Mycoremediation of Phosphorus in Agricultural Runoff using Mycorrhizal-Plant Associations

Progress report for ONE19-335

Project Type: Partnership
Funds awarded in 2019: $29,981.00
Projected End Date: 07/31/2021
Grant Recipient: University of Vermont
Region: Northeast
State: Vermont
Project Leader:
Dr. Josef Görres
University Of Vermont
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Project Information

Project Objectives:

Project Objectives:

This project aims to discover whether myco-phytoremediation restoration strategies can decrease P loads entering the lake while discerning how restoration strategies affect native pollinator habitat. Myco-phytoremediation is a form of mitigation of soil pollution through microbial, fungal and plant symbionts. In this case, the fungi are mycorrhizae. This research focuses on: phosphorus in soil, water, and leaves and habitat diversity amidst the research plots. Data is gathered through both field and laboratory mesocosm experiments.

Field Experiments

Phosphorus (P):

  1. Investigate whether the 3 treatments (control of original vegetation, native restoration plantings with and without mycorrhizae) within the riparian zone affect soil water SRP concentrations in the root zones (Figs. 1-7).Installation SF figures

Hypothesis: There is a difference in SRP concentrations among the following treatments: Original buckthorn dominated (OIV), Restored vegetation without mycorrhizae (RV), Restored vegetation with mycorrhizae (RVM). SRP concentrations vary in soil water in this order OIV>RV>RVM.

2. Determine whether mycorrhizae result in increased P concentrations in willow plant tissue.

Hypothesis: P plant tissue concentrations will be greater in the restored area with mycorrhizae than the area without mycorrhizae

Habitat diversity:

  1. Determine plant community composition and plant species richness in the three plots.

This is to provide a baseline of the plant community composition for future research.

Mesocosm Experiments

Phosphorus (P):

  1. To measure the difference in soluble reactive phosphorus concentrations in soil water of each mesocosm treatment (inoculated willow, uninoculated willow, buckthorn).

Hypothesis: The SRP concentrations in soil water will follow this order: inoculated willow<uninoculated willow< buckthorn.

2. To measure the difference in P plant concentrations and biomass between the three treatments.

Hypothesis:  P concentrations in plants and biomass will be in this order: RVM>RV>OIV.

If these strategies succeed, farmers can replicate this model to decrease legacy P runoff through low cost green infrastructure systems, which increase climate change resilience, diversify habitat, provide increased nutrient retention, and edible, medicinal, biomass products.

Introduction:

Phosphorus pollution from agriculture is one of multiple water quality issues threatening freshwater health. In Vermont there are concerns about the agricultural impact on Lake Champlain’s health. As a limiting nutrient in freshwater ecosystems, excessive P causes algal blooms, which can involve toxic algae that threaten aquatic trophic health and impairs tourism through beach closures and stymied water-related activities. According to the EPA’s 2016 Phosphorus Total Maximum Daily Load (TMDL) agriculture accounts for an estimated 41% of base phosphorus loading for the Vermont portion of the Lake. Decades of P imports as animal feed and fertilizer have caused a buildup of excessive P concentrations in the watershed, referred to as legacy P (Roy, 2018). Lake Champlain received a D+ in its cleanup report card, stating there is limited data on agricultural practices’ ability to remove phosphorus pollution. (Weber, 2018) This project directly addresses this. 

In a climate change adaptation survey (White, 2018), 74% of responding farmers demonstrated awareness of the problem, stating that they build soil health and use cover crops to manage heavy precipitation and flooding on their farms. The respondents emphasize holistic approaches, like regenerative agriculture, agroecology and permaculture (keyline, hugelkultur, berms, swales, and earthworks). This data indicates that farmers are open to alternative practices proposed in this pilot. 

Green storm-water infrastructure is now an accepted tool of urban pollution mitigation. This is not yet true for agriculture. In particular, incorporating fungi in mitigation of agricultural non-point pollution is not yet an accepted practice. Cover crops and nutrient recommendations are commonly practiced. However, these BMPs do not mitigate legacy P runoff from fields and drainage ways. One strategy involves incorporation of arbuscular and ectomycorrhizae fungi which increase plant nutrient uptake efficiency (Phillips, 2017; Smith and Read, 2008; Jones, 2009). Innoculating plantings on field and drainage way edges may help mitigate legacy P. 

