Resistance mechanism of Phomopsis longicolla to fludioxonil is associated with modifications in PlOS1, PlOS4 and PlOS5.

Phomopsis longicolla, a causal agent of soybean root rot, stem blight, seed decay, pod and stem canker, which seriously affects the yield and quality of soybean production worldwide. The phenylpyrrole fungicide fludioxonil exhibits a broad spectrum and high activity against phytopathogenic fungi. In this study, the baseline sensitivity of 100 P. longicolla isolates collected from the main soybean production areas of China to fludioxonil were determined. The result showed that the EC50 values of all the P. longicolla isolates ranged from 0.013 to 0.035 µg/ml. Furthermore, 12 fludioxonil-resistance (FluR) mutants of P. longicolla were generated from 6 fludioxonil-sensitive (FluS) isolates. and the resistance factors (RF) of 12 FluR mutants were >3500. Sequence alignment showed that multiple mutation types were found in PlOS1, PlOS4 or/and PlOS5 of FluR mutants. All the FluR mutants exhibited fitness penalty in mycelial growth, conidiation, virulence and osmo-adaptation. Under fludioxonil or NaCl treatment condition, the glycerol accumulation was significantly increased in FluS isolates, but was slightly increased in FluR mutants, and the phosphorylation level of most FluR mutants was significantly decreased when compared to the FluS isolates. Additionally, positive cross-resistance was observed between fludioxonil and procymidone but not fludioxonil and pydiflumetofen, pyraclostrobin or fluazinam. This is first reported that the baseline sensitivity of P. longicolla to fludioxonil, as well as the biological and molecular characterizations of P. longicolla FluR mutants to fludioxonil. These results can provide scientific directions for controlling soybean diseases caused by P. longicolla using fludioxonil.

Mining oomycete proteomes for phosphatome leads to the identification of specific expanded phosphatases in oomycetes.

Phosphatases are important regulators of protein phosphorylation and various cellular processes, and they serve as counterparts to kinases. In this study, our comprehensive analysis of oomycete complete proteomes unveiled the presence of approximately 3833 phosphatases, with most species estimated to have between 100 and 300 putative phosphatases. Further investigation of these phosphatases revealed a significant increase in protein serine/threonine phosphatases (PSP) within oomycetes. In particular, we extensively studied the metallo-dependent protein phosphatase (PPM) within the PSP family in the model oomycete Phytophthora sojae. Our results showed notable differences in the expression patterns of PPMs throughout 10 life stages of P. sojae, indicating their vital roles in various stages of oomycete pathogens. Moreover, we identified 29 PPMs in P. sojae, and eight of them possessed accessory domains in addition to phosphate domains. We investigated the biological function of one PPM protein with an extra PH domain (PPM1); this protein exhibited high expression levels in both asexual developmental and infectious stages. Our analysis confirmed that PPM1 is indeed an active protein phosphatase, and its accessory domain does not affect its phosphatase activity. To delve further into its function, we generated knockout mutants of PPM1 and validated its essential roles in mycelial growth, sporangia and oospore production, as well as infectious stages. To the best of our knowledge, this study provides the first comprehensive inventory of phosphatases in oomycetes and identifies an important phosphatase within the expanded serine/threonine phosphatase group in oomycetes.

Fusarium Wilt Invasion Results in a Strong Impact on Strawberry Microbiomes.

Plant-endophytic microbes affect plant growth, development, nutrition, and resistance to pathogens. However, how endophytic microbial communities change in different strawberry plant compartments after Fusarium pathogen infection has remained elusive. In this study, 16S and internal transcribed spacer rRNA amplicon sequencing were used to systematically investigate changes in the bacterial and fungal diversity and composition in the endophytic compartments (roots, stems, and leaves) of healthy strawberries and strawberries with Fusarium wilt, respectively. The analysis of the diversity, structure, and composition of the bacterial and fungal communities revealed a strong effect of pathogen invasion on the endophytic communities. The bacterial and fungal community diversity was lower in the Fusarium-infected endophytic compartments than in the healthy samples. The relative abundance of certain bacterial and fungal genera also changed after Fusarium wilt infection. The relative abundance of the beneficial bacterial genera Bacillus, Bradyrhizobium, Methylophilus, Sphingobium, Lactobacillus, and Streptomyces, as well as fungal genera Acremonium, Penicillium, Talaromyces, and Trichoderma, were higher in the healthy samples than in the Fusarium wilt samples. The relative abundance of Fusarium in the infected samples was significantly higher than that in the healthy samples, consistent with the field observations and culture isolation results for strawberry wilt. Our findings provide a theoretical basis for the isolation, identification, and control of strawberry wilt disease.

