Comprehensive Sclerotinia Stem Rot Screening of Soybean Germplasm Requires Multiple Isolates of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum population variability directly affects Sclerotinia stem rot (SSR) resistance breeding programs. In the north-central United States, however, soybean germplasm selection has often involved only a single isolate. Forty-four S. sclerotiorum isolates from Illinois, Michigan, Minnesota, Nebraska, Wisconsin, Poland, and across 11 different host species were evaluated for variation in isolate in vitro growth, in vitro oxalate production, and in planta aggressiveness on the susceptible soybean 'Williams 82'. Significant differences (P < 0.0001) were detected in isolate in planta aggressiveness, in vitro growth, and in vitro oxalate production. Furthermore, diverse isolate characteristics were observed within all hosts and locations of collection. Aggressiveness was not correlated to colony growth and was only weakly correlated (r = 0.26, P < 0.0001) to isolate oxalate production. In addition, the host or location of collection did not explain isolate aggressiveness. Isolate oxalic acid production, however, may be partially explained by the host (P < 0.05) and location (P < 0.01) of collection. Using a representative subset of nine S. sclerotiorum isolates and soybean genotypes exhibiting susceptible or resistant responses (determined using a single isolate), a significant interaction (P = 0.04) was detected between isolates and genotypes when SSR severity was evaluated. Our findings suggest that screening of S. sclerotiorum-resistant soybean germplasm should be performed with multiple isolates to account for the overall diversity of S. sclerotiorum isolates found throughout the soybean-growing regions of the United States.

Influence of Soybean Monoculture on Phialophora gregata f. sp. sojae IGS-Genotype B Isolate Aggressiveness.

Many soybean accessions described as resistant to brown stem rot (BSR) are preferentially colonized by isolates of Phialophora gregata f. sp. sojae genotype B. These isolates are generally considered less aggressive than isolates of P. gregata f. sp. sojae genotype A because they cause minor or no foliar symptoms characteristic of BSR. However, variation in aggressiveness has been observed among isolates of P. gregata f. sp. sojae genotype B. To determine if BSR-resistant soybean accessions would preferentially select for more aggressive isolates of P. gregata f. sp. sojae genotype B, monocultures of both BSR-resistant or BSR-susceptible accessions were established at the Arlington Agriculture Research Station, Arlington, WI. BSR-susceptible cv. Corsoy 79 and BSR-resistant plant introduction (PI) 567.157A were inoculated under greenhouse conditions with a total of 39 isolates of P. gregata f. sp. sojae genotype B obtained from the different monocultures. BSR severity was determined as the percentage of symptomatic foliar and internal stem tissue. Overall, BSR severity was low and did not exceed 20% for either foliar or stem symptoms. Isolates of P. gregata f. sp. sojae genotype B caused more severe foliar (P < 0.0001) and stem (P = 0.0008) symptoms of BSR on PI 567.157A than on Corsoy 79. Analysis of BSR stem symptom severity indicated an interaction (P = 0.0124) between soybean accession and the origin of isolates of P. gregata f. sp. sojae genotype B. Isolates of P. gregata f. sp. sojae genotype B obtained from the monoculture of a BSR-susceptible or -resistant accession were more aggressive than isolates from a mixed resistant and susceptible soybean monoculture. The relationship between the origin of isolate of P. gregata f. sp. sojae genotype B and isolate aggressiveness was more apparent for PI 567.157A than for Corsoy 79. Results of this study indicate that the monoculture of resistant or susceptible soybean favors an increase in the aggressiveness of isolates of P. gregata f. sp. sojae genotype B. Furthermore, results suggest that resistance to genotype A may be genetically different from resistance to genotype B.

Altered yolk structure and reduced hatchability of eggs from birds fed single doses of petroleum oils.

Yolk deposited by Japanese quail was abnormal for 24 hours after the oral administration of a single capsule containing 200 milligrams of bunker C oil. Both the structure and the staining properties of the yolk were affected. Fewer eggs were laid during the 4 days after dosing, compared to controls, and hatchability was drastically reduced. Hatchability returned to normal in 4 days. Three other reference oils also affected yolk structure. Canada geese given 2 grams and chickens given 500 milligrams of bunker C oil produced eggs with abnormal yolk rings.

