One of the major milestones in SCN biology is developing a completely annotated SCN reference genome, which we have recently accomplished for the TN10 SCN isolate (Masonbrink et al., 2019). Since then, we have made considerable progress and now have a chromosome level genome assembly ready and annotated. Simultaneously, we have developed a centralized, web-based repository called SCNBase (SCNBase.org). We have developed this web portal de novo and published it in November 2019 (https://doi.org/10.1093/database/baz111). This web portal now is the home to all bioinformatic, genetic, genomic, and molecular data generated for SCN, most of it through soybean check-off funding. Using this web portal, researchers and breeders from all over the world can access and analyze all public bioinformatic data generated in this proposal and curated from previous research at the nucleotide level. We are already seeing significant “web traffic” to this web portal suggesting that it is generating considerable interest in the SCN community from all over the world.

Our group is actively involved in developing, analyzing and comparing gene expression in virulent (i.e., able to infect resistant soybean cultivars) and avirulent (i.e., unable to infect resistant soybean cultivars) SCN populations with the goal to identify the genetic determinants of virulence. For that purpose, we are focusing on three individual gland cells, in which the nematode produces the effectors/tools required for infection and defense suppression, using a method we previously had developed. Toward that end, we have completed three independent biological replications of mRNA purification, library construction and sequencing (which is essentially an identification of all genes active in the gland cells at a given time) from each of the virulent (MM10) and avirulent (PA3) SCN populations. Furthermore, gene identities from all sequencing experiments have been generated utilizing the SCN TN10 genome produced by us (Masonbrink et al., 2019). We identified 13,617 unique transcripts from the PA3 population and 8,820 unique transcripts from the MM10 population, which represent a snapshot of gene activity during the early parasitic stage of SCN infection (parasitic J2). As a point of validation, we have identified all previously discovered SCN effectors expressed during this early parasitic stage within our gland cell-specific sequence data. Initial analysis of these sequence data has revealed intriguing gene expression differences between these virulent and avirulent SCN populations. Specifically, there are 32 genes upregulated in the SCN PA3 libraries versus the MM10 libraries. Protein products of 13 of these genes are predicted to be nuclear localized and likely have gene regulatory functions. This includes a diverse group of proteins involved in functions such as structural maintenance of chromosomes, splicing factors, a glycosyltransferase involved in increased virulence in other parasitic nematodes, DNA-binding proteins, and a ‘ran’-binding protein, among others. Conversely, we discovered 17 genes that are upregulated in SCN MM10 versus PA3. Protein products of eight of these genes are predicted to be nuclear localized. Among this group are previously reported effectors, a microtubule binding protein homolog from an animal-parasitic nematode, signal transduction proteins, and several unknown proteins. We are still at the initial stages of analysis but it is already clear that we have generated a rich source of critical data needed to understand SCN virulence. The MM26 parental population was prepared and sent for genome sequencing; the others will follow. RNA-seq data generated from early parasitic life stages of a virulent and avirulent population of SCN were used to conduct a reference-based transcriptomic analysis with the aid of the SCN genome. We identified 207 genes unique to the virulent SCN that could be turned on to overcome resistance mechanisms; conversely, 92 genes that could be turned off to evade triggering resistance mechanisms.

Being a sedentary endoparasite that relies exclusively on its host for survival, SCN has to suppress host defense responses for a significant duration in order to survive. The SCN achieves this by producing a large number of effector molecules and delivering them into the soybean cells via its mouth spear. These effectors specifically target host factors and modulate their functions, thereby also altering soybean defense responses. Generating an in-depth understanding of how individual effectors help SCN establish and maintain infection is a very difficult but necessary task that will reveal vulnerable “nodes” in host defense pathways that can be strengthened via either breeding or molecular approaches. As a part of this project, we are conducting an in-depth molecular characterization of SCN effectors specifically involved in host defense suppression. We are specifically focused on the effector named 28B03. We have identified that this particular effector is a robust host defense suppressor. We have observed that due to its function, plants become more susceptible to cyst nematodes. We have also identified that this effector specifically targets a previously uncharacterized plant protein kinase. By physically interacting with this novel protein kinase, the 28B03 effector suppresses phosphorylation of its substrate protein and completely alters the associated signal transduction pathway. This discovery and an in-depth molecular characterization of a novel defense response related kinase cascade in plants are breakthrough discoveries of this study. We are currently gearing up to conduct another high-impact protein interaction experiment in order to identify the complete ‘interactome’ of the proteins associated with this kinase cascade. As candidate SCN virulence genes are identified, gene function will be confirmed through biochemical and/or genetic analysis to better understand the mechanisms of virulence and to also evaluate these gene targets as vulnerable points of disruption in the SCN life cycle as a means to enhance resistance in soybean. We are focusing our efforts on validating the genes identified in 2.3 above to prioritize candidate virulence genes for further molecular functional studies.

Microplot field experiments were established in Illinois, Missouri, and Iowa during 2019 to evaluate how rotations of SCN resistance gene combinations affects SCN field population densities and virulence profiles. Treatments with resistance genes: rhg1-a, rhg1-b, Rhg4, cqSCN-006/cqSCN-007, and the chromosome 10 QTL, along with susceptible and resistant checks were designed to form 12 treatments with 3 replications in a randomized complete block design. The virulent SCN HG type 1.2.5.7 was selected for microplot inoculation at locations. At the end of the growing season, soil samples from all plots were obtained to determine egg density and SCN HG type at harvest. All resistance genotypes had a reduction in egg densities and the susceptible treatment had an significant increase in SCN egg density, while PI 90763 had the greatest percentage of egg count reduction followed by genotypes with resistance genes, rhg1-a + Rhg4. SCN HG type results at the end of the first growing season show no specific trend. These results are close to what we expected and show that the different sources influenced SCN reproduction in the field. During 2020, the second year of the rotation study will be grown and soil samples will be taken from each plot during the fall. The samples will be analyzed for SCN number and HG type to study the impact of rotations on these characteristics.

In 2020-2021, project co-PIs will continue to deliver farmer-friendly information about the ongoing research of the project through interviews requested by media and through interviews and other communications pieces developed by the SCN Coalition. Tylka and Mitchum were to organize, host, and lead in-person educational events for a group of select soybean farmers and national agriculture media near Ames, Iowa, on August 31st, 2020, and in Athens, Georgia, on January 26th, 2021. The research in our NCSRP-funded project was to be highlighted at both events. Many print stories and radio interviews were to emanate from the events as a means of informing growers of the research in the project. In addition, there was to be a National Soybean Nematode Conference held in Savannah, Georgia (immediately after the farmer-media event in Athens, Georgia) on January 28th, 2021. Results of the research in our NCSRP-funded project were to be highlighted at the conference as well. The COVID-19 pandemic and resultant restrictions on travel and large in-person events necessitated a change in these plans for the 3rd year of our project. The SCN Coalition and its communications firm redirected efforts in the spring of 2020 to making the planned educational events described above virtual by recording and distributing videos capturing much of the information that was to be delivered in the in-person events. Specifically, Tylka, Kaitlyn Bissonnette from the University of Missouri, and Sam Markell from North Dakota State University were interviewed while walking through demonstration plots by a video crew at an Iowa State University extension education farm on July 21, 2020. The video crew returned to that farm on August 31, 2020 to document via video Tylka plus in NCSRP executive director Ed Anderson and an Iowa farmer discussing additional aspects of SCN and its management. Plans currently are being made for the video crew and SCN Coalition communications staff to visit Georgia in early 2021 to collect similar video to represent virtually the information that Mitchum had originally planned to convey and demonstrate in her laboratory at the University of Georgia. All of this virtual educational content will be posted online at the SCN Coalition website, www.TheSCNCoalition.com, and the SCN Coalition YouTube channel, https://www.youtube.com/channel/UCaYpNqBx53-5MVmyYGHK7HQ. Some of the content already is available on the YouTube channel.

