Identification And Molecular Diagnostic Methods Of The Viruses And Viroids Infecting Chrysanthemum Morifolium

In this study, we focused on the viral diseases on chrysanthemum plants production, completed the census of diseases distribution and investigation for the first time; established multiple RT-PCR detection system; established the Real-time PCR detection system of chrysanthemum virus and viroid;established multiple RT-LAMP detection system of CVB and CSVd; determined two main types of pathogens and genome sequences which were compared the homology analysis.The results as following:1. Investigate the distribution of chrysanthemum virus and viroids disease was carried out and determined the kinds of pathogenic species in China. We got the conclusion that there were five kinds of viruses in chinese cultivation areas, including CVB, TAV, CMV, TMV and PVY, and two viroids in chinese cultivation areas, including CChMVd and CSVd, two kinds of pathogens of TSWV and PVX can't be detected.CVB and TAV were most important virused infected chrysanthemum, infection rate reached 26.55% and 19.27%, the other 3 viruses and 2 viroids infection incidence was relatively low,the infection rate is less than 5%. Thus, chrysanthemum virus and viroid existing certain difference occurred in different areas and distribution. At the same time, this study defines the distribution area of five viruses and two viroids virus in in our country, CVB was widely distributed in various regions of our country; TAV was detected in five areas, including Beijing, Henan, Heilongjiang, Jiangsu and Shaanxi province; PVY was detected in three regions, including Sichuan, Yunnan and Shaanxi province;CSVd was detected in three areas, including Henan, Jiangsu and Guangdong Province; CChMVd was detected in Jiangsu and Sichuan, and the CMV was only detected in Yunnan.2. In the work, a multiplex RT-PCR method using specific primer sets for each virus and viroid that produced seven distinct specific fragments of 218 bp, 354 bp, 399 bp, 425 bp, 593 bp, 634 bp and 852 bp was developed for the simultaneous detection and differentiation of PVY, CSVd, CChMVd,TMV, CMV, CVB and TAV, respectively. The method was validated by testing chrysanthemum samples collected from different regions of China, and showed high levels of reliability and sensitivity. Our results suggest that the new multiplex RT-PCR method has the potential to be used routinely for large-scale virus and viroid surveys.3. In order to detect viruses and viroids of import and export infected chrysanthemum, and developed the Real-time PCR method to detect TAV, CVB, CMV, TMV, CChMVd, CSVd and PVY system, the technique has high sensitivity and specificity, the chrysanthemum is the total RNA concentration 100 fg/uL can still be effective specific detection of these viruses, a new method provided more rapid accurate appraisal to detect chrysanthemum viruses and viroids.4. A multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assay was developed for the simultaneous detection of Chrysanthemum Virus B (CVB) and Chrysanthemum stunt viroid (CSVd), which are the major viral pathogens of chrysanthemum in worldwide. The method was verified by testing chrysanthemums samples showed high level of reliability and sensitivity. The developed mRT-LAMP assay offers an efficient, convenient and rapid tool for screening chrysanthemum virus and viroid, especially for CVB and CSVd, which can be diagnosed in a single reaction.5. The complete nucleotide sequence of Tomato Aspermy Virus (TAV) isolated from chrysanthemum in Henan province (TAV-Henan) was determined. TAV contains a tripartite genome of messenger plus-sense single-stranded RNAs designated RNA1, 2 and 3, The genome of TAV RNA1, 2 and 3 were 3409, 3097 and 2222 nucleotides in length, and contained five open reading frames (ORFs).Sequence analysis showed that the genomic sequence of TAV- Henan shared 98.2-98.6%?95.2-95.8%and 94.0-98.4% nucleotide identity with other TAV sequences reported previously. The complete nucleotide sequence of Chrysanthemum virus B (CVB) isolated from chrysanthemum in Beijing(CVB-Beiing) was determined. CVB contains single-stranded RNA genome of 8965 nucleotides in length, and contained six open reading frames (ORFs). Sequence analysis showed that the genomic sequence of CVB-Beiing shared 67.41-89.1% nucleotide identity with other CVB sequences reported previously.