Progress 09/01/23 to 08/31/24
Outputs
Target Audience:Target audiences reached by our research during this reporting period included soybean growers concerned about challenges posed by the SCN, industry representative, researchers, and farmers and general public attending the New York Ag Expo. Changes/Problems:We had significant challenges isolating good quality RNA directly from field soils and SCN cysts for Objective 1. Thus, we plan to focus instead on genome gene content of MAGs in the field-sampled metagenomes to assess genetic capability for producing secondary metabolites or CAZymes. We will still conduct transcriptomic analyses of microbial cultures or consortia in in-vitro co-culture bioassays to assess whether some of the genes we detect may be expressed during nematode parasitism. What opportunities for training and professional development has the project provided?The project has provided training and professional development for a postdoctoral researcher and an undergraduate student. The postdoctoral researcher has participated in workshops on computational biology for this project and learned skills in shotgun metagenome assembly cyst microbiome and presented their work at the APS NEA annual meeting.The undergraduate student has learned to culture fungi and bacteria and isolate DNA from them foridentification of isolates and is now working on atnhonor's thesis testing some of the bacteria and fungi they have isolated and learning how to analyze microbial communities in soil infested with SCN. How have the results been disseminated to communities of interest?The research results have been shared with academic and industry researchers through posters or oral presentation at professional society meetings. Both the postdoc and the PD have attended one or more meetings during this reporting period. Information on SCN was also disseminated at the NY Farm show in February 2024 to growers and the general public. Research Presentations: 1. Mycological Society of America, Markham, Ontario, CA. July, invited oral presentation Kathryn Bushley, David Showalter, Rodrigo Olarte, Stephen Rehner. Repertoires of Secondary Metabolite Biosynthetic Gene Clusters in Insect and Nematode Parasitic Fungi 2. Society of Invertebrate Pathology Annual Meeting, Vienna, Austria. July 28 - August 1, 2024. invited oral presentation Emily Green, Dong-gyu Kim, Deepak Haarith, Nin Knight, Senyu Chen, Kathryn Bushley. Fungal parasites of nematode eggs for integrated pest management of soybean cyst nematode 3. American Phytopathological Society, Northeast Area Meeting, Ithaca, NY. March 6-8, 2024. Invited oral presentation (Postdoctoral researcher). Emily Green and Kathryn Bushley. Investigating microbiomes for soybean cyst nematode biocontrol. Outreach Events: NY Farm Show - Participated with other USDA groups hosting a booth with one-page handouts and interactive exhibits on invasive species such as SCN. What do you plan to do during the next reporting period to accomplish the goals?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. During the next period, we plan to complete publication on key findings on taxa and functional genes enriched in cysts in suppressive soil treatments. We will also complete analysis of soil (bulk and rhizospheres) metagenomes 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. We plan to conduct additional network analysis of joint bacterial and fungal networks to identify potential interactions between bacteria and fungi and network analysis that includes taxa and their functional gene classes of secondary metabolites and CAzymes. 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. In the next reporting period, we plan to conduct the in-vitro bioassays in transwell plates for consortia of fungi and bacteria to test hypotheses of interactions mediated by secreted metabolites.We will also continue test key taxa in consortia alone in bioassays testing production of metabolites active against nematodes and direct parasitism of SCN for Objective 3 during the next reporting period.We plan to conductin-planta experiments or consortia in spring or summer of the next reporting period.
Impacts
What was accomplished under these goals?
