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This strategic plan summarizes the major accomplishments achieved in the last quinquennial by the soybean [Glycine max (L.) Merr.] genetics and genomics research community and outlines key priorities for the next 5 years (2024-2028). This work is the result of deliberations among over 50 soybean researchers during a 2-day workshop in St Louis, MO, USA, at the end of 2022. The plan is divided into seven traditional areas/disciplines: Breeding, Biotic Interactions, Physiology and Abiotic Stress, Functional Genomics, Biotechnology, Genomic Resources and Datasets, and Computational Resources. One additional section was added, Training the Next Generation of Soybean Researchers, when it was identified as a pressing issue during the workshop. This installment of the soybean genomics strategic plan provides a snapshot of recent progress while looking at future goals that will improve resources and enable innovation among the community of basic and applied soybean researchers. We hope that this work will inform our community and increase support for soybean research., (© 2024 The Author(s). The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

None of the current oomycota fungicides are effective towards all species of Phytophthora , Phytopythium , Globisporangium , and Pythium that affect soybean seed and seedlings in Ohio. Picarbutrazox is a new oomyceticide with a novel mode of action towards oomycete pathogens. Our objectives were to evaluate picarbutrazox to determine (i) baseline sensitivity (EC 50 ) to 189 isolates of 29 species, (ii) the efficacy with a base seed treatment with three cultivars with different levels of resistance in 14 field environments; and (iii) if the rhizosphere microbiome was affected by the addition of the seed treatment on a moderately susceptible cultivar. The mycelial growth of all isolates was inhibited beginning at 0.001 μg, and the EC 50 ranged from 0.0013 to 0.0483 μg of active ingredient (a.i.)/ml. The effect of seed treatment was significantly different for plant population and yield in eight of 14 and six of 12 environments, respectively. The addition of picarbutrazox at 1 and 2.5 g of a.i./100 kg seed to the base seed treatment compared to the base alone was associated with higher plant populations and yield in three and one environments, respectively. There was limited impact of the seed treatment mefenoxam 7.5 g of a.i. plus picarbutrazox 1 g of a.i./100 kg seed on the oomycetes detected in the rhizosphere of soybean seedlings collected at the V1 growth stage. Picarbutrazox has efficacy towards a wider range of oomycetes that cause disease on soybean, and this will be another oomyceticide tool to combat early season damping-off in areas where environmental conditions highly favor disease development., Competing Interests: The author(s) declare no conflict of interest.

The breeding of disease-resistant soybeans cultivars to manage Phytophthora root and stem rot caused by the pathogen Phytophthora sojae involves combining quantitative disease resistance (QDR) and Rps gene-mediated resistance. To identify and confirm potential mechanisms of QDR toward P. sojae , we conducted a time course study comparing changes in gene expression among Conrad and M92-220 with high QDR to susceptible genotypes, Sloan, and three mutants derived from fast neutron irradiation of M92-220. Differentially expressed genes from Conrad and M92-220 indicated several shared defense-related pathways at the transcriptomic level but also defense pathways unique to each cultivar, such as stilbenoid, diarylheptanoid, and gingerol biosynthesis and monobactam biosynthesis. Gene Ontology pathway analysis showed that the susceptible fast neutron mutants lacked enrichment of three terpenoid-related pathways and two cell wall-related pathways at either one or both time points, in contrast to M92-220. The susceptible mutants also lacked enrichment of potentially important Kyoto Encyclopedia of Genes and Genomes pathways at either one or both time points, including sesquiterpenoid and triterpenoid biosynthesis; thiamine metabolism; arachidonic acid; stilbenoid, diarylheptanoid, and gingerol biosynthesis; and monobactam biosynthesis. Additionally, 31 genes that were differentially expressed in M92-220 following P . sojae infection were not expressed in the mutants. These 31 genes have annotations related to unknown proteins; valine, leucine, and isoleucine biosynthesis; and protein and lipid metabolic processes. The results of this study confirm previously proposed mechanisms of QDR, provide evidence for potential novel QDR pathways in M92-220, and further our understanding of the complex network associated with QDR mechanisms in soybean toward P. sojae., Competing Interests: The author(s) declare no conflict of interest.

For soybean, novel single dominant Resistance to Phytophthora sojae ( Rps) genes are sought to manage Phytophthora root and stem rot. In this study, resistance to P. sojae was mapped individually in four recombinant inbred line (RIL) populations derived from crosses of the susceptible cultivar Williams with PI 407985, PI 408029, PI 408097, and PI424477 previously identified as putative novel sources of disease resistance. Each population was screened for resistance with five to seven isolates of P. sojae separately over multiple F 7 -F 10 generations. Additionally, three of the populations were screened with inoculum from the combination of three P. sojae isolates (PPR), which comprised virulence to 14 Rps genes. Over 2,300 single-nucleotide polymorphism markers were used to construct genetic maps in each population to identify chromosomal regions associated with resistance to P. sojae . Resistance segregated as one or two genes to the individual isolates and one gene toward PPR in each population and mapped to chromosomes 3, 13, or 18 in one or more of the four RIL populations. Resistance to five isolates mapped to the same chromosome 3 region are as follows: OH7 (PI 424477 and PI408029), OH12168, OH7/8, PPR (PI 407985), and 1.S.1.1 (PI408029). The resistance regions on chromosome 13 also overlapped for OH1, OH25, OH-MIA (PI424477), PPR (PI 424477, PI 407985, and PI 408097), PPR and OH0217 (PI 408097), and OH4 (PI 408029), but were distinct for each population suggesting multiple genes confer resistance. Two regions were identified on chromosome 18 but all appear to map to known loci; notably, resistance to the combined inoculum (PPR) did not map at this locus. However, there are putative new alleles in three of four populations, three on chromosome 3 and two on chromosome 13 based on mapping location but also known virulence in the isolate used. This characterization of all the Rps genes segregating in these populations to these isolates will be informative for breeding, but the combined inoculum was able to map a novel loci. Furthermore, within each of these P. sojae isolates, there was virulence to more than the described Rps genes, and the effectiveness of the novel genes requires testing in larger populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor AF and reviewer JJ declared a past co-authorship with the author AR., (Copyright © 2024 Clevinger, Biyashev, Schmidt, Song, Batnini, Bolaños-Carriel, Robertson, Dorrance and Saghai Maroof.)

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology., Competing Interests: The author(s) declare no conflict of interest.

Expression of quantitative disease resistance in many host-pathogen systems is controlled by genes at multiple loci, each contributing a small effect to the overall response. We used a systems genomics approach to study the molecular underpinnings of quantitative disease resistance in the soybean- Phytophthora sojae pathosystem, incorporating expression quantitative trait loci (eQTL) mapping and gene co-expression network analysis to identify the genes putatively regulating transcriptional changes in response to inoculation. These findings were compared to previously mapped phenotypic (phQTL) to identify the molecular mechanisms contributing to the expression of this resistance. A subset of 93 recombinant inbred lines (RILs) from a Conrad × Sloan population were inoculated with P. sojae isolate 1.S.1.1 using the tray-test method; RNA was extracted, sequenced, and the normalized read counts were genetically mapped from tissue collected at the inoculation site 24 h after inoculation from both mock and inoculated samples. In total, more than 100,000 eQTLs were mapped. There was a switch from predominantly cis -eQTLs in the mock treatment to an almost entirely nonoverlapping set of predominantly trans -eQTLs in the inoculated treatment, where greater than 100-fold more eQTLs were mapped relative to mock, indicating vast transcriptional reprogramming due to P. sojae infection occurred. The eQTLs were organized into 36 hotspots, with the four largest hotspots from the inoculated treatment corresponding to more than 70% of the eQTLs, each enriched for genes within plant-pathogen interaction pathways. Genetic regulation of trans -eQTLs in response to the pathogen was predicted to occur through transcription factors and signaling molecules involved in plant-pathogen interactions, plant hormone signal transduction, and MAPK pathways. Network analysis identified three co-expression modules that were correlated with susceptibility to P. sojae and associated with three eQTL hotspots. Among the eQTLs co-localized with phQTLs, two cis -eQTLs with putative functions in the regulation of root architecture or jasmonic acid, as well as the putative master regulators of an eQTL hotspot nearby a phQTL, represent candidates potentially underpinning the molecular control of these phQTLs for resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Million, Wijeratne, Karhoff, Cassone, McHale and Dorrance.)

