Soybean Pathogen Research Articles

Characterization of Soybean WRKY Gene Family and Identification of Soybean WRKY Genes that Promote Resistance to Soybean Cyst Nematode

WRKY proteins are a superfamily of plant transcription factors with important roles in plants. WRKY proteins have been extensively analyzed in plant species including Arabidopsis and rice. Here we report characterization of soybean WRKY gene family and their functional analysis in resistance to soybean cyst nematode (SCN), the most important soybean pathogen. Through search of the soybean genome, we identified 174 genes encoding WRKY proteins that can be classified into seven groups as established in other plants. WRKY variants including a WRKY-related protein unique to legumes have also been identified. Expression analysis reveals both diverse expression patterns in different soybean tissues and preferential expression of specific WRKY groups in certain tissues. Furthermore, a large number of soybean WRKY genes were responsive to salicylic acid. To identify soybean WRKY genes that promote soybean resistance to SCN, we first screened soybean WRKY genes for enhancing SCN resistance when over-expressed in transgenic soybean hairy roots. To confirm the results, we transformed five WRKY genes into a SCN-susceptible soybean cultivar and generated transgenic soybean lines. Transgenic soybean lines overexpressing three WRKY transgenes displayed increased resistance to SCN. Thus, WRKY genes could be explored to develop new soybean cultivars with enhanced resistance to SCN.

A tale of survival: Molecular defense mechanisms of soybean to overcome Soybean mosaic virus infection

Plant viruses represent some of the greatest contributors to crop losses worldwide. Viral disease symptoms often include mosaic leaf patterns, crinkled and yellowed leaves, plant stunting and even necrosis. As intracellular pathogens, viruses hijack host cell machinery for their own replication, which can elicit dramatic changes to their hosts at the cellular, molecular and physiological levels. Soybean mosaic virus (SMV) is a widely distributed soybean pathogen, which can cause severe stunting and yield losses to infected plants. A better understanding of the underlying interactions between SMV and soybean will aid in the establishment of disease management plans. In these studies, we implemented high throughput RNA sequencing approaches and targeted metabolite profiling to describe transcriptomic and metabolic changes occurring in soybean leaves ten days post-infection with SMV. A massive defense response was detected by the increased accumulation of transcripts associated with biotic stresses, including those involved in pathogen recognition, autophagy and defense inductors. Moreover, significant decreases in expression were detected for transcripts associated with fundamental growth and development processes, such as nutrient transport and photosynthesis. At the metabolomic level, significant changes were identified in correspondence with viral infection, particularly in amino acid concentrations. Overall, it appears as though SMV inoculated plants reroute their limited resources as a survival strategy, favoring defense over plant development.

Suppression or Activation of Immune Responses by Predicted Secreted Proteins of the Soybean Rust Pathogen Phakopsora pachyrhizi.

Rust fungi, such as the soybean rust pathogen Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are hyphal structures intimately associated with host-plant cell membranes. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characterization of effector proteins of rust fungi is important for understanding mechanisms that underlie their virulence and pathogenicity. Hundreds of candidate effector proteins have been predicted for rust pathogens, but it is not clear how to prioritize these effector candidates for further characterization. There is a need for high-throughput approaches for screening effector candidates to obtain experimental evidence for effector-like functions, such as the manipulation of host immune systems. We have focused on identifying effector candidates with immune-related functions in the soybean rust fungus P. pachyrhizi. To facilitate the screening of many P. pachyrhizi effector candidates (named PpECs), we used heterologous expression systems, including the bacterial type III secretion system, Agrobacterium infiltration, a plant virus, and a yeast strain, to establish an experimental pipeline for identifying PpECs with immune-related functions and establishing their subcellular localizations. Several PpECs were identified that could suppress or activate immune responses in nonhost Nicotiana benthamiana, N. tabacum, Arabidopsis, tomato, or pepper plants.

