First report of duranta leaf curl virus infecting Ficus virens showing leaf curl symptoms in Pakistan

Ficus virens (family: Moraceae) is a deciduous tree found in the Indian subcontinent, some other Southeast Asian countries, and northern Australia. It is valued for its latex, wood, and medicinal and edible products. In December 2018, during a routine survey to record begomoviruses other than cotton infecting viruses in Pakistan, curling of young leaves, which is a typical symptom of infection by viruses of the genus Begomovirus, was observed in F. virens trees in a park in Lahore, central Punjab, Pakistan. The total DNA of three trees was extracted using the Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and used as a template in PCR amplification with begomovirus-specific primers: Beg-F (5′-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3′) and Beg-R (5′- CTGCCACAACCATGGATTCACGCACAGGG-3′). The amplified 1-kb DNA was cloned into the pGEM-3Zf (+) vector (Promega, Madison, WI) and sequenced by a commercial sequencing service (Macrogen, Seoul, Korea). NCBI BLAST results revealed a nucleotide (nt) sequence identity of 95, 93, and 92% with chilli leaf curl India virus, rose leaf curl virus, and papaya leaf crumple virus, respectively. Based on the sequence obtained, new primers, DCP-F (5′-AGCACATTGGTAAAGTCATGTGTG-3′) and DCP-R (5′-GGATATCATGTCTGGACTCAAATG-3′), were designed to obtain full-length DNA-A of the begomovirus. An approximately 2.8-kb product was amplified and cloned. The resulting sequence (GenBank accession no. MN537564) showed 97.43% nt identity with duranta leaf curl virus (DLCV; KT948069), a Begomovirus species reported in the ornamental shrub Duranta erecta in Pakistan by Iram et al. (2005) and by Anwar et al. (2017). In the former case, only DNA-A of DLCV was found without any associated molecules such as DNA-B or DNA β, whereas, in the latter case, tomato leaf curl New Delhi virus DNA-B was found in association with the DNA-A of DLCV, like in many other Old World begomoviruses. The attempt to amplify DNA-B through PCR using newly designed primers NDB-F (5′-AAACACAGGAGGGATCGGAG-3′) and NDB-R (5′-CACAGATTTCCTTACGCGTATATT-3′) based on the sequence KT948071 was unsuccessful. Similarly, efforts to detect alphasatellite and betasatellite molecules in infected F. virens using universal primers (Bull et al. 2003; Briddon et al. 2002) were unsuccessful. Furthermore, Southern blot hybridization was carried out using [³²P]-radiolabeled PCR product of 1 kb obtained with Beg-F/R primers as a probe. This produced dsDNA bands of different conformations as well as a ssDNA band, which confirms the presence of DLCV in F. virens. Members of the family Geminiviridae, to which begomoviruses belong, mainly infect herbaceous hosts, but they have also been reported in a few woody hosts, such as citrus chlorotic dwarf associated virus in citrus (Loconsole et al. 2012), grapevine red blotch virus in grape (Al Rwahnih et al. 2013), jatropha mosaic virus in Jatropha multifida (Polston et al. 2014), and apple geminivirus 1 in apple tree (Liang et al. 2015). To our knowledge, this is the first report of DLCV infecting F. virens in Pakistan. The results obtained here indicate that F. virens may serve as an alternative host of DLCV and a possible source of infection to the commonly grown D. erecta ornamental garden hedge.