New Disease Reports
New Disease Reports (2017) 36, 6. [http://dx.doi.org/10.5197/j.2044-0588.2017.036.006]
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First report of Grapevine yellow speckle viroid-2 infecting grapevine (Vitis vinifera) in Thailand

P. Tangkanchanapas 1,2*, K. Reanwarakorn 3, H. Juenak 4 and K. De Jonghe 2

*Parichate.Tangkanchanapas@ilvo.vlaanderen.be

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Received: 26 Apr 2017; Published: 08 Aug 2017

Keywords: grapevine viroid disease 

Two viroids, Hop stunt viroid (HSVd) (Rakdang, 2001) and Grapevine yellow speckle viroid-1 (GYSVd-1) (Hannok & Reanwarakorn, 2005) have been reported to infect grapevine in Thailand. From February through March 2014, 22 grapevine leaf samples (Vitis vinifera cvs. Black Opal seedless, Black Queen and Pok Dum) were sampled from eight vineyards in Saraburi (4 samples) and Nakhon Ratchasima (18 samples) provinces, which are the major grapevine plantation regions in Thailand. Various symptoms were found, including leaf roll with red colour, leaf malformation, vein banding and yellowing, yellow spots, and yellow flecks (speckle) (Figs. 1-2). 

Total RNA from the 22 samples was extracted using a CTAB method (Doyle & Doyle, 1987) and subjected to RT-PCR for the detection of viroids known to occur in grapevine. For the detection of HSVd and Citrus exocortis viroid (CEVd), the methods of Shamloul et al. (2002) and Gross et al. (1982), respectively, were used. Additionally, two primer sets were designed to specifically detect the full-length genome of GYSPVd-1 [c-GYSVd1: 5′-CGAGGCTCACTCCCCCTCTGCC-3′ / h-GYSVd1: 5′-TCGTCGACGAAGGGGTGCACTCC-3′], and -2 [c-GYSVd2: 5′-GGTCCGCGGAGGCCTTCCGAGG-3′ / h-GYSVd2: 5′-TGCAGAGAAAAGAAGAAGGGCCCAG-3′] (Tangkanchanapas et al., 2015). The PCR products were cloned and two or three clones per sample were sequenced in one direction (First BASE Laboratories, Malaysia). 

The results revealed that 3/22 and 6/22 grapevine leaf samples were infected solely with GYSVd-1 and GYSVd-2, respectively. Five of the 22 samples had mixed GYSVd-1 and -2 infections. All GYSVd-1 and GYSVd -2 positive leaf samples showed only yellow spots and yellow speckles, yet none of the other symptoms. HSVd and CEVd were not detected in any sample. The genome variants ranged in size from 353-389 nucleotides for GYSVd-1 and 362-365 nucleotides for GYSVd-2. All individual sequences were submitted to GenBank (Accession Nos. KP010005-KP010012 for GYSVd-1 and KP010013-KP010023 for GYSVd-2). A nucleotide BLAST analysis using the GYSVd-1 and GYSVd-2 sequences showed that isolates from this study shared 93-99% and 98-99% similarity with corresponding GYSVd-1 (AF059712, DQ371471, GQ995468, X87913 and X87920) and GYSVd-2 isolates (FJ490172, FJ597943 and JQ686716), respectively. 

For phylogenetic analysis, maximum-likelihood method with 500 bootstrap replicates and a 50% cut-off value was used (MEGA version 7.0.21). For GYSVd-1 the Thai isolates cluster in two distinct phylogenic sub-clades indicating genetic variation, which cannot be linked to the geographic origin of sampling (Fig. 3). The Thai GYSVd-2 isolates formed a single clade indicating less variation compared to GYSVd-1. In addition, all of the GYSVd-1 and 2 infections were only found in Nakhon Ratchasima province, yet the viroids were found in all major grapevine cultivation sites in this province. However, disease incidence in all vineyards is very low (<1%). To our knowledge, this is the first report of natural GYSVd-2 infection in grapevine in Thailand.

Figure1+
Figure 1: Yellow speckle symptoms on grapevine leaves in which Grapevine yellow speckle viroid-1 and -2 were identified by RT-PCR and sequencing.
Figure 1: Yellow speckle symptoms on grapevine leaves in which Grapevine yellow speckle viroid-1 and -2 were identified by RT-PCR and sequencing.
Figure2+
Figure 2: Yellow spot symptoms on grapevine leaves in which Grapevine yellow speckle viroid-1 and -2 were identified by RT-PCR and sequencing.
Figure 2: Yellow spot symptoms on grapevine leaves in which Grapevine yellow speckle viroid-1 and -2 were identified by RT-PCR and sequencing.
Figure3+
Figure 3: Maximum-likelihood phylogenetic tree analysis of sequences of all Thai Grapevine yellow speckle viroid-1 and -2 isolates (indicated with blue and green dots, respectively) and those from other countries. The tree was constructed using MEGA version 7.0.21 with 500 bootstrap replicates and 50% cut-off value. Pepper chat fruit viroid (KM214229) was used as an outgroup.
Figure 3: Maximum-likelihood phylogenetic tree analysis of sequences of all Thai Grapevine yellow speckle viroid-1 and -2 isolates (indicated with blue and green dots, respectively) and those from other countries. The tree was constructed using MEGA version 7.0.21 with 500 bootstrap replicates and 50% cut-off value. Pepper chat fruit viroid (KM214229) was used as an outgroup.

References

  1. Doyle JJ, Doyle JL, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19, 11-15.
  2. Gross HJ, Krupp G, Domdey H, Raba M, Jank P, Lossow C, Alberty H, Sänger HL, Ramm K, 1982. Nucleotide sequence and secondary structure of Citrus exocortis and Chrysanthemum stunt viroid. European Journal of Biochemistry 121, 249-257. [http://dx.doi.org/10.1111/j.1432-1033.1982.tb05779.x]
  3. Hannok P, Reanwarakorn K, 2005. cDNA probe for Grapevine yellow speckle viroid detection. Kasetsart Journal (Natural Science) 39, 46-52.
  4. Rakdang W, 2001. Detection of Grapevine Viroid in Thailand. Nakorn Pathom, Thailand: Kasetsart University, Master's Thesis.
  5. Shamloul AM, Faggioli F, Keith JM, Hadidi A, 2002. A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. Journal of Virological Methods 105, 115-121. [http://dx.doi.org/10.1016/S0166-0934(02)00090-3]
  6. Tangkanchanapas P, Juenak H, Saelor N, Noochoo S, Reanwarakorn K, 2015. Development of detection technique for Grapevine yellow speckle viroid 1 and 2 (GYSVd-1 and 2) causing grapevine yellow speckle disease by RT-PCR method. Thai Agricultural Research Journal 33, 68-84.
To cite this report: Tangkanchanapas P, Reanwarakorn K, Juenak H, De Jonghe K, 2017. First report of Grapevine yellow speckle viroid-2 infecting grapevine (Vitis vinifera) in Thailand. New Disease Reports 36, 6. [http://dx.doi.org/10.5197/j.2044-0588.2017.036.006]

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