Final report for FNE19-943
Project Information
We performed an experiment in the summer of 2019 at Endless Farm LLC in Johnston, RI to measure direct impacts of indigenous microorganism (IMO) microbial spray, including soil microbial diversity, soil nutrient availability, and plant nutrient density in a lettuce crop. We prepared IMO inoculant according to the method of Llamelo et al (2016). Rice cooked with water 1:1 by weight was placed in a permeable basket in a nearby forest. The moldy rice was mixed with equal weight of brown sugar, mixed with well water at a 1:5 ratio by weight, strained, and sprayed on crops in the evening. The IMO spray was applied either four times during the one-month growing period, one time, or zero times with well water as a control. We tested plots for soil nutrient concentrations before and after the growing period, tested soil microbial communities using a phospholipid fatty acid (PLFA) analysis before and after the growing period, and tested plant leaves for leaf nutrient density at harvest. Our preliminary experiment found no impacts of IMO applications on the nutrient concentrations or microbial communities under the conditions the spray was tested. More research is needed to study the impacts of microbial inoculants on yield, pest and disease control, and soil microbial communities before recommendations can be made to farmers. We reached thousands of farmers by sending a summarized report to 9 local farmer newsletters, two local listservs, and holding two Q&A sessions on the Market Gardening Success Group and Korean Natural Farming farmer Facebook groups, where we directly interacted with nearly 100 farmers.
This project seeks to investigate how application frequency of indigenous microbial inoculant, also known as indigenous microorganism (IMO), may work to impact the size and diversity of the soil microbial community, soil nutrient availability, and leaf tissue nutrient density in lettuce production. If this method improves soil and plant nutrition by increasing soil microbial diversity, it will provide much needed evidence about how microbial amendments work, give farmers additional information on how often to apply microbial amendments, and allow farmers to make informed decisions about using microbial amendments.
In light of research suggesting overall nutrient density of vegetables has decreased in the US since the 1960s, many vegetable farmers have become interested in the effect of soil health on plant leaf nutrient density. With research suggesting tillage and soil drying negatively impact soil microbial populations, and commercial microbial inoculants commonly used to encourage beneficial fungal associations in large-scale soybean production, the concept of feeding the soil first has become a driving mindset for many vegetable farmers, who spend time and money trying out different methods to improve soil and plant health.
One inexpensive inoculant commonly used by small farmers is known as Indigenous Microorganism (IMO). IMO is distinguished from other microbial amendments in that, in theory, forest soils are used to culture a more diverse set of indigenous microbes. Some farmers report anecdotal benefits of IMO and related Korean Natural Farming practices, and they make broad-ranging claims including eradication of diseases and pest pressure on their farms, no longer a need to add nitrogen fertilizers to their crops, and improved crop quality and yield. Microbial diversity may well provide these benefits, but available research is limited on the impact of IMO application on microbial diversity, nutrient availability, and leaf nutrition.
While microbial inoculants are used by many vegetable farmers interested in improving the soil microbiome, limited research exists on IMO. Much of the existing research has focused on indirect effects of IMO, such as plant yield and size. With existing research showing both positive correlations and no correlations with plant yield and size, we aim to look at more direct impacts of IMO, including soil microbial diversity, soil nutrient availability, and plant nutrient density. This will allow us to better understand how IMO may work from non-anecdotal data, and contribute to standardized evidence to help farmers evaluate whether these practices might benefit them. Also, through testing both a single and repeated application of the microbial inoculant, we will also gain more knowledge of whether repeated applications are effective, unnecessary, or costly for the farmer.
Indigenous microorganisms (IMO) may provide an opportunity for farmers to inexpensively increase the microbial diversity of soils, with the possibility of many benefits following increased diversity, such as nutrient availability, increased plant size and nutrient quality, plant raw probiotic quality, reduced disease pressure, and nitrogen fixation by legume and non-legume denitrifying bacteria. This research may also give more information on how to increase nutrient density of plant leaf tissue.
IMO can be used on a farm of any size in any area of the world, with little cost to the farmer. Whether or not it is effective, or if it might take multiple applications or years to reach efficacy, is to be seen.
The Bionutrient Food Association in Massachusetts, dedicated to improving soil and plant nutrient quality, are influential among the up-and-coming young farmer community. Talks by farmers employing these microbial amendment practices are very popular at NOFA conferences, and farmers frequently discuss these topics on social media.
Endless Farm is a 2-acre organic no-till farm in Johnston, Rhode Island, producing greens and herbs since 2018.
Cooperators
- (Researcher)
Research
Experimental Design
Treatments were arranged as a randomized complete block design. Five 50' x 30" rows were established, with eight 8.2' x 2.5" plots established within each row. Plots were planted with three rows of lettuce (Lactuca sativa var. Cardinale) spaced 9” apart.
