Primarily Study On Detection And Seed Transmission Of Pepper Mild Mottle Virus

Pepper mild mottle virus (PMMoV) is a kind of seed transmission virus. Because of the characteristic of seed transmission, it can keep for long time and spread over long distances. Plants infected by PMMoV will appear the sysptoms including of inhibition of growth, petal fallen, and fruit decrescent. The virus is harmful to pepper production. PMMoV can survive for a long time and transmit through the seed for long distance. In this study, PMMoV is detected by extraction dsRNA and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), and the characteristic of seed transmission is also analyzed.1We extracted specific dsRNA fragments of PMMoV with modified dsRNA extraction method. We get purified dsRNA and reduce the loss of dsRNA by recovery of gel.2DOP-PCR systenm was developed by using dsRNA as a template. Because of the characteristics of dsRNA content was low and difficult to extract, the study would change the once conventional DOP-PCR reaction into many times, and amplify the positive bands with a small amount of dsRNA. The products were cloned and sequenced, and two PMMoV positive clones were respectively named DOP-PMMoV1and DOP-PMMoV2. BLAST results of DOP-PMMoV1in GenBank showed that the length of DOP-PMMoV1sequence was357nt and located in PMMoV coat protein gene which had a homology of100%with sequences of GenBank accession number AY859497, AB000709, AB069853, AB113117,AB113116, M81413, and AJ308228. DOP-PMMoV1sequence had the minimum homology of96%with sequences of GenBank accession number JN540023of PMMoV isolates. The length of DOP-PMMoV2sequence was502nt and also located in PMMoV coat protein gene. DOP-PMMoV1sequence had a homology of93%with sequences of GenBank accession number AY859497, AB000709,AB069853,AB113117,AB113116, and M81413.3Using modified CTAB method, total nucleic acid was ectracted from pepper leaves and seeds. Using PMMoV specific primers, PMMoV was detected by RT-PCR. Different primer combinations amplified the expected fragments which were testified by cloned and sequenced. The detection sensitivity of PMMoV using leaves as the sample was higher than that using seed as the sample. In this study, RT-PCR system can be effective, high sensitivity to detect the virus in the seeds. The high-sensitivity detection system for seed inspection and control of PMMoV by seed dispersal were great significance.4Separated the viral infection seed with tweezers and dissecting needle, PMMoV was detected by PCR in the seed surface, the seed coat, endosperm and embryo by, respectively. The results showed that the seed coat and endosperm contained large amounts of viruses, PMMoV could also be detected in the seed surface. PMMoV was not detected in the embryo. 5PMMoV infection statuses were analyzed in commercial seed by RT-PCR technology. The results showed that the seed of Beijing region had high rate of PMMoV infection, the seed of Shenyang had low rate of PMMoV infection, and the seed of Harbin had lowest rates of infection. PMMoV could be detected in the seeds that originated from Hebei,Shanxi, Netherlands, Lu Chang, and Beijing. All seeds originated from Beijing were infected by PMMoV. In the seed, PMMoV was also found from a few areas of Heilongjiang and Shanxi. The seed of Mongolia and Gansu did not find PMMoV-positave samples. Despite the current virus spread mediator in seed production and inspection process had been strict controled, but there was still part of the seed infected by PMMoV, indicating that the strengthening of the virus seed-borne still more urgent.6Small-scale hybridization experiments showed that if the female parent was infected by PMMoV, regardless of the male parent was infected by PMMoV, the seeds contained PMMoV. Female parent was not infected by PMMoV. regardless of the male parent was infected by PMMoV, then seeds did not contain PMMoV. Thus, the decision factor of the seeds with PMMoV was the female parent.