Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
APPLYING ENVIRONMENTAL FILTERING TO COLLECT AND FUNCTIONALLY COMPARE MICROBIAL ASSEMBLAGES WITH DIFFERENT ECOLOGICAL TRAITS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022062
Grant No.
2020-67013-30865
Cumulative Award Amt.
$480,000.00
Proposal No.
2019-06584
Multistate No.
(N/A)
Project Start Date
May 1, 2020
Project End Date
Apr 30, 2024
Grant Year
2020
Program Code
[A1102]- Foundational Knowledge of Agricultural Production Systems
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Plant Pathology and Environmen
Non Technical Summary
Agriculture tends to reduce biodiversity relative to environments that are not directly managed by humans. In general, this leads to a decrease in specialist organisms that survive under a narrow range of conditions, and promotes generalists that are able to tolerate a broader range of conditions. We do not know whether this occurs among soil microorganisms (e.g. bacteria and fungi), but certain management practices (e.g. nutrient applications) impose pressures that occur in cycles that may repeat seasonally or annually. Microorganisms must contend with these changes, and those that can thrive under a wider range of conditions can potentially remain active for a longer period each year. It is often assumed that many functions can be performed equally well by many different microorganisms, but some functions are not widespread, and studies of larger organisms (e.g. birds) suggest that specialist organisms in their home environment often outperform generalists. As a result, if human-imposed pressures cause soil communities to become dominated by generalist organisms, this could have consequences for microbial functions across landscapes.In this project, I will transfer communities between soil environments under different conditions in order to collect pools of microorganisms with different ecological traits. We will assess whether this selection leads to functional changes in these pools, with our main goal being to understand whether generalists can replace the function of specialists. A microorganism could be a generalist with respect to many aspects of the environment, but here we focus on microbes that thrive across a broad range of 1) temperatures, 2) resources, and 3) habitat types, which are all relevant to agriculture.In Aim 1, we will select pools of generalists or generalists + specialists by either maintaining a consistent environment (i.e. conditions do not change), or by imposing low or high degrees of environmental variability. In Aim 2, we will compare the function selected pools through assays of microbial growth, respiration, metabolism, and enzyme activity. In Aim 3, we will determine whether filtering for ecological types of microorganisms alters the pool that can be captured through culturing in the lab, and ultimately used for microbial product development.Our overall goals are to increase functional and ecological annotations of abundant sequence data, and to better inform microbial management in agriculture.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110107050%
1020110110050%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil;

Field Of Science
1070 - Ecology; 1100 - Bacteriology;
Goals / Objectives
The primary objective of this proposal is to determine how agriculturally-imposed selection pressures alter the composition and function of actively dispersing soil microbial pools. A secondary objective is to determine whether controlled manipulation of these pressures can facilitate cultivation of generalist microbial strains, which could allow for the production of more reliably effective microbial products for agriculture.These objectives will be achieved through three main Aims:Aim 1: Direct the microbial colonization of recipient soils by alternating environmental conditions after each community transfer event. Aim 1a. Alternating temperature Aim 1b. Alternating macronutrient concentrations Aim 1c. Alternating habitat typeAim 2: Assess the functional performance of selected microbial assemblages. Aim 2a. Habitat occupancy: biomass growth Aim 2b. Function: metabolic assays, respiration, and extracellular enzyme activity Aim 2c. Genetic differences: taxonomic comparison and genome projectionsAim 3: Determine whether filtering for particular functional subsets of a microbial community changes the cultivable pool of bacteria. Aim 3a. Contrast the composition of isolates from selected pools and initial soils using common media Aim 3b. Assess metabolic capacity of cultivable pool
Project Methods
Aim 1: Direct microbial colonization of recipient soils by alternating environmental conditions. We will use repeated transfer of microbial communities to sterile soils across consistent or variable abiotic environments. We will vary our definition of generalist in two ways: 1) extent of environmental change tolerated (low or high amplitude), and 2) aspect of the environment to which the organisms tolerate fluctuation (temperature, macronutrient concentration, habitat type).Recipient soils are the sterile soils in which microbes will grow. We will target soil with pH 6.0-7.0. ~40 L of soil will be collected, sieved to 2 mm, mixed 1:1 with sand, and placed in a walk-in freezer. When needed for use in microbial selection systems, batches of soil:sand mix will be sterilized. Source soils are non-sterile soils that provide the initial microbial inocula for selection under varied environmental conditions. We will allow microbes to colonize from multiple sources in our first Generation. Source