Detection And Identification Of Two Geminiviruses And The Associate Satellite DNA Infecting Tomato Plants And Development Of A Multiplex PCR Assay System

Geminiviruses constitute a large family of plant viruses with twin single-stranded circular DNA genomes. They occurred worldwide, and have devastating harm on crops in many countries. Since the 1990s, in Yunnan, Guangdong, Guangxi, Hainan and Fujian provinces of China, tomato, tobacco, papaya and other crops have been found to be infected by a variety of geminiviruses. This research investigated the distribution of Tomato yellow leaf curl virus (TYLCV) and Papaya leaf curl China virus (PaLCuCNV) in Shandong and Henan provinces and confirmed satellites associating with TYLCV.With total DNA isolated from diseased samples and the primers PA/PB specific to geminiviruses genome, a 535 bp fragment was obtained by PCR amplification. We also obtainted 16 complete genome sequences of TYLCV in samples collected from three cities of Henan province and five cities of Shandong province using primers (TY1-F/R, TY2-F/R, TY3-F/R) flanking full length of TYLCV. These nucleotide sequences of the fragment have the highest identity (99.1%) with that of Tomato yellow leaf curl virus isolate SDWF-TYLCV. And these isolates all cluster to TYLCV Israel strain, suggesting they belong to TYLCV Israel strain.We then amplified specific 2749 bp fragment using primers Fxyw-F/Fxyw-R of PaLCuCNV. In Zhengzhou, Jiaozuo, Anyang cities of Henan province, we screened seven positive samples infected by TYLCV and PaLCuCNV. When we detected these samples with universal primers ?01/?02 of geminiviral betasatellite, approximately 1.3 kb specific bands of DNAP could be amplified from those susceptible samples collected from Jiaozuo city of Henan province. Through BLAST and sequence alignment, we found that the amplified sequence was 89.7% identity with Tomato yellow leaf curl Vietnam betasatellite (TYLCVNB). This result confirmed that TYLCV was associated betasatellite in tomato planting area in Henan province.This research established a single and duplex PCR detection system of PaLCuCNV, TYLCV, TYLCVNB. The sensitivity of each system was determined. Results of the single PCR showed that the lowest concentration detecting the plasmid is 1 ng/mL for PaLCuCNV,1 ?g/mL for TYLCV,1 ng/mL for TYLCVNB. The lowest concentration for them in the duplex PCR is the same as that in the single PCR.