The Detection Of PMMoV And Study On The Related Elements Causing Symptom Difference Between PMMoV And TMV

Pepper mild mottle virus is a new emerging Tobamovirus disease on capsicum plant in the vicinity of Beijing in recent years. In order to control the damage and prevent the disease spreading。A digoxigenin-labelled cDNA probe PM, complementary to the regions between coat protein (CP) gene and 3'untranslated sequence of PMMoV genome, has been developed to detect the virus by dot-blot hybridization method. The specificity and sensitivity has been tested. The detection limit of the method was equivalent to 0.8μg fresh tissues infected by PMMoV, compared with DAS-ELISA and RT-PCR which was 39.06μg and 0.008μg detection limit respectively. The sensitivity of the dot-blot hybridization was higher than that of DAS-ELISA and lower than that of RT-PCR. 111 pepper samples including 93 field samples collected from pepper fields of Beijing and Baoding during 2006 and 18 commercial seed samples had been evaluated by dot-blot hybridization using this probe. The result has been identified by RT-PCR. The high sensitivity and reliability of the molecular hybridization assay described here, would provide an important alternative method for the detection of PMMoV in large-scale.In order to study the virus deeply, the infectious cDNA clone of PMMoV-CN, pMQC has been constructed. The completed genome sequence of the virus was constructed into a suitable vector by three steps. T7 promoter has been introduced into the 5'end point and two G also introduced in order to increase the efficiency of its in vitro transcription. The restricted enzyme site SmaI has been introduced into the 3'end point of the genome sequence in order to linearize the vector in the latter experiment. The pMD18-T simple vector was applied in constructing infectious cDNA clone, which has no any enzyme site, so we can introduce restriction enzyme site in primer by PCR according to the enzyme sites in the genome sequence of PMMoV-CN. The infectious cDNA clone was linearized and in vitro transcribed. The in vitro transcription product was inoculated onto three host plants, Chenopodium Amaranticolor, Chenopodium quinoa and Petunia hybrida. The infectivity of the infectious cDNA clone had been tested by biology method including RT-PCR, western blot and digoxigenin-labelled probe. The infectious cDNA clone of PMMoV-CN will provide an effective method to study the pathogenicity mechanism and gene function of the virus.In order to make sure the pathogenicity related elements Tobamovirus, two recombinant infectious cDNA clones have been constructed, pM-T-CP and pM-To-CP. The CP nucleotide sequence of pMQC was exchanged by that of TMV and ToMV respectively. The pathogenicity of pM-T-CP and pM-To-CP were tested on Petunia hybrida and Chenopodium amaranticolor. The result showed that pM-To-CP had no pathogenicity and pM-T-CP presented systematically symptom on Petunia hybrida, while PMMoV infected Petunia hybrida locally, so we suggested that TMV CP may help the virus move in long-distance in Petunia hybrida plant. The two recombinant infectious cDNA clones were also inoculated onto Chenopodium amaranticolor. pM-To-CP still had no pathogenicity and pM-T-CP infected Chenopodium amaranticolor showing local mottle symptom, which was as same as PMMoV-CN. So we suggested that the CP of PMMoV-CN may not play an important role in the symptom conformation of PMMoV-CN infecting Chenopodium amaranticolor and TMV CP may not be the avr gene in TMV infecting Chenopodium amaranticolor to show local necrotic lesion.In order to determine which sequence difference between TMV-U1 CP and PMMoV-CN CP plays an important role in causing their different symptom on Petunia hybrida, two recombinant infectious cDNA clones, including pM-T-CP1, pM-T-CP2 had been constructed. The two recombinant vectors including TMV CP sequence corresponding to 1-117,1-237 site in the PMMoV genome sequence. The pathogenicity of the two recombinant vectors had been tested by biology method. The result showed that both of them showed locally symptom on Petunia hybrida, which meaned that the difference of 1-117 and 1-237 sequence of CP were not related to the symptom difference between PMMoV-CN and TMV-U1 on Petunia hybrida. So the other sequence of CP will be exchanged to study the related elements causing different symptom between PMMoV and TMV on Petunia hybrida in the future.