Progress 01/01/18 to 12/31/22
Outputs
Target Audience:Target audience for this project are food producers and consumers, farm managers, fellow scientists, graduate and undergraduate students. Close interaction with members of the grape and tree fruit industry through extension activities will assure that results and recommendations are effectively disseminated to growers through several avenues. Outreach is conducted through existing websites, newsletters, blogs, and presentations at extension and scientific meetings. Furthermore, this project offers experiential learning opportunities for undergraduate and graduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Lily Cao, MS student, Ecology: Graduate student Cao accomplished tasks related to Objective 2. Cao was trained in and performed root analysis (e.g., separate roots by order, scan roots) and sample preparation prior to sequencing and metabolomic analysis,and is currently performing the required data analysis.Cao also presented her research findings at two academic conferences Suzanne Fleishman, Post-doctoral fellow:accomplished tasks related to publishing 3 manuscripts (Objective 1 and 3). Fleishman also presented her research findingsat several academic conferences. Meredith Persico, PhD Candidate, Plant Science:accomplished tasks related to publishing one manuscript(Objective 1). Persicoalso presented her research findings nt at one academic conference. Two undergraduate students (Hunter Tini and Grant Hoffer)assisted the graduate student(Cao) and PIs with field and laboratory tasks associated with the project. How have the results been disseminated to communities of interest?The results have been disseminated in the form of 11 presentations to the academic community (scientists, graduate and undergraduate students), one outreach presentation to stakeholders,one podcast, and 4 peer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: In 2022, major activities were focused on the analysis and summary of data collected tobetter define the role of major root traits (e.g., age, branching order, type) in structuring root and rhizosphere microbial composition, and publication of our findingsin three peer-reviewed manuscripts (see Products: Fleishman et al., 2022 and2023; Persico et al. 2023). Through this work and publications, we aimed to improve our understanding of factors contributing to variation in root-associated microbial communities. In Fleishman et al. 2022 (Functionally-explicit sampling can answer key questions about the specificity of plant-microbe interactions) we described a review of 377 "rhizosphere microbiome" research papers and demonstrate how matching a sampling method to the biological question can advance our understanding of host-microbe interactions in a functionally heterogeneous environment. Importantly, refined methods that are better matched to research questions will allow more sensitive detection of a) Heritability in microbial recruitment and b) Identification of the key rhizosphere microbes that shape plant health. In Fleishman et al. 2023 (Assessing microbial communities across the fine root landscape) we demonstrated how spatially explicit sampling yields unique insights into the relative importance of factors structuring root-associated microbial communities. For example, when comparing the factor categories in our prototype study, we found a higher importance of spatial factors (root clusters and spatial trends) than of the four root traits examined (age, branching order, and color). If our roots had been pooled rather than examined individually, the spatially derived variation at the scale of individual roots would not have been captured, leading to greater importance placed on root traits (i.e., a higher R2) than an individual root actually experiences. Our results suggest that the root traits that influence the microbial community of an individual root may be overshadowed by spatially structured factors, such as background soil conditions. In Persico et al. 2023 (The age of absorptive roots impacts root-adjacent microbial composition in grapevines) we investigated whether first-order root age impacts bacterial and fungal variation adjacent to the root surface, and if root age should be integrated into future studies on root functional traits and associated microbes. Overall, we found that microbial composition was distinct between young and old absorptive roots, with stronger evidence at the phylum and ASV taxonomic levels for fungi than bacteria.In contrast to the distinct shifts in microbial composition, there was little evidence of shifts in alpha diversity (i.e., observed ASVs and Shannon diversity) between young and old roots. Our study suggests that future work on the impacts of root functional traits on localized microbial composition may improve results interpretation and reduce some variation by accounting for root age at sampling. Collectively, our work improved our understanding of factors contributing to variation in root-associated microbial communities. While the high complexity of the rhizosphere creates logistical challenges,we suggested that unambiguous language and refined approaches will improve our ability to match methods to research questions and advance our understanding of the specificity of plant-microbial interactions. Objective 2: Our work (2021 and 2022, two-year study)addressed two major research questions: 1) How do cover crop and grapevine root interactions impact the soil microbiome and root traits and 2) How do soil nutrient levels affect the extent cover crop influence has on the soil microbiome and root traits? Briefly, in July 2021 and 2022, grapevine roots were dug up and placed into mesh bags with soil.Prepared grass cover crop patches were placed on top of the grapevine root mesh bags. This process was repeated but with different combinations of grapevine root and cover crop presence, giving 4 different treatment groups (Cover Crop x Grapevine Root, Cover Crop x No Grapevine Root, No Cover Crop x Grapevine Root, No Cover Crop x No Grapevine Root). This process was further repeated, in 2021 only, with the addition of organic nitrogen fertilizer. Sample collection occurred 5 weeks (2021) and 7 weeks (2022) after initiating the field experiment. Data analysis for the 2021ans 2022 experiments occurred in 2022. This study was led by Lily Cao (MS student in Ecology) who was supported by this grant in 2021 ad 2022. In 2022, she presented findings from this work at several conferences (see products) and she is currently summrizing this work in her MS thesis which will be defended in June 2023. Soil fertility test were done on bulk soil to examine shifts in soil conditions and nutrients. To analyze the microbial composition changes due to cover crop and nutrient addition treatments, 16s rRNA gene and ITS region amplicon sequencing was conducted to examine taxonomic and abundance shifts with bacteria and fungi. Metatranscriptomics was conducted on soil samples from 2022 to examine the functional changes in the soil microbiome that occurred due to cover crop treatment. In both years, we found that the alpha diversity of the soil microbiome did not differ between cover crop treatments when comparing within specific soil habitats, but beta diversity did. In the rhizosphere, cover crops shifted bacterial composition with no nitrogen amendments, while cover crop influence