Systemic foliar chlorosis of melon, watermelon, and cucumber plants grown in plastichouses was first observed in Shanghai, China in 2008. By the end of October 2009, this symptom had become prevalent across 13,000 ha of plastichouses in Shanghai, Ningbo in Zhejiang Province, and Shouguang in Shandong Province. By mid-October, disease incidence ranged from 50 to 100% and losses were estimated between 10 and 20% across 100 plastichouses. Initial disease symptoms were chlorosis beginning at the base and middle portion of the older leaves followed by development of chlorotic spots on the lamina. Within 4 to 5 days, the entire leaf lamina was bright yellow and the veins remained green. The whitefly, Bemisia tabaci, was frequently observed colonizing plants in all plastichouses included in this study. Leaf samples were collected from six symptomatic cucumber, melon, and watermelon plants from individual plastichouses in Shanghai, Ningbo, and Shouguang. A pair of degenerate primers, F-5′-GGN TTA GAN TTC GGN ACN AC-3′ and R-5′-TCA AAN GTN CCN CCN CCN AA-3′, that were specific for the genera Crinivirus and Closterovirus, family Closteroviridae (2) were used to amplify a 636-bp fragment of the viral heat shock protein 70 gene (Hsp70). A PCR product of the expected size was amplified from RNA extracted with TaKaRa RNAiso Reagent (TaKaRa, Dalian, China) from symptomatic leaf samples: 3 of 3 melon, 2 of 2 watermelon, and 1 of 1 cucumber, and from 5 of 5 Bemisia tabaci adults collected from plants in five plastichouses in Shanghai, Ningbo, and Shouguang. No PCR product was obtained from RNA extracted from cucumber leaves grown in a virus-free facility at the Fruit Research Institute, Zhengzhou. PCR products were sequenced from representative plants samples and the sequences were submitted to GenBank (Nos. GU721105 to GU721107, GU72118 to GU721110, and GU721111. The six Hsp70 sequences shared 99.8 to 100% identity with Cucurbit chlorotic yellows virus (CCYV) (GenBank No. AB523789) from Japan. Using the complete CCYV sequence (1), PCR primers were designed to amplify the complete CCYV coat protein (Cp): Cp F-5′-CGCAATCAATAAGGCGGCGACC-3′ and Cp R-5′-ACTACAACCTCCCGGTGCCAACT-3′ (804 bp), minor Cp (Cpm): Cpm-F-5′-TGATGAANTGCCANGCTNTGAAA-3′ and Cpm-R5′-ACAANTGATTCACATTNACAAT-3′ (1,632 bp); and Hsp70: Hsp F-5′-TGCAACCGATGTCAGGTTCAGCG-3′ with Hsp R-5′-TGGATAATTGGTCACGACCTCCAGT-3′ (1,947 bp). One PCR amplicon was obtained for each target gene using RNA extracted from a cucumber collected in Ningbo. Three of the PCR amplicons were cloned and the DNA sequence was determined. A representative sequence for each gene was deposited in GenBank as: cp (HM581658), cpm (HM581657), and hsp70 (HM581659). The cp, cpm, and hsp70 sequences shared 99.7, 99.9, and 99.9% nt identity with the respective genes of CCYV (AB523789), whereas they shared only 62.5, 49.9, and 69.6% identity with the respective gene sequences for Cucurbit yellow stunting disorder virus (CYSDV; NC004810), suggesting the two viruses are divergent crinivirus species. Although this virus was first reported to infect cucurbits in Japan in 2009 (1), to our knowledge, this is the first report of CCYV in China. Eradication and management efforts are therefore paramount to reducing the spread of the disease. References: (1) M. Okuda et al. Phytopathology 100:560, 2010. (2) T. Tian et al. Phytopathology 86:1167, 1996.
A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches
