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:

Project Objectives

After exploring the field, our farm partner cropping/tilling schedule, and learning that our intended inoculation technique of just applying soil from a wild area (hereafter referred to as: haphazard inoculation) is not a best practice, we revised our initial objectives and work plan.

Our original objectives were:

Overarching objective: to facilitate mycorrhizal colonization of tilled soils by creating “mycorrhizal banks” from where mycorrhizae spread into fields and increase crop phosphorus efficiency.

Specific Objectives:

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

           This will be tested by extracting and counting mycorrhizal hyphae in soil at different distances from the bank. This will offer information on optimal spacing between banks. We hypothesize mycorrhizae are more abundant close to banks but will spread further over time.

  1. Test whether plant phosphorus (P) content varies with distance from mycorrhizal banks.

We hypothesize plant P decreases with distance from banks and is positively correlated with mycorrhizal hyphal counts.

  1. Test whether soil (WEP-SRP) water extractable P varies with distance from bank edges and whether it is correlated with mycorrhizal densities and plant P uptake.

We hypothesize: a. water extractable P concentrations will be lower closer to banks b. mycorrhizal hyphal densities will be higher closer to banks. C. plants closer to banks will have increased P uptake.

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

We hypothesized based on mesocosm studies we conducted it will vary between species.

Our updated objectives were:

Overarching objectives:

  1. to facilitate mycorrhizal colonization of tilled soils by creating “mycorrhizal banks” (from adjacent wild buffers) of polyculture cover crops in which parsley is planted to test for P uptake efficiency
  2. to grow endemic mycorrhizae from wild buffers adjacent to the field for the farm to incorporate in year 2.

Year 1 Field Research Tested objective a:

  1. Effect of wild buffer inoculum (haphazard technique) on mycorrhizal bank in terms of mycorrhizal populations (spore counts, root colonization, extra radical hyphal length) by gathering baseline and end of harvest season data on mycorrhizal populations in N vs S wild buffers adjacent to field (from which mycorrhizal inoculum was gathered) and from corresponding N vs S field banks.
  2. Effect of mycorrhizal bank (haphazard inoculated) on soil P, & WEP-SRP concentrations by gathering baseline beginning and end of harvest season data on WEP-SRP concentrations in N & S wild and field buffers
  3. Effect of mycorrhizal bank (haphazard inoculated) on parsley P uptake by testing whether plant P content varies within bank treatment (inoculated vs uninoculated areas via haphazard technique)

Year 1 Greenhouse Research Tested objective b:

1.Effect of N wild buffer soil vs S wild buffer soil on inoculum viability by growing pot cultures of endemic mycorrhizae from buffers on either side of the field and testing viability at harvest.

  Year 2: Field Research will compare the effect on parsley P uptake and WEP-SRP concentrations of wild cultured endemic mycorrhizae from the farm’s wild buffers to commercial mycorrhizae and no mycorrhizae. The mycorrhizal bank we installed in year 1 will remain surrounding the field but not be part of our research.

Original objectives were changed because the farm’s tilling cycle would kill mycorrhizae growing into the field and the haphazard technique of inoculation is not considered effective. So we instead installed perennial cover crop banks on the field edges, applied the haphazard inoculation technique to see if it works and tested soil water (WEP-SRP), mycorrhizae, and parsley within this context. We eliminated the objective of woody shrubs since this was not practical for the farm machinery access needed. Justin Geibel and VYCC originally slated to participate in the project were replaced with intern support planting and scything banks installed along the field edges.

Attached is the revised mycorrhizal bank design (Image 1) and greenhouse endemic inoculant growing design (Image 2).

Image 1. Mycorrhizal Bank Design.

 

Image 2. Greenhouse random block design of growing endemic mycorrhizae.
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., 2005). 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 (P), 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 P accessibility by extending the roots’ reach within the soil, by promoting decomposition and in facilitating desorption of phosphorus.