was overshadowed by nitrogen amendments. Fungal composition in the rhizosphere did not change with cover crop treatments. In the bulk soil, cover crops shifted bacterial and fungal composition with and without nitrogen amendments. In both rhizosphere and bulk soil, nitrogen amendments changed bacterial and fungal composition regardless of cover crop treatment. In summary, we found that in a temperate vineyard in Pennsylvania, groundcover management practices substantially influenced rhizosphere bacteria and fungi. However, effects of cover crop on grapevine rhizosphere microbiome can be overshadowed by other factors like nitrogen amendments. Objective 3: In 2022, major activities were focused on the analysis and summary of data collected from our field (vineyard) studies which were summarized in a chapter of the PhD dissertation thesis of Suzanne Fleishman and in a peer-reviewed manuscript (see Products: Fleishman, 2022 PhD thesis, Fleishman et al., 2023). Briefly,little is known on how the groundcover practices, increasingly used in perennial agroecosystems, alter root-associated microbial community structure, particularly for bacteria or at deep depths. This study investigated how increasing plant diversity by planting groundcovers in vineyards can alter grapevine rhizosphere microbes with soil depth for two different rootstocks. Groundcover practices altered rhizosphere bacteria and fungi in contrasting ways and findings were dependent on the microbial taxonomic level, soil depth and growing season. Grapevine rhizosphere bacteria responded to groundcover practices by increasing diversity 20% and shifting composition in ways similar across the soil profile. In contrast, rhizosphere fungi had the strongest responses to groundcover practices in the shallowest soil zone where groundcover roots are densest. In this shallow zone there was marginal evidence that rhizosphere fungal diversity was substantially decreased by 39% in the first year of the study and fungal composition shifted in the second year of the study. In our study, soil depth (0-1 m) was a stronger and more consistent factor structuring grapevine rhizosphere microbial composition than groundcover management practices. Therefore, sampling soil (bulk and rhizophore) and roots exclusively from shallow soil depths (e.g., 0-30 cm) in vineyards or orchards might not provide a clear picture of the effects of management practices on soil microbial community abundance and diversity.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Belowground interactions at multiple scales in a grapevine-groundcover agroecosystem
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Fleishman, S. M., Eissenstat, D., Bell, T., & Centinari, M. (2022). Functionally-explicit sampling can answer key questions about the specificity of plant-microbe interactions. Environmental Microbiome. DOI: https://doi.org/10.1186/s40793-022-00445-x
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Fleishman, S. M., Eissenstat, D., Bell, T., & Centinari, M. (2023) Assessing microbial communities across the fine root landscape. Journal of Experimental Botany. DOI: https://doi.org/10.1093/jxb/erad019
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Fleishman, S. M., Eissenstat, D., Hoffer, G., & Centinari, M. (2023). Cover crops and a devigorating rootstock can impart substantial agroecosystem services to high-resource vineyards: a multi-year study. Agriculture, Ecosystems and the Environment. DOI: https://doi.org/10.1016/j.agee.2022.108287
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Persico, M., Fleishman, S. M., Eissenstat, D., Bell, T., & Centinari, M. (2023) The age of absorptive roots impacts root-adjacent microbial composition in grapevines. Phytobiomes. https://doi.org/10.1094/PBIOMES-11-22-0078-SC
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Centinari, M. (July 2022) "How under-vine cover crops could improve sustainable production practices," [Invited Presentation], International Cool Climate Wine Symposium, St. Catharines, Ontario, CA. International.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Fleishman, S., Eissenstat, D., Cantu, D., Bell, T., Centinari, M. (September 2022). "Agroecosystem management modifies the rhizosphere microbiome via soil and host responses: an integrated study of host physiology and root multiomics," [Oral presentation], International Phytobiomes Conference, Denver, Colorado.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Cao, Lily, Eissenstat, David, Centinari, Michela, and Bell, Terrence. (September 2022). "Manipulating the soil microbiome and root growth of grapevines with under-vine cover crops," [Flash Talk and Poster], International Phytobiomes Conference 2022
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Fleishman, S., Massonnet, M., Cantu, D., Eissenstat, D., Centinari, M. (July 24, 2022). "Direct comparisons of grapevine rootstock gene expression expand insights into root responses to nitrogen competition," [Oral presentation] Botany 2022, Anchorage, Alaska.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Cao, Lily, Eissenstat, David, Centinari, Michela, and Bell, Terrence. (July 2022). "Using under-vine cover crops to manipulate the soil microbiome and grapevine root physiology," [Oral Presentation], 8th International Symposium on Physiological Processes in Roots of Woody Plants.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Fleishman, S., Eissenstat, D., Cantu, D., Centinari, M. (July 2022). "A competitive groundcover alters grape rhizosphere and soil bacteria communities in an Eastern US Vineyard," [Oral presentation], 8th International Symposium on Physiological Processes in Roots of Woody Plants, Penn State, University Park.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Cao, L., Eissenstat, D., Centinari, M., Bell, T. (June 2022). "Manipulating the soil microbiome and root physiology through co-grown plant interactions," [Poster] The Changing Microbiome Symposium, Penn State, University Park.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Cao, L., Eissenstat, D., Centinari, M., Bell, T. (April 2023). Understanding the impacts of under-vine cover crop interactions with grapevines from a belowground microecological perspective [Oral Presentation]. 2023 Environmental Chemistry and Microbiology Student Symposium.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Extension Presentation, Pennsylvania Grape and Wine Industry Winter Conference, Pennsylvania Wine Marketing and Research Program Board, University Park, PA (March 2, 2022). Presentation title: "Review of practical considerations for under-trellis cover crops"
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Fleishman, S, W King, C Yates, M Persico, J Guo, M Centinari, T Bell, D Eissenstat."Opportunities to advance rhizosphere microbiome research with functionally and spatially explicit root sampling." Plant and Animal Genome Conference. 2022 Jan 12; Virtual. *Invited talk; PSU Microbiome Center Sponsored Speaker
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Persico, M., Fleishman, S., Eissenstat, D., Bell, T., Centinari, M. (July 2022). "Does root age impact microbial assemblage in the rhizoplane of absorptive grapevine roots?,"[Poster], 8th International Symposium on Physiological Processes in Roots of Woody Plants, Penn State, University Park. Awarded Second Place in Student Poster Competition.
