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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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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.