The 2020 tests have been organized and they include 194 entries from maturity group 00 to IV and will be grown in 32 environments in 10 states and one Canadian province. The seed for the tests has been shipped to collaborators and the tests are now being planted. During the growing season, soil samples will be taken from field sites and the samples will be evaluated for egg number and HG type of the SCN population at each location. The cooperators will maintain the plots, takes notes on agronomic traits and will harvest the plots to estimate yield. The data from the tests will be sent to a central location, the results will be analyzed and a summary will be reported to cooperators and other interested parties.

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Past research efforts to determine the inheritance of SCN virulence (i.e., the ability of the nematode to reproduce on resistant varieties) led to the identification of the reproduction on resistant varieties, or ror genes, which were shown to be inherited in both dominant and recessive manners. However, since the initial discovery of these genes there has been no further information published concerning their sequence identity or mechanism in conferring SCN virulence. Genome and transcriptome comparisons of SCN populations that differ in virulence on resistant soybean have the potential to identify genes underlying virulence, determine the mechanism/s of virulence, and lead to the development of molecular diagnostic tools to assess for virulence in field populations.

1.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum)
1.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum)
1.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)
1.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

Objective 2. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.
As described above, experimental lines with resistance gene combinations developed in Objective 1 during Phase I of this project were tested in four different rotation schemes with experimental lines containing various resistance gene combinations in a greenhouse study. SCN population increase was measured after each of 8 generations. Following the eighth generation of selection, the HG type of each population was determined. From this, we identified alternative resistance gene combinations that when used in rotation reduce the selection pressure on the SCN population thereby slowing nematode adaptation to resistant varieties.

2.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.
This project and the researchers involved in it produce much of the newest information being generated about the biology and management of SCN in the United States. As primary financial supporters of the work, it is critical that soybean farmers understand the rationale for the research and be made aware of the activities of the project and the new information being discovered. It also is important that individuals involved in soybean breeding in seed companies be made aware of the research results so that they are ready to adopt the new genetic resources for incorporation into commercially available soybean varieties. In addition to working with traditional university extension, a very efficient means to convey the results of this research to the farming and soybean breeding communities is through communication with the agriculture media. Project co-PIs will work to engage agriculture media directly and through the SCN Coalition.

3.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.
During 2019, the testing of SCN resistant experimental lines developed by breeders in 11 north central US states and Ontario was coordinated. This test is important to help breeders develop high yielding SCN resistant varieties. The tests included SCN resistant experimental lines and varieties that were grown in maturity group specific trials across 39 locations. These lines were also tested for SCN resistance and the soil samples from the environments were be evaluated for SCN population density and HG type. An initial report of the results from the 2019 test was sent to collaborators on December 19th, 2019 and the final report was delivered on January 6th, 2020.

4.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.

1.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum)
1.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum)
1.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)
1.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

Objective 2. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.

2.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.

3.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.

4.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

Updated April 19, 2021:
Objective 1.1:

The Mitchum group has continued to help develop and provide nematode materials for genome sequencing efforts.

The Hudson group has the multi-genome assembly project close to completion, with manually curated assemblies completed or under way for all seven of the target genotypes as well as the original TN10. For MM26, PA3 and OP50, we have finalized, frozen assemblies ready for annotation. For TN8, curated annotation is complete and a final step to remove duplicate haplotigs is under way. For TN7, TN20 and TN22, final steps of manual curation of the assemblies are in progress. The quality of each assembly is better than the previously published TN10 assembly and close to the quality of the current, manually curated TN10 assembly. Overall, we are very close to eight full high-quality assemblies of SCN lines corresponding to all of the major HG types.

For this reporting period the Baum and Severin groups would like to announce a new chromosome-level assembly of the TN10 H. glycines genome, with assembly and annotation statistics that are much improved over the previous version. We believe that this latest genome contains the highest confidence SCN gene models publicly available, since the gene models in this version were created from a consensus of nine separate annotations. The manuscript describing these latest results has been submitted and is currently under review. (Preprint DOI: 10.22541/au.161538368.83631935/v1). As part of this release, a large number of additional resources have become available on SCNBase to complement this genome assembly. We surveyed SNP and INDEL variations from 15 distinct H. glycines populations and have created SNP/INDEL-modified versions of the TN10 genome to represent pseudo-genomes from these 15 populations. With these new pseudo-genomes, we used the TN10 genes to identify genomic variation that affects gene structure and coding potential. These pseudo-proteomes were subjected to bioinformatic screens for secretion and can potentially lead to a greater understanding of how variation in signal peptide presence can vary across H. glycines populations, and perhaps how they could confer virulence. Simultaneously, we are also working on collecting and analyzing data to distinguish gene expression patterns between male and female H. glycines populations, which may provide new targets for resistance development. Additionally, differences in the male vs. female populations can be looked at as differences in a non-feeding (male) vs. feeding (female) population. Our most current analysis of this data has revealed multiple genes that are differentially expressed in males vs females.

Objective 1.2:

In Phase I of this project (under Obj 2.3 of the previous project) Mitchum identified a HG type 1.2.5.7 field population and continuously selected this population in the greenhouse on either a susceptible soybean line (SCN inbred population MM-BD1), a soybean line containing the rhg1 resistance gene from PI 88788 (SCN inbred population MM-BD2), a soybean line containing the rhg1 and Rhg4 resistance genes from PI 437654 (Hartwig) (SCN inbred population MM-BD3), and a soybean line containing resistance genes on chromosomes 15 and 18 from wild soybean G. soja (inbred population MM-BD4) for more than 12 generations. The original SCN field population and this series of SCN populations selected for virulence on each set of resistance genes has been continuously reared during this project period. A manuscript describing these populations was submitted and accepted to the scientific journal Plant Disease. https://doi.org/10.1094/PDIS-12-20-2556-RE

Objective 1.3:

