The Use of Nematodes and Enzyme Activities For On-Farm Soil Biological Health Tests

Final report for GNC20-308

Project Type: Graduate Student
Funds awarded in 2020: $10,875.00
Projected End Date: 12/31/2021
Grant Recipients: The Ohio State University ; Ohio State University
Region: North Central
State: Ohio
Graduate Student:
Faculty Advisor:
Dr. Christine Sprunger
Michigan State University
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Project Information


The Use of Nematodes and Enzyme Activities For On-Farm Soil Biological Health Tests

Soil degradation from intensive agricultural production can lead to many environmental issues, such as increased erosion, nutrient run-off, and crop disease. In addition, climate change will add to the environmental burden from agriculture as farmers adapt their agricultural practices to increased rainfall events and other climate change related phenomena. Enhancing soil health is a key strategy for farmers to maintain yields, reduce environmental issues, and adjust to climate change. Soil biological indicators can aid farmers in making comprehensive management decisions that can result in the improvement of soil health. Specifically, soil biological indicators map soil food webs and can therefore provide information on nutrient cycling and soil structure. Additionally, soil biological indicators are likely to be sensitive to changes in management practices, if this correlation was understood farmers could use this information to improve their soil health and therefore their productivity. Particularly, soil biological indicators of nematodes and enzyme activities can be used to provide farmers with a rapid assessment of the structure, enrichment, decomposition channel, disturbance, and nutrient cycling within a given cropping system. However, standard soil health tests currently do not utilize soil biological indicators to reflect soil food webs. Additionally, the sensitivity of nematode indices and enzyme activities to alterations in management practices has not been investigated. Thus, a knowledge base that may be able to inform farmer management decisions from these soil biological indicators will be created from the first part of this proposal.  This project aims to provide famers with answers to their prolonged question of “how we can increase the number of organisms that we have in the soil?” or “how are my biologicals doing?” (Sprunger, 2015). The goals of this project areto: 1) Determine if nematode indices and soil enzymes can serve as useful soil health indicators through the development of a knowledge base using soil biological indicator samples taken from two long-term no-till sites and 30 selected Ohio farms 2) Quantify nematode indices and enzyme activities across farmer fields in Ohio 3) Conduct on-farm interviews to understand farmers interest in soil biological indicators and how they may help farmers adjust best management practice decisions. The outcomes of this project can provide Ohio farmers with soil health tests that will help inform management decisions and therefore, improve yield, reduce run-off, and aid in the adaption to climate change.

Project Objectives:

This unique study will be the first to understand how long-term management practices of sustainable and conventional agriculture influence soil food webs. Particularly, soil biological indicators will be measured on two long-term no-till sites and 30 selected Ohio farms.  The measurement of microbial communities, nematode indices, and enzyme activities will be used to map soil food webs and therefore, provide unique information on the sensitivity of soil biological indicators to management change and the structure and function of the soil food web. The results from the investigation of these biological indicators will be able to provide farmers with information pertaining to the function of the soil food web under varying management practices

Farmers will learn how soil biological indicators can aid them in making comprehensive management decisions that will assist in the adaption to climate change, increase yield, and improve soil health. Specifically, nematode indices and enzyme activities can provide insight on the structure, decomposition channel, level of disturbance, enrichment, and nutrient cycling within their fields. After measuring the soil biological indicators on their fields, farmers will learn how to assess and interpret the results. The enhanced information and the knowledge base created through this study will be shared with the farmers and encourage the implementation of sustainable agriculture practices and enhanced soil health.


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Materials and methods:

On-farm Study


Our first objective was to quantify nematode indices and enzyme activities across farmer fields in Ohio and assess how such indicators informed soil biological health. To address this objective, farmers were recruited by extension agents and by university faculty and students at farmer-focused conferences. The farmers that expressed interest in the participation of this study were sent mail-in soil sample kits. Farmers were instructed to collect a stratified random sample from a field they perceive to be functioning the “best” and one that is functioning the “worst” in terms of soil health and productivity. In addition, farmers were asked to disclose their management strategies (crop type, tillage type, irrigation, etc.) within each of their best and worst fields. After the sample collection, the soil sample kit was mailed back to the lab for processing.  


