Foliar inoculation of Phyllachora maydis into corn induces infection and local spread in field environments.

Tar spot of corn (Zea mays L.) is a significant disease in the United States and Canada caused by Phyllachora maydis, an obligate biotroph fungus. However, field research critical for understanding and managing the disease has been hindered by a need for methods to inoculate corn with P. maydis in field environments. In this study, we developed and demonstrated the efficacy of a method to initiate tar spot in field settings using inoculations of corn leaves with P. maydis inoculum that had been stored at -20 °C for 10 months. Stromata of P. maydis were observed 19 days after inoculations in two field experiments, and stromata resulting from secondary spread were initially observed 39 to 41 days after the initial inoculations. Tar spot was not present in the fields beyond the inoculated areas or localized spread area, signifying that the establishment of initial disease resulted solely from inoculations. This study enhances our understanding of inoculation and infection of corn with P. maydis and tar spot development in field environments. The results will aid new research into understanding the corn tar spot pathosystem and improving management strategies.

Multistate Sensitivity Monitoring of Fusarium virguliforme to the SDHI Fungicides Fluopyram and Pydiflumetofen in the United States.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean (Glycine max). From 1996 to 2022, cumulative yield losses attributed to SDS in North America totaled over 25 million metric tons, which was valued at over US $7.8 billion. Seed treatments are widely used to manage SDS by reducing early season soybean root infection by F. virguliforme. Fluopyram (succinate dehydrogenase inhibitor [SDHI] - FRAC 7), a fungicide seed treatment for SDS management, has been registered for use on soybean in the United States since 2014. A baseline sensitivity study conducted in 2014 evaluated 130 F. virguliforme isolates collected from five states to fluopyram in a mycelial growth inhibition assay and reported a mean EC50 of 3.35 mg/liter. This baseline study provided the foundation for the objectives of this research: to detect any statistically significant change in fluopyram sensitivity over time and geographical regions within the United States and to investigate sensitivity to the fungicide pydiflumetofen. We repeated fluopyram sensitivity testing on a panel of 80 historical F. virguliforme isolates collected from 2006 to 2013 (76 of which were used in the baseline study) and conducted testing on 123 contemporary isolates collected from 2016 to 2022 from 11 states. This study estimated a mean absolute EC50 of 3.95 mg/liter in isolates collected from 2006 to 2013 and a mean absolute EC50 of 4.19 mg/liter in those collected in 2016 to 2022. There was no significant change in fluopyram sensitivity (P = 0.1) identified between the historical and contemporary isolates. A subset of 23 isolates, tested against pydiflumetofen under the same conditions, estimated an absolute mean EC50 of 0.11 mg/liter. Moderate correlation was detected between fluopyram and pydiflumetofen sensitivity estimates (R = 0.53; P < 0.001). These findings enable future fluopyram and pydiflumetofen resistance monitoring and inform current soybean SDS management strategies in a regional and national context.

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.

A new and effective method to induce infection of Phyllachora maydis into corn for tar spot studies in controlled environments.

BACKGROUND: Tar spot of corn is a significant and spreading disease in the continental U.S. and Canada caused by the obligate biotrophic fungus Phyllachora maydis. As of 2023, tar spot had been reported in 18 U.S. states and one Canadian Province. The symptoms of tar spot include chlorotic flecking followed by the formation of black stromata where conidia and ascospores are produced. Advancements in research and management for tar spot have been limited by a need for a reliable method to inoculate plants to enable the study of the disease. The goal of this study was to develop a reliable method to induce tar spot in controlled conditions. RESULTS: We induced infection of corn by P. maydis in 100% of inoculated plants with a new inoculation method. This method includes the use of vacuum-collection tools to extract ascospores from field-infected corn leaves, application of spores to leaves, and induction of the disease in the dark at high humidity and moderate temperatures. Infection and disease development were consistently achieved in four independent experiments on different corn hybrids and under different environmental conditions in a greenhouse and growth chamber. Disease induction was impacted by the source and storage conditions of spores, as tar spot was not induced with ascospores from leaves stored dry at 25 ºC for 5 months but was induced using ascospores from infected leaves stored at -20 ºC for 5 months. The time from inoculation to stromata formation was 10 to 12 days and ascospores were present 19 days after inoculation throughout our experiments. In addition to providing techniques that enable in-vitro experimentation, our research also provides fundamental insights into the conditions that favor tar spot epidemics. CONCLUSIONS: We developed a method to reliably inoculate corn with P. maydis. The method was validated by multiple independent experiments in which infection was induced in 100% of the plants, demonstrating its consistency in controlled conditions. This new method facilitates research on tar spot and provides opportunities to study the biology of P. maydis, the epidemiology of tar spot, and for identifying host resistance.

