Progress 05/01/24 to 04/30/25
Outputs
Target Audience:Our target audience has primarily been scientists, specifically agricultural, plant pathology, and microbial ecology researchers. We have reached them through several national, international, and local conference presentations and one peer-reviewed publication. However, we have also reached the public through an Auburn University press release describing the research. We have reached growers in Alabama by talking with them at the Alabama Soybean Producer Association. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have hired two new graduate students and one new post-doc in two labs to work on this project. Logan Luchs was hired in Dr. Noel's lab, and Arpan Parajuli was hired in Dr. Coleman's lab. In total, we have three graduate students (PSU: Theo Newbold, AU: Logan Luchs, Arpan Parajuli) and two post-docs (PSU: Eric Larson; AU: Emily Roggankamp) working on this project. Graduate students have been able to learn bioinformatics and analysis specific to microbiomes and work collaboratively for manuscript preparation. Students have gained grant writing experience through applying for state and national funding opportunities. Students have also given several presentations. Students have also gained experience in networking through requesting soil samples from professors in other states. How have the results been disseminated to communities of interest?1. Luchs, L., Larson, E., Newbold, T., Crandall, S. G., and Noel, Z.A. 2024. From seed to shining seedling: the soil steaming effects on spermosphere assembly and plant health. American Phytopathological Society Annual Meeting. Poster Presentation. 2. Noel, Z. A. 2024. The seed, the spermosphere, and the seedling: microbiome assembly in seedling establishment. International Phytobiomes Meeting. Poster Presentation. 3. Noel, Z.A., Seed, spermosphere, and seedling microbial assembly to combat seedling diseases. Athens GA, 2024. Invited seminar for Plant Pathology Department 4.Parajuli, A., Coleman, J.C., 2024. Auburn University Plants, Insects, and Microbes symposium. Poster Presentation. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We plan to analyze the microbiome data already obtained to select compositionally divergent communities to then shot-gun sequence. Post-doc Dr. Emily Roggenkamp will lead the shot-gun sequencing efforts, while PhD student Logan Luchs will handle the amplicon sequencing data. Further, we will use these data to inform which samples relate to plant health measurements like emergence, biomass, and soil properties. Possible compelling output from these data could include models that incorporate microbiome and soil data that predict soybean emergence. We will continue to analyze the microbiome data in preparation for publication. The samples are being re-sequenced with ITS primers to improve the quality of fungal data, which will allow for a more accurate taxonomic analysis. The prokaryotic data is currently being used to optimize analysis with DeSeq2 v.1.44.0 and BetaNRI. These methods will allow us to analyze the key differential taxa from planting to the resulting rhizosphere and between treatments (DeSeq2 v.1.44.0), as well as assess the stochasticity of selection (BetaNRI). We plan to submit a manuscript describing the effect of soil steaming on the stochasticity of the development within the spermosphere, the proceeding rhizosphere, and we will identify the key taxa in each developmental stage. We will then link these data to plant health metrics such as plant biomass and yield. We have found that steaming leads to more stochastic community assembly over time. The analysis and writing are being done by a research teaming consisting of a PhD student at Auburn and a postdoc and PhD student at Penn State. Additionally, we will use these data to inform which samples we will use in downstream shot-gun sequencing to decipher how steamed soil (low microbiome environment) alters the functional repertoire of the microbiome and these analyses will occur in over the next year. Objective 3: We will complete the transcriptomic analysis of the fungus in response to soybean seed exudates. Based on this data, we will generate mutants of the key genes in select BGCs that are significantly upregulated in response to exposure to soybean seed exudates. We anticipate a genome resource announcement concerning the two Fusarium soybean seedling pathogens will be submitted this upcoming reporting period.
Impacts
What was accomplished under these goals?
