M. D. Lyu,
M. J. Li,
X. M. Li, and
Y.-Q. Cheng, Department of Pomology/Laboratory of Stress Physiology and Molecular Biology for Tree Fruits, Key Laboratory of Beijing Municipality, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
Grapevine leafroll disease (GLD) is one of the most economically important diseases of cultivated grapevines (Vitis vinifera), causing decrease in yield, as well as decreasing the sugar levels and increasing the acidity of the berries (1). There are currently at least 10 serologically distinct viruses, referred to as grapevine leafroll-associated viruses (GLRaVs), from the family Closteroviridae that are associated with leafroll disease (4). China is one of the world's leading grape producers, and nearly 75% of the vineyards in China are located in Xinjiang Uygur Autonomous Region, and Hebei, Shandong, Gansu, Ningxia, and Yunnan provinces. Grapevine leafroll-associated virus 7 (GLRaV-7) isolates have been reported so far in Liaoning (GQ849392, GQ849393, and JF927943) and Henan (EF093187) provinces in China (3). The four Chinese isolates were isolated respectively from grape varieties, Cabernet Sauvignon (GQ849392, GQ849393), Centennial Seedless (JF927943), and Semillon (EF093187), and these grape varieties are introduced from abroad. Cow's Nipple and Dragon's Eye are old grape varieties native to China. Cow's nipple is extensively cultivated in Xinjiang Uygur Autonomous Region, while Dragon's Eye is widely planted in Heibei Province. To determine if GLRaV-7 was present in these two varieties, six samples (three per variety) were collected from six individual grapevines showing GLD-like symptoms in two vineyards in Xinjiang Uygur Autonomous Region and Hebei Province, respectively, in September 2011. Total RNA extracts obtained from phloem scrapings of samples using the RNeasy plant mini kit (QIAGEN) were tested by reverse transcription (RT)-PCR with primers F1 (5′-TATATCCCAACGGAGATGGC-3′) and R1 (5′-ATGTTCCTCCACCAAAATCG-3′) (2) specific to the heat shock protein 70 homologue (HSP-70 gene) of GLRaV-7. All samples produced a single band of the expected size of 502 bp. One GLRaV-7-specific amplicon per variety was cloned into pMD 18-T simple vector (TaKaRa). Plasmid DNA was purified using Column Plasmid DNAOUT (TIANDZ, Beijing, China) from three individual clones and sequenced from both directions. The sequence of the two isolates (GenBank Accession Nos. JX494722 and JX494723) shared 97.81% identity at the nucleotide level and 100% identity at the amino acid level. A pairwise comparison of HSP-70 sequences of the two isolates from this report with nine corresponding sequences of GLRaV-7 isolates (including four previously reported Chinese isolates) showed nucleotide sequence identities ranging from 91.24% (EF093187) to 98.80% (GQ849392). These samples were further analyzed by double antibody sandwich (DAS)-ELISA using antibody specific to GLRaV-7 (NEOGEN Europe, Ayr, Scotland) according to the manufacturer's instructions, and the results confirmed the presence of the virus in these samples that were positive by RT-PCR. To our knowledge, this is the first report of GLRaV-7 occurring in native grape varieties in China. These results could be helpful in developing sound diagnostic systems for implementing efficient disease management strategies.
References: (1) B. Akbas et al. Hort. Sci. 36:97, 2009. (2) E. Engel et al. Plant Dis. 92:1252, 2008. (3) X. Fan et al. Acta Hortic. Sinica 39:949, 2012. (4) G. P. Martelli. Extended Abstr. 16th Meet. International Council for the Study of Virus and Virus-like Diseases of Grapevines (ICVG). 15-23, 2009.