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Brown stem rot (BSR) reduces soybean [Glycine max (L.) Merr.] yield by up to 38%. The BSR causal agent is Phialophora gregata f. sp. sojae, a slow‐growing, necrotrophic fungus whose life cycle includes latent and pathogenic phases, each lasting several weeks. Brown stem rot foliar symptoms are often misdiagnosed as other soybean diseases or nutrient stress, making BSR resistance especially difficult to phenotype. To shed light on the genes and networks contributing to P. gregata resistance, we conducted RNA sequencing (RNA‐seq) of a resistant genotype (PI 437970, Rbs3). Leaf, stem, and root tissues were collected 12, 24, and 36 h after stab inoculation with P. gregata, or mock infection, in the plant stem. By using multiple tissues and time points, we could see that leaves, stems, and roots use the same defense pathways. Our analyses suggest that P. gregata induces a biphasic defense response, with pathogen‐associated molecular pattern (PAMP) triggered immunity observed in leaves at 12 and 24 h after infection (HAI) and effector triggered immunity detected at 36 h after infection in the stems. Gene networks associated with defense, photosynthesis, nutrient homeostasis, DNA replication, and growth are the hallmarks of resistance to P. gregata. While P. gregata is a slow‐growing pathogen, our results demonstrate that pathogen recognition occurs hours after infection. By exploiting the genes and networks described here, we will be able to develop novel diagnostic tools to facilitate breeding and screening for BSR resistance.

In an effort to identify new sources of resistance to brown stem rot, caused by Phialophora gregata, 829 soybean accessions originating from 14 provinces in central and southern China and ranging in maturity from group IV to group VIII were obtained from the USDA Soybean Germplasm Collection. All accessions were inoculated in sequential evaluations in the greenhouse with three isolates of P. gregata. Putatively resistant accessions were compared statistically with resistant and susceptible standards. Two hundred forty-one, or approximately 29% of the accessions evaluated, were resistant to all three isolates of P. gregata. The percentage of resistant accessions varied among provinces, with the highest percentages identified in the provinces of Anhui, Gansu, Jiangsu, and Sichuan. Resistance to brown stem rot was not associated with maturity of the accessions or presence of soybean mosaic virus. These resistant accessions could be utilized as sources of brown stem rot resistance through integration of northern and southern soybean germ plasm.

In 1992, a vascular disease was found in two fields of cultivar UC-15 garbanzo beans (Cicer arietinum). The fields were located on the central coast of California and Phialophora gregata was consistently isolated from the aboveground tissue in the host UC-15 plants. Diseased patches in the fields mimicked the more common Fusarium wilt with its discolored vascular tissue in the stems, but in these instances P. gregata was the sole fungus that grew out of the tissue after 5 to 8 days of incubation on isolation agar. Greenhouse pathogenicity tests yielded only minor symptoms whether a dip technique or stem puncture inoculations were used. In subsequent years it was noted that this disease was observed only in garbanzo fields where a pea crop (Pisum sativum) had been previously grown. Further isolation from small foot and root lesions of diseased field plants revealed that they were infected with Fusarium solani f. sp. pisi (Fsp) in addition to P. gregata. In greenhouse experiments with a combination of soilborne Fsp and/or stem inoculations with P. gregata, both pathogens infected the host. The symptoms from both pathogens, the strongest including occasional plant death, were similar to those observed in the fields. The effect was more than additive. Neither pathogen alone caused severe damage. In these tests Fsp was reisolated from root and foot cortical tissue along with P. gregata from stem vascular tissue near the top of the plants. Peas were an important crop along the central coast long before garbanzo beans were grown there. Fsp, a pathogen of both pea and garbanzo bean (1), has been known to attack garbanzos there since 1973 (2). The presence of yet another vascular disease of significance in this coastal area, besides those caused by Fusarium oxysporum f. sp. ciceri and Verticillium dahliae, has not been previously noted. Help in identification of P. gregata came from Diane Fogle, California Department of Food and Agriculture, Sacramento. References: (1) J. M. Kraft. Plant Dis. Rep. 53:110, 1969. (2) F. V. Westerlund Jr., et al. Phytopathology 64:432, 1974.

Genetic studies of Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR) of soybean, have led to the development of species-specific primers capable of separating isolates into two distinct genotypes, A and B. To determine whether genotypic characterization could be related to differences in BSR symptom expression, five soybean cultivars, Pioneer 9234, Corsoy 79 (both BSR susceptible), Williams, BSR 101, and Jack and plant introduction (PI) 437970 (all BSR resistant), were inoculated with a total of 27 isolates of each genotype in four greenhouse experiments conducted from February to November 2000. BSR severity was calculated as the percentage of symptomatic foliar, internal stem, and internal root tissue. Genotype A isolates caused significantly more severe (P < 0.0001) BSR foliar symptoms than genotype B isolates on Pioneer 9234, Corsoy 79, Williams, and BSR 101, while Jack and PI 437970 expressed minimal foliar symptoms regardless of isolate genotype. Overall, internal stem symptoms caused by genotype A isolates were more severe than those caused by genotype B isolates on Pioneer 9234, Corsoy 79, Williams, and BSR 101. Conversely, Jack and PI 437970 did not differ significantly in severity of stem symptoms when inoculated with isolates of genotype A or B. Internal root symptoms for genotype A isolates were generally more severe than for genotype B isolates on all soybean genotypes tested. Our data strongly suggest that A and B genotypes of P. gregata f. sp. sojae differ in the severity of symptoms they cause, and that these genotypes correspond to the Type I (defoliating) and Type II (nondefoliating), respectively, pathotypes previously proposed for this vascular pathogen of soybean.

