First Report of Tomato leaf curl New Delhi virus Infecting Common bean (Phaseolus vulgaris) in China.

Abstract

Common bean (Phaseolus vulgaris), the largest legume vegetable worldwide for its edible dry seeds or green pods, is widely cultivated in China. In March 2023, common bean (cultivar Yulong No.3) showing virus-like symptoms, including leaf curling and plant stunting, were found in Mengcheng (Anhui Province, China), with an incidence rate about 2% (n ≈ 3,000) in an area of about 700 m2 (Fig. S1A). To assess the causal agent of the suspected disease, symptomatic leaves from eight common bean plants were collected and mixed for high-throughput RNA sequencing (RNA-seq) at Novogene Bioinformatics Institute Co., Ltd. (Tianjin, China). Total RNA extraction was performed using TRIzol Reagent (Invitrogen; Carlsbad, CA, USA), followed by cDNA library preparation with the Illumina TruSeq RNA Library Prep Kit v2 (Illumina; San Diego, CA, USA). Barcoded libraries were subsequently subjected to 150-bp paired-end sequencing on an Illumina HiSeq 4000 platform. A total of 9,778,308 clean reads were obtained, which were assembled into 107,133 contigs using Trinity (Version 2.8.5). The contigs were compared with the NCBI viral RefSeq database using BLASTx. The results show that the presence of two viral contigs displaying 99.85% and 99.63% identity to the DNA-A (accession No. OQ190946) and DNA-B (accession No. OQ190952) of Tomato leaf curl New Delhi virus (ToLCNDV) respectively, and no other viruses were identified. The whole genome of this ToLCNDV isolate was then amplified by rolling circle amplification (RCA) using phi29 DNA polymerase, and digested by restriction endonuclease (Sac Ⅰ for DNA-A and Hind Ⅲ for DNA-B). The digested fragments were cloned into the Litmus 28i vector and confirmed by DNA sequencing. The DNA-A and DNA-B sequences of the common bean-infecting ToLCNDV (ToLCNDV-HF23BC) were deposited in GenBank under the accession No. PP937118 (DNA-A, 2739 nt) and No. PP937119 (DNA-B, 2693 nt). BLASTn analysis of DNA-A showed that it had the highest similarity (99.85%, 2735/2739) with the isolate infecting tomato from Zhejiang province, China (accession No. OP356207; Li et al. 2023) and isolate infecting cucumber from Shanghai, China (accession No. OQ190946; Zeng et al. 2023); DNA-B had the highest similarity (99.70%, 2685/2693) with the isolate infecting melon from Shanghai, China (accession No. OQ190952; Zeng et al. 2023). The phylogenetic tree constructed based on DNA-A sequences showed that the DNA-A of ToLCNDV-HF23BC is closely related to isolates from other Asian countries, but more distantly to most of those from European (Fig. S2). In addition, to verify the infectivity of ToLCNDV isolate HF23BC, we constructed its infectious clone (Fig. S1B), and agrobacterium-infiltrated into Nicotiana benthamiana and common bean (Phaseolus vulgaris). At 10 days and 25 days post-inoculation (dpi), Nicotiana benthamiana and common bean plants exhibited typical leaf curling symptoms (Fig. S1C). ToLCNDV belongs to the genus Begomovirus of the family Geminiviridae, infecting a variety of crops such as Solanaceae, Cucurbitaceae and Euphorbiaceae, causing serious yield and economic losses worldwide (Zaidi et al. 2017). To the best of our knowledge, this is the first report of ToLCNDV infecting common bean in China. As an important leguminous crop in China, further research on the transmission and pathogenicity mechanism of ToLCNDV is needed to ensure the safe production of common bean.