Real-time PCR assays for the quantification of Phialophora gregata f. sp. sojae IGS genotypes A and B.

Populations of Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR) of soybean, consist of two genotypes, designated A and B. These genotypes are differentiated by an insertion or deletion in the intergenic spacer region (IGS) of ribosomal DNA. The two genotypes differ in the type and severity of symptoms they cause and have displayed preferential host colonization. Methods to quantify populations of P. gregata f. sp. sojae and to distinguish between the two genotypes are essential to understanding this host-pathogen interaction and to improving control of BSR. A real-time, quantitative polymerase chain reaction (qPCR) assay was developed for the specific detection and quantification of P. gregata f. sp. sojae genotype A. This assay is specific to P. gregata f. sp. sojae genotype A, sensitive to 50 fg of DNA, and unaffected by the presence of soybean or soil DNA. When the P. gregata f. sp. sojae genotype A-specific primer/probe set is used in a multiplex qPCR assay with a previously developed primer/probe set which indiscriminately amplifies both genotypes, the quantity of P. gregata f. sp. sojae genotype B can be indirectly determined. This multiplex assay provides a rapid and robust method for studying both the population size and genetic structure of P. gregata f. sp. sojae in its soybean host and in the soil.

Soybean Stem Lignin Concentration Relates to Resistance to Sclerotinia sclerotiorum.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is an economically important disease of soybean (Glycine max) in the north-central United States and other temperate regions throughout the world. The occurrence and severity of Sclerotinia stem rot in the field is highly dependent upon prevailing environmental conditions, which can prove problematic when evaluating soybean accessions for resistance. The identification of an environmentally stable plant trait associated with resistance to S. sclerotiorum could be used to indirectly screen for resistance and would prove useful in the identification and development of resistant germplasm. Observations of the soybean-S. sclerotiorum interaction suggest a role for preformed stem lignin content in disease resistance. Although S. sclerotiorum produces numerous enzymes that degrade plant cell wall components, no lignin-degrading enzymes have been reported. Despite a hypothesized direct relationship between preformed lignin content and disease resistance, previous studies on soybean have correlated lignin content to nutritional value and not to disease resistance. We hypothesized that plants with low stem lignin are more susceptible and exhibit greater Sclerotinia stem rot severity than plants with high lignin concentrations. Six soybean accessions that varied in response to S. sclerotiorum were selected for study in a series of field experiments. Soybean stems were sampled at reproductive developmental stages that correspond to specific events in both soybean plant development and the Sclerotinia stem rot disease cycle. The lignin concentration of stem component samples was quantified. Soybean accessions expressed statistically different disease phenotypes in both 2004 and 2006. Lignin concentrations differed among accessions, growth stages, and plant parts. Results were contrary to our hypothesis, with positively ranked correlations observed between accession Sclerotinia stem rot severity and lignin concentration for all nodes and internodes assayed. For the R3 growth stage, lignin concentration of the internode between the fourth and fifth trifoliate leaves correlated best with disease severity data from each year (P = 0.005). These results indicate that resistance is related to low stem lignin concentration and that soybean stem lignin concentration can be used as a biological marker to select for resistance to S. sclerotiorum.

Characterizing Reaction of Soybean to Phialophora gregata Using Pathogen Population Density and DNA Quantity in Stems.

Evaluation of soybean germplasm for resistance to brown stem rot (BSR) is typically based on symptom severity. However, this approach may not reflect the level of colonization of soybean by the casual agent, Phialophora gregata. A potentially more accurate method to characterize resistance to BSR is to estimate pathogen quantity. The primary goal of this study was to evaluate soybean accessions for resistance to BSR based on the quantity of pathogen in stems. Plants were collected from experiments in field and controlled environments, and CFU and pathogen DNA quantity were determined using dilution plating techniques and real-time quantitative PCR (qPCR), respectively. In the field, the BSR-susceptible cultivars Corsoy 79 and Century 84 expressed greater than 73% foliar and stem symptom severity and had the highest pathogen population density, with a range from log10 4.3 to 4.7 CFU per gram of stem tissue. The resistant cultivar Bell expressed less than 10% foliar symptom severity, but had a pathogen population density that was not statistically different from the susceptible accessions. CFU measured in Dwight and L84-5873 were consistently lower than CFU in susceptible accessions and several resistant accessions. The amount of pathogen DNA differed among accessions in controlled environments. For example, Corsoy 79 and Century 84 had the highest pathogen DNA quantity, ranging from log10 6.19 to 6.65 copies, whereas the resistant cultivars Bell, Dwight, and L84-5873 had significantly lower DNA quantities, ranging from log10 2.04 to 2.91 copies. PI 437833 and IA2008R expressed low symptom severity but contained high DNA quantities. Pella 86, a highly symptomatic cultivar, had fewer CFU and lower DNA quantity in comparison to two other highly symptomatic cultivars and some cultivars with low symptom severity. These results suggest that some accessions express resistance to both pathogen colonization and symptom development, while others are resistant to symptom development but not to pathogen colonization. Results also indicate that resistant and susceptible accessions can be distinguished based on DNA quantity in controlled environments. In the field, differences between the pathogen population in resistant and susceptible cultivars were less distinct, possibly due to when plants were assayed.

