Soybean Cyst Nematode Heterodera Glycines Research Articles

N6-Methyladenosine (m6A) Sequencing Reveals Heterodera glycines-Induced Dynamic Methylation Promoting Soybean Defense.

Unraveling the intricacies of soybean cyst nematode (Heterodera glycines) race 4 resistance and susceptibility in soybean breeding lines-11-452 (highly resistant) and Dongsheng1 (DS1, highly susceptible)-was the focal point of this study. Employing cutting-edge N6-methyladenosine (m6A) and RNA sequencing techniques, we delved into the impact of m6A modification on gene expression and plant defense responses. Through the evaluation of nematode development in both resistant and susceptible roots, a pivotal time point (3 days postinoculation) for m6A methylation sequencing was identified. Our sequencing data exhibited robust statistics, successful soybean genome mapping, and prevalent m6A peak distributions, primarily in the 3' untranslated region and stop codon regions. Analysis of differential methylation peaks and differentially expressed genes revealed distinctive patterns between resistant and susceptible genotypes. In the highly resistant line (11-452), key resistance and defense-associated genes displayed increased expression coupled with inhibited methylation, encompassing crucial players such as R genes, receptor kinases, and transcription factors. Conversely, the highly susceptible DS1 line exhibited heightened expression correlated with decreased methylation in genes linked to susceptibility pathways, including Mildew Locus O-like proteins and regulatory elements affecting defense mechanisms. Genome-wide assessments, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and differential methylation peak/differentially expressed gene overlap emphasized the intricate interplay of m6A modifications, alternative splicing, microRNA, and gene regulation in plant defense. Protein-protein interaction networks illuminated defense-pivotal genes, delineating divergent mechanisms in resistant and susceptible responses. This study sheds light on the dynamic correlation between methylation, splicing, and gene expression, providing profound insights into plant responses to nematode infection.

The occurrence of cyst nematodes in agricultural fields of Southern Manitoba

In southern Manitoba, Canada, a survey was carried out in 2012 and 2013 to determine the presence of Heteroderidae cyst-forming plant-parasitic nematodes, with a focus on the soybean cyst nematode (SCN) (Heterodera glycines). A total of 48 fields having grown soybean were sampled. A modified Fenwick elutriation-flotation technique was used to extract cysts with a 75% cyst recovery efficiency. Cyst population density averaged 0.9 cysts kg−1 soil, with a total of 65 cysts recovered. Preliminary screening of cysts, based on general body shape and vulval cone top structure, showed the presence of cysts belong to circumfenestrate, and ambifenestrate groups of cyst-forming nematodes. Limited morphological data was accessible due to poor quality or insufficient cysts for analysis; however, generated DNA sequences for nuclear rDNA ITS and D2-D3 expansion region of the 28S rRNA were obtained for four samples and matched sequences in GenBank for the cyst nematodes Cactodera milleri, C. torreyanae, C. weissi, C. estonica and unknown Cactodera species. Only one of the ambifenestrate cysts with a cone top structure of Heterodera species yielded DNA for analysis and its identification was ambiguous for soybean cyst nematode (SCN). None of the cysts were positive through SCN diagnostic PCR. Cactodera is not a pest of soybean or other crops in Manitoba. These cyst nematodes are likely to be naturally associated with weeds and grasses in the sampled fields or may be introduced from neighbouring states of the USA. Further annual surveys are needed and recommended in the near future to encompass more soybean fields and corroborate the absence of the pest.

Effects of cover crops on hatching of and root penetration by Heterodera glycines

Summary The use of non-host cover crops can be a viable strategy for managing soybean cyst nematode (SCN) Heterodera glycines. Cover crops may affect SCN biology and reduce populations by acting as trap crops that promote the hatching and/or penetration by second-stage juveniles (J2) without supporting their development. Two growth chamber experiments were conducted to assess ten cover crops for their impacts on SCN hatching and penetration into the roots. For each experiment, with four replications per treatment, crops were planted in naturally-infested soil in two separate sets to be harvested 15 and 30 days after planting (DAP). SCN susceptible soybean ‘Barnes’ and non-planted natural soil (fallow) were used as controls. Faba bean ‘Petite’, a non-host of SCN, induced the greatest hatching among the cover crops and was statistically similar to soybean. Winter rye ‘ND Dylan’ also induced significant hatching compared to fallow. Root staining revealed that the highest number of J2 penetrated the faba bean roots at 15 DAP, followed by soybean and winter rye. While J2 penetrated all tested crops, they completed their development to become adult females only in soybean and turnip. Soybean cyst nematode development to adult females did not occur in faba bean, and the number of SCN inside the faba bean roots 30 DAP was significantly lower than at 15 DAP and also lower than in soybean. These results suggest that the faba bean affects SCN biology and has the greatest potential to act as a trap crop for managing SCN.

