Effect of drought stress during seed development on charcoal rot and yield of soybean.

The primary controls for charcoal rot in soybean, caused by the fungal pathogen Macrophomina phaseolina, are to avoid drought stress and to plant a moderately resistant cultivar. The effects of irrigation and cultivar were determined in 2011 and 2013 at the Lon Mann Cotton Research Station, Marianna, AR. Four soybean cultivars (Hutcheson, Osage, Ozark, and R01581F), were planted in plots with or without added M. phaseolina inoculum and subjected to three furrow irrigation regimes: full season irrigation (Full), irrigation terminated at R5 (CutR5), and non-irrigated (NonIrr). Normalized difference vegetative index (NDVI) was measured at R3 and R6. At harvest, plants and yields were collected. Roots and stems were split and the extent of visible colonization by M. phaseolina microsclerotia was assessed in the roots with a 1-5 scale (RSS) and the percent plant height stem discoloration (PHSD) measured. Precipitation in September and October was 54 and 65% below the 30-year average in 2011 and 2013, respectively. The CutR5 irrigation treatment resulted in one less irrigation than Full each year, but CutR5 NDVI's at R6 and yields were significantly lower than those with Full and not significantly different than those of NonIrr. The CutR5 RSS ratings were greater than either Full or NonIrr. Plant colonization by M. phaseolina was negatively correlated to yield in 2011 but not in 2013. No premature plant death caused by charcoal rot was observed in either year. These results indicated that late season drought stress may be more important to charcoal rot development than drought stress throughout the season, but other factors are needed to trigger early plant death and subsequent yield losses observed in grower fields.

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.

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.

First report of halo blight of hop (Humulus lupulus) caused by Diaporthe humulicola in Quebec, Canada.

Halo blight of hop caused by Diaporthe humulicola has recently been reported in Michigan and Connecticut (Higgins et al. 2021, Allan-Perkins et al 2020). In August 2020 growers in Quebec, Canada reported necrotic foliar lesions and desiccation of the hop strobile (cone) on Chinook and Nugget cultivars. The foliar lesions were dry concentric circles with a chlorotic halo surrounding the lesions; no pycnidia were observed on leaves or cones. Up to 100% of the infected bract tissue was dry and easily shattered, the grower estimated that more than 90% of the plants in the hopyard exhibited symptoms. Twenty-six isolates were obtained from surface-sterilized leaf and cone tissue by plating the leading edge of lesions on potato dextrose agar. Fungal isolates were hyphal tipped and were incubated at 22°C with a 12 h photoperiod. After 21-days, all cultures were white to beige with pycnidia. DNA was extracted from cultures using the MagMAX Plant DNA Isolation Kit (Applied Biosystems, Foster City, CA). DNA amplification of a representative isolate (CD6C) was performed with primers ITS1/ITS4 (White et al. 1990) for the internal transcribed spacer (ITS), CYLH3F/H3-1b (Glass and Donaldson 1995) for histone 3 (HIS), and Ef1728f/EF1-986R (Carbone and Kohn 1999) for translation elongation factor 1-α (TEF). Amplification primers were used for bidirectional Sanger sequencing, reads were assembled using Geneious Prime (Biomatters, New Zealand), and identified using NCBI BLAST. BLAST results showed that the sequences for TEF, ITS, and HIS all had 100% pairwise identity to Diaporthe sp. 1-MI (MT909101, MT909099, MT909093, OK001342, MZ934713, OK001341). Futhermore, BLAST results showed that ITS and HIS have 100% pairwise identity D. humulicola (MN152929, MN180214). The TEF sequence also had 99.7% pairwise identity to D. humulicola (MN180209). Koch's postulates were conducted by inoculating six 3-mo-old 'Chinook' plants with conidia harvested from 28-day-old cultures and spraying 50 ml of inoculum (6 x 105 conidia/ml) or water to each plant. Plants were then stored in a greenhouse at 100% relative humidity at 22°C with a 14-h photo period. Lesions appeared on the adaxial side of the leaf after 21 days. D. humulicola was re-isolated from all infected leaf tissue, but not from any water inoculated plants and identified by conidial morphology using descriptions from Higgins et al. (2021). So far, Diaporthe sp. 1-MI appears to be synonymous with Diaporthe humulicola, but currently two names are being utilized (i.e. Diaporthe leaf spot and halo blight). In Higgins et al., (2021) it was proposed that the name halo blight might be more appropriate because disease symptoms are not confined to the leaves and cause significant blighting of cones. Halo blight caused by D. humulicola appears widespread in Michigan and Canada and may become an issue in other eastern North American growing regions with humid conditions.

Draft Genome Sequence Resource for Blumeriella jaapii, the Cherry Leaf Spot Pathogen.

Blumeriella jaapii is the causal agent of cherry leaf spot (CLS), the most important disease of tart cherry in the Midwestern United States. Infection of leaves by B. jaapii leads to premature defoliation, which places trees at heightened risk of winter injury and death. Current management of CLS relies primarily on the application of three important fungicide classes, quinone outside inhibitors, sterol demethylation inhibitors, and succinate dehydrogenase inhibitors. Here, we present the first high-quality genome of B. jaapii through a hybrid assembly of PacBio long reads and Illumina short reads. The assembled draft genome of B. jaapii is 47.4 Mb and consists of 95 contigs with a N50 value of 1.5 Mb. The genomic information of B. jaapii, representing the most complete sequenced genome of the family Dermateaceae (Ascomycota) to date, provides a valuable resource for identifying fungicide resistance mechanisms of this pathogen and expands our knowledge of the phytopathogenic fungi in this family.

Fungal Endophytes of Populus trichocarpa Alter Host Phenotype, Gene Expression, and Rhizobiome Composition.

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa. This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata, but not I. europaea. Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth.

Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean.

Phytophthora root rot of soybean, caused by Phytophthora sojae, is one of the most important diseases in the Midwestern United States, and is estimated to cause losses of up to 1.2 million metric tons per year. Disease may also be caused by P. sansomeana; however, the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate, and sensitive detection of pathogens is crucial for management. Thus, multiplex qPCR and isothermal RPA (recombinase polymerase amplification) assays were developed using a hierarchical approach to detect these Phytophthora spp. The assays consist of a genus-specific probe and two species-specific probes that target the atp9-nad9 region of the mitochondrial genome that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assays run each probe independently with a plant internal control multiplexed in one amplification, obtaining a result in as little as 20 mins. The multicopy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora spp. The assays were highly specific when tested against a panel of 100 Phytophthora taxa and range of Pythium spp. The consistent detection level of the assay was 100 fg for the qPCR assay and 10 pg for the RPA assay. The assays were validated on symptomatic plants collected from Michigan (U.S.) and Ontario (Canada) during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.

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.