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

HomePlant DiseaseVol. 104, No. 3First Report of Alfalfa Leaf Curl Virus Infecting Alfalfa (Medicago sativa) in China Previous DISEASE NOTES OPENOpen Access licenseFirst Report of Alfalfa Leaf Curl Virus Infecting Alfalfa (Medicago sativa) in ChinaZ. P. Guo, J. X. Zhang, M. L. Wang, Y. Z. Guan, G. Qu, J. Y. Liu, Y. X. Guo, and X. B. YanZ. P. Guohttp://orcid.org/0000-0002-6550-3448College of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, J. X. ZhangCollege of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, M. L. WangCollege of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, Y. Z. GuanCollege of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, G. QuCollege of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, J. Y. LiuCollege of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, Y. X. Guo†Corresponding authors: Y. X. Guo; E-mail Address: yuxiaguo@163.com and X. B. Yan; E-mail Address: yxbbjzz@163.comCollege of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China, and X. B. Yan†Corresponding authors: Y. X. Guo; E-mail Address: yuxiaguo@163.com and X. B. Yan; E-mail Address: yxbbjzz@163.comCollege of Animal Science and Technology, Yangzhou University, Yangzhou 225000, China AffiliationsAuthors and Affiliations Z. P. Guo1 J. X. Zhang1 M. L. Wang1 Y. Z. Guan1 G. Qu1 J. Y. Liu1 Y. X. Guo1 † X. B. Yan2 † 1College of Animal & Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China 2College of Animal Science and Technology, Yangzhou University, Yangzhou 225000, China Published Online:30 Dec 2019https://doi.org/10.1094/PDIS-02-19-0318-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat 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.The author(s) declare no conflict of interest.References:Bejerman, N., et al. 2018. Arch. Virol. 163:799. https://doi.org/10.1007/s00705-017-3673-x Crossref, ISI, Google ScholarBernardo, P., et al. 2016. Virol. 493:142. https://doi.org/10.1016/j.virol.2016.03.016 Crossref, ISI, Google ScholarRoumagnac, P., et al. 2015. J. Virol. 89:9683. https://doi.org/10.1128/JVI.00453-15 Crossref, ISI, Google ScholarZerbino, D. R., and Birney, E. 2008. Genome Res. 18:821. https://doi.org/10.1101/gr.074492.107 Crossref, ISI, Google ScholarZhou, Q. Y., et al. 2016. Pratac Sci. 33:1297. https://doi.org/10.11829/j.issn.1001-0629.2015-0652 Google ScholarThe author(s) declare no conflict of interest.Funding: This work was supported by the National Key Research and Development Program of China (NO.2017YFD0502104), National Natural Science Foundation of China (NO.31502006), and Key Scientific Research of Higher Education Institutions of Henan Province (NO.15A230019).DetailsFiguresLiterature CitedRelated Vol. 104, No. 3 March 2020SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionPathogenicity of Lasiodiploidia pseudotheobromae in a coffee plant 3 days after inoculation (R. L. Freitas-Lopes et al.). Photo credit: U. P. Lopes. Seedling blight of soybean caused by soilborne pathogens (J. R. Lamichhane et al.). Photo credit: M. I. Chilvers. Metrics Downloaded 2,432 times Article History Issue Date: 3 Mar 2020Published: 30 Dec 2019First Look: 22 Oct 2019Accepted: 18 Oct 2019 Pages: 1001-1001 Information© 2020 The American Phytopathological SocietyFundingNational Key Research and Development Program of ChinaGrant/Award Number: NO.2017YFD0502104National Natural Science Foundation of ChinaGrant/Award Number: NO.31502006Key Scientific Research of Higher Education Institutions of Henan ProvinceGrant/Award Number: NO.15A230019Keywordsfirst reportalfalfa leaf curl virusalfalfaChinaThe author(s) declare no conflict of interest.Cited ByHigh-Throughput Sequencing for Deciphering the Virome of Alfalfa (Medicago sativa L.)11 September 2020 | Frontiers in Microbiology, Vol. 11Alfalfa leaf curl virus is transmitted by Aphis craccivora in a highly specific circulative mannerVirology, Vol. 546Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus13 March 2020 | Pathogens, Vol. 9, No. 3