A panel of qPCR assays to detect and quantify soybean soil-borne pathogens.

Fusarium oxysporum,F. graminearum,F. acuminatum,F. equiseti,F. proliferatum,F. solani, and Rhizoctonia solani are soil-borne fungal pathogens that cause substantial yield loss in a widespread list of crops worldwide. The objective of this study was to develop a panel of TaqMan assays for the detection and quantification of these six widespread soil-borne fungal species using real-time polymerase chain reaction (qPCR). The primers and probes were designed based on the intergenic spacer ribosomal RNA and translation elongation factor 1-alpha gene (tef1). These assays, although not multiplexed, can be performed simultaneously as they have similar reaction conditions, allowing more efficiency when targeting multiple pathogens in a sample. The assays presented high efficiency (94.3%-108.9%) and sensitivity, with a limit of detection of 0.05 picograms (50 femtograms) of target DNA. Results from an assay targeting 19 non-target and closely related species confirmed the specificity of the developed assays. The assays were also evaluated to detect the target species in different matrices, such as soil and plant material. This panel of qPCR assays is an additional tool that can be used by plant pathologists, microbiologists, plant breeders, diagnostic clinics, and other researchers interested in these fungal species.

Meta-Analytic Modeling of the Severity-Yield Relationships in Soybean Frogeye Leaf Spot Epidemics.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is an important foliar disease affecting soybean in the United States. A meta-analytic approach including 39 fungicide trials conducted from 2012 to 2021 across eight states (Alabama, Arkansas, Illinois, Iowa, Kentucky, Louisiana, Mississippi, Tennessee) was used to assess the relationship between FLS severity and soybean yield. Correlation and regression analyses were performed separately to determine Fisher's transformation of correlation coefficients (Zr), intercept (ß0) and slope (ß1). Disease pressure (low severity, ≤34.5; high severity, >34.5%) and yield class (low, ≤3,352; high, >3,352 kg/ha) were included as categorical moderators. Pearson's [Formula: see text], obtained from back-transforming the [Formula: see text]r estimated by an overall random-effects model, showed a significant negative linear relationship between FLS severity and yield ([Formula: see text] = -0.60). The [Formula: see text]r was affected by disease pressure (P = 0.0003) but not by yield class (P = 0.8141). A random-coefficient model estimated a slope of -19 kg/ha for each percent severity for a mean attainable yield of 3,719.9 kg/ha. Based on the overall mean (95% CI) of the intercept and slope estimated by the random-coefficients model, the estimated overall relative damage coefficient was 0.51% (0.36 to 0.69), indicating that a percent increase in FLS severity reduced yield by 0.51%. The best model included yield class as a covariate, and population-average intercepts differed significantly between low (3,455.1 kg/ha) and high (3,842.7 kg/ha) yield classes. This highlights the potential impact of FLS on soybean yield if not managed and may help in disease management decisions.

Efficacy and Profitability of Fungicides for Managing Frogeye Leaf Spot on Soybean in the United States: A 10-Year Quantitative Summary.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is an economically important disease of soybean in the United States. Data from 66 uniform fungicide trials (UFTs) conducted from 2012 to 2021 across eight states (Alabama, Arkansas, Illinois, Iowa, Kentucky, Louisiana, Mississippi, and Tennessee) were gathered and analyzed to determine the efficacy and profitability of the following fungicides applied at the beginning pod developmental stage (R3): azoxystrobin + difenoconazole (AZOX + DIFE), difenoconazole + pydiflumetofen (DIFE + PYDI), pyraclostrobin (PYRA), pyraclostrobin + fluxapyroxad + propiconazole (PYRA + FLUX + PROP), tetraconazole (TTRA), thiophanate-methyl (TMET), thiophanate-methyl + tebuconazole (TMET + TEBU), and trifloxystrobin + prothioconazole (TFLX + PROT). A network meta-analytic model was fitted to the log of the means of FLS severity data and to the nontransformed mean yield for each treatment, including the nontreated. The percent reduction in disease severity (%) and the yield response (kg/ha) relative to the nontreated was the lowest for PYRA (11%; 136 kg/ha) and the greatest for DIFE + PYDI (57%; 441 kg/ha). A significant decline in efficacy over time was detected for PYRA (18 percentage points [p.p.]), TTRA (27 p.p.), AZOX + DIFE (18 p.p.), and TMET + TEBU (19 p.p.) by using year as a continuous covariate in the model. Finally, probabilities of breaking even were the greatest (>65%) for the most effective fungicide DIFE + PYDI and the lowest (<55%) for PYRA. Results of this meta-analysis may be useful to support decisions when planning fungicide programs.