This reporting period, we analyzed both metagenome data for bulk soil and rhizosphere soil, SCN cysts, and soybean roots for Objective 1. A manuscript is in preparation describing key taxa and differences in functional gene content between four treatments outlined below. We also conducted co-occurrence network analysis for Objective 2 to identify several networks diagnostic of the suppressive soil treatment that will form the basis for selecting taxa for consortia being tested in Objective 3. 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. During this reporting period, we analyzed metabarcoding data and shotgun metagenomes of suppressive soils in long-term rotation fields for four treatments (Sa - annual rotation of soybean with corn, S3 - year three of soybean following five years of corn, and two long-term monocultures, Sr - resistant soybean with PI88788 resistance, and Ss - Susceptible soybean showing suppression of SCN). Key findings included identification of a core bacterial and fungal microbiome found within SCN cysts, as well as taxa that were either unique or showed greater relative abundance in one or more of these treatments. Bacteria that were present at >2% relative abundance and prevalence >70% across all treatments included Mesorhizobium (100% prevalence) and other members of the Rhizobium complex (97%), Pseudomonas (100%), Streptomyces (97%), Paenibacillus (74%), Steroidobacter (71%), and Aquabacterium (71%). Similarly, fungi present at > 1% relative abundance and showing >50% prevalence across all treatments included Mortierella (87% prevalence), Exophiala (83%), Orbiliales (O) (83%), Chytridiomycota (P) (67%), Aspergillaceae (F) (50%), Metapochonia (50%). These results were exciting as several of these fungal groups (Orbiliales, Metapochonia) are known nematode parasites, and previous work has isolated cultures of some species of Mortierella and Exophiala species that showed high rates of either egg-parasitism or egg-hatch inhibition in in-vitro assays (Haarith et al., 2020). The prevalence of the early-diverging group of fungi in Chytridiomycota was unexpected, but at least one species in this group is a known nematode parasite. We also identified taxa that showed differential abundance between treatments. The annual rotation (Sa) is the most highly conducive soil to SCN, while S3 showed more similarity to the suppressive long-term susceptible monoculture (Ss) than to Sa. We were particularly interested in taxa shared between S3, which our previous work shows may represent an intermediate step in the development of the suppressive long term Ss treatment. The Sr treatment, also a long-term monoculture of soybean, shared some microbial taxa with the Ss long-term monoculture. We were particularly interested in identifying taxa unique or having significantly higher relative abundance either in both the S3 and Ss, or only in the suppressive Ss long-term monoculture and not the Sr long-term monoculture. Among bacteria, four taxonomic groups had higher relative abundance in the S3 and Ss treatment, including members of the family Rhizobiaceae and the genera Chitinophaga, Acinetobacter, Niastella. The genus Pseudoxanthomonas showed higher relative abundance only in the Ss suppressive soil. For fungi, some taxa were shared between the two long-term monocultures (Sr and Ss), and one (Geranomyces) was shared between S3 and Ss. However, several taxonomic groups were unique or had higher abundance in the Ss treatment only, including Arnium, Neosetophoma, Ramicandelaber, Pyxidiophora, Mortierellaceae (F) Mortierellomycota (P), Rhizophydiales (O), and an unknown taxa mapping to Ascomycota (P). Finding of several early-diverging fungi, Geranomyces in Chytridiomycota and Ramicaldelaber (G) in Kickxellomycota were interesting. These groups of fungi are known to parasitize protistans, algae, as well as some vertebrates (e.g. frogs), but have not been well-studied or considered as potential biocontrol fungi for nematodes. Some are difficult to culture and may pose challenges for development of applied biocontrol agents. In terms of differences in functional content of the metagenomes, we focused on investigating secondary metabolite biosynthetic gene clusters (SMBCs) and carbohydrate active enzymes (CAzymes). We found that the PKS class of SMBCs was enriched in both S3 and Ss, while PKS-NRPS hybrids and a novel type of secondary metabolite cluster (RiPPs) that synthesize small bioactive peptides ribosomally were enriched in the Ss only treatment. For CAzymes, we found the Ss treatment had overall higher numbers of Glycoside Hydrolases (GH) class of enzymes. Among these, several families of fungal chitinases (GH18, GH19, GH23), which may also have a potential role in degrading chitin components of the nematode egg-shell and pharynx, were more abundant in the Ss treatment. Objective 2: Co-occurrence networks of taxa and genes We performed co-occurrence network analysis in Net-Comi for both bacterial and fungi. Key