Soil-health test processing


Soil health tests that comprised of free-living nematode identification, enzyme activities, mineralizable carbon, permanganate oxidizable carbon, protein, and soil texture were conducted on all collected samples. Additionally, routine soil health tests were conducted by Routine soil analyses were conducted by Spectrum Analytics (Washington Court, OH). Soil texture was measured using the protocol adapted from Gee et al., (1986)


Soil biological health indicators

Soil biological health indicator analyses that reflect C and N pools were conducted by the Soil Fertility Lab at The Ohio State University. Soils for POXC, protein, and mineralizable C were dried at 40°C. After drying samples were ground and sieved to 2 mm. The mineralizable C measurement reflects the pool of C that is most available to microbial communities. Mineralizable C was measured using the methods adapted from Franzluebbers et al., (2000) and Hurisso et al., (2018). The POXC analysis was adapted from Weil et al., (2003) and Culman et al., (2012) and reflects a more processed pool of labile C. Autoclave-citrate extractable (ACE) soil protein reflects an available pool of organic N and was measured through methods adapted from Hurisso et al. (2018).


Enzyme Assays

Enzyme activity was determined colometrically using the protocol adapted from Deng and Popova (2011) and Tabatabai (1994). In this study, enzyme activities of β-glucosidase (GLU), N-Acetyl-β-glucosaminidase (NAG) and aryl sulfatase (AS) were measured.


Nematode Analysis

Free-living nematodes were extracted using the elutriation method (Oostenbrink, 1960 The samples were then processed using the centrifugal sugar flotation method to bring the nematodes into solution (Hooper et al., 2005).


The total number of nematodes were counted in each sample under a microscope at 50x magnification and 100 individuals were identified to family at 100-400x. If the sample contained less than 100 nematodes all individuals were identified. Nematode counts were expressed as the number of nematodes in 100 g of dry soil. Each nematode was classified as an adult or dauer larvae to allow the determination of the population stage. Nematode taxa were assigned to trophic groups (Yeates et al., 1993) and colonizer-persister groups based on Bongers and Bongers (1998).  Soil food web indices were calculated using NINJA (Sieriebriennikov et al., 2014).


Farmer Interviews


We conducted farmer interviews to understand farmers’ interest in soil biological indicators and how indicators may help farmers adjust best management practices. Once soil health processing was completed, a soil health test report was compiled for each farmer, which included results of the soil health indicators that were measured and a comparison between their best and worst fields. These reports were sent to each of the participating farmers. After the compilation of their reports, individual farmer interviews were scheduled in which the researchers and farmers met to discuss and explain the results of their soil health test reports. Farmers were also asked additional questions related to the usefulness of the different soil health indicators, how indicators might be used to form future management decisions, and general questions about soil health.


Long-term Experimental Trial Study


Our third objective was to determine if nematode indices and soil enzymes could serve as useful soil health indicators through the development of a factor analysis model using soil biological indicator samples taken from two long-term no-till sites and 30 selected Ohio farms soil samples were collected during harvest from two long-term experimental trials with contrasting soil types (silt-loam and clay) within Ohio. Samples were collected within plots that were under varying combinations of tillage intensity and crop rotational diversity. The tillage factor was comprised of plots under no-till and chisel till. The crop rotational factor was comprised of plots under continuous corn, corn-soy crop rotations, and corn-forage-forage crop rotations. All soil health indicators mentioned above for the on-farm study, were also analyzed for this study. The major difference in terms of analyses was adding a microbial component, to explore the broader soil food web.