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.

Tabor, G. M., Tylka, G. L., Behm, J. E., and Bronson, C. R. 2003. Heterodera glycines infection increases incidence and severity of brown stem rot in both resistant and susceptible soybean. Plant Dis. 87:655-661. Growth chamber experiments were conducted to investigate whether parasitism by Heterodera glycines, the soybean cyst nematode, increases incidence and severity of brown stem rot (BSR) of soybean, caused by Phialophora gregata, in both resistant and susceptible soybean cultivars. Soybean genotypes with various combinations of resistance and susceptibility to both pathogens were inoculated with P. gregata alone or P. gregata plus H. glycines. In most tests of H. glycines-susceptible genotypes, incidence and severity of internal stem discoloration, characteristic of BSR, was greater in the presence than in the absence of H. glycines, regardless of susceptibility or resistance to BSR. There was less of an increasing effect of H. glycines on stem symptoms in genotypes resistant to both BSR and H. glycines; however, P. gregata colonization of these genotypes was increased. Stems of both a BSR-resistant and a BSRsusceptible genotype were colonized earlier by P. gregata in the presence than in the absence of H. glycines. Our findings indicate that H. glycines can increase the incidence and severity of BSR in soybean regardless of resistance or susceptibility to either pathogen. Additional keywords: BSR, pathogen interaction, Phialophora gregata, SCN

Growth chamber experiments were conducted to determine if extent of colonization of soybean stems by genotypes A and B of Cadophora gregata (Phialophora gregata), the causal agent of brown stem rot (BSR) of soybean, is similar in soybean plants resistant or susceptible to genotype A. Upon introduction of the two genotypes separately into the base of stems of 2-week-old seedlings, genotype A advanced with the growing tips of susceptible but not resistant genotypes. In contrast, genotype B did not advance with the growing tips of either resistant or susceptible soybean. In similar experiments, 5 weeks after introduction of genotype A, both mean percent stem length colonized by C. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. For genotype B, there was no or a slight difference between resistant and susceptible soybean genotypes in mean percent stem length colonized and no difference in mean percentage of symptomatic trifoliate leaflets 5 weeks after introduction of the pathogen. These results indicate that genotype A and genotype B differ not only in the severity of foliar symptoms they cause on genotype A-susceptible soybean plants, but also in how severely they colonize the stems of these soybean plants. In our experiments, genotype A and genotype B did not differ consistently in their ability to cause internal stem discoloration. The two genotypes of C. gregata can be distinguished based on how severely they colonize stems of genotype A-susceptible soybean. Thus, a BSR resistance screening method, which relies on assessment of stem colonization by C. gregata, works only for screening soybean lines resistance to genotype A. In light of these results, it is important to distinguish soybean resistance to genotype A versus genotype B of C. gregata. Whether genotype B causes yield loss and whether soybean plants can be distinguished as resistant or susceptible to genotype B needs to be investigated.

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.

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.

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.

Phialophora gregata, causal agent of brown stem rot of soybean and adzuki bean, causes internal stem browning and interveinal chlorosis and necrosis of leaves. Water relations and photosynthesis were studied in diseased and healthy soybeans to determine the physiological effects of brown stem rot. A susceptible (Pride B216) and a resistant (HSR 201) cultivar were grown in the greenhouse and growth chamber and injection-inoculated at vegetative stage 1. A significant amount of disease developed in both cultivars, but Pride B216 was more highly stressed by disease than BSR 201. Stem conductance was reduced, and stomatal conductance was increased in both inoculated cultivars compared to uninoculated plants. Transpiration was increased more in Pride B216 than in BSR 201, and photosynthesis was significantly increased in BSR 201 plants when inoculated. Disease-caused water stress can be attributed to a combination of reduced stem conductance and increased water loss resulting from increased stomatal conductance. Additionally, results indicate that photosynthesis may be directly affected by disease rather than by disruption of stomatal control. Preliminary data indicating tolerance to disease by a resistant cultivar, exclusive of pathogen inhibition, are presented.

Brown stem rot (BSR) of soybean [Glycine max (L.) Merr.], caused by Phialophora gregata, is an economically important disease prevalent in soybean producing regions of the northcentral U.S. and Canada. To date, all BSR resistant genes identified are located on chromosome 16 (formerly molecular linkage group J). The objective of this study was to determine if four plant introductions (PI) from south-central China identified as BSRresistant have resistance genes mapping to the same location on chromosome 16 as previously mapped BSR resistance genes. The four plant introductions, PI 594637, PI 594638B, PI 594650A, and PI 594858B were crossed to the BSR-susceptible cultivar ‘Century 84’ to develop four F2 populations. Each segregating population and the parental lines were screened for BSR resistance in growth chamber conditions. The F2:3 individual plants of each population were tested with the simple sequence repeat (SSR) markers Satt431

Brown stem rot, caused by the fungus Phialophora gregata, reduces soybean yield by up to 38%. Although three dominant resistance loci have been identified (Rbs1 to Rbs3), the gene networks responsible for pathogen recognition and defense remain unknown. Further, identification and characterization of resistant and susceptible germplasm remains difficult. We conducted RNA-Seq of infected and mock-infected leaf, stem, and root tissues of a resistant (PI 437970, Rbs3) and susceptible (Corsoy 79) genotype. Combining historical mapping data with genotype expression differences allowed us to identify a cluster of receptor-like proteins that are candidates for the Rbs3 resistance gene. Reads mapping to the Rbs3 locus were used to identify potential novel single-nucleotide polymorphisms within candidate genes that could improve phenotyping and breeding efficiency. Comparing responses to infection revealed little overlap in differential gene expression between genotypes or tissues. Gene networks associated with defense, DNA replication, and iron homeostasis are hallmarks of resistance to P. gregata. This novel research demonstrates the utility of combining contrasting genotypes, gene expression, and classical genetic studies to characterize complex disease resistance loci.