soils will be collected from 20 locations with moderate soil pH, with 10 from agricultural sites and 10 from unmanaged sites. Soils will be homogenized and added as inoculum in Generation 1. In parallel, we will use 3 soils as inoculum individually.For each sub-Aim, we will transfer microbial assemblages from one environment to the next every 3 weeks, in what we refer to as Generations, for 6 Generations. Microbes will either be passaged across Generations under consistent environmental conditions, or variable environmental conditions with low or high amplitude shifts. These are defined differently for each sub-Aim.Aim 1a. Alternating temperature conditions. Petri dishes will be filled with sterile recipient soil, and 1 g source soil will be spread at one side as inoculum. Recipient soil will be water saturated to near-capacity. After 3 weeks in temperature- and humidity-controlled chambers, we will remove recipient soil and transfer it to a new microcosm, where it will serve as source soil.Selection will occur under 7 prescribed conditions, each replicated 4 times. Each variable environment treatment will be performed in two sequences, with either the lowest temperature or highest temperature imposed first. Each will be repeated with four initial inocula.Following Generation 6, we will sample each recipient soil for DNA extraction and sequencing. We will perform a final transfer under standardized conditions, with all microcosms incubated at 20°C to facilitate cross-comparison of selected microbial pools.Aim 1b. Alternating macronutrient concentrations. For each generation, soils will receive no fertilizer, 175 mg/kg urea (Low fertilizer), or 500 mg/kg fertilizer (High fertilizer). Cycling will be as in Aim 1a, but with nutrient differences between Generations, and temperature held at 20°C.Aim 1c. Alternating habitat type. For each Generation, microbes will be added to sterile soil, a soil:water slurry, or river water. River water will be 0.2 μm filter-sterilized. Slurries will be 25% soil and 75% water (v/v), whereas water microcosms will be inoculated with soil at a ratio of 1:1000. When transfers occur from slurry to soil or water to soil, we will agitate to homogenize, and then add 1 ml to one side of the next microcosm.To assess microbial community composition, we will apply high-throughput sequencing of universal bacterial (16S rRNA gene) and fungal (ITS) DNA markers. DNA will be extracted with Nucleospin 96 Soil kits, followed by PCR amplification with bacterial and fungal primers, and sequencing on an Illumina MiSeq. Initial data processing will be performed with DADA2.Aim 2: Assess functional performance of selected microbial assemblages. We will test the multi-functionality of our collected assemblages to determine whether pools of generalists can replace the function of mixed generalist-specialist pools under different environmental conditions.To assess function, we will work with microbial pools generated from the mixed inoculum of 20 soils. Replicates will be combined to maximize richness (functional assays will be replicated, but seeded from one pool). For microbes selected under variable conditions, we will combine pools from the same selection treatment, but opposite sequence.Through initial assessment of sequence data in Aim 1, we will determine the difference in richness between pools and will use dilution-to-extinction to modify microbial richness in mixed generalist-specialist pools. Functional assays will be performed under the conditions imposed in Aim 1.Prior to performing functional assays, all assemblages will be grown under the test conditions for 4 weeks. We will use Qubit quantification to determine community biomass, and will normalize biomass transferred to new sterile environments. Following this transfer, pools will be grown for 8 weeks before functional assessment. Each pool will be replicated 4 times. Biomass will be assessed through Qubit quantification of extractable DNA at 2, 4, 8, and 24 weeks.To assess microbial pool transformation of available resources, we use three complementary approaches. First, we will assess in situ metabolism using MicroResp with various substrates added. Second, we will assess respiration without supplied resources. Finally, we will assay a selection of extracellular enzymes.Sequencing data from Aim 1 will be analyzed to 1) determine whether there is a phylogenetic signal of generalism, 2) infer putative community functions, and 3) assess the extent to which generalists dominate agricultural landscapes of different types.We will first broadly compare the phyla and classes of bacteria and fungi selected through each approach. We will then apply R packages that test for significant phylogenetic signal in traits. In addition to testing the phylogenetic signal of each type of generalism, we will identify taxa that appear to be multi-generalists, due to their appearance in response to multiple selection pressures.To infer putative functions, we will annotate sequences using FUNGuild and PICRUSt2, as well as manual annotations from the literature. PICRUSt2 also assigns closest genome matches in the IMG/M database, which we will use to estimate average genome size.Finally, we will compare our identified taxa to publicly available datasets. We will generate ratios of the abundance of taxa observed in our generalist pools with the abundance of taxa in the mixed pools not identified in generalist pools. This provides a ratio of known generalists and specialists that we can contrast across land and management types.Aim 3: Does filtering for functional