First Report on Wheat Dwarf India Virus Infecting Maize (<i>Zea mays</i>) in China

Maize (Zea mays) is among the most extensively cultivated crops in China, serving as a crucial source of food, feed, and biofuel. Viral diseases, such as maize lethal necrosis, contribute significantly to corn yield losses (Mahuku et al. 2015; Jiao et al. 2022). In 2022, an infected maize plant exhibiting virus-like symptoms, including yellowing and dwarfing, was observed in Hainan, China (Supplementary Figure S1). To identify the pathogen responsible, leaf tissues were collected and their total RNA was extracted and assessed for quality. Then the plant was tested by RT-PCR for the following six viruses reported in China causing yellowing and/or dwarfing, including sugarcane mosaic virus, barley yellow dwarf virus, maize yellow mosaic virus, southern rice black streaked dwarf virus, maize chlorotic mottle virus and sorghum mosaic virus (Chen et al. 2016; Gu et al. 2023). None of the viruses was detected (Supplementary Figure S2). Subsequently, a cDNA library was constructed using the Small RNA Sample Pre Kit (Illumina, San Diego, USA), and sequencing was performed on the Illiumia Novaseq platform (Biomarker Technologies Corporation, Beijing, China). After eliminating low-quality reads, non-coding RNAs and duplicate reads, the remaining reads were assembled into long contigs using Velvet 1.0.5 software with a k-mer value of 17, resulting in 14 contigs ranging from 55 nt to 931 nt in length, showing nucleotide identity ranging from 94.52% to 100% with the genome of wheat dwarf India virus (WDIV, GenBank accession no. NC_017828.1). WDIV belongs to the genus Mastrevirus, family Geminiviridae, and possesses a monopartite circular single-stranded DNA genome of 2.5 to 2.7 kb (Kumar et al. 2014a). To validate the findings, total DNA was extracted from three symptomatic leaves, and the coat protein (CP) gene of WDIV was amplified using the PCR protocol with primers CP01 (5'-ATGTCTCAGGTGAAGAAGAGGAC-3') and CP02 (5'-CTACTGGTTGCCGATACTCTTGA-3') (Kumar et al. 2014b). With 99% coverage, a 744 bp fragment obtained displayed 96.10% similarity to the coat protein gene of wheat dwarf India virus clone (GenBank accession no. MN240337.1) (Supplementary Figure S3). To obtain the complete genome sequence of the virus, PCR with our own designed primers WDIV-2F (5'-ACAAGCATCCGACGAAGCAGA-3') and WDIV-2R (5'-AAATATTCTAATACAGGCACAGGCT-3') yielded a circular DNA of 2,871 bp. The amplicon was cloned into the pEASY-T1 cloning vector (Transgen, Beijing, China), and three independent positive colonies of Escherichia coli DH5α carrying the viral amplicon were sequenced. The nucleotide sequences obtained had 96.73%, 96.80%, and 96.84% nt identity with the complete gene of wheat dwarf India virus clone (GenBank accession no. MN240328.1) and were deposited in the GenBank database under accession numbers OR282786 - OR282788. To identify the occurrence and distribution of WDIV infection, 12 maize samples were collected from 2 different regions of Hainan province (Sanya and Wuzhishan) and tested using PCR with primers CP01 and CP02, the results showed that 2 samples collected in Sanya were positive (Supplementary Figure S4). Previous reports confirmed that WDIV infects barley, wheat, and sugarcane (Boulton et al. 2002). This study represents the first report of WDIV infecting maize in China, providing valuable insights into its host range and offering potential guidance for managing viral diseases in maize.