Microbial communities in soils with different population densities of soybean cyst nematode

The relationship of abiotic factors with soybean cyst nematode (SCN), the most damaging pathogen of soybean, has been studied extensively, but characterization of microbial communities in infested soils and on soybean roots under field conditions remains unknown. This study investigated the relationship of different SCN population density levels with soil physical, chemical and microbial properties in soils and on soybean roots. A commercial soybean field with different levels of SCN densities (low, medium, high) was sampled, and microbial communities were characterized using both dilution plating assays and molecular approaches. Population densities of species of Trichoderma, actinomycetes, Pseudomonas and other bacteria were significantly higher (P ≤ 0.05) in soils with low SCN (SCN-C) compared with high (SCN-A) and medium levels (SCN-B). Diversity indices of Trichoderma communities on roots based on denaturing gradient gel electrophoresis (DGGE) were significantly higher in soils with SCN-C compared with SCN-A and SCN-B. Cluster analyses and canonical correspondence analysis (CCA) generally separated microbial communities into three groups based on different SCN levels, and CCA showed that low SCN population levels were positively correlated with high levels of nitrogen (N), potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), cation exchange capacity (CEC), base saturation (BS), the populations of Trichoderma and bacteria. Moreover, diverse Trichoderma, Pseudomonas, Bacillus and actinomycetes were found on roots in SCN-C soils based on dilution plating and DGGE, suggesting that the presence of these microbes on soybean roots might be associated with improved plant health and reduction of SCN.

First Report of the Soybean Cyst Nematode, Heterodera glycines, in New York

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the most damaging pathogen of soybean (Glycine max (L.) Merr.), causing more than $1 billion in yield losses annually in the United States (Allen et al. 2017). The SCN distribution map updated in 2014 showed that SCN were detected in all major soybean-producing states in the U.S. except West Virginia and New York (Tylka and Marett 2014). Soybean shows great economic promise in NY and its production area in the region has been expanding rapidly. In coordination with a statewide soybean disease survey, soil samples have been collected from seventeen counties throughout NY since 2013 to search for the presence of SCN. A post-harvest soil sample collected in the fall of 2016 from a soybean field in Cayuga County, NY, was processed using the sugar centrifugal-flotation method to extract nematodes and a few brown and lemon-shaped cysts, similar to those of SCN, were isolated. The lemon-shaped cysts were light to dark brown and had a zigzag patter...

Genome-wide association mapping of resistance to Phytophthora sojae in a soybean [Glycine max (L.) Merr.] germplasm panel from maturity groups IV and V.

Phytophthora sojae, an oomycete pathogen of soybean, causes stem and root rot, resulting in annual economic loss up to $2 billion worldwide. Varieties with P. sojae resistance are environmental friendly to effectively reduce disease damages. In order to improve the resistance of P. sojae and broaden the genetic diversity in Southern soybean cultivars and germplasm in the U.S., we established a P. sojae resistance gene pool that has high genetic diversity, and explored genomic regions underlying the host resistance to P. sojae races 1, 3, 7, 17 and 25. A soybean germplasm panel from maturity groups (MGs) IV and V including 189 accessions originated from 10 countries were used in this study. The panel had a high genetic diversity compared to the 6,749 accessions from MGs IV and V in USDA Soybean Germplasm Collection. Based on disease evaluation dataset of these accessions inoculated with P. sojae races 1, 3, 7, 17 and 25, which are publically available, five accessions in this panel were resistant to all races. Genome-wide association analysis identified a total of 32 significant SNPs, which were clustered in resistance-associated genomic regions, among those, ss715619920 was only 3kb away from the gene Glyma.14g087500, a subtilisin protease. Gene expression analysis showed that the gene was down-regulated more than 4 fold (log2 fold > 2.2) in response to P. sojae infection. The identified molecular markers and genomic regions that are associated with the disease resistance in this gene pool will greatly assist the U.S. Southern soybean breeders in developing elite varieties with broad genetic background and P. sojae resistance.