subsets of a microbial community change the cultivable bacterial pool? We will determine whether selection for assemblages that tolerate environmental fluctuations shifts the cultivable pool of bacteria.We will collect isolates from the mix of twenty soils used as inoculum in Aim 1 and from each of the generalist pools selected within the high amplitude variability environments. Isolates will be captured on 5 types of media targeted to maximize cultivable diversity.At sampling, all visible colonies will be picked and added to a low concentration saline buffer. This will be done for each medium type and dilution. A portion of this solution will be added to sterile soils and regrown for functional assessment and long-term storage. We will then extract the other portion for 16S rRNA gene sequencing.Since all microbes selected are cultivable, we can apply cultivation-dependent metabolic assays produced by Biolog. In addition, we will specifically compare the function of temperature generalist isolates with the initial isolate pool at 10°C, 20°C, and 30°C to determine whether they function more consistently across the temperature range.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:In this reporting period, we mainly reached academic audiences through a number of presentations, a paper published in FEMS Microbiology (led by Sarah Richards) and co-authored by Jeremy Sutherland. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, this project has provided training opportunities to 2 students (Sarah Richards, Ph.D. candidate and Keya Thumar, Professional master in biotechnology) and one post-doc (Nina Camillone). One undergrad student (Dylan Bellissimo) also had an opportunity to assist on projects and learn from the primary trainees. Sarah Richards and Keya Thumar presented a poster each at the One Health Microbiome Symposium on their results related to this project, this is an expert meeting focused on the role of microbiomes under the one health umbrella that was held in May 2024. How have the results been disseminated to communities of interest?Main dissemination in this period has been through the presentation of this work in two posters at the One Health Microbiome Symposium. What do you plan to do during the next reporting period to accomplish the goals?this is the last period of the award.

Impacts
What was accomplished under these goals? Please note that due to the change of PI the last report was submitted in August 2023 and this report only reports on the following and last 8 months of the award until the end of Arpil 2024. Aim 1 The habitat type publication by Sarah Richards was revised according to reviewers commenst and published during this period. Aim 2 This was the main focus of this last period: -Sarah Richards has worked with an undergrad student to optimize the phosphate solubilization assay that is used to measure the ability of Priesta megaterium (Pmeg) to solubilize phosphate. Pmeg is a plant growth-promoting bacterium, it action as such is linked to its ability to solubilize phosphate, making it bioavailable for the plant roots. It is important to measure it P solubilization capabilities through experimental evolution. Sarah optimized the assay, which is a plate assay along with the image analysis needed to quantitatively assess P solubilization. -Sarah Richards collected the second year of the microbial traps experiment, where she burried sterile soil in bags accross 10 cover crop treatments in a randomized block design within an organically managed agricultural research farm. She performed 16S and ITS analysis of the bulk soil and recolonized community to look at the influence of plants on the actively recolonizing data. Preliminary data indicate that in the first year the main effect was plants / no plant and this effect was also present in the second year. However the type of cover crop did not impact the recolonizing microbial community. Sarah is preparing a manuscript on this experiment to be submitted in fall 2024. -Keya Thumar a master in biotechnology student was hired and worked closely with Sarah Richards to perform a directed evolution experiment on a soil extract using 4 different carbon substrates (and mix of them) over 190 generations. The goal was to see if a complex community from soil would respond differently to selection by various substrates as a way to condition a soil inoculant with current soil members using the same substrate. After doing 16S analysis we found that for all carbon sources, the main bottleneck in terms of diversity section happened after 7 days in culture, all the community stabilized around 8-10 species. Surprisingly, the community selected with a large range of carbon substrate did not maintain more diversity than the other ones. Aim 3 -We hired a postdoc for a short-term project aiming at using fluorescent activated cell sorting (FACS) to profile cells populations from native soil before and after the soil has been incubated with 4 different carbon substrates. we were hoping to use the forward scatter channel as a proxy of cell size, however, we ran into some technical issues and had to step back and do some method development. We realized that the side scatter could be a better proxy for cell size unlike what is reported in the literature. we had to redo the experiment and optimize the incubation time therefore we did not get to sort within the time frame of the award. -Cultured assemblages (from high-throughput cultivation performed by Sean O'Rourke) and soil sources were sequenced during the previous period. This data is being analysed ?