Brown stem rot (BSR) of soybean [Glycine max (L.) Merr.], caused by Phialophora gregata (Allington & D.W. Chamb.) W. Gams 1971, is an economically important disease prevalent in soybean producing regions of the north-central United States and Canada. To date, all BSR resistant genes identified are located on chromosome 16 (formerly molecular linkage group J). The objective of this study was to determine if four plant introductions from south-central China identified as BSR resistant have resistance genes mapping to the same location on chromosome 16 as previously mapped BSR resistance genes. The four plant introductions, PI 594637, PI 594638B, PI 594650A, and PI 594858B, were crossed to the BSR-susceptible cultivar ‘Century 84’ to develop four F₂ populations. Each segregating population and the parental lines were screened for BSR resistance in growth chamber conditions. The F₂:₃ individual plants of each population were tested with the simple sequence repeat (SSR) markers Satt431 or Satt547, which map closely to BSR resistance quantitative trait loci (QTL) on chromosome 16. Associations between molecular data and phenotypic data used to validate QTL were analyzed using single factor ANOVA. Three of the four populations had markers on chromosome 16 significantly associated with BSR resistance with R ² values from 24 to 48%. However, when marker Satt547 was regressed on BSR resistance in population PI 594637 × Century 84, no significant association was observed. This result suggests that PI 594637 could have a new BSR resistance gene. Transgressive segregation also was observed in this population, and highly BSR resistant progeny could be used in the development of BSR resistant cultivars. Additional research and testing in this population will be conducted to identify resistance QTL(s) from this source.

Plants recognize invading pathogens and respond biochemically to prevent invasion or inhibit colonization in plant cells. Enhancing this response in crop plants could improve sustainable methods to manage plant diseases. To enhance disease resistance in soybean, the soybean phytoalexin glyceollin was assessed in soybean hairy roots of two soybean genotypes, Spencer and PI 567374, transformed with either soybean isoflavone synthase (IFS2) or chalcone synthase (CHS6) genes that were inoculated with the soybean pathogens Diaporthe phaseolorum var. meridionales, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Phytophthora sojae. The hairy-root-transformed lines had several-fold decreased levels of isoflavone daidzein, the precursor of glyceollin, and considerably lower concentrations of glyceollin induced by pathogens measured 5 days after fungal inoculation compared with the nontransformed controls without phenolic transgenes. M. phaseolina, P. sojae, and S. sclerotiorum grew much more on IFS2- and CHS6-transformed roots than on control roots, although there was no significant difference in growth of D. phaseolorum var. meridionales on the transformed hairy-root lines. In addition, glyceollin concentration was lower in D. phaseolorum var. meridionales-inoculated transformed and control roots than roots inoculated with the other pathogens. Glyceollin inhibited the growth of D. phaseolorum var. meridionales, M. phaseolina, P. sojae, S. sclerotiorum, and three additional soybean pathogens: Cercospora sojina, Phialophora gregata, and Rhizoctonia solani. The most common product of glyceollin conversion or degradation by the pathogens, with the exception of P. sojae, which had no glyceollin degradation products found in the culture medium, was 7-hydroxyglyceollin.

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.

Gram-positive bacterium BRF-1 isolated from the rhizosphere of root-rotten diseased soybean plants shows strong antifungal activity on plates. In the present study, the ability of BRF-1 to control brown stem rot disease and to promote plant growth was examined using soybean plants grown in pots. Brown stem rot is a severe soil-borne disease of soybean that is caused by the pathogenic fungus Phialophora gregata. Eighteen-day-old soybean seedlings were grown in a mixture of soil (40 mL) and BRF-1 suspension (10 mL) and were dipped in a suspension of P. gregata (106 c.f.u. mL−1), and the severity of the disease was judged 62 days after the fungal inoculation. Inoculation with BRF-1 at 107 and 108 c.f.u. mL−1 significantly lowered the severity of the disease. Soybean seeds incubated in 100 mL of the BRF-1 suspension (2 × 106 c.f.u. mL−1) were sown in pots filled with sand, and plants were collected 7 days after emergence. The dry weight increase in shoots and roots and mineral absorption were signifi...

The severity of brown stem rot (BSR) on soybean (Glycine max) is influenced by environment, genotype of the pathogen, and genetic resistance to BSR. BSR is caused by two genotypes of Phialophora gregata : genotype A causes pith browning and leaf necrosis, and genotype B typically causes only pith browning. The distribution of the genotypes and their interactions with resistant and susceptible soybean cultivars was investigated in multiple field environments. Plots were established with susceptible and resistant cultivars in the states of Illinois, Indiana, Iowa, Kansas, Minnesota, Michigan, Missouri, North Dakota, Ohio, and Wisconsin and in Ontario, Canada. Plants were sampled arbitrarily without regard to BSR symptoms at the R7 growth stage from the plots and from production fields in Illinois, Kansas, North Dakota, Nebraska, and South Dakota. DNA was extracted from stems and tested for the presence of P. gregata using a genotype-specific PCR assay. Phialophora gregata was detected in 26% of all samples,...

