Progress report for FNE24-078
Project Information
Our project is designed to
achieve several objectives:
- Experiment with Three Major Ecosystem Designs: Test three unique ecosystems to identify the most beneficial synergies for plant growth, disease and pest control. These will be tested sequentially to benefit insights gained after each experiment. For each iteration, we will compare an untreated control against an experimental ecosystem, sending each for root exudate analysis. Along with root exudate analysis, we will also study insect populations to assess the designed ecosystems’ abilities to promote their recycling.
- Experiment with Many Minor Ecosystem Designs: This objective is the same as the previous, except these experiments will not be analyzed for root exudates, thus saving significant costs, allowing for rapid iteration.
- Standardize Ecosystem Design Methodologies: Standardize methods for designing and deploying self-sustaining ecosystems that maintain their health and productivity, even in the absence of immediate pest threats.
Through this project, we anticipate not only advancing our scientific understanding of soil microbiology and plant-microbe interactions but also
delivering practical, tangible benefits to farming practices. Our goal is to pave the way for more sustainable, efficient, and productive agriculture, rooted in a deep understanding of the soil's living tapestry.
Problem:
Microgreens, herbs and subtropical plants, are a booming business worth billions and loved for their colors, flavors and nutrition. But they're under threat from tiny pests and diseases that can wipe out crops and profits. Most farms use chemical sprays to keep these pests away, but these aren't great for the planet or for people who prefer eating organic. There are some natural pest-fighting products out there, but they don't stick around long enough to keep protecting the plants. They're like a band-aid rather than a cure. And while some smart solutions use good bugs to fight the bad ones, they often don't stay for the long haul, dying from starvation or flying away, leaving plants vulnerable again.
Solution:
Our idea is like building a mini nature reserve right where we grow our microgreens and plants. We're not just focusing on getting rid of pests; we're creating an entire neighborhood of helpful creatures and good bacteria that take care of the plants from the ground up. Think of it as a tiny, bustling city under the leaves where everyone has a job to do, from tiny worms that make the soil rich for growing, to good bacteria that help plants absorb food better and grow stronger.
Here’s the clever part: by bringing together the right mix of these tiny helpers, we make a system that is self-sufficient. Like a well-tended garden, it can take care of itself. This means once we set it up, it keeps going, protecting and feeding the plants without us having to add more helpers later. This is good news for the farmer who won’t have to keep buying new products, and great for the environment because it's all natural.
For example, let’s say fungus gnats are the problem. We can introduce friendly nematodes that hunt down the gnat larvae. We'll also bring in parasitic fungi (the good kind) that take out the gnats' food source without harming the microgreens and plants. These fungi can also help the plants by extending their roots, which means the plants can reach more food and water in the soil. But how do the nematodes persist when their food’s gone? Well, we want to learn, through trial and error, how to add species as a secondary food source, that won’t harm the plant, so the nematodes can keep growing strong even when they’ve eaten all the pests in the environment.
But we’re not stopping there. We’ll add in a team of Bacillus bacteria, which are like tiny farmers themselves, helping to recycle nutrients and make the soil even better for plant growth. These bacteria also help keep the bad fungi away by living in the soil and roots, making it hard for the bad guys to take hold again. And because all these helpers are already approved by the EPA, we know they’re safe to use and won’t harm the environment.
In short, we're not just solving a pest problem; we're learning how to set-up a natural, self-sustaining system that’s a win-win for everyone: healthier plants, happier farmers, and a happier planet.
Cooperators
- - Technical Advisor
- - Producer (Researcher)
Research
Detailed methods and notes follow. These are a culmination of notes from multiple researchers on the farm and will continue to be updated as we progress. We additionally intend to further refine these records for our final report. Earthbarrier, a Boston-based biotechnology company that has been helping us perform science in our greenhouses, in partnership with We Grow Microgreens and our interns, have authored these notes.