Heterodera glycines , the soybean cyst nematode (SCN), and fungal pathogen Macrophomina phaseolina are economically important soybean pathogens that may coinfest fields. Resistance remains the most effective management tactic for SCN, and the rhg1-b resistance allele derived from plant introduction 88788 is most commonly deployed in the northern United States. The concomitant effects of SCN and M. phaseolina on soybean performance, as well as the effect of the rhg1-b allele in two different genetic backgrounds, were evaluated in three environments (during 2013 to 2015) and a greenhouse bioassay. Within two soybean populations, half of the lines had the rhg1-b allele, and the other half had the susceptible allele in the backgrounds of the cultivars IA3023 and LD00-3309. Significant interactions between soybean rhg1-b allele and M. phaseolina -infested plots were observed in 2014. In all experiments, initial SCN populations (Pi) and M. phaseolina in roots were associated with reduced soybean yield. SCN reproduction factor (RF = final population/Pi) was affected by SCN Pi, rhg1-b , and genetic background. A background-by-genotype interaction on yield was observed only in 2015, with a stronger rhg1-b effect in the LD00-3309 background, which suggested that the susceptible parent 'IA3023' is tolerant to SCN. SCN female index from greenhouse experiments was compared with field RF, and Lin's concordance and Pearson's correlation coefficients decreased with increasing field SCN Pi in soil. In this study, both SCN and M. phaseolina reduced soybean yield asymptomatically, and the impact of SCN rhg1-b resistance was dependent on SCN virulence but also population density.

Species of Phytophthora , Phytopythium , and Pythium affect soybean seed and seedlings each year, primarily through reduced plant populations and yield. Oxathiapiprolin is effective at managing several foliar diseases caused by some oomycetes. The objectives of these studies were to evaluate oxathiapiprolin in a discriminatory dose assay in vitro; evaluate oxathiapiprolin as a soybean seed treatment on a moderately susceptible cultivar in 10 environments; compare the impact of seed treatment on plant populations and yields in environments with low and high precipitation; and compare a seed treatment mixture on cultivars with different levels of resistance in four environments. There was no reduction in growth in vitro among 13 species of Pythium at 0.1 µg ml -1 . Soybean seed treated with the base fungicide plus oxathiapiprolin (12 and 24 µg a.i. seed -1 ) alone, oxathiapiprolin (12 µg a.i. seed -1 ) plus mefenoxam (6 µg a.i. seed -1 ), or oxathiapiprolin (24 µg a.i. seed -1 ) plus ethaboxam (12.1 µg a.i. seed -1 ) had greater yields in environments that received ≥50 mm of precipitation within 14 days after planting compared with those that received less. Early plant population and yield were significantly higher for seed treated with oxathiapiprolin (24 µg a.i. seed -1 ) + metalaxyl (13.2 µg a.i. seed -1 ) compared with nontreated for six of seven cultivars in at least one of four environments. Oxathiapiprolin combined with another Oomycota fungicide applied to seed has the potential to be used to protect soybean plant establishment and yield in regions prone to poor drainage after high levels of precipitation.

Phytophthora root and stem rot is a yield-limiting soybean disease caused by the soil-borne oomycete Phytophthora sojae . Although multiple quantitative disease resistance loci (QDRL) have been identified, most explain <10% of the phenotypic variation (PV). The major QDRL explaining up to 45% of the PV were previously identified on chromosome 18 and represent a valuable source of resistance for soybean breeding programs. Resistance alleles from plant introductions 427105B and 427106 significantly increase yield in disease-prone fields and result in no significant yield difference in fields with less to no disease pressure. In this study, high-resolution mapping reduced the QDRL interval to 3.1 cm, and RNA-seq analysis of near-isogenic lines (NILs) varying at QDRL-18 pinpointed a single gene of interest which was downregulated in inoculated NILs carrying the resistant allele compared to inoculated NILs with the susceptible allele. This gene of interest putatively encodes a serine-threonine kinase (STK) related to the AtCR4 family and may be acting as a susceptibility factor, based on the specific increase of jasmonic acid concentration in inoculated NILs. This work facilitates further functional analyses and marker-assisted breeding efforts by prioritizing candidate genes and narrowing the targeted region for introgression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WU declared a shared affiliation with the authors MM and MG to the handling editor at the time of review., (Copyright © 2022 Karhoff, Vargas-Garcia, Lee, Mian, Graham, Dorrance and McHale.)

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Phytophthora sojae , the causal agent of Phytophthora root and stem rot of soybean, has been managed with single Rps genes since the 1960s but has subsequently adapted to many of these resistance genes, rendering them ineffective. The objective of this study was to examine the pathotype and genetic diversity of P. sojae from soil samples across Illinois, Indiana, Kentucky, and Ohio by assessing which Rps genes were still effective and identifying possible population clusters. There were 218 pathotypes identified from 473 P. sojae isolates with an average of 6.7 out of 15 differential soybean lines exhibiting a susceptible response for each isolate. Genetic characterization of 103 P. sojae isolates from across Illinois, Indiana, Kentucky, and Ohio with 19 simple sequence repeat markers identified 92 multilocus genotypes. There was a moderate level of population differentiation between these four states, with pairwise F ST values ranging from 0.026 to 0.246. There were also moderate to high levels of differentiation between fields, with pairwise F ST values ranging from 0.071 to 0.537. Additionally, cluster analysis detected the presence of P. sojae population structure across neighboring states. The level of pathotype and genetic diversity, in addition to the identification of population clusters, supports the hypothesis of occasional outcrossing events that allow an increase in diversity and the potential to select for a loss in avirulence to specific resistance genes within regions. The trend of suspected gene flow among neighboring fields is expected to be an ongoing issue with current agricultural practices.

In this study, four recombinant inbred line (RIL) soybean populations were screened for their response to infection by Pythium sylvaticum , Pythium irregulare , Pythium oopapillum , and Pythium torulosum. The parents, PI 424237A, PI 424237B, PI 408097, and PI 408029, had higher levels of resistance to these species in a preliminary screening and were crossed with "Williams," a susceptible cultivar. A modified seed rot assay was used to evaluate RIL populations for their response to specific Pythium species selected for a particular population based on preliminary screenings. Over 2500 single-nucleotide polymorphism (SNP) markers were used to construct chromosomal maps to identify regions associated with resistance to Pythium species. Several minor and large effect quantitative disease resistance loci (QDRL) were identified including one large effect QDRL on chromosome 8 in the population of PI 408097 × Williams. It was identified by two different disease reaction traits in P. sylvaticum , P. irregulare , and P. torulosum . Another large effect QDRL was identified on chromosome 6 in the population of PI 408029 × Williams, and conferred resistance to P. sylvaticum and P. irregulare . These large effect QDRL will contribute toward the development of improved soybean cultivars with higher levels of resistance to these common soil-borne pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Clevinger, Biyashev, Lerch-Olson, Yu, Quigley, Song, Dorrance, Robertson and Saghai Maroof.)