Population genomic analysis reveals geographic structure and climatic diversification for Macrophomina phaseolina isolated from soybean and dry bean across the United States, Puerto Rico, and Colombia.

Macrophomina phaseolina causes charcoal rot, which can significantly reduce yield and seed quality of soybean and dry bean resulting from primarily environmental stressors. Although charcoal rot has been recognized as a warm climate-driven disease of increasing concern under global climate change, knowledge regarding population genetics and climatic variables contributing to the genetic diversity of M. phaseolina is limited. This study conducted genome sequencing for 95 M. phaseolina isolates from soybean and dry bean across the continental United States, Puerto Rico, and Colombia. Inference on the population structure using 76,981 single nucleotide polymorphisms (SNPs) revealed that the isolates exhibited a discrete genetic clustering at the continental level and a continuous genetic differentiation regionally. A majority of isolates from the United States (96%) grouped in a clade with a predominantly clonal genetic structure, while 88% of Puerto Rican and Colombian isolates from dry bean were assigned to a separate clade with higher genetic diversity. A redundancy analysis (RDA) was used to estimate the contributions of climate and spatial structure to genomic variation (11,421 unlinked SNPs). Climate significantly contributed to genomic variation at a continental level with temperature seasonality explaining the most variation while precipitation of warmest quarter explaining the most when spatial structure was accounted for. The loci significantly associated with multivariate climate were found closely to the genes related to fungal stress responses, including transmembrane transport, glycoside hydrolase activity and a heat-shock protein, which may mediate climatic adaptation for M. phaseolina. On the contrary, limited genome-wide differentiation among populations by hosts was observed. These findings highlight the importance of population genetics and identify candidate genes of M. phaseolina that can be used to elucidate the molecular mechanisms that underly climatic adaptation to the changing climate.

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species.

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.

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

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

Genetic Diversity and Aggressiveness of Fusarium virguliforme Isolates Across the Midwestern United States.

Sudden death syndrome (SDS) of soybean is a damaging disease caused by the fungus Fusarium virguliforme. Since this pathogen was first reported in the southern U.S. state of Arkansas in 1971, it has spread throughout the midwestern United States. The SDS pathogen primarily colonizes roots but also produces toxins that translocate to and damage leaves. Previous studies have detected little to no genetic differentiation among isolates, suggesting F. virguliforme in North America has limited genetic diversity and a clonal population structure. Yet, isolates vary in virulence to roots and leaves. We characterized a set of F. virguliforme isolates from the midwestern United States, representing a south to north latitudinal gradient from Arkansas to Minnesota. Ten previously tested microsatellite loci were used to genotype isolates, and plant assays were conducted to assess virulence. Three distinct population clusters were differentiated across isolates. Although isolates ranged in virulence classes from low to very high, little correlation was found between virulence phenotype and cluster membership. Similarly, population structure and geographic location were not highly correlated. However, the earliest diverging cluster had the lowest genetic diversity and was detected only in southern states, whereas the two other clusters were distributed across the Midwest and were predominant in Minnesota. One of the midwestern clusters had the greatest genetic diversity and was found along the northern edge of the known distribution. The results support three genetically distinct population clusters of F. virguliforme in the United States, with two clusters contributing most to spread of this fungus across the Midwest.

Sensitivity of Rhizoctonia solani Anastomosis Group 2-2 Isolates from Soybean and Sugar Beet to Selected SDHI and QoI Fungicides.