The low availability of P in soil is one part of the phosphorus story. The other is that P is often exported to water bodies as eroded sediments. In the water column of a lake P 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 P 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 P use efficiency of the plants which in turn holds more P 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 recently transitioned away from using high phosphorus composted chicken manure, and has fields excessive in P. This affords us the opportunity to test several research questions regarding mycorrhizal reserves. While originally, we were going to test how far into the field do mycorrhizae expand from the edge of the reserve, we realized this is no longer possible since their intermittent tilling practices (according to other studies and the literature) will kill mycorrhizae. A previous mesocosm research project we conducted in preparation for this project (Rubin and Görres, 2022) indicated that plant mycorrhizal symbiosis can still occur in excessive P conditions. We were curious to know three things. 1. Can soil from wild buffers laden with arbuscular mycorrhizal plants effectively inoculate a bank created on the field edge planted with a cover-crop polyculture? 2. Does endemic mycorrhizae grown from the N (shrubs and herbaceous species) vs S (trees and garlic mustard) wild buffers adjacent to the field would differ in their mycorrhizal populations and P cycling effects in soil, soil water, and parsley crop? 3. How will soil P. soil water P, and parley P uptake differ between treatments in which soil is uninoculated, or inoculated with endemic or commercial mycorrhizae?

Our objectives aim to refine arbuscular mycorrhizae cultivation techniques for farmer use, to investigate effects of endemic mycorrhizae on soil P, soil water P, and parsley P uptake, and to better understand how mycorrhizal banks can support farms in P cycling efficiency.

Sources Cited

Gosling, P., Hodge, A., Goodlass, G., Bending, G.D., 2006. Arbuscular mycorrhizal fungi and organic farming. Agriculture, Ecosystems & Environment 113, 17–35. doi:10.1016/j.agee.2005.09.009

Johnson, N., Anne--Cressey McGraw, 1998. The role of vesicular-arbuscular mycorrhizae in the reclamation of taconite mine tailings. Agriculture, Ecosystems & Environment 21, 135–142.

Plenchette, C., Clermont-Dauphin, C., Meynard, J.M., Fortin, J.A., 2005. Managing arbuscular mycorrhizal fungi in cropping systems. Canadian Journal of Plant Science 85, 31–40. doi:10.4141/P03-159

Rubin, J.A., Görres, J.H., 2022. Effects of mycorrhizae, plants, and soils on phosphorus leaching and plant uptake: Lessons learned from a mesocosm study. PLANTS, PEOPLE, PLANET 4, 403–415. doi:10.1002/ppp3.10263

Cooperators

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

Research

Materials and methods:

The revised research plan was to establish mycorrhizal banks of polyculture cover crops on the North and South sides of a field. 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 crops grown in the field this year were lacinato and green kale brussel sprouts, nappa cabbage, daikon, and baby bock choi which incidentally are all nonmycorrhizal.

Year 1 Field Installation & Testing

Image 3. North Field Bank.
Image 3. North Field Bank.

As indicated in the design (Image 1), the mycorrhizal bank is 5 ft wide and 970 ft long on the North and South Sides of the field. In year 1 field research objective 1, testing the haphazard technique of wild buffer inoculation involved first measuring out the mycorrhizal bank areas, gathering initial composite soil samples to test for Mehlich-3 extractable P, Total P, WEP-SRP (uploaded as SOPs here). The areas on both sides of the field (Images 3 & 4)

Image 4. South Field Bank.
Image 4. South Field Bank.

were then raked evenly and divided into 4 sections 238 ft long, flagged and separated by 1 ft wide strips that were planted with radish which serve as a buffer since radish does not partner with mycorrhizae. An area in both the N & S adjacent wild buffers to the field of 40’x 5’ was measured out (Images 5 & 6). Thirty sterilized trowels of soil were gathered into a 5-gallon bucket designated for the direction and mixed. Some of this soil was processed to determine Mehlich, WEP-SRP, & TP concentrations, mycorrhizal spore counts, hyphal length, and DNA. To determine mycorrhizal root colonization, roots of plants (i.e. weeds in the field, herbaceous or shrub roots from wild buffers, parsley crop) were dug up, and put in clean labelled bags.