Chemical Profiles of Heterodera glycines Suppressive Soils in Double Cropping Soybean Production.

We previously reported soybean fields double-cropped with winter wheat having reduced soybean cyst nematode (SCN) (Heterodera glycines) counts compared to fallow. A follow-up metagenomics study identified several fungal and bacterial taxa enriched in wheat fields, and some were reported to parasitize SCN. Knowing that phytocompounds with potential nematicidal activity are released via wheat roots and stubble, we implemented a dichloromethane-based extraction method and a gas chromatography-mass spectrometry (GCMS) system to investigate soil chemical profiles of samples collected from these fields and review the potential nematicidal activity of compounds with higher concentration in double cropping fields. 51 compounds were detected during the GCMS analysis, eight with unknown identification. Several compounds, including multiple fatty acids, had larger relative peak areas when double-cropped, compared to fallow samples. This study, along with our previously published one, provided a better understanding of the mechanisms that govern the effect of wheat on SCN populations. Rather than driven by a single mechanism, the suppression of SCN in soybean fields double-cropped with winter wheat was potentially linked to enriched microbial communities, increased populations of beneficial organisms, and higher concentrations of chemicals with potential nematicidal activity. To our knowledge, this is the first study using GCMS to characterize soil chemical profiles in soybean fields double-cropped with winter wheat regarding the suppression of SCN populations.

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.

Reduction of Pythium Damping-Off in Soybean by Biocontrol Seed Treatment.

Pythium spp. is one of the major groups of pathogens that cause seedling diseases on soybean, leading to both preemergence and postemergence damping-off and root rot. More than 100 species have been identified within this genus, with Pythium irregulare, P. sylvaticum, P. ultimum var ultimum, and P. torulosum being particularly important for soybean production given their aggressiveness, prevalence, and abundance in production fields. This study investigated the antagonistic activity of potential biological control agents (BCAs) native to the U.S. Midwest against Pythium spp. First, in vitro screening identified BCAs that inhibit P. ultimum var. ultimum growth. Scanning electron microscopy demonstrated evidence of mycoparasitism of all potential biocontrol isolates against P. ultimum var. ultimum and P. torulosum, with the formation of appressorium-like structures, short hyphal branches around host hyphae, hook-shaped structures, coiling, and parallel growth of the mycoparasite along the host hyphae. Based on these promising results, selected BCAs were tested under field conditions against six different Pythium spp. Trichoderma afroharzianum 26 used alone and a mix of T. hamatum 16 + T. afroharzianum 19 used as seed treatments protected soybean seedlings from Pythium spp. infection, as BCA-treated plots had on average 15 to 20% greater plant stand and vigor than control plots. Our results also indicate that some of these potential BCAs could be added with a fungicide seed treatment with minimum inhibition occurring, depending on the fungicide active ingredient. This research highlights the need to develop tools incorporating biological control as a facet of soybean seedling disease management programs. The harnessing of native BCAs could be integrated with other management strategies to provide efficient control of seedling diseases.

Trichoderma Isolates Inhibit Fusarium virguliforme Growth, Reduce Root Rot, and Induce Defense-Related Genes on Soybean Seedlings.

Sudden death syndrome (SDS) caused by Fusarium virguliforme is among the most important diseases affecting soybean in the United States. The use of biological control agents (BCAs) such as Trichoderma spp. can be a valuable resource to suppress F. virguliforme populations. Therefore, this research focused on screening possible BCAs against F. virguliforme and evaluating mycoparasitism and the induction of systemic resistance as mechanisms underlying the antagonistic activity of selected BCAs against F. virguliforme. In total, 47 potential BCAs, including 41 Trichoderma isolates and 6 Mortierella isolates, were screened in a dual-plate assay. The most effective isolates belonged to the Trichoderma harzianum species and were able to inhibit F. virguliforme radial growth by up to 92%. Selected Trichoderma isolates were tested in the greenhouse and in a microplot study. They reduced root rot caused by F. virguliforme when the plants were coinoculated with the pathogen and the BCA. The tested BCA's ability to reduce F. virguliforme growth may be related to several mechanisms of action, including mycoparasitism and induction of defense-related genes in plants, as revealed by monitoring the expression of defense-related genes in soybean. Our results highlight the potential of native Trichoderma isolates to inhibit F. virguliforme growth and reduce SDS severity, providing the basis for future implementation of biological control in soybean production. More efforts are needed to implement the use of these approaches in production fields, and to deepen the current knowledge on the biology of these highly antagonistic isolates.

