Leveraging RNA-Seq to Characterize Resistance to Brown Stem Rot and the Rbs3 Locus in Soybean

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.

Response of Soybean Pathogens to Glyceollin

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.

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.

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.

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.

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.

Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay

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.