Pythium, Phytophthora, and Phytopythium spp. Associated with Soybean in Minnesota, Their Relative Aggressiveness on Soybean and Corn, and Their Sensitivity to Seed Treatment Fungicides.

Pythium spp. cause seed decay, damping-off, and root rot in soybean and corn; however, their diversity and importance as pathogens in Minnesota are unknown. Our objectives were to identify the Pythium spp. present in Minnesota soybean fields, determine their aggressiveness on corn and soybean, and investigate their sensitivity to seed treatment fungicides. For identification, sequences obtained using internal transcribed space ITS4 and ITS1 primers were compared with reference sequences in the National Center for Biotechnology Information database. Seedling and soil samples yielded over 30 oomycete species. Aggressiveness was determined using two methods; a seed assay, which also examined temperature effects on aggressiveness, and a seedling assay. Of 21 species evaluated, seven Pythium spp. were pathogenic on both soybean and corn, reducing root growth by 20% or more while two Pythium and one Phytopythium spp. were pathogenic only on soybean. Aggressiveness of many isolates increased as temperature increased from 15°C to 25°C. The sensitivity of 10 pathogenic species to azoxystrobin, ethaboxam, mefenoxam, pyraclostrobin, or trifloxystrobin was tested. EC50 values for mefenoxam and ethaboxam were 10-2 of those to strobilurin fungicides. Pythium spp. in Minnesota are diverse and a significant cause of seedling disease on soybean and corn. Most Pythium spp. isolated in this study were more sensitive to mefenoxam and ethaboxam than to strobilurin fungicides.

Symptomatic and Asymptomatic Host Range of Fusarium virguliforme, the Causal Agent of Soybean Sudden Death Syndrome.

Sudden death syndrome, caused by Fusarium virguliforme, is an important disease of soybean in the United States. Fifteen species of crops, weeds, or prairie plants were evaluated for their potential as hosts of F. virguliforme. Root and foliar symptoms and plant biomass were assessed following greenhouse inoculation studies. Root colonization of F. virguliforme was determined with isolations and with polymerase chain reaction assays. Soybean, alfalfa, pinto and navy bean, white and red clover, pea, and Canadian milk vetch developed root necrosis. Soybean, alfalfa, and red clover also developed foliar symptoms following inoculation. Sugar beet and canola did not develop symptoms but had significant reductions in biomass, suggesting that they are also hosts of F. virguliforme. Corn, wheat, ryegrass, pigweed, and lambsquarters did not develop symptoms. However, these species appeared to be asymptomatic hosts because quantities of pathogen DNA detected in inoculated roots were similar to quantities detected in inoculated soybean roots. These results suggest that the number and diversity of hosts for F. virguliforme are greater than previously reported. The likely broad host range limits the efficacy of crop rotation and indicates that crops other than soybean can be damaged by F. virguliforme and maintain or increase inoculum in soil.

Soybean Rust Caused by Phakopsora pachyrhizi Detected in the State of Campeche on the Yucatan Peninsula, Mexico.

Soybean rust caused by Phakopsora pachyrhizi Syd. & P. Syd is a destructive foliar disease of soybean (Glycine max L), which was first confirmed in North America in Louisiana during 2004 (4). Soybean rust (SBR) has also been reported late in the growing season as far north as Illinois, Indiana, and Iowa. SBR was first confirmed in Mexico in 2005 in the state of San Luis Potosi on soybean (3) and subsequently reported in the states of Tamaulipas, Veracruz, and the southwestern coast of Chiapas. Symptoms of SBR were observed on leaves of multiple, nearly mature soybean plants near the city of Campeche (19.72796°N, 90.0771°W) on the Gulf Coast of the Yucatan Peninsula during November 2008. Angular and irregular chlorotic lesions on leaves contained necrotic spots and pale brown, erumpent, cone-like uredinia with a central opening. Ellipsoid to obovoid, echinulate, light tan urediniospores (10 to 13 × 16 to 18 µm) were observed microscopically. DNA was extracted from leaf tissue containing uredinia and from asymptomatic tissue with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA). P. pachyrhizi was confirmed in the symptomatic leaves by a PCR assay with Ppm1/Ppa2 primers, but not from the asymptomatic leaves (1). Subsequently, the DNA extracted from symptomatic and asymptomatic leaf tissues was tested again in another laboratory by a specific quantitative PCR assay (1), and positive results for the presence of soybean rust were obtained only from the symptomatic tissue. As a final confirmatory step, amplified DNA from the PCR assay was sequenced, and the results matched P. pachyrhizi sequences in the GenBank database. To our knowledge, these observations confirm for the first time the presence of P. pachyrhizi in the state of Campeche of southern Mexico. Although it was confirmed on soybean during 2008, it is not known how long the pathogen has been present or which other hosts may be infected there. The presence of SBR on the Yucatan Peninsula is significant because of its potential effects on local plant hosts. In addition, the climate allows possible year-round survival of the pathogen and long-distance transport of urediniospores to the United States. Potential transport of SBR spores from this part of Mexico to the United States has been reported through the application of NOAA's HYSPLIT (Hybrid Single Particle Lagrangian Integrated Transport) model and atmospheric back-trajectory analysis (2). References: (1) R. D. Frederick et al. Phytopathology 92:217, 2002. (2) S. V. Krupa et al. Plant Dis. 90:1254, 2006. (3) A. C. Rodriguez et al. Plant Dis. 90:1260, 2006. (4) R. W. Schneider et al. Plant Dis. 89:774, 2005.

