzyme conjugates simultaneously. Same results were obtained with PAS-ELISA and PTA-ELISA methods. ASGV in pears could be detected with dot immuno-binding assay (Dot-ELISA) after 512 times dilution. It indicates that Dot-ELISA is more sensitive than improved PAS-ELISA. The antibody from an apple isolate ACLSV-C could not be used to detect ACLSV from pears. It indicates that the isolates of ACLSV in pears might be distinguished in serological characteristic with the known ACLSV-C.Specific amplified products were obtained in the detection of ACLSV-C in Chenopodium quinoa by IC/TC -RT-PCR, but the copies obtained by IC-RT-PCR were less than those obtained by TC-RT-PCR. A targeted 499bp fragment was specifically amplified with IC/TC-RT-PCR and RT-PCR in the detection of ASGV-3 in Ch. quinoa. These two improved methods are more convenient and need less material and reagents compared with RT-PCR. Based on this, IC/TC-RT-PCR methods were successfully used to detect ASGV and ACLSV in pears and 499bp fragment was obtained for ASGV. In the same way, copies by IC-RT-PCR were less than TC-RT-PCR in the detection ACLSV. A TC-RT-PCR method basing on reverse transcription with random primer facilitated the detection for those samples mixed infected by ASGV and ACLSV. Meanwhile, we tried to detect ASPV with IC/TC-RT-PCR, but only two samples got the specific 316bp fragment.Four in vitro pears were obtained by tip culture with field samples, two of them mixed infected by ACLSV, ASGV and ASPV were treated by tip culture combined with virus inhibitor. Effects of virus elimination were identified by improved PAS-ELISA, Dot-ELISA and TC-RT-PCR methods. The primary results showed that ASGV and ASPV could be detected in two and one samples of nine subcultures, respectively, ACLSV was found in all samples.Molecular methods that were developed in our research were more reliable than serological methods for ASGV and ACLSV, and results obtained by molecular methods were consistent with indexing of indicators. But the quality of antibody affected the efficiency of IC-RT-PCR obviously.