Phytophthora sojae causes Phytophthora root and stem rot of soybean and has been primarily managed through deployment of qualitative Resistance to P. sojae genes (Rps genes). The effectiveness of each individual or combination of Rps gene(s) depends on the diversity and pathotypes of the P. sojae populations present. Due to the complex nature of P. sojae populations, identification of more novel Rps genes is needed. In this study, phenotypic data from previous studies of 16 panels of plant introductions (PIs) were analyzed. Panels 1 and 2 consisted of 448 Glycine max and 520 G. soja, which had been evaluated for Rps gene response with a combination of P. sojae isolates. Panels 3 and 4 consisted of 429 and 460 G. max PIs, respectively, which had been evaluated using individual P. sojae isolates with complex virulence pathotypes. Finally, Panels 5-16 (376 G. max PIs) consisted of data deposited in the USDA Soybean Germplasm Collection from evaluations with 12 races of P. sojae. Using these panels, genome-wide association (GWA) analyses were carried out by combining phenotypic and SoySNP50K genotypic data. GWA models identified two, two, six, and seven novel Rps loci with Panels 1, 2, 3, and 4, respectively. A total of 58 novel Rps loci were identified using Panels 5-16. Genetic and phenotypic dissection of these loci may lead to the characterization of novel Rps genes that can be effectively deployed in new soybean cultivars against diverse P. sojae populations., (© 2020 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Auxin (indole-3-acetic acid, IAA) has been implicated as a susceptibility factor in both beneficial and pathogenic molecular plant-microbe interactions. Previous studies have identified a large number of auxin-related genes underlying quantitative disease resistance loci (QDRLs) for Phytophthora sojae. Thus, we hypothesized that auxin may be involved the P. sojae -soybean interaction. The levels of IAA and related metabolites were measured in mycelia and media supernatant as well as in mock and inoculated soybean roots in a time course assay. The expression of 11 soybean Pin-formed ( GmPIN ) auxin efflux transporter genes was also examined. Tryptophan, an auxin precursor, was detected in the P. sojae mycelia and media supernatant. During colonization of roots, levels of IAA and related metabolites were significantly higher in both moderately resistant Conrad and moderately susceptible Sloan inoculated roots compared with mock controls at 48 h postinoculation (hpi) in one experiment and at 72 hpi in a second, with Sloan accumulating higher levels of the auxin catabolite IAA-Ala than Conrad. Additionally, one GmPIN at 24 hpi, one at 48 hpi, and three at 72 hpi had higher expression in inoculated compared with the mock control roots in Conrad. The ability of resistant cultivars to cope with auxin accumulation may play an important role in quantitative disease resistance. Levels of jasmonic acid (JA), another plant hormone associated with defense responses, were also higher in inoculated roots at these same time points, suggesting that JA also plays a role during the later stages of infection.

Key Message: Genomic prediction of quantitative resistance toward Phytophthora sojae indicated that genomic selection may increase breeding efficiency. Statistical model and marker set had minimal effect on genomic prediction with > 1000 markers. Quantitative disease resistance (QDR) toward Phytophthora sojae in soybean is a complex trait controlled by many small-effect loci throughout the genome. Along with the technical and rate-limiting challenges of phenotyping resistance to a root pathogen, the trait complexity can limit breeding efficiency. However, the application of genomic prediction to traits with complex genetic architecture, such as QDR toward P. sojae, is likely to improve breeding efficiency. We provide a novel example of genomic prediction by measuring QDR to P. sojae in two diverse panels of more than 450 plant introductions (PIs) that had previously been genotyped with the SoySNP50K chip. This research was completed in a collection of diverse germplasm and contributes to both an initial assessment of genomic prediction performance and characterization of the soybean germplasm collection. We tested six statistical models used for genomic prediction including Bayesian Ridge Regression; Bayesian LASSO; Bayes A, B, C; and reproducing kernel Hilbert spaces. We also tested how the number and distribution of SNPs included in genomic prediction altered predictive ability by varying the number of markers from less than 50 to more than 34,000 SNPs, including SNPs based on sequential sampling, random sampling, or selections from association analyses. Predictive ability was relatively independent of statistical model and marker distribution, with a diminishing return when more than 1000 SNPs were included in genomic prediction. This work estimated relative efficiency per breeding cycle between 0.57 and 0.83, which may improve the genetic gain for P. sojae QDR in soybean breeding programs.

Some Pythium spp. cause damping off and root rot in soybeans and other crop species. One of the most effective management tools to reduce disease is host resistance; however, little is known about resistance in soybean to Pythium spp. The soybean nested associated mapping (SoyNAM) parent lines are a set of germplasms that were crossed to a single hub parent to create recombinant inbred line populations for the purpose of mapping agronomic traits. The SoyNAM parents were screened for resistance to Pythium lutarium , Pythium oopapillum , Pythium sylvaticum , and Pythium torulosum in separate assays to evaluate seed and root rot severity. Of the 40 SoyNAM parents, only 'Maverick' was resistant to the four species tested; however, 13 were resistant to three species. Other lines were resistant to two, one, or none of the species tested. Correlations between seed and root rot severity for the lines assessed were weak or insignificant. Results indicate that mechanisms of resistance to seed and root rot caused by Pythium spp. may not necessarily be the same.

Background: Soybean seed treatment with fungicides is a well-established disease management strategy. However, the movement of these fungicides within seedlings is not always well characterized. Thus, the objectives of this study were to determine the pattern of translocation of three fungicides with different modes of action applied as a seed treatment, and the effect of soil type on translocation., Results: Most of the absorbed radioactivity was concentrated in the cotyledons and the maximum sum of the rates of absorption by roots, stems, and leaves of the plants was 15%. In most cases, absorption by roots, stems, and leaves were lower than 5% for 14 C-pyraclostrobin and 14 C-metalaxyl, and 1.6% for 14 C-carbendazim. Fungicides absorbed by the roots and the whole seedlings were higher when plants were grown in soil with lower organic matter content. Fungicides in the cotyledons are unlikely to be redistributed and are lost when cotyledons fall off the plants., Conclusion: Cotyledons are the part of the plant where fungicides are most absorbed, regardless of the fungicide. Soil type affects the absorption of fungicides, and in this study it was most likely caused by soil organic matter. These data improve knowledge of the movement of seed treatment fungicides in soybean seedlings and may help the development of seed treatment chemistry to manage seed and soilborne pathogens., (© 2020 Society of Chemical Industry.)

Phytophthora , Phytopythium , and Pythium species that cause early-season seed decay and pre-emergence and post-emergence damping off of soybean are most commonly managed with seed treatments. The phenylamide fungicides metalaxyl and mefenoxam, and ethaboxam are effective toward some but not all species. The primary objective of this study was to evaluate the efficacy of ethaboxam in fungicide mixtures and compare those with other fungicides as seed treatments to protect soybean against Pythium , Phytopythium , and Phytophthora species in both high-disease field environments and laboratory seed plate assays. The second objective was to evaluate these seed treatment mixtures on cultivars that have varying levels and combinations of resistance to these soilborne pathogens. Five of eight environments received adequate precipitation in the 14 days after planting for high levels of seedling disease development and treatment evaluations. Three environments had significantly greater stands, and three had significantly greater yield when ethaboxam was used in the seed treatment mixture compared with treatments containing metalaxyl or mefenoxam alone. Three fungicide formulations significantly reduced disease severity compared with nontreated in the seed plate assay for 17 species. However, the combination of ethaboxam plus metalaxyl in a mixture was more effective than either fungicide alone against some Pythium and Phytopythium species. Overall, our results indicate that the addition of ethaboxam to a fungicide seed treatment is effective in reducing seed rot caused by these pathogens commonly isolated from soybean in Ohio but that these effects can be masked when cultivars with resistance are planted.

Phytophthora sojae is one of the costliest soybean pathogens in the US. Quantitative disease resistance (QDR) is a vital part of Phytophthora disease management. In this study, QDR was measured in 478 and 495 plant introductions (PIs) towards P. sojae isolates OH.121 and C2.S1, respectively, in genome-wide association (GWA) analyses to identify genetic markers linked to QDR loci (QDRL). Populations were generated by sampling PIs from the US, the Republic of Korea, and the full collection of PIs maintained by the USDA. Additionally, a meta-analysis of QDRL reported from bi-parental studies was done to compare past and present findings. Twenty-four significant marker-trait associations were identified from the 478 PIs phenotyped with OH.121, and an additional 24 marker-trait associations were identified from the 495 PIs phenotyped with C2.S1. In total, 48 significant markers were distributed across 16 chromosomes and based on linkage analysis, represent a total of 44 QDRL. The majority of QDRL were identified with only one of the two isolates, and only a region on chromosome 13 was consistently identified. Regions on chromosomes 3, 13, and 17 were identified in previous GWA-analyses and were re-identified in this study. Five QDRL co-localized with P. sojae meta-QDRL identified from QDRL reported in previous biparental mapping studies. The remaining regions represent novel QDRL, in the soybean-P. sojae pathosystem and were primarily identified in germplasm from the Republic of Korea. Overall, the number of loci identified in this study highlights the complexity of QDR to P. sojae., Competing Interests: The authors have declared that no competing interests exist.