An indigenous microorganism (IMO) inoculant of aerobically fermented rice, cultured in a nearby forest soil, was prepared according to the method of Llamelo et al (2016). Inoculant was applied at a concentration of 200 mL IMO2 to 10 L water (Llamelo et al., 2016).
Plots were treated with no IMO, treated with IMO one time, or treated with IMO four times during the study. Control plots received an application of well water, and well water was also applied to plots not receiving IMO that week, to correct for the positive effect of a moist environment on microbial biomass. The treatments were as follows: 1) inoculant applied once after transplanting, and well water applied for the following three applications until harvest 2) inoculant applied approximately weekly after transplanting until harvest, and 3) well water applied approximately weekly after transplanting until harvest.
Field Site
The study was conducted at Endless Farm LLC in Johnston, Rhode Island (41°84’N, 71°54’W). The site was established in the spring of 2019 within a 1.4 acre field. The soil at the site is an Enfield silt loam (coarse-silty over sandy or sandy-skeletal, mixed, active, mesic Typic Dystrudepts) (http://websoilsurvey.nrcs.usda.gov/app/). The site has a 0-3% slope from northwest to southeast. There is no record of tile-drainage on the site. Before the initiation of the experiment in the spring of 2019, the site had been planted with leafy greens and not been treated with microbial inoculants, and in 2017 been planted with tulips. In 2016 and earlier, the site was fallow.
Soil samples were collected on March 31st for standard fertility analysis (pH, Modified Morgan extractable nutrients (P, K, Ca, Mg, Fe, Mn, Zn, Cu, B), lead, and aluminum, cation exchange capacity, and percent base saturation). Plots were weeded during the week prior to planting. On July 1st, feathermeal was added to each plot before planting at a rate of 75# N/acre, and calcitic limestone was added to amend the pH according to the recommendations for each test plot.
Crop Management
Lettuce (Lactuca sativa var. Cardinale) was seeded on May 15th and thinned to one germinated seed per cell. Plants of the same visual size were transplanted on July 2nd and were not root-bound. Crops were watered daily with an overhead rotating impact sprinkler system. Weeds were pulled on July 27-30th. Lettuce heads were harvested and weighed from August 16th through 18th. Each head in the plot was weighed and counted.
Microbial Inoculant
The indigenous microbial inoculant was prepared by cooking rice at a 1:1 ratio of rice to water by weight. Cooked rice was placed in on a forested hillside at Snake Den State Park in a permeable basket under the duff. Molded rice, with a majority of white mold, was collected and mixed with equal weight of brown sugar to make IMO2. IMO2 was mixed with well water at a ratio of 5:1, and the slurry was strained and applied with a sprayer in the evening to avoid strong sun. Attempts in July to inoculate moldy rice failed, and applications after July 30th were from a batch of IMO2 donated by a farmer from Orleans, Massachusetts, and prepared as described above. The microbial inoculant was applied on July 8th, 15th, 31st, and August 8th.
Soil Analysis
Soil samples were collected on March 31st and August 19th for standard fertility analysis (pH, Modified Morgan extractable nutrients (P, K, Ca, Mg, Fe, Mn, Zn, Cu, B), lead, and aluminum, cation exchange capacity, and percent base saturation). Six 2.4-cm diameter soil cores were sampled to 15 cm, composited, and air-dried before mailing for analysis.
Phospholipid Fatty Acid Analysis
Soil for PLFA was sampled to a depth of 15 cm. Five samples were taken per plot with a soil probe, composited, and subsampled to get 5-10 grams of soil per plot. The soil probe was cleaned with hydrogen peroxide between plots. Samples were placed in a freezer overnight at -10F until being shipped on ice. PLFA samples were taken after planting and prior to first microbial inoculant application on July 3rd, and on August 8th before harvest.
Leaf Tissue Analysis
Twenty mature lettuce leaves were collected at random from heads from each plot during harvest, and air-dried for leaf tissue analysis.
Statistics
Statistical analysis of treatments was performed using a Two-Way Analysis of Variance (ANOVA) using the ez package in R, and pairwise t tests were adjusted with the Bonferroni correction.
Impact on soil nutrient concentration
No meaningful significance was found in the data that suggests a linear impact of IMO treatments on any soil nutrient concentration tested (soil nutrient plots).
Impact on leaf tissue nutrient concentrations
As seen in the tissue test plots, no significant differences in leaf tissue concentrations of P, K, Ca, Mg, N, Cu, Mn, Fe, or B was measured between IMO treatments. The zinc concentration in leaf tissue for the single IMO application was significantly higher for plots treated with one application of IMO compared to the control or the four IMO applications samples. However, the control and four IMO application samples were not significantly different, so a linear effect cannot be confirmed.
Impact on PLFA concentration and microbial community composition
There were no significant effects of treatment by date for any PLFA measures of soil microbial population over time, including the composition of the soil microbiome, or the ratios of microbial groups to each other (soil PLFA plots).