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Leveraging aquatic-terrestrial interfaces to capture putative habitat generalists SC Richards, WL King, JL Sutherland, TH Bell FEMS Microbiology Letters 371, fnae025


Progress 05/01/20 to 04/30/24

Outputs
Target Audience:The target audience reached throughout the award was mostly academics through numerous formal (conferences talks and posters) and informal presentations, published articles and a master thesis published by Miranda DePriest. Changes/Problems:This project has switched to a new lead PI (Estelle Couradeau) in the third year, as the original PI moved to a new job at the University of Toronto, so was ineligible to hold this funding. Both the former PI and current PI continued to advise the main student working on this project (Sarah Richards) to help her in continuing towards project objectives. Sarah Richards was awarded a Nifa USDA Predoc in June 2023 and she switched her effort partially to this new award which goals were aligned this is why we ended up only spending about 60% of the award and are returning the rest. What opportunities for training and professional development has the project provided? Throughout the entire duration of the award, the project provided training and professional development opportunities for a total of 11 trainees: 7 students (Miranda DePriest, Sarah Richards, Caylon Yates, Jeremy Sutherland, Keya Thumar, and 2 undergraduate students: Cullen Wilson and Vedha Viddam) 2 technicians (Sean O'Rourke and Hanh Tran) 1 post-doc (Nina Camillone) These trainees had opportunities to present their work at various conferences, symposiums, and virtual events, as well as learn from each other and contribute to the overall success of the project. How have the results been disseminated to communities of interest? The project has actively disseminated its research findings and results through various channels during its duration. The main dissemination efforts include: Peer-reviewed publications in scientific journals, such as FEMS Microbiology Ecology. Presentations to industry partners, including a mini-symposium with Indigo Ag. Industry-run webinars, such as those organized by GALT Inc. Farmer-facing talks, including a series run by Stroud Water Center. Poster presentations at expert meetings, such as the One Health Microbiome Symposium, where Sarah Richards and Keya Thumar presented their results related to the project. Presentations and an accepted Master's thesis (Miranda DePriest). Presentations and a recent manuscript submission in another period. These dissemination efforts have helped to share the project's findings with a wide range of stakeholders, including industry partners, farmers, and the scientific community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aim 1: Direct the microbial colonization of recipient soils by alternating environmental conditions after each community transfer event. Year 1: Cycling experiments were performed under three different sets of conditions: temperature, pH, and habitat type (soil to water and back). Sequences were received and analyzed for the temperature-focused project, while DNA extraction and sequence preparation were planned for the other two. Year 2: Lab work and sequencing were completed for all cycling experiments. Preliminary analysis showed that the order of cycling (e.g., low to high temperature) impacts microbiome trajectories. For the habitat type experiment, results indicated that the richness of bacteria that can colonize water from soil is higher when the source soil is collected from a soil-water interface. Year 3: Sequencing analysis and manuscript writeup were completed for the soil-to-water cycling experiment. Results showed that only a few taxa (members of the Devosia and Micrococcaceae) were identified across all microcosms and demonstrated transitions from both soil to water and water to soil. Year 4: The habitat type publication was revised and published. Aim 2: Assess the functional performance of selected microbial assemblages. Year 1: The focus was solely on genetic analysis (shifts in microbiome composition). Limited access to the lab delayed functional assays. Year 2: Functional assays using MicroResp were initiated for the pH cycling experiment. More controlled assays in culture and soil systems were started to manipulate the diversity of environmental conditions. Year 3: A complex conditioning experiment was devised to have more precise control over conditioning of microbes prior to introduction to soils and functional testing. Sarah Richards, a Ph.D. student, led this work and validated assays for assessing biomass growth and function, including an assay to look at P solubilizing activity. Year 4: The main focus was on optimizing the phosphate solubilization assay used to measure the ability of Priesta megaterium (Pmeg) to solubilize phosphate. Sarah Richards also collected the second year of the microbial traps experiment and performed 16S and ITS analysis. Preliminary data showed that plants/no plant was the main effect on the recolonizing microbial community. A directed evolution experiment on a soil extract using 4 different carbon substrates was also conducted. Aim 3: Determine whether filtering for particular functional subsets of a microbial community changes the cultivable pool of bacteria. Year 1: Through a collaboration with GALT Inc, mass amounts of bacteria were cultivated from microbial pools grown up in soils of varying pH. The cultivable