The CAP superfamily protein PsCAP1 secreted by Phytophthora triggers immune responses in Nicotiana benthamiana through a leucine-rich repeat receptor-like protein.

The role of cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily proteins in the innate immune responses of mammals is well characterized. However, the biological function of CAP superfamily proteins in plant-microbe interactions is poorly understood. We used proteomics and transcriptome analyses to dissect the apoplastic effectors secreted by the oomycete Phytophthora sojae during early infection of soybean leaves. By transiently expressing these effectors in Nicotiana benthamiana, we identified PsCAP1, a novel type of secreted CAP protein that triggers immune responses in multiple solanaceous plants including N. benthamiana. This secreted CAP protein is conserved among oomycetes, and multiple PsCAP1 homologs can be recognized by N. benthamiana. PsCAP1-triggered immune responses depend on the N-terminal immunogenic fragment (aa 27-151). Pretreatment of N. benthamiana with PsCAP1 or the immunogenic fragment increases plant resistance against Phytophthora. The recognition of PsCAP1 and different homologs requires the leucine-rich repeat receptor-like protein RCAP1, which associates with two central receptor-like kinases BRI1-associated receptor kinase 1 (BAK1) and suppressor of BIR1-1 (SOBIR1) in planta. These findings suggest that the CAP-type apoplastic effectors act as an important player in plant-microbe interactions that can be perceived by plant membrane-localized receptor to activate plant resistance.

Divergent sequences of tetraspanins enable plants to specifically recognize microbe-derived extracellular vesicles.

Extracellular vesicles (EVs) are important for cell-to-cell communication in animals. EVs also play important roles in plant-microbe interactions, but the underlying mechanisms remain elusive. Here, proteomic analyses of EVs from the soybean (Glycine max) root rot pathogen Phytophthora sojae identify the tetraspanin family proteins PsTET1 and PsTET3, which are recognized by Nicotiana benthamiana to trigger plant immune responses. Both proteins are required for the full virulence of P. sojae. The large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and soybean cells in a plant receptor-like kinase NbSERK3a/b dependent manner. TET proteins from oomycete and fungal plant pathogens are recognized by N. benthamiana thus inducing immune responses, whereas plant-derived TET proteins are not due to the sequence divergence of sixteen amino acids at the C-terminal of EC2. This feature allows plants to distinguish self and non-self EVs to trigger active defense responses against pathogenic eukaryotes.

Fusarium-produced vitamin B6 promotes the evasion of soybean resistance by Phytophthora sojae.

Plants can be infected by multiple pathogens concurrently in natural systems. However, pathogen-pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae-soybean-Fusarium combinations, more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen co-inoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1 (Fpp1), encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B6 (VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of disease-resistant crop varieties and provide new strategies to control soybean root rot.

Phase-specific transcriptional patterns of the oomycete pathogen Phytophthora sojae unravel genes essential for asexual development and pathogenic processes.

Oomycetes are filamentous microorganisms easily mistaken as fungi but vastly differ in physiology, biochemistry, and genetics. This commonly-held misconception lead to a reduced effectiveness by using conventional fungicides to control oomycetes, thus it demands the identification of novel functional genes as target for precisely design oomycetes-specific microbicide. The present study initially analyzed the available transcriptome data of the model oomycete pathogen, Phytophthora sojae, and constructed an expression matrix of 10,953 genes across the stages of asexual development and host infection. Hierarchical clustering, specificity, and diversity analyses revealed a more pronounced transcriptional plasticity during the stages of asexual development than that in host infection, which drew our attention by particularly focusing on transcripts in asexual development stage to eventually clustered them into 6 phase-specific expression modules. Three of which respectively possessing a serine/threonine phosphatase (PP2C) expressed during the mycelial and sporangium stages, a histidine kinase (HK) expressed during the zoospore and cyst stages, and a bZIP transcription factor (bZIP32) exclusive to the cyst germination stage were selected for down-stream functional validation. In this way, we demonstrated that PP2C, HK, and bZIP32 play significant roles in P. sojae asexual development and virulence. Thus, these findings provide a foundation for further gene functional annotation in oomycetes and crop disease management.

