First Report of Cucurbit chlorotic yellows virus in Cucumber, Melon, and Watermelon in Greece.

In 2011, an outbreak of a yellowing disease causing chlorosis and Interveinal chlorotic spots on lower leaves was observed in cucumber (Cucumis sativus) and melon (C. melo) plants in two greenhouses on the island of Rhodes, Greece. Similar symptoms were observed in 2012 in open field watermelon (Citrullus lanatus) plants in Rhodes and in November 2013 in a cucumber greenhouse in Tympaki, Crete. Disease incidence ranged from 10 to 40%. The observed symptoms were similar to those caused by whitefly transmitted criniviruses (family Closteroviridae) Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo-yellows virus (BPYV), as well as Cucurbit chlorotic yellows virus (CCYV), a recently described crinivirus that infects cucurbits in Japan (4) and by the aphid transmitted polerovirus (family Luteoviridae) Cucurbit aphid-borne yellows virus (CABYV). Dense populations of whiteflies were present in all the affected crops. Leaf samples from cucumber (10 from Rhodes and 10 from Crete), melon (10), and watermelon (10) were collected and tested for the presence of the above viruses. Total RNA was extracted from the samples (2) and detection of BPYV, CYSDV, and CABYV was done as previously described (1,3) whereas detection of CCYV was conducted by herein developed two-step RT-PCR assays. Two new pairs of primers, 'CC-HSP-up' (5'-GAAGAGATGGGTTGGTGTAGATAAA-3')/'CC-HSP-do' (5'-CACACCGATTTCATAAACATCCTTT-3') and 'CC-RdRp-up' (5'-CCTAATATTGGAGCTTATGAGTACA-3')/'CC-RdRp-do' (5'-CATACACTTTAAACACAACCCC-3') were designed based on GenBank deposited sequences of CCYV for the amplification of two regions partially covering the heat shock protein 70 homologue (HSP70h) (226 bp) and the RNA dependent RNA polymerase (RdRp) genes (709 bp). Interestingly, CCYV was detected in all samples tested, while CYSDV was detected in 18 cucumbers (10 from Rhodes and 8 from Crete), 1 melon, and 3 watermelon plants. Neither BPYV nor CABYV were detected. In order to verify the presence of CCYV, the partial HSP70h and RdRp regions of a cucumber isolate from Crete were directly sequenced using the primers 'CC-HSP-up'/'CC-HSP-do' and 'CC-RdRp-up'/'CC-RdRp-do'. BLAST analysis of the obtained sequences (HG939521 and 22) showed 99% and 100% identities with the HSP70h and RdRp of cucumber CCYV isolates from Lebanon, respectively (KC990511 and 22). Also, the partial HSP70h sequence of a watermelon CCYV isolate from Rhodes showed 99% identity with the cucumber isolate from Crete. Whitefly transmission of CCYV was also carried out by using an infected cucumber from Crete as virus source. Four groups of 30 whitefly adults of Bemisia tabaci biotype Q were given an acquisition and inoculation access time of 48 and 72 h, respectively. Each whitefly group was transferred to a healthy cucumber plant (hybrid Galeon). Two weeks post inoculation, the plants, which have already been showing mild interveinal chlorosis, were tested for virus presence by RT-PCR. CCYV was successfully transmitted in three of four inoculated cucumbers, which was further confirmed by sequencing. In Greece, cucurbit yellowing disease has occurred since the 1990s, with CYSDV, BPYV, and CABYV as causal agents. To our knowledge, this is the first report of CCYV infecting cucurbits in Greece; therefore, our finding supports the notion that the virus is spreading in the Mediterranean basin and is an important pathogen in cucurbit crops. References: (1) I. N. Boubourakas et al. Plant Pathol. 55:276, 2006. (2) E. Chatzinasiou et al. J. Virol. Methods 169:305, 2010. (3) L. Lotos et al. J. Virol. Methods 198:1, 2014. (4) M. Okuda et al. Phytopathology 100:560, 2010.