The Mitchum group is focused on identifying SCN virulence genes used by the nematode to overcome the Peking-type (Rhg4-mediated) resistance. Our comparative transcriptomic analysis comprises (1) differential expression and (2) variant call analysis by utilizing RNA-seq data generated from the early parasitic stages of virulent and avirulent SCN populations adapted on soybean recombinant inbred lines (RILs) that only differ at the Rhg4 locus (i.e., a resistant RIL with a resistant Rhg4 allele and a susceptible RIL containing a susceptible Rhg4). With the aid of the newly annotated pseudomolecule SCN genome assembly, we have finalized the differential expression analysis which has allowed us to generate a list of potentially important differentially expressed genes (DEGs) containing genes that may be specific to Rhg4-mediated resistance as well as genes that may be important for virulent nematodes growing on resistant soybeans regardless of the type of resistance. This final list of DEGs was further prioritized for virulence effectors through cross-comparison with the nematode gland-specific RNA data sets (i.e., in silico subtraction for gland-specific genes) from the Baum group. Consequently, these DEGs will be subject to a high-throughput screening which will commence shortly. From our variant call analysis to discover single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) that may contribute to virulence, we previously found that the virulent SCN harbors 72,232 SNPs and 4,564 INDELs while the avirulent SCN contains 73,026 SNPs and 4,513 INDELs, relative to the reference genome with statistical significance. Since the last reporting period we have been able to filter out the majority of the SNPs and INDELs: 5,506 SNPs and 287 INDELs were unique to the avirulent nematode and 5,854 SNPs and 280 INDELs were exclusive to the virulent nematode, while those in common from both were 9,093 SNPs and 203 INDELs. We believe that SNPs and INDELs unique to the virulent nematode that are present at a relatively high or low frequency may be of special interest. Moreover, those that are common in both nematodes, but are significantly higher or lower in the virulent SCN relative to the avirulent SCN may be interesting as well. For this reporting period, we are now collaborating with the Severin group to predict the effect of these SNPs and INDELs on amino acid changes (e.g., synonymous vs. nonsynonymous mutations). Clearly, SNPs and INDELs that result in nonsynonymous mutations are more likely to contribute to virulence. As a complementary approach to our transcriptomic analysis, we are conducting Pool-seq which pools individual nematodes from the same population to increase the amount of DNA and accurately estimate the population allele frequencies. This sequencing strategy has successfully been utilized by potato cyst nematode (PCN) researchers to map to a region containing several candidate virulence genes from experimentally adapted PCN populations. Similarly, Pool-seq applied to our unique SCN populations will allow us to pinpoint candidate regions important for SCN virulence. In preparation for this strategy, we have been harvesting nematodes from these populations and optimizing protocols for DNA extraction and sequencing.

The Hudson’s group preliminary analysis of the genomes has already produced interesting results, with effector genes aligning to the same strand of the same chromosome across multiple lines (synteny). This result is important for understanding and tracking the evolution of virulence in SCN. Included among these effectors are Hgg23, G19B10, GLAND15, GLAND16 and GLAND17, glutathione peroxidase, G11A06, and GLAND5, these effectors aligned to the same chromosomes across all the different strain assemblies. A few effectors such as Hgg-25, G33E05 25A01, GLAND7, and G4G05, and GLAND18 had more variation in results including alignments to multiple chromosomes. These results need to be confirmed using the final, higher quality versions of the assemblies.

For this reporting period, the Baum and Severin groups continued to explore the RNA-seq dataset derived from a comparison of SCN gland cell transcriptomics of a virulent (MM10) and avirulent (PA3) population. We have developed this data analysis into a Resource Announcement paper, which we have submitted to the “Molecular Plant-Microbe Interactions”(MPMI) journal where it is currently under review. Briefly, with this submission, we have announced the availability of a unique gland-specific RNA-seq dataset for the SCN community, which provides an expression snapshot of gland cell activity during early infection of a virulent and avirulent SCN population. This represents a highly valuable resource for researchers examining effector biology and nematode virulence. Within the Resource Announcement, we have outlined a few initial intriguing gene expression differences between the two populations. Across all replications of these gland cell RNA-seq libraries, there are 96 mRNAs (which correlate to 115 genes) upregulated in the PA3 libraries versus the MM10 libraries. Conversely, there are 41 mRNAs (which correlate to 38 genes) that are upregulated in MM10 vs PA3. We are currently pursuing these differences and examining the data for the identification of novel effector candidates.

Objective 1.4:

As candidate SCN virulence genes are identified by the Mitchum group, gene function studies are being conducted using biochemical and/or genetic analysis to not only better understand the mechanisms of virulence, but to also evaluate these gene targets as vulnerable points of disruption in the SCN life cycle as a means to enhance resistance in soybean. The Mitchum group continued our characterization of novel stylet-secreted effectors of the soybean cyst nematode Heterodera glycines parasitome, 16B09 and 2D01. 16B09 and 2D01 belong to the same superfamily of effectors, highly expanded in the genome, share the same gene structure, harbor conserved protein domains, and exhibit the same spatial and temporal expression in the dorsal gland cell during parasitism. Host-induced gene silencing (RNAi) of 16B09 demonstrated a requirement of this effector protein for successful parasitism. Protein interaction studies identified a specific interaction of 2D01 with a plant plasma membrane-associated protein kinase. We further demonstrate that this protein kinase is expressed in feeding sites and plants unable to produce this kinase showed increased resistance to nematodes. The identified protein kinase is involved in the signaling pathway which is extremely important for both abscission and lateral root emergence and this process activates a number of cell wall modifying enzymes that are important for these processes. Both lateral root emergence and abscission require cell wall expansion and dissolution. These processes would be advantageous for the nematode as we know one of the key characteristics for syncytium formation is cell wall dissolution as it allows fusion of surrounding cells to form feeding site. We have now confirmed the subcellular localization of Hs2D01dSP in the cytoplasm of the plant cell and the protein kinase to the plasma membrane and demonstrated that that these two proteins interact in plants. A comparative genomics analysis of this effector family across populations of SCN differing in virulence on resistant soybean is currently under investigation.

The Baum group has been characterizing the robust defense suppression and comprehensive re-engineering of the feeding site, which are two of the hallmarks of the successful cyst nematode infection. As a part of this project, we are actively involved in conducting in-depth molecular characterization of the 28B03 effector family. Our analysis has shown that members of this effector family are robust defense suppressors. Our work has also revealed that a member of this family achieves its defense suppressive ability by interfering with a previously uncharacterized kinase cascade in plants. We continue to characterize the interactome associated with the kinases from this cascade as it will reveal signal transduction pathways that this particular effector modulates. To identify such an interactome, we are establishing a “proximity labeling assay system” in our laboratory, which is the latest and the most advanced technique to identify protein interactors in planta. In short, we have developed multiple fusion constructs in which we have fused an unmodified kinase, a dead version of this kinase as well as a truncated version of this kinase to a highly active derivative of the biotin ligase enzyme. As a control, we have fused the GUS marker gene to this biotin ligase enzyme in the same orientation. All these fusion constructs are expressed stably in Arabidopsis using the native promoter of the kinase in question. We have confirmed the expression of the GUS marker gene in the transgenic Arabidopsis lines showing that our fusion constructs are functional. Currently, we are in the process of assessing the activity of the biotin ligase enzyme in our transgenic plants. We will begin protein interaction work shortly after such confirmation. Proteins in the near vicinity of the active biotin ligase will be biotinylated and then will be purified using streptavidin beads. Biotinylated proteins will then be identified using mass-spectrometry, which will identify the complete ‘interactome’ of this particular kinase. We believe that establishing and characterizing such a system will prove pivotal for all our in planta protein interaction studies involving other effectors.