Influence of Monocropping Brown Stem Rot-Resistant and -Susceptible Soybean Accessions on Soil and Stem Populations of Phialophora gregata f. sp. sojae.

Brown stem rot (BSR)-resistant and -susceptible soybean accessions were continuously cropped in an area never previously seeded to soybean to study the influence of monocultures on soil and stem populations of Phialophora gregata f. sp. sojae. P. gregata f. sp. sojae population size and genotype composition were determined by dilution plating, isolation of P. gregata f. sp. sojae and standard polymerase chain reaction (PCR), and by quantitative real-time PCR (q-PCR. In general, the sizes of P. gregata f. sp. sojae populations in soil were similar regardless of monoculture. The percentage of P. gregata f. sp. sojae genotype B was greater than A in soil following the monoculture of both BSR-susceptible and -resistant soybean accessions. Following the monoculture of a BSR-resistant accession, the percentage of P. gregata f. sp. sojae genotype B was greater than A. Overall, P. gregata f. sp. sojae populations in stems of a BSR-susceptible accession were greater than those in stems of a BSR-resistant accession. P. gregata f. sp. sojae genotype B was detected more often than A in stems of both resistant and susceptible accessions planted following a BSR-resistant monoculture. P. gregata f. sp. sojae genotype B was also detected more often than A in stems of a BSR-resistant accession planted following a BSR-susceptible monoculture. P. gregata f. sp. sojae genotypes A and B were isolated at similar frequencies from stems of a BSR-susceptible accession planted following a BSR-susceptible monoculture. However, q-PCR results indicate that the percentage of P. gregata f. sp. sojae genotype A was greater than B in stems of a BSR-susceptible accession planted following a BSR-susceptible monoculture. Among BSR-susceptible accessions, those with the soybean cyst nematode (SCN)-resistant cv. Peking in their parentage had the largest populations of P. gregata f. sp. sojae and a greater percentage of P. gregata f. sp. sojae genotype B. Similar results were observed for BSR-resistant accessions derived from SCN-resistant PI 88788.

Integrated Management Strategies for Phytophthora sojae Combining Host Resistance and Seed Treatments.

Phytophthora sojae has re-emerged as a serious soybean pathogen in the past decade. This may be due in part to changes in resistance levels in current cultivars, adoption of P. sojae populations to deployed Rps genes, and highly favorable environments in the past decade. This multilocation study evaluated the effect of seed treatments on the incidence and severity of Phytophthora root and stem rot on soybeans with different combinations of Rps genes and levels of partial resistance. The efficacy of the seed treatments was highly variable across locations. Seed treatments (metalaxyl and mefenoxam) provided protection and increased yields across cultivars in locations where rain or irrigation occurred shortly after planting (Ohio, South Dakota, and Ontario). However, there were no significant differences in stand or yield consistently across cultivars in Iowa, Nebraska, Wisconsin, or Ohio, where heavy precipitation did not occur until later growth stages. The environment, levels of inoculum, and pathogen complex may have played a role in the different responses to the seed treatments and to the different combinations of Rps genes and levels of partial resistance to P. sojae in the cultivars. Fields that are poorly drained and have P. sojae populations with complex pathotypes may benefit the most from seed treatments. Individual fields where producers may see the greatest benefit to utilizing these integrated management strategies will need to be identified.