Contributing authors: Christopher Kenyon, Mikayla Von Ehrenkrook, Elson Ortiz, Michael Lembck, Lisa Evans, Tim Smith
Experiment 1
Wheat grass was studied to see if we could prevent infection of a naturally occurring greenhouse fungus by introduction of a fungal biocontrol agent. This was accomplished using 83 mg of Great White microbial mix because it contains a collection of Trichoderma species that are known to suppress other pest fungi via parasitism. We collected the fungi from a contaminated wheat grass specimen, suspended in a water solution, blended the solution fo 10 seconds in a standard nutribullet, and counted the resulting spore suspension with a hemacytometer. Fresh cultures of wheat grass were seeded in soil and the experiment was constructed thus:
- wheat grass alone
- wheat grass inoculated with fungus
- wheat grass inoculated with fungus and Great White microbial mix
- wheat grass with Great White microbial mix
Wheat grass was then allowed to grow for 7 days first in a germination chamber and then open in the greenhouse under the grow tables.
We found contamination in all conditions indicating some external source of fungal inoculation and insufficient control by the great white.
Experiment 2:
To follow up on our previous wheat grass experiment, we wanted to see where our contamination was coming from. We sterilized soil and water in an autoclave and surface sterilized wheat grass seeds with a 3% bleach 0.1% tween solution (30 minutes of shaking at 37C). We then repeated the previous experiment with the following conditions:
We performed a 2x2x2x2x2 factorial experiment with the following factors:
- wheat grass +/- surface sterilization
- soil +/- autoclaved sterilization
- water +/- autoclaved sterilization
- inoculation with fungus +/-
- inoculation with great white microbial mix +/-
We continued to find contamination in all conditions indicating some external source of fungal inoculation and insufficient control by the great white.
We planned to then repeat the experiment with increased amounts of great white microbial mix to see if we could improve the control but we ran out of time as the weather was getting too hot for the wheat grass to grow in the greenhouse.
Experiment 3:
Hydroponics shelves were prepared using captured rainwater where 40 L of rainwater in each condition was supplemented with Part A Part B nutrient salts per supplier's instructions. Control condition was left as is and experimental conditions were further supplemented with X grams of Mikro Myco. The shelves were then run with water flowing overnight to ensure complete mixing of nutrients and inoculants. It was noted the next day that the Mikro Myco inoculant formed a foamy consistency that was distributed across all of the shelves of the inoculated tower. Asian green seedlings were then planted in each well of each shelf of each tower and the plants were grown on a light dark cycle of 16:8. The plants were left to grow for 21 days.
Experiment 4:
Treatments
- Control: Radish grown in BX Pro-Mix growing medium
- Treatment 1: Radish grown in BX Pro-Mix growing medium mixed with 2.05 grams of Mikro-Root
- Treatment 2: Radish grown in BX Pro-Mix growing medium mixed with 6.21 grams of Great White
- Treatment 3: Radish grown in BX Pro-Mix growing medium mixed with 1 gram of Mikro-Root and 3.16 grams of Great White
Steps
- BX Pro-Mix growing medium moistened with warm water
- Mixed approximately a tray of BX Pro-Mix growing medium with varying amounts of Great White and Mikro-Root.
- Spread soil into trays and watered
- Measured radish microgreen seed for each treatment
- Control: 31.510 g
- Treatment 1: 31.220 g
- Treatment 2: 31 g radish seeds
- Treatment 3: 31.115 g radish seeds
- Seeded trays and put into germination chamber
Results: No visual difference between germination levels and growth of the microgreens.
Experiment 5:
We were interested in whether the results we were seeing were due to insufficient time for the fungus to colonize the soilless medium before plants were seeded. We therefore compared T0 (incubation time = 0 days) to T7 (incubation time = 7 days) allowing the fungus to colonize our soilless medium.
