First report of tomato leaf curl New Delhi virus infecting Abutilon theophrasti Medicus in China

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus in the family Geminiviridae, primarily transmitted by the whitefly Bemisia tabaci, although sap-mediated mechanical transmission has been reported for some isolates (Janssen et al. 2022). Since its first report in India in 1995 (Srivastava et al. 1995), ToLCNDV has spread to multiple countries and has been reported to infect at least 45 plant species, including economically important cucurbitaceous and solanaceous crops, and can cause yield losses exceeding 20% in open-field melon production (Hui et al. 2024; Cai et al. 2023). Because weeds may serve as alternative hosts and potential virus reservoirs, identifying new natural hosts is important for understanding ToLCNDV epidemiology. Abutilon theophrasti Medicus is an annual weed widely distributed in cultivated fields. In August 2025, during a field survey in Jingzhou, Hubei Province, China, a large population of A. theophrasti was observed near fields planted with pepper, tomato, and soybean. Among these plants, two exhibited virus-like symptoms, including leaf curling, wrinkling, and mosaic-like mottling. Total DNA from symptomatic leaves was extracted using the CTAB method and tested by PCR with ToLCNDV-specific primers. A 543-bp amplicon was obtained and confirmed by Sanger sequencing. Overlapping primers were then designed to amplify the complete viral genome, and the assembled DNA-A and DNA-B components were analyzed by BLASTn against the NCBI nucleotide database. The sequences were deposited in GenBank under accession numbers PX436854 (DNA-A) and PX436855 (DNA-B). DNA-A and DNA-B shared 99.60% and 99.44% nucleotide identity with ToLCNDV isolates GXBH-TG (PX021751.1) and FN (PV754986.1), respectively, supporting identification of the virus as ToLCNDV, designated ToLCNDV-JZ. Because sap transmissibility differs among reported ToLCNDV isolates, sap prepared from naturally infected A. theophrasti leaves was mechanically inoculated onto three Nicotiana benthamiana plants; No symptoms or ToLCNDV-specific PCR signals were detected at 15 dpi, indicating no mechanical transmission under the conditions tested. To further evaluate the susceptibility of A. theophrasti to ToLCNDV, healthy A. theophrasti plants were challenged using three independent inoculation methods: sap-mediated mechanical inoculation, viruliferous B. tabaci-mediated inoculation, and Agrobacterium-mediated inoculation with an infectious ToLCNDV-HB clone (DNA-A, PX962465; DNA-B, PX962467). No obvious disease symptoms were observed in A. theophrasti plants within 30 days. ToLCNDV-specific PCR products were detected in 4/10, 2/10, and 7/10 plants following mechanical, whitefly-mediated, and agroinfiltration inoculation, respectively. Viral CP was detected by Western blot in 1/2 mechanical and 1/3 agroinfiltration samples, but not in the two whitefly-mediated samples or Mock controls. These results indicate that A. theophrasti can support ToLCNDV infection without visible symptoms under the conditions tested. Together, the detection and complete genome sequencing of ToLCNDV from naturally symptomatic A. theophrasti, combined with experimental confirmation of the susceptibility of A. theophrasti to ToLCNDV, supports its status as a natural host of the virus. To the best of our knowledge, this is the first report of natural infection of A. theophrasti by ToLCNDV in China.