First report of Rhynchosia yellow mosaic virus (RhYMV) infecting butterfly pea (Clitoria ternatea) in India.

Abstract

Butterfly pea (Clitoria ternatea), also known as Asian pigeonwings is a perennial herbaceous plant belongs to the Fabaceae family has a great source of natural food colorants and antioxidants (Suarna et al., 2021). It is a multipurpose forage that produces bioactive compounds, acts as a cover crop and improves soil fertility through nitrogen fixation (Gomez et al, 2003). Rhynchosia Yellow Mosaic Virus (RhYMV) which is transmitted by the whitefly (Bemisia tabaci) was first reported from Pakistan in leguminous weed least snoutbean, Rhynchosia minima (Illias et al., 2009). There is no record on the infection of RhYMV in butterfly pea in India or anywhere in the world. In 2022 and 2023, 250 whole butterfly pea plants were collected from few home stead gardens from ten districts (Alipurduar, Darjeeling, Jalpaiguri, Cooch Behar, Malda, Nadia, Birbhum, Hoogly, Purulia and East Midnapore) within the West Bengal province in India, showed extensive yellow mosaic symptoms and distorted flower with bleaching of blue color of petals (Supplementary file 1). The detection of the virus was carried out in the Advanced Plant Virus Diagnostic Centre, Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. DNA extraction was conducted by using plant genomic DNA isolation kit (Sigma-Aldrich, St Louis, USA). The concentration and purity of DNA samples were determined by DeNovix-DS11 spectrophotometer. The presence of the virus was confirmed through polymerase chain reaction (PCR) using begomovirus-specific degenerated primer (Li et al., 2004) followed by RhYMV specific primer (Ilyas et al., 2009). Out of total 250 test samples, 241 were positive for begomovirus specific degenerated primer (96.40%) and out of 241 positive samples, 178 plants were positive to RhYMV specific primer which confirmed 73.85% samples infected to RhYMV. After confirmation of the virus whole genome obtained through Rolling Circle Amplification (RCA) (TempliPhi 100 Amplification Kit, Cytiva) followed by sequencing. After assembled and analyzing the data in CLC Genomics version Workbench 21.0.5 (Matvienko, 2015) whole genome of DNA-A sequence was depositd in the NCBI GenBank database (accession number- PP735226). The BLASTn analyses of the sequence indicated that the isolate from West Bengal, had the 95.44% identity (2616 bp out of 2741 bp) with a RhYMV isolate from snoutbean (Rhyncosia minima) from Pakistan (FM208847). For pathogenecity test (Supplementary file 2), whiteflies reared under polyhouse conditions, acquired the virus from infected plants over 24 hrs (AAP) then allowed viruliferous whiteflies to inoculate the healthy plants for 24 hrs (IAP) and symptoms were monitored under an insect-proof cage. Around two weeks of post- inoculation, all Clitoria plants exhibited mild yellow mosaic symptoms. At 28 dpi, bright yellow to green mosaic appearance, downward curling, and wrinkling of the leaves accompanied with thin tendril with severely distorted flowers were seen which was similar to the samples collected from the different districts. For further confirmation, the infected samples tested positive for RhYMV specific primer were subjected to RCA and whole genome sequencing and found same lineage of RhYMV (accession number- PQ360900). To the best of our knowledge, this is the first report on the incidence of RhYMV in butter fly pea which is to be considered as a new host of RhYMV in India could also act as a reservoir of the virus for further spread to other leguminous plants.