pool was found to vary substantially depending on the source soil in which bacteria are grown. Year 2: Extensive work was done to set up microcosms to passage microbiomes from source to recipient soils. High-throughput GALT cultivation was performed on 40+ samples, with the goal of comparing these outputs through sequencing. Year 3: Cultured assemblages and soil sources were sequenced. However, progress on this aim was limited due to a focus on Aim 2 and loss of project personnel. Year 4: A short-term project was initiated to use fluorescent activated cell sorting (FACS) to profile cells populations from native soil before and after incubation with 4 different carbon substrates. However, due to technical issues and method development, the experiment had to be redone and optimized, and sorting was not completed within the time frame of the award.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: TH Bell. 2024. The influence of near-term conditioning on microbial survival and function in phytobiomes. International Phytobiomes Conference, St. Louis, MI, USA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: TH Bell. 2024. The impacts of differential environmental conditioning on microbiome performance. Canadian Society of Microbiologists Meeting, London, ON, Canada
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: SC Richards, KHThumar, RJ Roman, WL King, TH Bell, E Couradeau, Directed Evolution of Beneficial Microbial Inoculant to Improve in-soil Survival, The ISME Conference, South Africa, 2024 - poster
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: SC Richards, LY Cao, E Couradeau, TH Bell, WL King, Differentiating active microbial colonizers under various cover crop mixes at the farm-scale, One Health Microbiome Symposium, PA, 2024 - poster
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: KH Thumar, SC Richards,J Roman, WL King, TH Bell, E Couradeau, Directed Evolution of Beneficial Microbial Inoculant to Improve in-soil Survival - One Health Microbiome Symposium, PA, 2024 - poster


Progress 05/01/22 to 04/30/23

Outputs
Target Audience: In this reporting period, we mainly reached academic audiences through a number of presentations, a paper published in Functional Ecology (led by Caylon Yates), and a recently submitted manuscript led by Sarah Richards and co-authored by Jeremy Sutherland. Changes/Problems: This project has switched to a new lead PI (Estelle Couradeau), as the original PI moved to a new job at the University of Toronto, so was ineligible to hold this funding. Both the former PI and current PI continue to advise the main student working on this project (Sarah Richards) to help her in continuing towards project objectives. In addition, the technician on the project (Sean O'Rourke) had been managing the culture-focused work, but moved to another job at Temple University. What opportunities for training and professional development has the project provided? This project has provided training opportunities for four students (Miranda DePriest, MS awarded; Sarah Richards, Ph.D. Candidate; Caylon Yates, Ph.D. awarded; Jeremy Sutherland, Ph.D. awarded) and two technicians (Sean O'Rourke and Hanh Tran). Two undergraduate students (Cullen Wilson and Vedha Viddam) also had the opportunity to assist on projects and learn from the primary trainees. Sarah Richards gave conference presentations on this work at multiple venues. How have the results been disseminated to communities of interest? Main dissemination in this period has been through presentations and a recent manuscript submission. What do you plan to do during the next reporting period to accomplish the goals? Aim 1 We will follow up on the habitat type publication that was recently submitted by Sarah Richards, with revisions as needed. Aim 2 This is our main focus moving forward. Specifically, Sarah Richards has begun mentoring an undergraduate to validate an assay that can detect changes in phosphate solubilization ability of a microbial strain that undergoes environmental conditioning in liquid culture. -We will hire a research assistant(20h / week) focusing on aim 2b. To assess how microbial assemblages' functional potential varies with environmental fluctuations, we will extract cells from agricultural soil and incubate them with four individual carbon substrates as well as a mixture of them, for a total of 5 incubation conditions and 5 replicate mixtures. We will passage the mixture every day for a total duration of 2 months. We will use the standard Biolog EcoPlate assay to assess the functional potential of the community before and after conditioning the various substrates. We will freeze a subset of the community once every week throughout the time of the experiment and will determine if 16 rRNA amplicon sequencing would be valuable to further characterize the evolution of the mix communities. We expect that microbial diversity will decrease along the course of the experiment as the members of the communities who can use the provided substrates more efficiently will have a fitness advantage. We expect that the multi-substrate condition will maintain a more diverse community of microbes as compared to single substrate. Finally, we will be able to see if the final community varies among replicates which would indicate that stochastic processes also come into play at the initial step of environmental filtering. This experiment has the potential to inform how the preconditioning of soil microbial inoculant in the context of