Project Details:

Title:
An integrated approach to enhance durability of SCN resistance for long-term, strategic SCN management (Phase II)

Parent Project: An integrated approach to enhance durability of SCN resistance for long-term strategic SCN management (Phase II)
Checkoff Organization:North Central Soybean Research Program
Categories:Soybean diseases, Nematodes, Breeding & genetics
Organization Project Code:00071398
Project Year:2021
Lead Principal Investigator:Andrew Scaboo (University of Missouri)
Co-Principal Investigators:
Thomas Baum (Iowa State University)
Andrew Severin (Iowa State University)
Gregory Tylka (Iowa State University)
Melissa Mitchum (University of Georgia)
Brian Diers (University of Illinois at Urbana-Champaign)
Matthew Hudson (University of Illinois at Urbana-Champaign)
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Keywords: Soybean Cyst Nematode

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

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.

Project Objectives

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)

Project Deliverables

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)

Progress of Work

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 PDF file

Final Project Results

Benefit to Soybean Farmers

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

Performance Metrics

Objective 1: Identify SCN virulence genes to better understand how the nematode adapts to reproduce on resistant varieties.
Accomplishments: In Phase I of the project we used innovative methods and new sequencing technologies to enhance and complete the current SCN reference genome assembly for the TN10 population (HG type 0) and the sequence of its bacterial endosymbiont. Additionally, we used state of the art bioinformatics tools to make substantial progress to annotate and curate the SCN genome. We generated a comprehensive SCN gene expression atlas from whole nematodes, which was also used in the annotation of the SCN genome. We created a genomic toolbox for SCN (SCN-Base) that facilitates the integration of very large sequence data sets, molecular markers, QTL data and genetic maps into an easy-to-use web interface. This resource will soon be released as a website that includes a Genome Browser for easy visualization of the SCN genome. We also developed lower-cost methods to produce reference genomes of additional HG types.

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

Objective 3. Translate the results of objectives 1-3 to the SCN Coalition to increase the profitability of soybean for producers.
Accomplishments: In Phase I of the project, an extension and outreach coordinator, advised by project Co-PI Dr. Tylka, was hired to provide farmer education and outreach for the project. A survey of extension and outreach educational materials about SCN biology and management in the NCSRP states was conducted by the coordinator. Materials from land-grant universities and private seed and chemical companies were gathered, analyzed, and compared.

Objective 4. Coordinate the testing of publicly developed SCN resistant experimental lines.
Accomplishments: During 2018, the testing of SCN resistant experimental lines developed by breeders in 11 north central US states and Ontario was coordinated. The tests include 182 SCN resistant experimental lines and varieties that are being grown in 104 maturity group specific trials across 39 locations. These lines are also being tested for SCN resistance and the soil samples from the environments will be evaluated for SCN population density and HG type.

Project Years