In May of 2004 and 2005, several diseased wheat (Triticum aestivum L.) plants showing extreme dwarfing, various types of yellowing, and reduced or no heading were found in the breeding fields of the Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan, Shanxi Province, China. On the basis of these symptoms, infection with Wheat dwarf virus (WDV) was suspected. Total DNA was extracted from diseased plants with the DNeasy Plant Mini kit (Qiagen, Hilden, Germany). Primers were designed based on the WDV-Enkoping1 genome sequence (NC_003326) (1), including: 245f, 5′-CGACTACGCCTGGCGAACATTTG-3′ (residues 245--267); 806r, 5′-TCTGGCATTGCCTGTTTCGG-3′ (complementary to residues 787--806); 1381f, 5′-CAGTGACATCTTCGCCGGAG-3′ (residues 1381--1400); and, 1886r, 5′-ACTCCGTAAGCCTCGAATCC-3′ (complementary to residues 1867--1886). With primer pairs 245f/806r, 1381f/1886r, 245f/1886r, and 1381f/806r, PCR products of 560, 506, 1642, and 2,275 bp were expected, respectively. After amplification, fragments of the expected sizes were seen on 1% (w/v) agarose gels. The fragments were purified by using a DNA gel extraction kit (TaKaRa, Dalian, China) and cloned into the pGEM-T vector (Promega, Madison, WI). The plasmids were transformed into E. coli strain DH5α and plasmid DNA was isolated from overnight cultures by alkaline lysis. Insert sequences were determined using the dideoxynucleotide chain termination method with an automated sequencer (ABI BigDye 3.1, Applied Biosystems, Foster City, CA). At least three independently isolated clones were analyzed for each PCR product. The compiled 2,750 nt sequence (GenBank Accession No. DQ868525) was 98.1, 98.5, 97.8, and 97.9% identical to WDV-Enkoping1 (NC_003326), WDV-SE (X02869), WDV-B (AM040732), and WDV-F (AM040733), respectively. Therefore, the virus isolate (WDV-TY) was identified as WDV (genus Mastrevirus, family Geminividae). Wheat samples collected from different provinces from 2004-2006 were also infected with WDV as indicated by PCR using the same primer pairs. For Shijiazhuang (Hebei Province), Yangling (Shanaxi Province), Kunming (Yunnan Province), Yuncheng (Shanxi Province), Tianshui (Gansu Province), Gangu (Gansu Province), and Zhenzhou (Henan Province), 13 of 14, 6 of 6, 5 of 5, 4 of 4, 2 of 3, 1 of 2, and 1 of 1 samples were positive, respectively, indicating a broad distribution of WDV in China. To our knowledge, this is the first report of WDV in wheat in China.
Reference: (1) A. Kvarnheden et al. Arch Virol. 147:205, 2002.
