Mycorrhizal Banks to Enhance Vegetable Yield and Reduce Water Quality Impairment by Mitigating Excessive Soil Phosphorus

Progress report for ONE21-391

Project Type: Partnership
Funds awarded in 2021: $29,994.00
Projected End Date: 07/31/2023
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:

From September 2021 to January 2022, we have been planning our experiments at Digger's Mirth Farm in the Intervale, Burlington, VT.  After exploring the field and our farm partner cropping schedule, we had to revise our initial objectives and plan of work. Originally our Objectives were

Overarching objective: We propose to help mycorrhizal colonization of tilled soils by providing “mycorrhizal banks” from where mycorrhizae spread into fields and thus increase crop phosphorus efficiency.

.Specific Objectives:

  1. Test whether mycorrhizae spread into a field from the mycorrhizae bank

               This will be tested by extracting and counting mycorrhizae hyphae in the soil at different distances from the bank. This will give us information on an optimal spacing between banks. The hypothesis is that mycorrhizae are more abundant close to the bank but will spread further as time goes by.

  1. Test whether plant phosphorus content varies with distance from the mycorrhizae bank

               Our hypothesis is that plant P decreases with distance from the bank and is positively correlated with soil hyphal counts .

  1. Test whether water extractable phosphorus varies with the distance from the edge of the bank and whether it is correlated with mycorrhizal densities and phosphorus plant uptake.

               Our hypothesis is that mycorrhizal symbiosis will still be established in soils with excessive soil test phosphorus. Hence the concentrations of water extractable phosphorus will be lower closer to the mycorrhizal buffers.

4: Test how much phosphorus is in coppiced woody vegetation grown in the mycorrhizal bank.

 

Our new objectives were: Overarching objective: We propose to help mycorrhizal colonization of tilled soils by providing “mycorrhizal banks” from where mycorrhizae spread into fields and thus increase crop phosphorus efficiency.

Year 1: Effect of Mycorrhizal bank on parsley

  1. Test whether mycorrhizae spread into a field from the mycorrhizae bank.
  2. Test whether plant phosphorus content varies with vegetated strip treatment (inoculated and uninoculated strip areas)
  3. Test whether water extractable phosphorus concentrations is less in the soil adjacent to the mycorrhizal bank than next to the unioculated verge and whether it is correlated with mycorrhizal densities and phosphorus plant uptake.

  Year 2: Effect on Cover Crops

A cover crop is maintained in the field for most of the growing season in year 2.

         4. Test whether cover crop has overwintered the mycorrhizal community.

After 6 consultations and two site visits with our farm partner the original objectives were no longer feasible. The sticking points were that the field was going to be opened, tilled and planted in sections and that the crops originally planned for this project were going to be planted later in the growing season. After some discussion, we agreed that we would use parsley as our experimental crop.  We replaced Original Objective 1, in which we proposed to measure the movement of mycorrhizae into the field to different distances, with New Objective 1: Test whether mycorrhizae spread into a field from the mycorrhizal bank. The difference here is that we are measuring the time course of mycorrhizal abundance in seed beds close to the bank.

Original objectives 2 and 3 remained the same, although the experimental design changed.

Original object 4 changed. We no longer use woody plants in the mycorrhizal bank so that there remains free access to the field by farm machinery. Instead of Original Objective 4, our New Objective 4, investigates whether a cover crop overwinters mycorrhizae effectively in the treatment plots. One personnel change had to be made here because planting of woody species was no longer necessary. Justin Geibel and the VT Youth Conservation Corps is no longer part of the project. Funds associated with the woody vegetation and the VTYCC were reallocated to student labor as the planting and maintenance of the seedbeds and the mycorrhizal bank are now done by Rubin and a small crew of students.

After 3 consultations with a statistician, the experimental design also changed to accommodate for the adjusted  objectives. See below.

