First Report of Lagenaria mild mosaic virus Infecting Bottle Gourd in India

Abstract

Bottle gourd or Lagenaria siceraria (Molina) Standl. (family: Cucurbitaceae) is an annual herbaceous crop grown worldwide. It is well-known for its use in Indian ayurvedic medicine. In India, during 2016 to 2017, total annual production of bottle gourd was 2,572,000 metric tons harvested from 157,000 ha (Government of India 2017). During December 2016 and February 2017, disease symptoms such as leaf mosaic and leaf curling were observed in the bottle gourd growing fields of sub-Himalayan West Bengal, India. In severe cases, blistering and deformation of leaf lamina were also observed. Twenty such symptomatic and three healthy-looking leaf samples were collected and analyzed using transmission electron microscopy. All symptomatic samples showed the presence of 500- to 700-nm-long filamentous rod-shaped particles resembling potexviruses (Kim et al. 2010). Total RNA was isolated from all the samples (Ghawana et al. 2011) and tested through reverse transcription polymerase chain reaction (RT-PCR) with Potex5 (5′-CAYCARCARGCMAARGAYGA-3′) and Potex2RC (5′-AGCATRGCNSCRTCYTG-3′) primers that specifically amplify the partial RNA-dependent RNA polymerase (RdRp) gene of the potexviruses (van der Vlugt and Berendsen 2002). RT-PCR conditions were set up as follows: initial incubation for 30 min at 50°C and initial denaturation for 5 min at 95°C; 35 cycles of denaturation at 95°C for 1 min, annealing at 51.5°C for 1 min, product extension at 72°C for 1 min; and final extension at 72°C for 10 min. The products were visualized under ultraviolet transilluminator after running on 1% agarose gel, and amplicon size was determined. All 20 infected samples produced an amplicon of ∼530 bp, whereas the three healthy-looking samples did not produce expected amplicons. All amplicons were cloned into pGEM-T vector (Promega, Madison, WI) and were sequenced. One such sequence of sample NE-02 was submitted to GenBank as Lagenaria mild mosaic virus (LaMMoV, accession no. MG752888) and showed 78% nucleotide (nt) identity and 91% amino acid (aa) identity with LaMMoV infecting bottle gourd in Myanmar (accession no. AB546335), as determined in a BLASTn analysis. To confirm the identity of the pathogen, another primer pair, RdRP1f (5′-CACCGGTGACTCAAAGCAAA-3′) and RdRP1r (5′-CCAATRGGCCTCTTTTCMAG-3′), was designed based on the genome of LaMMoV, papaya mosaic virus, and Alternanthera mosaic virus to amplify the RdRp gene of all the three potexviruses mentioned. For the primer set RdRP1f/RdRP1r, RT-PCR conditions were similar to those described above, except for the annealing step at 57°C for 1 min. RT-PCR conducted on all 20 symptomatic samples produced an amplicon of ∼810 bp, whereas none of the healthy-looking samples tested positive with the same primer pair. This new RT-PCR fragment of the sample NE-02 was also submitted to GenBank as LaMMoV (accession no. MH998222), because it showed 82% nt identity and 99% aa identity with LaMMoV (accession no. AB546335) following BLASTn analysis. The species demarcation criteria for the genus Potexvirus is less than 72% nt (or 80% aa) identity between their coat protein or RdRp genes (Adams et al. 2004). To the best of our knowledge, this is the first report of LaMMoV infecting bottle gourd in India. The finding of this study shall aid in developing effective management strategies.