Integration of Row Spacing, Seeding Rates, and Fungicide Applications for Control of Sclerotinia Stem Rot in Glycine max.

Soybean (Glycine max) farmers in the Upper Midwest region of the United States often experience severe yield losses due to Sclerotinia stem rot (SSR). Previous studies have revealed benefits of individual management practices for SSR. This study examined the integration of multiple control practices on the development of SSR, yield, and the economic implications of these practices. Combinations of row spacings, seeding rates, and fungicide applications were examined in multisite field trials across the Upper Midwest from 2017 to 2019. These trials revealed that wide row spacing and low seeding rates individually reduced SSR levels but also reduced yields. Yields were similar across the three highest seeding rates examined. However, site-years where SSR developed showed the highest partial profits at the intermediate seeding rates. This finding indicates that partial profits in diseased fields were reduced by high seeding rates, but this trend was not observed when SSR did not develop. Fungicides strongly reduced the development of SSR while also increasing yields. However, there was a reduction in partial profits due to their use at a low soybean sale price, but at higher sale prices fungicide use was similar to not treating. Additionally, the production of new inoculum was predicted from disease incidence, serving as an indicator of increased risk for SSR development in future years. Overall, this study suggests using wide rows and low seeding rates in fields with a history of SSR while reserving narrow rows and higher seeding rates for fields without a history of SSR.

Identification of Soybean (Glycine max) Check Lines for Evaluating Genetic Resistance to Sclerotinia Stem Rot.

Soybean production in the upper midwestern United States is affected by Sclerotinia stem rot (SSR) caused by the fungal pathogen Sclerotinia sclerotiorum. Genetic resistance is an important management strategy for this disease; however, assessing genetic resistance to S. sclerotiorum is challenging because a standardized method of examining resistance across genotypes is lacking. Using a panel of nine diverse S. sclerotiorum isolates, four soybean lines were assessed for reproducible responses to S. sclerotiorum infection. Significant differences in SSR severity were found across isolates (P < 0.01) and soybean lines (P < 0.01), including one susceptible, two moderately resistant, and one highly resistant line. These four validated lines were used to screen 11 other soybean genotypes to evaluate their resistance levels, and significant differences were found across genotypes (P < 0.01). Among these 11 genotypes, five commercial and public cultivars displayed high resistance and were assessed during field studies across the upper midwestern United States growing region to determine their response to SSR and yield. These five cultivars resulted in low disease levels (P < 0.01) in the field that were consistent with greenhouse experiment results. The yields were significantly different in fields with disease present (P < 0.01) and disease absent (P < 0.01), and the order of cultivar performance was consistent between environments where disease was present or absent, suggesting that resistance prevented yield loss to disease. This study suggests that the use of a soybean check panel can accurately assess SSR resistance in soybean germplasm and aid in breeding and commercial soybean development.

Diagnostic qPCR Assay to Detect Fusarium brasiliense, a Causal Agent of Soybean Sudden Death Syndrome and Root Rot of Dry Bean.

Species within clade 2 of the Fusarium solani species complex (FSSC) are significant pathogens of dry bean (Phaseolus vulgaris) and soybean (Glycine max), causing root rot and/or sudden death syndrome (SDS). These species are morphologically difficult to distinguish and often require molecular tools for proper diagnosis to a species level. Here, a TaqMan probe-based quantitative PCR (qPCR) assay was developed to distinguish Fusarium brasiliense from other closely related species within clade 2 of the FSSC. The assay displays high specificity against close relatives and high sensitivity, with a detection limit of 100 fg. This assay was able to detect F. brasiliense from purified mycelia, infected dry bean roots, and soil samples throughout Michigan. When multiplexed with an existing qPCR assay specific to Fusarium virguliforme, accurate quantification of both F. brasiliense and F. virguliforme was obtained, which can facilitate accurate diagnoses and identify coinfections with a single reaction. The assay is compatible with multiple qPCR thermal cycling platforms and will be helpful in providing accurate detection of F. brasiliense. Management of root rot and SDS pathogens in clade 2 of the FSSC is challenging and must be done proactively, because no midseason management strategies currently exist. However, accurate detection can facilitate management decisions for subsequent growing seasons to successfully manage these pathogens.

Laser ablation tomography for visualization of root colonization by edaphic organisms.