Introduction:

Mycorrhizae is an important part of agroecosystems although modern agriculture, including some forms of sustainable agriculture, impedes their effects. They affect mineral nutrition, crop disease resistance and drought tolerance and yet they are not promoted in agriculture (Plenchette et al 2004). They are symbionts with over 80% of crop plants (Gosling et al., 2006). Plants provide photosynthetic carbon compounds to mycorrhizal fungi and in return the fungi provide nutrients that are difficult for the plant to access. Phosphorus, a macro-nutrient needed in relatively large amounts for plant growth, is involved in the exchange between the plant and the mycorrhizal symbionts. However, phosphorus also gets tightly bound to Fe and Al oxides and upon application quickly becomes unavailable. Promoting mycorrhizae can help increase phosphorus accessibility by extending the roots’ reach within the soil, by promoting decomposition and in facilitating desorption of phosphorus.

The low availability of phosphorus in soil is one part of the phosphorus story. The other is that phosphorus is often exported to water bodies as eroded sediments. In the water column of a lake phosphorus can cause algal blooms which malaffect aquatic trophic web health, impair drinking water quality and decrease recreational value of the water. Often toxic blue-green algae are part of the blooms necessitating beach closures. Mycorrhizae can help reduce soil phosphorus and thus lower the risk of phosphorus water contamination.

One problem that we address is that many agroecosystems, in particular those that are tilled, have lower mycorrhizal densities which reduces the level of services they are able to provide. Short of transitioning fields to no-till systems, mycorrhizal crop plants may encounter fewer mycorrhizae rendering the symbiosis less effective. This then lowers the phosphorus use efficiency of the plants which in turn holds more phosphorus in the soil to be available for polluting lakes and streams through soil erosion. We propose to test the idea of mycorrhizal reserves, or banks, i.e., set aside lands that are maintained as a diverse natural community likely to support a diverse mycorrhizal community. These reserves will allow mycorrhizae to spread and recolonize fields after events that reduce mycorrhizal densities: such as tillage, fallow or flooding. This principle has been demonstrated in lands covered in mine tailings (Johnson, 1998), but has not been used in agriculture.

Our farm partner, Diggers Mirth, has just transitioned away from using high phosphorus composted chicken manure, and has fields excessive in phosphorus. This affords us the opportunity to test several research questions regarding mycorrhizal reserves. First, how far into the field do mycorrhizae expand from the edge of the reserve? Two, does the excessive phosphorus allow for a successful plant-mycorrhizae symbiosis? Three, how much nutrient and crop yield benefits do these mycorrhizal reserves provide in tilled fields?

Our objectives aim to measure not only the effectiveness of the mycorrhizal bank in providing mycorrhizae but also the effect of mycorrhizae on plant phosphorus uptake, crop yield and water extractable P concentrations associated with phosphorus losses to groundwater and by overland flow.

Cooperators

Click linked name(s) to expand/collapse or show everyone's info
  • Justin Geibel (Educator)
  • Hilary Martin - Producer
  • Jess Rubin (Educator and Researcher)

Research

Materials and methods:

The original research plan was this

Work under Objective 1

The field we selected (44.501 N, -72.325 W) is located on Winooski series very fine sandy loam. Soil in the Winooski series is moderately well drained. The area is level and becomes flooded occasionally in the spring time when heavy rains and snow melt coincide. The field is conventionally plowed and prepared with 4 foot wide seed beds stretching perpendicular from the edge of the planned mycorrhizae bank. The field is amended with organic fertilizers. The crop is potatoes, carrots or onions depending on the farmers plans that year.

Although the soil is mapped as level, previous research done under a SARE Partnership grant (ONE12 – 158) showed that there are many smaller depressions, common in other agricultural flood plain soils. These depressions are between 10 and 20 cm deep and between 3 to 5 m wide. Soil fertility varies as a function of elevation in these depressions (Ruhl et al., 2015). This effect has to be taken into account in our design to control for the topographic variation in soil fertility. A topographic survey of the experimental area will be done prior to the installation of the plots.

