Characterization of Tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of Watermelon and development of LAMP assay for its detection

V Venkataravanappa; K V Ashwathappa; C N Lakshminarayana Reddy; K S Shankarappa; M Krishna Reddy

doi:10.1007/s13205-020-02245-x

Characterization of Tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of Watermelon and development of LAMP assay for its detection

3 Biotech. 2020 Jun;10(6):282. doi: 10.1007/s13205-020-02245-x. Epub 2020 Jun 2.

Authors

V Venkataravanappa^{1

2}, K V Ashwathappa¹, C N Lakshminarayana Reddy³, K S Shankarappa^{4

5}, M Krishna Reddy¹

Affiliations

¹ ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore, 560089 Karnataka India.
² Division of Plant Pathology, Central Horticultural Experiment Station, Chettalli, ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bangalore, India.
³ Department of Plant Pathology, College of Agriculture, University of Agricultural Sciences, GKVK, Bangalore, 560065 Karnataka India.
⁴ Department of Plant Pathology, College of Horticulture, Bangaluru, 560065 India.
⁵ University of Horticultural Sciences, Bagalkot, Karnataka India.

Abstract

Diseases caused by begomoviruses are becoming the major limiting factors for the production of watermelon in India. Survey for the incidence of plants showing symptoms typical to begomovirus infection was conducted in watermelon fields. The study revealed that 40% of the watermelon plants were showing the yellowing and downward curling symptoms. Twenty infected samples were collected from the different farmer's fields to know the association of begomoviruses. The PCR amplification using begomovirus-specific primers resulted in an expected 1.2 kb PCR product indicating the begomovirus association with the watermelon samples. The sequence comparison results of 1.2 kb representing partial genome revealed that all sequences obtained from watermelon samples have a nucleotide (nt) identity of more than 98% among them and are maximum homology with Tomato leaf curl New Delhi virus (ToLCNDV). One watermelon sample (WM1) was selected for complete genome amplification using RCA method (rolling-circle amplification). Amplification of DNA B and no amplification of betasatellites and alphasatellite indicated this virus as bipartite. Sequence Demarcation Tool (SDT) analysis of the DNA A component of the WM1 isolate showed the maximum nt identity of 94.6-97.9% and 85.2-95.8% with ToLCNDV infecting cucurbits. The recombinant analysis showed that the genome was likely to be derived from the recombination of already reported begomoviruses (ToLCNDV, ToLCPalV, and MYMIV) infecting diverse crops. The whitefly cryptic species predominant in the begomovirus-infected watermelon fields were identified as Asia-II-5 group. The LAMP assay developed based on coat protein gene sequence was able to detect the ToLCNDV in the infected samples. Visual detection of the LAMP-amplified products was observed with the hydroxy naphthol blue. LAMP assay was also validated with ToLCNDV infected sponge gourd, spine gourd, ivy gourd, ridge gourd, and cucumber. This is the first report of ToLCNDV association with leaf curl and yellowing disease of watermelon from India and World based on complete genome sequencing.

Keywords: Phylogenetic analysis; Polymerase chain reaction; Recombination; Tomato leaf curl new delhi virus; Watermelon.