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 CCYV sequences were deposited in GenBank under accessions KC559449 to KC559451. The identity of the amplified CYSDV DNA could also be confirmed by sequencing of three PCR products. CCYV has first been proven to occur in different countries in East Asia and has recently been reported from Sudan (2) and Lebanon (1), indicating the putative spread of the virus wherever cucurbits are grown and its vector, the whitefly Bemisia tabaci, is present. Large populations of whiteflies were present in all surveyed areas. However, to our knowledge, this is the first report for the occurrence of CCYV in Iran. In conclusion, the presence of CCYV and CYSDV in the major cucurbit growing provinces and the large whitefly population pose a serious threat to cucurbit production in Iran. References: (1) P. E. Abrahamian et al. Plant Dis. 96:1704, 2012. (2) K. Hamed et al. Plant Dis. 95:1321, 2011. (3) R. Zeng et al. Plant Dis. 95:354, 2011.

Zucchini yellow mosaic virus (ZYMV; family Potyviridae, genus Potyvirus) causes high yield losses to cucurbits in many parts of the world. The virus was detected for the first time in Iran in 1988, but the isolates have not been characterized. To study the genetic and biological diversity among Iranian ZYMV isolates, a set of twelve isolates, obtained during an extensive survey conducted from 2003 to 2006 in the major cucurbitgrowing areas, were characterized. An experimental host range study of these isolates (referred as Iran-1 to Iran-12) revealed some variation in their biological properties. The nucleotide sequences of the genomic portion spanning the C-terminal part of NIb and N-terminal part of coat protein (CP) coding region were determined and compared with other available sequences. The identity among Iranian ZYMV isolates at the amino acid level reached 95.6-100%. The Iranian ZYMV isolates did not form a compact cluster in the phylogenetic tree, and the phylogenetic analyses and the estimation of genetic distance indicate that the Iranian ZYMV group consists of several independent introductions that evolved separately.

Although Watermelon mosaic virus (WMV) is one of the main cucurbit pathogens and has a worldwide distribution, reliable data on its molecular variability is still limited to some geographical regions. The genetic diversity of 36 WMV isolates from Slovakia and Iran were studied by sequence analysis targeting two opposite genomic regions (P1 and NIb-CP). Phylogenetic analysis using partial sequences of the P1 gene showed that Slovak WMV isolates had greater diversity, representing two groups (group 1 and group 2), whereas all Iranian isolates belonged to a single group (group 2), with relatively low divergeance. Interestingly, in the NIb-CP region, all analyzed Slovak and Iranian isolates clustered within the group 1, thereby illustrating the phylogenetic discrepancies between the two analyzed genomic regions. Based on these data, one-half of analyzed Slovak isolates and all Iranian WMV isolates showed a switch in affiliation based on considered genomic region, clearly indicating their recombinant nature. This work provides further evidence of the significant contribution of recombination to the evolutionary history of WMV and outlines the necessity to target more than a single genome fragment for accurate typing of WMV isolates.

The occurrence of Tomato yellow leaf curl virus (TYL-CV; genus Begomovirus, family Geminiviridae) in the major tomato-growing areas of Iran was determined using TAS-ELISA and PCR. The nucleotide sequences of the coat protein (CP) gene and intergenic region (IR) of eight Iranian isolates were determined. CP nucleotide identities among the Iranian isolates were 96-98%, and showed 94-96% identity with TYLCV-

During a survey of chickpea (Cicer arietinum L.) crops in western Iran in July 2009, leaf samples from yellow and stunted plants were collected from fields in the provinces of Kermanshah (n = 30) and Lorestan (n = 16). Symptoms suggested infections by luteoviruses, such as viruses of the Beet western yellows virus (BWYV) subgroup (e.g., Turnip yellows virus [TuYV]) (4) and Chickpea chlorotic stunt virus (CpCSV), a virus first described from Ethiopia (1) and recently reported from other countries of West Asia and North Africa (2). All 46 samples were analyzed by triple-antibody sandwich (TAS)-ELISA (3) using the luteovirus-specific monoclonal antibody (MAb) B-2-5G4 (1), a mixture of three MAbs (1-1G5, -3H4, and -4B12) to an Ethiopian (Eth) isolate of CpCSV (1), and six individual MAbs (5-1F10, -2B8, -3D5, -5B8, -6F11, and 6-4E10) to a CpCSV isolate from Syria (Sy) (2) in combination with a mixture of polyclonal antibodies to CpCSV and BWYV for plate coating. CpCSV-Eth and -Sy were used as positive controls. Six of the sixteen Lorestan samples and two of the thirty Kermanshah samples reacted with MAb B-2-5-G4, indicating infections with a luteovirus. While none of the 46 samples reacted with the mixture of the CpCSV-Eth specific MAbs, two (Lorestan No. 25 and Kermanshah No. 31) of the eight MAb B-2-5-G4-positive samples reacted strongly with each of the six individual MAbs to CpCSV-Sy. Since this indicated the presence of a serotype II isolate of CpCSV in these two chickpea samples from Iran, we tried to confirm this by reverse transcriptase (RT)-PCR. TRI-Reagent (Sigma, St. Louis, MO) was used for total RNA extraction from samples Nos. 25 and 31. RT-PCR was carried out using the primers 5′-CAC GTG AGA TCA ATA GTC AAT GAA TAC GGT CG-3′ (sense) and 5′-TTT GTA ATT ACC AAY ATT CCA-3′ (antisense) derived from the CpCSV coat protein (CP) gene and 5′ end of ORF5, the readthrough domain (RTD), respectively. In RT-PCR experiments, no amplification was observed from healthy plant extracts, but chickpea samples Nos. 25 and 31 yielded amplicons of ~1,100 bp, which were used for cloning and sequencing. The sequences of the complete CP gene and 5′ end of ORF5 (RTD) from the two samples were determined and deposited in GenBank (GU930837 and GU930838). Sequence analysis revealed that the two Iranian isolates were most similar to each other, sharing CP nucleotide and amino acid (aa) sequence identities of 97.8 and 99.1%, respectively. They differed from each other only in 3 of the 200 aa positions of their CP sequences and were indistinguishable in the 128 N-terminal aa positions of their RTD sequences. When using DNAMAN for phylogenetic analysis, they clustered with serogroup-II isolates of CpCSV from Egypt, Morocco, and Syria (2), with which they were most closely related (approximately 98% in CP aa sequence). While the two Iranian CpCSV isolates differed by approximately 10% in CP aa sequences from serotype-I isolates of CpCSV, they differed strikingly (by ~27%) in RTD aa sequences from CpCSV-Eth, a serotype-I isolate and the only CpCSV isolate for which RTD sequences are available. To our knowledge, this is the first report of the occurrence of CpCSV in Iran. The virus can cause yellowing and stunting of chickpea similar to symptoms caused by other viruses reported from this crop. References: (1) A. D. Abraham et al. Phytopathology 96:437, 2006. (2) A. D. Abraham et al. Arch. Virol 154:791, 2009. (3) A. Franz et al. Ann. Appl. Biol. 128:255, 1996. (4) K. M. Makkouk et al. J. Plant Dis. Prot. 110:157, 2003.