The Influence of Light on Relationships Between Sclerotinia Stem Rot of Soybean in Field and Controlled Environments.

Interaction phenotypes between soybean and Sclerotinia sclerotiorum observed in controlled and field environments often do not correlate. Resistant and susceptible accessions were selected to study light intensity as a variable that influences interaction phenotype. Objectives were to investigate whether light intensity within a controlled environment influences the ability to: i) distinguish resistant and susceptible accessions; ii) predict field interaction phenotypes; and iii) determine whether the method used for disease assessment influences the outcome of results. Six accessions were evaluated in growth chamber and field disease nursery trials. Five environments ranging from 146 to 434 µmol·m-2·s-1 of photosynthetically active radiation were established in the growth chamber. Inoculum was delivered to cut petioles at the R1 growth stage and interaction phenotypes were assessed for 11 days. For field trials, individual plants were rated at growth stage R7. The breeding line W04.1002 had less disease than NK S19-90 in field environments. Rank correlations of field and light environment interaction phenotypes indicate that light intensity affected the prediction of field performance. Evaluations conducted at 337 µmol·m-2·s-1 of light were most predictive of field interaction phenotypes (rs = 0.83 to 0.94; P = 0.05 to < 0.005). Controlling light intensity is critical to facilitate the identification of resistance to S. sclerotiorum in soybean.

Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa.

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.

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.

Population Dynamics of Phialophora gregata in Stem Residue of a Resistant and a Susceptible Soybean Cultivar.

Previous studies on the saprophytic survival of Phialophora gregata were conducted with soybean residue derived from a susceptible cultivar and did not address genotypes of P. gregata. This current study monitored the saprophytic population density of P. gregata in stem residue derived from a susceptible and a resistant soybean cultivar placed in the field. A second phase of the study followed the frequencies of genotypes A and B of P. gregata in stem residue derived from a susceptible cultivar. The population density of P. gregata declined 10-fold in stem residue from the initiation of sampling to the end of this 16-month study, regardless of cultivar or whether residue was positioned on the soil surface or buried. The population density of P. gregata was greater in buried residue of the resistant cultivar compared with the susceptible cultivar after 12 to 14 months, but equalized after 16 months. The population density of P. gregata was similar in residue derived from the susceptible and resistant cultivars if positioned on the soil surface. Genotype B was detected more frequently than genotype A of P. gregata at each sampling date regardless of residue placement.

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.

Assessing Heterodera glycines-Resistant and Susceptible Cultivar Yield Response.

The soybean cyst nematode Heterodera glycines (SCN) is of major economic importance and widely distributed throughout soybean production regions of the United States where different maturity groups with the same sources of SCN resistance are grown. The objective of this study was to assess SCN-resistant and -susceptible soybean yield responses in infested soils across the north-central region. In 1994 and 1995, eight SCN-resistant and eight SCN-susceptible public soybean cultivars representing maturity groups (MG) I to IV were planted in 63 fields, either infested or noninfested, in 10 states in the north-central United States. Soil samples were taken to determine initial SCN population density and race, and soil classification. Data were grouped for analysis by adaptation based on MG zones. Soybean yields were 658 to 3,840 kg/ha across the sites. Soybean cyst nematode-resistant cultivars yielded better at SCN-infested sites but lost this superiority to susceptible soybean cultivars at noninfested sites. Interactions were observed among initial SCN population density, cultivar, and location. This study showed that no region-wide predictive equations could be developed for yield loss based on initial nematode populations in the soil and that yield loss due to SCN in our region was greatly confounded by other stress factors, which included temperature and moisture extremes.

The Relationship Between Aphis glycines and Soybean mosaic virus Incidence in Different Pest Management Systems.

The soybean aphid, Aphis glycines, causes yield loss and transmits viruses such as Soybean mosaic virus (SMV) in soybean (Glycine max). Field experiments were designed to monitor the landing rate of A. glycines and transmission of SMV to soybean grown in six crop management environments. Management systems evaluated were the application of postemergence insecticide or no insecticide, and within each insecticide treatment no herbicide, glyphosate, or imazamox application. In 2001, early-season incidence of SMV was 2%, which increased to 80% within 18 days after the beginning of the A. glycines flight. In 2002, the incidence of SMV was 1% prior to the arrival of A. glycines, and increased to 44% within 21 days. The landing rate of A. glycines was fivefold higher in 2001 than in 2002. The incidence of SMV was lower in insecticide-treated plots in 2002, but no effect of insecticide was seen in 2001. Imazamox slowed the progression of SMV incidence, but the final incidence of SMV-infected plants was the same. Glyphosate had no consistent effect on SMV incidence. Yield was higher in the insecticide-treated plots in 2002, but not 2001. Insecticide and herbicide application had no substantial effects on seed quality.

