Progress 04/01/24 to 03/31/25
Outputs
Target Audience:1. Researchers, including faculty members, postdoctoral researchers, graduate students and undergraduate students. 2. Members of agricultural growers associations, including farmers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?As a result of this project, postdoctoral researcher, Dr. Laura Kaminsky continued to develop skills with arbuscular mycorrhizal fungi (AMF). She also developed several new techniques, including approaches to expose AMF hyphae to bacteria, harvest the hyphae and mount the samples under agar for hybridization. Additionally, she developed a modified in-growth core system to allow the capture of rhizosphere samples as well as hyphal samples from wheat. Dr. Kaminsky mentored an undergraduate student Giovanni Dimaiolo (Cornell Plant Science Major) who learned to harvest hyphae, and to isolate and purify myxobacteria. In November2024, Dr. Kaminskyaccepted a position with PASA, a Sustainable Agriculture Organization and she left our project in December 2024.Thus this project has contributed to work-force development, anda highly skilled researcher, with knowledge of AM symbioses and microbiome analyses has entered the US agricultural workforce. How have the results been disseminated to communities of interest?Data have been disseminated through seminars and presentations at Conferences and/or discussions with specific groups eg., Iowa Corn Growers Association. Postdoctoral researcher, Dr. Laura Kaminsky presented results of this project at the International Conference on Mycorrhizas, Manchester, UK, August 4-9, 2024 Discussions of this project occurred in seminars presented by Maria Harrison (PI of this project). CIFAR Fungal Kingdoms April 3-5, Toronto, Canada Penn State Univ. R. R. Nelson Memorial Lecture April 22, State College, PA Plant Microbiota Workshop, 15-17 April, Berlin, Germany Meeting at BTI with Iowa Corn Growers Association. What do you plan to do during the next reporting period to accomplish the goals?We will continue with the experiments as outlined in the proposal. There are no major changes to the experimental plan.
Impacts
What was accomplished under these goals?
Under Objective 1, we have recently completed the experimental phase of a large, multi-component experiment to document microbial communities associated with two genotypes of spring wheat (RHT1 RHT2 and rht1 rht2 genotypes). Our experimental design, which involves290 samples, will allow us to compare microbial communities recruited, over time,to the wheat rhizosphere, the AM fungal mycorrhizosphere and the AM fungal hyphosphere. Plant hosts includewheat (two genotypes) anda small model grass species, Brachypodium distachyon. The experimental phase, which included set-up, sampling, nucleic acid extraction,preparation of 16S amplicons and sequencing, is now complete. Computational and statistical analyses arein progress. The community composition datawill provide fundamental information that is valuable for researchers in the agricultural microbiome field. While the data will provide new information about the wheat rhizosphere and hyphosphere microbiomes, and will be anchored to our previous datasets via the Brachypodium data, our ultimate goal is to use this information to guide design of synthetic microbial communities (objective 3) which can increase plant-available P in agricultural soils. Consequently, ultimately, the information should be of interest to farmers and to the agriculturalinoculant industry. Under the second part of Objective 1, we have continued to isolate, culture and identify myxobacteria to supplement the culture collection. It is possible that myxobacteria are keystone species in the hyphae-associated communities and they may be significant to for hyphae-associated microbial community assembly and stability. For this reason, we aim to isolate representatives of the families identified as abundant on the hyphae in the 16S profiling data. To date, we have identified several new myxobacterial species but no members of the Sandaracinaceae family nor the Haliangium genus that were consistently enriched in the hyphosphere sequencing data in our prior experiments. We are continuing to explore isolation techniques and media that may allow us to isolate members of the myxococcales. Under Objective 2, we are mappingthe spatial organization of microbial communities on the hyphae using a technology called HiPR-FISH. The emergent properties of a microbial community may depend on the spatial organization of the community members and this knowledge (along with the knowledge generated in Objective 1) will inform the development of synthetic communities (Objective 3). As a first step, we have developed