Sudden death syndrome (SDS), caused by Fusarium virguliforme, has occurred in several US states and Ontario since 1985 but is a new problem in the northern states, including Minnesota. The characteristics and distribution of F. virguliforme and SDS in Minnesota and other northern soybean production areas were unknown. In 2006 and 2007, SDS was confirmed in 21 counties from the eastern to western borders of Minnesota and to 45°N based on symptoms and a polymerase chain reaction assay. It was also confirmed that F. virguliforme and Phialophora gregata, the cause of brown stem rot, can simultaneously infect a single plant. Isolates of F. virguliforme from soybeans in Minnesota were morphologically identical to those from other states and induced typical SDS symptoms on soybean seedlings. Isolates (n = 15) obtained from 15 fields in 10 Minnesota counties were compared genotypically and phenotypically to a group of 13 isolates of F. virguliforme from other states. Genetic similarity was very high among isolate...

Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.

Greenhouse, growth chamber, and field experiments were conducted to develop a method to assess resistance of soybeans to Cadophora gregata (Phialophora gregata), causal agent of brown stem rot (BSR). In the new method, C. gregata is introduced at the base of the stems of 2-week-old soybeans, and the presence of the fungus is assessed in the tips of the stems 5 weeks later. To test the effectiveness of the method, two populations of soybeans and 10 checks were inoculated at the stem base and then assayed for fungal colonization of the stem tips, percentage of symptomatic leaflets, and percent internal stem length discolored. The lines also were planted in naturally infested fields to assess for percent internal stem length discolored, and were tested for the presence/absence of a BSR-resistant molecular marker. Greenhouse, field, and molecular marker data were compared. Linear regression analysis suggested that percentage of plants with colonized stem tips explained 41 to 64% of the variability (P < 0.0001) in percent stem length discolored in the field and 58 to 85% of the variability (P < 0.0001) in molecular marker data for BSR resistance. Percent stem length discolored assessed in the greenhouse had the lowest correlation with percent stem length discolored in the field and with the molecular marker. Of three incubation temperatures tested, 22°C was the most conducive for distinguishing resistant/susceptible soybeans using the colonization method.

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.

Adzuki bean cultivar Acc259, which is resistant to races 1 and 2 of Phialophora gregata f. sp. adzukicola, was used as a breeding resource for resistance to brown stem rot (BSR). During the third year after two successive cultivations of Acc259, a severe outbreak of BSR occurred in an experimental plot at the Tokachi Agricultural Experiment Station, Hokkaido, Japan. The isolates obtained from diseased plants were virulent to Erimo-shozu (susceptible to all races) and Acc259 but avirulent to Kita-no-otome (resistant to race 1 but susceptible to race 2). The existence of a new race of P. gregata f. sp. adzukicola, designated race 3, was determined; and its frequency in the plot soil was shown to increase from 16.7% before planting Acc259 to 100% after the third year. Of 140 isolates from the commercial production area that were formerly identified as race 1, 13 were actually race 3 and were restricted to certain limited fields.

Many soybean [Glycine max (L.) Merr.] genotypes that carry resistance to soybean cystnematode (Heterodera glycines Ichinohe) (SCN) from plant introduction (PI) 88788 also carry resistance to brown stem rot (BSR) caused by the soilborne fungus Phialophora gregata (Allington & Chamberlain) W. Gams f. sp. sojae Kobayashi, Yamamoto, Negishi, and Ogoshi. The objectives of our research were to map and localize BSR resistance quantitative trait loci (QTL) from Bell, a BSR resistant cultivar with SCN resistance from PI 88788. Initial mapping was done with a population of 93 F 4 -derived lines developed from a cross between Bell and the SCN and BSR susceptible cultivar, Colfax. Lines were evaluated for BSR resistance in two field environments, a greenhouse, and with genetic markers from linkage group (LG) J. To confirm and further localize a resistance QTL, three near isogenic line (NIL) populations were created using an F 4 -derived line from the Bell X Colfax population. In the F 4 population, markers on LG J were significantly (P < 0.001) associated with BSR resistance in both the field (R 2 = 45%) and greenhouse (R 2 = 51%). Data from the NIL populations indicates the QTL is near the closely linked markers 21E22.spl, 21E22.sp2, and 35E22.spl, which is the same genetic region where Rbsl, Rbs2, and Rbs3, the three named BSR resistance genes, were previously mapped. In addition, the genetic region is tightly linked to a previously identified SCN resistance QTL from PI 88788. These results explain why many SCN resistant cultivars also carry BSR resistance and should assist breeders when selecting for resistance to both diseases.

Brown stem rot (BSR) of soybean [Glycine max (L.) Merr.], caused by Phialophora gregata (Allington & Chamberlain) W. Gams f. sp. sojae Kobayashi, Yamamoto, Negishi, and Ogoshi, can be effectively controlled with genetic resistance. Although three BSR resistance genes have been described, they all map to the same genetic region on linkage group (LG) J. In a previous screening of plant introductions (Pb) for BSR resistance, PI 567296B, PI 567323A, PI 567479, PI 567535B, and PI 567544 from central China were identified as having a high level of resistance. The objective of this study was to determine if resistance in these PIs maps to the same region on LG J where BSR resistance genes were previously identified. To map the resistance genes in these accessions, each PI was crossed to the susceptible cultivar Century 84 and 94 F 2-3 lines were developed from each cross. Lines were evaluated for BSR resistance in the greenhouse and screened with molecular markers from LG J. In all five populations, a quantitative trait locus (QTL) conferring BSR resistance was mapped to the same region on LG J where resistance has already been reported. These results suggest that the BSR resistance genes identified in these five PIs from central China are likely allelic with previously reported BSR resistance genes.

