Characterization of QoI-Fungicide Resistance in Cercospora Isolates Associated with Cercospora Leaf Blight of Soybean from the Southern United States.

Cercospora leaf blight (CLB) of soybean, caused by Cercospora cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae, is an economically important disease in the southern United States. Cultivar resistance to CLB is inconsistent; therefore, fungicides in the quinone outside inhibitor (QoI) class have been relied on to manage the disease. Approximately 620 isolates from plants exhibiting CLB were collected between 2018 and 2021 from 19 locations in eight southern states. A novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on two genes, calmodulin and histone h3, was developed to differentiate between the dominant species of Cercospora, C. cf. flagellaris, and C. cf. sigesbeckiae. A multilocus phylogenetic analysis of actin, calmodulin, histone h3, ITS rDNA, and transcription elongation factor 1-α was used to confirm PCR-RFLP results and identify remaining isolates. Approximately 80% of the isolates collected were identified as C. cf. flagellaris, while 15% classified as C. cf. sigesbeckiae, 2% as C. kikuchii, and 3% as previously unreported Cercospora species associated with CLB in the United States. PCR-RFLP of cytochrome b (cytb) identified QoI-resistance conferred by the G143A substitution. Approximately 64 to 83% of isolates were determined to be QoI-resistant, and all contained the G143A substitution. Results of discriminatory dose assays using azoxystrobin (1 ppm) were 100% consistent with PCR-RFLP results. To our knowledge, this constitutes the first report of QoI resistance in CLB pathogen populations from Alabama, Arkansas, Kentucky, Mississippi, Missouri, Tennessee, and Texas. In areas where high frequencies of resistance have been identified, QoI fungicides should be avoided, and fungicide products with alternative modes-of-action should be utilized in the absence of CLB-resistant soybean cultivars.

Breeding for disease resistance in soybean: a global perspective.

KEY MESSAGE: This review provides a comprehensive atlas of QTLs, genes, and alleles conferring resistance to 28 important diseases in all major soybean production regions in the world. Breeding disease-resistant soybean [Glycine max (L.) Merr.] varieties is a common goal for soybean breeding programs to ensure the sustainability and growth of soybean production worldwide. However, due to global climate change, soybean breeders are facing strong challenges to defeat diseases. Marker-assisted selection and genomic selection have been demonstrated to be successful methods in quickly integrating vertical resistance or horizontal resistance into improved soybean varieties, where vertical resistance refers to R genes and major effect QTLs, and horizontal resistance is a combination of major and minor effect genes or QTLs. This review summarized more than 800 resistant loci/alleles and their tightly linked markers for 28 soybean diseases worldwide, caused by nematodes, oomycetes, fungi, bacteria, and viruses. The major breakthroughs in the discovery of disease resistance gene atlas of soybean were also emphasized which include: (1) identification and characterization of vertical resistance genes reside rhg1 and Rhg4 for soybean cyst nematode, and exploration of the underlying regulation mechanisms through copy number variation and (2) map-based cloning and characterization of Rps11 conferring resistance to 80% isolates of Phytophthora sojae across the USA. In this review, we also highlight the validated QTLs in overlapping genomic regions from at least two studies and applied a consistent naming nomenclature for these QTLs. Our review provides a comprehensive summary of important resistant genes/QTLs and can be used as a toolbox for soybean improvement. Finally, the summarized genetic knowledge sheds light on future directions of accelerated soybean breeding and translational genomics studies.

Ecology and diversity of culturable fungal species associated with soybean seedling diseases in the Midwestern United States.

AIMS: To isolate and characterize fungi associated with diseased soybean seedlings in Midwestern soybean production fields and to determine the influence of environmental and edaphic factors on their incidence. METHODS AND RESULTS: Seedlings were collected from fields with seedling disease history in 2012 and 2013 for fungal isolation. Environmental and edaphic data associated with each field was collected. 3036 fungal isolates were obtained and assigned to 76 species. The most abundant genera recovered were Fusarium (73%) and Trichoderma (11.2%). Other genera included Mortierella, Clonostachys, Rhizoctonia, Alternaria, Mucor, Phoma, Macrophomina and Phomopsis. Most recovered species are known soybean pathogens. However, non-pathogenic organisms were also isolated. Crop history, soil density, water source, precipitation and temperature were the main factors influencing the abundance of fungal species. CONCLUSION: Key fungal species associated with soybean seedling diseases occurring in several US production regions were characterized. This work also identified major environment and edaphic factors affecting the abundance and occurrence of these species. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and characterization of the main pathogens associated with seedling diseases across major soybean-producing areas could help manage those pathogens, and devise more effective and sustainable practices to reduce the damage they cause.

