Characterizing the Diversity of Oomycetes Associated with Diseased Cotton Seedlings in Alabama.

Many oomycete species are associated with the seedlings of crops, including upland cotton (Gossypium hirsutum L.), which leads to annual threats. The diversity of oomycete species in Alabama needs to be better understood since the last survey of oomycetes associated with cotton in Alabama was 20 years ago-before significant updates to taxonomy and improvements in identification of oomycetes using molecular tools. Our current study aimed to identify oomycetes associated with Alabama cotton seedlings, correlate diversity with soil edaphic factors, and assess virulence toward cotton seed. Thirty symptomatic cotton seedlings were collected independently from 25 fields in 2021 and 2022 2 to 4 weeks after planting. Oomycetes were isolated by plating root sections onto a semiselective medium. The internal transcribed spacer (ITS) region was sequenced to identify the resulting isolates. A seed virulence assay was conducted in vitro to verify pathogenicity, and 347 oomycete isolates were obtained representing 36 species. Northern Alabama soils had the richest oomycete communities and a greater silt and clay concentration than sandier soils in the central and southern coastal plains. Globisporangium irregulare and Phytophthora nicotianae were consistently recovered from cotton roots in both years. Globisporangium irregulare was pathogenic and recovered from all Alabama regions, whereas P. nicotianae was pathogenic but recovered primarily in areas with lower sand content in northern Alabama. Many oomycete species have not been previously reported in Alabama or the southeastern United States. Altogether, this knowledge will help facilitate effective management strategies for cotton seedling diseases caused by oomycetes in Alabama and the United States.

Update on the state of research to manage Fusarium head blight.

Fusarium head blight (FHB) is one of the most devastating diseases of cereal crops, causing severe reduction in yield and quality of grain worldwide. In the United States, the major causal agent of FHB is the mycotoxigenic fungus, Fusarium graminearum. The contamination of grain with mycotoxins, including deoxynivalenol and zearalenone, is a particularly serious concern due to its impact on the health of humans and livestock. For the past few decades, multidisciplinary studies have been conducted on management strategies designed to reduce the losses caused by FHB. However, effective management is still challenging due to the emergence of fungicide-tolerant strains of F. graminearum and the lack of highly resistant wheat and barley cultivars. This review presents multidisciplinary approaches that incorporate advances in genomics, genetic-engineering, new fungicide chemistries, applied biocontrol, and consideration of the disease cycle for management of FHB.

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.

Soybean and Cotton Spermosphere Soil Microbiome Shows Dominance of Soilborne Copiotrophs.

The spermosphere is the transient, immediate zone of soil around imbibing and germinating seeds. It represents a habitat where there is contact between seed-associated microbes and soil microbes, but it is studied less than other plant habitats. Previous studies on spermosphere microbiology were primarily culture based or did not sample the spermosphere soil as initially defined in space and time. Thus, the objectives of this study were to develop an efficient strategy to collect spermosphere soils around imbibing soybean and cotton in nonsterile soil and investigate changes in microbial communities. The method employed sufficiently collected spermosphere soil as initially defined in space by constraining the soil sampled with a cork borer and confining the soil to a 12-well microtiter plate. Spermosphere prokaryote composition changed over time and depended on the crop within 6 h after seeds were sown. By 12 to 18 h, crops had unique microbial communities in spermosphere soils. Prokaryote evenness dropped following seed imbibition, with the proliferation of copiotrophic soil bacteria. Due to their long history of plant growth promotion, prokaryote operational taxonomic units (OTUs) in Bacillus, Paenibacillus, Burkholderia, Massilia, Azospirillum, and Pseudomonas were notable organisms enriched. Fungi and prokaryotes were hub taxa in cotton and soybean spermosphere networks. Additionally, the enriched taxa were not hubs in networks, suggesting that other taxa besides those enriched may be important for spermosphere communities. Overall, this study advances knowledge in the assembly of the plant microbiome early in a plant's life, which may have plant health implications in more mature plant growth stages. IMPORTANCE The central hypothesis of this research was that plant species and seed exudate release would alter the assembly of microbes in the spermosphere soil. Our research investigated the response of microbes to the initial burst of nutrients into the spermosphere soil, filling knowledge gaps from previous studies that pregerminated seeds under sterile conditions. We identified several copiotrophic bacterial lineages with a long history of plant growth promotion proliferating in response to the initial exudate release. With a comparative network approach, we show that these copiotrophic bacteria are not central to networks, demonstrating that other microbes (including fungi) may be important for community structure. This study improves knowledge on microbial dynamics in the understudied spermosphere and helps inform solutions for biologically or ecologically motivated solutions to spermosphere pathogens.