Field evaluation of three sources of genetic resistance to sudden death syndrome of soybean.

KEY MESSAGE: Despite numerous challenges, field testing of three sources of genetic resistance to sudden death syndrome of soybean provides information to more effectively improve resistance to this disease in cultivars. Sudden death syndrome (SDS) of soybean [Glycine max (L.) Merrill] is a disease that causes yield loss in soybean growing regions across the USA and worldwide. While several quantitative trait loci (QTL) for SDS resistance have been mapped, studies to further evaluate these QTL are limited. The objective of our research was to map SDS resistance QTL and to test the effect of mapped resistance QTL on foliar symptoms when incorporated into elite soybean backgrounds. We mapped a QTL from Ripley to chromosome 10 (CHR10) and a QTL from PI507531 to chromosomes 1 and 18 (CHR1 and 18). Six populations were then developed to test the following QTL: cqSDS-001, with resistance originating from PI567374, CHR10, CHR1, and CHR18. The populations which segregated for resistant and susceptible QTL alleles were field tested in multiple environments and evaluated for SDS foliar symptoms. While foliar disease development was variable across environments and populations, a significant effect of each QTL on disease was detected within at least one environment. This includes the detection of cqSDS-001 in three genetic backgrounds. The QTL allele from the resistant parents was associated with greater resistance than the susceptible alleles for all QTL and backgrounds with the exception of the allele for CHR18, where the opposite occurred. This study highlights the importance and difficulties of evaluating QTL and the need for multi-year SDS field testing. The information presented in this study can aid breeders in making decisions to improve resistance to SDS.

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.

FvSNF1, the sucrose non-fermenting protein kinase gene of Fusarium virguliforme, is required for cell-wall-degrading enzymes expression and sudden death syndrome development in soybean.

Fusarium virguliforme is a soil-borne pathogenic fungus that causes sudden death syndrome (SDS) in soybean. Its pathogenicity is believed to require the activity of cell-wall-degrading enzymes (CWDEs). The sucrose non-fermenting protein kinase 1 gene (SNF1) is a key component of the glucose de-repression pathway in yeast, and a regulator of gene expression for CWDEs in some plant pathogenic fungi. To elucidate the functional role of the SNF1 homolog in F. virguliforme, FvSNF1 was disrupted using a split-marker strategy. Disruption of FvSNF1 in F. virguliforme abolishes galactose utilization and causes poor growth on xylose, arabinose and sucrose. However, the resulting Fvsnf1 mutant grew similar to wild-type and ectopic transformants on glucose, fructose, maltose, or pectin as the main source of carbon. The Fvsnf1 mutant displayed no expression of the gene-encoding galactose oxidase (GAO), a secretory enzyme that catalyzes oxidation of D-galactose. It also exhibited a significant reduction in the expression of several CWDE-coding genes in contrast to the wild-type strain. Greenhouse pathogenicity assays revealed that the Fvsnf1 mutant was severely impaired in its ability to cause SDS on challenged soybean plants. Microscopy and microtome studies on infected roots showed that the Fvsnf1 mutant was defective in colonizing vascular tissue of infected plants. Cross and longitudinal sections of infected roots stained with fluorescein-labeled wheat germ agglutinin and Congo red showed that the Fvsnf1 mutant failed to colonize the xylem vessels and phloem tissue at later stages of infection. Quantification of the fungal biomass in inoculated roots further confirmed a reduced colonization of roots by the Fvsnf1 mutant when compared to the wild type. These findings suggest that FvSNF1 regulates the expression of CWDEs in F. virguliforme, thus affecting the virulence of the fungus on soybean.

FvSTR1, a striatin orthologue in Fusarium virguliforme, is required for asexual development and virulence.