A survey of 21 VT farmers (18 certified or operationally organic) conducted by MycoEvolve (co-PI Rubin) indicates high farmer interest in mycoremediation. Seventeen of the 21 farmers (81%) are interested in mycoremediation strategies. Six would attend workshops at current pilot demonstration sites, 8 would attend public workshop highlighting research, 12 would consult fact sheets, 9 would take a class geared specifically to farmers, and 10 would access results in peer reviewed journal articles. However, concerns about implementing mycoremediation included: effect on climate change resiliency (5 respondents), cost (9), long term maintenance (11), potential hydrologic changes (3), displacing space geared towards productivity (6), and lack of knowledge about the topic (4). 

At Shelburne farms total P concentrations in drainage water is 10 to 100 times greater than recommended P concentrations for that section of the lake (Dana Bishop, unpublished data). This is in- spite of sustainable farm practices. Legacy P is suspected as a source, but other sources cannot be ruled out. Our project modifies buffers around a drainage way with restored native species, half of which are mycorrhizally inoculated. 

Storm-water designers, environmental managers and policy makers have also expressed interest in mycoremediation. For example, the town of Colchester hired Mycoevolve, a company specializing in ecological resilience and watershed restoration harnessing  fungi and plants, to treat storm water in a tributary to Lake Champlain. Since completion of the project other private sector and community stakeholders have contacted MycoEvolve with interest in strategies to mitigate P runoff and enhance pollinator habitat.

Cooperators

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Research

Materials and methods:

The purpose of this grant is to test hypotheses that mycorrhizal vegetation replacing invasive species within and adjacent to agricultural drainage channels can remediate legacy P in drainage discharge (measured as soluble reactive phosphorus, SRP).

Pre-installation

Preliminary soil samples were collected to characterize pH, fertility and total P concentrations in soils. Preliminary soil and native tree root samples were gathered to determine original mycorrhizae (arbuscular (AMF) and ectomycorrhizae (ECM) presence in the three experimental plots. Our root sample microscopy studies from pre-installation samples indicate there was no ECM present before installation. Therefore, our qualitative studies on mycorrhizae will focus on AMF. During the month of January 2021 soil from pre and post installation from both the field and laboratory studies will be conducted.

Site preparation included removal of invasive plants of buckthorn (Rhamnus cathartica) from February through May 2020. We collaborated with Shelburne Farms staff in this effort. Removal techniques involved hand tools for root extraction where nonnative species were more than 2-ft of the drainage way. All nonnative species closer to drainageway banks were cut down at hip height and left to be cut back repeatedly throughout the season. This technique (Mike Bald of Got Weeds, personal correspondence) creates a 90% death rate without use of chemicals, mechanic equipment and disturbing soil microbial communities.

Meanwhile polyculture plantings and installation design were drafted by Jessica Rubin, ordered and purchased from VT Wetland Plants. Low-P perennial mix from VT compost was steam pasteurized and left to equilibrate for 6 weeks. All purchased native plants were potted up in this compost; half with Mycorrhizal Applications’ EndoEcto mix blended into the compost. Plants remained in pots for 2 months for acclimatization.

A 12-foot deer fence was erected around both experimental plots with corresponding latched doors to enter an exit. The control plot of nonnative buckthorn between the two experimental plots was not surrounded by a fence.

Six piezometer holes were dug in early spring (outside of) on either side of the channel adjacent to the treatment areas (RV, OIV, RVM). Using an auger I dug down 20 inches past where redox features appeared (approximate depth to seasonal high water table). I left the holes for a week to observe base flow water entering these holes. I then measured the depth to water from the top of the hole. Each auger hole was surveyed against a common elevation datum. Ground water elevation in the auger holes was calculated as the elevation of the auger hole minus the depth to ground water at that point. Triangulation was used to find the ground water elevation surface and ground water flow direction. We found that the flow of the water was from the NE to the SW, hydrologically connecting the composting area and the field below it with our study site.