|
Progress 01/01/21 to 12/31/21
Outputs
Target Audience:Target audience for this project are food producers and consumers, farm managers, fellow scientists, graduate and undergraduate students. Close interaction with members of the grape and tree fruit industry through extension activities will assure that results and recommendations are effectively disseminated to growers through several avenues. Outreach isconducted through existing websites, newsletters, blogs, and presentations at extension and scientific meetings. Furthermore, this project offersexperiential learning opportunities for undergraduate and graduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training opportunities to three graduate students (Lily Cao, Meredith Persico, and Suzanne Fleishman) and two undergraduate students (Raine Lookabilland Grant Hoffer) in the College of Agricultural Sciences at The Pennsylvania State University. Specifically: Lily Cao, MS student, Ecology: Graduate student Cao accomplished tasks related to Objective 2. Caowas trained in and performedroot analysis (e.g., separate roots by order, scanroots)and sample preparation prior to sequencing in Fall 2021 and is currently performing the required data analysis. Meredith Persico, PhD Candidate, Plant Science: Graduate student Persico accomplished tasks related to Objective 1, root age and microbial composition analysis. Persico was trained in and performed sample preparation prior to sequencing in Fall 2020 and is currently performing the required data analysis from Spring 2021 to date. Persico was enrolled in the course, "PPEM 440: Introduction to Microbiome Analysis" taught by co-PI Bell in spring 2021. She remains a member of the Data Analysis Working Group (DAWG) and Penn State and will be guest lecturing in PI Bell's "Microbial Analysis" course in April 2022. Suzanne Fleishman, PhD Candidate, Ecology: Graduate student Fleishman accomplished tasks related to drafting manuscripts "Functionally-explicit sampling can answer key questions about the specificity of plant-microbial interactions." and "Assessing microbial communities across the fine root landscape" (Objective 1). In Fall 2021, Fleishman was also selected as the "PSU Microbiome Center Sponsored Speaker" for the Plant and Animal Genome Conference which took place in January 2022. Graduate student Fleishman has also practiced peer-mentoring skills by advising graduate students Persico and Cao on their activities related to objectives 1 and 2, respectively. RaineLookabill and Grant Hoffer assisted the graduate students (Persico and Cao) and PIs with field and laboratory tasks associated with the project.?Grant Hoffer was the recipient of an undergraduate research award from the College of Agricultural Sciences (Fall 2021) to investigate how cover crops in vineyards influence juice chemistryand production parameters. How have the results been disseminated to communities of interest?The results have been disseminated in the form of one presentation to the academic community (scientists, graduate and undergraduate students), one outreach presentation to stakeholders, and onepeer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?In 2022, processed sequence data from root samples collected in 2021will be analyzed. We plan to finish data analysis for all objectives and submit four manuscripts to peer-reviewed journals based on this work and other data analyses we are conducting in relation to all objectives. We plan to present our work at least at one academic conference and at outreach meetings. The PIs and graduate students will meet in spring 2022 to decide if further field data collection is required based on results obtained.
Impacts
What was accomplished under these goals?
Objective 1: In Fall 2020, we began testing the research question, "How does root and rhizosphere microbial composition change in response to root age." We hypothesized microbial composition, with focus on bacteria and fungi, would shift as root characteristics change over root lifespan. The roots for this analysis were sampled in Summer 2018 from twenty-seven total vines, grafted on one of two rootstocks (medium and low vigor) and at one of two soil depths (topsoil and medium depth). Root preparation, DNA extraction, and sequencing was performed in Fall/Winter 2020. From Spring 2021 to date, bioinformatic analysis of the root rhizoplane 16S and ITS sequences has been conducted by graduate research assistant Meredith Persico, (PhD candidate in Horticulture) who was supported by this grant for the 2020-2021 academic year. A summary of the results was presented to the PIs and other students working on this project, Results are currently being summarized in a publication which will be submitted in 2022. Preliminary results suggest that fungal composition in the rhizoplane changes between "young" (<11 days) and "old" (11.5-40 days) first orderroots, but bacterial composition does not change among age categories. Persico is currently investigating which fungal phyla are responsible for the difference in composition between "young" and "old" age root categories. Results also suggest that "young" and "old" root categories do not affect the variability of root-associated bacterial or fungal communities (i.e., "young" roots do not have higher variation in bacterial composition than "old" roots). Overall, root age accounts for a small percentage of variation in bacterial and fungal composition, and the vine that the roots were sampled from more strongly influences both bacterial and fungal root-associated communities. Persico will be giving a guest lecture on research results in PI Bell's "Microbial Analysis" course at Penn State in April 2022 and submitting a poster abstract to the 8th International Symposium on the Physiology of Roots of Woody Plants in May 2022. During summer and fall 2021 two "Viewpoint" style manuscripts were drafted for winter 2022 submission. These manuscripts addressed the importance of accounting for the functional and spatial heterogeneity of root systems when sampling the rhizosphere microbiome. The manuscript "Functionally-explicit sampling can answer key questions about the specificity of plant-microbial interactions" reported and discussed a review of rhizosphere sampling methodologies and how current practices, which neglect functional heterogeneity (i.e., not based on root traits) within root systems, may not always capture the most useful information to address research questions. The manuscript "Assessing microbial communities across the fine root landscape" reported the results of the 2018 sampling of grapevine roots within a single experimental window from the field site to compare the importance of root traits (age, color, order, etc.) to spatial location for microbial community composition; the results of this study were used to suggest ways forward to account for functional and spatial heterogeneity in root systems. Objectives 2 and 3: In summer 2019 and 2020, bulk soils, grapevine roots and groundcover roots were sampled from root boxes for the full experimental design from the research vineyard at the Russel E. Larson Agriculture Research Center(Objectives 2 and 3). This included 3 depths up to 1 m, grapevines growing with and without groundcover, and on either medium or low rootstocks (32 grapevines total). Preliminary results from the 2019 sampling showed that rootstock had minimal impacts on grapevine rhizosphere