Soil biota have important effects on crop productivity, but can be difficult to study in situ. Laser ablation tomography (LAT) is a novel method that allows for rapid, three-dimensional quantitative and qualitative analysis of root anatomy, providing new opportunities to investigate interactions between roots and edaphic organisms. LAT was used for analysis of maize roots colonized by arbuscular mycorrhizal fungi, maize roots herbivorized by western corn rootworm, barley roots parasitized by cereal cyst nematode, and common bean roots damaged by Fusarium. UV excitation of root tissues affected by edaphic organisms resulted in differential autofluorescence emission, facilitating the classification of tissues and anatomical features. Samples were spatially resolved in three dimensions, enabling quantification of the volume and distribution of fungal colonization, western corn rootworm damage, nematode feeding sites, tissue compromised by Fusarium, and as well as root anatomical phenotypes. Owing to its capability for high-throughput sample imaging, LAT serves as an excellent tool to conduct large, quantitative screens to characterize genetic control of root anatomy and interactions with edaphic organisms. Additionally, this technology improves interpretation of root-organism interactions in relatively large, opaque root segments, providing opportunities for novel research investigating the effects of root anatomical phenes on associations with edaphic organisms.

Convergent Evolution of C239S Mutation in Pythium spp. ß-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to ß-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 µg ml-1), whereas 16% were insensitive (EC50 > 11 µg ml-1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in ß-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available ß-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Predicting Soybean Yield and Sudden Death Syndrome Development Using At-Planting Risk Factors.

In the United States, sudden death syndrome (SDS) of soybean is caused by the fungal pathogen Fusarium virguliforme and is responsible for important yield losses each year. Understanding the risk of SDS development and subsequent yield loss could provide growers with valuable information for management of this challenging disease. Current management strategies for F. virguliforme use partially resistant cultivars, fungicide seed treatments, and extended crop rotations with diverse crops. The aim of this study was to develop models to predict SDS severity and soybean yield loss using at-planting risk factors to integrate with current SDS management strategies. In 2014 and 2015, field studies were conducted in adjacent fields in Decatur, MI, which were intensively monitored for F. virguliforme and nematode quantities at-planting, plant health throughout the growing season, end-of-season SDS severity, and yield using an unbiased grid sampling scheme. In both years, F. virguliforme and soybean cyst nematode (SCN) quantities were unevenly distributed throughout the field. The distribution of F. virguliforme at-planting had a significant correlation with end-of-season SDS severity in 2015, and a significant correlation to yield in 2014 (P < 0.05). SCN distributions at-planting were significantly correlated with end-of-season SDS severity and yield in 2015 (P < 0.05). Prediction models developed through multiple linear regression showed that F. virguliforme abundance (P < 0.001), SCN egg quantity (P < 0.001), and year (P < 0.01) explained the most variation in end-of-season SDS (R2 = 0.32), whereas end-of-season SDS (P < 0.001) and end-of-season root dry weight (P < 0.001) explained the most variation in soybean yield (R2 = 0.53). Further, multivariate analyses support a synergistic relationship between F. virguliforme and SCN, enhancing the severity of foliar SDS. These models indicate that it is possible to predict patches of SDS severity using at-planting risk factors. Verifying these models and incorporating additional data types may help improve SDS management and forecast soybean markets in response to SDS threats.

Temporal Dynamics of Fusarium virguliforme Colonization of Soybean Roots.

Soybean sudden death syndrome (SDS) caused by Fusarium virguliforme is one of the most yield limiting soybean diseases in the United States. SDS disease symptoms include root rot and foliar symptoms induced by fungal toxins. Soybean cultivar resistance is one of the most effective SDS disease management options, but no cultivar displays complete resistance. Soybean SDS foliar symptoms are the primary phenotype used to screen and breed for SDS resistance. Root rot or root colonization measures are seldom utilized, partly due to the lack of convenient and accurate methods for quantification of F. virguliforme. In this study, greenhouse and field experiments were conducted to determine the temporal dynamics of F. virguliforme colonization of soybean roots using quantitative real-time PCR (qPCR). The infection coefficient (IC), or ratio of F. virguliforme DNA to soybean DNA, was determined in soybean cultivars with different SDS foliar resistance ratings. In greenhouse experiments, F. virguliforme was detected in all cultivars 7 days after planting (DAP), with a peak in IC at 14 DAP. All soybean cultivars developed SDS foliar symptoms, but F. virguliforme soybean root colonization levels did not significantly correlate with SDS foliar symptom severity. In field experiments, SDS foliar symptoms developed among soybean cultivars in alignment with provided foliar resistance ratings; however, the F. virguliforme IC were not significantly different between SDS foliar symptomatic and asymptomatic cultivars. F. virguliforme was detected in all cultivars at the first sample collection point 25 DAP (V3 vegetative growth stage), and the IC increased throughout the season, peaking at the last sample collection point 153 DAP (postharvest). Collectively, appearance and disease severity ratings of SDS foliar symptoms were not associated with F. virguliforme quantity in roots, suggesting a need to include F. virguliforme root colonization in breeding efforts to screen soybean germplasm for F. virguliforme root infection resistance. The findings also demonstrates root colonization of the pathogen on nonsymptomatic soybean cultivars leading to persistence of the pathogen in the field, and possible hidden yield loss.