precision ag approach where the inoculant would originate from the target soil. - Sarah Richards, PhD student on the project, previously devised an experiment to understand the effect of different plant species on microbial composition of active soil colonizers (which are more likely to represent the fraction of the soil microbiome contributing to soil functions). To accomplish this, she constructed and buried microbial traps (i.e., sterile soil enclosed by permeable mesh membranes) for in-field recolonization across 10 cover crop treatments in a randomized block design within an organically managed agricultural research farm. Moving forward her focus will be to collect these soil traps and perform amplicon sequencing of the 16S and ITS gene regions to observe how different soil conditions - influenced by various cover crops - drive patterns in microbial community assembly in soils. Aim 3 -We will hire a research assistant (20h/week) to advance aim 3. Building from our experience using high throughput cultivation from soil we realized that it would be more effective to profile the community as a whole and wait for it to shift before attempting to cultivate. We thereforewill use fluorescent activated cell sorting (FACS) to profile cells populations from native soil before and after the soil has been incubated with 4 different carbon substrates. We will use the forward scatter channel (FSC) as a proxy for cell size and sort 3 different cell fractions that we will plate on 0.1% R2A to perform colony count and point PCR on the colony using Sanger sequencing for taxonomic identification. We expect that as the soil gets conditioned, dormant cells will re-activate which may change the FSC pattern and in turn constrain the cultivability of microbes from the soil. The community composition of the original common soil source will be determined using 16S rRNA amplicon sequencing from the soil total DNA extract and soil total cell extract DNA (not accounting for soil DNA).

Impacts
What was accomplished under these goals? Aim 1 We completed sequencing analysis and manuscript writeup for experiment cycling microbiomes between soil and water; this work was recently submitted to FEMS Microbiology Letters While we showed that a large number of microbial types are capable of transitioning from soil -> water and vice versa, only a couple of taxa (members of the Devosia and Micrococcaceae) were identified across all microcosms and demonstrated transitions from both soil to water and water to soil Aim 2 Based on our initial work, we aimed to have more precise control over conditioning of microbes prior to introduction to soils and functional testing Sarah Richards, PhD student on the project, devised a complex conditioning experiment, that alternates a plant growth-promoting bacterium (one already shown in our group to be responsive to short-term environmental conditioning) between various resource and stressor states. Sarah completed a substantial amount of work in pre-testing this system and validating assays for assessing biomass growth and function, including an assay to look a P solubilizing activity, which is directly relevant to plant growth promotion. Sarah also wrote up and received a USDA predoctoral fellowship to support this and additional work looking at the impact of environmental resource manipulation on in-field microbiome assembly. We also published a paper on how switching bacteria to new soil types shifts their in-soil performance, with different effects on different taxa. This study was led by a PhD student, Caylon Yates. Aim 3 Cultured assemblages (from high-throughput cultivation performed by Sean O'Rourke) and soil sources were sequenced. Some analysis has occurred on this project, but progress on this aim has been more limited, due to a focus on Aim 2 and loss of project personnel.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Yates C, Trexler RV, Bonet I, King WL, Hockett KL, Bell TH. 2022. Rapid niche shifts in bacteria following conditioning in novel soil environments. Functional Ecology 36: 3085-3095.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Richards S. 2022. Differentiating active microbial colonizers under cover crop mixes at the farm-scale. International Phytobiomes; Denver, Calorado. USA. * Awarded 2nd place for poster and flash talk
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Richards S. 2022. Potential for Microbial Habitat Generalism at the Aquatic Terrestrial Interface. Changing Microbiomes Symposium; University Park, PA. USA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Bell TH. 2022. How directed evolution in complex environments can reshape bacterial niche breadth. Canadian Society of Microbiologists Conference, Guelph, ON, Canada.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bell TH. 2023. For better or worse, we can change microbes quickly. Western University, London, ON, Canada.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bell TH, Kaminsky LM, King WL, Yates C, Richards S, Cao L. 2023. Factors shaping the success and failure of microbial management in soils. Canadian Society of Microbiologists Meeting, Halifax, NS, Canada.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bell TH. 2023. Stretching microbial traits to meet soil management goals. Universit� de Sherbrooke, Sherbrooke, QC, Canada.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bell TH. 2023. Can we change a microbes idea of home? Agriculture and Agri-Food Canada, Ottawa, ON, Canada.