Installation

At the end of May 2020, once all plots were prepared and plants acclimatized, Vermont Youth Conservation Corps (VYCC) accompanied Jess and community volunteers in raking the soil in the experimental plots, seeding both restoration plots with a native riparian seed mix (RVM plot had inoculated seed), laid down jute erosion control mat and following the planting map planted all native species, inoculated and uninoculated according to their assigned plot. Everything was watered in and consistently watered with the help of Shelburne Farms staff throughout the summer and fall in between rain events, that were less than 1 inch over 24 hours, via water truck provided by the farm.

Data Gathering Post installation

Objective A: investigating whether the 3 treatments within the riparian zone affected soil water SRP concentrations in root zones. Collect soil – water with suction cup lysimeters in the root zone of willows to measure soil water SRP concentrations.  After each storm event  that delivered > .5  inches of rainfall, we collected soil water from 6 suction cup lysimeters in each treatment placed randomly near willow stems at restored sites and near buckthorn saplings of similar size in the plot with the original invasive vegetation. Each sample was filtered through .45 um syringe filters prior to analysis to remove any coloring agents. The samples were then stored frozen until analyzed for SRP by the Environmental Testing Lab at UVM.

Objective B: determining whether mycorrhizae increase P concentrations in willow plant tissue. To measure P concentration in willow leaves, 8 leaves per willow (total of 48 leaves) were gathered from the RV and RVM plots mid and late summer (July & August). Leaves were dried at 60 C, ground and then analyzed via the UVM AET Lab for P concentration in leaves.

Objective C: determining plant species richness and plant diversity in the three plots. All of this data was gathered once a month starting in July through November 2020, after plantings had 6 weeks to acclimatize to being transplanted.

 To determine plant species richness, I set up four transects parallel to the waterway which were placed at random distances from the water way.  Random distances from stream bank were generated using the Excel random number generator for each sampling time. All species along each transect were then recorded. Richness will be determined as the number of species present.. Richness will be recorded  as the compilation of all the different species found along the transects.

To capture plant diversity I set up five quadrats per plot (0.5 by 0.5 m quadrat). These were randomly placed (x steps along length and y steps across width). Random numbers to determine both X and Y were generated using the Excel random number generator each time. Species in each quadrat were identified and their abundance recorded using the Shannon-Weaver Diversty Index.

Both species richness and diversity from 2020 data will be calculated and analyzed in January 2021.

Objective D: Determining the difference in SRP concentrations in soil water of each mesocosm treatment (inoculated willow, uninoculated willow, buckthorn). Field plants were repotted in 6.5-cm diameter, 20-cm long conetainers  in pasteurized low-P compost mix which had acclimatized for 6 weeks. There were three treatments: Pussy willows (Salix discolor) were planted in 5 mesocosms inoculated with mycorrhizae, and 5 mesocosms that were not inoculated. Five more mesocosms were planted with buckthorn saplings harvested from the control plot that were

similar in size to the pussy willows; the medium in these pots were also not inoculated. The total soil volume in the conetainers was approximately 800 mL and pore volume was estimated as 400 mL (50% porosity). Twice a week 14 mg/L of SRP was added to the mesocosms via fertigation. The effect of plant treatments on soil water SRP was measured by leaching events induced every week or two over an eleven-week period. Amount of water added to and leaving each mesocosm as well as leachate color was recorded. All outflow was filtered through .45 um syringe filters. The samples were stored on ice and frozen until analyzed for SRP by the Environmental Testing Lab at UVM.

Since preferential flow existed as indicated by different collection volumes and  varying colors of leachate in each mesocosm, these trials will be conducted again in spring 2021 with the following adjustments: 1. The contact between the medium and the conetainer walls will be sealed with bentonite clay to prevent macropore flow along the mesocosm walls. 2. Leachate will be collected to represent the same pore volume for all containers.

Objective E: measuring the difference in P plant concentrations and biomass between the three treatments in mesocosm experiments.

To assess whether the mycorrhizae-plant association results in increased plant uptake of P, entire plants will be sampled at the end of the experiment in June 2021. At the end of the experiment, the plants will be harvested  and dried separately for stems, roots and leaves. Each sample thus obtained will be placed in paper bags and subsequently dried at 60 C. After drying, each sample will be weighed and ground prior to analysis. Then P concentrations will be measured separately for roots, shoots, and leaves. Biomass P will be calculated as the product of the dried biomass and P concentration for each sample.