findings include that the occurrence networks of bacteria within cysts showed that the network module with the strongest signal (eigen-value) included three taxa that were also more highly abundance in the S3 and Ss treatments (Rhizobiaceae, Niastella) or only Ss treatments (Pseudoxanthomonas). In the co-occurrence network analysis of cysts, many of these belonged to two subnetworks, one including Geranomyces (G), Rhizophydiales (O), Mortierellales (O), and Pleosporales (O) and another including Mortierellaceae (F), Ramicaldelaber (G), and the unknown Ascomycota (P) taxon. Cross-kingdom joint analysis of fungi and bacteria also identified some subnetworks unique to the Ss treatment. Most of our networks did not show many negative interactions that would be interpreted as potential competition among microbes. Thus, our data do not fully support the hypothesis that competition among microbes in soil may lead to expression of secondary metabolites that are incidentally toxic to nematodes. Objective 3: Test hypotheses of species interactions in microbial consortia We have identified fungal cultures of several of the taxa identified as belonging to a subnetwork with taxa more abundant in the Ss treatment for testing in microbial consortia, including Mortierellaceae, Neosetophoma, and Ramicandelaber that will form the first fungal consortia for testing in-vitro and in-plants. We are currently sequencing bacteria isolated from cysts to identify cultures corresponding to the four bacterial taxa (Rhizobiaceae, Niastella, Chitinophaga, Pseudoxanthomonas) that were more abundant in suppressive soil.
Publications
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Target audiences reached by our research during this reporting period include academic researchers, soybean growers, and industry representativesconcerned about challenges posed by the SCN. Othe audiences reached include the general public for outreach activities to educate about the SCN, especially in NY State where is has recently arrived. Changes/Problems:We had challenges in isolatingsufficient and good quality RNA from soybean cyst nematode cysts because of the extremely small sample size and difficulty inextracting sufficient numbers from soil. Because of this, we are planning to primarily focus on the metagenome analysis to identify pathways of interest and qPCR to verify expression of key genes implicated. We do not see this as a major impediment to dissecting pathways relevant to suppressive soils that should also be differentially abundant in metagenomes from conducive versus suppressive soils. What opportunities for training and professional development has the project provided?This project has provided opportunities for training of a postdoctoral researcher and an undergraduate student. The postdoctoral researcher has attended bioinformatic training workshops for R and python through the USDA's SCINet compute cluster and presented a poster of their research at the APS SCN Conference. The undergraduate student has participated in undergraduate research through Cornell University and will conduct an honors thesis investigating activity of fungal and bacterial cultures that were isolated for this project against SCN. How have the results been disseminated to communities of interest?Several research presentations were made at scientific and industry conferences: National Soybean Meeting. Dec. 14-16, 2023 Savannah, GA. Invited keynote presentation. Nematode Nematode parasitic fungi for biological control of soybean cyst nematode. Dr. Kathryn Bushley. Reached academic, industry, and grower target audiences. Presentation at Mycological Society of America Meetings. July 30-Aug. 3, 2023. Invited oral presentation.Killing Them Softly: Putting the Fungal Squeeze on Cyst Nematodes. Kathryn Bushley, Dong-gyu Kim, Deepak Haarith, Noah Strom, Zhou Dong. Reached academic target audiences. Several outreach presentations also reached both soybean grower groups and underrepresented groups in STEM: Participated in "Expanding your Horizons" to expose middle school girls to topics in STEM. Hosted a live demonstration of nematodes and fungal pathogens of soybean cyst nematode. April 1, 2023. Participated in 2023 Soil Health and Climate Resiliency Field Day - Seneca Falls, NY. Gave a talk on Biological Control of Soybean Cyst Nematode. Kathryn Bushley. Reached a target audience of ~100 soybean growers in NY state. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will complete analysis of genes and biochemical pathways in shotgun metagenomes for Objectives 1 and refine and finalizenetwork analysis for Objective 2. We plan to complete both these objectives and submit one or two manuscripts for publication. The next reporting period will focus primarily on Objective 3 to conduct both laboratory and plant-growth assays of microbial cultures both alone and in consortia for or their activity against SCN. A new researcher will be joining the project in fall 2024 to conduct this work.