Evaluation of Soil Suppressiveness of Various Japanese Soils against the Soybean Cyst Nematode Heterodera glycines and Its Relation with the Soil Chemical and Biological Properties

This study aimed to evaluate the suppressive potential of different soils on soybean cyst nematodes (SCN) and to estimate the suppressive mechanism. Fifteen soils (designated as soil A to O) from different agricultural fields with varying organic inputs were added with SCN-infested soil and grown with a green soybean variety. The SCN density in the soil at 6 weeks of soybean growth was markedly different depending on the soils used, indicating a different level of disease suppressiveness. No significant correlation was observed between the SCN density and any of the soil physicochemical and biological characteristics tested. Then, to estimate a suppression mechanism, F-soil that showed the lowest density of SCN was added to the SCN-infested soil with or without streptomycin to kill bacteria and grown with soybean. SCN density was not increased by the addition of streptomycin, indicating that soil bacteria may not be involved in the suppressiveness of F-soil. In total, 128 fungal strains were isolated from the rhizosphere of F-soil and inoculated in a combination or singly in the SCN-infested soil. After repeated screenings, five strains were selected since the SCN density was consistently decreased by them. Sequence analysis showed that they were closest to Clonostachys rosea, Aspergillus niger, Aspergillus fumigatus, Fusarium oxysporum, and Cylindrodendrum alicantinum. All five strains significantly reduced the mobility of second-stage juveniles (J2). Further, C. rosea a2, A. niger a8, and F. oxysporum a25 significantly decreased hatching. Overall, the present study demonstrated that soil fungi played an important role in SCN suppression in F-soil.

Full-Length Transcriptome Analysis of Soybean Cyst Nematode (Heterodera glycines) Reveals an Association of Behaviors in Response to Attractive pH and Salt Solutions with Activation of Transmembrane Receptors, Ion Channels, and Ca2+ Transporters.

Soybean cyst nematode (Heterodera glycines Ichinohe), a devastating pathogen in soybean, was chosen as a model system to investigate nematode behavior and gene expression changes in response to acidic and basic pH and salt signals (pH 4.5, 5.25, 8.6, and 10 and NaCl) through full-length transcriptome sequencing of 18 samples. An average of 4.36 Gbp of clean reads per sample were generated, and 3972 novel genes and 29,529 novel transcripts were identified. Sequence structural variation during or after transcription may be associated with the nematode's behavioral response. The functional analysis of 1817/4962 differentially expressed genes/transcripts showed that signal transduction pathways, including transmembrane receptors, ion channels, and Ca2+ transporters, were activated, but pathways involved in nematode development (e.g., ribosome) and energy production (e.g., oxidative phosphorylation) were inhibited. A corresponding model was established. Our findings suggest that these receptors and ion channels might be potential targets for nematicides or drug discovery.

Chemical Profiles of Heterodera glycines Suppressive Soils in Double Cropping Soybean Production.

We previously reported soybean fields double-cropped with winter wheat having reduced soybean cyst nematode (SCN) (Heterodera glycines) counts compared to fallow. A follow-up metagenomics study identified several fungal and bacterial taxa enriched in wheat fields, and some were reported to parasitize SCN. Knowing that phytocompounds with potential nematicidal activity are released via wheat roots and stubble, we implemented a dichloromethane-based extraction method and a gas chromatography-mass spectrometry (GCMS) system to investigate soil chemical profiles of samples collected from these fields and review the potential nematicidal activity of compounds with higher concentration in double cropping fields. 51 compounds were detected during the GCMS analysis, eight with unknown identification. Several compounds, including multiple fatty acids, had larger relative peak areas when double-cropped, compared to fallow samples. This study, along with our previously published one, provided a better understanding of the mechanisms that govern the effect of wheat on SCN populations. Rather than driven by a single mechanism, the suppression of SCN in soybean fields double-cropped with winter wheat was potentially linked to enriched microbial communities, increased populations of beneficial organisms, and higher concentrations of chemicals with potential nematicidal activity. To our knowledge, this is the first study using GCMS to characterize soil chemical profiles in soybean fields double-cropped with winter wheat regarding the suppression of SCN populations.