Heterodera glycines , the soybean cyst nematode, and Macrophomina phaseolina , causal agent of charcoal rot, are economically important soybean pathogens. The impact and effect of these pathogens on soybean yield in coinfested fields in the Midwest production region is not known. Both pathogens are soilborne, with spatially aggregated distribution and effects. Spatial regression analysis, therefore, is an appropriate method to account for the spatial dependency in either the dependent variable or regression error term from data produced in fields naturally infested with H. glycines and M. phaseolina . The objectives of this study were twofold: to evaluate the combined effect of H. glycines and M. phaseolina on soybean yield in naturally infested commercial fields with ordinary least squares and spatial regression models; and to evaluate, under environmentally controlled conditions, the combined effect of H. glycines and M. phaseolina through nematode reproduction and plant tissue fungal colonization. Six trials were conducted in fields naturally infested with H. glycines and M. phaseolina in Ohio. Systematic-grid sampling was used to determine the population densities of H. glycines and M. phaseolina , and soybean yield estimates. Though not used in any statistical analysis, M. phaseolina colony forming units from plant tissue, charcoal rot severity, and H. glycines type were also recorded and summarized. In two greenhouse experiments, treatments consisted of H. glycines alone, M. phaseolina alone, and coinfestation of soybean with both pathogens. Moran's I test indicated that the yield from five fields was spatially correlated ( P < 0.05) and aggregated. In these fields, to account for spatial dependence, spatial regression models were fitted to the data. Spatial regression analyses revealed a significant interaction effect between H. glycines and M. phaseolina on soybean yield for fields with high initial population densities of both pathogens. In the greenhouse experiments, H. glycines reproduction was significantly ( P < 0.05) reduced in the presence of M. phaseolina ; however, soybean tissue fungal colonization was not affected by the presence of H. glycines . The direct mechanisms by which H. glycines and M. phaseolina interact were not demonstrated in this study. Future studies must be conducted in the field and greenhouse to better understand this interaction effect.

Rhizoctonia solani AG-2-2IIIB is an important seedling pathogen of soybean in North America and other soybean-growing regions around the world. There is no information regarding the population genetics of field populations of R. solani associated with soybean seedling disease. More specifically, information regarding genetic diversity, the mode of reproduction, and the evolutionary factors that shape different R. solani populations separated in time and space are lacking. We exploited genotyping by sequencing as a tool to assess the genetic structure of R. solani AG-2-2IIIB populations from Illinois, Ohio, and Ontario and investigate the reproductive mode of this subgroup. Our results revealed differences in genotypic diversity among three populations, with the Ontario population having greatest diversity. An overrepresentation of multilocus genotypes (MLGs) and a rejection of the null hypothesis of random mating in all three populations suggested clonality within each population. However, phylogenetic analysis revealed long terminal multifurcating branches for most members of the Ontario population, suggesting a mixed reproductive mode for this population. Analysis of molecular variance revealed low levels of population differentiation, and sharing of similar MLGs among populations highlights the role of genotype flow as an evolutionary force shaping population structure of this subgroup.

Schwabe [teleomorph: Gibberella zeae (Schweintiz) Petch] has been identified as a pathogen of soybean [ (L.) Merr.] causing seed, seedling damping-off and root rot in North America. A major quantitative disease resistance locus (QDRL) that contributed 38.5% of the phenotypic variance toward in soybean was previously identified through mapping of a recombinant inbred line (RIL) population derived from a cross between 'Wyandot' and PI 567301B. This major QDRL mapped to chromosome 8 to a predicted 305 kb region harboring 36 genes. This locus maps near the locus for soybean cyst nematode (SCN) and the locus contributing to seed coat color. Long-read sequencing of the region was completed and variations in gene sequence and gene order compared with the 'Williams 82' reference were identified. Molecular markers were developed for genes within this region and mapped in the original population, slightly narrowing the region of interest. Analyses of the hybrid genome reassembly using three previously published bacterial artificial chromosome (BAC) sequences (BAC56G2, BAC104J7, and BAC77G7-a) combined with RNA-sequencing narrowed the region making candidate gene identification possible. The markers within this region may be used for marker-assisted selection (MAS). There were 10 differentially expressed genes between resistant and susceptible lines, with four of these candidates also located within the genomic interval defined by the flanking markers. These genes included an actin-related protein 2/3 complex subunit, an unknown protein, a hypothetical protein, and a chalcone synthase 3., (© 2019 The Author(s).)

Key Message: Two novel QTL for resistance to Pythium ultimum var. ultimum were identified in soybean using an Illumina SNP Chip and whole genome re-sequencing. Pythium ultimum var. ultimum is one of numerous Pythium spp. that causes severe pre- and post-emergence damping-off of seedlings and root rot of soybean [Glycine max (L.) Merr.]. The objective of this research was to identify quantitative trait loci (QTL) for resistance to P. ultimum var. ultimum in a recombinant inbred line population derived from a cross of 'Magellan' (moderately resistant) and PI 438489B (susceptible). Two different mapping approaches were utilized: the universal soybean linkage panel (USLP 1.0) and the bin map constructed from whole genome re-sequencing (WGRS) technology. Two genomic regions associated with variation in three disease-related parameters were detected using both approaches, with the bin map providing higher resolution. Using WGRS, the first QTL were mapped within a 350-kbp region on Chr. 6 and explained 7.5-13.5% of the phenotypic variance. The second QTL were positioned in a 260-kbp confidence interval on Chr. 8 and explained 6.3-16.8% of the phenotypic variation. Candidate genes potentially associated with disease resistance were proposed. High-resolution genetic linkage maps with a number of significant SNP markers could benefit marker-assisted breeding and dissection of the molecular mechanisms underlying soybean resistance to Pythium damping-off in 'Magellan.' Additionally, the outputs of this study may encourage more screening of diverse soybean germplasm and utilization of genome-wide association studies to understand the genetic basis of quantitative disease resistance.

High levels of genetic diversity have been described within the Pythium irregulare complex from several host plants; however, little is known about the population structure in fields used for grain production. Therefore, the objective of this study was to evaluate the genetic diversity and population structure of 53 isolates baited from 28 soybean and corn production fields from 25 counties in Ohio. Genetic diversity was characterized based on sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and with 21 simple sequence repeat (SSR) markers. In addition, aggressiveness on soybean, optimum growth temperature, and sensitivity to metalaxyl fungicide were determined. ITS sequence analysis indicated that four isolates clustered with P. cryptoirregulare, whereas the remaining isolates clustered with P. irregulare that was subdivided into two groups (1 and 2). Cluster analysis of SSR data revealed a similar subdivision, which was also supported by structure analysis. The isolates from group 2 grew at a slower rate, but both groups of P. irregulare and P. cryptoirregulare recovered in this study had the same optimum growth at 27°C. Variability of aggressiveness and sensitivity toward metalaxyl fungicide was also observed among isolates within each group. The results from this study will help in the selection of isolates to be used in screening for resistance, assessment of fungicide efficacy, and disease management recommendations.

Germplasm screening programs have primarily relied on inoculation with mycelia to determine the resistance reaction of soybean genotypes to Sclerotinia sclerotiorum. However, under field conditions, ascospores are the primary source of inoculum. Therefore, the objective of this study was to determine which components most accurately differentiate the resistance reaction of soybean genotypes inoculated with ascospores of S. sclerotiorum. Ascospores were produced in the laboratory and all of the experiments were carried out under controlled conditions with inoculations at flowering stage. Initially, inoculum densities of 1 × 10 4 , 1 × 10 5 and 1 × 10 6 ascospores ml -1 were compared on six soybean genotypes with known resistance reactions. Disease symptoms developed on all genotypes and at all inoculum densities. The highest ascospore concentration increased infection efficiency but it was not correlated with an increase in lesion length. Components of resistance were then measured on a set of 17 cultivars with known resistance reactions at 1 × 10 5 ascospores ml -1 . Resistance reactions could be differentiated based on the level of infection efficiency and lesion length on the main stem. Although inoculation with ascospores presents some limitations such as the time required for inoculum production as well as the time and space required for plant growth, it has the potential to be used to complement other methods for the characterization of resistance of soybean genotypes.