The SET domain protein PsKMT3 regulates histone H3K36 trimethylation and modulates effector gene expression in the soybean pathogen Phytophthora sojae.

Plant pathogens secrete effector proteins to overcome host immunity and promote colonization. In oomycete plant pathogens, the expression of many effector genes is altered upon infection; however, the regulatory mechanisms are unclear. In this study, we identified a su(var)3-9, enhancer of zeste, and trithorax (SET) domain protein-encoding gene, PsKMT3, that was highly induced at early infection stages in Phytophthora sojae. Deletion of PsKMT3 led to asexual development and pathogenicity defects. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and western blot analyses demonstrated that histone H3K36 trimethylation (H3K36me3) was significantly reduced genome-wide in mutants. RNA-seq analysis identified 374 genes encoding secreted proteins that were differentially expressed in pskmt3 at the mycelium stage. The significantly altered genes encompassed the RxLR (Arg-x-Lys-Arg) effector gene family, including the essential effector genes Avh23, Avh181, Avh240, and Avh241. Transcriptome analysis at early infection stages showed misregulation of effector gene expression waves in pskmt3. H3K36me3 was directly and indirectly associated with RxLR effector gene activation. Our results reveal a role of a SET domain protein in regulating effector gene expression and modulating histone methylation in P. sojae.

Convergent evolution of immune receptors underpins distinct elicitin recognition in closely related Solanaceous plants.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

Functional Characterization of Two Cell Wall Integrity Pathway Components of the MAPK Cascade in Phomopsis longicolla.

The pathogenic fungus Phomopsis longicolla causes numerous plant diseases, such as Phomopsis seed decay, pod and stem blight, and stem canker, which seriously affect the yield and quality of soybean production worldwide. Because of a lack of technology for efficient manipulation of genes for functional genomics, understanding of P. longicolla pathogenesis is limited. Here, we developed an efficient polyethylene glycol-mediated protoplast transformation system in P. longicolla that we used to characterize the functions of two genes involved in the cell wall integrity (CWI) pathway of the mitogen-activated protein kinase (MAPK) cascade, including PlMkk1, which encodes MAPK kinase, and its downstream gene PlSlt2, which encodes MAPK. Both gene knockout mutants ΔPlMkk1 and ΔPlSlt2 displayed a reduced growth rate, fragile aerial hyphae, abnormal polarized growth and pigmentation, defects in sporulation, inadequate CWI, enhanced sensitivity to abiotic stress agents, and significant deficiencies in virulence, although there were some differences in degree. The results suggest that PlMkk1 and PlSlt2 are crucial for a series of growth and development processes as well as pathogenicity. The developed transformation system will be a useful tool for additional gene function research and will aid in the elucidation of the pathogenic mechanisms of P. longicolla. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Phytophthora sojae nuclear effector PsAvh110 targets a host transcriptional complex to modulate plant immunity.

Plants have evolved sophisticated immune networks to restrict pathogen colonization. In response, pathogens deploy numerous virulent effectors to circumvent plant immune responses. However, the molecular mechanisms by which pathogen-derived effectors suppress plant defenses remain elusive. Here, we report that the nucleus-localized RxLR effector PsAvh110 from the pathogen Phytophthora sojae, causing soybean (Glycine max) stem and root rot, modulates the activity of a transcriptional complex to suppress plant immunity. Soybean like-heterochromatin protein 1-2 (GmLHP1-2) and plant homeodomain finger protein 6 (GmPHD6) form a transcriptional complex with transcriptional activity that positively regulates plant immunity against Phytophthora infection. To suppress plant immunity, the nuclear effector PsAvh110 disrupts the assembly of the GmLHP1-2/GmPHD6 complex via specifically binding to GmLHP1-2, thus blocking its transcriptional activity. We further show that PsAvh110 represses the expression of a subset of immune-associated genes, including BRI1-associated receptor kinase 1-3 (GmBAK1-3) and pathogenesis-related protein 1 (GmPR1), via G-rich elements in gene promoters. Importantly, PsAvh110 is a conserved effector in different Phytophthora species, suggesting that the PsAvh110 regulatory mechanism might be widely utilized in the genus to manipulate plant immunity. Thus, our study reveals a regulatory mechanism by which pathogen effectors target a transcriptional complex to reprogram transcription.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of a C2H2 Transcription Factor (PsCZF3) Associated with RxLR Effectors and Carbohydrate-Active Enzymes in Phytophthora sojae Based on WGCNA.

