First Report of Alfalfa mosaic virus and Soybean dwarf virus on Soybean in North Dakota

Soybean (Glycine max L.) is the major oilseed crop in North Dakota, with production concentrated in the eastern half of the state. Only one virus, Soybean mosaic virus, has been reported from soybean in North Dakota (4). In July and August of 2010, 200 soybean fields from 25 counties were surveyed for Alfalfa mosaic virus (AMV) and Soybean dwarf virus (SbDV). AMV and SbDV have been detected infecting soybean in multiple Midwestern states and are reported to reduce yields in soybean (1,3). Each field was sampled with a grid pattern across the area with at least 8 km between fields. From each field, leaves were collected from 20 plants without regard for symptoms along a transect of approximately 170 m. Leaves from each field were bulked and sap was extracted in phosphate buffer and stored at -80°C until tested using double-antibody sandwich (DAS)-ELISA with positive controls and reagents and protocols from Agdia Inc. (Elkhart, IN). Using DAS-ELISA, AMV was detected in eight of the 200 soybean fields. For sequence-based virus detection, total RNA was extracted from all field samples using a Qiagen RNeasy Plant Mini Kit (Germantown, MD), pooled, depleted of ribosomal RNA (RiboZero Epicentre, Madison, WI), reverse transcribed, sequenced using an Illumina HiSeq2000 (San Diego, CA), and compared to all available viral amino acid and nucleotide sequences. The analysis detected AMV and SbDV sequences in the pool of 200 fields. The presence of AMV and SbDV was confirmed by quantitative real-time reverse transcription (qRT)-PCR (1,3). For AMV, total RNA extracted from bulked leaves from each of the 200 fields was tested using AMVspecific primers (5'-ATGCTACCCAGGCATGTATATTT-3' and 5'-GCTGCATCTTTCGCCAGAA-3') and a FAM-labeled minor-groove binding TaqMan probe (5'-TGGACGTTACCCCCGGA-3'). One field sample from Cass county positive for AMV by ELISA was also positive for AMV by qRT-PCR, confirming the presence of AMV in the field sample. For SbDV, an RNA pool representing all 200 fields, subpools, and individual field samples was analyzed by qRT-PCR (1) and DAS-ELISA. One field sample from Grand Forks County tested positive for SbDV by qRT-PCR and DAS-ELISA, confirming the presence of SbDV in the field sample. Because leaf samples were collected and pooled prior to analysis, the symptom phenotypes of individual field plants could not be correlated with positive ELISA or qRT-PCR results. AMV was reported by the American Phytopathological Society Virus Working Group (2007 to 2008) to be widely prevalent in North Dakota, but we found no peer-reviewed reports of verified AMV identification on any crop in the state. To our knowledge, this is the first confirmed report of AMV and SbDV infecting soybean in North Dakota. Serious infestations by the soybean aphid, Aphis glycines, requiring chemical control, have occurred in recent years in North Dakota. Because A. glycines is a vector for both viruses (1,2), the distribution, incidence, and agronomic impact of AMV and SbDV could be affected in years when A. glycines infestations are high. In addition, AMV is seedborne in soybean and may cause seed mottling, a concern for the food-grade soybean industry where production is primarily for export. References: (1) V. D. Damsteegt et al. Plant Dis. 95:945, 2011 (2) J. H. Hill et al. Plant Dis. 85:561, 2001. (3) H. A. Hobbs et al. Plant Health Progress doi:10.1094/PHP-2010-0827-01-BR, 2010. (4) B. D. Nelson and L. L. Domier. Plant Dis. 93:760, 2009.