Non-target impacts of fungicide disturbance on phyllosphere yeasts in conventional and no-till management.

Fungicides reduce fungal pathogen populations and are essential to food security. Understanding the impacts of fungicides on crop microbiomes is vital to minimizing unintended consequences while maintaining their use for plant protection. However, fungicide disturbance of plant microbiomes has received limited attention, and has not been examined in different agricultural management systems. We used amplicon sequencing of fungi and prokaryotes in maize and soybean microbiomes before and after foliar fungicide application in leaves and roots from plots under long-term no-till and conventional tillage management. We examined fungicide disturbance and resilience, which revealed consistent non-target effects and greater resiliency under no-till management. Fungicides lowered pathogen abundance in maize and soybean and decreased the abundance of Tremellomycetes yeasts, especially Bulleribasidiaceae, including core microbiome members. Fungicide application reduced network complexity in the soybean phyllosphere, which revealed altered co-occurrence patterns between yeast species of Bulleribasidiaceae, and Sphingomonas and Hymenobacter in fungicide treated plots. Results indicate that foliar fungicides lower pathogen and non-target fungal abundance and may impact prokaryotes indirectly. Treatment effects were confined to the phyllosphere and did not impact belowground microbial communities. Overall, these results demonstrate the resilience of no-till management to fungicide disturbance, a potential novel ecosystem service provided by no-till agriculture.

Endophytic Fungi as a Promising Biocontrol Agent to Protect Wheat from Fusarium graminearum Head Blight.

The search for beneficial endophytes that can be part of a constructed microbial community has increased in recent years. We characterized three endophytic fungi previously isolated from wheat for their in vitro and in planta antagonism toward the Fusarium head blight pathogen, Fusarium graminearum. The endophytes were phylogenetically characterized and shown to be Alternaria destruens, Fusarium commune, and Fusarium oxysporum. Individual fungal endophytes significantly increased seed weight and lowered the accumulation of the mycotoxin deoxynivalenol compared with F. graminearum-infected wheat heads without endophyte pretreatment. Investigation into the mechanism of competition in vitro showed that endophytes competitively excluded F. graminearum by preemptive colonization and possible inhibition over a distance. Investigations on the use of these endophytes in the field are in progress. Identification of these three endophytes highlights a common quandary in searching for beneficial microbes to use in agriculture: species definitions often do not separate individual isolates' lifestyles. A greater understanding of the risks in using intraspecies variants for biocontrol is needed and should be examined in the context of the ecology of the individuals being investigated.

Phytophthora sojae Pathotype Distribution and Fungicide Sensitivity in Michigan.

Identifying the pathotype structure of a Phytophthora sojae population is crucial for the effective management of Phytophthora stem and root rot of soybean (PRR). P. sojae has been successfully managed with major resistance genes, partial resistance, and fungicide seed treatments. However, prolonged use of resistance genes or fungicides can cause pathogen populations to adapt over time, rendering resistance genes or fungicides ineffective. A statewide survey was conducted to characterize this pathotype structure and fungicide sensitivity of P. sojae within Michigan. Soil samples were collected from 69 fields with a history of PRR and fields having consistent plant stand establishment issues. Eighty-three isolates of P. sojae were obtained, and hypocotyl inoculations were performed on 14 differential soybean cultivars, all of which carry a single Rps gene or no resistance gene. The survey identified a loss of effectiveness of Rps genes 1b, 1k, 3b, and 6, compared with a previous survey conducted in Michigan from 1993 to 1997. Three effective resistance genes were identified for P. sojae management in Michigan; Rps 3a, 3c, and 4. Additionally, the effective concentration of common seed treatment fungicides to inhibit mycelial growth by 50% (EC50) was determined. No P. sojae isolates were insensitive to the tested chemistries with mean EC50 values of 2.60 × 10-2 µg/ml for ethaboxam, 3.03 × 10-2 µg/ml for mefenoxam, 2.88 × 10-4 µg/ml for oxathiapiprolin, and 5.08 × 10-2 µg/ml for pyraclostrobin. Results suggest that while there has been a significant shift in Rps gene effectiveness, seed treatments are still effective for early season management of this disease.

A ß-lactamase gene of Fusarium oxysporum alters the rhizosphere microbiota of soybean.