The field is adjacent to a 60-foot-long, 30 feet wide strip of an early succession old field. This strip is going to be set aside as the mycorrhizal bank. The adjacent field managed by the Intervale Community Farm will be under cruciferous crops, that are not known to have mycorrhizal associations.  Digger’s Mirth field is to the northeast of the bank, so that the vegetation and its arrangement in the mycorrhizal bank have to be considered for their potential light and wind shading effect. For this reason, woody species will be at the center of the strip (and cyclically coppiced), while forbs and herbaceous vegetation will be closer to the fields. See plant palette attached (Table 1).

The mycorrhizal bank will be the only experimental source of mycorrhizae for the field. We will set up 3 transects, each one with four 4 feet by 4 feet monitoring plots. Plots will be on the seed beds. The transects will be oriented roughly parallel to and about 6 feet, 12 feet and 24 feet from the edge (Figure 1). Plot locations will be chosen with a stratified random scheme. Stratification will be on ground elevation, so that the plots are at the same elevation to control for topography related fertility variations. Seedbeds will be prepared and planted by the partner farmer. No P is going to be applied on these plots. However, both N and K will be applied according to soil test recommendations. Fertilizer applications to the plots and weeding in the plots will be done by the UVM team. There are two variables to consider here. First, the distance from the edge of the field. According to mine tailing research, the further away from a source of mycorrhizae the later the mycorrhizae will colonize (Johnson and McGraw, 1988). Although our hypothesis is that the instantaneous availability of P rules colonization and thus we expect that both fields will equally benefit from the mycorrhizal bank. Second, time is a factor that may determine how far into the field mycorrhizae spread from the edge of the field.

Work under objectives 1 and 3:

We will collect three soil samples from each plot prior to the planting (May), 6 weeks after planting (July) and at the end of the growing season (September). Soil samples from the same plot will be homogenized. The soil will be split into two fractions. One will be used for measuring water-extractable phosphorus (work under Objective 4), the other will be used for determining the prevalence of mycorrhizae. The latter sample will be air dried and mycorrhizal hyphae extracted using the method of Miller et al (1995) modified by Juice (2016). Then hyphal length in the soil sample will be assessed microscopically using the gridline intersect method (GLIM) (Tennant, 1975).

Hyphal length determined by the gridline intersect method is likely described by a Poisson distribution. These data will thus be assessed with a generalized linear model with assuming a Poisson distribution and a log link function. GLMs do not use pairwise comparisons like those in Tukey’s honestly significant difference test (HSD). Instead they use contrasts. Contrasts will be set up to compare the GLIM determined hyphal length for high and low phosphorus soils at the same distance from the mycorrhizal bank. This will test the effect of high and low phosphorus on hyphae production. In addition, contrast will be set up to test whether there are differences in hyphal densities as a result of distance. This will be done for each of the three time points separately. Further contrasts will be set up looking at the effect of time on each transect.

Work under Objective 2

Six weeks after planting and at harvest (end of growing season) we will collect new leaves from several plants in the plots to estimate the phosphorus concentration of the plants. The tissue material will be dried in paper bags at 60oC (one bag per plot). The samples will subsequently be ground in a coffee mill and submitted for phosphorus analysis.

In addition, at the end of the season we will measure the harvestable crop yield as mass per plot. We will also measure the phosphorus concentrations in coppiced woody vegetation.

These data will be checked for normality, transformed if necessary, and analyzed with ANOVA. Leaf P will be tested with a three-way ANOVA with soil P level, distance from bank, and time as variables. Harvestable crop yield will be analyzed with a two-way ANOVA. This is followed by comparisons with Tukey’s HSD test if ANOVA detects differences. In the case that transformations cannot assure normality, we will utilize non-parametric tests.

