Location: Emerging Pests and Pathogens Research
Project Number: 8062-22410-007-016-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2021
End Date: Jul 31, 2025
Objective:
The goal of this research is to identify key taxa, genes, and metabolic pathways involved in soybean cyst nematode (SCN) suppression. Also, to elucidate how species-interactions within microbial communities drive nematode antagonistic properties of SCN suppressive soil microbiomes that can be harnessed to manage these plant parasitic nematodes.
Objective 1: Identify genes and secondary metabolite biosynthetic clusters (SMBCs) enriched or upregulated in microbial communities in bulk soil, rhizosphere soil and SCN cysts of soybean in SCN suppressive soils. This project will focus on a known SCN suppressive soil, located in Waseca, MN, which shows much lower SCN densities than surrounding fields [2, 4, 11] and a paired nearby non-suppressive field. Shotgun metagenomic and meta-transcriptomic approaches will be used to profile the diversity and abundance of taxa, gene families, and metabolic pathways involved in nematode parasitism (e.g. proteases, carbohydrate degrading enzymes, and SMBCs). Microbes will also be cultured from suppressive soils or identified within a large collection of fungi previously cultured by the Bushley lab [12]. Two Hypotheses will be tested: 1) H1.1: The SCN suppressive soil microbiome will be enriched in taxa, genes, and metabolic pathways (proteases, Cases, or specific toxins) with known roles nematode parasitism and 2) H1.2: The SCN suppressive soil microbiome will be enriched in taxa and genes (SMBCs) with functions in secondary metabolism and antibiosis.
Objective 2: Identify interactions between species, genes, and metabolic pathways using co-occurrence networks. Metagenome and metatranscriptome data collected in Obj. 1 will be analyze using co-occurrence networks to identify differences in network structure between suppressive versus non-suppressive communities and highly connected taxa, genes, or metabolic pathways. From this objective, we will identify several (1-3) candidate microbial consortia (of 2-6 taxa) for functional testing. Two hypotheses will be tested: H2.1: Nematode suppression will be mediated directly by interactions between microbes with known roles in nematode parasitism and H2.2: Nematode suppression will be mediated indirectly by antagonistic competitive interactions between non nematode-parasitic microbes that induces production of secondary metabolites incidentally toxic to nematodes.
Objective 3: Test hypotheses of species interactions in microbial consortia. Taxa comprising microbial consortia will be grown alone and in microbial consortia by systematically adding or removing taxa in the presence and absence of the nematode host. We will use several in-vitro bioassays as a high-throughput screen for production of metabolites active against nematodes. For selected consortia (1-3) with high bioactivity, we will conduct transcriptomics experiments to identify upregulated genes and SBGCs and bioactivity-guided fractionation to identify active metabolites. For selected consortia, the raw filtrates as well as live microbial consortia will also be tested in-planta in greenhouse assays for ability to control SCN reproduction in the presence of the plant host.
Approach:
Objective 1. Overall Approach: We will sample a known SCN suppressive soil, located in Waseca, MN, and a paired nearby non-suppressive field. Bulk and rhizosphere soil and SCN cysts will be sampled and a combination of parallel ‘omics’ approaches and culturing will be used to compare microbial communities between suppressive and non-suppressive soils.
1.1 Sampling Bulk soil, rhizosphere soil, and SCN cysts will be sampled at 1) mid-season (July/early August) and 2) harvest (late September/October). 1.2 Metagenomics: Shotgun metagenomics will identify taxa, genes, and metabolic pathways that are enriched in suppressive soils. Taxonomic diversity will be assessed using a marker gene approach. Gene functions will be assigned using PFAM, KEGG, and GO databases and manual curation of several genes families (CAZymes, P450s, proteases, and secondary metabolite genes). 1.3 Metatranscriptomics: RNA-Seq will be performed on rhizosphere soil and cysts. Assembled metagenomes will be pooled into a single reference metagenome for analyses of differentialA expression between treatments. 1.4 Culturing: Bacteria and fungi will be cultured from bulk and rhizosphere soil using dilution plating on several media, while cysts will be surface sterilized and placed water agar. Fungi or bacteria that grow out will be subculture into pure culture and identified by morphology, microscopy, and/or sequencing of rDNA sequences.
Objective 2: Overall Approach: Metagenome data collected in Obj. 1 will be analyzed using co-occurrence networks to 1) characterize differences in network structure between suppressive versus non-suppressive communities and 2) identify highly connected taxa, genes, or metabolic pathways or network ‘hubs’ to select taxa for microbial consortia in Obj. 3.
2.1 Analyze taxa, genes, and metabolic pathways using co-occurrence networks: Taxonomic, Gene, and metabolic pathway co-occurrence networks and meta-transcriptome data, gene co-expression networks will be created an compared. Networks of taxa, genes and metabolic pathways, and gene expression will be analyzed to calculate measures of topological network properties such as degree, betweenness, and centrality, and network ‘hubs’.
Objective 3: Overall approach: To test hypotheses of species interactions using microbial consortia, we will use bioassays to test culture filtrates produced by consortia for inhibition of SCN egg hatch and toxicity towards J2. We plan to screen ~20-30 consortia of 2-3 taxa.
3.1 Test consortia in in-vitro competition experiments with direct physical contact: We will conduct several in-vitro bioassays using SCN HG1: 1) a cyst co-culture competition bioassay, and 2) a liquid co-culture with consortia grown in minimal media +/- SCN added.
3.2 Chemical communication via ‘exometabolites’ between consortia members: Exo-metabolite experiments of consortia grown in both a defined minimal medium and a rich media in the absence of the nematode. Consortia will be grown in modified Transwell plate system.
3.3 Transcriptome and chemical analysis of consortia producing nematicidal compounds: RNA-Seq will be done on the best ~1-3 consortia using the in-vitro assays.