For Mehlich 2 gloved handfuls of soil from each bucket were placed in labelled clean plastic bags and submitted to UVM’s Agricultural and Environmental Testing Lab (AETL) who shipped it to University of Maine’s Soil Testing lab (NE Coordinating Committee for Soil Testing, 2011) for Mehlich-3-extractable nutrient concentration analysis.

For WEP - SRP, 25 g of fresh soil were dried. 2 grams of dry soil were sieved (2mm mesh), put in a 40-ml centrifuge tube with  20 ml of distilled water, shaken at 25 rpm for one hour, centrifuged at 6000 rpm for 10 minutes and filtered through a 0.45 μm nylon 33 mm syringe membrane filter (Fisherbrand, Suwanee, GA, USA) into labelled centrifuge tubes to prepare the sample for SRP measurement. This was submitted to UVM’s Agriculture Environment Testing Lab (AETL) where SRP concentration was determined colorimetrically on a Lachat Quick Chem Series 2 (Hach, Loveland, CO, USA) (US EPA, 2015) at 880 nm.

For TP the remaining dried soil was sieved. 1.5 grams added to a labelled scintillation vial were submitted to AETL and analyzed, using Microwave assisted digestion utilizing Nitric acid (U.S. Environmental Protection Agency, 1996) followed by ICP analysis (Avio 200, Perkin-Elmer Corp., Shelton, CT, USA).

Mycorrhizae presence was determined through enumeration of extra radical hyphae length, spore density, and root colonization percentage. Microscope slides for extra radical hyphal counts were prepared through a modified soil extraction using sodium metaphosphate, soil stirring, dilution, and vacuum pumped through a .2 µm filter stained with acid fuchsin (Juice, 2016 unpublished). Hyphal length was determined using the line intersect method described by Tennant (1975). Spores were extracted through modified protocols involving 25 g of soil, soap, a blender, sieves, sugar solution, and centrifuging to obtain 50 ml of supernatant spores in solution. 0.5 ml of the solution is pipetted onto a clean, labelled slide and counted under a microscope. Spore density (Images 5,6,7) was calculated from the counts, dilution and amount of soil used (Gerdemann and Nicolson, 1963). 

Image 7
Image 7. Spores at 10x extracted from field bank soil.
Image 6.
Image 6. Spores at 10x, extracted from wild buffer soil.
Image 5.
Image 5. Spores under 10x, extracted from wild buffer soil.

 

 

 

 

 

 

 

 

 

 

 

Root colonization was determined through a modified protocol first developed by (Deguchi et al., 2017). Washed roots with diameter of 0.5–1 mm were placed in 10% KOH solution, autoclaved (Consolidated Stilts and Sterilizers, Boston MA), rinsed, acidified with 1%HCl and stained with Acid Fuchsin. After being destained they were stored at 4C in water before being examined under a compound microscope (Olympus CX41, Olympus Corporation NY, NY). 4 roots were mounted with Polyvinyl-Lacto-Glycerol on a labeled slide. 10 randomized fields within one slide were selected to examine for presence of AMF structures (arbuscules, vesicles or spores). Percent colonization was calculated as the: number of microscope fields containing colonized roots * 10.

An Illumina HiSeq1500 was used to sequence DNA extracted from soil samples from soil gathered from wild buffers, field banks, and the field. High quality (phred score >30) reads of 150 bp were generated, which ranged in number from just over 971,000 up to almost 10 million per sample. Poorer quality bases were trimmed to improve alignment to the MaxiKraken database using Kraken2 (Wood et al., 2019).