The rhizosphere is a multitrophic environment, and for soilborne pathogens such as Fusarium oxysporum, microbial competition in the rhizosphere is inevitable before reaching and infecting roots. This study established a tritrophic interaction among the plant growth-promoting rhizobacterium Burkholderia ambifaria, F. oxysporum and Glycine max (soybean) to study the effects of F. oxysporum genes on shaping the soybean microbiota. Although B. ambifaria inhibited mycelial growth and increased bacterial propagation in the presence of F. oxysporum, F. oxysporum still managed to infect soybean in the presence of B. ambifaria. RNA-Seq identified a putative F. oxysporum secretory ß-lactamase-coding gene, FOXG_18438 (abbreviated as Fo18438), that is upregulated during soybean infection in the presence of B. ambifaria. The ∆Fo18438 mutants displayed reduced mycelial growth towards B. ambifaria, and the complementation of full Fo18438 and the Fo18438 ß-lactamase domain restored mycelial growth. Using the F. oxysporum wild type, ∆Fo18438 mutants and complemented strains with full Fo18438, Fo18438 ß-lactamase domain or Fo18438 RTA1-like domain for soil inoculation, 16S rRNA amplicon sequencing revealed that the abundance of a Burkholderia operational taxonomic unit (OTU) was increased in the rhizosphere microbiota infested by the strains with Fo18438 ß-lactamase domain. Non-metric multidimensional scaling and PICRUSt2 functional analysis revealed differential abundance for the bacterial ß-lactam-related functions when contrasting the genotypes of F. oxysporum. These results indicated that the Fo18438 ß-lactamase domain provides F. oxysporum with the advantage of growing into the soybean rhizosphere, where ß-lactam antibiosis is involved in microbial competition. Accordingly, this study highlights the capability of an F. oxysporum gene for altering the soybean rhizosphere and taproot microbiota.

Influence of Soybean Tissue and Oomicide Seed Treatments on Oomycete Isolation.

Soybean seedlings are vulnerable to different oomycete pathogens. Seed treatments containing the two antioomycete (oomicide) chemicals, metalaxyl-M (mefenoxam) and ethaboxam, are used for protection against oomycete pathogens. This study aimed to evaluate the influence of these two oomicides on isolation probability of oomycetes from soybean taproot or lateral root sections. Soybean plants were collected between the first and third trifoliate growth stages from five Midwest field locations in 2016 and four of the same fields in 2017. Oomycetes were isolated from taproot and lateral root. In 2016, 369 isolation attempts were completed, resulting in 121 isolates from the taproot and 154 isolates from the lateral root. In 2017, 468 isolation attempts were completed, with 44 isolates from the taproot and 120 isolates from the lateral roots. In three of nine site-years, the probability of isolating an oomycete from a taproot or lateral root section was significantly different. Seed treatments containing a mixture of ethaboxam and metalaxyl significantly reduced the probability of oomycete isolation from lateral roots in Illinois in 2016 and 2017, but not in other locations, which may have been related to the heavy soil type (clay loam). Among the 439 isolates collected from the two years sampled, 24 oomycete species were identified, and community compositions differed depending on location and year. The five most abundant species were Pythium sylvaticum (28.9%), P. heterothallicum (14.3%), P. ultimum var. ultimum (11.8%), P. attrantheridium (7.9%), and P. irregulare (6.6%), which accounted for 61.7% of the isolates collected. Oomicide sensitivity to ethaboxam and mefenoxam was assessed for >300 isolates. There were large differences in ethaboxam sensitivity among oomycete species, with effective concentrations to reduce optical density at 600 nm by 50% compared with the nonamended control (EC50 values) ranging from <0.01 to >100 µg/ml and a median of 0.65 µg/ml. Isolates with insensitivity to ethaboxam (>12 µg/ml) belonged to the species P. torulosum and P. rostratifingens but were sensitive to mefenoxam. Oomicide sensitivity to mefenoxam ranged from <0.01 to 0.62 µg/ml with a median of 0.03 µg/ml. The mean EC50 value of the five most abundant species to ethaboxam ranged from 0.35 to 0.97 µg/ml of ethaboxam and from 0.02 to 0.04 µg/ml of mefenoxam. No shift in sensitivity to mefenoxam or ethaboxam was observed as a result of soybean seed treatment or year relative to the nontreated seed controls. In summary, this study contributed to the understanding of the composition of oomycete populations from different soybean root tissues, locations, years, and seed treatments. Finally, seed treatments containing mefenoxam or metalaxyl plus ethaboxam can be effective in reducing the probability of oomycete isolation from soybean roots.

Crop Management Impacts the Soybean (Glycine max) Microbiome.