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:In this reporting period, we mainly reached academic audiences through a number of presentations at institutes in both the US and Canada, as well as a Master's thesis published by Miranda DePriest, a trainee who worked on the project. Changes/Problems:We are still working to get back to where we would like to be, given the work lag created by COVID. There is also some turnover on the project, with our newly hired technician moving to a new position and a graduating student (Miranda DePriest) moving off the project. These personnel changes have led us to prioritize completion of existing work, as opposed to beginning certain assays (e.g. functional tests) that we had otherwise planned for this period. What opportunities for training and professional development has the project provided?This project has provided training opportunities for two students (Miranda DePriest, MS thesis accepted; Sarah Richards, Ph.D. Candidate) and a technician (Sean O'Rourke). Two undergraduate students (Cullen Wilson and Vedha Viddam) also had the opportunity to assist on projects and learn from the primary trainees. Sarah Richards has had an abstract accepted to give a conference presentation on this work, which will occur in May 2022. How have the results been disseminated to communities of interest?Main dissemination in this period has been through presentations and an accepted MS thesis (Miranda DePriest). What do you plan to do during the next reporting period to accomplish the goals?Aim 1 - We are working to more closely control conditions in hybrid culture/soil systems. These systems are currently being proofed to understand how conditions impact microbial growth over various time periods. - A publication is in development for the habitat type cycling experiment (Sarah Richards) and we expect this to be completed within the next reporting period. Aim 2 - In the systems described above, we will also look to assess functional shifts in microbes and microbiomes in response to manipulated conditions. Initially we are focusing on growth rate, but will also assess other factors such as carbon resource use. - In addition, we will assess functional change in previously collected microbiomes, such as those produced in the pH cycling experiment. Aim 3 - We are focused on completing sequencing for this Aim so that we can assess the extent to which pre-filtering impacts cultivable diversity in bacteria. Sequencing prep is ongoing.

Impacts
What was accomplished under these goals? Aim 1 - Lab work and sequencing completed for temperature, pH, and habitat type cycling experiments. - Sequencing analysis for temperature, pH, habitat type (soil to water and back) was mostly completed. - Results for sequencing analysis for temperature and pH cycling are written up in a preliminary form in the Master's thesis of Miranda DePriest. One of our more interesting findings was that order of cycling (e.g. going from low to high temperature as opposed to the reverse) impacts microbiome trajectories. This implies that the sequence of environmental change or farm manipulations can lead to different outcomes for soil microorganisms. - Sequencing results for the habitat type experiment are being written up for a manuscript by Sarah Richards, Ph.D. Candidate. Interestingly, we found that the richness of bacteria that can colonize water from soil appears to be higher when the source soil is collected from a soil-water interface. Aim 2 - initial genomic projections are ongoing. - Functional assays using MicroResp have begun for the pH cycling experiment. - Have started initiating more controlled assays in culture and soil systems that allow us to manipulate the diversity of environmental conditions (Sarah Richards). Aim 3 - We have done extensive work here in setting up microcosms to passage microbiomes from source to recipient soils. - High-throughput GALT cultivation has been done on 40+ samples, as well as direct DNA extractions from soils, with the goal of comparing these outputs through sequencing. Work on this project carried out by Sean O'Rourke.

Publications

  • Type: Theses/Dissertations Status: Accepted Year Published: 2022 Citation: DePriest M. 2022. The effect of environmental heterogeneity on bacteria within an agricultural soil community. Master's Thesis, Ecology IGDP, Penn State University
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH. 2021. Microbial niche space constrains microbiome manipulation. Cornell University, Ithaca, NY, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH. 2021. Microbiome manipulation depends entirely on microbial niche space. Millersville University, Millersville, PA, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH. 2021. Exploring and extending microbial niche space. The Microbiome Centers Consortium Seminar Series.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH. 2021. Microbiomes are changing, so where do we want them to go? University of New Hampshire, Durham, NH, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Bell TH. 2022. How niche breadth can unlock microbial solutions to global change. University of Toronto, Scarborough, ON, Canada.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Bell TH. 2022. Leveraging microbial niche breadth to enhance food system security. Wilfrid Laurier University, Waterloo, ON, Canada.