Objective 2.1:

At the conclusion of the 2020 growing season, two separate multi-core soil samples were collected from each microplot in the experiments conducted in central Iowa and in north central Iowa by the Tylka group. One set of soil samples from each experiment were processed at Iowa State University to determine the end-of-season population density of SCN in each microplot. The second set of soil samples from each experiment were sent to the University of Missouri for HG Type testing to determine if and how the soybean genotypes grown in the microplots in 2020 might have shifted the virulence phenotypes (HG types) of the SCN populations originally added to the microplots in the spring of 2019. Preliminary data analysis was completed for each of the two field experiments in Iowa, and some trends in SCN population density were observed. In both experiments, the highest SCN population densities occurred in microplots in which the susceptible soybean variety was grown. The microplots that had continuous cropping of the same resistance in 2019 and 2020 had higher SCN population densities than the microplots that had different resistant varieties grown in them in 2019 and 2020. The lowest population densities were found in microplots where soybeans with SCN resistance from PI90763 were grown. Also, the microplots in which soybeans with PI90763 resistance were grown also were lower than those that previously had soybeans with rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10 SCN resistance grown in 2019 and then were rotated to soybeans with PI90763 SCN resistance in 2020. The results of the HG Type tests on the SCN populations in the soil samples collected from the microplots at harvest in 2019 revealed that almost all of the SCN populations in the soil at both experimental locations Iowa had an HG Type of 1.2 with a range of female indices of 20-69% on PI88788 and 3-36% on Peking. The plots in which soybeans with rhg1-a + rhg4 and PI90763 SCN resistance were grown had the highest female indices on Peking and PI90763, but the SCN population in every plot had a female index greater than 10% on PI88788. No elevated reproduction on PI437654 was detected. HG Type test results of the SCN populations in soil samples collected at harvest in 2020 are not yet available. Preparation and planning for the 3rd growing season is currently underway.

In IL, the Diers group is preparing and distributing seed for all collaborators for the 2021 SCN resistance source rotation study. This will be the third year of the rotation study and we will be rotating the plots back to what was grown in them during 2019. We recently received the HG type results from the fall 2019 plots as the results had been delayed until now because of Covid. Although the results have not been fully analyzed, a few observations can be reported. One is that the nematode population in all plots could overcome PI 88788 resistance and the female index (FI) on this source ranged from 10.1% to 96%. The fact that the nematodes could overcome PI 88788 resistance was expected because this was the HG type of the nematodes used to inoculate the plots. There were six plots that had nematodes that could overcome Peking resistance and the two plots with the highest FI on Peking (37-23%) were planted to a line with Peking type resistance.

In Missouri, fall 2020 soil samples were taken for SCN egg counts and HG type tests and were processed. The egg count data has shown that the susceptible plots have the highest egg densities followed by the rhg1-b plots. The PI 90763, rhg1-a + Rhg4, and rhg1-b+G.soja+ch.10 treatments had the lowest population densities and the highest reduction in SCN egg densities compared to initial levels. In continuous treatments, the highest reduction in egg counts was observed in PI 90763 and rhg1-a + Rhg4. Similarly, the highest reduction in percentage change in the egg counts is in those treatments rotated with PI 90763. SCN HG type data has shown that in continuous PI 90763 treatment, an increase in virulence on Peking and PI 88788 has been observed. The seeds obtained for the third-year field season experiment have been checked, packaged and an entry list has been prepared.

Objective 3.1:

The Mitchum lab helped to produce The SCN Coalition’s “Let’s Talk Todes” Research Collection video series now released online https://www.thescncoalition.com/lets-talk-todes/research-collection. In this video series, soybean growers and scientists (nematologists, breeders, plant geneticists, extension pathologists) who are battling SCN explain the checkoff-funded research they are conducting that's focused on bringing new tools to soybean growers in the fight against parasitic nematodes.

Tylka conducted 18 radio and newspaper/magazine interviews from October 2020 through March 2021. The loss of effectiveness of PI88788 SCN resistance was discussed and this current NCSRP-funded research project was mentioned and described whenever time/space permitted. In the fall of 2020 The SCN Coalition began posting short videos about SCN biology, management, and research on the project website www.TheSCNCoalition.com. The first seven videos of the SCN Coalition’s “Let’s Talk Todes” video collection were made available online at www.thescncoalition.com/lets-talk-todes in October 2020 and received >930,000 views from October to November 2020. Our current NCSRP-funded SCN research project is described by Tylka in one of these videos (see https://youtu.be/4PpvvavwwHc).

Objective 4.1:

The results from the 2020 SCN Regional Test were received from cooperators and summarized in a report by the Diers group. The initial version of the report was sent to cooperators on December 10th and the final version was delivered on January 12th. These timely deliveries of results are important so cooperators can make decisions on selections in time for winter crosses and nurseries. Plans have been made for the 2021 SCN Regional Test. This test will include 242 entries that range from MG 0 to IV. The tests have been organized, the seed has been received from cooperators and will be shipped to cooperators soon. Arrangements also have been made to test the lines for SCN resistance in a greenhouse at the University of Illinois.

View uploaded report

Updated November 14, 2021:
A description of relevant progress for principle and co-principle investigators is below for each objective and sub objective in our proposal. Our team has made tremendous progress in accomplishing our research goals, conducting field experiments, publishing refereed journal articles, and communicating our results to scientists and soybean producers even during the challenges faced due to the current COVID-19 pandemic.

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.

1.1: Sequence, curate and annotate SCN reference genomes for each common HG type (Severin, Hudson, Baum, Mitchum)

The Baum and Severin groups have already reported previous H. glycines genome-related publications (SCN reference genome, Masonbrink et al., 2019; SCN chromosomal assembly, Masonbrink et al., 2021 and SCNBase, Masonbrink et al., 2019) that resulted from collaborative work. For this reporting period, we further assessed the gene family expansions and male/female transcriptomic/genomic data, and released an update to SCNBase. Software updates of SCNBase have increased user access to genomics though improvements to the Features Database. The user now has direct access to dozens of different gene characteristics from the published SCN genomes, which now simultaneously displays a genome browser for the gene of interest, https://db.scnbase.org/feature/?feature_id=2275432. Previously we surveyed genomic variation from 15 H. glycines populations and modified versions of the TN10 genome to represent pseudo-genomes from these 15 populations. We've incorporated this information into a larger work that assesses the extent of gene family duplication and contraction that has occurred in SCN and related species at three phylogenetic nodes. Overall, the comparisons include sedentary vs mobile parasites, root-knot vs cyst nematodes, and Globodera species vs SCN. We identified 551 gene family expansions in sedentary nematodes vs mobile outgroups, 124 gene expansions in cyst nematodes vs root-knot nematodes, and 479 gene family expansions/contractions in SCN vs Globodera species. This work is currently being written up for submission and will result in the release of 323 columns of data describing every gene in SCN, across 15 SCN populations, and gene families among 13 related parasitic nematodes. We are continuing to assess gene expression patterns and genomic structure between male and female H. glycines genomes and transcriptomes, which may provide new targets for resistance development. We previously assessed gene expression differences in a non-feeding (male) vs. feeding (female) population, revealing 6,039 upregulated genes in males and 5,881 upregulated genes in females. To complement this approach, we obtained long sequencing reads from male and female nematodes, and have completed preliminary sex-specific gene predictions. Though further work is needed, this data will display the genomic and transcriptomic differences between male and female nematodes, further enhancing our understanding of SCN sex determination and SCN genes involved in manipulating the host.

The Mitchum group has continued to help develop, provide, and utilize nematode materials for genome sequencing, RNA-seq, and Pool-seq project objectives.

1.2: Generate sufficient genetic material of virulent SCN populations selected on different types of resistance (Mitchum, Baum)

The Mitchum group has finished harvesting sufficient genetic material of the above-mentioned four SCN populations and optimized their DNA extraction procedure to meet the stringent requirements necessary for the Pool-Seq strategy. For this reporting period, we have completed the sequencing run and its bioinformatic analysis is currently underway. We are confident that our Pool-Seq approach will not only help guide us toward the candidate virulence regions important for breaking the Peking-type (Rhg4-mediated) resistance, but also confirm the candidate genes identified from our transcriptomic analysis (outlined below).

