First Report of Cucurbit chlorotic yellows virus on Cucumber in Lebanon

Abstract

In the summer of 2011, whiteflies (Bemisia tabaci) were collected from cucumbers plants showing interveinal yellowing on the lower leaves growing in commercial greenhouses in Jiyye area, South Lebanon. About twenty whiteflies per seedling were placed on healthy cucumber (Cucumis sativus cv. Beit alpha) seedlings at the second-leaf stage and grown in insect-proof cages in the university glasshouse. After 4 days of feeding, the whiteflies were sprayed with an insecticide (Imidacloprid). Three weeks post inoculation, three out of eight inoculated plants developed typical yellowing symptoms on old leaves. Symptoms started with chlorotic spots that later coalesced into interveinal chlorosis similar to that induced by Cucurbit yellow stunting disorder virus (CYSDV), a widely occurring virus in Lebanon. Total RNA was extracted from the three symptomatic plants using TRI reagent (Sigma-Aldrich, St Louis, MO). The three symptomatic plants, however, tested negative for CYSDV by tissue blot immuno assay and reverse transcription (RT)-PCR (2). Another Crinivirus, Cucurbit chlorotic yellows virus (CCYV), was suspected (1). Analysis of the RNA extracts of the three symptomatic plants by RT-PCR using the CCYV heat shock protein (HSP70h) specific primer pair (CCYV-HSP-F1/CCYV-HSP-R1) (3) showed a band of the expected size (462 bp). A small scale survey from February to April 2012, covering the two major cucumber growing areas on the Lebanese coastal areas, Byblos (North) and Jiyye (South), was conducted. Eight out of 10 cucumber leaf samples collected from Jiyye and seven out of 38 from Byblos area tested positive for CCYV by RT-PCR. Therefore, RNA extracts from cucumber which were positive for CYSDV and stored at –80°C since spring 2011 were tested again for CCYV. Fourteen randomly selected RNA extracts out of 76 samples were all found to have mixed infections with CCYV. For partial molecular characterization of the Lebanese CCYV isolates collected during summer 2011, regions of the HSP70h and coat protein (CP) genes from CCYV were amplified using specific primers (CCYV-CPs/CCYV-CPas) (1, 3). Sequences of the purified amplicons of 462 and 725 bp respectively, were submitted to GenBank (Accession Nos. JX014262 and JX014263). The HSP70h and CP genes were highly homologous to the Japanese CCYV isolate (AB523789), sharing nucleotide sequence identities of 99.8% and 99.7%, respectively. The CCYV HSP70h gene of the Lebanese isolate is distantly related to other criniviruses sharing nucleotide sequence identities of 76.4, 75.8, and 74.7% with Lettuce chlorosis virus (FJ380119), Bean yellow disorder virus (EU191905), and CYSDV (AY242078), respectively, whereas the CCYV CP gene shares 77, 76, and 70% nucleotide sequence identities with the latter viruses, respectively. CCYV has been reported only in Japan, China, Taiwan, and Sudan (1). To our knowledge, this is the first report of CCYV in Lebanon and the Mediterranean basin. CCYV may have also spread to neighboring countries but has not been reported yet. The rapid outbreak of new whitefly-transmitted viruses poses a serious threat to cucurbit production in the Mediterranean region and Europe. References: (1) K. Hamed et al. Plant Dis. 95:1321, 2011. (2) H. Hourani and Y. Abou-Jawdah J. Plant Pathol. 85:1, 2003 (3) R. Zeng et al. Plant Dis. 95:354, 2011.