Evaluation Soybean Cultivars for Reaction to Heterodera glycines Populations HG Types 7 and 1.3.4.7 in Northeast China.

Soybean cyst nematode Heterodera glycines (SCN) is a major threat to global soybean production. Effective management of this disease is dependent on the development of resistant cultivars. Two SCN HG Types, 7 and 1.3.4.7. were previously identified as prevalent H. glycines populations in Northeast China. In order to evaluate soybean cultivars resistant to local SCN populations, 110 domestic commercial soybeans from different regions of Northeast China were assessed in the greenhouse to determine their potential as novel sources of resistance. The results suggested that cultivars responded differently to the two HG types. Of the 110 soybean cultivars evaluated, 24 accessions were classified as resistant or moderately resistant to HG Type 7, and five cultivars were classified as resistant or moderately resistant to HG Type 1.3.4.7. Among the tested cultivars, Kangxian 12 and Qingdou 13 had resistance response to both HG types 7 and 1.3.4.7. Thus, these broad-based SCN cultivars will be the valuable materials in the SCN resistance breeding program.

Registration of ‘S11‐16653C’ soybean: A high‐yielding conventional cultivar with broad resistance to diseases and nematodes

AbstractThe soybean [Glycine max (L.) Merr] line S11‐16653C (Reg. no. CV‐554, PI 700000) was developed and released as a cultivar by the University of Missouri, Fisher Delta Research, Extension, and Education Center (MU‐FDREEC). S11‐16653C is an early maturity group V (relative maturity 5.3), high‐yielding conventional (non‐genetically modified) cultivar with determinate growth habit. It is a chloride excluder and has a broad disease resistance package, including soybean cyst nematode (Heterodera glycines Ichinohe) races 1 (HG type 2.5.7), 2 (HG type 1.2.5.7), 3 (HG type 5.7), 5 (HG type 2.5.7), southern root‐knot nematode [Meloidogyne incognita (Kofold & White) Chitwood], peanut root‐knot nematode [Meloidogyne arenaria (Neal) Chitwood], reniform nematode (Rotylenchulus reniformis Linford & Oliveira), stem canker (Diaporthe aspalathi Jansen, Castlebury & Crous), Phytophthora root rot (caused by Phytophthora sojae M.J.Kaufmann & J.W.Gerdemann), and sudden death syndrome (Fusarium virguliforme O'Donell & T. Aoki). S11‐16653C was tested in 78 locations across nine states (Alabama, Arkansas, Kansas, Louisiana, Missouri, Mississippi, North Carolina, Tennessee, and Virginia) from 2012 to 2016. It was entered in internal yield tests at the MU‐FDREEC (2012–2014), USDA Uniform Soybean Tests (2014–2016), and soybean state variety tests (2015). It showed consistent high‐yielding performance and broad adaptability throughout the trials and locations. Overall, S11‐16653C is a high‐yielding conventional soybean cultivar that has potential for commercial production and serves as an exceptional germplasm for soybean breeding.

Fluopyram activates systemic resistance in soybean

The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a significant yield-limiting factor in soybean production in the Midwestern US. Several management practices are implemented to mitigate yield losses caused by SCN, including using SDHI (succinate dehydrogenase inhibitors) fungicides delivered as seed treatments. A set of studies was conducted to evaluate the effect of two seed-applied succinate dehydrogenase inhibitors (SDHI) compounds, fluopyram and pydiflumetofen, on SCN population densities, plant injury, and plant growth. Cyst counts in untreated control and pydiflumetofen treated plants were 3.44 and 3.59 times higher than fluopyram, respectively, while egg counts were 8.25 and 7.06 times higher in control and pydiflumetofen. Next-generation sequencing was later employed to identify transcriptomic shifts in gene expression profiles in fluopyram and pydiflumetofen -treated seedlings. RNA expression patterns of seed treatments clustered by sampling time (5 DAP vs. 10 DAP); therefore, downstream analysis was conducted by timepoint. At 5 DAP, 10,870 and 325 differentially expressed genes (DEG) were identified in fluopyram and pydiflumetofen, respectively. These same treatments generated 219 and 2 DEGs at 10 DAP. Multiple DEGs identified in soybean seedlings treated with fluopyram are linked to systemic resistance, suggesting a potential role of systemic resistance in the suppression of SCN by fluopyram, in addition to the known nematicidal activity. The non-target inhibition of soybean succinate dehydrogenase genes by fluopyram may be the origin of the phytotoxicity symptoms observed and potentially the source of the systemic resistance activation reported in the current study. This work helps to elucidate the mechanisms of suppression of SCN by fluopyram.