1.3: Resequence the genomes and transcriptomes of virulent SCN populations described in 2.2 and conduct comparative analyses (Severin, Hudson, Mitchum, Baum)

Upon the completion and analysis of our Resource Announcement of comparative transcriptomics of virulent (MM10) and avirulent (PA3) SCN populations during early infection, the Baum and Severin group realized the need for an additional time point to strengthen this analysis. Therefore, we are currently working to generate a later stage infection comparison of these two populations. This will to enable us to monitor gene expression changes across a larger portion of the critical feeding site formation and maintenance steps in the parasitic life cycle of SCN. For this work, we are using a new approach to gland cell collection, which we feel will improve throughput and allow us to decrease the time it takes to generate this labor-intensive collection step. Initial indications are promising and we are looking towards generating RNA-seq libraries, using newly developed kits designed for single-cell RNA-seq analysis, from these samples shortly.

The Mitchum group has been conducting the qRT-PCR validation of differentially expressed genes (identified from the first part of our transcriptomic analysis), including several from the 14 genes of special interest, and were able to confirm upregulation in the virulent population. Simultaneously, we have also made progress on the variant call analysis (the second part of our transcriptomic studies) by further filtering out the majority of SNPs and INDELs and only keeping those with higher (and the highest) variant allele frequencies, which may be present in potentially important virulence genes. This selection has resulted in 682 SNPs and 28 INDELs unique to the avirulent nematode, and 690 SNPs and 19 INDELs exclusive to the virulent counterpart. Interestingly, and as expected, some of these SNP- or INDEL-harboring genes were those also identified from the differential expression analysis, a strong indicative that these genes are most likely to be involved in virulence for overcoming the Peking-type (Rhg4-mediated) resistance.

1.4: Validate and characterize genes associated with SCN virulence and evaluate their utility as novel resistance targets (Mitchum, Baum)

The Baum group has worked on the characterization of the function of 28B03 effector family, which has now been completed, and the results are being finalized for publication submission. This manuscript is a very lengthy and complete functional assessment of this effector and its ability to interfere with a plant signal transduction pathway. In short, 28B03 targets a novel plant kinase that in turn cooperatively with another plant kinase leads to the initiation of signal transduction processes that initiate a subset of defense responses. We have shown that the 28B03 effector interferes with this signal transduction, thus, compromising plant defenses and leading to increased host susceptibility. This work pinpoints a potential plant target to increase plant resistance. I.e., one can now devise mechanisms to interfere with the inhibitory function of 28B03 to prevent the nematode from inactivating plant defenses. In addition to this direct characterization of the 28B03 function, we also started work to understand the full extent of the signal transduction cascade that is targeted by this effector. I.e., what other plant responses are governed by this cascade. For this purpose we are conducting protein-protein interaction assays using a novel proximity labelling approach, as we have outlined in the previous progress reports. Progress towards these proximity labeling assays for 28B03 and the targeted kinases continues in the Baum lab. We have generated homozygous lines for several of the required constructs and are ready for the next steps. Through our confocal experiments utilizing co-localization studies of 28B03 and the identified kinases, we can infer that our proposed cascade model is valid, as the effector and kinases are co-localized together in these assays.

During this reporting period the Mitchum group has focused our efforts on finalizing a manuscript describing the results of the functional characterization and genome analysis of this effector family. We also continued additional functional work on new candidates identified from the gland-enriched RNA-seq analysis described under 1.3 above.

Objective 2. Determine what combinations of resistance genes would be beneficial in variety rotations to enhance the durability of SCN resistance in soybean.

2.1 Evaluate how rotations of various resistance gene combinations impact SCN field population densities and virulence profiles (Diers, Scaboo, Tylka, Mitchum)

The 2021 growing season was year 3 of the microplot experiments for the project in Iowa led by the Tylka group. The experiments (including two in Iowa) were planted with the 3rd year treatment scheme, in which six of the 12 treatments were resistance genotypes that remained constant or continuous over the three years, and the other six treatments were genotypes that were rotated back to the genotypes that were grown in year 1. Throughout the 2021 growing season, weeds were controlled via hand weeding and mowing, and insecticides were applied to control Japanese beetles. By the end of September 2021, soybean plants were beginning senescence in Iowa, but were not fully mature and ready for harvest at the time of this report. Once the plants are fully mature, two separate multi-core soil samples will be collected from each microplot. One set of soil samples will be used to determine final SCN population densities and the other set will be used to assess virulence of the SCN populations through HG type testing. The HG type data from fall 2020 were received from Missouri in June of 2021 and compared to the HG type data previously received from fall 2019. Statistical analysis has not yet been conducted on changes in SCN population density or virulence over the 2019 and 2020 growing seasons.

In Missouri, the Scaboo group managed the microplots which were harvested using a mechanical thresher on 18th October 2021 and the weight of the seeds from each microplot were taken. Fall 2021 soil samples for SCN egg counts and HG type test were collected on 21st October 2021. For the HG type test, the increase pots were set up using four Williams-82 seedlings for the soil collected from each microplot. The HG type test for the samples from the 3rd year will be conducted on 22nd November 2021. The soil samples for determining egg density have been set up for drying and the samples will be processed during November of 2021.

In Illinois, the Diers group planted the third year of the microplots in Urbana, Illinois and maintained these plots through the growing season. The plots are maturing now and will be harvested to provide seed for next year. Soil cores will also be taken from the plots soon and shipped to the nematology lab at the University of Missouri to determine both egg numbers and HG types of samples from each plot. We recently received the HG type analysis results from most of the plots from the 2020 tests. Although a full analysis will not be done until all data are received, we generally found increases in female index values for differentials that have the same resistance genes as the genotypes grown in the sampled plots. For example, large increases in FI values on PI 90763 were observed in plots grown continuously with this genotype. Smaller increases were observed on plots that only had PI 90763 one year.

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.

3.1: Inform growers on effective rotation schemes designed to protect our resistant sources (Tylka, Mitchum)

Tylka gave 2 presentations and conducted 8 radio and newspaper/magazine interviews in the last 3 months. The loss of effectiveness of PI 88788 SCN resistance often was discussed and this NCSRP-funded research project was mentioned and described.

Mitchum conducted an interview with Successful Farming Radio highlighting the research under this project directed at developing new modes of genetic resistance and how we are using the developed SCN populations to better understand how the nematode acquires the ability to reproduce on resistant varieties

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.

4.1: Organize tests of experimental lines developed by public breeders in the north central US states and Ontario (Diers)

The 2021 SCN Regional Test was grown by cooperators and most of the harvest has been completed. The 2021 test includes 242 entries that range from MG 0 to IV. Data sheets have been sent to the cooperators and they will add their results onto these sheets and return the results by early December and we plan to provide a preliminary test report to cooperators by the end of that month.