The first and third sections of the banks on each side were haphazard inoculated by adding 1/3 of the 5-gallon bucket to the area and raked in. A polyculture cover crop seed mix (Italian Rye grass, Hairy vetch, medium red clover, white clover, oats) was raked into the four plots and 1/6 lb of radish was raked into the dividing buffer strips (Images 8 & 9).

Image 8.
Image 8. North field bank with radish strip.
Image 9
Image 9. South field bank with radish strip.

Italian rye grass. As the banks grew, they were scythed every few weeks to avoid seed heads forming and blowing into the field, per farmer’s request. After scything the cover crop in the field banks (Image 10) quite low and once the parsley started from seed in early spring in the greenhouse was ready, we transplanted them in the field buffers in late June. Sixteen parsley starts were transplanted in each of the 8 plots in a cluster 5” apart from each other and these areas were surrounded by flags (Image 11). Throughout the summer, we hand scythed the cover crop around the parsley so that it could grow (Image 12).

In late August parsley and soil was harvested (Image 13). 2 clumps of parsley plants were dug up (Image 14). Soil around the roots was shaken into a bag where soil was processed for mycorrhizal spores and extra radical hyphae . Roots were cut off and processed in the lab for root colonization. Leaf and stem were weighed fresh, then dried in a labelled clean, brown paper bags for 7days in a drying room (37.78 C). after bags were tared for calculations to determine P removal/ harvest. Material was ground in the UDY Cyclone Mill (.8mm screen). 1.5g of dried plant material were added to sterile, labeled and weighed scintillation vials. They were submitted to AETL and analyzed, using Microwave assisted digestion utilizing Nitric acid (U.S. Environmental Protection Agency, 1996) followed by ICP analysis (Avio 200, Perkin-Elmer Corp., Shelton, CT, USA). P mass recovered in plant tissue was calculated: P concentration * dry biomass. Soil around the plants was also harvested and analyzed for WEP-SRP, TP, and Mehlich-3 extractable P.

Image 10
Image 10. Parsley planted amidst scythed cover crop.
Image 11
Image 11. Flagged area of parsley in one of the field bank sections.
Image 12
Image 12. Close up of parsley amidst polyculture cover cropped field bank.
Image 13
Image 13. Jess harvesting soil and parsley from field banks.
Image 14
Image 14. Harvested parsley leaf and root in Luca's hand.

The data for soil TP, Mehlich-3 extractable P, WEP-SRP, plant P uptake, and mycorrhizal counts were analyzed via a General Linear Model (GLM) with treatment and in some cases date as predictors for P concentrations or mycorrhizal counts respectively. If Levine test’s model assumptions were not met, then data were log transformed prior to non-parametric analysis. Where the model was statistically significant (p < 0.05) for one predictor with more than two levels, Analyses were conducted via SPSS28.0.0. (IBM Corp, Armonk, NY, USA). Summary statistics (using Graph Pad Prism 9.2.0, San Diego, Ca, USA) are shown in graphs to allow comparisons with literature values.

For DNA, order level counts were used to calculate relative abundance in each sample (Table 3). Additionally, Principal Components Analysis (PCA) applied a model-free data reduction technique to examine the relationship among samples and primary taxa influencing the relationships via PCA (Ringner, 2008) loadings (Figure 3). Ampvis2 used in the R environment generated heatmaps (Table 3) for microbial and fungal taxa of interest (Andersen et al., 2018). In addition, sequences were examined in One Codex targeted analysis using 5S, 16S, 18S, 23S, 28S, ITS, and gyrB (Minot et al., 2015) to identify species level taxa. Data was sorted for a limited number of P solubilizing bacteria and mycorrhizae taxa likely found in this soil.

Year 1 Greenhouse Research

Image 2. Random Block Design of Inoculum Growing Pot Cultures in Greenhouse.