Soybean ( Glycine max L. Merr.) white mold (SWM), caused by Sclerotinia sclerotiorum (Lib) de Barry), is a devastating fungal disease in the Upper Midwest of the United States and southern Canada. Various methods exist to evaluate for SWM resistance and many quantitative trait loci (QTL) with minor effect governing SWM resistance have been identified in prior studies. This study aimed to predict field resistance to SWM using low-cost and efficient greenhouse inoculation methods and to confirm the QTL reported in previous studies. Three related but independent studies were conducted in the field, greenhouse, and laboratory to evaluate for SWM resistance. The first study evaluated 66 soybean plant introductions (PIs) with known field resistance to SWM using the greenhouse drop-mycelium inoculation method. These 66 PIs were significantly ( P < 0.043) different for resistance to SWM. However, year was highly significant ( P < 0.00001), while PI x year interaction was not significant ( P < 0.623). The second study compared plant mortality (PM) of 35 soybean breeding lines or varieties in greenhouse inoculation methods with disease severity index (DSI) in field evaluations. Moderate correlation (r) between PM under drop-mycelium method and DSI in field trials ( r = 0.65, p < 0.0001 ) was obtained. The PM under spray-mycelium was also correlated significantly with DSI from field trials ( r = 0.51, p < 0.0018 ) . Likewise, significant correlation ( r = 0.62, p < 0.0001) was obtained between PM across greenhouse inoculation methods and DSI across field trials. These findings suggest that greenhouse inoculation methods could predict the field resistance to SWM. The third study attempted to validate 33 QTL reported in prior studies using seven populations that comprised a total of 392 F 4 : 6 lines derived from crosses involving a partially resistant cultivar "Skylla," five partially resistant PIs, and a known susceptible cultivar "E00290." The estimates of broad-sense heritability ( h 2 ) ranged from 0.39 to 0.66 in the populations. Of the seven populations, four had h 2 estimates that were significantly different from zero ( p < 0.05). Single marker analysis across populations and inoculation methods identified 11 significant SSRs ( p < 0.05) corresponding to 10 QTL identified by prior studies. Thus, these five new PIs could be used as new sources of resistant alleles to develop SWM resistant commercial cultivars.

Background: Virus induced gene silencing (VIGS) is a powerful genomics tool for interrogating the function of plant genes. Unfortunately, VIGS vectors often produce disease symptoms that interfere with the silencing phenotypes of target genes, or are frequently ineffective in certain plant genotypes or tissue types. This is especially true in crop plants like soybean [ Glycine max (L.) Merr]. To address these shortcomings, we modified the inoculation procedure of a VIGS vector based on Apple latent spherical virus (ALSV). The efficacy of this new procedure was assessed in 19 soybean genotypes using a soybean Phytoene desaturase ( GmPDS1 ) gene as the VIGS target. Silencing of GmPDS1 was easily scored as photo-bleached leaves and/or stems., Results: In this report, the ALSV VIGS vector was modified by mobilizing ALSV cDNAs into a binary vector compatible with Agrobacterium tumefaciens -mediated delivery, so that VIGS-triggering ALSV variants could be propagated in agro-infiltrated Nicotiana benthamiana leaves. Homogenate of these N. benthamiana leaves was then applied directly onto the unifoliate of young soybean seedlings to initiate systemic gene silencing. This rapid inoculation method bypassed the need for a particle bombardment apparatus. Among the 19 soybean genotypes evaluated with this new method, photo-bleaching indicative of GmPDS1 silencing was observed in nine, with two exhibiting photo-bleaching in 100% of the inoculated individuals. ALSV RNA was detected in pods, embryos, stems, leaves, and roots in symptomatic plants of four genotypes., Conclusions: This modified protocol allowed for inoculation of soybean plants via simple mechanical rubbing with the homogenate of N. benthamiana leaves agro-infiltrated with ALSV VIGS constructs. More importantly, inoculated plants showed no apparent virus disease symptoms which could otherwise interfere with VIGS phenotypes. This streamlined procedure expanded this functional genomics tool to nine soybean genotypes.

Recent outbreaks of Sclerotinia stem rot (SSR) of soybean in Ohio, along with new fungicides and cultivars with resistance to this disease, have led to a renewed interest in studies to update disease management guidelines. The effect of host resistance (in moderately resistant [MR] and moderately susceptible [MS] cultivars) and chemical control on SSR and yield was evaluated in 12 environments from 2014 to 2016. The chemical treatments evaluated were an untreated check, four fungicides (boscalid, picoxystrobin, pyraclostrobin, and thiophanate-methyl), and one herbicide (lactofen) applied at soybean growth stage R1 (early flowering) alone or at R1 followed by a second application at R2 (full flowering). SSR developed in 6 of 12 environments, with mean disease incidence in the untreated check of 2.5 to 41%. The three environments with high levels of SSR (disease incidence in the untreated check >20%) were used for further statistical analysis. There were significant effects (P < 0.05) of soybean cultivar and chemical treatment on SSR levels. Significantly lower levels of SSR were observed in MR cultivars. Both boscalid and lactofen reduced SSR but did not increase yield. Pyraclostrobin increased SSR compared with the untreated check in the three environments with high levels of disease. In the six fields where SSR did not develop, chemical treatment did not increase yield, nor was the yield from the MR cultivar significantly different from the MS cultivar. For Ohio, MR cultivars alone were effective for management of SSR in soybean fields where this disease has historically occurred.

Key Message: A major novel quantitative disease resistance locus, qRfg&#95;Gm06, for Fusarium graminearum was genetically mapped to chromosome 6. Genomic-assisted haplotype analysis within this region identified three putative candidate genes. Fusarium graminearum causes seed, root rot, and seedling damping-off in soybean which contributes to reduced stands and yield. A cultivar Magellan and PI 567516C were identified with low and high levels of partial resistance to F. graminearum, respectively. Quantitative disease resistance loci (QDRL) were mapped with 241 F 7:8 recombinant inbred lines (RILs) derived from a cross of Magellan × PI 567516C. Phenotypic evaluation for resistance to F. graminearum used the rolled towel assay in a randomized incomplete block design. The genetic map was constructed from 927 polymorphic single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers. One major QDRL qRfg&#95;Gm06 was detected and mapped to chromosome 6 with a LOD score of 20.3 explaining 40.2% of the total phenotypic variation. This QDRL was mapped to a ~400 kb genomic region of the Williams 82 reference genome. Genome mining of this region identified 14 putative candidate disease resistance genes. Haplotype analysis of this locus using whole genome re-sequencing (WGRS) of 106 diverse soybean lines narrowed the list to three genes. A SNP genotyping Kompetitive allele-specific PCR (KASP) assay was designed for one of the genes and was validated in a subset of the RILs and all 106 diverse lines.

Background: Phytophthora root and stem rot is one of the most yield-limiting diseases of soybean [Glycine max (L.) Merr], caused by the oomycete Phytophthora sojae. Partial resistance is controlled by several genes and, compared to single gene (Rps gene) resistance to P. sojae, places less selection pressure on P. sojae populations. Thus, partial resistance provides a more durable resistance against the pathogen. In previous work, plant introductions (PIs) originating from the Republic of Korea (S. Korea) have shown to be excellent sources for high levels of partial resistance against P. sojae., Results: Resistance to two highly virulent P. sojae isolates was assessed in 1395 PIs from S. Korea via a greenhouse layer test. Lines exhibiting possible Rps gene immunity or rot due to other pathogens were removed and the remaining 800 lines were used to identify regions of quantitative resistance using genome-wide association mapping. Sixteen SNP markers on chromosomes 3, 13 and 19 were significantly associated with partial resistance to P. sojae and were grouped into seven quantitative trait loci (QTL) by linkage disequilibrium blocks. Two QTL on chromosome 3 and three QTL on chromosome 19 represent possible novel loci for partial resistance to P. sojae. While candidate genes at QTL varied in their predicted functions, the coincidence of QTLs 3-2 and 13-1 on chromosomes 3 and 13, respectively, with Rps genes and resistance gene analogs provided support for the hypothesized mechanism of partial resistance involving weak R-genes., Conclusions: QTL contributing to partial resistance towards P. sojae in soybean germplasm originating from S. Korea were identified. The QTL identified in this study coincide with previously reported QTL, Rps genes, as well as novel loci for partial resistance. Molecular markers associated with these QTL can be used in the marker-assisted introgression of these alleles into elite cultivars. Annotations of genes within QTL allow hypotheses on the possible mechanisms of partial resistance to P. sojae.

