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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.

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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.

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

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

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Sudden death syndrome (SDS), caused by members of Fusarium solani species complex (FSSC) clade 2, is a major and economically important disease in soybean worldwide. The primary causal agent of SDS isolated to date in North America has been F. virguliforme. In 2014 and 2016, SDS symptoms were found in two soybean fields located on the same farm in Michigan. Seventy Fusarium strains were isolated from roots of the SDS-symptomatic soybeans in two fields. Phylogenetic analysis of partial sequences of elongation factor-1α, the nuclear ribosomal DNA intergenic spacer region, and the RNA polymerase II beta subunit revealed that the primary FSSC species isolated was F. brasiliense (58 and 36% in each field) and the remaining Fusarium strains were identified as F. cuneirostrum, F. phaseoli, an undescribed Fusarium sp. from FSSC clade 2, and strains in FSSC clade 5 and FSSC clade 11. Molecular identification was supported with morphological analysis and a pathogenicity assay. The soybean seedling pathogenicity assay indicated that F. brasiliense was capable of causing typical foliar SDS symptoms. Both root rot and foliar disease severity were variable by strain, just as they are in F. virguliforme. Both FSSC 5 and FSSC 11 strains were also capable of causing root rot, but SDS foliar symptoms were not detected. To our knowledge, this is the first report of F. brasiliense causing SDS in soybean in the United States and the first report of F. cuneirostrum, F. phaseoli, an as-yet-unnamed Fusarium sp., and strains in FSSC clade 5 and FSSC clade 11 associated with or causing root rot of soybean in Michigan.

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