approaches to visualize bacteria, one species as a time, on the surface of the hyphae using fluorescence in situ hybridization (FISH). So far, this has allowed us to identify specific community members that associate with dead hyphae while other members consistently associate with living hyphae. When considering the development of synthetic communities, both groups are of potential importance; isolates that associate with dead hyphae may recyclenitrogen, while those on the living hyphae may provide specific services to the AMF. The single species FISH experiments also allowed us to develop methods to handle the hyphae, which we have now optimized. For the next stage, the spatial organization of a larger community (ie., knowledge of each microbes nearest neighbors), we are using a more advanced FISH technique (HiPR-FISH) that allows the simultaneous identification of multiple microbes at one time. This year, colleagues who developed HiPR-FISH (Hao Shi, Kanvas Biosciences), have developed bar-coded probes for an initial 12-member community. We chose these 12 members based on their abundance and occurrence in our 16S profiling datasets. Over the past year, we have shown that these bar-coded fluorescent probes distinguish the 12 species in mixtures. Furthermore, the analysis indicates that we can quantify the different species and quantification data arereproducible across replicate experiments. Hybridization to extraradical hyphae exposed to the 12 species is in progress. The data generated under this objective will tell us which microbes live in close proximity (micron scale resolution) on the hyphae and these data will inform synthetic community development (Objective 3). Data generated from experiments under Objectives 1 and 2 are required for the development of robust synthetic communities. Additionally, synthetic community development will also be guided by the hydrolytic capacities of the species, either alone or in consortia. Potential hydrolytic capacitycan be predicted from thegenome sequences and we couple these predictions with experiments to test the predictions and to evaluate the full hydrolytic potential. Over the past year,we tested our isolates for their abilities to solubilize phosphorus from two key complex sources common in the soil, phytate and calcium phosphate. We also evaluated their ability solubilize chitin, which results in the release and recycling ofnitrogen. In each case, we used an opaque screening medium containing the compound of interest which turns clear if the plated bacterial isolate has the enzymatic potential to solubilize that nutrient. For each nutrient, we identified several isolates with the capability to hydrolyze phytate, calcium phosphate and chitin. This information will be used when considering which isolates to include in the synthetic communities.
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Progress 04/01/23 to 03/31/24
Outputs
Target Audience:Researchers, including faculty members, postdoctoral researchers, graduate students and undergraduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training: In the course of this work, postdoctoral researcher, Dr. Laura Kaminsky learned many techniques that were new to her, includingextracting AMF spores and inoculating wheat, collecting AMF hyphae under the microscope, clearing and staining plant roots to check AMF colonization levels, conducting FISH on hyphal and bacterial samples, microscopy work on FISH and other samples, development of primers and isolate tracking,isolating and maintaining cultures of myxobacteria, and determining the phosphorous content of plant tissues. Mentoring: Dr Kaminsty mentored a rotating graduate student who assisted with aspects of severalexperiments, including inoculating plants with AM fungi,growing bacteria and calibrating OD600 data to cell suspension concentrations, designing PCR primers, harvesting hyphae, and running FISH hybridization. How have the results been disseminated to communities of interest?Discussions of this project occurred in seminars presented by Maria Harrison (PI of this project) at the following venues: Roger E. Koeppe Endowed Lecture, Oklahoma State University, March 3, 2023 Caltech RSI Ecology and Biosphere Engineering Seminar Series, May 8, 2023 BTI Scientific Advisory Board, May 10, Ithaca, NY VIB Conference "Translational Research in Crops" June 23-26, Ghent, Belgium Presentation to the Iowa Corn group, November 30, 2023. Presentation to the American Association of University Women, January 22, 2024 What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on the experiments asoutlined in the original proposal. There are no major changes to the experimental plan.
Impacts
What was accomplished under these goals?