Work under Objective 3:

Soil samples collected under Objective 1 will be extracted with deionized water by the method described by Self-Davis et al. (2000). In brief, 2 g of soil dried at 60 C for 48 hours and sieved through a 2 mm sieve are placed shaken for one hour. The solution is then filtered either through a 0.45 um membrane filter and acidified to pH 2 with HCl to inhibit phosphate forming secondary compounds. This solution is then spun down in a centrifuge and the supernatant analyzed using colorimetric methods.

For this objective we wondered whether the experimental factors influence the amount of water extractable phosphorus in the soil. This is important because water-extractable phosphorus is subject to various fates including its loss to ground water. This is particularly important because the water table can reach so high into the soil profile in the Intervale floodplain, providing a conduit for dissolved phosphorus to Lake Champlain.

However, we have to consider that there is an obvious relationship between mycorrhizal length and soil concentrations of water extractable phosphorus. We propose to use ANCOVA (Analysis of Covariance) which allows to simultaneously analyze the treatment factors (distance from bank, time and soil test P concentration) and the relationship among response variables (hyphal length, plant uptake and water extractable P).

Objective 4: Willow will be coppiced in the first year of its growth. The samples will be dried, ground and then submitted for phosphorus analysis to obtain an estimate of how much phophous can be recovered. Supporting Material

Year 1: Effect of Mycorrhizal bank on parsley

  1. Test whether mycorrhizae spread into a field from the mycorrhizae bank.
  2. Test whether plant phosphorus content varies with vegetated strip treatment (inoculated and uninoculated strip areas)
  3. Test whether water extractable phosphorus concentrations is less in the soil adjacent to the mycorrhizal bank than next to the unioculated verge and whether it is correlated with mycorrhizal densities and phosphorus plant uptake.

  Year 2: Effect on Cover Crops

A cover crop is maintained in the field for most of the growing season in year 2.

         4. Test whether cover crop has overwintered the mycorrhizal community.

 This will be tested early in the growing season (May) by extracting and counting mycorrhizae hyphae in the soil adjacent to a vegetated field verge that is inoculated with mycorrhizae (the bank) and comparing it with counts in soils adjacent to a verge that is not inoculated.  The hypothesis is that mycorrhizae are more abundant close to the bank than next to the uninoculated verge.

The new research plan is

Year 1: Effect of mycorrhizal bank on mycorrhizal colonization of parsley

Work in Year 1: Field Preparation

The field we selected (44.501 N, -72.325 W) is located on Winooski series very fine sandy loam. Soil in the Winooski series is moderately well drained. The area is level and becomes flooded occasionally in the spring time when heavy rains and snow melt coincide. The field is conventionally plowed and prepared with 4-foot wide seed beds stretching parallel to the edge of the planned mycorrhizae bank. The field is amended with organic fertilizers. The crop in year 1 will be parsley. In year 2 the field will be under a cover crop (likely oats).

Although the soil is mapped as level, previous research done under a SARE Partnership grant (ONE12 – 158) showed that there are many smaller depressions, common in other agricultural flood plain soils. These depressions are between 10 and 20 cm deep and between 3 to 5 m wide. Soil fertility varies as a function of elevation in these depressions (Ruhl et al., 2015). This effect has to be taken into account in our design to control for the topographic variation in soil fertility. A topographic survey of the experimental area will be done prior to the installation of the plots.

Year 1: Objectives 1 to 3 New

After working with UVM statistician Maria Sckolnick, we made limited changes to the project and the experimental plan was adjusted to meet the new objectives. In particular the experiment is set on two opposing sites of the field. Both side will receive two replicates of two treatments, for a total of 4 replicates of each treatment. Treatment 1 is inoculated by mycorrhizae (M), treatment 2 (the control) is not inoculated by mycorrhizae (C). Using two sides of the field, allows us a little bit of control over spatial variability in the field. Here are the new specifics:

The field is surrounded by a 4-foot wide vegetated buffer, currently mainly under grasses. This strip will be plowed in the spring and prepared for reseeding. On both the southern and the northern edges of the field, a 160-feet long stretch of the buffer will be set aside for two experimental treatments, one with mycorrhizal inoculant (M) and the other without (C ). The 160- foot long strip on both sides of the field will be split into 40 foot plots. Each plot will be designated either as M or C. Two of each treatment will be randomly placed at either side of the field (total of 4 replications). The M plots will be inoculated with soils from a nearby wild area to provide native, local mycorrhizal inoculant. The C plots will not receive any inoculant. Then the entire field verge so prepared will be planted with a six to eight species cover crop cocktail including mycorrhizal legumes, buckwheat and grasses. Any abutting C and M plots will be separated by a 2-foot buffer of radishes (Raphanus sativa) which is non-mycorrhizal to provide protection against mycorrhizae creeping from the M into the C plots. In year 1, adjacent to both the northern and southern field edges a 160-foot long field section on which our project is located will be planted to curly leaf parsley (4-foot seed beds on both sides of the field) after an extensive regimen of plowing, disking and seed bed preparation. We will set up two transects parallel to the vegetated strip, one on each side of the field in a single seed bed. Plots will be on the seed beds and opposite the M and C plots. Phosphorus is alread high in this field and will not be applied on these plots. However, both N and K will be applied according to soil test recommendations. All field maintenance of the plots and weeding in the plots will be done by the UVM team (reflected fin the budget as additional funds for personnel). There are two variables to consider here. First, which treatment the plots are next to (M and C). We hypothesize that the plots adjacent to the M plot will have more mycorrhizae than those adjacent to the C plots. Second, mycorrhizal colonization is dependent on the sampling date. When samples are taken early during the season, there will be few mycorrhizae; later in the season there will be more. According to mine tailing research, the further away from a source of mycorrhizae the later the mycorrhizae will colonize (Johnson and McGraw, 1988).

Seed mixes for the mycorrhizae bank was designed with specialists at High Mowing Seeds in Wolcott Vermont from December 2021 to January 2022, and were ordered and received in March 2022. We also ordered radishes, non myorrhizal plant, that will be planted in between the M and C plots on the field verge to prevent mycorrhizae colonizing the C plots from the M plots. .

Objective 1: Test whether mycorrhizae have spread into the a field from the mycorrhizal bank.

We will collect three soil samples from each plot prior to the planting (June), 4 weeks after planting (July) and at the end of parsley cropping cycle (end of July). Soil samples from the same plot will be homogenized. The soil will be split into two fractions. One will be used for measuring water-extractable phosphorus, the other will be used for determining the prevalence of mycorrhizal propagules. At the end of the growing season, parsley will be harvested, roots and stems separated. The plant sample will be air dried and mycorrhizal hyphae extracted using the method of Miller et al (1995) modified by Juice (2016). Then hyphal length in the soil sample will be assessed microscopically using the gridline intersect method (GLIM) (Tennant, 1975). We may also employ a method to determine the amount of root length colonized.

Hyphal length per gram of oven-dry soil determined by the gridline intersect method is likely described by a Poisson distribution. These data will thus be assessed with a generalized linear model with assuming a Poisson distribution and a log link function. GLMs do not use pairwise comparisons like those in Tukey’s honestly significant difference test (HSD). Instead they use contrasts. Contrasts will be set up to compare the GLIM determined hyphal length for high and low phosphorus soils at the same distance from the mycorrhizal bank. This will test the effect of high and low phosphorus on hyphae production. In addition, contrast will be set up to test whether there are differences in hyphal densities as a result of distance. This will be done for each of the three time points separately. Further contrasts will be set up looking at the effect of time on each transect.

We have nothing to report here yet, because the experimental work will begin at the end of April 2022..

Objective 2: test whether plant phosphorus content is greater in plots adjacent to the M than the C treatment.

Four weeks after planting and at harvest we will collect new leaves from several plants in the plots to estimate the phosphorus concentration of the plants. The tissue material will be dried in paper bags at 60oC (one bag per plot). The samples will subsequently be ground in a coffee mill and submitted for phosphorus analysis.

