First Report of Tomato leaf curl New Delhi virus and a Tomato yellow leaf curl Thailand betasatellite Causing Severe Leaf Curl Disease of Potato in Pakistan

In April 2011, during a field survey, a severe leaf curl disease was observed on potato (Solanum tuberosum) in potato-growing areas of Punjab, Pakistan (approximate coordinates 30.3551°N, 72.5321°E). Severe upward and downward leaf curling, vein swelling, and leaf thickening, resulting in conspicuous bushy appearance suggested that the disease might be caused by begomoviruses. The disease symptoms were widespread throughout the potato growing districts with an incidence of 5 to 10%. To confirm the association of begomoviruses with the disease, total DNA was extracted from 10 symptomatic samples using CTAB protocol. Samples without any symptoms were also collected as negative controls. Universal primers designed on conserved sequences of begomoviruses DNA A were used in PCR (Shahid et al. 2007). A PCR product of expected size (∼2.8 kb) was obtained in three out of 10 symptomatic samples and confirmed the association of begomoviruses with the disease. The amplified products were cloned and sequenced. Complete nucleotide sequence of two clones were submitted in GenBank (accession numbers LN908935 and LN908936). NCBI BLASTn analysis showed maximum nucleotide identity with the isolates of Tomato leaf curl New Delhi virus (ToLCNDV), a highly prevalent bipartite begomovirus in the Indian subcontinent reported from several crops such as cotton, chilli, potato, etc. (Zaidi et al. 2016). Sequence demarcation tool (SDT) analysis reveals that the ToLCNDV isolates from Pakistan have >99% sequence identity with the ToLCNDV isolates identified from potato plants in India (KC874503 and KC874505). Previously, the association of ToLCNDV with leaf curl disease of potato has been reported from India (Usharani et al. 2004). For infectivity analysis, all efforts to mechanically transmit the ToLCNDV isolates in potato were unsuccessful under our conditions. In order to confirm the presence of begomoviruses, Southern hybridization was performed with ToLCNDV AV2 probe (a DIG labeled probe, target specificity to AV2 gene of ToLCNDV). All PCR positive plants showed hybridization with ToLCNDV AV2 probe in Southern blot analysis while in healthy plants, no hybridization signals were observed. The symptom phenotype on potato suggested that DNA satellites might be associated with the severe disease in some of symptomatic samples. In order to check the presence of DNA satellites, universal primers for alphasatellites and betasatellites were used in PCR (Briddon et al. 2002). No PCR product was amplified by alphasatellite primers, but use of betasatellite primers in PCR resulted in amplification of a product of desirable size (∼1.4 kb) in two out of three symptomatic DNA A positive samples. The amplified product of one sample was cloned, sequenced, and submitted in GenBank (LK933548). BLASTn showed that the sequence shared maximum sequence identity (>96%) with Tomato yellow leaf curl Thailand betasatellite (TYLCTHB) isolates FR819710, EF068245, and HG531760. To induce systemic infection, DNA A of ToLCNDV does not necessarily require DNA B as the association of DNA A of ToLCNDV as a helper virus with betasatellite has been proven (Saeed et al. 2007). To our knowledge, this is the first report of a bipartite begomovirus, ToLCNDV, with leaf curl disease of potato in Pakistan. In addition, this is the first report of the association of ToLCNDV and TYLCTHB infecting potato in Pakistan. This is an indication of the increased incidence of and expansion in the host range of begomovirus disease complexes, which can pose serious future threat to agriculture in Pakistan. © 2017, American Phytopathological Society. All rights reserved.