First Report of Sudden Death Syndrome of Soybean in Wisconsin.

In August of 2006, soybean (Glycine max (L.) Merr.) plants collected from Columbia, Dane, Green Lake, Walworth, Jefferson, and Waushara counties in southern Wisconsin exhibited symptoms typical of sudden death syndrome (SDS) caused by Fusarium virguliforme O'Donnell & Aoki [synonym F. solani (Mart.) Sacc. f. sp. glycines] (1). Foliar symptoms ranged from chlorotic spots to severe interveinal chlorosis and necrosis. Taproots of symptomatic plants were necrotic and stunted and stems exhibited a light tan discoloration, but never the dark brown discoloration typical for brown stem rot, a disease with similar foliar symptoms. Isolations from root and crown tissue of symptomatic plants were made using one-quarter-strength potato dextrose agar (PDA) amended with 100 ppm of streptomycin. Slow-growing, white-to-cream fungal colonies with blue and turquoise sporodochia were observed. Spores produced in sporodochia grown on PDA ranged in size from 32.5 to 70 µm long (average 53.1 µm) and 3 to 6 µm wide (average 4.4 µm) and with 3-5 septa (mode of 3). Isolates were characteristic of F. virguliforme based on colony morphology, spore morphology and size, and the absence of microconidia (3). The identity of F. virguliforme was confirmed by PCR amplification and DNA sequencing of the ITS, BT1, Act, and EF1B regions. All isolate sequences exhibited single nucleotide polymorphisms that matched the sequences of these regions of F. virguliforme. Koch's postulates were conducted to confirm that the causal agent of the observed symptoms was F. virguliforme. Inoculum of single-spore isolates was produced on sterilized sorghum seed. After 14 days of incubation at 20 to 22°C and a 12-h photoperiod, the sorghum seed was assayed to determine colonization incidence by transferring seeds to PDA. In all trials, sorghum seed was 100% infested. Infested sorghum seeds (35) were placed in potting soil at 2 cm beneath each seed of the susceptible soybean cv. Williams 82 (4). Noninfested sorghum seed was used for a noninoculated control. Three trials were performed, each using 15 replicates of several fungal isolates and 15 replicates of the noninoculated control. Plants were grown in water baths located in a greenhouse (trial 1) and in a growth chamber (trial 2) and both maintained at an average temperature of 25°C with a 14-h photoperiod (2). The third trial was conducted in the growth chamber without a water bath with the same temperature and light regimen. In all environments, inoculated plants developed chlorotic spots 14 days after planting. After 21 days, symptoms progressed to a range of chlorotic mottling to interveinal chlorosis and necrosis. Foliar and root symptoms that resembled those on the original plant samples infected with F. virguliforme appeared on 88% of inoculated plants. Isolates that resembled the original F. virguliforme were recovered from 75% of inoculated plants and from 88% of plants showing symptoms. No symptoms were observed and no isolates were recovered from noninoculated plants. There was a statistically significant difference between inoculated and control plants (P < 0.001) based on the presence of symptoms and isolation success using the Goodman χ2 analysis. The confirmation of the presence of SDS in five counties suggests that the disease is widespread in Wisconsin and could become a serious threat to soybean production in the future. References: (1) T. Akoi et al. Mycoscience 46:162, 2005. (2) R. Y. Hashmi et al. Online publication. doi:10.1094/PHP-2005-0906-01-RS. Plant Health Progress, 2005. (3) K. W. Roy et al. Plant Dis. 81:259, 1997. (4) J. C. Rupe et al. Can. J. Bot. 79:829, 2001.

First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Wisconsin.

Brown root rot (BRR) has been associated with winterkill of alfalfa (Medicago sativa L.) in the temperate regions of North America where winters are severe (1). Although suspected, BRR has not been associated with winterkill of alfalfa in the upper Midwestern United States. Alfalfa plants exhibiting symptoms resembling those induced by the causal agent Phoma sclerotioides G. Preuss ex Sacc. were collected from fields in Marinette, Pierce, and Marathon counties in Wisconsin during the spring and early summer of 2003. Symptoms included stunting and decline in 1- to 3-year-old plants that were slow to break dormancy in the early spring. Roots frequently exhibited dark brown lesions or were entirely decayed. Advanced lesions often formed dark bands around the circumference of tap and secondary roots. Beaked pycnidial structures typical of P. sclerotioides were also observed on many samples with advanced lesions. Plants with symptoms of BRR were also observed in Clark, Langlade, Lincoln, Oconto, Shawno, Taylor, and Wood counties. Several lesion areas of tissue on the tap and lateral roots of each sample were excised with a sterile scalpel. Total DNA was extracted using the Fast DNA kit (Bio 101, Carlsbad, CA). In addition, soil samples were collected in the root rhizosphere of symptomatic plants from four fields in two counties. Soil DNA was extracted with the Ultra-Clean DNA soil extraction kit (Mo Bio, Solana Beach, CA). DNA extractions were diluted 1:10 or 1:50, and samples were evaluated for the presence of P. sclerotioides using polymerase chain reaction (PCR)-based sequence-characterized amplified region (SCAR) markers according to the method described previously (4). Amplicons of the expected size (499 bp) were detected from alfalfa roots sampled from Marathon (4 of 4), Marinette (4 of 5), and Pierce (4 of 4) counties but not in roots from healthy controls produced in the greenhouse at Prosser, WA. PCR amplicons were also produced from all field soil samples in Marathon and Marinette counties. Proof of pathogenicity via Koch's postulates for this host-pathogen system was not attempted because of the extensive time period required (1). However, characteristic beaked pycnidia were present, and the pathogen was identified using PCR on DNA from roots symptomatic of BRR. Detection of BRR has been limited in the United States to Wyoming (2), but has been thought to occur in other states with severe winters (3). To our knowledge, this is the first report of P. sclerotioides in Wisconsin. References: (1) J. G. N. Davidson. Brown root rot. Pages 29-31 in: Compendium of Alfalfa Diseases. 2nd ed. D. L. Stuteville and D. C. Erwin, eds. The American Phytopathological Society, St. Paul, MN, 1990. (2) F. A. Gray et al. Pages 27-28 in: Proc. 10th Western Alfalfa Improv. Conf., 1997. (3) C. R. Hollingsworth et al. Can. J Plant Pathol. 25:215, 2003. (4) R. C. Larsen et al. Plant Dis. 86:928, 2002.

Detection and description of spatial patterns of bacterial brown spot of snap beans using cyclic samples.

ABSTRACT Snap bean plants within seven-row segments that ranged from 65 to 147 m were sampled, using a cyclic sampling plan. In the cyclic sampling plan, only 6 of every 31 plants were sampled, but sampled plants were spaced such that pairs of plants that were 1, 2, 3, 4,..., 1,525 plants apart could be identified within each sample. Every leaflet on every sampled plant was assessed for bacterial brown spot, and the proportion of disease leaflets per plant was determined. Arcsine square-root-transformed disease incidence values were analyzed for spatial patterns by autocorrelation and spectral analyses. Disease patterns were detected at several different scales within a single snap bean row, at distances that ranged from 20 to 100 m. Approximately 23 to 53% of the disease variability in the samples could be described by sine and cosine curves, indicating a substantial component of regularity in the disease patterns. Possible origins for these regular patterns, including cultural practices and seed infestation, are discussed.