A. Ahmad, M. Ashfaq, M. Ahsan
doi: 10.4454/jpp.v99i3.3977
Viral diseases hamper successful production of solanaceous crops in Pakistan (Ahmad and Ashfaq, 2017; Ashfaq and Ahmad, 2017). In August 2014, 25 chilli pepper leaf samples with symptoms of mottling, interveinal chlorosis and dark green vein-banding were collected from three sites in the Multan district. All diseased samples and two healthy samples were screened against potyvirus infection by indirect PTA-ELISA using “potyvirus group test” kit (Bioreba AG, Switzerland). Only 16 samples were positive for potyvirus infection and were further tested for the presence of specific potyviruses by RT-PCR. Total RNAs extracted using TRIzol reagent (Life Technologies) were subsequently subjected to RT-PCR using primers Poty3 and Oligo(dT)18 (Tsai et al., 2008). All 16 RT-PCR amplicons (ca. 0.8 kb) were purified using QIAquick® PCR purification kit (Qiagen) and sequenced. Sequencing results confirmed the presence of chilli veinal mottle virus (ChiVMV), potato virus Y (PVY) and chilli ringspot virus (ChiRSV) in 11, three and two (Capsicum annuum var. sanam) samples, respectively. ChiRSV and PVY presence were confirmed in chilli fields in Basti Malook and Basti Islampur, respectively, while ChiVMV presence was recorded in all three sites of Multan district. Aphids were also observed (2-4 aphids per plant) on a few plants in the fields from where ChiRSV positive samples were collected. ChiRSV was further confirmed using ChiRSV-specific forward primer (5’- AAGAAGCTGTACACAGGAGAGGA-3’, designed from GenBank Accession No. JQ234922) and Oligo(dT)18. A total of 997 nucleotides (nt) were obtained from each amplicon including the full coat protein cistron and 181 nt of the 3’ nontranslated region, and the sequences deposited in GenBank (KX816566, KX816567); these two sequences shared >98% nt identity. BLASTn revealed 92-98% sequence identities with chilli (C. annuum) isolates of ChiRSV from China (KX258620, KX379001, JN008909) and Vietnam (DQ925438, DQ925439). This virus, first reported in 2008 in Vietnam and China in 2009 from chilli pepper, has not been found elsewhere in the world (Wang et al., 2012). To the best of our knowledge, this is the first confirmed report of ChiRSV on chilli pepper in Pakistan.