Impacts
What was accomplished under these goals?
Summary: Research during this project period focused on completion of analysis of microbial taxa characterizing suppressive soil microbiomes for Objective 1. Shotgun metagenomic data were assembled and analysis of gene families, functional categories, and pathways and network analysis in progress. Samples collected during 2023 field season of additional suppressive soils with different pH and crop treatment, including a long-term susceptible soybean rotation and rotation with two soybean lines with different resistance sources (PI88788 and Peking) have been processed and DNA sequencing is in progress. 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. During this reporting period, we completed analysis of 16S and ITS barcodes in metagenomic data to identify taxa enriched in long-term suppressive soil treatment compared to annual rotation and conducive soils. Among fungi, we found that several key taxonomic groups are found enriched. Members of the order Hypocreales, particularly the genera Metarhizium and Metacordyceps, which include known nematode and insect pathogens, were enriched in suppressive plots in bulk soil, rhizosphere soil, or roots. Members of the family Mortierellaceae were ubiquitous as common soil fungi in both conducive and suppressive bulk and rhizosphere soils, but were enriched in cysts only of suppressive soils. Laboulbeniomycetes, which are typically a ectoparasite of insects, were found in bulk, rhizosphere soil and cysts, with one genus Pyxidiophora enriched in both bulk soil and cysts in suppressive soil treatments. In cyst metagenomes, several early diverging and often unculturable groups of fungi, including members of classes Kickxellomycetes (genus Ramicaldelaber) and Chytridiomycetes (order Rhizophidiales), were abundant in the suppressive soil treatments and also formed part of a core network of fungi in cysts (see below). Among Bacteria, genus Flavobacterium was enriched in bulk soil and cysts, while Chitinophaga and Devosia were enriched in both roots and cysts, Rhizobiaceae, Niastella, Pseudoxanthomonas were enriched in cysts in suppressive treatments. Shotgun metagenomes of cysts were assembled and yielded and average of 37 metagenome assembled genomes (MAGs) of bacteria that could be identified to a bacterial genus and showed >25% completeness based on orthologous clusters and <10% contamination. analysis of gene families (secondary metabolite clusters, CAZymes, and Proteases) is underway. We also conducted and nearly completed culturing and identification of additional fungi (~60 species/400 strains) and bacteria (~25 species/100 strains) for use in laboratory bioassays and consortia experiments. Objective 2:Identify interactions between species, genes, and metabolic pathways using co-occurrence networks. Network analysis was performed using Net-CoMI and yielded networks for bacterial and fungal samples from cysts and bulk soils. For cysts, the strongest signal in the bacterial network included several taxa that were most abundant in the suppressive soil treatment, including Rhizobiaceae (F), Pseudoxanthomonas, and Niastella (Fig. 1;A), as well as subnetworks for Bradyrhizobium and Mesorhizobium and two species of Clostridium sensu stricto, where while not more abundant in cysts showed enrichment in either rhizosphere (Bradyrhizobium) or bulk (Clostridium) soils in suppressive soils. For the fungal cyst network, the largest and most strongly supported network (Fig. 1;B, red) included several taxa (Pseudocoleophoma, Vishniacozyma, and Bullera) among others that were found in both long-term soybean plots, including both resistant and susceptible soybean varieties, and were thus not specifically associated with suppressive soil. However, two smaller networks contained many of the taxa enriched in cysts in suppressive treatments (Fig. 1; B, orange and green). The first contained several early diverging fungi (Orders Rhizophydiales, Spizellomycetales (Geranomyces), and Mortierellales) as well an ascomycete order (Pleosporales), with the second containing an unknown Ascomycete taxa, members of the Family Mortierellaceae, and another early divering fungus (Ramicaldelaber) in order Kickxellales.The presence of Mortierellomycetes, which are known to include nematode and specifically egg parasitic taxa and other early diverging fungi is intriguing and may have been missed by previous culture-based studies. Current work is working on constructing a combined network of fungal and bacterial taxa. These networks are being refined but at least for some bacteria (Pseudoxanthomonas, Niastella, Rhizobiaceae, and Bradyrhizobium) and fungi (Mortierelllaceae) that we have also identified through culturing approach, provide the initial basis for selecting groups of consortia for testing in Objective 3. Objective 3:Test hypotheses of species interactions in microbial consortia. We have selected several initial consortia including Bacteria in genera Pseudoxanthomonas, Niastella, and Rhizobium and fungi in order Mortierellales for testing and work will begin on laboratory bioassays and transcriptomic analyses.