Discussion
Our results revealed no significant impact of IMO applications on the nutrient concentrations in soil and in mature lettuce leaf tissue. Neither did our results find a significant impact of IMO applications on the concentration of various PLFAs and composition of the soil microbial community.
Though plots were arranged in a randomized complete block design, plots at times showed significant differences before any treatments were applied. A larger study with larger subplots might correct for variation seen in this small-scale plot.
The experiment was conducted on a no-till but unmulched area of field. Hot, drought-like summer conditions in 2019 combined with insufficient soil cover could significantly have significantly reduced soil microbial populations and negate the impact of microbial sprays. In addition, an irrigation malfunction exacerbated drought-like conditions in all plots for several days, which may have also reduced overall soil microbial communities.
Soil and PLFA samples were taken randomly within plots to test the overall microbial community, but soil microbial populations are the most concentrated near plant roots. This experiment could be repeated, but with samples taken intentionally in the rhizosphere of plants to observe the impact of microbial inoculants in the root system. This experiment could be repeated on a different crop with greater soil coverage.
More research is needed to study the impacts of microbial inoculants on yield, pest and disease control, and soil microbial communities before recommendations can be made to farmers.
Our results revealed no significant impact of IMO applications on the nutrient concentrations in soil and in mature lettuce leaf tissue. Neither did our results find a significant impact of IMO applications on the concentration of various PLFAs and composition of the soil microbial community.
More research is needed to study the impacts of microbial inoculants on yield, pest and disease control, and soil microbial communities before recommendations can be made to farmers. KNF farmers recommended attempting this experiment again by sidedressing with IMO4 (a mix of grain and soil inoculated with IMO2, following the guidelines documented in Cho's Natural Farming), rather than a foliar spray of IMO2. This research does not delve into the potential benefits to pest and disease control which some farmers have anecdotally noted. Though the cost of creating and applying IMO is low, little research exists on how microbial sprays influence crop yield and resilience to pests. Additional research is needed on a larger scale to determine the cost preparing and applying the spray compared to the benefits.
Education & Outreach Activities and Participation Summary
Participation Summary:
We created a summarized report (IMO-Informational-sheet) for farmers, extension educators, and researchers and submitted it to 9 New England-area newsletters, including NOFA/RI, Bionutrient Food Association, Young Farmers Network, URI Extension, UMass Extension, RI Division of Ag, NE Ag exchange, and two listserves, emasscraft and NCTFA. We held two question-answer sessions on farmer facebook groups, in which we directly interacted with 97 farmers and reached thousands more. We also consulted with four farmers interested in applying to SARE grants, and 6 people interested in IMO research.
Learning Outcomes
Researchers gained knowledge, skills, and awareness of sustainable agriculture methods, microbial inoculants, Korean natural farming methods, measures of soil microbial life, and gained information on future research questions.
Project Outcomes
Through this study, the PI learned much more about the diversity of preparations of microbial inoculants used by farmers around the world, the long history of microbial amendments in Korean farming, and connected with several farmers interested in performing research on microbial inoculants. There is a great interest in the farming community about organic and locally derived IPM, and during the Q&A sessions in the Korean Natural Farming and Market Gardening Success Group facebook groups, we interacted with nearly 100 farmers, learned more about the impact of plant variety on seed, and received lots of interest and information on improving future research.
Our results revealed no significant impact of IMO applications on the nutrient concentrations in soil and in mature lettuce leaf tissue, nor on the concentration of various PLFAs and composition of the soil microbial community under the conditions of the experiment. More research is needed to study the impacts of microbial inoculants on yield, pest and disease control, and soil microbial communities before recommendations can be made to farmers. We also recommend carrying out this study on a larger scale, to better assess the cost-benefit of application on a larger-scale.
A paper is being drafted for publication. Though no significant results appeared, the PI and Advisor feel the research will be useful for future research on microbial inoculants.
Though plots were arranged in a randomized complete block design, plots at times showed significant differences before any treatments were applied. A larger study with larger subplots might correct for variation seen in this small-scale plot.
Many farmers using homemade microbial inoculants are invested in following KNF practices specifically outlined by Master Cho, which this experiment did not cover. Future research on using the IMO4 microbial inoculant and layering multiple practices would better test and explain relationships between variables in the natural farming approach.
The experiment was conducted on a no-till but unmulched area of field. Hot, drought-like summer conditions in 2019 combined with insufficient soil cover could significantly reduce soil microbial populations and negate the impact of microbial sprays. In addition, an irrigation malfunction exacerbated drought-like conditions in all plots for several days, which may have also reduced overall soil microbial communities.
Soil and PLFA samples were taken randomly within plots to test the overall microbial community, but soil microbial populations are the most concentrated near plant roots. This experiment could be repeated, but with samples taken intentionally in the rhizosphere of plants to observe the impact of microbial inoculants in the root system. This experiment could be repeated on a different crop with greater soil coverage.
More research is needed to study the impacts of microbial inoculants on yield, pest and disease control, and soil microbial communities before recommendations can be made to farmers.