Participation Summary
1 Farmer participating in research

Education & Outreach Activities and Participation Summary

8 Curricula, factsheets or educational tools
1 Journal articles
2 On-farm demonstrations
1 Published press articles, newsletters
6 Tours
2 Webinars / talks / presentations
13 Workshop field days
4 Several folks walking the trail during installation or when I was gathering data that asked about the project and received impromptu infomration. A few individuals emailed me who had seen the project or heard about the work and asked questions about the field of myco-phytoremediation.

Several educational photos were taken throughout the spring, summer and fall highlighting the project and different aspects of it. These were posted on instagram and facebook.

Participation Summary

5 Farmers
4 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Shelburne Farms participated by providing site preparation (cutting buckthorn trunks) in late winter-early spring and in helping with water assistance throughout the summer and fall. Participation by local groups was discouraged by the University due to Covid-19 crisis and in agreement with Vermont Governor’s orders. MycoEvolve did host a series (13) of physically distant work parties over late winter and early spring to assist in site preparation of the Shelburne Farms project site. 

August/September 2019: Site Analysis: Jess Rubin walked site with UVM Extension consultant, Dana Bishop Farmer, and Water Resource Manager Consultant Lauren, and soil scientist Josef Gorres. Coordinate with Shelburne Farms on the final research and landscape design. Secured project with VYCC and walked site with Justin Geibel. Output: Drafted design outline and created series of maps: site map, contours, hydrology, installation plan.

October/November 2019: Ground truthed measurement, ground truthed soil morphology through soil pit study, laid out design, took initial water and soil samples in existing drainage way and throughout site. Conducted soil morphology and hydrological analysis. Output: identified SRP and TP hotspot for installation location. Created an initial site analysis report with recommendations and site limitations.

November/December 2019: Coordinate with Shelburne Farms and VYCC installation details, spring/summer schedule. Began intern search. Began plant palette and mycorrhizae species orders. Located companies to order plants, mycorrhizae and erosion materials from. Output: solid plan for spring summer installation. Planting sketch and initial palette. Rough draft of methodology data tables created.

January- April 2020: Shelburne Farms Staff chainsawed the trunks off all buckthorn species in the restoration plots. Jess via MycoEvolve (since UVM research was shut down to COVID) organized 12 physically distant work parties in which 11 community members and two homeschool families helped remove the trunks and limbs into piles to be chipped and then removed roots of all buckthorn species in the restoration sites more than 2 ft from the waterways. Some of these community members also helped to erect the deer fence and construct the doors.

March 2020: polyculture planting designs were completed. Plants were ordered from VT Wetland Supply. Low P perennial soil mix was pasteurized and brought to sit at the UVM Horticulture Farm. Jess offered an educational workshop 3 days before COVID broke out in Vermont at Fletcher Free Library in Burlington sharing with the local community about this project. Article in ‘7Days’ came out which highlighted the project.

https://www.sevendaysvt.com/vermont/vermonters-face-stormwater-threat-with-fungi-rain-gardens-and-more/Content?oid=29872924&fbclid=IwAR3RiKTw7HJe7IgH9CuT-a0_SOXJ_cugAB32SsKv31vXHR7lysn7vEi4UJY

April 2020: half of pasteurized soil mix was inoculated with Mycorrhizal Applications Endo/Ecto mix and the other half were kept uninoculated.  Bare roots plants from VT Wetland Supply were repotted in their corresponding treatment soil types for both field and mesocosm experiments.

May 2020: Jess and an intern gathered soil and native tree roots samples for pH, soil Total P concentrations and to evaluate mycorrhizal presence pre-installation in treatment plots. All soil and roots for mycorrhizal analysis were placed them in the fridge to be analyzed over December 2020 break via microscopy. In late May VYCC and community volunteers assisted with installation and watering. A short video was created by Red Fox Media that circulated facebook.