Phytophthora sojae is a destructive soybean pathogen that orchestrates various secreted proteins (effectors) to modulate plant immunity and facilitate infection. Although a number of effectors have been identified and functionally studied in P. sojae, the way these molecules are regulated is marginally known. In this study, we performed a weighted gene correlation network analysis (WGCNA) based on digital RNA-seq, which enabled the identification of a transcription factor (PsCZF3) in P. sojae. This transcription factor is a C2H2-type zinc finger protein that regulates the transcription of 35 RxLR effectors during the early infection stage. Phylogenetic analysis revealed that PsCZF3 is a highly conserved protein across oomycetes, suggesting that this regulation mechanism may broadly exist in oomycete species. In addition, by building a subnetwork of PsCZF3 and correlated genes, we also found that PsCZF3 contributed to the transcriptional regulation of carbohydrate-active enzymes. Our findings suggest that the activation of PsCZF3 facilitates P. sojae infection by up-regulating RxLR effectors and carbohydrate-active enzymes.

Whole-Genome Metalloproteases in the Wheat Sharp Eyespot Pathogen Rhizoctonia cerealis and a Role in Fungal Virulence.

Rhizoctonia cerealis is the causal agent of sharp eyespot, a devastating disease of cereal crops including wheat. Several metalloproteases have been implicated in pathogenic virulence, but little is known about whole-genome metalloproteases in R. cerealis. In this study, a total of 116 metalloproteases-encoding genes were identified and characterized from the R. cerealis Rc207 genome. The gene expression profiles and phylogenetic relationship of 11 MEP36/fungalysin metalloproteases were examined during the fungal infection to wheat, and function of an upregulated secretory MEP36 named RcFL1 was validated. Of 11 MEP36 family metalloproteases, ten, except RcFL5, were predicted to be secreted proteins and nine encoding genes, but not RcFL5 and RcFL2, were expressed during the R. cerealis infection process. Phylogenetic analysis suggested that MEP36 metalloproteases in R. cerealis were closely related to those of Rhizoctonia solani but were remote to those of Bipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum, and Pyricularia oryzae. Expression of RcFL1 was significantly upregulated during the infection process and induced plant cell death in wheat to promote the virulence of the pathogen. The MEP36 domain was necessary for the activities of RcFL1. Furthermore, RcFL1 could repress the expression of wheat genes coding for the chitin elicitor receptor kinase TaCERK1 and chitinases. These results suggest that this MEP36 metalloprotease RcFL1 may function as a virulence factor of R. cerealis through inhibiting host chitin-triggered immunity and chitinases. This study provides insights on pathogenic mechanisms of R. cerealis. RcFL1 likely is an important gene resource for improving resistance of wheat to R. cerealis through host-induced gene silencing strategy.

Transcriptome reveals BCAAs biosynthesis pathway is influenced by lovastatin and can act as a potential control target in Phytophthora sojae.

AIMS: Lovastatin has been indicated to impair growth and development of Phytophthora sojae. Therefore, this study was performed to understand the inhibitory mechanism of lovastatin and investigate the metabolic pathway potentially served as a new control target for this plant pathogen. METHODS AND RESULTS: Whole transcriptome analysis of lovastatin-treated P. sojae was performed by RNA-sequencing. The results revealed that 84 genes were upregulated and 58 were downregulated with more than fourfold changes under treatment. Kyoto Encyclopaedia of Genes and Genomes analysis indicated that the branched-chain amino acids (BCAAs) biosynthesis pathway was abundantly enriched. All enzymes in the BCAAs biosynthesis pathway were identified in the P. sojae genome. Moreover, the study found that the herbicide flumetsulam targeting acetohydroxyacid synthase (AHAS) of the BCAAs biosynthesis pathway could effectively inhibit mycelial growth of P. sojae. CONCLUSIONS: Lovastatin treatment significantly influences the BCAAs biosynthesis pathway in P. sojae. Moreover, the herbicide flumetsulam targets AHAS and inhibits growth of P. sojae. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study revealed that BCAAs biosynthesis pathway was influenced by lovastatin treatment and its key enzyme AHAS was identified as a potential new control target, which provides clues for exploring more oomycetes to control plant diseases caused by P. sojae.