Integration of sudden death syndrome resistance loci in the soybean genome.

KEY MESSAGE: Complexity and inconsistencies in resistance mapping publications of soybean sudden death syndrome (SDS) result in interpretation difficulty. This review integrates SDS mapping literature and proposes a new nomenclature system for reproducible SDS resistance loci. Soybean resistance to sudden death syndrome (SDS) is composed of foliar resistance to phytotoxins and root resistance to pathogen invasion. There are more than 80 quantitative trait loci (QTL) and dozens of single nucleotide polymorphisms (SNPs) associated with soybean resistance to SDS. The validity of these QTL and SNPs is questionable because of the complexity in phenotyping methodologies, the disease synergism between SDS and soybean cyst nematode (SCN), the variability from the interactions between soybean genotypes and environments, and the inconsistencies in the QTL nomenclature. This review organizes SDS mapping results and proposes the Rfv (resistance to Fusarium virguliforme) nomenclature based on supporting criteria described in the text. Among ten reproducible loci receiving our Rfv nomenclature, Rfv18-01 is mostly supported by field studies and it co-localizes to the SCN resistance locus rhg1. The possibility that Rfv18-01 is a pleiotropic resistance locus and the concern about Rfv18-01 being confounded with Rhg1 is discussed. On the other hand, Rfv06-01, Rfv06-02, Rfv09-01, Rfv13-01, and Rfv16-01 were identified both by screening soybean leaves against phytotoxic culture filtrates and by evaluating SDS severity in fields. Future phenotyping using leaf- and root-specific resistance screening methodologies may improve the precision of SDS resistance, and advanced genetic studies may further clarify the interactions among soybean genotypes, F. virguliforme, SCN, and environments. The review provides a summary of the SDS resistance literature and proposes a framework for communicating SDS resistance loci for future research considering molecular interactions and genetic breeding for soybean SDS resistance.

Fungal biotechnology: From yesterday to tomorrow.

Fungi have been used to better the lives of everyday people and unravel the mysteries of higher eukaryotic organisms for decades. However, comparing progress and development stemming from fungal research to that of human, plant, and bacterial research, fungi remain largely understudied and underutilized. Recent commercial ventures have begun to gain popularity in society, providing a new surge of interest in fungi, mycelia, and potential new applications of these organisms to various aspects of research. Biotechnological advancements in fungal research cannot occur without intensive amounts of time, investments, and research tool development. In this review, we highlight past breakthroughs in fungal biotechnology, discuss requirements to advance fungal biotechnology even further, and touch on the horizon of new breakthroughs with the highest potential to positively impact both research and society.

A global-temporal analysis on Phytophthora sojae resistance-gene efficacy.

Plant disease resistance genes are widely used in agriculture to reduce disease outbreaks and epidemics and ensure global food security. In soybean, Rps (Resistance to Phytophthora sojae) genes are used to manage Phytophthora sojae, a major oomycete pathogen that causes Phytophthora stem and root rot (PRR) worldwide. This study aims to identify temporal changes in P. sojae pathotype complexity, diversity, and Rps gene efficacy. Pathotype data was collected from 5121 isolates of P. sojae, derived from 29 surveys conducted between 1990 and 2019 across the United States, Argentina, Canada, and China. This systematic review shows a loss of efficacy of specific Rps genes utilized for disease management and a significant increase in the pathotype diversity of isolates over time. This study finds that the most widely deployed Rps genes used to manage PRR globally, Rps1a, Rps1c and Rps1k, are no longer effective for PRR management in the United States, Argentina, and Canada. This systematic review emphasizes the need to widely introduce new sources of resistance to P. sojae, such as Rps3a, Rps6, or Rps11, into commercial cultivars to effectively manage PRR going forward.

Effectors of Plant Necrotrophic Fungi.

Plant diseases caused by necrotrophic fungal pathogens result in large economic losses in field crop production worldwide. Effectors are important players of plant-pathogen interaction and deployed by pathogens to facilitate plant colonization and nutrient acquisition. Compared to biotrophic and hemibiotrophic fungal pathogens, effector biology is poorly understood for necrotrophic fungal pathogens. Recent bioinformatics advances have accelerated the prediction and discovery of effectors from necrotrophic fungi, and their functional context is currently being clarified. In this review we examine effectors utilized by necrotrophic fungi and hemibiotrophic fungi in the latter stages of disease development, including plant cell death manipulation. We define "effectors" as secreted proteins and other molecules that affect plant physiology in ways that contribute to disease establishment and progression. Studying and understanding the mechanisms of necrotrophic effectors is critical for identifying avenues of genetic intervention that could lead to improved resistance to these pathogens in plants.