Objective 1: We sequenced the seed epiphyte microbial communities and isolated plus identified the fungi and bacteria on the seed for comparison to the community data. These data are fundamental because they are the basis for the other objectives. We use the same seed for different objectives, so the same seed epiphyte community is input into each experiment. We collected three soils from Alabama and five other states 1-2 weeks after soybean planting to determine the functional genes consistently recruited to a spermosphere. We have leveraged these funds for additional funding to conduct an amplicon sequencing run, ensuring we select the most taxonomically divergent samples for shotgun sequencing. This way, we will determine which functional genes are recruited to the spermosphere despite the taxonomic divergence. A graduate student has collected all samples for objective 1 (n = 400), and the graduate student and a post-doc have extracted DNA from all samples, constructed bacterial and fungal amplicon libraries, and sequenced them on an Illumina NextSeq. Plant health measurements were taken for soybeans growing in each soil (i.e., germination percentage, emergence percentage, soil edaphic factors). We are currently processing these data on the Alabama Supercomputer Authority's High-Performance Computing system. Based on these analyses, we will select samples input for shot-gun sequences at greater than or equal to 6 Gb per sample. We have used this type of sequencing for other projects and have completed the bioinformatic pipeline, which has worked well. Objective 2: We have completed amplicon-based sequencing of the bacterial and fungal soil communities to select the samples for shotgun sequencing. We are analyzing these data by visualizing correlations and significant trends in R (phyloseq package). These analyses are being undertaken primarily by a graduate student at Auburn, with assistance from a postdoc and a doctoral candidate at Penn State. Preliminary results indicate that the prokaryotic compositions of the steamed soil communities differed from those of non-steamed soil communities and soybean epiphytes. Moreover, we found that the bacterial communities found in the steamed soil treatment (low-microbiome environment) fluctuated significantly overtime; this may suggest that the non-steamed/control treatment maintained steady state community dynamics throughout the experiment. We are continuing to complete and improve analyses while prepping a manuscript for submission to a peer-reviewed journal. Objective 3: We have screened 23 members of the Fusarium solani species complex for virulence on soybean. Several Fusarium isolates from three species can cause pre-emergent damping-off of soybean seeds, and the most virulent representatives of the F. falciforme and F. solani species were selected for genome sequencing. After sequencing these two genomes, we identified all the biosynthetic gene clusters (BGCs) that are capable of producing a secondary metabolite. Interestingly, both of these species can produce cyclosporin, a secondary metabolite with known antimicrobial properties. As a goal of this proposal is to determine how pathogens are able to manipulate the other microbes in the soybean spermosphere, we have begun generating mutants of the nonribosomal peptide synthetase responsible for the synthesis of this compound. Additionally, we have evaluated the ability of F. falciforme and F. solani to grow in the presence of only the soybean seed exudates and found both species are capable of growing and using these exudates as their sole carbon and nitrogen sources.
Publications
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:Our target audience has primarily been scientists, specifically agricultural, plant pathology, and microbial ecology researchers. We have reached them through conference presentations and peer review publications. However, we have also reached the general public through Auburn University press releases describing the research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have hired two new graduate students in two labs to work on this project. Logan Luchs was hired in Dr. Noel's lab, and Arpan Parajuli was hired in Dr. Coleman's lab. In total, we have three graduate students (PSU: Theo Newbold, AU: Logan Luchs, Arpan Parajuli) and one post-doc (PSU: Eric Larson) working on this project. Graduate students have been able to learn bioinformatics and analysis specific to microbiomes and work collaboratively for manuscript preparation. Students have gained grant writing experience through applying for state and national funding opportunities for graduate students. Students have also given several presentations. Students have also gained experience in networking through requesting soil samples from professors in other states. How have the results been disseminated to communities of interest?1. Noel, Z.A., 2023. Cotton and Soybean spermosphere microbiome. International Congress of Plant Pathology. Lyon, France. Aug. 23 2023 Press: https://agriculture.auburn.edu/research/microbiome-exploration-represents-a-new-frontier-of-research/ Additionally, Auburn University Graduate students (Logan Luchs and Arpan Parajuli) have successfully delivered their proposal seminars to the department. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We plan to sequence the microbiome of spermosphere soils from three Alabama-derived soils and four other soils from different states to determine the taxonomic core spermosphere microbiome. Further, we will use these data to inform which samples relate to plant health measurements like emergence, biomass, and seedling photosynthetic efficiency. Next, we will use these data to select samples for shot-gun sequencing to test the hypothesis that there is also a functional core microbiome. We will hire an additional post-doc for this project in the coming year. Objective 2: We will continue analyzing the microbiome data in preparation for publication. We plan to submit a manuscript describing the effect of soil steaming on the transmission of the microbiome from a seed to the spermosphere to the rhizosphere and link it to plant health metrics like biomass and yield. The analysis and writing are being undertaken by a team of two postdocs and a PhD student at Penn State. Additionally, we will use these data to inform which samples we will use in the shot-gun sequence to determine how steaming soil alters the functional repertoire of the microbiome, and we plan to analyze that data in the coming year. Objective 3: We will sequence the genomes of two Fusarium isolates capable of causing pre-emergent damping-off on soybeans. These isolates will be exposed to soybean seed exudates and determine which genes involved in secondary metabolite production are upregulated in response to soybean seed exudates. We then plan to generate null mutants of at least 5 genesin one of the Fusarium isolates.