Objective 1.Profile HMCs and microbial communities associated with spores of two AM fungi during AM symbiosis with wheat andBrachypodium distachyon, and culture abundant HMC members currently missing from the culture collection. Under objective 1, we have initiated experiments to determine the AM fungal hyphae-associated microbial communities ( referred to hereafter as HMCs) during AM symbiosis of Rhizophagus irregularis with two genotypes of wheat (RHT1 RHT2 and rht1 rht2 genotypes). Over the past year, we have obtained and amplified wheat germplasm and optimized inoculation conditions to enable robust establishment of AM symbioses in thesewheat genotypes. Having accomplished this, we are now setting up the main profiling experiment. The data generated will allow us to assess the impact of plant species and genotype on HMC composition; the former will occur through comparisons with our earlier published data.These data are fundamental in nature and will be useful to other researchers in the agricultural microbiome field. Ultimately, the data will support the development and use of synthetic communities in experiments outlined under objective 3. Under objective 1, we have also initiated experiments to determine if the HMCs are transferred to subsequent generations of AM fungi through passge on the spore surfaces. If microbes are transmitted on spore surfaces, this will have practical value when developing or managing microbial community inoculants. Assessing spore transmission is a multi-generational experiment and involves exposing AM plants to live soil and then profiling HMCs associated with the extraradical hyphae and subsequently the microbial communities on the spores that develop from the extraradical hyphae. Half of the spore sample will then be used to establishe new AM symbioses and profiling the HMCs on the hyphaewill allow us to determine whether the hyphae receive their HMCs fromthe spores. Currently, we are half-way through this experiment; extraradical hyphae have been harvested and samples stored at -80C. Sporulation is in progress. The data generated from this experiment is fundamental in nature and will be of interest to AM symbiosis reseachers. However, it will also will inform microbial community application experiments. The final experiments under Objective 1 include culturing members of the HMCs that were missing from our initial culture collection. Members of the myxoccocales are abundant in the HMCs as assessed by 16Sprofiling. Using new cultivation efforts, we have obtained five myxobacterial isolates. Three belong to the Corallococcus genus and two belong to the Nannocystis genus. These are new isolates and will be included in future synthetic community experiments under objective 3. So far, we have not identified all of the myxoccocales genera that we identified in the 16S profiling. Consequently, we are continuing our culturing efforts and will includebait microbes from the HMC collection. Objective 2.Map the spatial organization of the HMCs on the ERH with micron-scale resolution using HiPR-FISH. Members of the microbial community interact with their neighbors and the emergent properties of the community may depend on the spatial organization of the community members. To determine this spatial organization (knowledge of each microbes nearest neighbors) we are collaborating with colleagues who developed HiPR-FISH, a technique that allows fine-scale visual resolution of the location of different bacterial taxa in biological samples. This is a new technique and the initial work involves protocol development. Over the past year, we developed three different experimental approaches to inoculate, capture and prepare fixed, bacteria-coated AMF hyphae onto slides in a manner compatible with HiPR-FISH protocols. Briefly, AM fungal hyphae were grown and exposed to a 20 member test community. Hyphae were extracted and arranged on slides with care taken to preserve the spatial structure of hyphae. A thin overlay of 0.75% agar was then applied to hold hyphae in place. The presence of bacteria coating the hyphae harvested in this way was confirmed with a universal bacterial FISH probe. These slides are now ready for HiPR-FISH. In the meantime, our collaborators developed bar-coded probes to distinguish the 20 member community and are completing probe quality checks. The data from these initial experiments should begin to illuminate spatial locations of the different community members. The data generated under this objective are fundamental in nature and will be of interest to researchers in the agricultural microbiome research field. Ultimately, the data will inform synthetic community development (Objective 3). Objective 3. Test the hypothesis that HMCs contribute to plant P nutrition during AM symbiosis. We hypothesize that microbial communities adept at phosphorus solubilization may further boost P transfer to the plant through AM symbiosis. However, there may also be other activities that enhance symbiotic performance, possibly through as yet unknown functions. Experiments under this objective will be largely guided by the data from Objective 2. However, even in the absence of the spatial data, we have started to test some initial hypotheses. To guide the selection of isolates for initial experiments, we analyzed our HMC culture collection and 16S profiling data to determine which of our isolates were the most ubiquitous on AMF hyphae. We then used these data to test the hypothesis that the more commonly occurring and abundant P-solubilizing isolates, arethe most important for hyphosphere P solubilization and plant growth promotion. To test this hypothesis, a synthetic community with four isolates that included the most ubiquitous HMC isolates was applied to Brachypodium distachyon-Rhizophagus irregularis mycorrhizalplants. 11 weeks after bacterial addition, no differences were observed in biomass or plant P content between Rhizophagus irregularis only and the Rhizophagus irregularis plus synthetic community treatment. Based on this experiment, we reject the hypothesis that the most commonly occurring isolates are the most important for P solubilization. Although this initial experiment didn't yield increases in plant growth, it was nevertheless useful as we also used it to evaluate isolate persistence through the experiment. We developed primers for genes specific to each isolate and were able to track 75% of the isolates on the hyphae and in the growth substrate. Future synthetic community experiments will be guided by phytase and phosphatase expression levels of each isolate, as well as the isolate co-occurrence data generated through the experiments in Objective 2.
