First Report of Alfalfa Leaf Curl Virus Infecting Alfalfa (Medicago sativa) in China

Abstract

Alfalfa (Medicago sativa L.) is one of the most important leguminous forage crops globally and has grown in importance in China with ∼4.72 million hectares in 2015. A previous study reported the occurrence of several common viruses (e.g., alfalfa mosaic virus [AMV], white clover mosaic virus, bean yellow mosaic virus, and cowpea mosaic virus) in alfalfa fields in China, in Anning District and Gaolan County of Lanzhou, and Jingtai County of Baiyin City, Gansu Province (Zhou et al. 2016). In April 2017 a survey was conducted to identify viruses infecting alfalfa in a temperate region: Yuanyang County, Henan Province (N35°01′, E113°43′). From one field, 117 alfalfa leaf samples with symptoms of macular mosaic (n = 26), mottle mosaic (21), etiolation (1), shrinkage (27), mosaic shrinkage (25), and dwarfism (17) were collected. Total RNA was extracted from a composite sample comprising six leaves exhibiting each above-mentioned symptom using an EASYspin Plant Micro RNA Rapid Extraction Kit (Aidlab Biotechnologies, Beijing, China) following the manufacturer’s guidelines. The RNA was then subjected to high-throughput sequencing of small RNA to detect any viruses using the Illumina Hiseq4000 platform at Biomarker Technologies (Beijing, China). Over 20 million clean reads between 18 and 35 nt were obtained. Using Velvet 1.0 software (Zerbino and Birney 2008), 6,942 contigs from 33 to 416 nt were assembled. Although the majority of the contigs were mapped to the host genome, 58, 13, and 10 contigs were mapped to AMV (Alfamovirus, Bromoviridae, reference sequences [RSs] AMV-RNA1 NC_001495, AMV-RNA2 NC_002024, and AMV-RNA3 NC_002025), alfalfa dwarf virus (ADV, Rhabdovirus, Rhabdoviridae, RS KP205452), and alfalfa leaf curl virus (ALCV, Capulavirus, Geminiviridae, RS KX574859), respectively. The 10 projected ALCV contigs totaled 1,113 nt, accounting for 40.47% of the complete ALCV RS of 2,750 nt. The nucleotide sequence identity between these contigs and the ALCV RS ranged from 96 to 100%. Total DNA was then extracted from the composite sample using a New Plant Genomic DNA Rapid Extraction Kit (Aidlab Biotechnologies) following the manufacturer’s guidelines. The presence of ALCV in the pooled sample was confirmed by PCR using an ALCV-specific primer pair, ALCV-1F 5′-TGGAATATTGTGCTGCTTGG-3′ and ALCV-1R 5′-ATTTTGGGACTTGTGCTCCA-3′ (Roumagnac et al. 2015), and subsequent cloning and Sanger sequencing. The 785-nt-long amplicon, deposited in GenBank as MK422439, exhibited a sequence identity of 96% to the ALCV RS. To further confirm the result, the full genome of ALCV in the pooled sample was amplified by fusion PCR using improved primer pair ALCV-F 5′-CCCTGGCCTGCTAAAGTGGCCCAATTCAACATGG-3′ and ALCV-R 5′-CCAGGGGGCCTTATTCCTCTGGGACCG-3′ adapted from Bernardo et al. (2016), cloned, and sequenced. The complete 2,750-bp sequence was deposited in GenBank as MK422438. BLASTn analysis indicated the sequence shared 90 and 98% identity with previously reported ALCV isolates from France (KT214370) (Roumagnac et al. 2015) and Argentina (MG792039) (Bejerman et al. 2018), respectively, thus confirming the ALCV identity of the virus. To reveal whether ALCV was associated with the symptoms, all samples were subjected to PCR with the primer pair ALCV-1F/ALCV-1R. ALCV was detected in 38 of 117 samples: in 8 of 26 samples with macular mosaic, 5 of 21 with mottle mosaic, 11 of 27 with shrinkage, 8 of 25 with mosaic shrinkage, and 6 of 17 with dwarfism, but not in the single plant showing etiolation. Interestingly, AMV was detected in the pooled sample as well as all 117 individual samples by RT-PCR, whereas ADV failed to be detected by RT-PCR in any of the samples. These results demonstrated that ALCV was present in alfalfa in China, and it often coinfected alfalfa plants with AMV. This is the first report of ALCV infecting alfalfa in China. The distribution of ALCV within alfalfa-growing regions, the identity of potential virus reservoirs, and the economic impact have yet to be determined. The results help expand understanding of this virus and help in selection of virus-free propagation material for resistant breeding of alfalfa and production of high-quality forage.