Development and Evaluation of Glycine max Germplasm Lines with Quantitative Resistance to Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen of soybean that can cause significant yield losses to growers when environmental conditions are favorable for the disease. The development of resistant varieties has proven difficult. However, poor resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. The objective of this project was to develop soybean germplasm lines that have a high level of Sclerotinia stem rot resistance to be used directly as cultivars or in breeding programs as a source of improved Sclerotinia stem rot resistance. Sclerotinia stem rot-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to Heterodera glycines and Cadophora gregata in addition to favorable agronomic traits. Following greenhouse evaluations of 1,076 inbred lines derived from these crosses, 31 lines were evaluated for resistance in field tests during the 2014 field season. Subsequently, 11 Sclerotinia stem rot resistant breeding lines were moved forward for field evaluation in 2015, and seven elite breeding lines were selected and evaluated in the 2016 field season. To better understand resistance mechanisms, a marker analysis was conducted to identify quantitative trait loci linked to resistance. Thirteen markers associated with Sclerotinia stem rot resistance were identified on chromosomes 15, 16, 17, 18, and 19. Our markers confirm previously reported chromosomal regions associated with Sclerotinia stem rot resistance as well as a novel region of chromosome 16. The seven elite germplasm lines were also re-evaluated within a greenhouse setting using a cut petiole technique with multiple S. sclerotiorum isolates to test the durability of physiological resistance of the lines in a controlled environment. This work presents a novel and comprehensive classical breeding method for selecting lines with physiological resistance to Sclerotinia stem rot and a range of agronomic traits. In these studies, we identify four germplasm lines; 91–38, 51–23, SSR51–70, and 52–82B exhibiting a high level of Sclerotinia stem rot resistance combined with desirable agronomic traits, including high protein and oil contents. The germplasm identified in this study will serve as a valuable source of physiological resistance to Sclerotinia stem rot that could be improved through further breeding to generate high-yielding commercial soybean cultivars.

Effect ofBeauveria bassiana(Hypocreales: Clavicipitaceae) and Fungicide Applications onMegacopta cribraria(Hemiptera: Plataspidae) in Soybean

Core IdeaRoutine fungicide applications to soybean did not consistently affect kudzu bug suppression by Beauveria bassiana.Declining populations of kudzu bug,Megacopta cribraria(F.), in the southeastern United States have been observed since 2013 and are generally attributed to increased prevalence of natural enemies.Beauveria bassiana(Bals.‐Criv.) Vuill. infects kudzu bugs in North America, but the level of pest suppression has not been reported.Beauveria bassianais sensitive to classes of fungicides used to manage foliar pathogens in soybean. Here, the degree to whichB. bassianasuppressed kudzu bugs in soybean and changes in pest abundance as a result of routine fungicide applications were assessed in soybean plots located in Alabama and Georgia. In Alabama, seed and foliar treatments ofB. bassianaspores and treatment with fungicides were tested. In Georgia, conservation tillage and fungicide applications were tested for their effect on kudzu bug populations andB. bassianarelated mortality. Kudzu bug suppression due toBeauveriadiffered between sites, with 20 to 75% cadavers to total bugs observed in Alabama and 1 to 6% observed in Georgia. Tillage had no effect on kudzu bug numbers or suppression, though fungicide applications decreased suppression compared with the non‐treated control on one sample date.Beauveria bassianaseed and foliar treatments did not affect kudzu bug suppression. Effects of fungicides on ratios of cadavers to total bugs were variable, though lessBeauveriawas observed in plots receiving a fungicide tank mix compared with non‐treated controls. Fungicides had no consistent effect on kudzu bug populations at either site.

Characterization of dark septate endophyte fungi associated with cultivated soybean at two growth stages

Dark Septate Endophytes (DSE) is a diverse group of Ascomycetes that colonize the roots of a wide range of plants species. They can be found in all ecosystems, from deserts to the tropics, but also in agroecosystems associated to the crops. Despite the ubiquitous of these fungi, only a few major crops were assessed for this association in field conditions. In general terms, a complex consortium of DSE fungi were observed to colonize the roots of some crops. In this work we study the DSE community associated to soybean in the most productive area of this crop in Argentina. We hypothesized that DSE root colonization level, and the identity/frequency of the isolates switch according to the growth stage of the crop. A total of 34 dark fungal colonies were isolated from two growth stages, being only six isolates asyntomatic in the resynthesis assay. Some taxa as Boeremia sp., Cadophora sp., Coniothyrium sp., Corynespora cassicola, Peyronellaea sp. and Phaeosphaeria sp. were exclusively isolated from seedlings, supporting the hypothesis of DSE fungal consortium switching. The soybean pathogens Corynespora cassicola and Macrophomina phaseolina were isolated from young and mature plants respectively. Additional studies should focus on a fine analysis of the dynamics of these fungi, considering also the driving factors that could determine these changes. To understand these mechanisms may be fundamental for a better sustainable management of the crop.