Responses of infective juveniles of the soybean cyst nematode (Heterodera glycines) and the root-knot nematodes (Meloidogyne hapla, M. incognita) to amino acids

Summary Plant-parasitic nematode infective juveniles (J2) use phytochemical signals released into the rhizosphere to locate host roots. Amino acids are the second most abundant metabolites of root exudates, but it is unknown if they are associated with J2 chemotaxis. In this study, J2 chemotaxis and mortality of the soybean cyst nematode (Heterodera glycines) and root-knot nematodes (Meloidogyne incognita and M. hapla) were examined in response to 15 amino acids and the corresponding pH values for tested amino acid solutions were measured. Responses varied by amino acid and among the species. Significant attraction, determined by J2 count within amino acid solution dispensers after 24 h exposure, occurred with 19 out of 45 J2-amino acid combinations. Heterodera glycines, M. hapla and M. incognita were attracted to nine, three and seven amino acids, respectively. Strongest attractions were to acidic polar amino acids aspartate and glutamate (H. glycines, M. hapla) and basic polar arginine (M. hapla), as previously reported, acid and basic pH attracting nematodes, thereby indicating that pH might be one of the attraction factors for these amino acids. All three nematodes exhibited clustering behaviours, such as halo or balling formations, just outside amino acid solution dispensers, with H. glycines, M. hapla and M. incognita responding to four, 12 and two amino acids, respectively. Six of 15 amino acid solutions, representing a range of pH values, caused increased mortality. Certain aspartate and glutamate affected both H. glycines and M. hapla; arginine, aspartate, cysteine, lysine, methionine affected M. incognita; and cysteine caused complete mortality in M. hapla. All the results suggest that amino acids affect nematode attraction and mortality.

A novel sugar beet cyst nematode effector 2D01 targets the Arabidopsis HAESA receptor-like kinase.

Plant-parasitic cyst nematodes use a stylet to deliver effector proteins produced in oesophageal gland cells into root cells to cause disease in plants. These effectors are deployed to modulate plant defence responses and developmental programmes for the formation of a specialized feeding site called a syncytium. The Hg2D01 effector gene, coding for a novel 185-amino-acid secreted protein, was previously shown to be up-regulated in the dorsal gland of parasitic juveniles of the soybean cyst nematode Heterodera glycines, but its function has remained unknown. Genome analyses revealed that Hg2D01 belongs to a highly diversified effector gene family in the genomes of H. glycines and the sugar beet cyst nematode Heterodera schachtii. For functional studies using the model Arabidopsis thaliana-H. schachtii pathosystem, we cloned the orthologous Hs2D01 sequence from H. schachtii. We demonstrate that Hs2D01 is a cytoplasmic effector that interacts with the intracellular kinase domain of HAESA (HAE), a cell surface-associated leucine-rich repeat (LRR) receptor-like kinase (RLK) involved in signalling the activation of cell wall-remodelling enzymes important for cell separation during abscission and lateral root emergence. Furthermore, we show that AtHAE is expressed in the syncytium and, therefore, could serve as a viable host target for Hs2D01. Infective juveniles effectively penetrated the roots of HAE and HAESA-LIKE2 (HSL2) double mutant plants; however, fewer nematodes developed on the roots, consistent with a role for this receptor family in nematode infection. Taken together, our results suggest that the Hs2D01-AtHAE interaction may play an important role in sugar beet cyst nematode parasitism.

Biocontrol of Three Severe Diseases in Soybean

Three damaging soybean diseases, Sclerotinia stem rot caused by a fungus Sclerotinia sclerotiorum (Lid.) de Bary, Phytophthora root rot caused by a fungus Phytophthora sojae, and soybean cyst nematode (Heterodera glycines Ichinohe), are destructive to soybean growth and yield and cause huge economic losses. Biocontrol is an effective way to control soybean diseases with the advantage of being environmentally friendly and sustainable. To date, few reviews have reported the control of these three soybean diseases through biocontrol measures. In this review, the biological characteristics of the three pathogens and the incidence features of the three soybean diseases were first introduced. Then, biocontrol agents containing fungi and bacteria capable of controlling the three diseases, as well as their control abilities, were emphasized, followed by their mechanisms of biocontrol action. Bacillus and Streptomyces were found to possess the ability to control all three soybean diseases under greenhouse or field conditions. Finally, suggestions about screening new biocontrol species and deeply studied biocontrol molecular mechanisms are provided for further research on the biocontrol of soybean diseases.

