Natural Infection of Citrullus colocynthis by Papaya ringspot virus-W in Iran.

Abstract

Papaya ringspot virus (PRSV, genus Potyvirus, family Potyviridae) is economically important due to its worldwide distribution and because it can cause serious losses in both cucurbit crops and papaya (3). PRSV has been previously reported from cucurbit crops in Iran (2). In Khuzestan Province, southwest Iran, cucurbit crops, including cucumber, melon, squash, pumpkin, and watermelon, are grown on about 30,000 ha with 720,000 t production annually. To identify possible alternative hosts that may serve as PRSV reservoirs, samples of 36 different common weed species (17 symptomatic and 19 asymptomatic) including Amaranthus sp. (slim amaranth), Carthamus sp. (safflower), Chenopodium album L. (lamb squarters), Citrullus colocynthis (L.) Schrad (colocynth), Convolvulus arvensis L. (field bindweed), Datura stramonium L. (jimson weed), Euphorbia sp. (wart weed), Malva sylvestirs L. (common malva), Solanum nigrum L. (black nightshade), and Sonchus asper (L.) Hill (prickly sow-thistle) were collected in cucurbit open fields during 2012 to 2013 in Khuzestan Province, where PRSV symptoms were observed. Symptoms on weed samples included mottling, mosaic, blistering, cholorosis, vein clearing, interveinal yellowing, yellows, necrosis, leaf distortion, and curling. Samples were tested by DAS-ELISA with specific antisera against PRSV using reagents from Bioreba (Switzerland). Three of the 36 weed samples belonging to C. colocynthis (Cucurbitaceae) with mottling and chlorosis symptoms were positive for PRSV by ELISA. Leaf extracts from PRSV ELISA-positive samples were mechanically inoculated onto indicator host plants, causing local lesions on Chenopodium amaranticolor and systemic symptoms on Cucumis melo, Cucumis sativus, and Cucurbita pepo, but could not produce symptoms on Nicotiana glutinosa, N. tabacum cv. White Burley, or N. tabacum cv. Xanthi. Total RNA was extracted from infected leaves using Tri-reagent (Sigma) and first-strand cDNA synthesis was performed using M-MuLV reverse transcriptase (Fermentas, Lithuania), according to the manufacturer's instructions. The presence of PRSV was confirmed by RT-PCR using primers for the complete coat protein (CP) gene of PRSV-W (forward 5'-GCAGCAATGATAGAGTCATG-3' and reverse 5'-AACACACAAGCGCGAGTATTCA-3') (1). The complete CP nucleotide sequence of three Iranian PRSV isolates consisted of 864 nt, coding for a 288 amino acid (aa) protein. Subsequent analysis showed that the CP nucleotide sequences of Iranian isolates (GenBank Accession Nos. KM047884 to KM047886) from C. colocynthis samples were identical. Furthermore, BLAST analysis of the nucleotide sequence comparisons revealed that the Iranian isolates shared the highest identity (96%) with the Chinese PRSV isolate (DQ449533). PRSV-W has been previously reported from different cucurbits using serological and biological detection (2); however, this result provides the first molecular demonstration, to our knowledge, of PRSV-W on C. colocynthis. C. colocynthis is a perennial weed in West and South Iran. This information on the natural infection of C. colocynthis with PSRV-W will help to better understand PRSV epidemiology and to develop a successful management program for reducing the impact of this disease.