June-November 2020: treatment plots (1760 ft2 of the two treatment areas ) were watered via Shelburne Farms’ water truck in between adequately sized storms (>1inch over 24 hr period) with 510 gallons once a week. This calculation is based on the maximum evaporation rate of 1 inch with .5 crop coefficient. Field data of monthly pH tests, plant species richness and diversity data, storm based lysimeter testing, and two Pleaf tissue sampling were gathered. During this time mesocosm experiments were also conducted. Jessica shared about this project to Dr. Eric Roy’s Bionutrient lab via a zoom presentation. An article for the International Journal of Environmental Research and Public Health was written by Jess with assistance from advisor Josef during this time.

August 2020: Abenaki members of the Alnobaiwi, a 503C dedicated to preserving Abenaki cultural heritage conducted a ceremony at the restoration research site with clan mothers. 12 community members (several of who helped in site preparation) attended the event. Six individual tours were offered to individuals who expressed interest in the project. Interview with DC Extinction rebellion covered the project when interviewing Jess Rubin about restoration work with which she is involved through a youtube video.

September 2020: a group of students from a UVM introductory class in environmental science were given a tour of the project. They gathered data to use for their class project through which they learned the basics about mycoremediation and data analysis.

December 2020: The journal article (https://www.mdpi.com/1660-4601/18/1/7) was accepted after a few rounds of edits by the IJERP and published on 12/20. All 2020 data is being analyzed via JUMP software over school vacation. Mycorrhizal presence is also being quantified during this time via microscopy. 2020 final report is written and submitted. New SARE partnership grant is being written to continue the research beyond a year.

Learning Outcomes

4 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation
Key areas in which farmers reported changes in knowledge, attitude, skills and/or awareness:

The following is more about what we learned. We are still waiting for more data before we can share something tangible to farmers.

Our 2020 field season revealed a few patterns.

In terms of soil, there was no significant difference in soil pH between the plots. The pH values varied between 3.13 –6.28. The average pH was 4.82 However over time the soil pH decreased by an average of 1.20 pH units in all plots. This may be due to seasonal biogeochemical changes.

In terms of soluble reactive phosphorus (SRP) concentrations in sol water, the variations within treatments is less than the variations between the treatments suggesting a real treatment effect. There was a significant difference in the SRP between the inoculated and uninoculated plots; the mean SRP of the inoculated plot was greater than of the uninoculated plot. This is opposite of what we expected and may be due to various field variables we are still in the process of understanding such as differing levels of shade, and P inputs from uplands. or greater differences in legacy P

In terms of P concentrations in the willows in the two restoration plots, data from two separate samplings indicated the leaves in the inoculated plots had smaller concentrations of P than the uninoculated plots. This is opposite of what we expected and may be due to various field variables we are still in the process of understanding, including the potential that stores of P in the roots have not been evaluated,different inputs of P from uplands. and legacy P. Over time from early summer to mid autumn in both plots the leaf P-leaf concentrations increased.

We hope the second field season in 2021 will shed more light on the processes in the plots. We are planning to investigate soil nutrient content in the plots to evaluate any difference in soil fertility that could explain the unexpected effect of treatments on P concentrations in soil water and leaves.

In terms of our mesocosm studies, the overall trend was that leachate from the buckthorn had the least SRP while the inoculated had the most SRP. This may be due to the fact that the buckthorn may have still had its AMF partners in its roots while the restored willows either did not in case of the uninoculated or were just being established in terms of the inoculated conetainer. Over time the SRP concentrations seemed to decrease until the end of the early fall when the concentrations started to rise. This may be due to the seedlings growth beginning to take up P and then releasing it once they began entering senescence.

In the second year, we will make slight changes to the mesocosm experiment. For example we will seal the soil around the edges of the mesocosms to prevent leachate from finding macropores along the conetainer walls bypassing the soil. We hope the second lab season in 2021 and our improved methodology will shed more light on the processes in the mesocosms.

Project Outcomes

2 Grants applied for that built upon this project
2 Grants received that built upon this project
3 New working collaborations
Project outcomes:

Josef and Jess published the following paper: Citation: Rubin, J.A.; Görres, J.H. Potential for Mycorrhizae-Assisted Phytoremediation of Phosphorus for Improved Water Quality. Int. J. Environ. Res. Public Health 2021, 18, 7. https://dx.doi.org/doi:10.3390/ ijerph18010007

https://www.mdpi.com/1660-4601/18/1/7

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.