10/7/24
T7 treatments mixed with approximately two trays worth of Pro-Mix BX growing medium in buckets
- Control: Peat-based Pro-Mix
- Great white: 4 grams
- Mikro-root: 4 grams
- Mikro-myco: 4 grams
- Plant thrive: 4 grams
10/8, 10/9, 10/12/2024
T7 Buckets shaken
10/15/24
T0 treatments mixed with approximately two trays worth of Pro-Mix growing medium in buckets
- Control: Peat-based Pro-Mix
- Great white: 4 grams
- Mikro-root: 4 grams
- Mikro-myco: 4 grams
- Plant thrive: 4 grams
Pea shoots seeded using 8 treatments of Pro-Mix
- Pea shoots soaked
- Treatments spread in labeled trays and watered
- Pea shoots seeded
- Pea shoots covered with non-treated Pro-Mix
- Trays put under the table with a tray on top to germinate
10/21/24
Removed pea shoots from under the table and watered. No observable difference in germination rates between trays.
10/25/24
Measured trays:
- Picked 10 shoots from each tray at random
- Measured shoots from the seed to the top of the leaf
- Data (not yet analyzed): https://docs.google.com/spreadsheets/d/1bGroC6gi39QZjhQz4WvN2MoxBqkBIdORfnW_CU9z1LM/edit?gid=0#gid=0
Jan 9th - notes by Michael
In 4 grams of Great White:
Ectomycorriza:
● Pisolithus tinctorius.........751,500 propagules
● Rhizopagan luteolus.............20,876 propagules
● Rhizopagan fulvigleba..........20,876 propagules
● Rhizopagan villosullus..........20,876 propagules
● Rhizopagan amylopogon......20,876 propagules
● Scleroderma citrinum............20,876 propagules
● Scleroderma cepa...................20,876 propagules
Endomycorrhiza:
● Glomus aggregatrum...................332 propagules
● Glomus intraradices....................332 propagules
● Glomus mosseae..........................332 propagules
● Glomus etunicatum.....................332 propagules
● Glomus clarum.............................44 propagules
● Glomus monosporum.................44 propagules
● Glomus deserticola......................44 propagules
● Paraglomus brasilianum.............44 propagules
● Gigaspora margarita....................44 propagules
As well, contains 2,100,000 CFU's of the each of the following species:
Azobacter chroococcum, Bacillus subtilis, Bacillus licheniformis, Bacillus azotoformans, Bacillus megaterium, Bacillus coagulens, Bacillus pumilis, Bacillus amyloliquefaciens, Paenibacillus polymyxa, Paenibacillus durum, Saccharomyces cerevisiae, Pseudomonas aureofaciens, Pseudomonas fluorescens,
and,
Trichoderma:
● Trichoderma koningii.........751,500 propagules
● Trichoderma harzianum......501,000 propagules
In 4 grams of Mikro Myco:
4 species of Endomycorrhizae (260 cfu):
● Glomus intraradices..................260 cfu
● Glomus mosseae......................260 cfu
● Glomus aggregatum..................260 cfu
● Glomus etunicatum....................260 cfu
7 species of Ectomycorrhizae (872,000 cfu):
● Rhizopogon villosulus.............124,572 cfu
● Rhizopogon luteolus...............124,572 cfu
● Rhizopogon amylopogon........124,572 cfu
● Rhizoporon fulvigleba.............124,572 cfu
● Pisolithus tinctorius................ 124,572 cfu
● Scleroderma cepa...................124,572 cfu
● Scleroderma citrinum..............124,572 cfu
4 species of Rhizobacteria (1.6×10⁹ cfu):
● Bacillus licheniformis................4×10⁸ cfu
● Bacillus pumilis.........................4×10⁸ cfu
● Bacillus subtilis.........................4×10⁸ cfu
● Bacillus megaterium.................4×10⁸ cfu
3 species of Beneficial Fungi (3.0×10⁶ cfu):
● Trichoderma harzianum...............1,000,000 cfu
● Trichoderma viride.......................1,000,000 cfu
● Trichoderma longibrachiatum......1,000,000 cfu