Rhizoctonia solani causes root and stem diseases on soybean and sugar beet, and fungicides are commonly used to manage these diseases. Quinone outside inhibitor (QoI) fungicides (pyraclostrobin and azoxystrobin) have been used for in-furrow and postemergence application since 2000. Succinate dehydrogenase inhibitor (SDHI) fungicides (sedaxane, penthiopyrad, and fluxapyroxad) became popular seed treatments after their registration in Minnesota and North Dakota between 2012 and 2016. Periodic monitoring of sensitivity to these fungicides in R. solani anastomosis group (AG) 2-2 is important to detect potential shifts in sensitivity over time. R. solani AG 2-2 isolates (n = 35) collected from soybean and sugar beet in Minnesota and North Dakota were evaluated in vitro for sensitivity. Isolates were considered as baseline or nonbaseline for the above-mentioned fungicides based on previous potential exposure. The effective concentration (EC50) required to suppress radial fungal growth by 50% was determined. The mean EC50 values for sedaxane, penthiopyrad, fluxapyroxad, and pyraclostrobin were 0.1, 0.15, 0.16, and 0.25 (µg ml-1), respectively. The mean EC50 value for azoxystrobin for 22 isolates was 0.76 to 1.56 µg ml-1; and EC50 could not be determined for 13 isolates because of <50% inhibition at the highest concentrations used. The EC50 values for the QoI fungicides did not differ significantly between baseline and nonbaseline isolates. EC50 values for SDHI fungicides were significantly higher for isolates collected from soybean than from sugar beet, and isolates collected from both crops had similar EC50 values for pyraclostrobin. All SDHI fungicides and pyraclostrobin effectively suppressed R. solani isolates from soybean and sugar beet at low concentrations in vitro.

Meta-Analysis of Soybean Yield Response to Foliar Fungicides Evaluated from 2005 to 2018 in the United States and Canada.

Random-effect meta-analyses were performed on data from 240 field trials conducted between 2005 and 2018 across nine U.S. states and Ontario, Canada, to quantify the yield response of soybean after application of foliar fungicides at beginning pod (R3) stage. Meta-analysis showed that the overall mean yield response when fungicide was used compared with not applying a fungicide was 2.7% (110 kg/ha). Moderator variables were also investigated and included fungicide group, growing season, planting date, and base yield, which all significantly influenced the yield response. There was also evidence that precipitation from the time of planting to the R3 growth stage influenced yield when fungicide was used (P = 0.059). Fungicides containing a premix of active ingredients from multiple groups (either two or three ingredients) increased the yield by 3.0% over not applying a fungicide. The highest and lowest yield responses were observed in 2005 and 2007, respectively. Better yield response to fungicides (a 3.0% increase) occurred when soybean crops were planted not later than 21 May and when total precipitation between planting and the R3 application date was above historic averages. Temperatures during the season did not influence the yield response. Yield response to fungicide was higher (a 4.7% increase) in average yield category (no spray control yield 2,878 to 3,758 kg/ha) and then gradually decreased with increasing base yield. Partial economic analyses indicated that use of foliar fungicides is less likely to be profitable when foliar diseases are absent or at low levels.

Meta-Analytic and Economic Approaches for Evaluation of Pesticide Impact on Sclerotinia Stem Rot Control and Soybean Yield in the North Central United States.

As complete host resistance in soybean has not been achieved, Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum continues to be of major economic concern for farmers. Thus, chemical control remains a prevalent disease management strategy. Pesticide evaluations were conducted in Illinois, Iowa, Michigan, Minnesota, New Jersey, and Wisconsin from 2009 to 2016, for a total of 25 site-years (n = 2,057 plot-level data points). These studies were used in network meta-analyses to evaluate the impact of 10 popular pesticide active ingredients, and seven common application timings on SSR control and yield benefit, compared with not treating with a pesticide. Boscalid and picoxystrobin frequently offered the best reductions in disease severity and best yield benefit (P < 0.0001). Pesticide applications (one- or two-spray programs) made during the bloom period provided significant reductions in disease severity index (DIX) (P < 0.0001) and led to significant yield benefits (P = 0.0009). Data from these studies were also used in nonlinear regression analyses to determine the effect of DIX on soybean yield. A three-parameter logistic model was found to best describe soybean yield loss (pseudo-R2 = 0.309). In modern soybean cultivars, yield loss due to SSR does not occur until 20 to 25% DIX, and considerable yield loss (-697 kg ha-1 or -10 bu acre-1) is observed at 68% DIX. Further analyses identified several pesticides and programs that resulted in greater than 60% probability for return on investment under high disease levels.

