Complete nucleotide sequence of a new isolate of tomato leaf curl New Delhi virus infecting cucumber, bottle gourd and muskmelon in Thailand

Abstract

Geminiviruses have monopartite or bipartite circular sin-gle-stranded DNA (ssDNA) genomes, encapsidated ingeminate particles. The family Geminiviridae is composedof four genera, distinguished by their host range, insectvector, and genome organization [2]. Most of the charac-terized geminiviruses are transmitted by whiteflies(Bemisia tabaci Genn.) and belong to the genus Begomo-virus. Begomoviruses generally have bipartite genomes(designated as DNA-A and DNA-B) and infect dicotyle-donous plants. Some begomoviruses, however, have asingle genomic DNA molecule. Yellow leaf curl diseasecauses heavy losses to cucurbit plants in Thailand.Recently, we reported that yellow leaf curl disease ofpumpkin (Cucurbita pepa L.) is associated with squash leafcurl China virus [4]. Cucumber plants showing severeyellow leaf disease symptoms were observed during 1996.The disease was transmitted by grafting and whiteflyinoculations (unpublished results). To date, only partialDNA fragments of begomoviruses from diseased cucum-ber, muskmelon (Cucumis melo L.), cantaloupe (Cucumismelo var. reliculatus) and wax gourd (Benincase hispidaCogn.) have been sequenced [6–8]. The complete nucleo-tide sequences of DNA-A and DNA-B were obtained fromcucumber plants. DNA-A shares 97% with isolates oftomato leaf curl New Delhi virus (ToLCNDV) and thusnamed tomato leaf curl New Delhi virus-[Cucum-ber:Thailand] (ToLCNDV-[Cuc:Tha]). This virus has alsobeen detected on bottle gourd (Lagenaria leucantha Rosby)and muskmelon exhibiting yellow leaf symptoms collectedfrom fields in the same region. The sequences obtainedfrom bottle gourd and muskmelon isolates share more than99% sequence identity with ToLCNDV-[Cuc:Tha]. Thispaper describes for the first time an isolate of ToLCNDVinfecting cucumber, bottle gourd and muskmelon samplesand reports its complete nucleotide sequence. However, noDNA-B component has been described for ToLCNDVoriginating from Thailand. Here, we describe briefly thatyellow leaf curling of cucumber is associated with andcaused by a bipartite begomovirus.In 1996, Cucumis sativus L. plants exhibiting yellow leafsymptoms were collected from fields at Kamphaengsaen,Nakorn Pathom, Thailand. In addition, samples exhibitingyellow symptoms on bottle guard and muskmelon were alsocollected from nearby fields and were maintained on sus-ceptible cucumber plants by grafting/whitefly inoculation inan insect-proof glasshouse. Leaf tissue was desiccated withsilica gel for 2 weeks and stored at 4 C until use. Totalnucleic acids were extracted from the leaf tissues of infectedcucumber plants, as described by Kon et al. [5]. The poly-merase chain reaction (PCR) was carried out as describedby Sharma et al. [9]. Initially, 2.6 kbp viral DNA fragmentswere obtained from diseased cucumber plants using be-gomovirus DNA-A-specific primers UPV1 and UPC2 [1]and cloned into pGEM-T Easy Vector (Promega, Madison,WI) and sequenced. Recombinant plasmids containing anapproximately full-length insert of the PCR product were