Molecular Identification And Detection Methods Of Pepper Mild Mottle Viruses & Cucumber Mosaic Viruses

Sweet( hot)pepper were wildly cultivated all over the world and it was one of most important vegetables.General speaking,the losses caused by pepper virus account for 25~50 percent of total yield ,but total yield can be lost when the disease was severe.The pepper virus threatened the cultivation of pepper in Beijing,Baoding and Ningxia,so some sample were collected to identify the viruses by serological methods.The following problems were studied and discussed.Six plant viruses (TMV,CMV,PMMV,AMV,ACMV,PVY)were detected by ELISA kit.The detection results show Ningxia and Baoding pepper virus disease were mainly caused by CMV, PMMV respectively and Beijing pepper virus disease were mainly caused by PMMV, CMV.The host range experiment show PMMV systemically infected Capsicum annuum, Capsicum frutescens, Gomphrena globosa, Petunia hybrida,but it doesn't infect Cucumis sativus, Lycopersicon esculentum, Brassica pekinensis, Cucumis melo, Lactuca sativa and Momordica charantia.When Datura stramonium , Chenopodium amaranticolor , Chenopodium quinoa , Nicotiana tabacum, N. glutinosa, N.tabacum cv. Xanthic nc were Inoculated , local lesion were only found in the inoculated leaves. The 16 pepper varietie was inoculated with PMMoV in greenhouse and all the tested varieties were susceptible to PMMV.PMMV was purified with the procedure including PEG precipitation and differential centrifugation.Lots of Rod-shaped particles typical of Tobamovirus were seen by electron microscopy. It was showed that the coat protein of the virion consisted of only one subunit with the molecular weight of 20 KD by SDS-PAGE.Rabbit was immunized using the purified virus and the antiserum was obtained after five immune. Double sandwich enzyme-linked immunosorbent assay showed the antiserum had titer of 1:32768 with high specificity to PMMV.The cDNA of PMMV was obtained by RT-PCR, after the total plant RNA was extracted. The coat protein gene,move protein gene and replication associated protein gene were amplified by three paires of specific primers.lt was suggested that PMMV -Bei (beijing) and PMMV -Bao (baoding) are the same strain and they are most related to the Japan isolation (AB000709) according to the nucleotide sequece analysis .The serological methods and Nucleotide sequence analysis of the coat protein gene show Ningxia and Beijing isolate both belong to CMV subgroupl. Ningxia isolate was most closet to AJ271416 and Beijing isolate was most closet to AF127977.The percentage of seed transmission of PMMV and CMV is almost 100%,but the transmission of PMMV and CMV from seed into seedling has not found so far by DAS-ELISA assay.The high titer antiserum and nucleotide sequence of PMMV and CMV coat protein...