The soil-borne fungus Fusarium virguliforme causes sudden death syndrome (SDS), one of the most devastating diseases of soybean in North and South America. Despite the importance of SDS, a clear understanding of the fungal pathogenicity factors that affect the development of this disease is still lacking. We have identified FvSTR1, a F. virguliforme gene, which encodes a protein similar to a family of striatin proteins previously reported to regulate signalling pathways, cell differentiation, conidiation, sexual development, and virulence in filamentous fungi. Striatins are multi-domain proteins that serve as scaffolding units in the striatin-interacting phosphatase and kinase (STRIPAK) complex in fungi and animals. To address the function of a striatin homologue in F. virguliforme, FvSTR1 was disrupted and functionally characterized using a gene knock out strategy. The resulting Fvstr1 mutants were largely impaired in conidiation and pigmentation, and displayed defective conidia and conidiophore morphology compared to the wild-type and ectopic transformants. Greenhouse virulence assays revealed that the disruption of FvSTR1 resulted in complete loss of virulence in F. virguliforme. Microtome studies using fluorescence microscopy showed that the Fvstr1 mutants were defective in their ability to colonize the vascular system. The Fvstr1 mutants also showed a reduced transcript level of genes involved in asexual reproduction and in the production of secondary metabolites. These results suggest that FvSTR1 has a critical role in asexual development and virulence in F. virguliforme.

Fluopyram activates systemic resistance in soybean.

The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a significant yield-limiting factor in soybean production in the Midwestern US. Several management practices are implemented to mitigate yield losses caused by SCN, including using SDHI (succinate dehydrogenase inhibitors) fungicides delivered as seed treatments. A set of studies was conducted to evaluate the effect of two seed-applied succinate dehydrogenase inhibitors (SDHI) compounds, fluopyram and pydiflumetofen, on SCN population densities, plant injury, and plant growth. Cyst counts in untreated control and pydiflumetofen treated plants were 3.44 and 3.59 times higher than fluopyram, respectively, while egg counts were 8.25 and 7.06 times higher in control and pydiflumetofen. Next-generation sequencing was later employed to identify transcriptomic shifts in gene expression profiles in fluopyram and pydiflumetofen -treated seedlings. RNA expression patterns of seed treatments clustered by sampling time (5 DAP vs. 10 DAP); therefore, downstream analysis was conducted by timepoint. At 5 DAP, 10,870 and 325 differentially expressed genes (DEG) were identified in fluopyram and pydiflumetofen, respectively. These same treatments generated 219 and 2 DEGs at 10 DAP. Multiple DEGs identified in soybean seedlings treated with fluopyram are linked to systemic resistance, suggesting a potential role of systemic resistance in the suppression of SCN by fluopyram, in addition to the known nematicidal activity. The non-target inhibition of soybean succinate dehydrogenase genes by fluopyram may be the origin of the phytotoxicity symptoms observed and potentially the source of the systemic resistance activation reported in the current study. This work helps to elucidate the mechanisms of suppression of SCN by fluopyram.

Impact of Wheat on Soybean Cyst Nematode Population Density in Double-Cropping Soybean Production.

Double-cropping is defined as producing more than one crop on the same parcel of land in a single growing season. It is reported to have many benefits when incorporated in cropping systems, including improving soil health. In some double-cropping systems, soybean is planted following winter wheat. The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a major soybean pathogen, and several reports suggest suppressive effects of wheat on SCN populations. Field trials were conducted from 2017 to 2018 to investigate the effect of wheat on SCN populations in double-cropping soybean. Nine fields with three levels of initial SCN populations (low, moderate, and high) were selected in Illinois. Wheat was planted in strips alternating with strips-maintained weed-free and under fallow over winter and early spring. Soybean was planted in all strips after wheat harvest. SCN egg densities were acquired at four time points: wheat establishment, post-wheat/pre-soybean, mid-soybean (R1 growth stage or beginning of flowering), and post-soybean harvest. Wheat strips reduced SCN egg densities compared with fallow strips at the R1 stage (-31.8%) and after soybean harvest (-32.7%). Double-cropping soybean with wheat has the potential to suppress SCN field populations and is a system with the potential to provide additional farm income. This study is meant to be a first step toward a better understanding of the mechanisms that govern the suppression of SCN by wheat.