Pathotype diversity of Phytophthora sojae was assessed in 11 states in the United States during 2012 and 2013. Isolates of P. sojae were recovered from 202 fields, either from soil samples using a soybean seedling bioassay or by isolation from symptomatic plants. Each isolate was inoculated directly onto 12 soybean differentials; no Rps gene or Rps 1a, 1b, 1c, 1k, 3a, 3b, 3c, 4, 6, 7, or 8. There were 213 unique virulence pathotypes identified among the 873 isolates collected. None of the Rps genes were effective against all the isolates collected but Rps6 and Rps8 were effective against the majority of isolates collected in the northern regions of the sampled area. Virulence toward Rps1a, 1b, 1c, and 1k ranged from 36 to 100% of isolates collected in each state, while virulence to Rps6 and Rps8 was less than 36 and 10%, respectively. Depending on the state, the effectiveness of Rps3a ranged from totally effective to susceptible to more than 40% of the isolates. Pathotype complexity has increased in populations of P. sojae in the United States, emphasizing the increasing importance of stacked Rps genes in combination with high partial resistance as a means of limiting losses to P. sojae.

Phytophthora root and stem rot, caused by Phytophthora sojae, is an economically important disease of soybean throughout the Midwestern United States. This disease has been successfully managed with resistance (Rps) genes; however, pathogen populations throughout the Midwest have developed virulence to many Rps genes, including those that have not been deployed. To gain a better understanding of the processes that influence P. sojae evolution, the population genetic structure was compared among populations using one isolate collected from 17, 33, and 20 fields in Iowa, Ohio, and South Dakota, respectively, as well as multiple isolates from individual fields in Iowa, Ohio, and Missouri. Genotypic diversity was measured using 21 polymorphic microsatellite (simple-sequence repeat) markers. and pathotype diversity using 15 soybean differentials. For all but three of the populations with low sample size, there was a high level of pathotype diversity and a low to moderate level of genotypic diversity among the populations for both comparisons between states and within-field variation. None of the Rps-gene differentials were resistant to all of the isolates. There were 103 unique multilocus genotypes identified in this study and only 2 were identified from the same field. Although no clones were identified in more than one field, pairwise F ST indicated that some gene flow within neighboring fields does occur but not across the region, including fields from neighboring states. These results suggest that there is a strong probability that each state may have their own or several regional populations, as well as provide further evidence of high diversity within this homothallic pathogen which may be due, in part, to limited gene flow, mutation, or outcrossing, and this likely affects the success of deployment of resistance.

Vertically transmitted bacterial symbionts are common in arthropods. Aphids undergo an obligate symbiosis with Buchnera aphidicola, which provides essential amino acids to its host and contributes directly to nymph growth and reproduction. We previously found that newly adult Aphis glycines feeding on soybean infected with the beetle-transmitted Bean pod mottle virus (BPMV) had significantly reduced fecundity. We hypothesized that the reduced fecundity was attributable to detrimental impacts of the virus on the aphid microbiome, namely Buchnera. To test this, mRNA sequencing and quantitative real-time PCR were used to assay Buchnera transcript abundance and titre in A. glycines feeding on Soybean mosaic virus-infected, BPMV-infected, and healthy soybean for up to 14 days. Our results indicated that Buchnera density was lower and ultimately suppressed in aphids feeding on virus-infected soybean. While the decreased Buchnera titre may be associated with reduced aphid fecundity, additional mechanisms are probably involved. The present report begins to describe how interactions among insects, plants, and plant pathogens influence endosymbiont population dynamics., (© 2015 The Royal Entomological Society.)

Key Message: A major novel QTL was identified in a recombinant inbred line population derived from a cross of 'Wyandot' × PI 567301B for Fusarium graminearum, a seed and seedling pathogen of soybean. Fusarium graminearum is now recognized as a primary pathogen of soybean, causing root, seed rot and seedling damping-off in North America. In a preliminary screen, 'Wyandot' and PI 567301B were identified with medium and high levels of partial resistance to F. graminearum, respectively. The objective of this study was to characterise resistance towards F. graminearum using 184 recombinant inbred lines (RILs) derived from a cross of 'Wyandot' × PI 567301B. The parents and the RILs of the mapping population were evaluated for resistance towards F. graminearum using the rolled towel assay in a randomized incomplete block design. A genetic map was constructed from 2545 SNP markers and 2 SSR markers by composite interval mapping. One major and one minor QTL were identified on chromosomes 8 and 6, respectively, which explained 38.5 and 8.1 % of the phenotypic variance. The major QTL on chromosome 8 was mapped to a 300 kb size genomic region of the Williams 82 sequence. Annotation of this region indicates that there are 39 genes including the Rhg4 locus for soybean cyst nematode (SCN) resistance. Based on previous screens, PI 567301B is susceptible to SCN. Fine mapping of this locus will assist in cloning these candidate genes as well as identifying DNA markers flanking the QTL that can be used in marker-assisted breeding to develop cultivars with high levels of resistance to F. graminearum.

Background: Root system architecture is important for water acquisition and nutrient acquisition for all crops. In soybean breeding programs, wild soybean alleles have been used successfully to enhance yield and seed composition traits, but have never been investigated to improve root system architecture. Therefore, in this study, high-density single-feature polymorphic markers and simple sequence repeats were used to map quantitative trait loci (QTLs) governing root system architecture in an inter-specific soybean mapping population developed from a cross between Glycine max and Glycine soja., Results: Wild and cultivated soybean both contributed alleles towards significant additive large effect QTLs on chromosome 6 and 7 for a longer total root length and root distribution, respectively. Epistatic effect QTLs were also identified for taproot length, average diameter, and root distribution. These root traits will influence the water and nutrient uptake in soybean. Two cell division-related genes (D type cyclin and auxin efflux carrier protein) with insertion/deletion variations might contribute to the shorter root phenotypes observed in G. soja compared with cultivated soybean. Based on the location of the QTLs and sequence information from a second G. soja accession, three genes (slow anion channel associated 1 like, Auxin responsive NEDD8-activating complex and peroxidase), each with a non-synonymous single nucleotide polymorphism mutation were identified, which may also contribute to changes in root architecture in the cultivated soybean. In addition, Apoptosis inhibitor 5-like on chromosome 7 and slow anion channel associated 1-like on chromosome 15 had epistatic interactions for taproot length QTLs in soybean., Conclusion: Rare alleles from a G. soja accession are expected to enhance our understanding of the genetic components involved in root architecture traits, and could be combined to improve root system and drought adaptation in soybean.

Unlabelled: Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction., Importance: Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus.

Partial resistance to Phytophthora sojae in soybean is controlled by multiple quantitative trait loci (QTL). With traditional QTL mapping approaches, power to detect such QTL, frequently of small effect, can be limited by population size. Joint linkage QTL analysis of nested recombinant inbred line (RIL) populations provides improved power to detect QTL through increased population size, recombination, and allelic diversity. However, uniform development and phenotyping of multiple RIL populations can prove difficult. In this study, the effectiveness of joint linkage QTL analysis was evaluated on combinations of two to six nested RIL populations differing in inbreeding generation, phenotypic assay method, and/or marker set used in genotyping. In comparison to linkage analysis in a single population, identification of QTL by joint linkage analysis was only minimally affected by different phenotypic methods used among populations once phenotypic data were standardized. In contrast, genotyping of populations with only partially overlapping sets of markers had a marked negative effect on QTL detection by joint linkage analysis. In total, 16 genetic regions with QTL for partial resistance against P. sojae were identified, including four novel QTL on chromosomes 4, 9, 12, and 16, as well as significant genotype-by-isolate interactions. Resistance alleles from PI 427106 or PI 427105B contributed to a major QTL on chromosome 18, explaining 10-45% of the phenotypic variance. This case study provides guidance on the application of joint linkage QTL analysis of data collected from populations with heterogeneous assay conditions and a genetic framework for partial resistance to P. sojae.