A protoplast generation and transformation method for soybean sudden death syndrome causal agents Fusarium virguliforme and F. brasiliense.

BACKGROUND: Soybean production around the globe faces significant annual yield losses due to pests and diseases. One of the most significant causes of soybean yield loss annually in the U.S. is sudden death syndrome (SDS), caused by soil-borne fungi in the Fusarium solani species complex. Two of these species, F. virguliforme and F. brasiliense, have been discovered in the U.S. The genetic mechanisms that these pathogens employ to induce root rot and SDS are largely unknown. Previous methods describing F. virguliforme protoplast generation and transformation have been used to study gene function, but these methods lack important details and controls. In addition, no reports of protoplast generation and genetic transformation have been made for F. brasiliense. RESULTS: We developed a new protocol for developing fungal protoplasts in these Fusarium species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of F. virguliforme and F. brasiliense are sensitive to the antibiotics hygromycin and nourseothricin, but strains transformed with resistance genes displayed resistance to these antibiotics. In addition, integration of fluorescent protein reporter genes demonstrates that the foreign DNA is expressed and results in a functional protein, providing fluorescence to both pathogens. CONCLUSIONS: This protocol provides significant details for reproducibly producing protoplasts and transforming F. virguliforme and F. brasiliense. The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of F. virguliforme and F. brasiliense. Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species.

Fluopyram Sensitivity and Functional Characterization of SdhB in the Fusarium solani Species Complex Causing Soybean Sudden Death Syndrome.

The succinate dehydrogenase inhibitor (SDHI) fungicide, fluopyram, is used as a soybean seed treatment to manage Fusarium virguliforme, the casual agent of sudden death syndrome (SDS). More recently, other species within clade 2 of the Fusarium solani species, F. tucumaniae in South America and F. brasiliense in America and Africa, have been recognized as additional agents capable of causing SDS. To determine if fluopyram could be used for management of SDS caused by these species, in vitro sensitivity tests of the three Fusarium species to fluopyram were conducted. The mean EC50 values of F. brasiliense and F. virguliforme strains to fluopyram were 1.96 and 2.21 µg ml-1, respectively, but interestingly F. tucumaniae strains were highly sensitive (mean EC50 = 0.25 µg ml-1) to fluopyram compared to strains of the other two species. A sequence analysis of Sdh genes of Fusarium strains revealed that the F. tucumaniae strains contain an arginine at codon 277 in the SdhB gene instead of a glycine as in other Fusarium species. Replacement of glycine to arginine in SdhB-277 in a F. virguliforme wild-type strain Mont-1 through genetic transformation resulted in increased sensitivity to two SDHI fungicides, fluopyram and boscalid. Similar to a F. tucumaniae strain, the Mont-1 (SdhBG277R) mutant caused less SDS and root rot disease than Mont-1 on soybean seedlings with the fluopyram seed treatment. Our study suggests the amino acid difference in the SdhB in F. tucumaniae results in fluopyram being efficacious if used as a seed treatment for management of F. tucumaniae, which is the most abundant SDS causing species in South America. The establishment of baseline sensitivity of Fusarium species to fluopyram will contribute to effective strategies for managing Fusarium diseases in soybean and other pathosystems such as dry bean.

MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network.

Large-scale high-throughput plant phenotyping (sometimes called phenomics) is becoming increasingly important in plant biology and agriculture and is essential to cutting-edge plant breeding and management approaches needed to meet the food and fuel needs for the next century. Currently, the application of these approaches is severely limited by the availability of appropriate instrumentation and by the ability to communicate experimental protocols, results and analyses. To address these issues, we have developed a low-cost, yet sophisticated open-source scientific instrument designed to enable communities of researchers, plant breeders, educators, farmers and citizen scientists to collect high-quality field data on a large scale. The MultispeQ provides measurements in the field or laboratory of both, environmental conditions (light intensity and quality, temperature, humidity, CO2 levels, time and location) and useful plant phenotypes, including photosynthetic parameters-photosystem II quantum yield (ΦII), non-photochemical exciton quenching (NPQ), photosystem II photoinhibition, light-driven proton translocation and thylakoid proton motive force, regulation of the chloroplast ATP synthase and potentially many others-and leaf chlorophyll and other pigments. Plant phenotype data are transmitted from the MultispeQ to mobile devices, laptops or desktop computers together with key metadata that gets saved to the PhotosynQ platform (https://photosynq.org) and provides a suite of web-based tools for sharing, visualization, filtering, dissemination and analyses. We present validation experiments, comparing MultispeQ results with established platforms, and show that it can be usefully deployed in both laboratory and field settings. We present evidence that MultispeQ can be used by communities of researchers to rapidly measure, store and analyse multiple environmental and plant properties, allowing for deeper understanding of the complex interactions between plants and their environment.