community composition. However, groundcover competition increased grapevine rhizosphere bacterial diversity. In order to verify and expand upon the findings from the 2019 growing season, the experiment was replicated during the growing season of 2020. Sequences 16s and ITS sequences were received in December 2021. Currently, 2019 and 2020 sequences are being analyzed together and preliminary results suggest that the year of sampling, groundcover practices, rootstock practices, and soil depth each impact rhizosphere microbiomes differently; however, in a highly variable manner. In summer 2021, we set up a field experimentat the Russel E. Larson Agriculture Research Center research vineyard to further investigate how cover crop - grapevine root competition and nutrient dynamics affect the rhizppsphere and soil microbiome and root traits. This study has beenled by Lily Cao, MS student in Ecology, whohas been supported by this grant since July 2021. We used a field study system of Noiret grapevines and grass cover crops, where cover crop grass was transplanted on top of grapevine roots and nutrient amendments added in the form of organic nitrogen fertilizer. We hypothesize that 1) grapevine and grass cover crop root competition will 1) shift the soil microbiome, 2) change grapevine root growth and induce unique root metabolite production, and 3) have decreased impact on soil microbiome composition and root traits with soil nutrient additions. Briefly, in July 2021, grapevine roots were dug up and placed into mesh bags with soil. Prepared grass cover crop patches were placed on top of the grapevine root mesh bags. This process was repeated but with different combinations of grapevine root and cover crop presence, giving 4 different treatment groups (Cover Crop x Grapevine Root, Cover Crop x No Grapevine Root, No Cover Crop x Grapevine Root, No Cover Crop x No Grapevine Root). This process was further repeated with the addition of organic nitrogen fertilizer in the form of blood meal, giving a total of 8 treatment combintationand 96 mesh bags (12 bags for each treatment combination). Photos of the grapevine roots were taken prior to inserting into the mesh bags. In August 2021, after 5 weeks of the initial field set up, bulk soil, rhizosphere soil, and grapevine root samples were collected to perform soil microbial community, root traits, and nutrient amendment analysis. In September 2021, DNA was extracted from the bulk and rhizosphere samples. PCR amplification of the 16S rRNA gene and ITS1 gene regions were done. Between October 2021-November 2021, library prep for the 16S and ITS amplicon sequencing was done. In December 2021, the samples were sent to PSU Genomics Core for 16S and ITS amplicon sequencing.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Fleishman, S., Bock, H., Eissenstat, D., Bell, T., Centinari, M. (August 2021). "Replacing herbicides with vegetation in vineyards alters root distributions, edaphic factors, and microbial diversity in a depth-dependent manner," Annual Virtual Meeting of the Ecological Society for America, Ecological Society for America, On-Line.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Centinari, M. Research Trials on Marquette: Spring frost avoidance and under vine cover crops, Nebraska Wine and Grape Growers Association Annual Meeting, On-Line
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Vanden Heuvel J and Centinari M (2021). Under-vine vegetation mitigates the impacts of excessive precipitation in vineyards. Frontiers in Plant Science, 12:713135. https://doi.org/10.3389/fpls.2021.713135
|
Progress 01/01/20 to 12/31/20
Outputs
Target Audience:Target audience for this project are food producers and consumers, farm managers, fellow scientists, graduate and undergraduate students. Close interaction with members of the grape and tree fruit industry through extension activities will assure that results and recommendations will be effectively disseminated to growers through several avenues. Outreach will be conducted through existing websites, newsletters, blogs, and presentations at extension and scientific meetings. Furthermore, this project will offer experiential learning opportunities for undergraduate and graduate students. Changes/Problems:Due to the complete halt on all non-essential research activities at The Pennsylvania State University following the arrival of Covid-19 in Pennsylvania, we have experienced a significant impact on the timeline of this project. We had no access to labs from March through June 2020, with only one person per lab for months after that, only recently increasing to a possibility of two per lab, but with the expectation that we limit overlap time. This has really limited our access to laboratory space to perform laboratory analyses and to prepare samples for microbiome sequencing. In addition, we could not hire as much help in 2020 as anticipated because of Covid-19 restrictions. We are working with the grants and contracts office at our institution to submit no-cost project extension to December 31, 2022. An extension of the period of performance will allow us to hire the personnel necessary to complete field work in spring and summer of 2022 and all laboratory analyses for samples already collected. What opportunities for training and professional development has the project provided?The project provided training opportunities to three graduate students (Suzanne Fleishman, Meredith Persico, and Caylon Yates) and two undergraduate students (Hayden Bock and Grant Hoffer) in the College of Agricultural Sciences at The Pennsylvania State University. Specifically: Meredith Persico, PhD Candidate, Horticulture: Graduate student Persico accomplished tasks related to Objective 1, root age and microbial composition analysis. Persico was trained in and performed sample preparation prior to sequencing in Fall 2020 and is currently performing the required data analysis. To prepare for research activities associated with Objective 1, Persico completed the course, "PPEM 456: Microbial Ecology" in Fall 2020 and is currently enrolled in "PPEM 440: Introduction to Microbiome Analysis." In addition, she is an active member of the graduate student-led bioinformatics group, "Data Analysis Working Group (DAWG)," organized by the Penn State Microbiome Center; hands-on lab training and data-analysis, have also enhanced the skillset of Persico. Suzanne Fleishman, PhD Candidate, Ecology: From fall 2019-Summer 2020, graduate student Fleishman acted as a co-instructor for the Data Analysis Working Group (DAWG) in the Penn State Microbiome Center which involved collaborating with and educating other graduate students on microbiome data analysis skills. This training has also enabled Fleishman to practice peer-mentoring skills by advising graduate student Persico in tasks related to accomplishment of objective 1. Hayden Bock and Grant Hoffer assisted the graduate student (Fleishman) and PIs with field and laboratory tasks associated with the project. Grant Hoffer was the recipient of an undergraduate research award from the College of Agricultural Sciences (submitted in Fall 2020) to investigate how cover crops in vineyards influence soil nutrient and water availability and production parameters. How have the results been disseminated to communities of interest?The results have been disseminated in the form of one presentation to the academic community (scientists, graduate and undergraduate students), one outreach presentation to stakeholders, and three peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?In winter 2021, processed sequence data from root samples collected for objective 1 will be analyzed. We plan to submit three manuscripts to peer-reviewed journals based on this work and other data analyses we are conducting in relation to all objectives. We plan to present our work at least one academic conference and at outreach meetings. The PIs and graduate students will meet in spring 2021 to decide if further field data collection is required based on results obtained to date and approval of a no-cost project extension (see changes/problems section).