View uploaded report

Updated August 17, 2022:
Sub-objective 1.1:
The Baum group has accomplished an analysis of gene expansions and contractions, as well as the presence and absence of gene families in H. glycines compared to 13 related species in the Tylenchomorpha. We are functionally characterizing these gene families and their association with previously published H. glycines effectors, as well as their secretory status, expression, nuclear localization, and impact of their variability across 15 populations of H. glycines. We have shown that 551 gene expansions in sedentary nematodes differentiate them from migratory nematodes, 124 gene expansions in cyst nematodes differentiate them from root-knot nematodes (Meloidogyne), and 1,100 gene expansions in H. glycines differentiates it from Globodera cyst nematode species. We have also shown a number of gene gain and loss events that have directly impacted the H. glycines genome, many of which have shown an atypical inheritance pattern across related species. In one instance, we show 175 gene families that are only shared between H. glycines and Meloidogyne species, 15 of which are targeted to the secretory pathway, including two predicted H. glycines effectors. Using effectors from relatively closely related species, we have identified 405 putative effector families present in H. glycines, though only 137 of these families contain a gene that produces a protein with a signal peptide for secretion. Of these 137 putative effectors, 11 were associated with gene expansions in sedentary nematodes compared to migratory nematodes, 6 were associated with the expansions of gene families in cyst nematodes versus root-knot nematodes, and 24 were associated with the expansion of Heterodera gene families in comparison to Globodera. Using population variation from 15 distinct populations of H. glycines, we were able to show which genes typically produce secreted proteins across the assayed SCN lines. In this respect, we show that 59 gene expansion families have consistent signals for secretion across multiple populations, even though they were not predicted to be secreted in the previously published TN10 genome. These results warrant further research into gene evolution. We also have developed plans to update SCNBase with new RNAseq data from the recently published J2 gland RNAseq and will integrate gene family criteria. To complement the methods of identifying genes involved in the parasitism of H. glycines, we have undertaken a genome assembly and annotation project of male and female specimens. Males and females dramatically change their parasitic behavior with the onset of adulthood and, thus, studying their genomes and transcriptomes will reveal insights into gene functions, particularly for effectors. We sequenced male and female genomes using nanopore long reads and added male and female specific RNA-seq to this analysis to better predict the gene variation among the sexes. Since the last report we have scaffolded these male and female genomes with HiC to obtain nine pseudomolecules for each genome. Interestingly, we found large differences in genome size between these nanopore genomes (male: 115.5Mb, female: 112.6Mb) and our previously published TN10 genome (158Mb), which is likely attributable to the differences in sequencing technology and assembly software. Subsequently, we used male and female RNAseq with Braker to annotate genes in each genome, finding that the disparity continues between these nanopore genomes and TN10 genome at the genic level. In the male and female gene annotations, we found 16,421 genes and 16,530 genes, respectively. These gene annotation totals are substantially reduced from our previous annotation of the TN10 genome at 22,465 genes, though the gene copy number reduction is in proportion with the genome size reduction. We have begun to compare the genomes at the level of gene structure and expression using Orthofinder and differential expression analyses. Thus far we have found that 9-10,000 of differentially expressed genes cluster to the same orthologue family, leaving slightly more than half of the genes with significant divergence between the sexes. Continuation of gene expansion and contraction as well sex-specific genomic differences will further refine these early findings.

The Mitchum group submitted the following manuscript during the past quarter - Verma A, Lin M, Smith D, Lee C, Walker JC, Hewezi T, Davis EL, Hussey RS, Baum TJ, Mitchum MG. A novel cyst nematode effector (2D01) targets the Arabidopsis HAESA receptor-like kinase. Mol. Plant-Microbe Interact.

Sub-objective 1.2:
Phase II of this project saw the Baum group developing further resources to expand the toolbox that will aid us in understanding SCN virulence. Focusing once again on the three gland cells that SCN uses to produce the tools (effectors) required for establishment of successful infection, as well as defense suppression, we have improved on the technology. Taking advantage of recent developments toward single cell sequencing, and specifically new technology available for the generation of single cell RNA-seq libraries, we have applied these technologies to our work with gland cell isolation and transcriptomics in SCN. We successfully generated single cell RNA-seq libraries for our avirulent (PA3) and virulent (MM10) SCN populations at different time points. These libraries represented four biological replications of 5 dorsal and subventral gland cells per rep. This was our group’s first attempt at applying single cell sequencing technologies to SCN transcriptomics and required a fair amount of optimization. Through this process, we developed a technique for live gland cell collection, versus fixed tissue collection, which improved the quality of RNA collected and allowed us to collect picogram quantities of RNA from individual gland cells. The overall coverage of genes identified within these new J3 single cell libraries, with reference to the SCN TN10 genome is slightly greater than the coverage of genes identified with our previous pooled gland cell parasitic J2 libraries. We identified a total of 14,667 and 14,000 genes at a normalized read count of at least 5 counts from the SCN PA3 J3 and MM10 J3 libraries, respectively. This is compared to a total of 12,495 and 12,289 genes at the same read count cutoff for the respective SCN populations with our older technology. While we cannot rule out that this could be due to life stage differences, it certainly points to the fact that this new technology can yield similar, if not better numbers of identified genes from lesser amounts of material. We are currently working on the transcriptomics to generate both broad and specific comparisons of both the life stages and virulence differences that exist in our two SCN reference populations. This will provide an extensive and novel look at SCN virulence at this level.

All seven of the genomes of the additional Hg types are now assembled, and the assemblies are completed and frozen. They are ready to distribute or submit to NCBI, however we need to annotate and analyze them before publishing a paper on the genomes, and to make them useful to more people in the group.

The Hudson group has been proceeding with analysis in several ways. Firstly, genome synteny for the 7 newly assembled SCN strains was compared with mummer (dotplots) and circos (BLAST hits) to the chromosome level SCN assembly TN10 (Masonbrink et al 2021). These data showed that the assembled 9 chromosomes were in similar structure with the previously reported TN10, although not identical. We started the genome functional and structural annotation. Repetitive elements modeled with RepeatModeler and quantified repetitive content with RepeatMasker. The repeat analysis showed repeat content was similar across strains with respect to repeat class and quantity. Subsequently, we began gene prediction using RNAseq evidence (previously reported Gardner et al 2018 & Lian et al 2019) and protein models (from previously reported TN10 Masonbrink et al 2021) and other cyst nematodes including Globodera pallida (Cotton et al 2014) and Globodera rostochiensis (Eves-van den Akker 2016). Preliminary gene models show similar structure to the previous assemblies’ gene models. Our future work will include refinement of gene models and subsequent functional annotation of the gene models.

The Mitchum group has concluded the RNA-seq analysis for the purpose of identifying soybean cyst nematode genes potentially responsible for overcoming the Peking-type resistance. This led us to identify 14 genes of special interest categorized into three subgroups: (1) putative effectors involved in defense suppression, (2) putative enzymes related to reactive oxygen species, and (3) putative vitamin B-associated genes. Additionally, the Mitchum lab collected sufficient genetic material of two pairs of SCN populations (unadapted or adapted to reproduce on resistant soybeans) and optimized their DNA extraction procedure to meet the stringent requirements necessary for the Pool-Seq strategy. The Pool-Seq approach should help guide us toward the candidate virulence regions in the SCN genome important for breaking the Peking-type (Rhg4-mediated) resistance. We have completed the Pool sequencing and bioinformatic analysis is currently underway. We expect that some of the identified from the RNAseq analysis may also appear in the Pool-seq analysis providing a stronger support for their potential importance in virulence. For the most promising genes, we are currently testing correlation to virulence in other SCN populations with known HG types, prior to embarking on functional characterization.