The detailed methodology of how the inoculum was cultured, tested for viability and stored through the pot cultures is written out in the guide that is an educational output for this project (draft attached). In short, when soil was gathered from the N and S wild buffers, they were added to sterilized and prepared medium in pots that were then planted with the same cover crop mix as the edge of field banks. These were organized in a random block design (Image 2) and grown in a greenhouse at the farm. As they grew, (Images 15, 16, 17), they were watered once a day and fertilized once a week (Image 18) with a modified fertilizer solution (340 ml per pot) which was a mix of 15g North Country Organics (6-0-6), 0.9g Epsom salt, 6ml Neptune's Harvest (2-3-1), 2 gallons of water. After 14 weeks watering and fertilization stopped. Week 15 plants were cut. Week 16 samples were tested for viable spores. Once deemed viable (Image 19), they were cut in small pieces, mixed into the medium and stored in sterilized bins with holes covered with athletic tape (Image 20) and stored in a fridge until they are applied later this spring to soil hosting fresh parsley seeds.

Closeup of week 1 pot culture inoculum
Image 15. Week 1 of Inoculum growing in pot cultures.
Image 16
Image 16. Week 3 of Inoculum growing in pot cultures.
Image 17
Image 17. Week 11 of inoculum growing in pot cultures.
Image 18
Image 18. Luca fertigating the pot cultures.
Image 20.
Image 20. Viable harvest inoculum ready for storage.
Image 19
Image 19. Colonized parsley roots showing vesicles at 20x.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sources Cited

Andersen, K.S., Kirkegaard, R.H., Karst, S.M., Albertsen, M., 2018. ampvis2: an R package to analyse and visualise 16S rRNA amplicon data (preprint). Bioinformatics. doi:10.1101/299537

Deguchi, S., Matsuda, Y., Takenaka, C., Sugiura, Y., Ozawa, H., Ogata, Y., 2017. Proposal of a New Estimation Method of Colonization Rate of Arbuscular Mycorrhizal Fungi in the Roots of Chengiopanax sciadophylloides. Mycobiology 45, 15–19. doi:10.5941/MYCO.2017.45.1.15

Gerdemann, J.W., Nicolson, T.H., 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Transactions of the British Mycological Society 46, 235–244. doi:10.1016/S0007-1536(63)80079-0

Minot, S.S., Krumm, N., Greenfield, N.B., 2015. One Codex: A Sensitive and Accurate Data Platform for Genomic Microbial Identification (preprint). Bioinformatics. doi:10.1101/027607

Ringner, M., 2008. What is principal component analysis? Computational Biology Primer. Nature Biotechnology 26, 303–304. doi:https://doi.org/10.1038/nbt0308-303

Tennant, D., 1975. A Test of a Modified Line Intersect Method of Estimating Root Length. Journal of Ecology 63, 995–1001. doi:10.2307/2258617

Wood, D.E., Lu, J., Langmead, B., 2019. Improved metagenomic analysis with Kraken 2. Genome Biology 20, 257. doi:10.1186/s13059-019-1891-0

 

 

 

 

Research results and discussion:

In Year 1 we aimed to test the haphazard technique of wild buffer inoculum on field edge mycorrhizal banks consisting of polyculture cover crops to see if soil & soil water P, mycorrhizal populations, and parsley would indicate differences due to inoculation. We found the technique does not work. While there were no statistically significant differences in treatment (haphazard inoculated and uninoculated), substantial findings arose regarding how farm management and location in a field may affect microbial and mycorrhizal populations, soil P and soil water P concentrations.

A. Data that demonstrated the haphazard inoculation technique was ineffective include all of our mycorrhizal, parsley P uptake, soil, and soil water P data. There was no significant difference in spore counts, extra radical hyphae length, or root colonization percentages, total P, Mehlich-3 extractable P, WEP- SRP, parsley P uptake (data not shown) between inoculated and uninoculated banks.     