Phialophora gregata f.sp. sojae, a soilborne vascular pathogen causing brown stem rot of soybean, has been divided into A and B populations based on variation in the intergenic spacer region of nuclear rDNA (rDNA marker). The A and B populations correlate with defoliating and nondefoliating pathotypes, respectively. In this study, eight additional polymorphic anonymous marker loci (five inter simple sequence repeat loci and three long-primer random amplified polymorphic DNA loci) were identified and applied to a total of 189 isolates. Alleles of these eight loci were invariant within, but different between the A and B populations, providing further evidence that the rDNA marker identifies genetically distinct populations. The two populations were sympatric, residing not only in the same field, but also in the same plants under field conditions. Representative strains of the two populations, when used individually in inoculations, infected both resistant cv. Bell and susceptible cv. Sturdy. However, when the same representatives of the two populations were mixed in a 1 : 1 ratio and used as a mixed inoculum in a competitive bioassay, differential cultivar preference was revealed using PCR detection of populations in infected plants. Population A was detected significantly more often (18 out of 24 plants) in the susceptible cv. Sturdy, whereas population B was detected significantly more often (17 out of 24 plants) in the resistant cv. Bell, corroborating earlier field studies. This is the first controlled experiment to demonstrate a differential host preference of P. gregata f.sp. sojae toward different cultivars of the same host species. Unification of terminologies used in P. gregata f.sp. sojae is discussed.

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.

Growth chamber experiments were conducted to determine if resistance to Phialophora gregata, the causal agent of brown stem rot (BSR) of soybean, is expressed in the stems of resistant soybean genotypes. Upon introduction of the pathogen into the base of stems of 2-week-old seedlings, the fungus advanced with the growing tips of plants of susceptible genotypes but lagged behind in resistant genotypes. Five weeks after introduction of the pathogen, both mean percent stem length colonized by P. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. These results indicate that resistance can be expressed in the stems of resistant soybean plants and suggest that stem inoculation methods may be useful for assessing resistance to P. gregata. Also, in our experiments, internal stem discoloration was not as useful as colonization and foliar symptoms in discriminating resistant from susceptible genotypes.

Genetic variation and variation in aggressiveness in Phialophora gregata f. sp. sojae, the cause of brown stem rot of soybean, was characterized in a sample of 209 isolates from the north-central region. The isolates were collected from soybean plants without regard to symptoms from randomly selected soybean fields. Seven genotypes (A1, A2, A4, A5, A6, M1, and M2) were distinguished based on DNA fingerprinting with microsatellite probes (CAT)5 and (CAC)5, with only minor genetic variation within the A or M genotypes. Only the A1, A2, and M1 genotypes were represented by more than one isolate. The A genotypes dominated in the eastern Iowa, Illinois, and Ohio samples, whereas the M genotypes were dominant in samples from western Iowa, Minnesota, and Missouri. In growth chamber experiments, isolates segregated into two pathogenicity groups based on their aggressiveness toward soybean cvs. Kenwood and BSR101, which are relatively susceptible and resistant, respectively, to brown stem rot. In both root dip inoculation and inoculation by injecting spores into the stem near the ground line (stab inoculations), isolates of the A genotypes caused greater foliar symptoms and more vascular discoloration than isolates of the M genotypes on both cultivars of soybean. All isolates caused foliar symptoms in both cultivars and in three additional cultivars of soybean with resistance to brown stem rot. Greater differences between the A and M genotypes were seen in foliar symptoms than in the linear extent of xylem discoloration, and greater differences were seen in Kenwood than in BSR101. Inoculation of these genotypes into five cultivars of soybean with different resistance genes to brown stem rot showed a genotype × cultivar interaction. A similar distinction was found in an earlier study of the adzuki bean pathogen, P. gregata f. sp. adzukicola, and consistent with the nomenclature of that pathogen, the soybean pathogens are named the aggressive race (race A) and the mild race (race M) of P. gregata f. sp. sojae.

The distribution of two races (1 and 2) of Phialophora gregata f. sp. adzukicola, the causal agent of brown stem rot of adzuki bean, was examined using a total of 483 isolates obtained from 39 fields in 19 locations on Hokkaido, Japan between 1997 and 1999. Race 1 was predominant (416 isolates or 86.1%) in the commercial fields tested. Race 2 was found in 25 fields (64.1%), including two fields of cultivar Kita-no-otome (resistant to race 1, but susceptible to race 2), indicating that race 2 was widely distributed in most of the production areas in Hokkaido. Using amplified fragment length polymorphisms (AFLP), a total of 67 polymorphic AFLP markers was recorded among 72 representative isolates (37 and 35 isolates of races 1 and 2, respectively), and 57 distinct haplotypes were detected. Cluster analysis revealed no close correlation between races and AFLP groups. Thus, no difference was observed between values of gene diversity in each race (0.253 and 0.284 in races 1 and 2, respectively), and the coefficient of gene differentiation was very low (G ST =0.015). Gene differentiation between both races by analysis of molecular variance was not significantly different from zero (Φ=−0.001; p=0.403). However, the results of gene differentiation among regional populations (G ST =0.290, Φ=0.292; p

A cultivar de soja BRS 153, oriunda do cruzamento EMBRAPA 1 x Braxton, é de ciclo médio, possui plantas de crescimento determinado, flor branca, pubescência cinza e grãos de hilo marromclaro. É resistente ao cancro-da-haste (Diaporthe phaseolorum f. sp. meridionalis), à podridão-parda-da-haste (Phialophora gregata), à mancha-olho-de-rã (Cercospora sojina) e ao oídio (Microsphaera diffusa). O rendimento médio de grãos, em 19 ambientes do Rio Grande do Sul, foi 12% e 14% superior aos das cultivares RS 7-Jacuí e BR-16, respectivamente. É indicada para semeaduras em novembro, em todo o Rio Grande do Sul.