Soybean (Glycine max) is an important leguminous crop that is grown throughout the United States and around the world. In 2016, soybean was valued at $41 billion USD in the United States alone. Increasingly, soybean farmers are adopting alternative management strategies to improve the sustainability and profitability of their crop. Various benefits have been demonstrated for alternative management systems, but their effects on soybean-associated microbial communities are not well-understood. In order to better understand the impact of crop management systems on the soybean-associated microbiome, we employed DNA amplicon sequencing of the Internal Transcribed Spacer (ITS) region and 16S rRNA genes to analyze fungal and prokaryotic communities associated with soil, roots, stems, and leaves. Soybean plants were sampled from replicated fields under long-term conventional, no-till, and organic management systems at three time points throughout the growing season. Results indicated that sample origin was the main driver of beta diversity in soybean-associated microbial communities, but management regime and plant growth stage were also significant factors. Similarly, differences in alpha diversity are driven by compartment and sample origin. Overall, the organic management system had lower fungal and bacterial Shannon diversity. In prokaryotic communities, aboveground tissues were dominated by Sphingomonas and Methylobacterium while belowground samples were dominated by Bradyrhizobium and Sphingomonas. Aboveground fungal communities were dominated by Davidiella across all management systems, while belowground samples were dominated by Fusarium and Mortierella. Specific taxa including potential plant beneficials such as Mortierella were indicator species of the conventional and organic management systems. No-till management increased the abundance of groups known to contain plant beneficial organisms such as Bradyrhizobium and Glomeromycotina. Network analyses show different highly connected hub taxa were present in each management system. Overall, this research demonstrates how specific long-term cropping management systems alter microbial communities and how those communities change throughout the growth of soybean.

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.

A High-Throughput Microtiter-Based Fungicide Sensitivity Assay for Oomycetes Using Z'-Factor Statistic.

Current methods to quantitatively assess fungicide sensitivity for a diverse range of oomycetes are slow and labor intensive. Microtiter-based assays can be used to increase throughput. However, many factors can affect their quality and reproducibility. Therefore, efficient and reliable methods for detection of assay quality are desirable. The objective of this study was to develop and validate a robust high-throughput fungicide phenotyping assay based on spectrophotometric quantification of mycelial growth in liquid culture and implementation of quality control with Z' factor and growth curves. Z' factor was used to ensure that each isolate grew enough in the absence of fungicides compared with the negative control, and growth curves were used to ensure active growth at the time of concentration of a fungicide that reduces growth by 50% (EC50) estimation. EC50 and relative growth values were correlated in a side-by-side comparison with values obtained using the amended medium (gold standard) assay. Concordance correlation indicated that the high-throughput assay is accurate but may not be as precise as the amended medium assay. To demonstrate the utility of the high-throughput assay, the sensitivity of 216 oomycete isolates representing four genera and 81 species to mefenoxam and ethaboxam was tested. The assay developed herein will enable high-throughput fungicide phenotyping at a population or community level.

Annotation resource of tandem repeat-containing secretory proteins in sixty fungi.

Fungal secretory proteins that interact with host plants are regarded as effectors. Because fungal effectors rarely contain conserved sequence features, identification and annotation of fungal effectors from predicted secretory proteins are difficult using outward comparison methods such as BLAST or hidden Markov model. In desire of more sequence features to prioritize research interests of fungal secretory proteins, this study developed a pipeline to identify tandem repeat (TR) domain within putative secretory proteins and tested a hypothesis that at least one type of TR domain in non-orthologous secretory proteins has emerged from convergent evolution for plant pathogenicity. There were 2804 types of TR domains and a total of 2925 TR-containing secretory proteins found from 60 fungi. There was no conserved type of TR domain shared only by plant pathogens, indicating functional divergence for different types of TR domain and TR-containing secretory proteins. The annotation resource of putative fungal TR-containing secretory proteins provides new sequence features that will be useful for the community interested in fungal effector biology.

Significant Influence of EC50 Estimation by Model Choice and EC50 Type.

The effective control to 50% growth inhibition (EC50) is a standard statistic for evaluating dose-response relationships. Many statistical software packages are available to estimate dose-response relationships but, recently, an open source package ("drc") in R has been utilized. This package is highly adaptable, having many models to describe dose-response relationships and flexibility to describe both hormetic relationships and absolute and relative EC50. These models and definitions are generally left out of phytopathology literature. Here, we demonstrate that model choice and type of EC50 (relative versus absolute) can matter for EC50 estimation using data from Pythium oopapillum and Fusarium virguliforme. For some P. oopapillum isolates, the difference between absolute and relative EC50 was significant. Hormetic effects changed F. virguliforme EC50 distributions, leading to higher estimates than when using four- or three-parameter log-logistic models. Future studies should pay careful attention to model selection and interpretation in EC50 estimation and clearly indicate which model and EC50 measure (relative versus absolute) was used. We provide guidelines for model choice and interpretation for those wishing to set up experiments for accurate EC50 estimation.