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.

Sequence diversity of readthrough proteins of Soybean dwarf virus isolates from the Midwestern United States.

The amino acid sequence diversity of readthrough proteins (RTPs) of 24 dwarfing isolates of Soybean dwarf virus (SbDV) from Wisconsin and Illinois was analyzed. The RTP, a minor component of viral capsids, has a significant role in specificity of aphid transmission of luteovirids. Among the isolates, nucleotide sequence identities ranged from 95 to 100%. The predicted amino acid sequences differed at 56 amino acid positions in the 54 kDa RTD compared to only five positions in the 22 kDa CP. Phylogenetic analysis of both amino acid and nucleotide sequences showed three distinct clusters of SbDV isolates.

First Report of Soybean dwarf virus in Soybean in Northern Illinois.

Soybean dwarf virus (SbDV), a member of the Luteoviridae, is transmitted persistently by colonizing aphids and causes significant yield losses in soybean (Glycine max L.) in Japan. In the United States, SbDV is endemic in red and white clover (Trifolium pratense L. and T. repens L.) (1,3). Even so, SbDV has been detected in soybean only in Virginia (2) and Wisconsin (4). A study conducted in Illinois during 2001 and 2002 detected SbDV in clover but not soybean (3). During August of 2006, two surveys for virus diseases in soybean were conducted in Illinois. In the first survey, 30 soybean leaf samples were collected without regard for symptoms from each of 10 fields in each of five northern Illinois counties (Carroll, Jo Daviess, Ogle, Stephenson, and Winnebago). In the second survey, 10 random soybean leaf samples and 10 samples with virus-like symptoms were collected from each of 30 soybean rust sentinel plots spread throughout Illinois. Total RNA was extracted from pools of 90 to 100 plants and analyzed by quantitative real-time reverse transcriptase (QRT)-PCR using a fluorescently labeled minor groove binding probe (VIC-5'-AGCATATCCAAAGACGC-3'-MGBNFQ, nt 2358-2374) and flanking primers (5'-TGGCTATTATAGAATGGTGCGTAAAC-3', nt 2327-2351; and 5'-GCCATGGAAATGAGGGAATG-3', nt 2395-2376). From the first survey, pools from Carroll, Jo Daviess, and Ogle were positive for SbDV. Analysis of individual leaf samples from positive pools by double-antibody sandwich-ELISA (Agdia, Elkhart, IN) showed that one sample in each county was positive for SbDV. On the basis of the number of randomly sampled plants, the incidence of SbDV infection in northern Illinois was approximately 0.3%. In the second survey, SbDV was detected in one pool containing symptomatic plants from five soybean rust sentinel plots. Further QRT-PCR analysis showed that the sentinel plot in Bureau County was positive for SbDV. Because of the sampling protocols used, it was not possible to determine symptom phenotypes of SbDV-positive samples. Sequence analysis of the combined coat protein (CP) and readthrough domain (RTD) encoding region (nt 3019-5094) of SbDV isolates from Bureau (GenBank Accession No. EU095847) and Carroll (GenBank Accession No. EU095846) counties showed that the predicted amino acid sequences were 96 and 95% identical to a Japanese dwarfing isolate of SbDV (GenBank Accession No. AB038150), respectively. The predicted CP amino acid sequences of the Illinois isolates were identical and RTD amino acid sequences differed at six positions. To our knowledge, this is the first report of infection of soybean plants in Illinois with SbDV. References: (1) V. D. Damsteegt et al. Phytopathology 89:374, 1999. (2) A. Fayad et al. Phytopathology (Abstr.) 90(suppl.):S132, 2000. (3) B. Harrison et al. Plant Dis. 89:28, 2005. (4) A. Phibbs et al. Plant Dis. 88:1285, 2004.

Green Stem Disorder of Soybean.

