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Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus affecting tomato cultivation on the Indian subcontinent. Recently, however, a new strain of the virus, named ToLCNDV-ES, has spread to Mediterranean countries such as Spain, Italy, and Tunisia, and occurred in Cucurbita crops, causing economic damage. Although ToLCNDV is spread by the sweet potato whitefly (Bemisia tabaci), like other begomoviruses, it has not been clear how ToLCNDV suddenly spread from the Indian subcontinent to the Mediterranean region. In 2017, ToLCNDV was diagnosed in young seedlings germinated naturally from fruits fallen in the prior year on a farm located in Giugliano in Campania, Naples, Italy, suggesting a possible role of the seeds in vertical transmission of the virus. Because sweet potato whiteflies were widespread naturally in that region, it was necessary to verify that in an artificial insect vector-free condition. Seeds were harvested from two ToLCNDV-infected zucchini squash cultivars in Naples in 2017 and 2018 to examine whether ToLCNDV can be transmitted from zucchini squash seeds to young plants. Viral DNA was amplified from these seeds and 1- to 3-week-old seedlings germinated from them with a ToLCNDV-specific primer set. According to PCR results, viral contamination was confirmed from all harvested seeds and dissemination was proven from 61.36% of tested seedling samples. Mechanical transmission from seed-borne virus-infected seedlings to healthy zucchini squash plants was also succesful, demonstrating that seedlings from ToLCNDV-infected seeds did act as inoculum. This is the first report demonstrating that ToLCNDV is a seed-transmissible virus in zucchini squash plants in Italy.

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.

Recently, chili pepper (Capsicum annuum) plants in Indonesia have been devastated by a notorious bipartite begomovirus infection named Pepper yellow leaf curl Indonesia virus (PepYLCIV), which causes a distinct decrease in chili pepper production. Pepper yellow diseases have been known since early 2000; however, the spread of this virus thus far is distressing. These diseases can reduce chili yields by 20-100% in Indonesia. As previously known, begomovirus can be transmitted through whitefly to several host plants from the families Solanaceae, Compositae, and Leguminosae. In the field, a single plant was observed with severe symptoms of pepper yellow leaf curl disease, while other plants in the same field were asymptomatic and healthy. The observation leads to the possibility that the virus can be transmitted from previously infected chili pepper plants through seeds, as begomovirus transmission through seeds has been reported before. This study was conducted using seeds from chili peppers infected with viruses from different places in Indonesia. Whole seeds, embryos, and seedlings from PepYLCIV infected seeds were investigated in this study by performing viral genome DNA extraction, uracil DNA glycosylase-PCR, and sequencing analysis. Results revealed that both DNA-A and DNA-B of PepYLCIV in seeds and embryos of infected chili pepper plants were detected. The results also showed that 25-67% of PepYLCIV DNA-A and 50-100% of DNA-B were detected from seedlings grown from infected chili pepper seed collected from different location, thus confirming PepYLCIV as a seed-transmissible virus in chili pepper plants.