First report of pepper leaf curl virus causing leaf curl of castor bean (Ricinus communis) in India

Castor bean (Ricinus communis, Euphorbiaceae), has a wide range of medicinal properties and grows in diverse agroclimatic zones in India. During a survey in May-June 2023, 30 diseased castor bean plants, were sampled from three locations (ten per location) in the district of Pune, Maharashtra: Wakad (18.6011° N, 73.7641° E), Baner (18.5590° N, 73.7868° E) and Pimpri (18.6298° N, 73.7997° E). The plants exhibited symptoms consistent with begomovirus infection, i.e. leaf curling and crinkling with occasional yellowing of the lamina around leaf veins (Figure 1). Total DNA was isolated from leaves of two diseased and two asymptomatic plants from each of the collection sites, using the CTAB method (Doyle & Doyle 1987). As the diseased symptoms resembled begomovirus infection, the presence of viruses in this genus was checked by PCR using the Deng A/Deng B primer pair (Deng et al., 1994). An amplicon of the expected size (c. 550 bp) was obtained in all diseased, but not asymptomatic, plants. To identify the begomovirus to species, primers to detect Chilli leaf curl virus (Kumar et al., 2016), Euphorbia leaf curl virus (Ye et al., 2022) and eight other begomoviruses were used (Sinha et al., 2011). The primers designed for Pepper leaf curl virus (PepLCV) yielded the expected 1044 bp product from all six diseased samples. The amplicon was sequenced and submitted to Genbank (Accession No. OR360532). A BLASTn search indicated the sequence had 99.71% and 98.28 % nucleotide identities to PepLCV isolates from Palampur (JN663870) and Varanasi (EF190217), respectively. No amplification was achieved using primers DNA B(f) /DNA B(r) (Reddy et al., 2005), indicating that the virus is monopartite. To investigate the presence of a β-satellite, primers designed specifically for betasatellites of the Varanasi isolate of PepLCV (β-PepLCV(f) 5′-ACCGTGGGCGAGGGGGGTCTTTG-3′, β-PepLCV(r) 5′-CACACCGCCGCGCGTGTCGTAAA-3′) were used. These amplified a 1289 bp product which was sequenced and submitted to Genbank (PP092204.1). The MEGA 11 software was used to perform neighbour-joining analysis on sequences of the protein coding regions of the 1044 bp amplicon (DNA-A) together with 32 different begomoviruses. The analysis indicates that the current isolate is closely related to other isolates of PepLCV. Furthermore, the betasatellite region associated with the current isolate has similarities with the betasatellite of other PepLCV isolates (Figure 2). To demonstrate pathogenicity, 1 gramme of symptomatic, PepLCV-positive castor bean leaves were homogenised in phosphate-buffered saline. The leaf suspension was used to mechanically inoculate leaves of 10 three-week-old castor bean seedlings, grown in pots containing sterile media in a polyhouse with a 16/8 hr light/dark cycle at 26 ±2°C. Control seedlings were mock-inoculated with phosphate-buffered saline only. Three weeks after inoculation, seven of the 10 inoculated plants started displaying leaf curling symptoms, while leaves from mock-inoculated plants showed no symptoms (Figure 3). DNA was isolated from inoculated seedlings and tested using the primers of Sinha et al. (2011) and the novel primers described above. Amplicons of 1044 bp (PepLCV DNA-A) and 1289 bp (β-satellite PepLCV) were produced from all symptomatic samples. This is the first report of PepLCV associated with castor bean leaf curl disease in India. R. Chetia and N. Aggarwal contributed equally to this work. The authors are thankful to Dr. D.Y. Patil Vidyapeeth, Pune, India for financial support in the form of a DPU-Seed money grant (DPU/906-3/2022). R. Chetia is thankful to the Science and Engineering Research Board, Department of Science and Technology, Government of India for providing project assistance through a startup research grant (File number: SRG/2022/000070).