Sub-objective 1.3:
The Baum group has indicated that characterization of the function of 28B03 effector family has been advanced to a natural stopping point to publish the first extensive report on this effector’s function during parasitism. A complete manuscript has been written and all data compiled. We are waiting on last additions and reviews from co-authors and then will submit the manuscript to start the publication process. Given the thorough study of this effector, this manuscript is a very extensive and thorough functional assessment of this effector and its ability to interfere with a plant signal transduction pathway. In short, 28B03 targets a novel plant kinase protein, which in turn cooperatively with another plant kinase leads to the initiation of signal transduction processes that initiate a subset of plant defense responses. We have shown that the 28B03 effector interferes with this signal transduction, thus, compromising plant defenses and leading to increased host susceptibility. Through our confocal experiments utilizing co-localization studies of 28B03 and the identified kinases, we can infer that our proposed cascade model is valid, as the effector and kinases are co-localized together in these assays. This work identifies a potential plant target to increase plant resistance (i.e., one can now devise mechanisms to interfere with the inhibitory function of 28B03 to prevent the nematode from inactivating a plant defense signal transduction kinase).

The Mitchum group has characterized the function of the 2D01 effector, which was found to represent a novel, highly diversified effector gene family in the soybean cyst nematode genome that may function in SCN virulence. A potential plant protein target of this nematode effector protein was identified, which is a well-known component of a signaling pathway regulating the expression of cell wall modifying genes important for various aspects of plant development. We determined that the plant target protein is expressed in the developing nematode feeding sites where cell wall modification is critical for their establishment and the parasitic success of the nematode. Our findings indicate the nematode may be using this effector as a means to co-opt this host signaling pathway to promote parasitism. Thus, this work has identified a nematode effector-host protein interaction for which targeted disruption has the potential to enhance plant resistance. The following manuscript describing this work has been submitted for publication- Verma A, Lin M, Smith D, Walker JC, Hewezi T, Davis EL, Hussey RS, Baum TJ, Mitchum MG. A novel cyst nematode effector (2D01) targets the HAESA receptor-like kinase.

Objective 2:
The Diers group is preparing and distributing seed for all collaborators for the 2022 SCN resistance source rotation study. This will be the fourth year of the rotation study and we will be rotating the plots back to what was grown in them during 2020. We now have egg numbers from the plots grown in 2019-2021 and HG type values from 2019-2020. These results show that continuous planting of PI 90763 had the lowest egg number increases in plots across the three years. However, the continuous production of this source of resistance is selecting nematodes that can overcome this resistance and the female index (FI) in plots grown with PI 90763 was 41 after 2020, which indicates that the nematode population in these plots may start increasing. The rotation that had the lowest increase in egg numbers over the three years is rhg1b+soja+ch10 in 2019 followed by PI 90763 in 2020 and then rhg1b+soja+ch10 in 2021. This rotation also showed a low increase in egg numbers in other states and did not increase the FI on PI 88788 or Peking. The study will be repeated in 2022 to provide further insight into these resistance sources.

The Scaboo group processed soil samples for determining egg density and HG types for each microplot. The egg density results showed that there is an increasing trend in SCN population density in the third year of rotation except for plots with continuous PI 90763 and rhg1-b+G. soja+10 rotated with PI 90763. There was also a significant reduction in the percentage change in the egg count for these two treatments. The HG type data also showed that the continuous rhg1-a + rhg4 and continuous PI 90763 treatments facilitated the development of more virulent nematode populations with HG type (HG type: 1.2.3-; race 4), where nematode had adapted on the Peking type resistance sources (PI 90763, Peking and Pickett). Similarly, nematode populations from treatments involving rotations of rhg1-b (and/or stacked resistances) with PI 90763 showed to have reduced female index on PI 88788, PI 90763, and Peking indicator lines. We look forward to the upcoming planting season where we plan on rotating an additional cycle of continuous and rotated schemes as conducted previously in 2020.

The Mitchum group was sent SCN material recovered from the continuous and rotation microplots and they were increased, processed for eggs, and archived as part of a wormplasm collection for future sequencing efforts to pinpoint virulence genes.

At the conclusion of the 2021 growing season, The Tylka group collected two separate multi-core soil samples from each microplot in the experiments conducted in central Iowa and north central Iowa. One set of soil samples from each experiment were processed at Iowa State University to determine the end-of-season SCN egg population density in each microplot. The second set of soil samples were sent to the University of Missouri for HG Type testing to determine how the soybean genotypes grown in the microplots in 2021 have affected or shifted the virulence profiles (HG types) of the SCN populations from the 2020 growing season and from the initial SCN populations that were added to the microplots in the spring of 2019.

Preliminary data analysis show some trends in changes SCN population densities. In both experiments, the greatest SCN population densities occurred in microplots in which the susceptible soybean variety was grown. Most of the microplots that had continuous cropping of the same resistance in 2019, 2020, and 2021 had greater SCN population densities than microplots in which resistant genotypes were rotated in 2019, 2020, and 2021. The lowest population densities occurred in microplots where soybeans with SCN resistance from PI 90763 were grown. The microplots in which soybeans with rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10 SCN resistance were grown (collectively referred to as genotypes having “PI88788-type” resistance) in 2019, rotated to soybeans with PI 90763 SCN resistance in 2020, and then rotated back to the same resistance as in 2019 had increased SCN population densities in 2021 compared to population densities at the end of both previous years. Even though SCN population densities declined after rotating from genotypes with PI88788-type resistance in 2019 to rhg1-a + rhg4 (or “Peking-type”) resistance in 2020, population densities increased to levels greater than in 2019 and 2020 when genotypes with PI88788-type resistance were again grown in the microplots in 2021. Similarly, plots in which rhg1-a + rhg4 or Peking-type resistance was grown in 2019 then rotated to rhg1-b, rhg1-b + soja, or rhg1-b + soja + ch10 SCN (PI88788-type resistance) resistance in 2020, and then back to rhg1-a + rhg4 resistance in 2021 had greater SCN population densities at the end of the 2021 growing season than in the previous two years.

Objective 3:
Greg Tylka conducted 19 interviews with radio and newspaper/magazine journalists and gave 12 presentations (in person and virtual) from October 2021 through March 2022. The loss of effectiveness of PI88788 SCN resistance was discussed in every interview and presentation, and this current NCSRP-funded research project was mentioned and described whenever time/space permitted.

Melissa Mitchum is serving as the Chair of the organizing committee for the 2022 National Soybean Nematode Conference (NSNC). The location, venue, and draft schedule was developed. Save the date flyers were distributed and the development of the scientific program was initiated.

Objective 4:
The Diers group has continued to organize these tests. During the period of the grant we managed the Northern Regional Soybean Cyst Nematode Tests, which it is a cooperative test of publicly developed experimental lines with SCN resistance. The lines were developed by university breeding programs and these breeders grew locations of the test. Our role was to organize the tests; distribute seed to the breeders who grew the tests; obtain, organize, and analyze the data from the test locations; and assemble and distribute reports based on these test results. The results from these tests are important for breeders to help them decide what experimental lines to release as new varieties. During 2020 these tests included 184 lines that were evaluated in 27 locations, in 2021 there were 242 lines evaluated in 30 locations, and in 2022 there are 225 lines being evaluated in 29 locations.

Objective 5:
The Diers and Scaboo groups have continued to advance breeding efforts towards the development of cultivars with novel SCN resistance. For this reporting period, we are excited to report that we have now completed successful crossing attempts (3 backcrosses) using PI 90763 as a donor parent, and LD11-2170 and SA13-1385 as recurrent parents, for three major genes associated with resistance to virulent nematode populations (rhg1-a, rhg2, and Rhg4). For each crossing attempt, we have identified desirable F1 plants using marker assisted selection, and we have sped up the process by utilizing our winter nurseries in Hawaii and Puerto Rico for the last two years. As I type this report, our staff and student are in Kekaha Kauai Hawaii tissue sampling BC3F2 plants to identify homozygous individuals with desired combinations of our target genes. During the summer of 2022, we will grow plant rows derived from selected plants, and our first yield trials of this material will be in the summer of 2023.