Distinct Mechanisms of Biotic and Chemical Elicitors Enable Additive Elicitation of the Anticancer Phytoalexin Glyceollin I.

Phytoalexins are metabolites biosynthesized in plants in response to pathogen, environmental, and chemical stresses that often have potent bioactivities, rendering them promising for use as therapeutics or scaffolds for pharmaceutical development. Glyceollin I is an isoflavonoid phytoalexin from soybean that exhibits potent anticancer activities and is not economical to synthesize. Here, we tested a range of source tissues from soybean, in addition to chemical and biotic elicitors, to understand how to enhance the bioproduction of glyceollin I. Combining the inorganic chemical silver nitrate (AgNO3) with the wall glucan elicitor (WGE) from the soybean pathogen Phytophthora sojae had an additive effect on the elicitation of soybean seeds, resulting in a yield of up to 745.1 µg gt−1 glyceollin I. The additive elicitation suggested that the biotic and chemical elicitors acted largely by separate mechanisms. WGE caused a major accumulation of phytoalexin gene transcripts, whereas AgNO3 inhibited and enhanced the degradation of glyceollin I and 6″-O-malonyldaidzin, respectively.

Effects of Fertilizer, Nematicide, and Tillage on Plant–Parasitic Nematodes and Yield in Corn and Soybean

Core Ideas Tillage decreased plant–parasitic nematode population densities during portions of the study. Fertilizers did not help manage plant–parasitic nematode population densities. Manure application increased both corn and soybean yields. Soybean response to NPK and NPKS was more positive in a lower yielding environment. Soybean cyst nematode (SCN, Heterodera glycines) is the most damaging pathogen of soybean [Glycine max (L.) Merr.] in the United States and plant–parasitic nematodes such as Pratylenchus (lesion nematode) and Helicotylenchus (spiral nematode) commonly infect corn (Zea mays L.). There are few management options for these nematodes, so understanding how common agronomic practices affect these nematodes is critical. Therefore, a series of 2‐yr experiments were conducted in corn and soybean production. Long‐term conventional tillage (CT) and minimum tillage (MT) treatments were in place at the study site. Fertilizer and granular nematicide—for assessing crop damage by nematodes—treatments were applied: (i) untreated control, (ii) NPK, (iii) NPKS, (iv) liquid swine manure, (v) nematicide without fertilizer, and (vi) NPKS with nematicide. Lesion nematode and SCN densities were greater in MT than CT during portions of the study, but tillage did not affect spiral nematodes. Corn yield was 8% greater in CT than MT in the first phase of the rotation, but tillage did not significantly affect soybean yield. None of the fertilizers—manure, NPK, or NPKS—reduced plant–parasitic nematode densities compared to control. Manure consistently increased corn and soybean yields. The NPK and NPKS were beneficial for corn, but inconsistent for soybean yields. There was some evidence that soybean yield response to fertilizers was greater in more stressed environments, such as with more SCN pressure. Because nematicide efficacy was inconsistent, it was not possible to make a definitive measurement of crop damage by plant–parasitic nematodes in this study.

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.

Ozonation Efficacy in the Treatment of Soil-Borne Phytophthora sojae in Cultivating Soybeans

Ozonation was studied for inactivating Phytophthora sojae, a predominant soybean pathogen that causes root and stem rot, and pre-and post-emergence soybean damping-off. Typically, fungicides are used to treat soils to control the damage from P. sojae to soybean production. An environmentally friendly method of ozonation was studied for inactivating P. sojae, a model Phytophthora pathogen that affects a wide range of high-value crops. Assays of artificially inoculated soil samples with P. sojae were treated with different doses of gaseous ozone. This study showed that a dosage of 0.47 g.kg-1 O3 in the soil totally prevented root and stem-rot disease incidence by P. sojae. The findings of this research clearly indicate that ozonation is an efficient alternative to chemical fungicides in the inhibition of Phytophthora diseases in the soil, hence a balancing feedback loop reinforcing the soil system as natural capital.