Seasonal Progression and Agronomic Impact of Tobacco streak virus on Soybean in Wisconsin.

Pod necrosis and dieback of terminal portions of stems, commonly called bud blight, are observed in soybean fields and associated with Tobacco streak virus (TSV), an understudied virus of soybean. Furthermore, many TSV-infected plants are asymptomatic. The objectives of this study were to characterize the distribution and seasonal progress of TSV-infected plants in both natural and controlled epidemics, and the agronomic impact of TSV on soybean in plots with controlled introduction of inoculum. Incidence of TSV-positive samples ranged from 17 to 56% in a general survey. In the presence of natural sources of inoculum, the incidence of TSV-infected plants ranged from 10 to 95% depending on cultivar and location, and peaked at growth stage R2, but detection dropped dramatically at R5. During 2001, significant yield loss and incidence of mottled seed were associated with TSV, but results were confounded by a high incidence of SMV. In 2002, SMV was controlled by cultivar selection, and a 25% reduction in grain yield was attributable to TSV. The incidence of mottled seed and green stem syndrome were low in the presence of TSV. Reductions in plant density and final plant height contributed to reduced yields. However, no significant differences were found in seed number per plant and 100-seed weight. Data indicate that plant mortality was the main mechanism by which TSV caused yield loss in induced epidemics.

A Molecular Marker Identifying Subspecific Populations of the Soybean Brown Stem Rot Pathogen, Phialophora gregata.

ABSTRACT A molecular marker was developed to separate and identify subspecific populations of Phialophora gregata, the causal agent of soybean brown stem rot. A variable DNA region in the intergenic spacer of the nuclear rDNA was identified. Two specific primers flanking the variable region were developed for easy identification of the genotypes using polymerase chain reaction (PCR). These two specific primers amplified three DNA products. The three PCR products were used to separate isolates of P. gregata into distinct genotypes: A (1,020 bp), B (830 bp), and C (660 bp). Genotype C was found in isolates obtained from Adzuki beans from Japan, whereas all 292 isolates obtained from soybean and the 8 isolates from mung bean belonged to either genotype A or B. The original nondefoliating (type II) strain ATCC 11073 (type culture of P. gregata) belonged to genotype B. The difference between genotypes A and B was due only to an 188-bp insertion or deletion; genotype C, however, differs from genotypes A and B at 58 point mutations, in addition to the length difference. Isolates of both genotypes A and B were widespread in seven Midwestern states. Genotype A was found mostly in certain susceptible soybean cultivars like Sturdy and Pioneer 9305, whereas genotype B was found predominately in brown stem rot-resistant soybean cvs. Bell, IA 3003, and Seiben SS282N. The specific primers were also used to directly detect cultivar-preferential infection by the two genotypes in infected soybean stems growing in the same field. Data from direct detection in soybean stems showed that cultivar-preferential infection by the two genotypes of P. gregata was significant.

Regression Analyses for Evaluating the Influence of Bacillus cereus on Alfalfa Yield Under Variable Disease Intensity.