B. Location (defined by both in which field feature and cardinal directions) affects baseline microbial & mycorrhizal communities present, P indicators (Mehlich-3 extractable P) in the soil, mycorrhizal root colonization and extra radical hyphae lengths. Field trial data from June indicate location was a factor affecting WEP-SRP data. Similarly, a positive correlation between extra radical hyphal lengths and WEP-SRP was found. Starting with baseline data comparing mycorrhizal root colonization percentages statistically significant differences between the wild buffers and the field (p = 0.002)  and between north and south (p = 0.003) was found with more colonization occurring in the field compared to the wild and in the North compared to the South (Figure 1).

Figure 1 indicates statistically significantly more mycorrhizal root colonization is occurring in the field compared to wild buffers which was surprising considering the density of herbaceous species, shrubs and trees in the wild buffers compared to the few weeds growing in the field at the time of sampling. This data highlights why the haphazard technique does not work, which may be because propagule density in the wild buffers is likely not enough for effective colonization. It has long been known that flooding and long fallow can disrupt the propagation of mycorrhizae (Ellis, 1998). The presence of weeds may actually maintain mycorrhizal populations. The trend of more extra radical hyphae found in N compared to S wild buffer may be due to differences in plant species composition or difference in seasonal high water table. Species in the N wild buffer consisted of: grasses, willow, primrose, dandelion, jewelweed, vetch, ash, sensitive fern, goldenrod, milkweed, mosses while in the S wild buffer: stinging nettle, goldenrod, box elder, ash, burdock, horsetail, grasses, horsetail, Virginia creeper, garlic mustard, mosses. These species vary with which mycorrhizae they partner. This area merits further research.

Figure 1. Baseline Mycorrhizal Root Colonization
Figure 1. Baseline % mycorrhizal root colonization.
Figure 2 Baseline Mycorrhizal Extra Radical Hyphal Length
Figure 2. Baseline mycorrhizal extra-radical hyphal length

Baseline mycorrhizal hyphal lengths were statistically significantly different for location (p = 0.010) with more mycorrhizal found in the wild buffers compared to the field. There was no statistically significant for direction (p = 0.947) between N and S (Figure 2 ).

Figure 2 demonstrates why wild buffers can be sources of inoculum for growing endemic mycorrhizae. Since there are fewer disturbances in wild buffers the mycorrhizal populations and propagule counts are likely high, and certainly more than in areas (field) experiencing disturbance such as tilling and nutrient additions. This is well documented in the literature.

Baseline Mehlich-3 extractable P concentrations were statistically significant for location(p = 0.012) but not for direction (p = 0.105) however the trend indicates lower Mehlich-3 concentrations in the N compared to the S (Figure 3).

Figure 3 highlights how farm management practices likely affect P concentrations in the soil. From least disturbed to most disturbed Mehlich – 3 extractable P concentrations were: Wild buffer< Field bank<Field. 

Figure 3 Baseline Mehlich - 3 Concentrations
Figure 3. Baseline Mehlich-3 extractable P concentrations in different field areas

Baseline DNA data from looking at both mycorrhizal and P solubilizing bacteria data indicate differences due to location.  PC2 distinguishes microbial communities by location (N versus S) for the wild buffers. It is interesting to note that there is a distinction between the N (field and wild buffer) and the S (field and wild buffer) which are separated by PC2 (Figure 4). The PC1 separates the wild buffers clearly from the field bank and field. 

Figure 4 highlights substantial differences both between the N & S areas of the field and between the distinct areas of wild buffer, field banks, and field in terms of mycorrhizal and P solubilizing microbes. These differences may affect P cycling in soil, soil water, and plants.

Figure 4. DNA PC Mycorrhizal & Microbial Data
Figure 4. Principle component analysis of mycorrhizae and phosphorus solubilizing bacteria.

The heat map (Figure 5) indicates baseline differences in the Bacillales order between the wild buffers and the field (including field bank and field).

Figure 5 highlights distinct differences in at least one bacterial order (Bacillales) between the wild buffer, field bank, and field. This is an important reminder about baseline composition of microbial and fungal communities differs based on management strategy and species present.