Cultivar Preference and Genotype Distribution of the Brown Stem Rot Pathogen Phialophora gregata in the Midwestern United States.

Brown stem rot (BSR), caused by Phialophora gregata f. sp. sojae, is an important yield-limiting disease of soybean (Glycine max) in the midwestern United States. Midwestern populations of P. gregata are separated into genotypes A and B based on intergenic spacer sequences of nuclear ribosomal DNA. Genotype A causes both leaf and stem symptoms, and genotype B typically causes internal stem symptoms only. Data are limited on the geographic distribution of genotypes A and B. It is not well understood whether cultivars may be infected preferentially by a genotype. Field plots were established at five sites in Illinois, three sites in Wisconsin, and two sites in Minnesota in two different years. Soybean cvs. Bell, BSR101, Dwight, Sturdy, Williams 82, LN92-12033, and LN92-12054 were sown with two to four replications at each field site. From each plot, 5 to 10 stems were harvested arbitrarily at the R8 growth stage and assayed by polymerase chain reaction to detect the A and B genotypes. Both pathogen genotypes were detected at all locations except Urbana, where only genotype A was detected, and St. Paul, where only B was detected. Genotype A was the predominant genotype detected in susceptible cvs. Williams 82 and LN92-12054, with 70 and 78% of infected stems, respectively, positive for A. The other susceptible cultivar, Sturdy, yielded predominantly genotype A at four of the seven Illinois and Wisconsin locations where both pathogen genotypes were present, but yielded predominantly B at the Minnesota location where both genotypes were detected. Genotype B was the predominant type detected in partially resistant cvs. Dwight, LN92-12033, and Bell, with 56, 85, and 99% of the infected stems, respectively, testing positive for B.

First Report of Charcoal Rot (Macrophomina phaseolina) on Soybean in Minnesota.

In August 1999, soybean (Glycine max (L.) Merr.) plants exhibiting symptoms of charcoal rot were observed near Zumbrota, MN. Symptoms included shrunken, unfilled pods, and brown, wilted leaves attached to dead petioles and stems (1). When stems of symptomatic soybean plants were split, areas of gray-to-black discoloration where present in the stem cortex (1). Black, spherical microsclerotia 77 to 90 µm in diameter and elongated microsclerotia 77 to 120 µm long (1) were found in vascular tissue. Stem tissue placed on potato dextrose agar (PDA) yielded fungal colonies identified as Macrophomina phaseolina (Tassi) Goid. based on gray colony color, colony morphology, and the presence of microsclerotia 70 to 90 µm in diameter. In 2000, M. phaseolina was isolated from plant samples gathered from 11 of 90 fields sampled in a statewide soybean disease survey. More studies are needed to determine the distribution of charcoal rot in Minnesota; however, the occurrence of symptoms at one location and the presence of M. phaseolina in soybean-growing areas of Minnesota suggest that charcoal rot may occur in susceptible soybean cultivars under favorable environmental conditions. Reference: (1) G. S. Smith and T. D. Wyllie. Charcoal rot. Pages 29-30 in: Compendium of Soybean Diseases, 4th ed. G. L. Hartmann, J. B. Sinclair, and J. C. Rupe, eds. The American Phytopathological Society, St. Paul, MN, 1999.

First Report of Sudden Death Syndrome (Fusarium solani f. sp. glycines) of Soybean in Minnesota.