Soybean vein necrosis-associated virus (SVNaV), a newly discovered tospovirus that infects soybean, was first described as widespread in a number of southern and midwestern states, but so far has not been reported in Ohio (1). Here we describe its occurrence in six different soybean leaf samples collected from five Ohio counties: Champaign, Hardin, Sandusky, Seneca, and Wyandot. Specifically, SVNaV was initially identified through a comprehensive survey during the summer of 2011 that used high throughput sequencing to detect genome sequences of viruses present in a pool of 110 field samples collected from 24 Ohio counties. Three assembled contigs, with sizes of 7,551, 4,937, and 1,554 nucleotides (nt) respectively, share 99% nt identity with the three SVNaV genomic RNAs (L, M, and S), and thus constitute partial sequences of the SVNaV Ohio (OH) isolate. The distribution of this virus was further delineated using reverse transcription (RT)-PCR with primers SVNaV-1734F (5' CCATCTTTCTTTCCAGGCATTTCA 3') and SVNaV-S-2421R (5' GATTCAAGTTCAGCGAGTTCTACAA 3'). All plants from which the SVNaV-positive samples were collected showed typical virus symptoms, including systemic mosaic accompanied by leaf deformation, chlorosis, vein necrosis, and rusty spots on mature leaves. These symptoms are largely consistent with the previous report by Zhou and colleagues (1). Intriguingly, further analysis with RT-PCR revealed that five out of the six SVNaV-positive samples also contained a second virus, with Bean pod mottle virus found in four of the samples, and Tobacco ringspot virus in the fifth. Since it is not yet possible to initiate SVNaV infection mechanically, it is difficult to determine whether the co-infecting viruses contribute to the disease symptoms and yield losses. It should be noted that SVNaV may have been in Ohio for some time since symptoms similar to those reported by Zhou and colleagues (1) have been observed in soybean fields of this state since at least 2009. Furthermore, while in 2011 these symptoms were observed in only a few fields, as reflected by the detection of SVNaV in six of the 110 samples, the 2012 growing season has seen a big jump of symptomatic plants and fields. The current report confirms its presence with molecular evidence and lays the groundwork for further assessment of its impact on soybean production. Reference: (1) J. Zhou et al. Virus Genes 43:289, 2011.

Phytophthora root and stem rot caused by Phytophthora sojae Kaufmann and Gerdemann is one of the most severe soybean [Glycine max (L.) Merr] diseases in the USA. Partial resistance is as effective in managing this disease as single-gene (Rps gene)-mediated resistance and is more durable. The objective of this study was to identify quantitative trait loci (QTL) associated with partial resistance to P. sojae in PI 398841, which originated from South Korea. A population of 305 F7:8 recombinant inbred lines derived from a cross of OX20-8 × PI 398841 was used to evaluate partial resistance against P. sojae isolate C2S1 using a tray test. Composite interval mapping using a genome-wide logarithm of odd (LOD) threshold detected three QTL on chromosomes 1, 13, and 18, which individually explained 4-16 % of the phenotypic variance. Seven additional QTL, accounting for 2-3 % of phenotypic variance each, were identified using chromosome-wide LOD thresholds. Seven of the ten QTL for resistance to P. sojae were contributed by PI 398841. Seven QTL co-localized with known Rps genes and previously reported QTL for soil-borne root pathogens, isoflavone, and seed oil. Three QTL on chromosomes 3, 13, and 18 co-localized with known Rps genes, but PI 398841 did not exhibit an Rps gene-mediated resistance response following inoculation with 48 different isolates of P. sojae. PI 398841 is potentially a source of novel genes for improving soybean cultivars for partial resistance to P. sojae.

Background: Phytophthora sojae is the primary pathogen of soybeans that are grown on poorly drained soils. Race-specific resistance to P. sojae in soybean is gene-for-gene, although in many areas of the US and worldwide there are populations that have adapted to the most commonly deployed resistance to P. sojae ( Rps) genes. Hence, this system has received increased attention towards identifying mechanisms and molecular markers associated with partial resistance to this pathogen. Several quantitative trait loci (QTL) have been identified in the soybean cultivar 'Conrad' that contributes to the expression of partial resistance to multiple P. sojae isolates., Results: In this study, two of the Conrad QTL on chromosome 19 were dissected through sequence and expression analysis of genes in both resistant (Conrad) and susceptible ('Sloan') genotypes. There were 1025 single nucleotide polymorphisms (SNPs) in 87 of 153 genes sequenced from Conrad and Sloan. There were 304 SNPs in 54 genes sequenced from Conrad compared to those from both Sloan and Williams 82, of which 11 genes had SNPs unique to Conrad. Eleven of 19 genes in these regions analyzed with qRT-PCR had significant differences in fold change of transcript abundance in response to infection with P. sojae in lines with QTL haplotype from the resistant parent compared to those with the susceptible parent haplotype. From these, 8 of the 11 genes had SNPs in the upstream, untranslated region, exon, intron, and/or downstream region. These 11 candidate genes encode proteins potentially involved in signal transduction, hormone-mediated pathways, plant cell structural modification, ubiquitination, and basal resistance., Conclusions: These findings may indicate a complex defense network with multiple mechanisms underlying these two soybean QTL conferring resistance to P. sojae. SNP markers derived from these candidate genes can contribute to fine mapping of QTL and marker assisted breeding for resistance to P. sojae.

Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots.

Two new species of Pythium, pathogens of corn and soybean in Ohio, are described. Pythium schmitthenneri sp. nov. and Pythium selbyi sp. nov. both have morphological and sequence characteristics that place them in clade E1 of the genus Pythium. Morphology and sequence analysis of the ITS1-5.8S-ITS2 regions of these species were different from previously described species. The ITS region of Pythium schmitthenneri was 99.9% similar to P. acrogynum and 99.8% similar to P. hypogynum. All three species are characterized by globose to limoniform sporangia and plerotic oospores. Pythium schmitthenneri has mostly diclinous antheridia, compared to the strictly hypogynous antheridia of P. acrogynum and P. hypogynum. The temperature for growth of P. schmitthenneri is below 4 C to 32 C, and optimum growth is 18-25 C compared to 31-34 C for P. hypogynum. The ITS region of P. selbyi was 97.1% similar to P. longandrum and 97.5% similar to P. longisporangium. All three species are characterized by globose sporangia, mostly plerotic oospores, with one to two oospores per oogonium, and hypogynous or monoclinous antheridia. The temperature for growth of P. selbyi is below 4 to 32 C, with an optimum 18-25 C. These new species were widely dispersed throughout the soybean- and corn-producing regions in Ohio, making their characterization critical for managing the Pythium complex that causes seedling and root-rot disease in Ohio soybean and corn fields.

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

Analysis of an organism's genetic diversity requires a method that gives reliable, reproducible results. Microsatellites are robust markers, however, detection of allele sizes can be difficult with some systems as well as consistency among laboratories. In this study, our two laboratories used 219 isolates of Phytophthora sojae to compare three microsatellite methods. Two capillary electrophoresis methods, the Applied Biosystems 3730 Genetic Analyzer and the CEQ 8000 Genetic Analysis system, detected an average of 2.4-fold more alleles compared to gel electrophoresis with a mean of 8.8 and 3.6 alleles per locus using capillary and gel methods, respectively. The two capillary methods were comparable, although allele sizes differed consistently by an average of 3.2 bp across isolates. Differences between capillary methods could be overcome if reference standard DNA genotypes are shared between collaborating laboratories.