Publications
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Target audiences reached by our efforts during this reporting period include industry and academic researchers and growers interested in challenges posed by the soybean cyst nematode. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training and professional development for a postdoctoral researcher and an undergraduate student. The postdoctoral researcher has participated in workshops on computational biology for this project. The undergraduate student is obtaining their first experience working in a laboratory and learning to culture fungi and isolate DNA. How have the results been disseminated to communities of interest?The research to date from initial cyst microbiome is being shared with researchers in academic, non-profit, and industry and grower groups at the APS National Soybean Cyst Nematode conference (Dec. 14-16, 2022). Both the postdoc and the PD will attend the conference and PD Bushley will give an invited talk on biocontrol of SCN using fungi at the conference. What do you plan to do during the next reporting period to accomplish the goals?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. During the next period, we plan to complete shotgun metagenome and metatranscriptome sequencing of data from season 1 of the project. We plan to complete analyses of diversity and abundance of taxa, gene families, and metabolic pathways involved in nematode parasitism (e.g. CAZymes, secondary metabolites) or others we uncover. We will also complete a second season of field sampling of two of the fields to capture the rotation from resistant to susceptible soybean, where we have observed a rapid increase in nematode populations in conducive soil for an additional comparison with susceptible soybean. 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. We plan to conduct network analysis of season 1 data during the next reporting period. 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. Based on the data gathered from Obj. 1, we will begin in-vitro bioassays for candidate fungi cultured in 2022 for production of metabolites active against nematodes in the next reporting period and direct parasitism of SCN. We will begin forming hypotheses regarding consortia to analyze and testing methodologies for metabolite production of consortia in transwell assays.
Impacts
What was accomplished under these goals?
Accomplishments: The project was initiated during this project period and research efforts to date have focused on collecting and processing field samples for Objective 1 for initial metagenomic and metatranscriptomic characterization of suppressive soil microbiomes in Objective 1. 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. A postdoctoral researcher was hired in March of 2022 to conduct sampling and metagenomic and metatranscriptomic analyses for objectives 1 and 2. During the 2022 growing season three fields that contain plots with long term suppressive soil treatmentsand rotation treatments of either soybean with a nonhost crop (corn) or resistant and susceptible soybean that will be compared to suppressive soil plots were sampled. Plots were sampled both at midseason (July) and at harvest (September) to collect bulk soil, rhizosphere soil, soybean roots (midseason only) and soybean cyst nematode (SCN) cysts. These samples were processed for both DNA and RNA extractions and culturing. We conducting culturing of fungi and bacteria from these plots and are working on sequence identification using sanger sequencing of the ITS and 16S regions. All of the midseason 2022 samples have been processed for isolation of DNA and submitted for next-generation sequencing. The DNA extraction of fall 2022 samples and RNA extractions of all samples are currently underway.
Publications
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