Green stem disorder of soybean (Glycine max) is characterized by delayed senescence of stems with normal pod ripening and seed maturation. Three different field research approaches were designed to determine the relationship of green stem disorder to Bean pod mottle virus (BPMV) and other potential factors that may be involved in causing this disorder. The first research approach surveyed green stem disorder and BPMV in individual plants monitored in several commercial soybean fields during three growing seasons. Leaf samples from maturing plants (growth stage R6) were tested by enzyme-linked immunosorbent assay (ELISA) for BPMV. The percentage of monitored plants infected with BPMV at growth stage R6 in some fields was higher than the incidence of green stem disorder at harvest maturity. Many plants infected with BPMV did not develop green stem disorder, and conversely, many plants that had green stem disorder were not infected with BPMV. According to a chi-square test of independence, the data indicated that green stem disorder was independent of BPMV infection at growth stage R6 (P = 0.98). A second research approach compared green stem disorder incidence in an identical set of soybean entries planted in two locations with different levels of natural virus infection. Despite differences in virus infection, including BPMV incidence, 20 of 24 entries had similar green stem disorder incidence at the two locations. A third research approach completed over two growing seasons in field cages showed that green stem disorder developed without BPMV infection. BPMV infection did not increase green stem disorder incidence in comparison to controls. Bean leaf beetle, leaf hopper, or stinkbug feeding did not have an effect on the incidence of green stem disorder. The cause of the green stem disorder remains unknown.

Occurrence of Seed Coat Mottling in Soybean Plants Inoculated with Bean pod mottle virus and Soybean mosaic virus.

Soybean seed coat mottling often has been a problematic symptom for soybean growers and the soybean industry. The percentages of seed in eight soybean lines with seed coat mottling were evaluated at harvest after inoculating plants during the growing season with Bean pod mottle virus (BPMV), Soybean mosaic virus (SMV), and both viruses inside an insect-proof cage in the field. Results from experiments conducted over 2 years indicated that plants infected with BPMV and SMV, alone or in combination, produced seed coat mottling, whereas noninoculated plants produced little or no mottled seed. BPMV and SMV inoculated on the same plants did not always result in higher percentages of mottled seed compared with BPMV or SMV alone. There was significant virus, line, and virus-line interaction for seed coat mottling. The non-seed-coat-mottling gene (Im) in Williams isoline L77-5632 provided limited, if any, protection against mottling caused by SMV and none against BPMV. The Peanut mottle virus resistance gene Rpv1 in Williams isoline L85-2308 did not give any protection against mottling caused by SMV, whereas the SMV resistance gene Rsv1 in Williams isoline L78-379 and the resistance gene or genes in the small-seeded line L97-946 gave high levels of protection against mottling caused by SMV. The correlations (r = 0.77 for year 2000 and r = 0.89 for year 2001) between virus infection of the parent plant and seed coat mottling were significant (P = 0.01), indicating that virus infection of plants caused seed coat mottling.

Distribution of Leaf-Feeding Beetles and Bean pod mottle virus (BPMV) in Illinois and Transmission of BPMV in Soybean.

Bean leaf beetles (BLB; Cerotoma trifurcata) were collected in soybean (Glycine max) fields in 58 and 99 Illinois counties surveyed during the 2000 and 2001 growing seasons, respectively. In 2000, BLB counts were highest in the central portion of the state. BLB counts were lower the following year, but were more uniformly distributed throughout the state. BLB tested positive for Bean pod mottle virus (BPMV) in 37 of 41 counties assayed in 2000. In 2001, BLB tested positive for BPMV in 86 of 99 counties sampled. In 2000 and 2001, western corn rootworm (WCR; Diabrotica virgifera virgifera) adults were abundant in soybean fields only in east central Illinois. WCR adults tested positive for BPMV in 21 of 21 east central Illinois counties in 2000 and 20 of 24 sampled in 2001. BPMV was detected in soybean plants in 38 of 46 counties sampled in 2000. Field-collected WCR adults transmitted BPMV to potted soybean plants at low rates either directly from BPMV-infected soybean fields or with prior feeding on BPMV-infected plants. This is the first report of the distribution of BLB, WCR adults, and BPMV in Illinois and of BPMV transmission by adult WCR.

Prevention of morphological changes in alginate microcapsules for islet xenotransplantation.