In vitro sensitivity assessment of late season soybean pathogens to fungicide mixtures

Late season diseases cause yield reductions to soybean grown worldwide. In Argentina, fungicide mixtures composed of quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs), and the newly introduced succinate dehydrogenase inhibitors (SDHIs), have been effective in managing these diseases. Nevertheless, the risk of selecting strains with resistance to these classes of fungicides is considered to be high. This preliminary study was carried out to determine in vitro sensitivities as determined by the effective concentration that inhibited 50% of radial mycelial growth (EC50 values) of Cercospora kikuchii, Colletotrichum truncatum and Phomopsis phaseoli to selected QoI and DMI fungicide mixtures. The results indicated that EC50 values ranged from 0.0065 to 0.0402 μg/ml for C. kikuchii, from 0.0344 to 0.1744 μg/ml for C. truncatum and from 0.0001 to 0.1974 μg/ml for P. phaseoli. To better study the possible resistance against these pathogens, future tests should consider several isolates for each pathogen from different production areas and different fungicide active ingredients.

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode Heterodera glycines.

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled "Parthenon pillars." In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.

A Phytophthora Effector Manipulates Host Histone Acetylation and Reprograms Defense Gene Expression to Promote Infection

Immune response during pathogen infection requires extensive transcription reprogramming. A fundamental mechanism of transcriptional regulation is histone acetylation. However, how pathogens interfere with this process to promote disease remains largely unknown. Here we demonstrate that the cytoplasmic effector PsAvh23 produced by the soybean pathogen Phytophthora sojae acts as a modulator of histone acetyltransferase (HAT) in plants. PsAvh23 binds to the ADA2 subunit of the HAT complex SAGA and disrupts its assembly by interfering with the association of ADA2 with the catalytic subunit GCN5. As such, PsAvh23 suppresses H3K9 acetylation mediated by the ADA2/GCN5 module and increases plant susceptibility. Expression of PsAvh23 or silencing of GmADA2/GmGCN5 resulted in misregulation of defense-related genes, most likely due to decreased H3K9 acetylation levels at the corresponding loci. This study highlights an effective counter-defense mechanism by which a pathogen effector suppresses the activation of defense genes by interfering with the function of the HAT complex during infection.

Influence of continuous cropping on corn and soybean pathogens

ABSTRACT The objective of this study was to evaluate the influence of two tillage programs (conventional and no-tillage) and different rotations with soybeans and corn on the occurrence of Fusarium species. The work was conducted in the experimental field and Seed Laboratory at Iowa State University. The treatments were: tillage (no-tillage and conventional tillage), crop (corn and soybeans) and three different cropping sequences for corn and soybeans, respectively. Treatment with corn: (1) the first year after four years of soybeans (1C); (2) alternating corn and soybeans each year (C / SB); (3) Continuous corn (C). Treatment with soybeans; (1) the first year after four years of corn (1S); (2) alternating soybeans and corn each year (SB / C); (3) Continuous soybeans (S). Two plant counts were performed in two stages in soybean (VC and V3) and corn (V1 and V3). The root system of ten plants were collected in the vegetative stages V2 and V5 for soybeans and corn. The fungi were isolated from the roots, and the Fusarium species were identified based on the most distinctive morphological characteristics. Nine species were identified in both soybeans and corn, namely F. acuminatum, F. equiseti, F. graminearum, F. oxysporum, F. proliferatum, F. solani, F. subglutinans, F. verticillioides and F. virguliforme. There were no significant differences among the treatments concerning the Fusarium species composition, for both soybeans and corn. In soybeans Fusarium oxysporum was the most frequently observed saprophyte species, followed by F. solani. For corn the main trends in the data was the predominance in all treatments of F. solani, whose highest frequency was in the no-tillage system.

Papers of note in Science355 (6326).

This week's articles identify a requirement for lipid transport between the endoplasmic reticulum and plasma membrane in insulin release, a mechanism in which contact with a host cell reprograms bacterial gene expression, a signal from a soybean pathogen that impairs soybean's defense response, and a mechanism by which hair follicles may be leveraged to reduce scar formation.

Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae.

To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast.

Efficient Genome Editing in the Oomycete Phytophthora sojae Using CRISPR/Cas9.

Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community. Here, we describe a detailed protocol for CRISPR/Cas9-mediated genome editing in P. sojae, including single guide RNA (sgRNA) design and construction, efficient gene replacement, and mutant-screening strategies. This protocol should be generally applicable for most culturable oomycetes. We also describe an optimized transformation method that is useful for other Phytophthora spp. including P. capsici and P. parasitica. © 2017 by John Wiley & Sons, Inc.