In August 2002, soybean (Glycine max (L.) Merr.) plants exhibiting foliar and root symptoms typical of sudden death syndrome were observed in Blue Earth and Steele counties in south-central Minnesota. Leaf symptoms ranging from small chlorotic spots to prominent interveinal necrosis were present on soybean plants at the R6 to R7 growth stage. As plants matured, complete defoliation took place with only petioles remaining. Symptomatic plants had necrotic secondary roots, truncated taproots, and discolored cortical tissue at the soil line. Blue sporodochia containing macroconidia were observed on the taproot of affected plants at both locations (3,4). Multiple cultures from both locations were obtained by transferring macroconidia from the sporodochia to potato dextrose agar (PDA) and modified Nash-Snyder Medium (NSM) (3). After 14 days, isolations were made from fungal colonies exhibiting bluish pigmentation and masses of bluish macroconidia (4). The isolates grew slowly, developed a bluish color, and formed sporodochia containing abundant macroconidia on NSM. These isolates were identified as Fusarium solani (Mart.) Sacc. f. sp. glycines based on colony characteristics and morphology of macroconidia (2). Pathogenicity tests were conducted with a single isolate from each location. The isolate from Blue Earth County was inoculated as mycelia in a plug of media onto taproots of plants of susceptible cvs. Williams 82 and Spencer at the V2 growth stage. Chlorotic spots appeared on leaves after 12 days of growth at 22 to 25°C in the greenhouse. Interveinal necrosis appeared after 15 days (4). The isolate from Steele County was used to inoculate the susceptible cv. Great Lakes 3202. Sorghum seed (3 cm3) infested with mycelia of the isolate were placed 2 to 3 cm below soybean seed planted in Cone-Tainers. Noninfested sorghum seed was used as a control. Plants were maintained for 21 days at 22 to 28°C in the greenhouse. Chlorotic spots appeared on leaves of inoculated plants within 21 days after planting followed by the development of interveinal chlorosis and necrosis (1). Molecular analysis further supported the identification of the Steele County isolate as F. solani f. sp. glycines. Polymerase chain reaction with specific primers Fsg1 and Fsg2 of total genomic DNA extracted from the Steele County isolate amplified a 438-bp DNA fragment identical with that extracted from previously identified isolates of F. solani f. sp. glycines (1). In 2002, symptoms of sudden death syndrome were also reported in Olmsted, Freeborn, and Mower counties. Although studies are needed to determine the distribution of sudden death syndrome in the state, the occurrence of the symptoms at multiple locations suggests that F. solani f. sp. glycines is widely distributed in southeast and south-central Minnesota. The counties where sudden death syndrome symptoms were reported are located in the most productive soybean-growing region of Minnesota. Sudden death syndrome could be a serious threat to soybean production in this area since poorly drained, heavy, clay soils are common, and soil temperatures 18°C or less are normal before the end of May. References: (1) S. Li et al. Phytopathology 90:491, 2000. (2) K. W. Roy. Plant Dis. 81:566, 1997. (3) K. W. Roy et al. Plant Dis. 81:1100, 1997. (4) K. W. Roy. Plant Dis. 81:259, 1997.

Efficacy of Fungicides on Sclerotinia sclerotiorum and Their Potential for Control of Sclerotinia Stem Rot on Soybean.

Sclerotinia stem rot of soybean, caused by Sclerotinia sclerotiorum, is a major disease in the north central region of the United States. One approach to managing Sclerotinia stem rot on soybean is the use of fungicides. S. sclerotiorum was assayed for sensitivity to benomyl, tebuconazole, thiophanate methyl, and vinclozolin in pure cultures on agar medium, inoculated soybean seedlings, detached inoculated leaves, and in experimental field plots. To evaluate the inhibitory effect of four fungicides on growth of S. sclerotiorum in vitro, potato dextrose agar (PDA) was amended with the fungicides at six concentrations. Based on measurements of fungal radial growth, vinclozolin was the most effective in inhibiting S. sclerotiorum mycelial growth at 1.0 µg a.i./ml of PDA. Ranges of reduction of radial growth of 91 isolates of S. sclerotiorum on PDA amended with thiophanate methyl and vinclozolin were 18 to 93% and 93 to 99%, respectively, when compared with the nonamended agar control. Benomyl, thiophanate methyl, and vinclozolin applied to greenhouse-grown seedlings prevented S. sclerotiorum from expressing symptoms or signs on leaf tissue. Detached leaves sprayed with thiophanate methyl and then inoculated with mycelial plugs of S. sclerotiorum did not express symptoms or signs. Of 13 different environments in Illinois, Indiana, Ohio, and Wisconsin from 1995 through 2000, six had low Sclerotinia stem rot incidence (<1%), three environments had low to moderate Sclerotinia stem rot incidence (5 to 25%), and four environments had high Sclerotinia stem rot incidence (>25%). When disease incidence was high, no consistent control of Sclerotinia stem rot was observed with benomyl or thiophanate methyl using different application systems. However, under low disease incidence, spray systems that were able to penetrate the canopy reduced the incidence of Sclerotinia stem rot an average of 50%.