A BRS 205, cultivar de soja de ciclo semiprecoce, obtida do cruzamento [BR-16(2) x Ocepar 8] x Tracy-M, é indicada para o Rio Grande do Sul, em semeaduras de novembro. Possui crescimento determinado, plantas de flor branca, pubescência marrom, porte baixo e grãos de hilo preto. Tem resistência ao cancro-da-haste (Diaporthe phaseolorum f. sp. meridionalis), à podridão-parda-da-haste (Phialophora gregata), à mancha-olho-de-rã (Cercospora sojina) e à pústula bacteriana (Xanthomonas axonopodis pv. glycines). É moderadamente resistente ao oídio (Microsphaera diffusa). O rendimento médio de grãos foi 6% e 11% superior ao das cultivares RS 7-Jacuí e BR-16, respectivamente.

Rhynchosporium secalis causes an economically important foliar disease of barley, rye, and other grasses known as leaf blotch or scald. This species has been difficult to classify due to a paucity of morphological features; the genus Rhynchosporium produces conidia from vegetative hyphae directly, without conidiophores or other structures. Furthermore, no teleomorph has been associated with R. secalis, so essentially nothing is known about its phylogenetic relationships. To identify other fungi that might be related to R. secalis, the 18S ribosomal RNA gene and the internal transcribed spacer (ITS) region (ITS1, 5.8S rRNA gene, and ITS2) were sequenced and compared to those in databases. Among 31 18S sequences downloaded from GenBank, the closest relatives to R. secalis were two species of Graphium (hyphomycetes) and two other accessions that were not identified to genus or species. Therefore, 18S sequences were not useful for elucidating the phylogenetic relationships of R. secalis. However, analyses of 76 ITS sequences revealed very close relationships among R. secalis and species of the discomycete genera Tapesia and Pyrenopeziza, as well as several anamorphic fungi including soybean and Adzuki-bean isolates of Phialophora gregata. These species all clustered together with 100% bootstrap support. On the basis of these results, the teleomorph of R. secalis, if it exists, most likely will be a small apothecium produced directly on dead, infected host tissue. The ITS analysis also indicated that higher-level classifications within the discomycetes need to be revised, and that Tapesia and Pyrenopeziza probably do not belong in the Dermateaceae.

Flow cytometry was used to characterize isolates of Phialophora gregata, the causal organism of brown stem rot (BSR) in soybean, using the fluorescence intensity of YOYO-1 stained conidia. The conidia were derived from isolates of P. gregata isolated from field-grown soybean vascular tissue from two locations in Wisconsin. At each location, four soybean cultivars were grown. Two cultivars were susceptible to BSR and two cultivars were resistant to BSR. Ninety-six isolates, one per plant, were characterized for DNA content. The stained conidia were analyzed with an EPICS XL (Coulter Corporation, Hialeah, Florida, USA) flow cytometer cell sorter with CICERO software (San Rafael, CA, USA). The isolates differed in their mean DNA content, ranging from 78.0 to 116.8 arbitrary units. Statistical analysis showed a significant difference in the DNA content variability of the isolates between the two locations. A notable difference in DNA content variability was observed between the isolates collected from the resistant cultivars between the two locations. However, no significant variation occurred in the DNA content of the isolates collected from susceptible cultivars within or between locations. The observed variation among the isolates is hypothesized to be due to the occurrence of separate populations of P. gregata and selection for mean DNA content that could colonize specific plant genotypes.

A microsatellite-enriched library was constructed from genomic DNA of Phialophora gregata f. sp. sojae, the causal agent of soybean brown stem rot. Specific oligonucleotide primer pairs were designed to flank individual microsatellite loci, and used in PCR to detect polymorphisms among 118 contemporary and archival isolates of P. gregata from several countries, which represent two distinct, but sympatric populations delineated by rDNA genotypes. In general, low levels of genetic variation were observed and manifested in two ways. First, the number of polymorphic loci was low. Among 24 microsatellite loci screened, four polymorphic loci were detected, and two of them were located on the same clone. Second, the number of alleles in the polymorphic loci was low. Only two alleles were observed in each polymorphic locus. Alleles of the polymorphic microsatellite loci were invariant within, but different between, the two populations of P. gregata. From all evidence available, P. gregata f. sp. sojae exhibits several genetic features characteristic of clonal genetic structure, namely widespread distribution and stability of identical genotypes, absence of recombinant genotypes, and correlation of independent genetic traits including three sets of molecular markers, defoliating and nondefoliating pathotypes, and differential pathogenic fitness.

AFLP (amplified fragments length polymorphism) and ISSR (inter simple sequence repeat) analyses were used to detect genetic diversity among 46 Phialophora gregata isolates including 41 from soybean, four from mung bean, and one from adzuki bean. Five AFLP primers amplified 55 fragments, of which 20 bands were polymorphic. Thirteen ISSR primers amplified 66 fragments, of which 45 bands were polymorphic. The ISSR technique detected more polymorphism than the AFLP technique did. A UPGMA (unweighted pair-group arithmetic mean) dendrogram was constructed based on the 121 amplified fragments with AFLP and ISSR primers to depict genetic diversity and relationships among the 46 isolates. Various levels of DNA polymorphism were detected among P. gregata isolates from soybean and mung bean. The estimated average genetic diversity is 0.079 for the population of 45 isolates from soybean and mung bean. The ISSR technique was shown to be more effective and economic than the AFLP technique in detecting genetic variation in P. gregata.

