First report of zoysia mosaic virus infecting zoysiagrass in China

Zoysiagrass (Zoysia spp.) is a widely used warm-season turfgrass on lawns, sports fields, and golf courses owing to its low-input management requirements (Loch et al., 2017). It tolerates abiotic stresses including drought, salinity, and foot traffic (Chandra et al., 2017), but remains susceptible to diseases like large patch caused by Rhizoctonia solani (Koehler and Miller 2019), and rust caused by Puccinia zoysiae (Zhang et al., 2022). Viral infections were rarely reported until 2023, when zoysia mosaic virus (ZoMV, Poacevirus; Potyviridae) was first identified in Z. matrella (Merrill) cultivar ‘Fox’ imported from Japan to the United States (Adhikari et al. 2023). In 2023, mild mosaic symptoms were observed on Z. japonica in a zoysiagrass germplasm evaluation field at China Agricultural University Experiment Station in Zhuozhou, Hebei Province (39°28′58.9″N, 115°58′7.9″E). To investigate the suspected viral agent, symptomatic leaves were fixed in 4% paraformaldehyde, embedded in Spurr’s resin, sectioned, and examined under a Hitachi transmission electron microscope (Yan et al., 2015). Flexuous, filamentous viral particles (~280 nm) and pinwheel-like inclusions were observed in the cytoplasm, indicating Potyviridae infection, whose genome carries a 3′ poly(A) tails (López-Moya and García 2008). RNA from infected leaves was sequenced at the Wuhan iGeneBook Biotechnology Co., Ltd. for virus identification. Polyadenylated RNA was enriched using oligo(dT) beads, fragmented, and reverse-transcribed. After end repair, A-tailing, and adaptor ligation, the library was PCR-amplified. Double-stranded DNA was denatured and circularized to form single-stranded circles, followed by rolling circle amplification to generate DNA nanoballs (DNBs). Sequencing on the DNBSEQ-T7 platform (BGI, China) produced 7.7 Gb of high-quality data, comprising 51,567,374 reads (150 bp, PE150). Clean reads were assembled de novo using MEGAHIT v1.1.3 (Li et al., 2016), and contigs were refined with CAP3 (v02/10/15) using the overlap-layout-consensus strategy. Redundant sequences were removed using CD-HIT-EST (v4.6, 95% identity), resulting in 73,295 non-redundant contigs. BLASTn and BLASTx searches (E ≤ 1e−5) against NCBI databases showed that Contig_4514 (8,758 bp) shared 84.9% identity with ZoMV (OP425115.1). The 5′ and 3′ viral ends were confirmed using the HiScript-TS 5′/3′ RACE Kit (Vazyme, RA101) and Sanger sequencing. The complete genome was amplified in three overlapping fragments using specific primers (ZoMV5′-F/ZoMV-CI-R, ZoMVCI-F/ZoMVCP-R, ZoMV-CP-F/3′-R). These fragments were assembled in yeast using the pCB301-2μ-HDV vector (Yang et al., 2024) and Sanger sequenced. The acquired 9,794-nt genome shared 84.7% nucleotide and 95.1% polyprotein amino acid identity with strain ZM864 (OP425115.1). This isolate was designated ZoMV ZS101 (PV643905). Notably, ZM864’s CP was 939 bp (313 aa), while ZS101’s was 984 bp (328 aa), with 80.2% nucleotide and 88.4% amino acid identity. Seventeen zoysiagrass samples were randomly collected from the same field and tested by RT-PCR using the primer pair ZoMV-CP-F2/ZoMV-CP-R2, with 3 (17.6%) testing positive. To our knowledge, this is the first report of ZoMV in Z. japonica and in China. Given that mite transmission is characteristic of Poacevirus triticum mosaic virus (TriMV) (Fellers et al., 2009), identifying the potential vector of ZoMV is essential for evaluating the risks of turfgrass health.