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.

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.

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.

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.

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.

Evaluation of Sclerotinia Stem Rot Resistance in Oilseed Brassica napus Using a Petiole Inoculation Technique Under Greenhouse Conditions.

A petiole inoculation technique was adapted for evaluating resistance of oilseed Brassica napus seedlings to Sclerotinia sclerotiorum. In the first of four experiments, four isolates of S. sclerotiorum were tested, two originating from soybean and two from B. napus. In all, 10 to 47 B. napus accessions were inoculated in the seedling stage and responses to isolates were evaluated using days to wilt (DW) and a lesion phenotype index (LP). There were no significant differences in virulence among the four isolates for DW and only slight differences for LP. However, significant differences (P < 0.0001) were observed among the B. napus accessions for DW and LP in this experiment and in subsequent experiments using one isolate. The responses of accessions were consistent among experiments and among evaluation criteria. Higher levels of resistance were found among winter-type than spring-type accessions, and among rapeseed-quality compared with canola-quality accessions. The most resistant accessions identified also were the most resistant when inoculated at the flowering stage. Terminal stems were inoculated immediately below the lowest flower and stem lesion length (SLL) was used to characterize the interaction phenotype of each accession. The petiole inoculation technique can be used successfully to differentiate oilseed B. napus germ plasm for response to S. sclerotiorum. This inoculation technique and the sources of resistance identified in this study may be used to determine inheritance resistance to S. sclerotiorum and for improving oilseed B. napus cultivars for resistance to this important pathogen.

Resistance to Brown Stem Rot in Soybean Germ Plasm with Resistance to the Soybean Cyst Nematode.

The soybean cyst nematode (SCN) and Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR), are two pathogens of soybean commonly found in the same field throughout the north-central United States. Field experiments designed to study the role of SCN-resistant germ plasm in soybean production have led to data suggesting that some sources of SCN resistance also may provide resistance to BSR. Soybean germ plasm with resistance to SCN was evaluated in greenhouse and field environments for resistance to BSR development based on the percentage of host tissue symptomatic of BSR. Comparison of SCN-resistant cultivars and plant introductions (PI) to standard BSR-resistant and -susceptible checks were conducted in two greenhouse experiments using a root-dip inoculation with a single isolate of P. gregata. For both greenhouse experiments, PI 209332 was the only source of SCN resistance with resistance to BSR similar to standard BSR-resistant checks. Nine other sources of SCN resistance, including PI 88788 and Peking, expressed BSR symptom severity similar to BSR-susceptible checks. Cultivars derived from most SCN-resistant sources, including PI 209332, also were susceptible to BSR development, while four of the five cultivars derived from PI 88788 were highly resistant to BSR development. SCN-resistant cultivars derived from PI 88788, Peking, and PI 209332 were planted along with standard BSR-resistant and -susceptible checks at two field locations naturally infested with P. gregata and SCN or P. gregata alone. As in greenhouse experiments, four of the five cultivars derived from PI 88788 expressed resistance to BSR development equal to or better than standard BSR-resistant checks at both locations. In contrast, cultivars derived from PI 209332 and Peking expressed varying levels of disease development depending on field environment. Yields observed for PI 88788-derived cultivars were higher than BSR-resistant checks regardless of the presence of SCN. Data from both greenhouse and field experiments suggest that cvs. Williams and Williams 82 may contain a gene or genes for BSR resistance that require one or more modifier genes, possibly located in the genome of PI 88788, for complete expression.

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.

Occurrence of Soybean Stem Canker (Diaporthe phaseolorum var. meridionalis) in Wisconsin.

Soybean (Glycine max) developed symptoms characteristic of stem canker during the 2000 to 2003 growing seasons in Wisconsin. Symptoms were widespread in 2003 and were associated with yield losses of ≈1% statewide and as much as 25% in individual fields. Affected plants expressed dieback of foliage beginning at growth stage R3 and progressed until the R6 growth stage. Dark brown lesions were frequently observed at a single node on the lower portion of stems of plants expressing foliage dieback. Fungi were isolated from symptomatic plants collected from seven growers' fields in Rock, Sauk, Veron, and Walworth counties and the Arlington and Marshfield Agricultural Research Stations. Stems with lesions were cut into ≈5-mm pieces, surface disinfested with a 0.5% NaOCl solution for 3 min, rinsed three times in sterile distilled water, and placed on water agar (WA) or potato dextrose agar (PDA) at pH 4.5. Hy-phal tips from colonies of interest were excised and placed on acidified PDA at 25°C under continuous light for 25 to 30 days. In addition to Diaporthe phaseolorum var. caulivora (the cause of northern stem canker), four isolates of D. phaseolorum var. meridionalis (the cause of southern stem canker) were isolated. Colonies of D. phaseolorum var. meridionalis isolates were white, lanose, and became tan with age as previously described for D. phaseolorum var. meridionalis (1). Pycnidia with alpha conidia (no beta conidia) and perithecia with 3.1 to 3.4 × 9.5 to 9.8 µm ascospores formed on oat flakes on acidified WA after 30 days. Stromata were brown to black and irregularly shaped. Four isolates of D. phaseolorum var. meridionalis were tested for pathogenicity in a controlled environment using a cut stem inoculation method (2). Stems of 3-week-old seedlings of cv. Sturdy were cut at the midpoint between the second and third node, and a PDA mycelial plug (4 mm in diameter) was placed on the surface of the cut stems. This method was used to inoculate 15 plants in three replicates for each isolate tested. Inoculated plants were placed in a mist chamber in the dark at 25°C for 4 days and later moved to a greenhouse with a 16-h photoperiod at 24 ± 3°C for 3 days. All plants challenged by this method exhibited stem lesions that were 2 to 3 cm long and of similar color to lesions observed in field-grown plants. For each isolate tested, D. phaseolorum var. meridionalis was reisolated from three randomly selected symptomatic plants. Negative controls with a PDA plug did not produce lesions. To our knowledge, this is the first report of D. phaseolorum var. meridionalis on soybean in Wisconsin. The significance of this report relates to the potential spread of D. phaseolorum var. meridionalis beyond its known southern range in the United States. References: (1) F. A. Fernandez et al. Stem Canker. Pages 33-35 in: Compendium of Soybean Diseases, 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999. (2) S. Li et al. Plant Dis. 85:1031, 2001.

Impact of Herbicides on Heterodera glycines Susceptible and Resistant Soybean Cultivars.

Several abiotic and biotic stresses can affect soybean in a growing season. Heterodera glycines, soybean cyst nematode, reduces yield of soybean more than any other pathogen in the United States. Field and greenhouse studies were conducted to determine whether preemergence and postemergence herbicides modified the reproduction of H. glycines, and to determine the effects of possible interactive stresses caused by herbicides and H. glycines on soybean growth and yield. Heterodera glycines reproduction factor (Rf) generally was less on resistant than susceptible cultivars, resulting in a yield advantage for resistant cultivars. The yield advantage of resistant cultivars was due to more pods per plant on resistant than susceptible cultivars. Pendimethalin reduced H. glycines Rf on the susceptible cultivars in 1998 at Champaign, Illinois, and in greenhouse studies reduced dry root weight of H. glycines-resistant and susceptible cultivars, therefore reducing Rf on the susceptible cultivars. The interactive stresses from acifluorfen or imazethapyr and H. glycines reduced the dry shoot weight of the resistant cultivar Jack in a greenhouse study. Herbicides did not affect resistant cultivars' ability to suppress H. glycines Rf; therefore, growers planting resistant cultivars should make herbicide decisions based on weeds present and cultivar tolerance to the herbicide.