Figure 5. DNA Heat Map.
Figure 5. Heat map of relative abundances for three orders of mycorrhizae and ten orders of phosphorus solubilizing bacteria.

Pooling baseline data by direction, there was no statistically significant difference in WEP-SRP concentrations in April between the wild buffer, field bank and field, but there  was a trend of SRP concentrations from highest to lowest being field>field bank>wild buffer (Figure 6).

Figure 6. SRP-WEP Concentrations on the Farm
Figure 6. Baseline data of WEP-SRP in the three field treatment types.

A positive correlation was found between WEP-SRP concentrations and root colonization where for each 1% root colonization increased, WEP-SRP concentrations increased by 6.11 μg/L.

Figure 6 demonstrates how WEP-SRP concentrations correlate with Mehlich 3 concentrations in correspondence to areas of management strategies with least to most disturbance; wild buffer> field bank> field. This highlights that moving field conditions closer to wild buffer conditions, i.e. through establishing field banks may help decrease soil water P concentrations.

It was surprising to find a positive correlation between mycorrhizal root colonization and WEP-SRP concentrations. Our hypothesis, the literature, and other studies (Rubin and Görres, 2022b) indicate the correlation would be negative, i.e. the more mycorrhizal root colonization is, the lower WEP-SRP concentrations would be. This is counterintuitive because we expected that plants would drive down soil water P (here represented by WEP-SRP). However, a more active mycorrhizosphere of P solubilizing bacteria may increase WEP-SRP.

In June when WEP-SRP data was log transformed (due to unequal variance) there was a statistically significant difference in WEP-SRP concentrations between N vs S with lower WEP-SRP in the north in the wild buffers. This graph shows the untransformed data (Figure 7).

Figure 7. Baseline SRP-WEP Concentrations in Wild Buffer
Figure 7. Baseline SRP-WEP Concentrations in Wild Buffer

Figure 7 indicates a difference between the wild buffers in WEP-SRP concentrations. The N wild buffer’s lower WEP-SRP concentrations compared to the S wild buffer could be due to differences in spatial variability, drainage, or species composition.

C. Growing viable endemic mycorrhizae is a relatively easy and accessible practice for farmers to adopt is indicated by the guide that we are publishing. In terms of data, we found that there was no statistically significant difference in viability between the N & S inoculum pot cultures (data not shown).

Sources Cited

Ellis, J.R., 1998. Post Flood Syndrome and Vesicular-Arbuscular Mycorrhizal Fungi. Journal of Production Agriculture 11, 200–204. doi:10.2134/jpa1998.0200

Rubin, J.A., Görres, J.H., 2022b. The effects of mycorrhizae on phosphorus mitigation and pollinator habitat restoration within riparian buffers on unceded land. Restoration Ecology 31, e13671. doi:10.1111/rec.13671

Research conclusions:

In year 1 the main findings from our field research were that A. the haphazard inoculation technique does not work (which is likely why it is not recommended in the literature and instead pot culture techniques are (i.e. McCoy, 2016; Phillips, 2017); hence growing endemic mycorrhizae is a valuable practice for farmers wanting to inoculate their crops B.  Location in the field of where the research is conducted is a crucial variable that likely affects microbial, fungal communities and corresponding P cycling due to respective conditions such as drainage, sunlight, microbial/fungal biome, and vegetation. Mycorrhizal banks remain an intriguing concept to support edge of field mycorrhizal reserves though their maintenance and function for crop productivity requires further research. C. Growing viable endemic mycorrhizae is a relatively easy and accessible practice for farmers to adopt. It will be interesting entering year 2 to determine the effects of endemic mycorrhizae on parsley crop P uptake in one location so that location does not affect results. 

In conclusion this field study demonstrated why endemic inoculum needs to be grown in pot cultures rather than directly applied from wild areas. Propagule density/gram of soil is an important requirement for mycorrhizae to activate its nutrient cycling activity.