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:- Academic audiences - Industry collaborators - Farming professionals Changes/Problems:Obviously COVID work has been a huge challenge. We had no lab access until July and after that, could only have 1 person per lab and only 3 total per week (each week we could choose which 3 people had access). Given that I had 9 people to cycle through, this was a huge barrier. We've been allowed 2 people per lab since the beginning of 2021, with no limit on number of people per week. One of the students supported by the project (Sarah Richards) started in September and has yet to be trained to the degree that I would like. With labs opening up more, her skills will grow, as will her ability to help move projects ahead. We've also noticed that one of the experiments (temperature switching) provided too many opportunities for contamination of sterile soil, so we are rethinking our approach to limit the opening of microcosms, or to change where and how we do this. What opportunities for training and professional development has the project provided?This project is training two Ph.D. students (Miranda DePriest and Sarah Richards). So far, neither student has been substantially supported by the project, as both had additional outside awards. However, student stipend will be charged to the award starting in the next reporting year. These students have had opportunities to present virtually in the past year, including through a mini-symposium with Indigo Ag and a farmer-facing web series run by Stroud Water Center. We have also been delayed in hiring technical help for this project due to hiring freezes at Penn State and smallapplicant pools. However, we have finally identified a technician to assist on the project, who will be split 50% with another lab, allowing us to stretch their time of contribution to the project. How have the results been disseminated to communities of interest?- Publications in peer-reviewed articles (FEMS Microbiology Ecology). - Presentations with industry (Indigo Ag mini-symposium). - Industry-run webinars (GALT Inc.). - Farmer-facing talks (Stroud Water Series). What do you plan to do during the next reporting period to accomplish the goals?Aim 1 - In addition to completing our ongoing cycling experiments, we are going to look at opportunities to perform more controlled cycling. For example, we are thinking about cycling between soil and culture environments, which impacts the survival and success of isolates in establishing in agricultural systems. Aim 2 - Continue DNA extraction and sequencing prep for projects for which this has not been completed. - Assuming greater lab access, perform functional assays, including habitat occupancy and extracellular enzyme activity under varying conditions. We have been validating our EEA protocols through other related projects. Aim 3 - Since the cultivation experiment we performed was based on a few frozen samples, we expect to look at this in a more robust experimental design. We have recently purchased a GALT isolation platform for our Department, and plan to use it for this purpose.

Impacts
What was accomplished under these goals? Aim 1 - We have performed cycling under three different sets of conditions: temperature, pH, and habitat type (soil to water and back). - For the temperature-focused project, we have received sequences, which we are currently analyzing. For the other two, DNA extraction and sequence prep will happen over the next few months. Aim 2 - At this point, we are solely focused on the genetic analysis (shifts in microbiome composition). Limited access to the lab has made it hard to initiate and conduct functional assays, so those have been delayed. Aim 3 - Through a collaboration with GALT Inc, we were actually able to cultivate mass amounts of bacteria from microbial pools grown up insoils of varying pH. Originally our goal was to do this using standard agar plates, but this collaboration dramatically enhanced our throughput. These pools were then contrasted with cultivable bacteria from the source soil using LoopSeq sequencing. This assay showed that the cultivable pool does vary substantially, depending on the source soil in which bacteria are grown.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Bell TH, Bell T. 2021. Many roads to bacterial generalism. FEMS Microbiology Ecology 97: fiaa240.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH. 2021. Defining, shifting, and leveraging the niche breadth of microorganisms. McMaster University, Hamilton, ON, Canada
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH. 2021. Using environmental filtering to collect ecologically-relevant microbes. General Automation Lab Technologies Webinar.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH, Kaminsky LM, DePriest M. 2021. Can we alter the survival range of microbial products? Indigo Ag, Boston, MA, USA.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Bell TH, Kaminsky LM, Richards S. 2021. The present and future of boosting soil health through microbial management. Stroud Water Research Center, Avondale, PA, USA.