Impacts
What was accomplished under these goals?
Objective 1: In spring 2019, rhizoplane (i.e., soil adherent to the root surface) samples collected from individual roots (n = 103) from one root box window (20 cm wide and 30 cm long) were extracted, prepped, and submitted for sequencing. By spring 2020, both bacterial and fungal sequences were received and were examined for the relative contributions of root traits (i.e., color, branching order, age) and space on determining the composition of bacterial communities. The results from this analysis suggest that rhizoplane bacterial communities are highly variable and that root traits are less important than spatial factors in determining community composition. Interestingly, we also found that theamount of variation explained varies depending on the taxonomic classificationused, suggesting that phylogenetic scale is important in addition to spatiotemporal scale when examining rhizoplane bacterialcommunities. A manuscript discussingthe challenges and opportunities to disentangle the relative importance of ecological processes driving rhizosphere community assembly is currently in preparation for submission in spring 2021. In Fall 2020, we began addressing the research question "How does root and rhizosphere microbial composition change in response to root age?" We hypothesized that microbial composition, with focus on bacteria and fungi, would shift as the morphology and functions of roots change over their root lifespan. The roots for this analysis were sampled during the summer of 2018 at the research vineyard located at the Russell E. Larson Agricultural Research Center (The Pennsylvania State University) where the same Vitis genotype is grown on two different rootstocks with and without cover crops. Twenty-seven vines in the bare soil (without cover crop) treatment were selected for this experiment and all the roots were collected on the same day. Roots were accessed through the root boxes and sampled from both rootstocks (medium and low vigor) and two soil depths (shallow depth, 0-30 cm, and medium depth, 31-60 cm). At sampling, roots were classified by order (pioneer, 1st, 2nd, or 3rd) and ranged in age from 0.5 to 40 days old. In Fall 2020, roots from the medium vigor and shallow soil treatment were selected for subsequent microbial composition analysis; out of possible rootstock and soil sampling depths, this treatment combination (high vigor + topsoil) contained the highest number of total root samples, highest diversity of roots sampled from different vines, and largest number of 1st order roots, which was chosen for analysis to mitigate complications of introducing other factors, like root order, into the analysis. Root samples preparation, DNA extraction from rhizoplane soil, and sequencing was all performed in Fall 2020. One-hundred 1st order roots, representing various ages, were weighed, and sonicated to remove rhizoplane soil, from which subsequent microbial composition sequencing was performed. Primers were selected to amplify the 16S rRNA and ITS gene regions of each sample to identify bacterial and fungal taxa, respectively. After amplification, sample preparation, including bead cleanup and indexing, was performed before samples were sent for sequencing at the Cornell Genomics Core Facility. Bioinformatic analysis of the root rhizoplane 16S and ITS sequences is currently being conducted by Meredith Persico, PhD candidate in Horticulture, supported by this grant for the 2020-2021 academic year. In 2020 we also published a manuscript which focused on the effects of root branching orders (1st &2nd, 3rd, and 4th) on root-associated bacterial composition and abundance. Roots were collected in 2018 from six woody tree species (Acer saccharum, Carya glabra, Juglans nigra, Liriodendron tulipifera, Pinus strobus, Quercus rubra) grown for 23 years at a common garden located at the Russell E. Larson Agricultural Research Center (The Pennsylvania State University). We hypothesized that microbial filtering would be greatest in the most distal roots (1st & 2nd). We found that root order strongly impacted composition across tree species, with absorptive lower order roots exerting the greatest selective pressure. Microbial carrying capacity was higher in absorptive roots in two of three tested tree species. This study indicates lower order roots as the main point of microbial interaction with fine roots, suggesting that root homogenization could mask microbial recruitment signatures. Objectives 2 and 3: In 2020, we analyzed soil and root-associated bacterial (16S rRNA) sequencing data from bulk soils, grapevine roots and cover crop roots samples collected in summer 2019 from the research vineyard. Roots and soil were sampled from root boxes for the full experimental design. This included 3 depths up to 1 m, grapevines growing with and without cover crop, and on either medium or low-vigor rootstocks (32 grapevines total). In late 2019 DNA was extracted from bulk soils and rhizosphere soils from the roots and 16s (bacteria) sequences were received from the Cornell Genomics facility in spring 2020. Preliminary results show that rootstock had minimal impacts on grapevine rhizosphere community composition. However, presence of cover crop increased grapevine rhizosphere bacterial diversity. Overall, there was little evidence that cover crops influenced grapevines differently across depth or that the cover crop rhizosphere outcompeted the grapevine rhizosphere microbiome when roots were in close proximity. These results were presented at the Ecological Society of America meeting in August 2020. To gain further insight into the full microbial community for the study, the ITS (fungal) gene for all of the samples was amplified in 2020 and sequences were received in winter 2021. Grapevine and cover crop roots and soils collected in summer 2019 from soil cores were analyzed from fall 2019 to spring 2020 for root mass distributions (RMD), root length distributions (RLD), soil mineral macronutrients, total soil carbon (TC), and total soil nitrogen (TN). Results showed that RLD and RMD shifted in response to both cover crop and rootstock management practices. Cover crop root presence drastically increased TC and TN in shallow soil layers, but not deeper soil layers. Results from this study were published in the Agriculture Ecosystems and the Environment journal in January 2021.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
King WL*, Yates CF*, Guo J, Fleishman SF, Trexler RV, Centinari M, Bell TH, Eissenstat DM, (In prss). The hierarchy of root branching order determines bacterial composition, microbial carrying capacity and microbial filtering. Communications Biology.