Summary • Dark-septate endophytic (DSE) fungi are ubiquitous in the roots of Arctic and alpine plants, yet very little is known about their phylogenetic identities or effects on their host plants. • Several such fungi were isolated from the alpine snowbed plant Ranunculus adoneus in the Front Range of Colorado, USA; one isolate was chosen for detailed study. The ability of this isolate to re-colonize plant roots in pot cultures was assessed, and phylogenetic analyses were performed using small-subunit (SSU), 5.8S and internal transcribed spacer (ITS) 2 ribosomal DNA sequences. • This isolate had the ability to produce root endophytic structures in pot cultures similar to those reported from other sources and observed in R. adoneus roots. SSU phylogenetic analyses showed this isolate to be related to a clade within the Euascomycetes containing the Leotiales and Erysiphales. In addition, SSU and 5.8S–ITS2 sequences showed high phylogenetic similarity to a variety of isolates reported from other plants of diverse geographical origins. Although most of these isolates remain unidentified, one closely related isolate was the anamorphic taxon Phialophora gregata. • The results suggest that this DSE isolate might belong to the fairly closely related group of plant endophytes that have varied effects on the plants that they inhabit.

Brown stem rot (BSR) of soybean [Glycine max (L.) Merr.] is caused by the fungal pathogen Phialophora gregata (Allington & D.W. Chamberlain) W. Gams and occurs in soybean production areas around the world, Brown stem rot resistance genes Rbs 1 , Rbs 2 , and Rbs 1 have been identified in soyhean germplasm and plant introductions through traditional genetic analyses. Resistance to BSR has been shown to reduce yield losses in soybean, but selection for this trait is laborious and confounded by environmental variation, The objectives of this study were to identify molecular markers linked to BSR resistance genes Rbs 1 and Rbs 2 , and map these genes in the soybean genome, Genetic families of populations segregating for Rbs 1 and Rbs 2 were evaluated in the greenhouse for BSR phenotypic reaction and identified as resistant, segregating, or susceptible. Leaf tissue collected from members of F 2:3 families was bulked and DNA simple sequence repeat (SSR) marker analysis was used to identify markers that cosegregated with BSR reaction phenotypes. Five pairs of Rbs 2 near-isogenic lines were subjected to a similar analysis to verify results obtained from marker analysis conducted on the population segregating for Rbs 2 . Results of marker analyses indicated that SSR markers Satt215 and Satt431 were linked to Rbs 1 and that Satt244 and Satt431 were linked to Rbs 2 . Marker-assisted selection in the Rbs 1 (using Satt431) and Rbs 2 (using Satt244) populations would have correctly predicted 88 and 82%, respectively, of the BSR reaction phenotypes. The Rbs 1 and Rbs 2 loci map to Molecular Linkage Group J and lie in a region known to contain Rbs 1 . This region also contains loci conditioning resistance to taxonomically diverse fungal pathogens and a locus affecting nodulation in response to a bacterial symbiont.

Brown stem rot (BSR) causes vascular and foliar damage in soybean [Glycine max (L.) Merr.]. Identification of plants resistant to BSR by inoculation with Phialophora gregata (Allington & W.W. Chamberlain) W. Gams is laborious and unreliable because of low heritability. Molecular markers linked to the resistance gene could he used to screen for resistant individuals and hasten the development of BSR resistant genotypes. The objective of this study was to develop molecular markers for efficient identification of BSR resistant plants in a breeding program. Seventeen resistant and 29 susceptible cultivars and plant introductions as well as recombinant inbred lines derived from a cross between BSR 101 and PI 437.654 were assayed by PCR-based markers derived from RFLPs K3751-1 and RGA2V-1, Satt244, or developed from bacterial artificial chromosome (BAC) sequences. The DNA markers that were developed tag the BSR locus and are informative in a diverse range of soybean germplasm. Markers detected different banding patterns between resistant and susceptible genotypes. The PCR-based markers will most likely be useful in screening for BSR resistance and allow soybean breeders to transfer rapidly resistance derived from Rbs 3 to improved cultivars or soybean lines. The markers are relatively easy-to-use, inexpensive, and highly informative. Soybean breeding efforts can now be designed to incorporate the use of marker information when parental genotypes possess contrasting banding patterns.

Harrington, T. C., Steimel, J., Workneh, F., and Yang, X. B. 2000. Molecular identification of fungi associated with vascular discoloration of soybean in the north central United States. Plant Dis. 84:83-89. Brown stem rot is a common but poorly understood vascular wilt disease of s oybean. In order to more clearly delimit the causal agent ( Phialophora gregata) and distinguish it from other morphologically similar fungi from discolored soybean stems, fungi were isolated on a semi-selective medium from discolored and non-discolored soybean stems collected at random across Iowa, Illinois, Minnesota, Missouri, and Ohio. A total of 11 fungi were commonly isolated and characterized based on colony morphology and DNA sequences of the internal transcribed spacer region of the rDNA operon. Phomopsis longicolla was the most frequently isolated fungus, but it was isolated more commonly from lightly discolored or non-discolored stems than from discolored stems. Phialophora gregata was the next most frequently isolated fungus and was isolated more commonly from discolored stems and more commonly in 1996 than in 1995, which had a warm growing season and relatively little brown stem rot. In inoculation experiments, only P. gregata was capable of causing the vascular discoloration and leaf symptoms typical of brown stem rot; none of the seven isolates could be considered non-defoliating. Two other fungi, Plectosphaerella cucumerina and Gliocladium roseum, were similar in colony morphology to Phialophora gregata but were not pathogenic to soybean, and these may be the same species as those referred to by earlier workers as Acremonium spp. or the non-defoliating form of P. gregata. Additional keywords: Cladosporium tenuissimum, Diaporthe phaseolorum, Fusarium spp., Glycine max, Macrophomina phaseolina

2,3-Dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-4-one, was isolated from 3 week old rice cultures of Phialophora gregata , a soybean pathogen, grown at 25°C. This material in repeated bioassays prevented callus formation on soybean hypocotyl sections at 100 and 200 mg/ml callus culture medium. This is the first report of the production of this metabolite by a member of the Phialphora genus and of its toxicity to soybeans.