Microbial Analysis


Soil samples from the long-term trials were sequenced to quantify the effect various tillage intensities and rotational diversity have on microbial community structure. Soil DNA was extracted from homogenized soil (0.25 g) using the MO BIO PowerSoil®DNAIsolationKit. PCR was run for each soil sample to amplify the DNA. The DNA amplicons were cleaned using the SPRI bead, quantified using the Quant It Pico Greenkit, and the libraries pooled at equal molar ratios. The Illumina MiSeq platform, using paired end reads (Illumina Reagent Kit v2, 500 reaction kit) at the MICIC (Wooster OH) will be used to sequence the pooled libraries.



Creation of a soil health framework


To understand how soil health indicators can inform underlying soil health traits and to investigate if long-term agricultural trials and on-farm trials effects soil health differently a factor analysis was conducted. In summary, all data that was compiled that described management practices and the measurement of all soil health indicators. An exploratory factor analysis was then conducted to synthesize the underlying soil health traits that impact soil health indicators. The effect of management practice and type of agricultural trial was then tested on the soil health traits to understand if soil health traits are impacted by management practice and if there was a difference between long-term and on-farm trials an analysis of variance was conducted to test the effect of management practice and trial type on the soil health traits.


Research results and discussion:

Nematode Analysis


The quantification of free-living nematodes on-farm and across two long-term experimental trials indicated that nematode feeding group abundances rather than nematode indices may serve as a valuable tool for soil health assessment within midwestern cropping systems. Within experimental trials only one nematode index was significantly affected by variance within tillage and crop rotation. Specifically, systems with perennial crop rotation and under no-till systems had a significantly greater channel index, indicating decomposition through the fungal microbial channel. In contrast, nematode feeding group abundances of bacterivores were greater in systems with increased management intensity of annual legume rotation and increased tillage. Sensitive nematode feeding group abundances of predator-omnivore nematodes were found to be enhanced in systems of reduced tillage intensity. Within on-farm systems, sensitive nematode feeding groups of predator-omnivore abundances were greater in farmers chosen “best” fields. Additionally, like that of the long-term experimental trials nematode indices commonly did not indicate differences of soil biological health between farmers “best” and “worst” fields.  These results indicate that nematode feeding groups rather than nematode indices may be more so suited as indicators of soil food web health in varying agricultural management practices.


Soil biological health indicators


Soil health indicators that represented the stabilization of the C pool (permanganate oxidizable carbon (POXC)), organic nitrogen (N) (protein), and microbial activity (respiration) proved to be sensitive in indicating changes in management practices both on-farm and in experimental trials. Within experimental trials protein, respiration, and POXC were found to be significantly greater in systems with increased crop rotation diversity and decreased tillage intensity. Similar to the experimental trials, on-farm systems that incorporate sustainable management practices such as crop rotation, organic farming, and reduced tillage intensity were also found to enhance POXC, protein, and respiration. Additionally, when comparing farmers “best’ and “worst” fields POXC, protein, and respiration were usually enhanced in the “best” field. These soil biological indicators have the potential to serve as valuable indicators of the effect of management intensity on carbon storage, organic nitrogen storage, and microbial activity.


Enzyme Assays


Enzyme activities that measured carbon, phosphorus, sulfur, and nitrogen nutrient cycling were found to be sensitive soil health indicators of sustainable management practices both on-farm and in experimental trials. Specifically, all enzyme activities that were measured were enhanced in systems that incorporated more sustainable management practices. This study indicated that enzyme activities are valuable soil biological health indicators of nutrient cycling within varying management practices.  


Farmer Interviews


Farmer interviews were conducted as a one-on-one meeting between the researchers and farmers where soil health test reports were explained to the farmer and the researchers were able to understand what part of the soil health reports were most interesting and most useful for farmers to understand the health of their soil on both their “best” and “worst” fields. Most farmers found the common soil health measurements such as organic matter to first be the most interesting and useful as these are soil health indicators that were most familiar to them. However, once novel soil biological indicators that were more sensitive to management practice differences in their “best and “worst” fields were explained many farmers grew extremely interested in understanding the health of the soil food web in their soil. Additionally, in many of the interviews farmers asked considerable follow-up questions such as “what management practices can we adopt to enhance the health of the soil food web in our systems?”. Overall, farmers supported and wanted to learn more about the novel soil biological health indicators that were introduced to them within the soil health test reports.