The heterologous expression of conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs suppresses Meloidogyne incognita parasitism in Gossypium hirsutum (upland cotton)

Two conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs function in defense to the parasitic soybean cyst nematode Heterodera glycines. Gene Ontology analyses of RNA seq data obtained from MAPK3-1-overexpressing (OE) and MAPK3-2-OE roots compared to their control, as well as MAPK3-1-RNA interference (RNAi) and MAPK3-2-RNAi compared to their control, hierarchically orders the induced and suppressed genes, strengthening the hypothesis that their heterologous expression in Gossypium hirsutum (upland cotton) would impair parasitism by the root knot nematode (RKN) Meloidogyne incognita. MAPK3-1 expression (E) in G. hirsutum suppresses the production of M. incognita root galls, egg masses, and second stage juveniles (J2s) by 80.32%, 82.37%, and 88.21%, respectfully. Unexpectedly, egg number increases by 28.99% but J2s are inviable. MAPK3-2-E effects are identical, statistically. MAPK3-1-E and MAPK3-2-E decreases root mass 1.49-fold and 1.55-fold, respectively, as compared to the pRAP15-ccdB-E control. The reproductive factor (RF) of M. incognita for G. hirsutum roots expressing MAPK3-1-E or MAPK3-2-E decreases 60.39% and 50.46%, respectively, compared to controls. The results are consistent with upstream pathogen activated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI) functioning in defense to H. glycines. The experiments showcase the feasibility of employing MAPK3, through heterologous expression, to combat M. incognita parasitism, possibly overcoming impediments otherwise making G. hirsutum’s defense platform deficient. MAPK homologs are identified in other important crop species for future functional analyses.

A Biochar Improves the Efficacy of Green Manure-Based Strategies to Suppress Soybean Cyst Nematode (Heterodera glycines) and Promotes Free-Living Nematode Populations

A Biochar Improves the Efficacy of Green Manure-Based Strategies to Suppress Soybean Cyst Nematode (Heterodera glycines) and Promotes Free-Living Nematode Populations

Production of Purpureocillium lilacinum and Pochonia chlamydosporia by Submerged Liquid Fermentation and Bioactivity against Tetranychus urticae and Heterodera glycines through Seed Inoculation.

Pochoniachlamydosporia and Purpureocillium lilacinum are fungal bioagents used for the sustainable management of plant parasitic nematodes. However, their production through submerged liquid fermentation and their use in seed treatment have been underexplored. Therefore, our goal was to assess the effect of different liquid media on the growth of 40 isolates of P. lilacinum and two of P. chlamydosporia. The most promising isolates tested were assessed for plant growth promotion and the control of the two-spotted spider mite (Tetranychus urticae) and the soybean cyst nematode (Heterodera glycines). Most isolates produced > 108 blastospores mL−1 and some isolates produced more than 104 microsclerotia mL−1. Microsclerotia of selected isolates were used to inoculate common bean (Phaseolus vulgaris L.) seeds in greenhouse trials. All fungal isolates reduced the T. urticae fecundity in inoculated plants through seed treatment, while P. chlamydosporia ESALQ5406 and P. lilacinum ESALQ2593 decreased cyst nematode population. Purpureocillium lilacinum was more frequently detected in soil, whereas P. chlamydosporia colonized all plant parts. Pochonia chlamydosporia ESALQ5406 improved the root development of bean plants. These findings demonstrate the possibility of producing submerged propagules of P. chlamydosporia and P. lilacinum by liquid culture, and greenhouse trials support the applicability of fungal microsclerotia in seed treatment to control P. vulgaris pests.

AAC McRae soybean

AAC McRae is a high yielding food grade soybean [Glycine max (L.) Merr.] cultivar with yellow hilum, high protein concentration, and acceptable processing quality for foreign and domestic tofu, soymilk, and miso markets. It has excellent soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) and soybean sudden death syndrome (SDS) (Fusarium virguliforme O'Donnell & T. Aoki) resistance. AAC McRae was developed at the Agriculture and Agri-Food Canada (AAFC) Harrow Research and Development Centre (Harrow-RDC), Harrow, Ontario, and is adapted to areas of southwest Ontario with 3100 or more crop heat units and has a relative maturity group of 2.2 (MG 2.2).