*Authors contributed equally to the publication
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Fleishman SF*, Bock HW*, Eissenstat DM, Centinari M (In press) Undervine groundcover substantially increases shallow but not deep soil carbon in a temperate vineyard. Agric Ecosyst Environ. https://doi.org/10.1016/j.agee.2021.107362.
*Authors contributed equally to the publication.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Fleishman SF, Bell TH, Eissenstat DM, Centinari M. A competitive groundcover alters grape rhizosphere and soil bacteria communities in an Eastern US Vineyard. Ecological Society for America, 2020 Conference. 2020 August 3-6; Virtual Presentation
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Centinari M. How to utilize under-vine cover crops to reach your vineyard production goal. Cornell University, Lake Erie Regional Grape Program, Virtual Coffee Pot Meetings, On-Line, 25 participants.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
Yates CF*, Guo J*, Bell TH, Fleishman SF, Bock HW , Trexler RV, Eissenstat DM, Centinari M (In press). Tree-induced alterations to soil properties and root-associated microorganisms following 23 years in a common garden. Plant Soil. https://doi.org/10.1007/s11104-021-04846-8
*Authors contributed equally to the publication
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Progress 01/01/19 to 12/31/19
Outputs
Target Audience:Target audience for this project are food producers and consumers, farm managers, fellow scientists, graduate and undergraduate students. Close interaction with members of the grape and tree fruit industry through extension activities will assure that results and recommendations will be effectively disseminated to growers through several avenues. Outreach will be conducted through existing websites, newsletters, blogs, and presentations at extension and scientific meetings.Furthermore, this project will offer experiential learning opportunities for undergraduate and graduate students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided training opportunities to three graduate students (Suzanne Fleishman, Jing Guo, and Caylon Yates) and six undergraduate students (Hayden Bock, Sarah Brau, Erica Laveaga, Powen Pai, Alexa Sebastianelli, Ben Smith) in the College of Agricultural Sciences at The Pennsylvania State University. Specifically: Hayden Bock was the recipient of an undergraduate research award from the College of Agricultural Sciences to investigate how cover crops in vineyards influence soil nutrient availability, soil organic matter, and organic carbon (objectives 2 and 3). Erica Laveaga and Alexa Sebastianelli assisted the graduate student (Fleishman) and PIs with field and laboratory tasks associated with the project all year long. During the summer both Laveaga and Sebastianelli conducted an in-depth search in Web of Science for peer-reviewed articles on "rhizosphere microbiome." Over 160 articles were organized based on several parameters including plant species and type (e.g., annual, perennial) studied and sampling methods used. This information will be used for a review paper on rhizosphere microbiome sampling methods (objective 2). Powen Pai was trained to examine photographs of root box windows and evaluate feasible approaches for estimating root production and turnover (objective 3). Sarah Brau was trained to trace grapevine roots into ArcGIS software for mapping and spatial analysis (objective 1). Ben Smith conducted soil and root length measurements from 2019 soil core samples (objectives 2 and 3). How have the results been disseminated to communities of interest?The results have been disseminated in the form offour presentations to the academic community (scientists, graduate and undergraduate students) and one outreach publication for the general public. What do you plan to do during the next reporting period to accomplish the goals?In winter 2020, processed sequence data from root and soil samples collected in 2019 will be analyzed. The PIs and graduate students will meet in spring 2020 to plan field data collection for the 2020 season based on results obtained in 2019. We also plan to submit three manuscripts to peer-reviewed journals based on work conducted in 2018 and 2019 and present our work at several academic conferences and outreach meetings.
Impacts
What was accomplished under these goals?