In 4 grams of Mikro Root:
Beneficial Fungi
● Trichoderma harzianum.....................8.0 x 10⁷ cfu
● Trichoderma viride.............................8.0 x 10⁷ cfu
In 4 grams of Plant Thrive:
7.00 × 10⁸ CFU each of:
● Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium
6.00 × 10⁸ CFU each of:
● Pseudomonas fluorescens, Pseudomonas putida
5.00 × 10⁸ CFU each of:
● Azospirillum amazonense, Azospirillum lipoferum
2.40 × 10⁸ CFU each of:
● Bacillus firmus, Bacillus pumilus, Bacillus azotoformans, Bacillus coagulans, Bacillus pasteurii, Geobacillus stearothermophilus
6.00 × 10⁷ CFU each of:
● Pseudomonas aureofaciens, Streptomyces coelicolor, Streptomyces lydicus, Streptomyces griseus, Trichoderma reesei, Trichoderma hamatum, Trichoderma harzianum
28 spores of Rhizophagus intraradices
Experiment 6:
Our grant proposal intended to investigate the conditions necessary to create self-sustaining cycles of predatory insects. We predicted one of our initial obstacles to be the food sources of those predators and the food sources of the prey. In response to this obstacle, we chose to focus on greenhouse farmers most prevalent pest, aphids.
11/25/24
Treatments
- Control: Pea shoots grown in sand
- GW 4: Pea shoots grown in a mixture of sand and a solution of 4 grams of GW and water
- GW 24: Pea shoots grown in a mixture of sand and a solution of 24 grams of GW and water
Setup
- Soaked pea shoots in warm water
- Drained pea shoots
- Created two solutions of Great White mixed with warm water
- 4 grams of Great White
- 24 grams of Great White
- Mixed solutions with sand in trays
- Saturated sand with water
- Measured pea shoots for 3 trays
- 0.265 kg (265 g) of pea shoots per treatment
- Seeded pea shoots and covered in a thin layer of sand
- Put trays under the table with a tray on top of the sand
12/3/24
Pea shoots removed from under the table. Visual notes on trays:
- Control: Growing the best by far. Sand is the driest in this tray.
- GW 4: Growing the worst. Inconsistent germination and lower height. Sand is the wettest in this tray.
- GW 24: Has more germination than GW4, but less than Control. Sand moisture is somewhere between Control and GW4.
Is the inconsistent germination in the GW 4 & GW 24 because of GW, sand moisture, or both? Did GW make the sand retain moisture. Did GW inhibit growth, causing less pea shoots to take up water? Or were the trays watered inconsistently, and levels of moisture inhibited growth.
12/10/24
Visual notes on trays:
- Control:
- GW 4:
- GW 24:
Measured pea shoots
- Rinsed sand out of trays and pulled up the mat of pea shoots
- Drained pea shoots of water and weighed
- Control: 0.915 kg
- GW 4: 0.73 kg
- GW 24: 0.95 kg
- Randomly selected 50 shoots from each treatment and photographed the roots
Experiment 8:
11/25/24
Treatments
- Control: Pea shoots grown in sand
- GW 4: Pea shoots grown in a mixture of sand and a solution of 4 grams of GW and water
- GW 24: Pea shoots grown in a mixture of sand and a solution of 24 grams of GW and water
Setup
- Soaked pea shoots in warm water
- Drained pea shoots
- Created two solutions of Great White mixed with warm water
- 4 grams of Great White
- 24 grams of Great White
- Mixed solutions with sand in trays
- Saturated sand with water
- Measured pea shoots for 3 trays
- 0.265 kg (265 g) of pea shoots per treatment
- Seeded pea shoots and covered in a thin layer of sand
- Put trays under the table with a tray on top of the sand
12/3/24
Pea shoots removed from under the table. Visual notes on trays:
- Control: Growing the best by far. Sand is the driest in this tray.