ABSTRACT We developed and tested regression methods to exploit the variability in disease inherent in field experiments, and applied the methods to evaluate strains of Bacillus cereus for biocontrol efficacy. Four B. cereus strains were tested for their effect on alfalfa (Medicago sativa) performance in 16 field trials planted during 1993 to 1996 at multiple sites in Wisconsin. To evaluate performance of the strains, we used the ratio of (metalaxyl response)/(untreated control response) as a measure of disease intensity within the experiments. The ratio of (Bacillus response)/(untreated control response) was then regressed as a function of disease intensity. The slope of the resulting line provides a statistical test to compare performance of the Bacillus strain with that of the untreated seed (H(o): slope = 0) and metalaxyl controls (H(o): slope = 1). Under conditions in which disease occurred, forage yield of plots planted with seed treated with B. cereus strain AS4-12 exceeded yield from the untreated control plots (P = 0.002) and was similar to yield of plots planted with metalaxyl-treated seed (P = 0.14). Yield gain associated with AS4-12 and metalaxyl seed treatment averaged 6.1 +/- 2.8% (+/-standard error) and 3.0 +/- 2.8%, respectively. In contrast to the regression approach, means analysis by analysis of variance did not detect differences among treatments. Three other B. cereus strains either did not increase alfalfa yield or increased yield less than did AS4-12. Metalaxyl and three of the Bacillus strains increased seedling emergence, but the improved stands were not predictive of increased forage yield. In six additional studies conducted for one season in 1997, AS4-12 enhanced yield of two cultivars at diverse locations in Wisconsin, but there was an apparent cultivar-location interaction. A strong correlation between response to AS4-12 and metalaxyl treatment suggests that these treatments controlled similar pathogens, most likely the oomycete pathogens Phytophthora medicaginis and Pythium spp.

Inoculation with Nonpathogenic Fusarium solani Increases Severity of Pea Root Rot Caused by Aphanomyces euteiches.

Aphanomyces euteiches is an important root-rotting pathogen of pea. When recovering isolates of A. euteiches from infested soils in Wisconsin using pea as a bait host, isolates of Fusarium solani often were recovered. Experiments were established to compare disease symptoms of pea seedlings inoculated with isolates of A. euteiches and F. solani alone or in combination. Inoculation of pea seedlings with either of two isolates of A. euteiches produced typical root rot symptoms. However, inoculation of pea seedlings with an isolate of F. solani resulted in no disease symptoms, indicating that the isolate was nonpathogenic to pea. Co-inoculation of pea seedlings with A. euteiches and the nonpathogenic isolate of F. solani resulted in significantly (P = 0.05) greater disease severity than inoculation with A. euteiches alone. Both A. euteiches and F. solani could be reisolated, individually or together, from pea seedlings following individual or co-inoculations, respectively. Although the mechanisms of interaction between these two species are unknown, the synergism documented in this study indicates that the interactions of pathogens with nonpathogens may affect development of disease symptoms.

Characteristics and Frequency of Aphanomyces euteiches Races 1 and 2 Associated with Alfalfa in the Midwestern United States.

Aphanomyces root rot of alfalfa, caused by Aphanomyces euteiches, kills seedlings and causes decline of established plants in slowly drained soils. Two races of A. euteiches that are pathogenic to alfalfa have been identified. Despite the contribution of race 1 resistance to establishment and yield of alfalfa, race 1-resistant alfalfa cultivars perform poorly in some fields infested with A. euteiches. Many isolates of A. euteiches obtained from the soils of problematic fields are of a race 2 phenotype. The purpose of this study was to determine distribution, frequency, and pathogenic and genotypic characteristics of race 1 (R1) and race 2 (R2) isolates from 21 fields: 13 in Wisconsin, 7 in Minnesota, and 1 in Kentucky. A. euteiches was successfully isolated from the soil of 16 of the 21 fields; 405 isolates were obtained from Wisconsin, 4 from Minnesota, and 48 from Kentucky. Pathogenicity and race phenotype of isolates were characterized on Saranac (susceptible to R1 and R2 isolates) and WAPH-1 (resistant to R1 and susceptible to R2 isolates) alfalfa populations. One Wisconsin field with no recent history of alfalfa production had a high frequency (51%) of R2 isolates, and 43% of all isolates were R2 from fields with a history of alfalfa production. In a location that was planted continuously to pea for 30 years, 27% of the isolates were R2. Random amplified polymorphic DNA (RAPD) analysis of three R1 and three R2 isolates with eight primers generated 43 total polymorphic bands; however, none of the bands were uniquely associated with race phenotype. Cluster analysis based on RAPD bands revealed no consistent genotypic distinctions between R1 and R2 isolates of A. euteiches. Evaluation of eight commercial alfalfa cultivars for resistance to two R1 and two R2 isolates demonstrated that most are susceptible to R2 isolates; however, those selected for R2 resistance express resistance to R2 isolates. The results suggest that R2 isolates represent a widespread risk to alfalfa cultivars having resistance only to R1 isolates in fields with varied cropping histories.