First Report of Soybean Vein Necrosis Disease Caused by Soybean vein necrosis-associated virus in Wisconsin and Iowa.

Several viral diseases of soybean (Glycine max) have been identified in the north-central U.S. soybean production area, which includes Wisconsin and Iowa (2). Previously, Soybean vein necrosis disease (SVND) caused by Soybean vein necrosis-associated virus was reported in Arkansas, Tennessee, and other southern states (4). In September 2012, soybean plants with symptoms similar to those reported for SVND (4) were observed in fields across Wisconsin and Iowa. Symptoms included leaf-vein and leaf chlorosis, followed by necrosis of the leaf veins and eventually necrosis of the entire leaf. Six samples with symptoms indicative of SVNaV were collected from research plots located at the West Madison Agricultural Research Station located in Madison, WI. An additional three samples were collected from three locations in central Iowa. Total RNA extracted from each sample using the Trizol Plus RNA purification kit (Invitrogen, Carlsbad, CA) was used to generate complementary DNA (cDNA) using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturers' suggested protocols. The resulting cDNA was used as template in a PCR with SVNaV-specific primers, SVNaV-f1 and SVNaV-r1 (3). PCRs of two of the six Wisconsin samples and two Iowa samples were positive. Amplification products were not detected in the other five samples. The amplification products from the four strongly positive samples were purified using the Wizard SV Gel and PCR Purification Kit (Promega, Madison, WI) following the manufacturer's suggested protocol and were subjected to automated sequencing (University of Wisconsin Biotechnology Center or Iowa State University, DNA Sequencing Facilities). BLASTn (1) alignments of the 915-bp consensus sequence revealed 98% and >99% identity of the Wisconsin and Iowa samples, respectively, with the 'S' segment of the SVNaV 'TN' isolate (GenBank Accession No. GU722319.1). Samples from the same leaf tissue used above, were subjected to serological tests for SVNaV using antigen coated-indirect ELISA (3). Asymptomatic soybeans grown in the greenhouse were used as a source of leaves for negative controls. These tests confirmed the presence of SVNaV in eight symptomatic soybean leaflets collected in Wisconsin and Iowa. The asymptomatic control and one Iowa sample, which was also PCR-negative, were also negative by serological testing. Six additional samples from soybean fields in as many Wisconsin counties (Fond Du Lac, Grant, Green, Juneau, Richland, Rock) tested positive for SVNaV using specific primers that amplify the 'L' segment (4). The sequenced amplification products (297-bp) showed 99 to 100% homology to the L segment of the TN isolate (GU722317.1). To our knowledge, this is the first report of SVNaV associated with soybean and the first report of SVND in Wisconsin and Iowa. Considering that little is known about SVNaV, it is assumed that it is like other Tospoviruses and can cause significant yield loss (4). Soybean is a major cash crop for Wisconsin and Iowa, and infection by SVNaV could result in potential yield loss in years where epidemics begin early and at a high initial inoculum level. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) G. L. Hartman et al. Compendium of Soybean Diseases, 4th ed, 1999. (3) B. Khatabi et al. Eur. J. Plant Pathol. 133:783, 2012. (4) J. Zhou et al. Virus Genes 43:289, 2011.

First Report of Cactodera milleri in Wisconsin.

During the summer of 2005, lemon-shaped cysts and second-stage juveniles of a cyst nematode were recovered from soil at the University of Wisconsin Agricultural Research Station in Hancock, WI. Samples were collected on multiple dates from a plot (61 × 12 m) in continuous potato production for 20 years with significant weed pressure. PCR-restriction fragment length polymorphism profiles of the internal transcribed spacer (ITS) 1 region using restriction enzyme HhaI indicated Cactodera spp. (2). Morphological observations and morphometrics made on cysts, males, J2s, and eggs were consistent with Cactodera milleri Graney and Bird, 1990 (1). Host range studies were conducted in a growth chamber. Soybean, potato, and beet did not support nematode development and reproduction. Common lambsquarters (Chenopodium album), a known host of C. milleri, was an excellent host. No obvious aboveground disease symptoms were evident on lambsquarters in the growth chamber assay. This detection represents the first record of C. milleri in Wisconsin. Unless detailed morphological or molecular measurements are made, C. milleri may be easily confused with the soybean cyst nematode, Heterodera glycines. The presence of lambsquarters in fields planted with glyphosate-resistant soybeans makes the recovery of both nematode species in a single soil sample possible. References: (1) L. S. O. Graney and G. W. Bird. J. Nematol. 22:457, 1990. (2) A. L. Szalanski et al. J. Nematol. 29:255, 1997.

First Report of Soybean dwarf virus on Soybean in Wisconsin.

Soybean dwarf virus (SbDV) causes widespread economic losses on soybean (Glycine max (L.) Merr.) in Japan (4), and has been reported on soybean in Virginia (2), in various legumes in the southeastern United States (1), and in peas in California (3). During late July and early August of 2003, soybean plants in Wisconsin were surveyed for SbDV. In 286 soybean fields at the R2-R4 growth stage, the uppermost fully unfurled leaf was collected from 10 plants at each of five sites. Samples were collected at random without regard to symptoms. SbDV symptom information was not recorded. Samples were stored on ice until frozen at -80°C. Five fields in four Wisconsin counties (Columbia, Lafayette, Sauk, and Waushara) tested positive for SbDV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). DAS-ELISA testing was conducted with reagents from Agdia, Inc (Elkhart, IN) following the manufacturer's protocol. Absorbance was read at 405 nm with a Stat Fax 2100 microplate reader (Awareness Technology, Inc., Palm City, FL) or visually evaluated. DAS-ELISA did not discriminate between strains of SbDV. The presence of SbDV was confirmed, and strain identity was inferred as dwarfing strain using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from homogenized leaf tissue, reverse transcribed, and amplified with the SuperScript One Step RT-PCR System (Invitrogen, Carlsbad, CA) and SbDV-specific primers (5'-CTGCTTCTGGTGATTACACTGCCG-3' and 5'-CGCTTTCATTTAACGYCATCAAAGGG-3'). Size of the RT-PCR products (110 bp) was consistent with the dwarfing strain, SbDV-D. All locations that tested positive for SbDV showed soybean aphids, Aphis glycines Matsumura (Homoptera: Aphididae), on 100% of soybean plants. Several aphid species have been reported to vector SbDV, but at this time, vector relations in the Wisconsin infections are unknown. To our knowledge, this is the first report of SbDV infecting soybean in Wisconsin. References: (1) V. D. Damsteegt et al. Plant Dis. 79:48, 1995. (2) A. Fayad et al. Phytopathology (Abstr.) 90(Suppl.):S132, 2000. (3) G. R. Johnstone et al. Phytopathology (Abstr.) 74:795(A43), 1984. (4) T. Tamada et al. Ann. Phytopathol. Soc. Jpn. 35:282, 1969.