Identification Of Pathogens Associated With Mulberry Mosaic Dwarf Disease

Mulberry mosaic dwarf disease,a major disease in mulberry in the past decades, seriously influence the development of silkworm industry. Infected mulberry plants show symptoms like dwarf, mosaic, leaf deformation, crinkling, leaf margins curling. Although studies on this disease have lasted for nearly a century, its pathogen is not yet clear. In view of this, the purpose of this study is to identify the pathogens associated with mulberry mosaic dwarf disease.Initially, a total of 101 mulberry samples were collected from sericultural scientific research base of Ankang University in Shaanxi Province. And 92 of them show typical symptoms of mulberry mosaic dwarf disease and 9 samples are symptomless. Geminivirus, nepovirus and mulberry small circular RNA(msc RNA), were detected from one symptomatic sample and no pathogens were detected from one healthy sample using high-throughput sequencing. RT-PCR and PCR were performed to confirm the existence of the three pathogens in the infected sample.Secondly, incidence of three pathogens in all infected samples were surveyed by RT-PCR、PCR and southern blot hybridization. The results showed the incidences of geminiviruses, nepovirus and msc RNA were 92.4%, 52.17% and 40.2%, respectively. Therefore, the results revealed a close association of the geminivirus with diseased mulberry trees. However, a relative high incidence of nepovirus indicated the probable mixed infection between geminivirus and nepovirus. Msc RNA is not responsible for the disease because we detected the existence of msc RNA from healthy mulberry samples. Although our experiments have confirmed the close relationship between geminivirus and mosaic dwarf disease, bioassay experiments are still needed according to Koch’s postulates to determine the pathogen ultimately.Finally, the geminivirus associated with mulberry mosaic dwarf disease was identified and tentatively named Mulberry mosaic dwarf associated virus(MMDa V). The full length genomic sequence of the geminivirus were cloned by PCR and RCA(rolling circle amplification) methods. Sequencing results showed that this geminivirus has a monopartite circular DNA genome(2952 nt). Aside from the conservative geminiviruses genome structure(stem-loop, TATA box, and nonanucleotide TAATATTAC), the number and location of ORF(open reading frame) are different from other geminiviruses ORFs. Viral strand contains 5 ORFs and complementary strand contains 2 ORFs, and the genome organization is unique among all the other genome structures belonging to geminiviruses. One intron(101 nt) was predicted on the complementary strand using software, which was followed by RT-PCR validation. Moreover, a total of 136 small RNA reads spanning the exon-exon junctions predicted for the spliced Rep transcripts were also identified. These findings provide additional indirect evidence that unspliced and spliced transcripts are actually generated in vivo. Based on the judgement of sequence features, the intron belonged to type U2 introns. Phylogenetic analyses showed that Mulberry mosaic dwarf associated virus(MMDa V)and Citrus chlorotic dwarf associated virus(CCDa V)clustered together in the same branch, clearly separated from all the other geminiviruses. Therefore, MMDa V and CCDa V appear to be closely related to each other, thus supporting their inclusion in a putative novel genus in the family Geminiviridae.Due to the limitations of traditional detection methods, pathogens of mulberry mosaic dwarf disease have not been clear yet. Innovation of this thesis is that we break the “Bottleneck” of traditional detection technology by using high-throughput sequencing technology. This technology can detect all the viral pathogens existing in infected samples, and we, subsequently, will find the virus which is associated with this disease by incidence survey of all candidate pathogens. Bioassay are performing to identify the pathogenicity of MMDa V according to Koch’s postulate. Eventually, all the above results will uncover the real pathogen of mulberry mosaic dwarf disease and we will solve the crucial scientific problem.