Castor bean (Ricinus communis) is a member of the family Euphorbiaceae and is native to tropical regions. The seed is a source of castor oil which has a variety of pharmaceutical and industrial uses. In India, castor oil is used in alternative medicine as a laxative, purgative, and for curing arthritis. Castor bean is affected by pathogens, both fungal (Karan, 1966) and viral, including Cucumber mosaic virus (Raj et al., 2010) and Olive latent virus 2 (Grieco et al., 2002). During a survey in January 2020 in the Jalandhar region of Punjab, northern India, R. communis plants were found to show begomovirus-like leaf curling symptoms (Figure 1).
Enlarge this image.FIGURE 1. Ricinus communis leaves showing curling symptoms in the Jalandhar region of Punjab, northern India
Three diseased leaf samples were collected from different locations around Jalandhar though most plants observed during the survey appeared to have symptoms. Total DNA was extracted from the samples using a DNeasy® Plant Mini Kit (Qiagen, Germany). The DNA samples were subjected to PCR using begomovirus-specific primers AV494 and AC1048 (Wyatt & Brown, 1996). PCR was performed with an initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 40 seconds, annealing at 55°C for 40 seconds, extension at 72°C for 1 minute, and final extension at 72°C for 10 minutes. Products of the expected size (c. 570 bp) were obtained with all three samples. The amplicons were sent to Eurofins Genomic India (Bangalore) for direct sequencing. A BLAST search showed that the sequences had 99% identity with a number of ToLCNDV isolates (GenBank Accession Nos. MT185657 and MT185656).
Novel primers were designed to amplify the complete DNA-A of the isolate, ToLCNDV-F (5'-GAGGTCAGCAATCTGCCAAC-3')/ToLCNDV-R (5'-CTCTAGCTGATCTGCCATCG-3'). PCR was done with initial denaturation at 94°C for 3 minutes, thirty cycles of denaturation at 94°C for 60 seconds, annealing at 56°C for 40 seconds, extension at 72°C for 3 min 15 seconds, followed by final extension at 72°C for 7 minutes. A product of the expected size (c. 2.7 kb) was obtained. The PCR product was ligated into pGEM®-T Easy vector (Promega, USA), transformed into Escherichia coli DH5α competent cells and three recombinant clones were selected. The plasmid was isolated from the colonies using the PureYield™ Plasmid Miniprep System (Promega, USA) and sequenced by Eurofins Genomic India. The end sequences were assembled to give a 994 nt long sequence (MW574990). A BLAST search revealed 99% identity with an Indian isolate of ToLCNDV (MT185657) infecting bitter gourd. A total of 19 sequences (Table 1) belonging to distinct begomovirus species were selected based on maximum sequence identity with the present isolate for phylogenetic analysis. Pairwise alignment of the nucleotide sequences was performed using the ClustalW program and a phylogenetic tree was constructed with the neighbour-joining method (using 1000 bootstrap replicates) available in MEGA X. The analysis showed that the isolate was closely related to other ToLCNDV isolates, with the highest identity (99%) to an Indian ToLCNDV isolate from okra (MN728677; Fig. 2). Only a 994 nt long sequence was used for this phylogenetic analysis and analysis of the complete genome may draw a different conclusion.
TABLE 1 Begomovirus species used for phylogenetic analysis
| Begomovirus species | Abbreviations | Accession Number |
| Present isolate | MW574990 | |
| Tomato leaf curl new Delhi virus-[India:Okra:2019] | ToLCNDV-[Ind:Okr:19] | MN728677 |
| Bitter gourd yellow vein virus-[India:Lentin:2014] | BiYVV-[Ind:Len:14] | KM190927 |
| Tomato leaf curl Taiwan virus-[Taiwan:Tomato:1997] | ToLCTWV-[Tai:Tom:97] | U88692 |
| Chayote enation yellow mosaic virus-[India:Chayote:2016] | ChaEYMV-[Ind:Cha:16] | KX259339 |
| Squash leaf curl China virus-[Combodia:Melon:2018] | SqLCCV-[Com:Mel:18] | MK064240 |
| Loofa yellow mosaic virus-[Vietnam:Luffa:2003] | LoYMV-[Vie:Luf:03] | AF509739 |
| Melon leaf curl virus-[Pakistan:Cucumis:2007] | MeLCV-[Pak:Cuc:07] | AM494976 |
| Squash leaf curl Philippines virus-[ Philippines:Cucurbita:2003] | SqLCPV-[Phi:Cuc:03] | AB085793 |
| Pumpkin yellow mosaic Malaysia virus-[ Malaysia:Pumpkin:2006] | PumYMMV-[Mal:Pum:06] | EF197941 |
| Tobacco curly shoot virus-[China:Tobaco:2013] | ToCSV -[Chi:Tob:13] | HG003650 |
| Chilli leaf curl virus-[India:Papaya:2005] | ChiLCV-[Ind:Pap:05] | DQ989326 |
| Papaya leaf curl virus-[India:Tomato:2005] | PapLCV-[Ind:Tom:05] | DQ629103 |
| Ageratum yellow vein Sri Lanka virus-[Srilanka:Ageratum:2000] | AgYVSLV-[Sri:Age:00] | AF314144 |
| Pepper leaf curl virus-[India:Chilli:2011] | PepLCV-[Ind:Chi:11] | JN663870 |
| Croton yellow vein mosaic virus-[India:Acalypha:2012] | CroYVMV-[Ind:Aca:12] | FN645898 |
| Tomato yellow leaf curl virus-[Egypt:Tomato:2006] | ToYLCV-[Egy:Tom:06] | AY594174 |
| Tomato leaf curl Joydebpur virus-[India:Chilli:2007] | ToLCJV-[Ind:Chi:07] | HM007098 |
| Malvastrum yellow vein virus-[China:Ageratum:2004] | MaYVV-[Chi:Age:04] | AJ744881 |
| Tomato leaf curl Karnataka virus-[Pakistan:Solanum lycopersicum:2015] | ToLCKV-[Pak:Sol:15] | LN878122 |
Enlarge this image.FIGURE 2. Phylogenetic tree constructed using ClustalW program showing relationship between the present isolate and related begomovirus species taken for phylogenetic analysis. Accession numbers are indicated for each virus isolate. The present isolate showed the closest relationship with an Indian isolate of ToLCNDV (MN728677)
ToLCNDV is a widespread virus infecting more than 43 plant species including vegetables, ornamentals, fibre crops and weeds. To the best of our knowledge this is the first report of ToLCNDV infecting R. communis. It is important to maintain surveillance to understand the distribution of ToLCNDV and to assist in developing long-term management control strategies.
ACKNOWLEDGEMENTSThe authors are thankful to the competent authorities of DAV University, Jalandhar Punjab, India for providing necessary research facilities, and the Council of Scientific and Industrial Research, Government of India for financial support [38 (1466)/18/EMR-II].
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; Lager, P 1 ; Kumar, Y 1
1 Department of Biotechnology, DAV University, Jalandhar, Punjab, India