- GW 4: Growing the worst. Inconsistent germination and lower height. Sand is the wettest in this tray.
- GW 24: Has more germination than GW4, but less than Control. Sand moisture is somewhere between Control and GW4.
Is the inconsistent germination in the GW 4 & GW 24 because of GW, sand moisture, or both? Did GW make the sand retain moisture. Did GW inhibit growth, causing less pea shoots to take up water? Or were the trays watered inconsistently, and levels of moisture inhibited growth.
12/10/24
Visual notes on trays:
- Control:
- GW 4:
- GW 24:
Measured pea shoots
- Rinsed sand out of trays and pulled up the mat of pea shoots
- Drained pea shoots of water and weighed
- Control: 0.915 kg
- GW 4: 0.73 kg
- GW 24: 0.95 kg
- Randomly selected 50 shoots from each treatment and photographed the roots
Experiment 9:
General notes:
Rearing Aphidius ervi
Objective
What are you trying to achieve or test? Clearly define the purpose of the experiment.
Background
● Understanding population cycles between aphids and wasps brings us closer to unraveling the necessary conditions for sustainable predator environments.
● Managing pest populations is ultimately the priority in our farm and testing how effective 250 wasps are in managing aphids answers a question relevant to real farmers.
● We purchased 250 Aphidius ervi wasps and released them on 17 pepper plants to fight an ongoing aphid infestation. Since then we have observed the establishment of a population of wasps that has cycled tightly with the aphid populations. We began labeling branches and recording aphid populations
Methods
1. Location and Setup
○ Location: Flood benches in Greenhouse
i. 17 pepper plants
ii. Plants between 1-3 feet
○ 250 wasps were spread evenly among 17 plants
2. Data Collection
○ Individual aphids were counted periodically by hand
○ As populations were discovered, branches were labeled with hole-punched notecard paper
i. Notecards occasionally fell, leading to gaps in the data. Branches were relabeled uniquely to capture trends. For example, Branch 1 may suddenly stop receiving data inputs but the same branch data may be represented by a new branch label 45.
Data table
https://docs.google.com/spreadsheets/d/1R5xwZrRFOl2XxJE1lqHjEYdk8ObmOozwj7ssnBSVjzQ/edit?usp=sharing
Experimental Activities Conducted to Date:
Wheat Grass Pathogen Suppression:
We found during humid and hot Summer growing conditions contamination from a local greenhouse fungus.
- Tested whether fungal biocontrol agents (e.g., Trichoderma spp. in Great White microbial mix) could suppress greenhouse fungal contamination.
- Conditions: Wheat grass alone, inoculated with fungus, inoculated with fungus and Great White microbial mix, and inoculated with Great White microbial mix alone.
- Process: Fungus was blended and quantified with a hemocytometer; treatments were applied to sterilized soil and monitored for 7 days under greenhouse conditions.
- Results: Contamination persisted across all conditions, indicating external fungal sources and insufficient control by Great White microbial mix.
Source Tracking of Fungal Contamination:
- Sterilized soilless medium, water, and wheatgrass seeds (surface-sterilized with 3% bleach and 0.1% Tween).
- Conducted a 2x2x2x2x2 factorial experiment to isolate contamination sources.
- Results: Contamination persisted, suggesting external inoculation sources and insufficient biocontrol by Great White.
Hydroponic Trials:
- Asian greens were grown in hydroponic systems using rainwater with and without Mikro Myco supplementation.
- Observations: Mikro Myco condition formed foam across inoculated shelves. Plant growth was monitored for 21 days under controlled light-dark cycles.
- Mikro Myco-treated plants had a statistically significant growth advantage over untreated plants in early growth stages, but differences were washed out after continued growing.
Radish Microgreens in Pro-Mix BX:
- Tested different microbial supplements (Great White, Mikro-Root, and combinations) on radish germination and growth.
