Progress 07/01/23 to 06/30/24
Outputs
Target Audience:Agricultural researchers through poster presentations at scientific conferences. The target audience also includes co-investigators, scientists, and studentsworking on the project, as well as colleagues engaged in microbiome research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Hands-on instruction and technical training are taking place in the Halverson and Castellano laboratories. Collectively, these labs have two undergraduate students, two graduate students, one post-doctoral researcher, and one high-school science teacher (participating in a Research Experience for Teachers-[RET] program) participating in laboratory components of the research. Two graduate students participated in professional, scientific meetings to share results and learn more about their discipline, technical advancements, and career opportunities while also providing networking opportunities. How have the results been disseminated to communities of interest?Our work has been disseminated through classroom instruction in Castellano's Agroecology course serving both undergraduate and graduate students, as well as Halverson's Laboratory in Microbial Physiology, Genetics and Diversity for undergraduate students, where students learn techniques for characterizing rhizosphere microbial communities. Portions of the results have been presented as posters at international conferences and to peers in the graduate programs/seminar series at ISU that are open to the general public.One graduate student gave a public seminar open to ISU faculty and students on his research as a requirement for defending his master's thesis, which will be submitted/completed in the near future. What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we plan to complete the construction of libraries to characterize the bacterial, archaeal, and fungal communities of the bulk soil, rhizosphere, and hyperspheres of high- and non-high NUE germplasm grown in soil with different (low, medium, high) N-fertilization histories. We will also complete the quantitative PCR measurements of total bacterial, archaeal, and fungal community sizes. We will also complete the isolation of DNA from plant tissue for microbiome characterizations of the endophytic community. Once amplicon sequencing is completed, we will begin bioinformatic analyses. For Aim 2, we will complete the inorganic N measurement of bulk soil, rhizosphere, and hyphosphere compartments of samples collected from the microbiome analyses described in Aim 1. We will also perform isotopic N mass spectrometry measurements to determine how much N is acquired by AMF. For Aim 3, we will continue to perform root architectural analyses of high and non-high NUE genotypes, assess the effects of Massilia to stimulate root development in low N conditions and complete the isolation of root exudates for metabolomic analyses. If the 15N analyses indicate differences in AMF-mediated N acquisition under low fertilization, we will explore developing tools to ascertain differences in plant transporters for incorporating N from AMF.
Impacts
What was accomplished under these goals?
Aim 1: Assess how high-nitrogen Use Efficiency (NUE) germplasm influences rhizosphere and endophytic microbiome assembly. In this reporting period, we implemented our redesigned rhizotrons (mesocosms) to better enable higher throughput for comparing differences between high- and non-high NUE mazie genotypes on i)rhizosphere, ii) endosphere, and iii) hyphosphere bacterial, archaeal and fungal communities. Plants were grown in soils of contrasting low, middle, and high nitrogen fertilization to the V5/V6developmental stage with sufficient replication (n=8) for robust co-occurrence (network) analyses. Collected all the samples and extracted high quality DNA from bulk soils, rhizosphere, and endosphere samples and are in the process of isolating DNA from root tissue. We have processed all the bulk soil, rhizosphere, and hyphosphere samples for inorganic N pool sizes and for measuring the extent of Arbuscular Mycorrhizal fungi (AMF) contributing to NUE by the high-NUE genotypes. Samples are being processed for generating libraries for near full-length sequencing of the small subunit rRNA genes. Aim 2: Quantify how high-NUE maize germplasm influences rhizosphere N-transformations. In this reporting period, we completed collecting rhizosphere soil solution samples through various maize developmental stages (up to V5/V6) and conducted experiments to measure the gross rates of ammonia/nitrate production and consumption, potential nitrification rates, and inorganic N pool sizes at the V5/V6 developmental stage. Samples have been processed, and most of the data has been analyzed. The data supports the prediction that some high-NUE genotypes stimulate ammonia production (N-supply) in the rhizosphere and that some linesenhance nitrogen retention in the rhizosphere by inhibiting nitrification compared to non-high NUE germplasm. Additional statistical analyses are in progress. Aim 3: Assess how maize root properties are influenced by genotype and N-fertilization. In this reporting period, we have expanded on our initial observations that high-NUE genotypes vary in root architectural properties, differing in how they influence microbes around roots. We show that the root architecture of high-NUE genotypes generates roots that are more branched and with finer roots, in particular more lateral roots, compared to non-high NUE genotypes. Determined that some maize root-associated Massilia species isolates produce more biofilm when exposed to root exudates and are better at stimulating lateral root formation. This indicates that recruitment of Massilia on roots under low N fertilizer conditions could enhance nitrogen use efficiency by stimulating root development to better capture nitrogen. Stimulation of root development by Massilia could contribute to the observation of increased biomass in low N soils when plants are inoculated with a 31-member (29 species) synthetic community that includes twoMassilia isolates.