We refined techniques to grow endemic mycorrhizae from wild buffers adjacent to a field and produced a guide for farmers. We found no statistically significant difference in inoculum viability between wild buffers (N vs S) though they had distinct floral communities. 

Sources Cited

McCoy, P., 2016. Radical Mycology: A Treatise on Seeing & Working with Fungi. Chthaeus Press, Portland, OR.

Phillips, M., 2017. Mycorrhizal Planet: How Symbiotic Fungi Work with Roots to Support Plant Health and Build Soil Fertility. Chelsea Green Publishing.

Participation Summary
1 Farmers participating in research

Education & Outreach Activities and Participation Summary

1 Curricula, factsheets or educational tools
1 Webinars / talks / presentations
2 Other educational activities: Occasional informal farm tours were given to friends that are farmers or agricultural workers. Activities and findings from our research have been shared both in the MycoEvolve website as well as the rootsandtrails_mycoevolve instagram account.

Participation Summary:

14 Farmers participated
207 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Winter 2021 we shared our preliminary research and plan for this research at the VT NOFA conference which had 216 participants. Jess Rubin’s MycoEvolve (www.mycoevolve.net) website has updates from our activities and findings as does her instagram account rootsandtrails_mycoevolve. 14 farmers in the circles of Diggers’ Mirth and Pitchfork Farm know about this research. The guide we are publishing will hopefully reach many farmers and earth tending professionals. We will be sharing it at this winter VT NOFA conference in a workshop. We are applying techniques we honed in creating the guide to an ecological restoration project on another farm in the same bioregion.Guide to Growing Mycorrhizal Inoculum, draft form

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:

It may be too early for us to determine this yet. The farmers we worked with were willing to add a bank around their field that was planted with covercrop polycultures in the area of our study and wildflowers surrounding the rest of the farm field. They said that this was a practice they would likely continue.

Project Outcomes

4 Farmers changed or adopted a practice
4 Grants applied for that built upon this project
1 New working collaboration
Project outcomes:

It may be too early for us to determine this yet. 

One change is that the Diggers' Mirth farmers are retaining the field banks we installed on the north side. They say the banks are useful to them because they have switched to having beds with multiple species that they mow next to the fence line instead of a blank bed they till to manage weeds. They hope these borders are easier to manage than tilling for weed control and that they provide habitat, food source for insects.... (farmer Hilary Martin Correspondence, January 2023).

Assessment of Project Approach and Areas of Further Study:

When we realized the haphazard inoculation technique was not considered best practices and the complex tilling cycle of the farm, our modification of the study to maintain the gist of it while incorporating it into areas that would not be affected by tilling worked. In hindsight I would allow more time and personnel for the scything of the field banks since they grew very tall and once the parsley was planted in them, more frequent hand scything may have facilitated more growth in the parsley crop. The concept of mycorrhizal banks still has merit, especially in terms of hosting diverse mycorrhizae if left over seasons undisturbed but for this farm’s crop production systems it is not certain they can be a supportive practice. All of our SOP for P and mycorrhizae were honed. The questions for year 1 we set out to study were answered. Additional work that remains unanalyzed in spreadsheets is a more thorough taxonomical analysis on the different genera and species of mycorrhizae found in the different areas of the farm. We were able to record differences in spore shape, diameter, and even color with 3-5 guesses next to each one, based on guides we could find but this is as far as we got due to time and budgetary constraints. Similarly, there are limited mycorrhizal genome sequences so it was unfortunate how little mycorrhizal data we were able to get through our DNA analysis as we hypothesize there are distinct genera between areas in the farm due to management and other environmental conditions. This gap is worth pursuing as it can further inform farmers about which particular mycorrhizae can grow where and with which plant species. This would require an entirely separate study. We think all farmers and land tenders in bioregions concerned with mitigating P pollution from upland terrestrial activity could benefit from this research. We hope the guide, once it is finalized, and our findings in year 2 will be supportive to farmers and the regenerative agricultural community.

 

Information Products

    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.