Wheat Production Alters Soil Microbial Profiles and Enhances Beneficial Microbes in Double-Cropping Soybean

Plant-parasitic nematodes represent a substantial constraint on global food security by reducing the yield potential of all major crops. The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is widely distributed across important soybean production areas of the U.S., being the major soybean yield-limiting factor, especially in the Midwestern U.S. Double cropped (DC) soybean is commonly planted following winter wheat. We previously reported double-cropping soybean fields with reduced SCN counts compared to fallow at both R1 growth stage (beginning of flowering) (−31.8%) and after soybean harvest (−32.7%). To test if higher counts of beneficial and SCN antagonistic microorganisms could be correlated with the suppression of SCN in fields previously planted with wheat, three field locations with noted SCN suppression were selected for a metagenomics study. Ten subplots were selected (5 wheat and 5 fallow pre-soybean) from each location. A total of 90 soil samples were selected: 3 fields ×2 treatments × 3 timepoints × 5 replications. Three DNA markers targeted distinct microbial groups: bacteria (16S V4-V5), fungi (ITS2), and Fusarium (tef1). Amplicons were sequenced using an Illumina MiSeq platform (300 bp paired-end). Sequencing datasets were processed in R using the DADA2 pipeline. Fungal populations were affected by location in all sampling periods and differed significantly between DC and fallow plots at soybean planting and after harvest (P < 0.001). Several enriched fungal and bacterial taxa in wheat plots, including Mortierella, Exophiala, Conocybe, Rhizobacter spp., and others, were previously reported to parasitize SCN and other plant-parasitic nematodes, suggesting a potential role of beneficial microbes in suppression of SCN in soybean fields double-cropped with wheat.

A fragment of a 70-kDa Heterodera glycines heat shock protein (HgHSP70) interacts with soybean cyst nematode-resistant GmSHMT08

Soybean cyst nematode (SCN) Heterodera glycines is considered as the major constraint to soybean production. GmSHMT08 at Rhg4 locus on chromosome 08, encoding a serine hydroxylmethyltransferase, is a major gene underlying resistance against H. glycines in Peking-type soybeans. However, the molecular mechanism underpinning this resistance is less well characterized, and whether GmSHMT08 could interact with proteins in H. glycines remains unclear. In this study, yeast two-hybrid screening was conducted using GmSHMT08 as a bait protein, and a fragment of a 70-kDa heat shock protein (HgHSP70) was screened from H. glycines that exhibited interaction with GmSHMT08. This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays. Our finding reveals HgHSP70 could be applied as a potential candidate gene for further exploring the mechanism on GmSHMT08-mediated resistance against SCN H. glycines.

Kinase-dead mutation: A novel strategy for improving soybean resistance to soybean cyst nematode Heterodera glycines.

Protein kinases phosphorylate proteins for functional changes and are involved in nearly all cellular processes, thereby regulating almost all aspects of plant growth and development, and responses to biotic and abiotic stresses. We generated two independent co‐expression networks of soybean genes using control and stress response gene expression data and identified 392 differentially highly interconnected kinase hub genes among the two networks. Of these 392 kinases, 90 genes were identified as “syncytium highly connected hubs”, potentially essential for activating kinase signalling pathways in the nematode feeding site. Overexpression of wild‐type coding sequences of five syncytium highly connected kinase hub genes using transgenic soybean hairy roots enhanced plant susceptibility to soybean cyst nematode (SCN; Heterodera glycines) Hg Type 0 (race 3). In contrast, overexpression of kinase‐dead variants of these five syncytium kinase hub genes significantly enhanced soybean resistance to SCN. Additionally, three of the five tested kinase hub genes enhanced soybean resistance to SCN Hg Type 1.2.5.7 (race 2), highlighting the potential of the kinase‐dead approach to generate effective and durable resistance against a wide range of SCN Hg types. Subcellular localization analysis revealed that kinase‐dead mutations do not alter protein cellular localization, confirming the structure–function of the kinase‐inactive variants in producing loss‐of‐function phenotypes causing significant decrease in nematode susceptibility. Because many protein kinases are highly conserved and are involved in plant responses to various biotic and abiotic stresses, our approach of identifying kinase hub genes and their inactivation using kinase‐dead mutation could be translated for biotic and abiotic stress tolerance.

Developing a Real-Time PCR Assay for Direct Identification and Quantification of Soybean Cyst Nematode, Heterodera glycines, in Soil and Its Discrimination from Sugar Beet Cyst Nematode, Heterodera schachtii.

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the Heterodera schachtii sensu stricto group is not only difficult and time-consuming but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes and to quantify SCN directly from DNA extracts of field soils. SCN- and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated on the basis of target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and they may serve as useful diagnostic tools in research, diagnostic laboratories, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.