Effects of Soybean Cultivar, Foliar Application of Azoxystrobin, and Year on Seed Vigor and Microflora Under Delayed Harvest Conditions.

The effects of cultivar and foliar fungicide applications on soybean seed germination, vigor, microflora, and yield after delayed harvest were determined at the University of Arkansas Vegetable Research Station in Kibler, AR, from 2008 to 2010. Seven cultivars with varying levels of resistance to Diaporthe spp. or Cercospora spp. were treated or not treated with a foliar application of azoxystrobin at the R5 growth stage. Plots were harvested 3 weeks after the plants had reached harvest maturity. Yields were recorded, samples of seed were collected, and standard germination (SG) and accelerated aging (AA) were assessed. Seeds were also assayed for infection by fungi on modified potato dextrose agar and by bacteria on nutrient agar. Seed vigor was significantly reduced by infection with Diaporthe spp., Fusarium spp., and Bacillus subtilis, but not with Cercospora spp. Cultivar had a significant impact on yield, seed vigor, and seed infection levels. The cultivar Osage had consistently high seed vigor and low overall seed infection incidence throughout the study. MO/PSD-0259, AG 4403, and UA 4805 also had relatively high seed vigor and low seed infection rates. PI 80837 had a low incidence of seed infection by Diaporthe spp. and Fusarium spp. in 2008 and 2010, but high levels in 2009, when environmental conditions were especially favorable for these pathogens. AP 350 and Suweon97 had relatively high seed infection incidences, particularly of Diaporthe spp. and Fusarium spp., and relatively low seed vigor. Application of the foliar fungicide azoxystrobin at the R5 growth stage significantly increased AA across years and cultivars and increased seed infection by Diaporthe spp. in 2009 across cultivars. There were significant negative correlations between yield and seed infection by Diaporthe spp. and Bacillus subtilis during 1 year and with Fusarium spp. during all 3 years. Overall, resistance to seed infection can persist even when harvest is delayed. In addition to Diaporthe spp., other seedborne pathogens may reduce seed vigor and yield.

Effectiveness of a Seed Plate Assay for Evaluating Charcoal Rot Resistance in Soybean and the Relationship to Field Performance.

Charcoal rot of soybean, caused by Macrophomina phaseolina, is a disease of economic significance in the United States. Although there are soybean cultivars with moderate resistance, identifying and quantifying resistance is challenging. Existing assays are time consuming, and results are often highly variable. The objectives of this research were to (i) create a reproducible seed plate assay (SPA) for charcoal rot resistance and (ii) correlate field-based disease assessments with SPA results on diverse soybean accessions. To develop the SPA, surface-disinfected seeds from eight soybean genotypes (representing three susceptible and five resistant cultivars) were placed on water agar plates inoculated with M. phaseolina. After incubation at room temperature in darkness for 7 days, percent germination was determined for each cultivar relative to the germination on noninoculated plates. Results from SPA were in general agreement with published responses. None of the soybean genotypes showed complete resistance to M. phaseolina. For the second objective, charcoal rot resistance in 18 soybean accessions was assayed with SPA, and results were analyzed for correlation with field disease assessments from Stuttgart, AR, from 2011 to 2014 and from Rohwer, AR, in 2011 and 2012. SPA consistently categorized soybean genotype resistance compared with field disease assessment averages, and results were consistent with previously published resistance determinations. SPA was significantly correlated with percent height of internal stem discoloration (PHSD) at Stuttgart from 2011 to 2013 and in 2012 at Rohwer, with root and stem severity (RSS) at Rohwer in 2012, and with tap root colonization (CFU) at Stuttgart in 2012. SPA was significantly correlated to yield at Stuttgart in 2011, 2013, and 2014, and in 2011 and 2012 at Rohwer. Yield was not correlated to RSS, PHSD, or CFU at either location or in any year. Therefore, SPA is a reproducible and rapid assay for charcoal rot resistance in soybean and is significantly associated to field performance.