View uploaded report

Unraveling the genes and their activities in different populations and sexes of the soybean cyst nematode, the number 1 pathogen of soybean production, has provided the research community with unprecedented information needed to understand this pathogen’s biology.

In particular, these advancements now put the research community in a position to understand how some populations of the soybean cyst nematode cannot be controlled by resistant soybean cultivars while other populations are completely controlled and rendered ineffective. In other words, we are getting closer to answering the question how some nematodes are more virulent than others. Answering this and other related questions will advance the fight against this pathogen on many levels. For one, understanding the genetic bases of nematode virulence will allow scientists to devise molecular nematode tests to assess which soybean cultivar should be chosen to manage a nematode population found in a given agricultural field. Furthermore, understanding the basic mechanisms of soybean cyst nematode infection biology and virulence will empower scientists to specifically tailer novel control mechanisms against the nematode. For example, this current NCSRP project has revealed one mechanism how a nematode infection strategy impacts the plant host to make it more susceptible. Such discoveries now allow scientists to counteract this nematode action to make plants more resistant. Due to the findings of this NCSRP project there now are numerous novel molecular targets that can be exploited for the engineering of soybean plants that are more resistant to this pathogen.

The Mitchum group has concluded a comparative RNA-sequencing analysis for the purpose of identifying soybean cyst nematode genes potentially responsible for overcoming soybean resistance mediated by the Rhg1 and Rhg4 resistance genes derived from Peking. This led us to identify 14 genes of special interest categorized into three subgroups: (1) putative effectors involved in defense suppression, (2) putative enzymes related to reactive oxygen species, and (3) putative vitamin B-associated genes. Additionally, the Mitchum lab collected sufficient genetic material of two pairs of SCN populations (unadapted or adapted to reproduce on resistant soybean) and optimized their DNA extraction procedure to meet the stringent requirements necessary for the Pool-Sequencing strategy. The Pool-Sequencing approach will guide us toward the candidate virulence regions in the SCN genome. The Pool-Sequencing was completed and bioinformatic analysis is currently underway. It is expected that some of the identified genes from the RNA-sequencing analysis may also appear in the Pool-sequencing analysis providing a stronger support for their potential importance in virulence. For the most promising genes, correlation testing to virulence in other SCN populations with known HG types is underway, prior to embarking on functional characterization. In addition, the function of the 2D01 effector gene, which was found to represent a novel, highly diversified effector gene family in the soybean cyst nematode genome and implicated in SCN virulence, was studied in more detail. A potential plant protein target of this nematode effector protein was identified, which is a well-known component of a signaling pathway regulating the expression of cell wall modifying genes important for various aspects of plant development. We determined that the plant target protein is expressed in nematode feeding sites in roots where cell wall modification is a critical aspect of feeding site establishment, and ultimately, parasitic success of the nematode. Our findings indicate the nematode may be using this effector as a means to co-opt this host signaling pathway to promote parasitism. Thus, this work has identified a nematode effector-host protein interaction for which targeted disruption has the potential to enhance plant resistance. The following manuscript describing this work has been submitted for publication- Verma A, Lin M, Smith D, Walker JC, Hewezi T, Davis EL, Hussey RS, Baum TJ, Mitchum MG. A novel cyst nematode effector (2D01) targets the HAESA receptor-like kinase.

The Diers, Scaboo, and Tylka groups will finish the 4th year of our rotation study during 2022, and we already have impressive and interesting results from the first three years, and we intend to publish and communicate our results of this study during 2023. In Illinois, we now have egg numbers from the plots grown in 2019-2021 and HG type values from 2019-2020. These results show that continuous planting of PI 90763 had the lowest egg number increases in plots across the three years. However, the continuous production of this source of resistance is selecting nematodes that can overcome this resistance and the female index (FI) in plots grown with PI 90763 was 41 after 2020, which indicates that the nematode population in these plots may start increasing. The rotation that had the lowest increase in egg numbers over the three years is rhg1b+soja+ch10 in 2019 followed by PI 90763 in 2020 and then rhg1b+soja+ch10 in 2021. This rotation also showed a low increase in egg numbers in other states and did not increase the FI on PI 88788 or Peking.

In Missouri, the egg density results showed that there is an increasing trend in SCN population density in the third year of rotation except for plots with continuous PI 90763 and rhg1-b+G. soja+10 rotated with PI 90763. There was also a significant reduction in the percentage change in the egg count for these two treatments. The HG type data also showed that the continuous rhg1-a + rhg4 and continuous PI 90763 treatments facilitated the development of more virulent nematode populations with HG type 1.2.3-; race 4, where nematode had adapted on the Peking type resistance sources (PI 90763, Peking and Pickett). Similarly, nematode populations from treatments involving rotations of rhg1-b (and/or stacked resistances) with PI 90763 showed to have reduced female index on PI 88788, PI 90763, and Peking indicator lines.

The Mitchum group was sent SCN material recovered from the continuous and rotation microplots and they were increased, processed for eggs, and archived as part of a wormplasm collection for future sequencing efforts to pinpoint virulence genes.

At the conclusion of the 2021 growing season, The Tylka group collected two separate multi-core soil samples from each microplot in the experiments conducted in central Iowa and north central Iowa. One set of soil samples from each experiment were processed at Iowa State University to determine the end-of-season SCN egg population density in each microplot. The second set of soil samples were sent to the University of Missouri for HG Type testing to determine how the soybean genotypes grown in the microplots in 2021 have affected or shifted the virulence profiles (HG types) of the SCN populations from the 2020 growing season and from the initial SCN populations that were added to the microplots in the spring of 2019.

Preliminary data analysis show some trends in changes SCN population densities. In both experiments, the greatest SCN population densities occurred in microplots in which the susceptible soybean variety was grown. Most of the microplots that had continuous cropping of the same resistance in 2019, 2020, and 2021 had greater SCN population densities than microplots in which resistant genotypes were rotated in 2019, 2020, and 2021. The lowest population densities occurred in microplots where soybeans with SCN resistance from PI 90763 were grown. The microplots in which soybeans with rhg1-b, rhg1-b + soja, and rhg1-b + soja + ch10 SCN resistance were grown (collectively referred to as genotypes having “PI88788-type” resistance) in 2019, rotated to soybeans with PI 90763 SCN resistance in 2020, and then rotated back to the same resistance as in 2019 had increased SCN population densities in 2021 compared to population densities at the end of both previous years. Even though SCN population densities declined after rotating from genotypes with PI88788-type resistance in 2019 to rhg1-a + rhg4 (or “Peking-type”) resistance in 2020, population densities increased to levels greater than in 2019 and 2020 when genotypes with PI88788-type resistance were again grown in the microplots in 2021. Similarly, plots in which rhg1-a + rhg4 or Peking-type resistance was grown in 2019 then rotated to rhg1-b, rhg1-b + soja, or rhg1-b + soja + ch10 SCN (PI88788-type resistance) resistance in 2020, and then back to rhg1-a + rhg4 resistance in 2021 had greater SCN population densities at the end of the 2021 growing season than in the previous two years.

This project will benefit soybean producers by creating a long term management strategy for SCN through knowledge and soybean germplasm development.