BACKGROUND: Alginate microcapsule swelling, which occurs as a result of increased hydrophilicity owing to the Ca++ that remains after rapid chelation of the inner alginate core, is a problem in encapsulation. We have previously shown that exchange of the residual divalent Ca++ with the monovalent Na+ through the use of 6 mmol/L Na2SO4 decreases swelling in chelated alginate-polylysine-alginate microcapsules, and this process enhances their durability. The purpose of the present study was to examine the morphology of Na2SO4-treated microcapsules in long-term incubation with the use of serum-supplemented culture medium. METHODS: Spherical beads of purified alginate (3%) that were gelled with 1.1% CaCl2 were first coated with polylysine, and then with 0.24% alginate. After rapid chelation of the inner alginate core with 55 mmol/L sodium citrate, the capsules were either incubated for 30 minutes in 6 mmol/L Na2SO4 or left untreated (control). Each group of capsules was then placed in a flask containing Ham's culture medium supplemented with 20% porcine serum and incubated at 37 degrees C. RESULTS: The diameters of Na2SO4-treated capsules only increased modestly from a mean +/- SD of 635 +/- 22.08 to 684.53 +/- 17.86 microm (P<0.0001) by day 7, with no further increases thereafter. In contrast, control capsules showed a steady increase in their mean diameters, which changed from 639.55 +/- 21.44 to 735.48 +/- 108.85 microm (P < 0.0001) by day 66. In addition, whereas treated capsules remained spherical, control capsules showed progressive polymorphism. CONCLUSION: We have developed a new method of making more durable and stable microcapsules that can be used for islet cell xenotransplantation.

Durability of sodium sulfate-treated polylysine-alginate microcapsules.

Chelated hollow microcapsules are unstable under in vitro conditions because of their hygroscopic nature. Nongel inducing cations, such as Na+, stabilize the outer membrane of the alginate-polylysine-alginate microcapsules leading to more stable beads. We made different batches of empty capsules with a mean +/- SEM diameter of 607+/-11 microns, and found that within 1 week of incubating these capsules in normal saline at 37 degrees C, they increased to 718+/-10 microns (p < 0.05, n = 5). In initial experiments, we made different batches of capsules and divided them into two groups. One group was left untreated (control) whereas the other was treated with 6 mM Na2SO4 for 30 min, before incubation in saline at 37 degrees C. Control capsules increased in weight and size, before beginning to melt in less than 1 week. In contrast, treated capsules rapidly lost weight and remained intact during 1 month of follow-up. In perifusion experiments, we found no deleterious effect of sodium sulfate treatment on the function of islets enclosed in the capsules.

Solanaceous Weeds as Possible Sources of Cucumber mosaic virus in Southern Illinois for Aphid Transmission to Pepper.

Over 5,000 individual plants representing approximately 55 species from an area in southern Illinois where Cucumber mosaic virus (CMV) has been a major problem in pepper (Capsicum annuum) were tested for the presence of CMV by enzyme-linked immunosorbent assay (ELISA). Representative ELISA-positive samples were checked by western blot tests to confirm virus-specific reactions. Nearly all of the infected plants detected were either Solanum ptycanthum (eastern black nightshade) or Physalis spp. (principally P. heterophylla, groundcherry). Over 1,000 pepper transplants and approximately 500 tomato transplants, collected prior to planting, were negative for CMV by ELISA. In aphid transmission (arena) experiments, all five aphid species tested were capable of transmitting CMV from nightshade to pepper: Aphis fabae subsp. solanella, Aphis gossypii, Myzus persicae, Rhopalosiphum padi, and Sitobion avenae. Aphis fabae subsp. solanella, A. gossypii, and A. nerii were able to transmit CMV from P. heterophylla to pepper. Aphis fabae subsp. solanella was commonly found colonizing nightshade from May through October in southern Illinois.

Seed Transmission of Indian Peanut Clump Virus (IPCV) in Peanut and Millets.

An enzyme-linked immunosorbent assay (ELISA) procedure was developed to test peanut seed for Indian peanut clump virus (IPCV). A double antibody sandwich form of ELISA detected the Hyderabad isolate (IPCV-H) in seed of peanut. Correlation was established between the results from ELISA performed on cotyledons of peanut seed and grow-out tests. Seed transmission in the field-infected peanut plants ranged from 3.5 to 17%, depending on the genotype. The transmission frequency was 48 to 55% in seed collected from plants infected through seed. Because testae of all seed contained viral antigen, their removal was essential for the determination of frequency of seed transmission. Apparently the virus present only in cotyledons and embryo contributed to the seed transmission. For the first time, IPCV-H was shown to be seed transmitted in finger millet (Eleusine coracana), foxtail millet (Setaria italica), and pearl millet (Pennisetum glaucum) at frequencies of 5.2, 9.7, and 0.9%, respectively. Seed transmission was not observed in sorghum (Sorghum bicolor). Significance of seed transmission in millet crops is discussed.