Benefits and Profitability of Fluopyram-Amended Seed Treatments for Suppressing Sudden Death Syndrome and Protecting Soybean Yield: A Meta-Analysis.

A meta-analytic approach was used to summarize data on the effects of fluopyram-amended seed treatment on sudden death syndrome (SDS) and yield of soybean (Glycine max L.) in over 200 field trials conducted in 12 U.S. states and Ontario, Canada from 2013 to 2015. In those trials, two treatments-the commercial base (CB), and CB plus fluopyram (CBF)-were tested, and all disease and yield data were combined to conduct a random-effects and mixed-effects meta-analysis (test of moderators) to estimate percent control and yield response relative to CB. Overall, a 35% reduction in foliar disease and 295 kg/ha (7.6%) increase in yield were estimated for CBF relative to CB. Sowing date and geographic region affected both estimates. The variation in yield response was explained partially by disease severity (19%), geographic region (8%), and sowing date (10%) but not by the resistance level of the cultivar. The probability of not offsetting the cost of fluopyram was estimated on a range of grain prices and treatment cost combinations. There was a high probability (>80%) of yield gains when disease level was high in any cost-price combinations tested but very low when the foliar symptoms of the disease were absent.

Oomycete Species Associated with Soybean Seedlings in North America-Part I: Identification and Pathogenicity Characterization.

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.

Evaluation of Commercial Soybean Cultivars for Reaction to Phomopsis Seed Decay.

Phomopsis seed decay (PSD), caused by Phomopsis longicolla (syn. Diaporthe longicolla), is an economically important soybean disease causing poor seed quality. Planting resistant cultivars is one of the most effective means to control PSD. In this study, 16 commercially available maturity groups IV and V soybean cultivars, including two previously identified PSD-resistant and two PSD-susceptible checks, were evaluated for seed infection by P. longicolla in inoculated and noninoculated plots, and harvested promptly or with a 2-week delay in harvest. The test was conducted at Stoneville, Mississippi, in 2012 and 2013. Seed infection by P. longicolla ranged from 0.5 to 76%, and seed germination ranged from 18 to 97%. One MG IV cultivar (Morsoy R2 491) and five MG V cultivars (Progeny 5650, Progeny 5706, Asgrow 5606, Asgrow 5831, and Dyna-Gro33C59) had significantly (P ≤ 0.05) lower percent seed infected by P. longicolla than their respective susceptible checks and other cultivars in the same tests. Information obtained from this study will be useful for soybean growers and breeders for selection of cultivars for planting or breeding and future genetic studies in the development of cultivars with improved resistance to PSD.

Oomycete Species Associated with Soybean Seedlings in North America-Part II: Diversity and Ecology in Relation to Environmental and Edaphic Factors.

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.

Screening a Diverse Soybean Germplasm Collection for Reaction to Purple Seed Stain Caused by Cercospora kikuchii.

Purple seed stain (PSS), caused by Cercospora kikuchii, is a prevalent soybean disease that causes latent seed infection, seed decay, purple seed discoloration, and overall quality deterioration. The objective of this research was to screen soybean accessions from the United States Department of Agriculture germplasm collection for resistance to PSS. In total, 123 plant introductions (PI) from 28 different countries, representing maturity groups (MG) III, IV, and V, were screened. Incidence of Cercospora leaf blight (% CLB), visual PSS (% PSS), and seed infected by C. kikuchii (% C. kikuchii) in harvested seed were determined. In 2007, % C. kikuchii was 2 to 51% for MG III, 2 to 35% for MG IV, and 0 to 33% for MG V. In 2008, % C. kikuchii was 0 to 45% for MG III, 1 to 71% for MG IV, and 0 to 15% for MG V. In total, four and ten PI from MG III and IV, respectively, were identified as resistant to PSS in both years. Highly positive correlations were found for inoculated versus noninoculated treatments and for % PSS versus % C. kikuchii infection. The PSS-resistant PI identified in this study will be valuable to breeders in developing resistant cultivars.

Evaluation of Diverse Soybean Germplasm for Resistance to Phomopsis Seed Decay.

