RT-PCR Detection And Sequence Analysis For Virus And Viroid Inducing Spot Symptoms

Grapevine fleck or Grapevine yellow speckle diseases caused by Grapevine fleck virus (GFkV) and Grapevine yellow speckle viroids (GYSVds) ubiquitous occurred in grapevine production areas and causes important economic loss. GFkV is the type species of genus Maculavirus in family Tymoviridae. GYSVds, mainly including GYSVd-1 and GYSVd-2, are the members of genus Apscarviroid in family Pospiviroidae. The grapevine yellow speckle disease caused by these two viroids is the only known grapevine disease caused by viroid. In order to realize the infection features of these pathogens in grapevine grown in China, RT-PCR was performed for the detection of GFkV and GYSVd and their sequences were analyzed in this study. The obtained results provided valuable information for the establishment of effective control measures and detection systems. Results are as follows:1. Totally,46 grapevine samples collected from Fruit Research Institute in Zhengzhou, Institute of Fruit and Tea Science in Hubei and Huazhong Agricultural University in 2004-2010 were used for the detection of GFkV by RT-PCR. Results showed that 21 samples (the sample numbers from the mentioned three areas were three, 17 and one, respectively) were positive for GFkV, accounting 45.7% of detected samples. The amplified products from six samples were cloned and sequenced. The results showed that the size of amplified products was 179 bp. Sequence analysis revealed that these cloned gene fragments share 96.6%-100% of nucleotide similarities with each other, 96.6%-98.9% and 95.0%-98.3% similarities with the reported sequences from Italy (GeneBank accession no. AJ309022) and America (GeneBank accession no. GU372372-4), respectively.2. The viral RNAs were extracted from buds and phloem tissues of the dormant canes of grapevine cv. Shengbao, Victoria and Centennial Seedless by magnetic nano-particles (MNP). The effects of plant tissues on the detection efficiency of GFkV by RT-PCR were analyzed. Results showed that buds were suitable for used as detection materials, from which expected products were amplified, and the phloem tissues from the same dormant grapevine canes sometimes give a negative result. The TaqMan probe and primers were designed based on the obtained sequences. The MNP combined with quantitative RT-PCR (qRT-PCR) method was primarily established for the detection of GFkV. The results showed that the qRT-PCR was more sensitive than RT-PCR and it could detect the GFkV in buds and phloem tissues of the dormant grapevine canes. GFkV titers were relatively higher in Centennial Seedless than in the other two samples. Results also confirmed that the GFkV titer in dormant buds was 10 times higher than that in phloem. The results also showed that the designed TaqMan probe had good specificificity.3. RT-PCR using the reported degenerate primers of GYSVds was employed for the detection of GYSVd-1 and GYSVd-2. Amplicons with expected size were obtained from grapevine cv. White Rose and Yan73. Those products were cloned and sequenced. Results showed that the size of amplified products was 366-367 bp covering the full length of the viroid. There were some nucleotide diversities between two isolates and within clones from the same isolate. Four clones Z1-24 of products from White rose shared the highest similarities with the GYSVd-1'Type 2'variant (GenBank accession no. Z17225), belonging to symptom-inducing variant. Two clones from Yan73 shared the highest similarities with the GYSVd-1'Type 1'variants (GenBank accession no. X87913), belonging to the non-symptom inducing variant. The nucleotide variations of clones Z1-Z4 mainly occurred in Pathogenic region and Central conserved region when their sequences were compared with Z17225. The effects of plant tissues on the detection efficiency of GYSVd-1 by RT-PCR were analyzed. Results showed that leaves were the best materials used for the detection of GYSVd-1, and its efficiency decreased with phloem tissues and petiole.