Fusarium graminearum causes seed decay and damping-off of soybean. This study evaluated the effect of inoculum density of F. graminearum, temperature, and fungicide seed treatments on disease development. To determine the optimum conditions for disease development, individual soybean seed was inoculated with 100 μl of a suspension of 2.5 × 10 2 , 2.5 × 10 3 , 2.5 × 10 4 , or 2.5 × 10 5 macroconidia/ml in a rolled-towel assay at temperatures of 18, 22, and 25°C. Inoculum concentrations of 2.5 × 10 4 macroconidia/ml or higher were necessary for optimum disease development at all temperatures. The efficacy of captan, fludioxonil, mefenoxam + fludioxonil, azoxystrobin, trifloxystrobin, and pyraclostrobin as seed treatments was then evaluated with the same assay at 2.5 × 10 4 and 2.5 × 10 5 macroconidia/ml. Seed treated with captan at 61.9 g a.i. or fludioxonil at 2.5 or 5.0 g a.i. per 100 kg developed smaller lesions than other seed treatments and the nontreated control. Based on these results, there are limited choices in fungicide seed treatments for managing this seedling disease, and it is possible that shifts in seed treatment products may have played a role in the recent emergence of this soybean pathogen.

Purpose of Work: A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required. A protocol for DNA isolation from Phytophthora and Pythium suitable for the evaluation of a large set of molecular markers was modified from one previously developed for soybean seed. There was a one to three fold increase in the amount of DNA that was extracted using the modified protocol compared to a commercial kit. The DNA obtained using the modified protocol was suitable for the amplification of microsatellite markers as well as the ITS region. This protocol is inexpensive, easy, quick, and efficient in terms of the volume of reagents and the number of steps involved in the procedure. The method may be applicable to other oomycetes and effectively implemented in other laboratories.

Brown spot, caused by Septoria glycines, is the most common foliar disease of soybean in Ohio, but its economic impact has not been assessed on modern cultivars. Therefore, the objectives of this study were to (i) evaluate the effect of S. glycines on soybean yield and (ii) evaluate the efficacy of strobilurin- and triazole-based fungicides on the control of brown spot. Yield loss associated with S. glycines was determined using weekly applications of chlorothalonil. The efficacy of azoxystrobin, pyraclostrobin, tebuconazole, and flutriafol alone and in combinations were also assessed using applications at the R3 and R5 growth stages at two locations over 3 years. Significantly different levels of brown spot developed following applications of chlorothalonil, with mean yield differences between treated and nontreated plots ranging from 196 to 293 kg/ha. Pyraclostrobin and azoxystrobin applied at the R3 growth stage significantly reduced final levels of brown spot; however, significant increases in yield occurred in only three of the six location-years. Triazoles, flutriafol and tebuconazole, applied at R3 or R5 did not significantly decrease levels of brown spot or impact yield. More data on the accurate timing of fungicides are still required to establish a long-term management program for this disease, and resistance to brown spot should be monitored in soybean cultivar development to prevent future yield losses.

Bean pod mottle virus (BPMV) infection reduces yield and seed quality in soybean. To test the hypothesis that virus incidence and movement within plots would be reduced in soybean with resistance to feeding by the virus' bean leaf beetle (Cerotoma trifurcata) vector, BPMV spread was evaluated in five soybean genotypes at two inoculum levels over 2 years at two locations in Ohio. Soybean genotypes included two insect-feeding-susceptible genotypes (Williams 82 and Resnik), two insect-feeding-resistant, semidwarf genotypes (HC95-15 and HC95-24), and an insect-feeding-susceptible, semidwarf genotype (Troll). BPMV incidence was assessed in individual plants at growth stages R5/R6 and R7/R8 using enzyme-linked immunosorbent assay. Beetle feeding was visually assessed in 2004. Data for infection of individual plants were analyzed using a generalized linear mixed model, with a binomial distribution and logit-link. Within plots, BPMV incidence was highest in Resnik and Williams 82 and significantly lower in Troll. Incidence in HC95-15 was not significantly different than in Williams 82 and Resnik but incidence in HC95-24 was lower than in Resnik. BPMV incidence was also significantly (P < 0.05) affected by year, location, inoculum level and sampling date, with increasing incidence over time and higher incidence at the higher inoculum level. Beetle feeding damage was affected by the interaction of location-genotype. Significant spatial aggregation of infected plants was found for most plots but aggregation was independent of host genotype and inoculum level. Although the results indicate that BPMV infection varied by genotype, they do not support the hypothesis that insect-feeding resistance is sufficient to reduce the incidence and spread of BPMV.

During the spring of 2004, corn seedlings with symptoms of wilting and stunting were observed in corn fields with emergence problems in Madison and Brown counties, Ohio. Phytophthora isolates were recovered from sections of root tissue of diseased seedlings placed on dilute V8 media amended with pentachloronitrobenzene, iprodione, benlate, neomycin sulfate, and chloramphenicol. Colonies were rosaceous on potato dextrose agar, with a growth rate of 5 mm per day. Homothallic isolates with paragynous antheridia were observed on lima bean agar (LBA); oogonia were 35 to 50 μm in diameter. Sporangia were ovoid to obpyriform, nonpapillate, with an average size of 49 × 30 μm. Pathogenicity was tested on corn seeds using a petri dish assay with 3-day-old cultures on LBA and a sand-cornmeal cup test amended with inoculum from 7-day-old cultures on LBA (1). After 1 week in the petri dish assay, the seeds failed to germinate completely and were covered with white, fungal-like, aerial mycelia and the pathogen was recovered from brown discolored radicle roots. In the cup assay, 2-week-old seedlings developed the same symptoms observed in the field; the pathogen was also isolated from brown discolored roots. In both assays, no symptoms developed in the noninoculated controls. Both pathogenicity tests were repeated two times. Genomic DNA was extracted from mycelia of two isolates and the internal transcribed spacer (ITS) region was amplified and sequenced using ITS6/ITS4 primers (2). Both isolates had identical ITS sequences (GenBank Accession No. GQ853880). A BLAST search of the NCBI database showed 100% homology with the sequence of the haplotype isolate of Phytophthora sansomeana (Accession No. EU925375). P. sansomeana is a new species characterized principally by a large oogonial diameter (37 to 45 μm), rapid growth rate (7 to 10 mm/day), and an ITS sequence falling in Cooke's clade 8 (4). Pathogenicity tests, morphological characteristics, and the ITS sequence analysis indicate that P. samsomena is the causal agent of the symptoms observed on corn seedlings. P. sansomeana has been reported as a pathogen of soybean in Indiana, Douglas-fir in Oregon, and weeds in alfalfa fields in New York (4). To our knowledge, this is the first report of P. sansomeana infecting corn in Ohio, albeit other isolates have previously been recovered from soybean in the state. There are four previous reports of Phytophthora spp. affecting corn in the United States and Mexico (3). Crop rotation will have little effect in management of this pathogen since corn and soybean are produced in the same fields continuously throughout the state. References: (1) K. E. Broders et al. Plant. Dis. 91:727, 2007. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN. 1989. (4) E. M. Hansen et al. Mycologia 101:129, 2009.

Phytophthora sojae has re-emerged as a serious soybean pathogen in the past decade. This may be due in part to changes in resistance levels in current cultivars, adoption of P. sojae populations to deployed Rps genes, and highly favorable environments in the past decade. This multilocation study evaluated the effect of seed treatments on the incidence and severity of Phytophthora root and stem rot on soybeans with different combinations of Rps genes and levels of partial resistance. The efficacy of the seed treatments was highly variable across locations. Seed treatments (metalaxyl and mefenoxam) provided protection and increased yields across cultivars in locations where rain or irrigation occurred shortly after planting (Ohio, South Dakota, and Ontario). However, there were no significant differences in stand or yield consistently across cultivars in Iowa, Nebraska, Wisconsin, or Ohio, where heavy precipitation did not occur until later growth stages. The environment, levels of inoculum, and pathogen complex may have played a role in the different responses to the seed treatments and to the different combinations of Rps genes and levels of partial resistance to P. sojae in the cultivars. Fields that are poorly drained and have P. sojae populations with complex pathotypes may benefit the most from seed treatments. Individual fields where producers may see the greatest benefit to utilizing these integrated management strategies will need to be identified.