First report of mixed infection of Zucchini yellow mosaic virus and Tomato leaf curl New Delhi virus in bittergourd in India.

Cucurbits are cultivated in the plains of North-western India. Recently, bittergourd plants showing severe yellow mosaic, blistering and curling of leaves were observed at Research farms of Punjab Agricultural University, Ludhiana (India). Tender leaves of symptomatic plants were subjected to ELISA using commercial kits (Agdia, USA) against common cucurbit viruses such as Cucumber mosaic virus subgroups I and II, Potato virus X, Potato virus Y (PVY)o, PVYN, and Zucchini yellow mosaic virus (ZYMV). Samples were only positive for ZYMV in ELISA. Total RNA from ZYMV infected samples was subjected to cDNA synthesis using oligo-dT primers, and DNA amplification using primers ZY-2/ZY- 3 designed in the coat protein (CP) and Nib genomic region (Thomson et al., 1995), yielding a product of approximately 1200 bp in RT-PCR. This amplicon was cloned in pGEM-T easy vector (Promega, USA) and sequenced. Sequence analysis indicated 99.5% nucleotide and 97.8% amino acid identities between the ZYMV isolate from bittergourd (KJ614229) and form Cucumis anguria (GQ482976) in India. The presence of whiteflies during sampling suggested the presence of a begomovirus. To confirm the occurrence of a begomovirus, total DNA was isolated from symptomatic bittergourd leaves and subjected to PCR using universal degenerate primers AV494 /AC1048 designed in the core CP gene region (Wyatt and Brown, 1996). The 575 bp DNA product was cloned and sequenced. Sequence analysis showed that the begomovirus isolate from bittergourd in India (KJ44258) had 100% nucleotide and amino acid identity with Tomato leaf curl New Delhi virus reported from bittergourd in Pakistan (AJ854186). To our knowledge this is the first report of mixed infection of the aphid- transmitted potyvirus ZYMV and the whitefly-transmitted begomovirus ToLCNDV in bittergourd.