Impacts
What was accomplished under these goals?
Objective 1: We sequenced the seed epiphyte microbial communities and isolated plus identified the fungi and bacteria on the seed for comparison to the community data. These data are fundamental because they are the basis for the other objectives. We use the same seed for different objectives, so the same seed epiphyte community is input into each experiment. To determine the functional genes consistently recruited to a spermosphere, we collected all soils from Alabama and several other states 1-2 weeks after planting. We have leveraged these funds for additional funding to conduct an amplicon sequencing run, ensuring we select the most taxonomically divergent samples for shotgun sequencing. This way, we will determine which functional genes are recruited to the spermosphere despite the taxonomic divergence. We have collected all samples for objective 1 (~300 samples) minus one soil and plan to start DNA extraction, library preparation, and sequencing in the coming months. Plant health measurements were taken for soybeans growing in each soil (i.e., germination, emergence, photosynthetic efficiency, seed necrosis). We will begin analysis of these data concerning taxonomy in the coming months. Based on this analysis, we will select samples to the shot-gun sequence at greater than or equal to 6 Gb per sample. We have used this type of sequencing for other projects and have completed the bioinformatic pipeline, which has worked well. Objective 2: We investigated how spermosphere assembly differs with different starting soil microbial compositions and abundances (steamed vs non-steamed soil). We were interested in quantifying whether the microbial communities are similar in the rhizosphere of seedlings and bulk soil. We conducted a greenhouse trial at Penn State from June to October 2023. We collected spermosphere, rhizosphere, bulk soil microbiome samples, and soybean physiology data. The study was conducted with 20 replicates (n=40). Microbiome samples were collected at planting, 17 hours post-planting, and once the soybeans reached the V2 growth stage. Soybean yield data was collected from each plant as was the foliar biomass. Foliar biomass was not different between steamed and non-steamed treatments, but the steamed soils yielded higher soybean pod weights (p=0.005). A postdoc oversaw this greenhouse trial, and an exemplary undergraduate research assistant conducted daily maintenance. DNA was extracted from the samples at Penn State in tandem with DNA extracted from soybean seed epiphytes by a postdoc at Auburn. The DNA that was then prepped was sequenced by the postdoc at Auburn. We have completed sequencing of the bacterial and fungal communities to select the samples for shotgun sequencing. We are currently analyzing the resulting amplicon-based microbiome data. This analysis is being undertaken by both the aforementioned postdocs and a graduate student at Penn State. Preliminary results indicate that the prokaryote and fungal compositions of the steamed soil communities differed from those of non-steamed soil communities and soybean epiphytes. Moreover, the non-steamed soil was more stable in composition. We are prepping a manuscript based on these initial results and will submit it by the end of the summer. Objective 3: We have screened 23 members of the Fusarium solani species complex for virulence in soybeans. Several Fusarium isolates from three species can cause pre-emergent damping-off of soybean seeds, and the most virulent representatives of the F. falciforme and F. solani species were selected for genome sequencing. After sequencing these two genomes, we will assess the expression of the core biosynthetic gene of the secondary metabolite gene cluster after exposure to soybean seed exudates via RNA sequencing. We have also evaluated the diversity of soybean seed exudates by untargeted mass spectrometry. Many amino acids, carbohydrates, and other metabolites were identified, which can serve as a basis for determining which metabolite(s) are responsible for inducing the expression of the genes of interest in the fungal pathogen.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Olofintila OE, Noel ZA. 2023. Soybean and Cotton Spermosphere Soil Microbiome Shows Dominance of Soilborne Copiotrophs. Microbiol Spectr 11:e00377-23.
https://doi.org/10.1128/spectrum.00377-23
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