An Oomycete-Specific Leucine-Rich Repeat-Containing Protein Is Involved in Zoospore Flagellum Development in Phytophthora sojae.

A leucine-rich repeat (LRR) is a widespread structural motif of 20 to 30 amino acids with characteristic repetitive sequences rich in leucine. LRR-containing proteins are critical for ligand recognition and binding, participating in plant development and defense. Like plants, oomycetes also harbor genes encoding LRR-containing proteins, but their functions remain largely unknown. We identified a zoospore-upregulated gene from Phytophthora sojae with LRRs and an extra structural maintenance of chromosomes-like domain. We generated knockout and complemented knockout strains of this LRR protein and found that its deletion resulted in a pronounced reduction in zoospore mobility and chemotaxis, cyst germination, and virulence. Interestingly, micro-examination of zoospores under a scanning electron microscope revealed irregularly shaped zoospores without flagella in these deletion mutants. In addition, the reintroduction of this LRR protein into the knockout mutant reversed all the deficiencies. Our data demonstrate a critical role for the Phytophthora LRR protein in modulating zoospore development, which impairs migration to the host soybean and affects the spread of Phytophthora pathogens.

Phytophthora sojae Transformation Based on the CRISPR/Cas9 System.

Phytophthora sojae is a model species for the study of plant pathogenic oomycetes. The initial research on gene function using Phytophthora was mainly based on gene silencing technology. Recently, the CRISPR/Cas9-mediated genome editing technology was successfully established in P. sojae and widely used in oomycetes. In this protocol, we describe the operating procedures for the use of CRISPR/Cas9-based genome editing technology and PEG-mediated stable transformation of P. sojae protoplasts. Two plasmids were co-transformed into P. sojae: pYF515 expressing Cas9 and the single guide RNA, and the homologous replacement vector of the candidate gene. Finally, the ORF of candidate gene were replaced with the ORF of the entire hygromycin B phosphotransferase gene (HPH), to achieve precise knockout.

Diaporthe Diversity and Pathogenicity Revealed from a Broad Survey of Soybean Stem Blight in China.

Many species in the fungal Diaporthe (anamorph Phomopsis) genus have become a group of the most important pathogens that cause seed decay, stem and pot blight, and stem canker in soybean production worldwide, resulting in significant yield loss. Due to increased disease prevalence but a lack of research, we performed an extensive field survey to isolate and identify the Diaporthe species associated with soybean stem blight in six provinces of China between 2019 and 2020. A total of 92 Diaporthe isolates were identified based on morphological and multilocus phylogenetic analysis and classified into six species: D. longicolla, D. unshiuensis, D. sojae, D. caulivora, D. tectonigena, and an unknown Diaporthe sp. The most frequently identified species was D. longicolla with 57 isolates. High genetic diversity was observed for the D. longicolla isolates, and haplotype network analysis revealed a mixed structure among the population in the six provinces. In comparative pathogenicity assays, different virulence levels were observed among the 92 Diaporthe isolates. The results of this study provide new insights into the Diaporthe spp. associated with soybean stem blight in China and can help in the development of effective management strategies.

Wheat Straw Return Influences Soybean Root-Associated Bacterial and Fungal Microbiota in a Wheat-Soybean Rotation System.

Roots hold complex microbial communities at the soil-root interface, which can affect plant nutrition, growth, and health. Although the composition of plant microbiomes has been extensively described for various plant species and environments, little is known about the effect of wheat straw return (WSR) on the soybean root microbiota. We used Illumina-based 16S rRNA and ITS amplicon sequencing to track changes in bacterial and fungal microbiota in bulk soil and soybean rhizosphere, rhizoplane, s1and endosphere during the third and fourth years after implementing WSR in a wheat-soybean rotation system. The results revealed that WSR had a greater impact on fungal communities than bacterial communities, particularly in bulk soil, rhizosphere, and rhizoplane. WSR enriched the relative abundance of cellulose-degrading fungi (e.g., Acremonium, Trichoderma, and Myrmecridium, among which Trichoderma also had antimicrobial activity), saprotroph (e.g., Exophiala), and nitrogen cycling bacteria (e.g., Chryseolinea). Furthermore, WSR depleted the relative abundance of pathogenic fungi (e.g., Fusarium and Alternaria). These data revealed for the first time that WSR had diverse effects on soybean root-associated microbial community composition, not only in soil but also in the rhizosphere, rhizoplane, and endosphere.