In addition, at the end of the season we will measure harvestable crop yields as mass per plot. We will also measure phosphorus concentrations in the aboveground plant tissue.

These data will be checked for normality, transformed if necessary, and analyzed with ANOVA. Leaf P will be tested with a three-way ANOVA with soil P level, distance from bank, and time as variables. Harvestable crop yield will be analyzed with a two-way ANOVA. This is followed by comparisons with Tukey’s HSD test if ANOVA detects differences. In the case that transformations cannot assure normality, we will utilize non-parametric tests.

We have nothing to report here yet as the experimental work will begin at the end of April 2022.

Objective 3: Test whether soil phosphorus concentrations were affected by the mycorrhizal banks

Soil samples collected under Objective 1 will be extracted with deionized water by the method described by Self-Davis et al. (2000). In brief, 2 g of soil dried at 60 C for 48 hours and sieved through a 2 mm sieve are placed shaken for one hour. The solution is then filtered either through a 0.45 um membrane filter and acidified to pH 2 with HCl to inhibit phosphate forming secondary compounds. This solution is then spun down in a centrifuge and the supernatant analyzed using colorimetric methods (WEP-SRP).

For this objective we wondered whether the experimental factors influence the amount of water extractable phosphorus (WEP_SRP) in the soil. This is important because WEP-SRP is subject to various fates including its loss to ground water. This is particularly important because the water table can reach so high into the soil profile in the Intervale floodplain, providing a conduit for dissolved phosphorus to Lake Champlain.

However, we have to consider that there is an obvious relationship between mycorrhizal length and soil concentrations of water extractable phosphorus. We propose to use ANCOVA (Analysis of Covariance) which allows to simultaneously analyze the treatment factors (distance from bank, time and soil test P concentration) and the relationship among response variables (hyphal length, plant uptake and water extractable P).

We have nothing to report here yet as the experimental work will begin at the end of April 2022.

Year 2: Overwintering of the mycorrhizae in a cover crop.

After the end of the growing season, the field is tilled and a cover crop (oats and peas) is sown. We hypothesize that the mycorrhizal propagules will still be able to infect the cover crop and so will be carried over to the next year.

We have nothing to report here yet as the experimental work will begin at the end of April 2022.

Objective 4: Test whether the cover crop overwintered the mycorrhizae

At the beginning of the growing season (June), we will revisit the plots set up in year 1, take soil samples in the same way, and test for mycorrhizae as well as for WEP-SRP.

We have nothing to report here yet as the experimental work will begin at the end of April 2022.

 

Participation Summary
1 Farmer participating in research

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

We will share our project results through two on-site, educational workshops, one in year 1 and one in year 2, where we distribute fact sheets for farmers. We are also in discussion with NOFA-Vermont to present at their winter conference. In addition we will prepare a peer reviewed journal article. The final report will be shared, with permission of SARE, on the PI’s web page. Jess Rubin also facilitates an ecological restoration business known as MycoEvolve, which focuses on research, education, and earthworks. This organization has experience in providing educational workshops as well as providing educational material.  MycoEvolve has education partners through which some of the outreach will be facilitated. These include NOFA VT, VT Agricultural Water Quality Partnership, Vermont Youth Conservation Corps, local farms such as Bread & Butter, Intervale, UVM extension, and local conservation districts. Farmers attending workshops will receive factsheets. MycoEvolve’s website will also feature this research with pictures, objectives and project updates for interested audiences. We will also submit a factsheet publication to SARE and share it at any conference we present at so that other interested stakeholders have access. We intend to reach 40 farmers through workshops, 30 farmers through factsheets, 20 regenerative agriculture practitioners through our SARE publication, 30 regenerative agricultural academics, 15 youth working on the conservation crew, and 5 other agricultural workers through word of mouth.

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.