First Report of Alfalfa Mosaic Virus on <i>Cynanchum rostellatum</i> in China

Alfalfa mosaic virus (AMV), a globally distributed plant pathogen in the genus Alfamovirus (family Bromoviridae), was first identified as the causative agent of alfalfa mosaic disease in 1931 (Hull R., 1969; Moreira AG et al., 2010). This virus has affected over 600 species across 70 families under natural and experimental conditions (Wang X et al., 2023), and is transmitted by aphids, seeds and pollen, and mechanical inoculation (Hull R., 1969). Typical infection symptoms include mild mottling, chlorotic local lesions, mosaic patterns, mottled appearance, necrosis and reduced yield. Cynanchum rostellatum (Apocynaceae), a medicinal plant widely distributed in China (Wei LL et al., 2019), was observed with virus-like symptoms in August 2022. Approximately 20% of C. rostellatum plants in a natural field near the Sijiazi Village, Kuangdonggou Town, Gaizhou City of Liaoning Province, exhibited chlorotic ringspot, yellowing mosaic and white mosaic symptoms (Fig. S1). Two symptomatic leaves (samples B5 and B6; Fig. S1 c-d) were subjected to RNA sequencing at BGI Genomics (Wuhan, China). Ribosomal RNA-depleted libraries were sequenced on the MGISEQ-2000 platform, yielding 43.21 M (B5) and 42.54 M (B6) clean reads. De novo assembly using CLC Genomics Workbench 11 (Qiagen, USA) and Trinity v2.0.6 (Haas et al., 2013) generated contigs with 64,497.08 to 121,895.91×coverage. Blastx analysis revealed three AMV-associated contigs (2,023 nt-3,641 nt) sharing 98.5%-99.2% nucleotide identity with reference sequences (GenBank accession no. OL521600, MT596937 and OL521627). Sample B5 was selected for full-genome amplification using seven specific primers (Table S1) following the protocol of the PrimeScriptTM One-Step RT-PCR Kit (Takara, Dalian, China). The RNA 1 (3,643 nt), RNA2 (2,593 nt) and RNA3 (2,027 nt) sequences of isolate LNMJ (GenBank accession no. PP079913-PP079915) shared the highest identity with Chinese AMV isolates from Medicago sativa (98.5%-99.2%). Phylogenetic analysis of RNA3 further supported close clustering with isolate XH1-XJ1 (Fig S2). Three specific primer pairs (LM-AMV-TJ-1F/1R, LM-AMV-TJ-2F/2R, LM-AMV-TJ-3F/3R) were developed to test 14 samples collected within 50 meters of B5/B6. The detection rates for RNA1-3 were 78.57% (11/14), 57.14% (8/14), and 100% (14/14), respectively. The mechanical inoculation of LNMJ to eleven Nicotiana benthamiana plants confirmed infectivity, with all plants testing positive for all three RNAs via RT-PCR. Additionally, seventeen plants (including Capsicum annuum, Apium graveolens, Ipomoea batatas, Sicyos angulatus, Glycine max, Abutilon theophrasti, Arachis hypogaea, Cucumis sativus, Luffa acutangula, Agastache rugosa, Chelidonium majus, Glycine max, Cucurbita moschata, Helianthus tuberosus, Prunus pseudocerasus, Malus pumila and Prunus persica) exhibiting virus-like symptoms nearby the B5 and B6 plants were screened, revealing AMV-positive results in cucumber and Abutilon theophrasti plants. More field testing is needed to understand the role of this virus host.