- Results: No visual differences in germination or growth between treatments.
Pea Shoots in Sand and Great White:
- Evaluated the effect of increasing concentrations of Great White on pea shoot germination and growth in sand.
- Process: Pea shoots were grown in trays with sand supplemented with 0 g, 4 g, or 24 g of Great White.
- Results: Control conditions outperformed treatments with Great White. Higher moisture retention in treated sand may have inhibited germination, but experimental conditions seem to exhibit significant root growth improvements over control. Repeated this experiment in 72-well trays to more carefully assess individual plant root growth.
Aphid Rearing and Biocontrol with Aphidius ervi:
- Investigated population cycles of aphids and Aphidius ervi on pepper plants.
- Process: Released 250 wasps onto 17 pepper plants and monitored aphid populations weekly, using labeled branches for consistent tracking.
- Results: Population monitoring is still ongoing. Population dynamics demonstrated the need for stable aphid prey populations to sustain predator cycles. Overall, aphid populations are holding steady, suggesting insufficient wasp populations to achieve complete control, but interestingly, a stable predator-prey dynamic may be achievable over sustained periods even in greenhouses.
- We do find that the peppers have become banker plants and are surviving the Winter, where without the wasps the aphid populations may have exploded. We are not having aphid explosions this year that we have seen in the past.
Scale Biocontrol using Aphytis melinus:
- Investigated scale populations on guanabana trees and their response to release of a market-available scale biocontrol. Aphytis melinus is known to be an ectoparasite of certain scale species.
- Process: Released 200,000 Aphytis melinus on guanabana trees due to inconclusive pest identification by the University of Massachusetts Amherst.
- Results: Observed scale populations have yet to decline. Aphytis melinus was observed briefly following release (2–3 days) but has since escaped observation, likely due to cold temperatures and an improper fit between pest and biocontrol agent.
Data Collection:
Plant Growth Assessment:
- Measuring biomass using precision milligram scales.
- Evaluating root and leaf health microscopically.
- Assessing plant root and shoot growth computationally with large batch sizes and computer-automated pipelines.
- Developing software to measure insect populations using computer vision analysis and track populations in real time.
Upcoming Molecular and Chemical Analyses:
- Sequencing soil DNA using Nanopore technology to map microbial population dynamics.
- Sending plant samples to Lifeasible for root exudate profiling.
Analysis:
Statistical Evaluation:
- Comparing microbial diversity using Shannon indices.
- Conducting t-tests for biomass, root length, microbial populations, and root exudate differences.
Iterative Refinement:
- Adjusted experimental designs based on insights from early trials, attempting to find measurable metrics where plants exhibited physiological differences relative to controls.
Challenges and Adjustments:
Insect Attrition:
- Observed higher-than-expected mortality in early insect releases. Addressed this by integrating secondary food sources, especially for lacewings.
Weather Impact:
- Temperature fluctuations in the greenhouse prompted adjustments in experimental direction.
Quality of Microbial Products:
In December 2024 we learned of an interesting research article comprising a metaanalysis of soil amendment microbial products suggesting that mycorrhiza products largely are not of the quality they are advertised as. This may be due to extended shelf time or poor mixing of the product in packaging. We examined the Great White under our microscope and found no presence of living mycorrhizae. We had been using this product for the duration of our work. This calls into question the effects we observed on root size. We expect this was due to the other microbes present in Great White such as Bacillus spp. We will continue to microscopically verify the quality of the microbial products we use in the future because of this development.
At this point in our project we are hesitant to draw strong conclusions as we have only recently reached the point where we are starting to see differentiable results between our experimental conditions and controls. Additionally we are still in the midst of analyzing the results from the experiments that did yield noticeable differences. We are confident though at the least that microbial amendments do indeed induce a marked beneficial effect in root length in pea sprouts. We intend to investigate this in detail further and expand these inquiries to other microbial combinations and plants.