First Report of Alfalfa Mosaic Virus ( <i>Alfamovirus</i> AMV) in Alfalfa ( <i>Medicago sativa</i> L.) in Yunnan, China

Alfalfa (Medicago sativa) is the world’s most important feed crop and is known as “the Queen of forage”, providing animal feed with high protein, a balanced amino acid profile, as well as rich vitamins and minerals (Ye et al. 2025). Alfalfa has been cultivated in about 80 countries around the world, mainly concentrated in the United States, Argentina, and China, with a total area of over 30 million hectares (Zhang et al. 2025). In June 2025, alfalfa plants showing yellowing, mosaic, and dwarfing symptoms, suspected to be caused by viral infection, were found in fields in Qujing City, Yunnan Province. The observed area showing disease symptoms was approximately 0.2-0.33 hectares and the disease incidence was calculated to be about 40% from 20 randomly selected plants. For viral pathogen identification, symptomatic leaves from 10 alfalfa plants were pooled for small RNA deep sequencing (sRNA-Seq). Total RNA extraction from plant samples was performed using the VAMNE Magnetic Universal Plant Total RNA Kit (Vazyme Biotech, Nanjing, China). Small RNA library construction was performed using the small RNA Sample Pre Kit (Genepioneer, Nanjing, China), and high-throughput sequencing was performed on the Illumina NovaSeq platform by Genepioneer (Nanjing, China). A total of 0.23 G Reads were obtained from small RNA sequencing, and the Q30 was above 95.33%. Redundant reads were removed by using the cutadapt software (version 1.18) to obtain 10,316,710 high-quality Clean Read sequences (between 18 and 26 nucleotides) for subsequent analysis. The sRNA clean reads were aligned to reference virus sequences using bwa (version 0.7.17-r1188). A total of 846,069 reads were aligned to the reference virus genome, with a mapping ratio of 8.3%. Then, velvet (version 1.1.07) was used to de novo assemble the viral contigs of the unaligned sRNA sequences. After merging the contig sequences and using cd-hit to remove redundancy, a total of 509 unique contigs were obtained. The redundant contig sequences were sequentially aligned with the viral nucleic acid database using BLASTN; a total of 55 small contigs, ranging from 51 to 1033 nt, were identified, with nucleotide sequence identities of 91.82 to 95.07% to Alfamovirus AMV (Alfalfa mosaic virus, genus alfamovirus, family Bromoviridae). To confirm the sRNA-Seq results, PCR was performed with the specific primers AMV-RNA3-F/AMV-RNA3-R (5'-GTTTTAATACCATTTTCAAAATATTCC-3'/5'-GCATCCCTTAGGGGCATTCATGC-3'). The primer were originally designed from the complete sequence of Alfamovirus AMV strain HZ segment RNA3 (GenBank accession numbers: HQ316637.1). Amplicons approximately 2,100 bp in size were amplified, sequenced (Tsingke, Beijing, China), and obtained the complete AMV-QJ RNA3 genome sequence (accession no. PX412439.1). Blastn analysis revealed 99.65% sequence identity with the Alfamovirus AMV isolate AMV_R3_ALF1071 (ON669107.1) based on the complete RNA3 genome. Alfamovirus AMV is distributed worldwide and highly harmful. To our knowledge, it has not been reported in Yunnan, China. This study enriches the distribution of Alfamovirus AMV in China and supports comprehensive prevention and control of forage virus diseases.