The objective of this study was to map the gene(s) conferring resistance to brown stem rot in the soybean cultivar BSR 101. A population of 320 recombinant inbred lines (RIL) was derived from a cross of BSR 101 and PI 437.654. Seedlings of each RIL and parent were inoculated by injecting stems with a suspension of spores and mycelia of Phialophora gregata, incubated in a growth chamber at 17 °C, and assessed for resistance by monitoring the development of foliar and stem symptoms. The population also was evaluated with 146 RFLPs, 760 AFLPs, and 4 probes for resistance gene analogs (RGAs). Regression analysis identified a significant association between resistance and several markers on Linkage Group J of the USDA-ARS molecular marker linkage map. Interval analysis with Mapmaker QTL identified a major peak between marker RGA2V-1 and AFLP marker AAGATG152M on Linkage Group J. A second peak, associated only with stem symptoms, was identified between the RFLP B122I-1 and RGA2V-1, also on Linkage Group J. When composite interval mapping with QTL Cartographer was used, two linked QTL were identified with both foliar and stem disease assessment methods: a major QTL between AFLP markers AAGATG152E and ACAAGT260, and a minor QTL between RGA3I-3 and RGA3I-2. These results demonstrate that composite interval mapping gives increased precision over interval mapping and is capable of distinguishing two linked QTL. The minor QTL associated with the cluster of RGA3I loci is of special interest because it is the first example of a disease resistance QTL associated with a resistance gene analog.

An insertion sequence was detected near the 3′ end of the nuclear small subunit rDNA in isolates ofPhialophora gregata f. sp.adzukicola, the causal agent of the brown stem rot disease of adzuki bean. This insertion sequence was absent in isolates ofP. gregata, f. sp.sojae which causes brown stem rot of soybean. The insertion sequence is 304 bp long and contains all the characteristics of group I introns. These characteristics include, the four conserved sequence elements (P, Q, R, and S), a U at the 5′ splice site of the exon, a G at the 3′ splice site of the intron, a putative internal guiding sequences; the sequence also fits a secondary structure model for group I introns. Similar to most group I introns found in nuclear small subunit rDNA, the intron was located in a highly conserved region and is devoid of long open reading frames. This intron provides a convenient marker for use in conventional PCR to separateP. gregata f. sp.adzukicola fromP. gregata f. sp.sojae.

Summary-Experiments were carried out in pots to study the influence of the soybean cyst nematode (Heterodera g/y&es) on the incidence of brown stem rot (BSR) in soybeans (Glycine max) and adzuki beans (Vigna angularis) caused by the soilbome fungi, Phialophora gregata fsp. sojae and P. g. f.sp. adzukicola. The presence of H. glycines increased the incidence of BSR in susceptible soybean (cv. Nakate-Hikarikuro) and adzuki bean (cv. Erimoshozu) for both pathogens. The disease-enhancing effects of H. glycines were not shown in the soybean cultivar Peking, which is resistant to the nematode. In a soybean cultivar resistant to BSR (cv. BSR302), the incidence of BSR was low, even under the presence of H. glycines. In split-root experiments using the two BSR susceptible cultivars, the diseaseenhancing effects of H. glycines were not observed in either soybeans or adzuki beans, whose roots were inoculated separately with P. gregata and H. glycines. 0 1997 Elsevier Science Ltd

Flow cytometry was used to characterize isolates of Phialophora gregata using the fluorescence intensity of propidium iodide-stained conidia. The isolates differed in their mean fluorescence intensity, ranging from 100.0 to 129.7 arbitrary units (AU). When the number of fluorescent events was plotted against intensity of fluorescence, a single peak was observed. Fluorescent patterns of Acremonium isolates from soybean vascular tissue were compared with those of P. gregata. Their mean fluorescence intensity ranged from 76.4 to 88.0 AU. With some of these isolates, multiple peak histograms were observed, corresponding to multiple spore sizes as well as single and double nucleated conidia. Using flow cytometry, we were able to distinguish P. gregata isolates from those of Acremonium, based on mean fluorescence intensity and/or the presence of multiple peaks. Flow cytometric analysis of propidium-iodide stained conidia of Phialophora isolates should prove to be useful for determining the relative DNA content of different isolates collected from different geographic areas.

Soybean accessions from China were screened in an attempt to identify unique sources of resistance to Phialophora gregata, the cause of brown stem rot. In 1994, over 500 accessions from the USDA Soybean Germplasm Collection, University of Illinois, Urbana-Champaign, were evaluated in the field at Urbana, IL, for reaction to brown stem rot. The accessions originated from nine provinces in central China and ranged in maturity from groups II to IV. Disease assessment was based on incidence of foliar symptoms and severity of stem symptoms produced by infection with natural inoculum. Based on field results, 64 putatively resistant lines were selected and evaluated in the greenhouse by a root-dip inoculation method. Thirteen accessions with levels of resistance equal to those of resistant standards were identified from five provinces. These lines may have value as donors of unique sources of resistance to brown stem rot.