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Progress 04/01/22 to 03/31/23
Outputs
Target Audience:Researchers, including faculty members, postdoctoral researchers, graduate students and undergraduate students. Changes/Problems:The main challenge that I met during the first year of the award was the hiring of personnel for the project. In the first half of 2022, the applicant pools were sparse and I was unable to identify a postdoctoralcandidate with thenecessary skills for the project. This changed in the Fall 2022 and I made an offer to a strong candidate, Laura Kaminsky. However, she was still finishing her Ph.D so it took a few more months before she was able to join the project. She joined the lab in February 2023 and is making rapid progress. We wil not change any aspects of the project but it is likely that we will eventually request a no-cost extension in order to complete the experiments proposed under the three objectives outlined in the proposal. What opportunities for training and professional development has the project provided?Postdoctoral researchers Dr. Laura Kaminsky has received training in the establishmentof AM symbiosis in Brachypodium distachyon and the in vitro co-culture of Rhizophagus irregularis with carrot roots.She has learned approaches to culture Myxobacteria from hyphae andtechniques for Fluorecence In Situ Hybridization (FISH) of bacteria on the extraradical hyphae of R. irregularis. How have the results been disseminated to communities of interest?Discussions of this project and the initial data have been included in seminars presented by Maria Harrison (PI of this project) at the following venues: 31st Fungal Genetics meeting, Asilomar, CA, March 15-19 Plant Biochemistry Symposium and Biodiscovery Institute Seminar Series, UNT, Denton, Texas, October 7. Whetzel, Wescott, Dimmock lecture, Plant Path. and Plant Microbe Biol., SIPs, December 7. University of California at Riverside, Seminar in the joint seminar series of the GGB/CMDB/MCBL programs, Feb 21-22nd, 2023 Oklahoma State University, Roger E. Koeppe Endowed Lectureship, March 3, 2023 What do you plan to do during the next reporting period to accomplish the goals?Over the next reporting period, we will continue with the experiments outlined in the proposal.
Impacts
What was accomplished under these goals?
Objective 1. We initiated various preparative experiments to enable the comparative hyphae-associated microbe (HMC) profiling experiments. It was necessary to increase production of Gigaspora gigantea spores to develop a single large batch of spores sufficient for several experiment to ensure consistency across experiments. We maintain the species in the lab but not at the scale required for the experiments in Obj 1. This species sporulates profusely on Bahia grass stock plants but takes approximately 6-9 months to sporulate. This preparative step is now complete and we have sufficient spores to enable the comparative profiling experiments. A pilot experiment is in progress to ensure that the mesh core size used with previous AM fungi is suitable for this species. Additional experiments under objective 1 include characterizing the microbes in the culture collection. These data, along with data from the spatial profiling experiments, will guide the development of the SynComs. Initial characterization experiments included growth characteristics on several liquid and solid media, assays for phytase and phosphate solubilization and biofilm formation. Characterization of 12 previously uncharacterized species, has revealed 4 strains with phytate solubilizing activity, one of which also has strong phosphate solubililizing activity from insoluble calcium phosphate. Six of the strains are strong biofilm producers. Objective 2. Under objective 2, we have initiated experiments to evaluate methods for capturing and mounting extraradical hyphae on slides in preparation for the HiPR FISH experiments. There are several challenges to overcome to ensure that the capture and mounting approaches are compatible with the HiPR FISH technologies. We have also optimized hyphal growth in liquid media in sterile co-culture with carrot roots. This culture system provides one source of raw materials for these experiments. Exposure of hyphae to microbes is in progress. Objective 3. Ultimately, the design of SynComs will be driven by data generated under Obj. 2 as well as the microbe characterization experiments outlined in objective 1. However, we decided to initiate some simple SynCom experiments in order to test and optimize the experimental set-up. The first experiment used two Syncoms, each with 4 HAMs species designed to maximize taxonomic diversity while also considering phytase and phosphate solubilizing activities. Brachypodium distachyon plants were inoculated with Rhizophagus irregularis and grown together in low P growth conditions to allow establishment of the mycorrhizal association. At 7 days post planting, 1 x 106 colony forming units (CFU) of each strain were applied to the pots and plants were then supplied with phosphate in the form of calcium phosphate (sparingly soluble). At 10 weeks post planting, tiller number in the mycorrhizal plants was double that of the controls but was not affected either positively or negatively by the SynComs. Mycorrhizal plants showed a significant increase in shoot biomass (dry weight) relative to the mock-inoculated plants However, shoot biomass showed a small but significant increase in mycorrhizal plants that received SynCom 2 relative to the mycorrhiza plants that did not receive microbes. This effect was not seen in plants receiving SynCom 1. SynCom2 included the HAM strain which exhibited high phosphate solubilizing activity. These initial results are very encouraging and we are continuing to enhance our experimental design with new approaches to evaluate responses to localized sources of phytate.
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