Characterization of the Response of Ornamental Graminoids to Attempted Infection by Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum causes Sclerotinia stem rot (SSR) (also called white mold), resulting in stem rot and death of many common herbaceous ornamental plant species. Resistant plants would be useful to manage SSR; however, the host range of S. sclerotiorum is unclear. The goal of this study was to determine how the ornamental graminoids Pennisetum glaucum, Setaria italica, Juncus inflexus, Carex flagellifera, Isolepis cernua, and Acorus gramineus respond to inoculation with S. sclerotiorum. Plants were inoculated in the field and in controlled environments at 13, 16, 19, or 22°C with or without wounding, and evaluated for SSR. Inoculated detached leaves were stained to examine infection and oxalate oxidase production. A. gramineus developed SSR in field and controlled environments. Sclerotinia stem rot was not observed on P. glaucum in field environments. The disease developed on P. glaucum and S. italica in controlled environments, and severity increased with decreasing temperature and wounding. J. inflexus, C. flagellifera, and I. cernua developed no or minor symptoms of SSR in field and controlled environments. Mycelia penetrated A. gramineus leaves 24 h after inoculation (HAI) and P. glaucum at 48 HAI, but did not penetrate J. inflexus at 24, 48, or 96 HAI. Oxalate oxidase was not detected in inoculated leaves of these ornamental graminoids. The results illuminate S. sclerotiorum's interactions with monocots and broaden the understanding of SSR resistance in ornamental grasses.

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

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

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

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

Effects of Pathogen Population Levels and Crop-Derived Nutrients on Development of Soybean Sudden Death Syndrome and Growth of Fusarium virguliforme.

Sudden death syndrome (SDS) of soybean, caused by Fusarium virguliforme, is a significant disease of soybean. The suite of factors that influence disease development is incompletely understood. The goal of this study was to determine the effects of pathogen population levels, crop residues, seed exudates, and their interactions on development of SDS and growth of F. virguliforme. Studies were conducted in a greenhouse with cultivars susceptible and partially resistant to SDS, four pathogen population levels, and six crop residue treatments (none; ground corn seed, stalks, and roots; ground soybean stems; and sorghum seed). Root rot was assessed 15 and 50 days after inoculation (dai) and foliar disease and plant biomass were assessed 50 dai. Population level increases and crop residues had significant interacting effects on increasing foliar disease severity and root rot and on biomass reduction. Disease severity was positively correlated with population and biomass was negatively correlated. Plants grown with no crop residues exhibited low or no root rot or foliar disease 15 dai, and severity was greatest with corn and sorghum seed. In vitro studies were conducted to test the effects of exudates collected from germinating soybean and corn seed on growth of F. virguliforme and F. solani. Growth of these fungi was greater in exudates than in water. More growth occurred in exudates collected during soybean radicle emergence than those sampled at other times during germination. These studies show that pathogen population levels and crop-derived nutrients in soil interact and influence severity of SDS. Results have implications for gaging disease risk and managing SDS.

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

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

PCR-Mediated Detection and Quantification of the Goss's Wilt Pathogen Clavibacter michiganensis subsp. nebraskensis Via a Novel Gene Target.

Goss's leaf blight and wilt of maize (corn) is a significant and reemerging disease caused by the bacterium Clavibacter michiganensis subsp. nebraskensis. Despite its importance, molecular tools for diagnosing and studying this disease remain limited. We report the identification of CMN_01184 as a novel gene target and its use in conventional PCR (cPCR) and SYBR green-based quantitative PCR (qPCR) assays for specific detection and quantification of C. michiganensis subsp. nebraskensis. The cPCR and qPCR assays based on primers targeting CMN_01184 specifically amplified only C. michiganensis subsp. nebraskensis among a diverse collection of 129 bacterial and fungal isolates, including multiple maize bacterial and fungal pathogens, environmental organisms from agricultural fields, and all known subspecies of C. michiganensis. Specificity of the assays for detection of only C. michiganensis subsp. nebraskensis was also validated with field samples of C. michiganensis subsp. nebraskensis-infected and uninfected maize leaves and C. michiganensis subsp. nebraskensis-infested and uninfested soil. Detection limits were determined at 30 and 3 ng of pure C. michiganensis subsp. nebraskensis DNA, and 100 and 10 CFU of C. michiganensis subsp. nebraskensis for the cPCR and qPCR assays, respectively. Infection of maize leaves by C. michiganensis subsp. nebraskensis was quantified from infected field samples and was standardized using an internal maize DNA control. These novel, specific, and sensitive PCR assays based on CMN_01184 are effective for diagnosis of Goss's wilt and for studies of the epidemiology and host-pathogen interactions of C. michiganensis subsp. nebraskensis.

Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

Anatomical response and infection of soybean during latent and pathogenic infection by type A and B of Phialophora gregata.

Growth and anatomical responses of plants during latent and pathogenic infection by fungal pathogens are not well understood. The interactions between soybean (Glycine max) and two types of the pathogen Phialophora gregata were investigated to determine how plants respond during latent and pathogenic infection. Stems of soybean cultivars with different or no genes for resistance to infection by P. gregata were inoculated with wildtype or GFP and RFP-labeled strains of types A or B of P. gregata. Plants were sectioned during latent and pathogenic infection, examined with transmitted light or fluorescent microscopy, and quantitative differences in vessels and qualitative differences in infection were assessed using captured images. During latent infection, the number of vessels was similar in resistant and susceptible plants infected with type A or B compared to the control, and fungal infection was rarely observed in vessels. During pathogenic infection, the resistant cultivars had 20 to 25% more vessels than the uninfected plants, and fungal hyphae were readily observed in the vessels. Furthermore, during the pathogenic phase in a resistant cultivar, P. gregata type A-GFP was limited to outside of the primary xylem, while P. gregata type B-RFP was observed in the primary xylem. The opposite occurred with the susceptible cultivar, where PgA-GFP was observed in the primary xylem and PgB-RFP was limited to the interfascicular region. In summary, soybean cultivars with resistance to BSR produced more vessels and can restrict or exclude P. gregata from the vascular system compared to susceptible cultivars. Structural resistance mechanisms potentially compensate for loss of vessel function and disrupted water movement.

Contributions of Fusarium virguliforme and Heterodera glycines to the disease complex of sudden death syndrome of soybean.

BACKGROUND: Sudden death syndrome (SDS) of soybean caused by Fusarium virguliforme spreads and reduces soybean yields through the North Central region of the U.S. The fungal pathogen and Heterodera glycines are difficult to manage. METHODOLOGY/PRINCIPAL FINDINGS: The objective was to determine the contributions of H. glycines and F. virguliforme to SDS severity and effects on soybean yield. To quantify DNA of F. virguliforme in soybean roots and soil, a specific real time qPCR assay was developed. The assay was used on materials from soybean field microplots that contained in a four-factor factorial-design: (i) untreated or methyl bromide-fumigated; (ii) non-infested or infested with F. virguliforme; (iii) non-infested or infested with H. glycines; (iv) natural precipitation or additional weekly watering. In years 2 and 3 of the trial, soil and watering treatments were maintained. Roots of soybean 'Williams 82' were collected for necrosis ratings at the full seed growth stage R6. Foliar symptoms of SDS (area under the disease progress curve, AUDPC), root necrosis, and seed yield parameters were related to population densities of H. glycines and the relative DNA concentrations of F. virguliforme in the roots and soil. The specific and sensitive real time qPCR was used. Data from microplots were introduced into models of AUDPC, root necrosis, and seed yield parameters with the frequency of H. glycines and F. virguliforme, and among each other. The models confirmed the close interrelationship of H. glycines with the development of SDS, and allowed for predictions of disease risk based on populations of these two pathogens in soil. CONCLUSIONS/SIGNIFICANCE: The results modeled the synergistic interaction between H. glycines and F. virguliforme quantitatively in previously infested field plots and explained previous findings of their interaction. Under these conditions, F. virguliforme was mildly aggressive and depended on infection of H. glycines to cause highly severe SDS.