Creation of a soil health framework


To understand the relationships between all soil health indicators that were measured within this study an exploratory factor analysis was utilized to understand if the suite of soil biological health indicators that were measured share similar underlying soil health traits. Results indicated that soil health indicators of enzyme activities and nematode feeding groups shared similar soil health traits. However, nematode indices did not integrate with any other soil biological indicators. Additionally, the soil health traits that described nematode indices were not significantly affected by management practices. These results demonstrate that soil health indicators of nematode feeding groups and enzyme activities may better describe changes in soil health from management practices than compared to nematode indices. Thus, the use of exploratory factor analysis can aid in discerning what soil health indicators to use when assessing soil health.

Participation Summary
40 Farmers participating in research

Educational & Outreach Activities

40 Consultations
2 Curricula, factsheets or educational tools
1 Journal articles
1 Webinars / talks / presentations

Participation Summary:

40 Farmers participated
3 Ag professionals participated
Education/outreach description:

Farmer soil health consultations took place virtually during the winter of 2021. The consultations included one-on-one discussions between university researchers and farmers, whereby soil health test reports were explored in a participatory action research framework. Specifically, from February-March farmers were emailed their soil health report package and a meeting was set up to explain the results of the soil heath report.


Two factsheets and educational tools have been created. The first was a factsheet given to farmers in the soil heath test kits that were mailed to them at the beginning of the project, which described which soil health indicators were analyzed after they sent their samples in. The second educational tool was the individual soil health report package that was created for the farmer to reference and understand the results of their soil health test. This soil health report was discussed with the farmer during the consultation.


Currently, one manuscript highlighting work from the long-term experimental trials is under review in the journal of Soil Science and Plant Nutrition. A second manuscript is under review in the  Journal of Ecological Indicators 2021. The second manuscript features a factor analysis that was conducted to understand the differences between on-farm and long-term trials on soil health indicators, as well as to discern the relationship between different soil health traits under varying management practices with a special emphasis on how soil food web structure and function can infer soil health.


Results from the Factor Analysis was presented at the Ecological Society of America meeting on August 4th 2021. The title of the talk that was presented was “A conceptual framework for assessing the suitability of novel soil biological health indicators in varying agroecosystems”. This talk aimed to explain the results of the quantitative factory analysis to other researchers to explain the novelty behind quantitatively understanding the relationships between soil health indicators, and how underlying soil health traits may govern certain groups of soil health indicators.

Project Outcomes

2 New working collaborations
Project outcomes:

This project provided farmers with a conclusive soil health test report on both farmers “best” and “worst” fields. The conclusive report directly affected agricultural sustainability as this report could then be utilized for farmers to shift their management practices to enhance the health of their soils. This project has contributed to the future sustainability of agriculture as the potential of novel soil biological indicators to function as sensitive indicators of soil biological health was assessed. This project found that on both experimental and on-farm sites nematode community feeding groups, enzyme activities, POXC, protein, and respiration may be the best soil biological health indicators to inform the affect of agriculture management practices on soil food web health. Thus, moving forward efforts can now be made to enhance the efficiency and reliability of these specific soil biological health indicators. This data and assessment comprises one published peer-reviewed publication in the Journal of Ecological Indicators. 

Knowledge Gained:

This project substantially changed my views on which agricultural management practices are the most sustainable for enhancing soil health. Specifically, for many of the on-farm fields that were surveyed it was more common for conventional agricultural systems that adopted sustainable practices to have enhanced soil health than compared to organic systems. This finding was surprising to me as organic agriculture is thought to be more sustainable, however, it became apparent that this form of agriculture can still be quite intensive and disruptive to soil ecosystems. Additionally, this project made me more aware of the soil biological health indicators that are the most sensitive and useful to farmers. This information can now help me gauge which soil health indicators may be most practical to perfect for wide-scale utilization of these novel soil health indicators.

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.