In winter 2019, we tested the feasibility of characterizing root-associated bacterial composition at the centimeter scale (all objectives). We extracted DNA from varying amounts of root material taken from grapevines and grass (red fescue), two plant species that differ greatly in both root morphology and rooting density. We sought to understand: 1) if standard molecular approaches enable examination of root-associated bacterial communities at a centimeter scale? and 2) does the amount of root material used influence bacterial community composition and diversity metrics? Results suggested that there is not a methodological limitation to studying small amounts of root material (0.25 cm root length was the minimum amount tested). However, both the diversity and composition of the root-associated bacterial communities may differ with the amount of material used. Our results suggest that there should be an emphasis on standardizedsample size. These findings will guide us on how to sample roots for all obejctives and anlayze the data. Based on the results, a publication (short communication) will be submitted to Soil Biology and Biogeochemistry in 2020 (Challenges of estimating rhizosphere bacteria on a centimeter scale; Fleishman, S, T Bell, D Eissenstat, M Centinari). In winter 2019, we also analyzed root-associated bacterial (16S rRNA) sequencing data of three root branching orders (1st & 2nd, 3rd, and 4th) collected from six woody tree species (Acer saccharum, Carya glabra, Juglans nigra, Liriodendron tulipifera, Pinus strobus, Quercus rubra) grown for over twenty years at a common garden located at the Russell E. Larson Agricultural Research Center (The Pennsylvania State University). This experiment was conducted to confirm that root order is a trait that influences root-associated microbes across species (objective 1). The PIs and graduate students involved in the project (Suzanne Fleishman, Jing Guo, and Caylon Yates) are currently preparing two peer-reviewed publications based on the results of this work. In spring 2019, the rhizoplane (i.e., soil adherent to the root surface) samples collected from individual roots (n = 103) from one root box window (20 cm wide and 30 cm long) were extracted, prepped, and sent for bacterial (16S rRNA) sequencing. The rhizoplane bacteria samples were examined for the relative contributions of root traits (i.e. color, branching order, age) and space on determiningthe composition of bacterial communities (objective 1). Thus far, preliminary results suggest that, when examining rhizoplane bacteria on a fine scale, space may be a stronger predictor of the community composition than root characteristics. Spatial variation in bacterial composition was better explain when communities were examined at higher (i.e. phylum) rather than lower (i.e. ASV) taxonomic level. Among the root characteristics analyzed, root color may be the most important contributor to structuring rhizoplane bacterial composition. Furthermore, it is unclear if random or unmeasured factors (e.g. microhabitat, soil chemistry) may account for more variation than the factors analyzed, which warrants further investigations. Based on findings from "one window" rhizoplane bacteria composition, in fall 2019 the rhizoplane samples were prepped for fungal (ITS) sequencing to expand the examination to other microbial types. Sequencing data are expected to be received in early 2020. Preliminary results of this experiment were presented at the American Society for Enology and Viticulture - Eastern Section annual conference and at the seminar series of the Penn State Microbiome center. In summer 2019, root and soil sampling was conducted at the research vineyard located at the Russell E. Larson Agricultural Research Center (The Pennsylvania State University) where the same Vitis genotype is grown on two different rootstocks with and without cover crops (all objectives). Specifically: 1. Grapevine roots growing on one-side of each root box in the bare soil treatment were traced and sampled to replicate sampling conducted in 2018 (objective 1). 2. Cover crop (red fescue, Festuca rubra) and grapevine roots were sampled in all treatments from one side of all the boxes across all depths down to 1 m (objectives 2 and 3). Rhizoplane soil from red fescue and grapevine and bulk soil samples were prepped and sent for bacterial (16S rRNA) sequencing in the fall. Sequencing data are expcted to be recived in early (January, February) 2020. These results will be presented at the Ecological Society of America annual meeting in August 2020. 3. Sixty-four one-meter deep soil cores were taken in the area beneath the vines from all treatments. The cores were divided into six depth increments, and each depth was split in half vertically. One-half was set aside and frozen for microbial community analyses to allow for a methodological comparison between coring and root box methods (objectives 2 and 3). The other half was used for analysis of grapevine and grass root length, root mass, soil organic carbon, organic matter, and mineral nutrients in fall 2019. Preliminary results suggest that groundcover and rootstock practices can substantially alter rooting and resource patterns in vineyard soils. Cover crop (red fescue) improved parameters of soil health, such as soil organic carbon, shift soil-resource availability, and increase deep root growth. PIs, graduate student Suzanne Fleishman, and undergraduate student Hayden Bock are summarizing the main findings in a peer-reviewed publication.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Fleishman, S, D Eissenstat, T Bell, M Centinari. Do rootstock genotype, soil depth, and root traits structure community composition of bacteria associated with grapevine roots? American Society for Enology and Viticulture - Eastern Section Conference. 16 July 2019; Geneva, NY.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Fleishman, S, D Eissenstat, T Bell, M Centinari. Do root characteristics and space organize rhizosphere bacteria communities? Penn State University Microbiome Center Seminar. 15 November 2019; University Park, PA.
- Type:
Other
Status:
Published
Year Published:
2019
Citation:
"Fortifying fruit: harnessing soil and root microbiomes to boost crop productivity." Our research project was featured in: Retaking the Field Volume 4: Science Breakthroughs for Thriving Farms and a Healthier Nation (https://supportagresearch.org/assets/pdf/retaking-the-field-vol-4.pdf). Retaking the field is a collaborative report from 20 Fed By Science universities and the SoAR Foundation.
