First Report of Cucurbit chlorotic yellows virus Infecting Cucumber, Melon, and Squash in Iran

Yellowing diseases of field- and greenhouse-grown cucurbits are becoming increasingly important in many cucurbit cultivation areas in Iran. Virus surveys were conducted from 2011 to 2012 in greenhouse-grown cucumber (Cucumis sativus L.) and field-cultivated cucumber, squash (Cucurbita sp.) and melon (Cucumis melo L.) in Tehran, Semnan, Bushehr, Hormozgan, Isfahan, Yazd, and Fars provinces, the major cucurbit-growing areas in Iran. Leaf samples with various symptoms, e.g., chlorosis, interveinal chlorotic spots on lower leaves, bright yellow color or sever yellowing on older leaves, were collected and screened for the presence of the whitefly transmitted criniviruses (family Closteroviridae) Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) through double-antibody sandwich (DAS)-ELISA, using CCYV and CYSDV specific antisera (DSMZ, Germany). The ELISA results showed that of 347 cucumber leaf samples originating from cucumber greenhouses, 170 and 65 were positive for CCYV and CYSDV, respectively, and 45 samples were infected with both viruses. In addition, of 147 leaf samples collected from melon, cucumber, and squash grown in open fields, 57 and 53 were infected with CCYV and CYSDV, respectively, and 14 were infected with both viruses. These results indicate that these two viruses are widely distributed on these cucurbit crops in Iran. CCYV was not detected in Bushehr and CYSDV was not detected in Isfahan and Hormozgan provinces. To confirm the presence of CCYV and CYSDV, total RNA was extracted (Sigma Chemical, St. Louis, MO) from 18 samples that reacted positive in DAS-ELISA originating from different surveyed provinces. RT-PCR was carried out using specific primers Crini-s2 (5'-CATTCCTACCTGTTTAGCCA-3') (2) and Crini-as1 (5'-ATCCTTCGCAGTGAAAAACC-3') to amplify a 460-bp fragment of the HSP70 gene and CCYV using specific primers CCYV-HSP-F1 (5'-TGCGTATGTCAATGGTGTTATG-3') and CCYV-HSP-R1 (5'-ATCCTTCGCAGTGAAAAACC-3') to amplify a 462-bp fragment of the HSP70 gene (latter 3 primers from [3]). Expected DNA fragments for CYSDV and CCYV were amplified from 11 (CCYV 7/11, CYSDV 4/11) of 18 samples but not from any of the healthy controls. Further analysis by sequencing three selected PCR products amplified with primers CCYV-HSP-F1/R1 showed complete consensus among the sequences, and in comparison with sequences available at GenBank, the highest identities were obtained to Asian CCYV isolates (94% nt/98% aa identity). The C