Phomopsis seed decay (PSD), caused primarily by the fungal pathogen Phomopsis longicolla, is one of the most important diseases reducing seed quality and yield of soybean. Few cultivars have been identified as resistant. To identify new sources of resistance to PSD, 135 soybean germplasm accessions, originating from 28 countries, were field screened in Arkansas, Mississippi, and Missouri in 2009. Based on seed assays of natural field infection by P. longicolla in 2009, 42 lines, including the most resistant and susceptible lines, were reevaluated in the field in 2010, 2011, and 2012 with P. longicolla-inoculated and noninoculated treatments. Six maturity group (MG) III (PI 189891, PI 398697, PI 417361, PI 504481, PI 504488, and PI 88490), four MG IV (PI 158765, PI 235335, PI 346308, and PI 416779), and five MG V (PI 381659, PI 381668, PI 407749, PI 417567, and PI 476920) lines had significantly lower percent seed infection by P. longicolla than the susceptible checks and other lines in the same test (P ≤ 0.05). They appeared to have some levels of resistance to PSD. These new sources of PSD resistance can be used in developing soybean breeding lines or cultivars with resistance to PSD, and for genetic mapping of PSD resistance genes.

Mating-type distribution and genetic diversity of Cercospora sojina populations on soybean from Arkansas: evidence for potential sexual reproduction.

Cercospora sojina causes frogeye leaf spot of soybean, which can cause serious economic losses in the United States. In this study, 132 C. sojina isolates were collected from six fields (from two counties, Cross and Crawford) in Arkansas. To determine mating type, a multiplex polymerase chain reaction assay was developed with primers specific for C. sojina. Of the 132 isolates, 68 isolates had the MAT1-1-1 idiomorph and 64 isolates had the MAT1-2 idiomorph; no isolates possessed both idiomorphs. Both mating types were present in a variety of spatial scales, including separate lesions on individual leaves. Clone-corrected data from eight microsatellites indicated that mating-type loci were present in approximately equal proportions in all populations analyzed, which suggests that Arkansas populations of C. sojina are undergoing cryptic sexual reproduction. All six populations evaluated had high genotypic diversity of 26 to 79%. In addition, among strains isolated from a single leaf, multiple and distinct haplotypes were associated with both mating types, supporting the hypothesis that sexual reproduction occurs within the populations. Most populations showed significant gametic disequilibrium but levels of disequilibrium were relatively low, particularly in populations from Crawford County. A low differentiation index (GST) was observed for all simple-sequence repeat markers across all populations. Furthermore, the value of G statistics between populations suggests that significant genetic exchange exists among the populations. Taken together, these results demonstrate that C. sojina populations from Arkansas are genetically diverse and most likely undergoing sexual reproduction.

Agrobacterium tumefaciens-mediated transformation of the soybean pathogen Phomopsis longicolla.

To facilitate functional genomics in the soybean pathogen Phomopsis longicolla, we developed a robust Agrobacterium tumefaciens-mediated transformation system that yielded 150-250 transformants per 1×10(6) conidia of P. longicolla. This first report of P. longicolla transformation provides a useful tool for insertional mutagenesis in an increasingly important pathogen of soybean.

Effect of Lactofen, Azoxystrobin, and Genotypes on Charcoal Rot, Phomopsis Seed Decay, and Pod and Stem Blight in Soybean.

Yield-limiting diseases such as charcoal rot and Phomopsis seed decay have a significant impact on the economic potential for soybean because there are few methods for management of these diseases. The objectives of this study were to determine the development of charcoal rot, infection of seed by Phomopsis spp., and severity of pod and stem blight on Asgrow 4403, Delta Pine 5806, United States Department of Agriculture-introduced DT 97-4290 and plant introduction (PI) number PI 567562A, and Asgrow 4403 treated and not treated with lactofen or azoxystrobin. This is the first report of high levels of resistance in PI 567562A to charcoal rot, and resistance in this PI was greater than for DT 97-4290. Application of lactofen at growth stage R1 and azoxystrobin at either planting, R3, or R6 had no significant impact on severity of charcoal rot, percentage of harvested seed infected by Phomopsis spp., or severity of pod and stem blight on genotype Asgrow 4403. Of four genotypes evaluated, none were resistant to infection by Phomopsis spp. The genotypes Asgrow 4403, DP 5806, and DT 97-4290 were susceptible to pod and stem blight and PI 567562A was resistant.