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Progress 01/01/18 to 12/31/18
Outputs
Target Audience:Target audience for this project are food producers and consumers, farm mconsumersanagers, fellow scientists, graduate and undergraduate students. Close interaction with members of the grape and tree fruit industry through extension activities will assure that results and recommendations will be effectively disseminated to growers through several avenues. Outreach will be conducted through existing websites, newsletters, blogs, and presentations at extension and scientific meetings. Furthermore, this project will offer experiential learning opportunities for undergraduate students, as for example the development of a research class project for the PPEM 497 course " Environmental Microbiomes: Concepts and Analysis Tools" instructed by co-PI Bell at The Pennsylvania State University. Changes/Problems:We decided to postpone root sampling for objective 2 (examine how proximity to grass cover crop roots shift the root-associated microbiome of a woody fruit crop species) to the 2019 growing season to allow for full, complete establishment of cover crops near the root boxes windows. We also agreed that, before proceeding with further root sampling, we needed to have a better understating on how the major root traits shape microbial composition. This knowledge will allow us to optimize technique for labor intensive root sampling in the 2019 season. What opportunities for training and professional development has the project provided?The project provided training opportunities to three graduate students (Suzanne Fleishman, Jing Guo and Caylon Yates) and three undergradute students (Franco Acevedo Lugo, Amanda Seow, Anastaysia Kowal). Franco Acevedo Lugo is an undergraduate student from the University of Puerto Rico at Mayagüez. He assisted the graduate student (Fleishman) with field work associated with the project for 8 weeks (02/06 - 07/28). Franco was enrolled in the SROP program (Summer research Opportunities Program), an 8-week gateway to graduate education at Big Ten Academic Alliance universities, which includes The Pennsylvania State University. The goal of the SROP program is to increase the number of under-represented students who pursue graduate study and research careers. Amanda Seow and Anastaysia Kowal are two undergraduate students in the College of Agricultural Sciences at The Pennsylvania State University; they both assisted the graduate student (Fleishman) and PIs with field and laboratory tasks associated with the project. All the graduate students associated with the project (Fleishman, Guo, Yates) participated in the course 'PPEM440 - Environmental Microbiomes: Concepts and Analysis Tool' developed and led by co-PI Bell. 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?In winter/ spring 2019, processed sequence data from root and soil smaples collected in 2018will be analyzed. Based on these results we will plan field data collection for the 2019 season to address objectives 1-3. We also plan to submit a manuscript to a peer-reviewed journal based on our work on the six woody tree species at the common garden.
Impacts
What was accomplished under these goals?
Winter/Spring 2018 A graduate student (Suzanne Fleishman, Ph.D. level) was recruited to work on the project. Suzanne Fleishman was admitted to the Ecology Intercollege Graduate Degree PhD Program at The Pennsylvania State University and awarded a University Graduate Fellowship (UGF) for the 2018-2019 academic year. Suzanne started her PhD in fall 2018; she is co-advised by PIs Centinari and Eissenstat and PI Bell is a member of her thesis committee. In winter 2018 we analyzed preliminary data from samples collected in 2017 from the experimental site (vineyard at the Russell E. Larson Agricultural Research Center). We performed 16S rRNA gene sequencing on root and soil samples, cover crop and no cover crop treatments, various soil depths, and different root orders. These data were central to student projects in a new microbiome analysis course at The Pennsylvania State University (designed and led by Co-PI Bell), which is now titled PPEM440 - Environmental Microbiomes: Concepts and Analysis Tools. Working with these data gave students first-hand experience in analyzing raw data, and their insights helped us to determine which factors had substantial impacts on microbiome composition. We observed decreases in bacterial diversity with increasing soil depth and were encouraged to observe differences in bacterial composition between different root orders (Objective 1 and 3). Based on these preliminary results, we decided to extend our 2018 root and soil sampling to other six woody tree species to confirm that root order is a trait that influences root-associated microbes across species (Objective 1). Jing Guo, a visiting PhD scholar from the School of Life Science, Northeast Normal University, China has been responsible for field and lab work associated with this research project, under the supervision of the PIs. Caylon Yated, PhD student in the Ecology Intercollege Graduate Degree PhD Program at The Pennsylvania State University adivsed by co-PI Bell, assisted with sample preparation for bacterial (16S rRNA gene) sequencing. Information on experiment set up and sample collection is reported in the 'Summer 2018' section. Finally, in spring of 2018, the technician and graduate student prepared the 64 root box sides/windows for monitoring root growth (photographs and tracing; all objectives) Summer 2018 At the beginning of the summer a method for photographing root boxes windows for all treatment combinations (32 vines) was developed. Photographs were then taken every other week throughout the summer. Photographs will be used to monitor root production and turnover for grapevines with and without cover crop and grafted on two rootstocks (Objective 3). Above-ground growth and production measurements were also conducted for all treatments as well as soil moisture measurements. To define how major root traits (e.g., age, branching order, type) and soil depth structure root and rhizosphere microbial composition (objectives 1 and 3), grapevine roots in the no cover crop treatment (16 vines; two rootstocks) were traced on 11 dates between June 21 and July 28, 2018. On July 28, 886 roots were sampled at two depths (0-30; 31-60cm), totaling 11 different age groups (from 1-day to 60-day old) and 3 types (pioneer, first order, second order). Thirty-two bulk soil samples were also collected on the same day for microbiome and nutrient analysis. To confirm that root order is a trait that influences root-associated microbes across species, we also sampled roots from six woody tree species (Acer saccharum, Carya glabra, Juglans nigra, Liriodendron tulipifera, Pinus strobus, Quercus rubra) grown at a common garden located at the Russell E. Larson Agricultural Research Center (Objective 1). Each tree species was planted in 1996 in eight replicated blocks. In each block, six individual trees were planted in monospecific plots, aligned in two rows and spaced 3 m apart with 5 m of spacing between neighboring plots. Roots from the six woody tree species and adherent soil were sampled over two days (July 3 and July 13) for a total of 108 root samples, divided in 3 branching orders (1st & 2nd, 3rd, and 4th) for each species. Bulk soil samples were also collected for each block to characterize soil environment (nutrient and chemical characteristics). Fall 2018 In Fall 2018, the graduate students associated with the project (Suzanne Fleishman, Jing Guo and Caylon Yates) tested methods for separating soil and roots in sonic water bath (all objectives). After developing a standardized method for soil-root separation, root-adhering soil samples from the six woody tree species were prepared for bacterial (16S rRNA) sequencing (objective 1). The graduate students also conducted a test to identify the minimum amount of root material required for quality sequencing information.
Publications
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