Publications
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Agricultural researchers- posters were presented at two conferences, International Phytobiomes Conference 2022 (13-15 September 2022, Denver, CO, USA) and the 2022 ASA-CSSSA-SSSA International Annual Meeting (November 6-9, Baltimore, MD, USA). Undergraduate and graduate students at ISU through formal classroom and laboratory instruction. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Hands-on instruction and technical training are taking place in the Halverson and Castellano laboratories. Collectively, these labs have two undergraduate students, two graduate students, and one post-doctoral researcher participating in laboratory components of the research. One graduate student and one post-doctoral researcher participated in professional, scientific meetings to share results and to learn more about their discipline, technical advancements, and career opportunities while also providing networking opportunities. How have the results been disseminated to communities of interest?Our work has been disseminated through classroom instruction in Castellano's Agroecology course serving both undergraduate and graduate students, and Halverson's Laboratory in Microbial Physiology, Genetics and Diversity for undergraduate students, where students learn techniques for characterizing rhizosphere microbial communities. Part of the results has been presented as posters at international conferences and to peers in the graduate programs/seminar series at ISU that are open to the general public. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to continue analyzing samples for Aim 1 that were collected from bulk soil and rhizosphere of high- and low- NUE maize germplasm grown in soil with differing N-fertilization histories. Analyses include 15N isotope pool dilution measurement, an isotope ration mass spectrometer, and inorganic N pool sizes by colorimetric assays. DNA will be extracted from soil and rhizosphere samples for measuring the abundance of taxa involved in key N-cycling processes. For Aim 2, we will propagate plants in a modified rhizotron system to collect bulk soil, rhizosphere, hyphosphere, and endosphere microbial communities of high- and low-NUE maize germplasm grown in soil with differing N-fertilization histories. DNA will be obtained from these samples and processed for quantitative PCR and/or amplicon profiling of bacterial and fungal communities. We anticipate DNA will be submitted for amplicon sequencing by the end of this reporting period. For Aim 3, we will begin the propagation of plants for the collection of root exudates for metabolomic analyses once we finalize the development of an experimental system that is appropriate for the goal.
Impacts
What was accomplished under these goals?
Aim 1. Assess how high-NUE maize germplasm influences rhizosphere and endophytic microbiome assembly. We have completed the design, test, build, and evaluationphase of the rhizotron system to complete the objectives of this Aim. This refined rhizotron(mesocosm) system will permit a more robust assessment of the potential contribution of Arbuscular Mycorrhizal Fungi (AMF) in nitrogen acquisition by the high-NUE maize germplasm, utilize less variable nitrogen fertilizer soil, and provide compartments to evaluate root-free soil and the hyposphere. Presently, we are propagating plants to examine rhizosphere and endophytic microbiome assembly. Aim 2. Quantify how high-NUE maize germplasm influences rhizosphere N-transformations. In this reporting period, a graduate student has collected all the plant and soil samples necessary for the completion of this Aim. Significantly,he has developed the expertise to prepare samples labeled with 15N for mass spectral analysis for determining rates of nitrate and ammonia production in bulk compared to rhizosphere soil. Moreover, he obtained samples for inorganic N-pool size determinations and has conducted experiments to assess potential nitrification and ammonification. Aim 3. Assess how maize root properties are influenced by genotype and N-fertilization We are taking a hiatus on the continuation of additional root architectural analyses until we complete the analysis of samples obtained for Aim 2and have completed obtaining microbiome samples for Aim 1. We have also designed and tested a system for collecting root exudates and anticipate making new versions based on our earlier outcomes. The genome sequencesof 4 Masillia species (Oxalobacteraceae) have been obtained using both long-read Nanopore and short-read Illumina sequencing platforms. Assembly and polishing of the genomes are nearly complete, as well as initial bioinformatic analysis. These strains show different abilities to attach to plant roots and form biofilms and likely represent at least two and possibly three species. Reports by others suggest that these bacteria contribute to maize nitrogen use efficiency by promoting root development.
Publications
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The target audience reached by our efforts during this reporting period include co-investigators, scientists, and students working on the project as well as colleagues engaged in microbiome research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Hands-on instruction and training is taking place in the Halverson and Castellano laboratories where collectively one undergraduate researcher and two graduate students are participating in laboratory components of the research. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to initiate experiments to begin collecting the bulk soil, rhizosphere, and endosphere samples necessary for generating profiles of the microbial community and how N-fertilization levels and maize genotype influence microbial communities. We also plan to initiate experiments to measure rates of nitrification and ammonia production in the rhizosphere and endosphere, as well as to finalize development of the microfluidic-based quantitative PCR platform to measure ammonia mono-oxygenase gene abundance. Lastly, we will begin developing protocols for isolating maize root exudates for metabolomic analyses.
Impacts
What was accomplished under these goals?
We successfully generated the high NUE germplasm required for this project in October2021, which was required to begin the proposed studies. We obtained sufficient amount of seed for all germplasm that will be needed for the duration of the project. Aim 1. Assess how high-NUE maize germplasm influences rhizosphere and endophytic microbiome assembly We successfully recruited a post-doctoral fellow to work on this aim. Aim 2. Quantify how high-NUE maize germplasm influences rhizosphere N-transformations In this reporting period, a graduate student initiated studies to assess the proposed methodologies for quantifying rhizosphere N-transformations. This work has led to several key changes in the experimental plan to accommodate the robust root development of the high NUE maize germplasm, as well as a strategy for measuring the non-radioactive nitrogen isotope for these experiments. Moreover, we have validated new molecular tools (PCR primers) specific for different groups (clades) of bacteria for measuring the abundance of genes involved in nitrification that are amendable to microfluidic, high-throughput analyses. Aim 3. Assess how maize root properties are influenced by genotype and N-fertilization We did a preliminary screen of root architectural features of the high-NUE maize germplasm and observed substantive differences in genotypes that exhibit greater grain yields than the parental lines under low and under low and high nitrogen fertilization levels. This suggests that differences in yield performance cannot be simply attributed to differences in one or more root architectural features. These results were obtained when plants were grown under in soil from the low and moderate N fertilization levels of the long-term nitrogen-fertilization study site: studies are planned for examining root architectural features under high N fertilization. Lastly, we isolated several Oxalobacteriaceae from maize roots that we are currently characterizing (genomic and phenotypic): these organisms were recently shown to contribute to maize nitrogen use efficiency.
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