Optimizing Anaerobic/Biological Soil Disinfestation Amendment Composition Through Soil Fermentation Experiments

Progress report for GS22-266

Project Type: Graduate Student
Funds awarded in 2022: $16,500.00
Projected End Date: 08/31/2024
Grant Recipient: University of Tennessee
Region: Southern
State: Tennessee
Graduate Student:
Major Professor:
Dr. David Butler
University of Tennessee, Knoxville
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Project Information


Anaerobic (or biological) soil disinfestation (ASD) is a promising technique for control of soilborne plant pathogens, including fungal pathogens. Treated soil is amended with easily decomposable organic amendments, covered with plastic film, and saturated via irrigation for a brief time period to induce anaerobic conditions. ASD was developed as an alternative to chemical fumigation, which has severe ecological and human health hazards. Known mechanisms of ASD fungitoxicity include release of fungicidal volatile fatty acids (VFAs) and dissolved Fe2+ and Mn2+ because of anaerobic soil conditions, and changes to the soil microbial communities. However, little is known about how soil amendments (substrates) used in ASD, soil texture, and soil pH affect the balance of VFAs produced and the soil concentrations of dissolved Fe2+ and Mn2+, important components in suppressing soilborne fungal pathogens. In this proposed study, we will perform soil incubation studies on Fusarium oxysporum (Fo)-infested soils of varying soil texture to examine how substrate composition influences VFA and reduced metal balance. We will first perform soil incubation studies on soils of varying texture amended with varying levels of soybean protein isolate and dried molasses (to vary amendment protein to carbohydrate ratio), followed by greenhouse studies utilizing strawberries grown in soil infested with Fo and amended with a range of amendment types, and either limed or not limed (to alter soil pH during treatment). Data from this experiment will be used to identify the optimal amendments for not only Fo suppression, but also for strawberry plant health.

Project Objectives:
  1. Evaluate the effects of ASD amendment substrate composition (protein-to-carbohydrate ratio) and lime application on Fo suppression and soil VFA, Fe2+ and Mn2+ balance in soils of varying texture.

Hypothesis: High-protein amendments and liming are expected to favor increased production of long-chain VFAs such as n-butyric and isovaleric acids compared to high-carbohydrate amendments and therefore enhance Fo suppression. Additionally, the efficacy of VFAs in suppressing Fo is expected to be lower in fine-textured than in course-textured soil due to greater levels of VFA adsorption by clay and silt particles. However, this is also expected to be counterbalanced by the release of greater concentrations of Fe2+ and Mn2+ into soil solution as silt and clay minerals are typically richer in Fe and Mn than sand minerals.

  1. Evaluate VFA and reduced metal balance, plant health, and response of soil fungal populations (Fo, Trichoderma spp., mycorrhizal fungi) associated with different ASD amendments.

Hypothesis: High-protein/high-metal amendments such as spent brewer’s yeast and Azolla meal will both be expected to enhance production of long-chain VFAs such as n-butyric and isovaleric acid in addition to Fe2+ and Mn2+ compared to carbohydrate-based amendments such as wheat bran and dried molasses. Therefore, high-protein/high-metal amendments are expected to not only be more effective for suppressing Fo but also maximize benefits to plant health and beneficial soil fungi by supplying diverse biochemical substrate types as well as macro- and micronutrients.


Materials and methods:

Soil Conditions

Silty clay soils werebe collected in east Tennessee, sieved (10-mm mesh) to remove large rocks and debris, then air-dried. After texture determination via the hydrometer method, soil samples will be mixed in varying weight ratios with sand to achieve silt-loam, sandy loam, and sandy soils. Soil soluble Ca, Mg, K, P, Fe, Mn, and Al concentrations will be measured by Mehlich-1 extraction and inductively-coupled plasma atomic absorption spectroscopy (ICP-AES). The sum of Mehlich-1 extractable Ca, Mg, K, Fe, Mn, and Al, and H+ concentrations determined by soil pH measurements will be used to calculate the CEC for each soil texture. Total soil C and N concentrations will be measured by the dry combustion method.


Inoculum Preparation

Inoculum will be prepared as described by Momma et al. (2006). An Fo isolate previously collected from diseased strawberry roots from a commercial Tennessee field was grown on potato dextrose broth (PDB) for seven days at 25oC with shaking. To obtain Fo chlamydospores, the mycelial mass grown in PDB was suspended in potato sucrose broth (PSB) for seven days, homogenized, and added to sterilized soil water extract and incubated four weeks at 25oC.


Soil Fermentation Studies

Centrifuge tubes (50-mL) were filled with 30 g of soil of silty clay, sandy loam, or sand texture (Table 1). Each soil sample within centrifuge tubes was inoculated with 4 mL of chlamydospore suspension with ~25,000 chlamydospores/ml, mixed with amendment (different ratios of protein and carbohydrate using soy protein isolate and dried molasses; Table 1) at 1% w/w basis, then saturated with water and incubated for two weeks at 30-35°C. Inorganic N fertilizers was used to adjust the C:N ratios of all amendment mixtures to 5:1, the C:N ratio of the soybean protein isolate (Yoshiki et al., 2013). Limed (0.1% w/w dolomitic lime applied) and no lime treatments will be included alongside nonamended controls. For each treatment combination, there were 4 replicates and the experiment was repeated.


Table 1. ASD amendment trials: the effect of protein to carbohydrate ratio and soil texture on VFA balance and disinfestation efficiency





1. 100% soybean protein isolate


2. 75% soybean protein isolate, 25% dried molasses


3. 50% soybean protein isolate, 50% dried molasses


4. 25% soybean protein isolate, 75% dried molasses


5. 100% dried molasses


Soil Texture


1. Silty clay


2. Sandy loam


3. Sand




1. no lime


2. 0.1% w/w dolomitic lime


Factorial combinations


# of experimental units total (2 trials)



Greenhouse Studies

Pots will be filled with approximately 2.5-L sand, sandy loam, or silty clay soil and inoculated with the chlamydospore-containing suspension at a rate of ~10000 chlamydospores per gram soil. For each soil texture type, there will be five treatments with three replicates per treatment; control (inorganic N added) and four amendment types added at 1% w/w to soil (dried molasses, wheat bran, Azolla meal, and spent brewer’s yeast; both with 0.1% calcium carbonate lime and no lime replicates (Table 2). Each pot was wetted to field (container) capacity, covered with plastic tarp, and incubated for ASD treatment at 25-35°C for 14 days in the greenhouse. Post treatment, one strawberry transplants (plugs) was  planted in each pot.


Table 2. ASD amendment greenhouse trials: the effect of ASD amendment type and soil texture on VFA balance and disinfestation efficiency





1. Control


2. Dried molasses


3. Wheat bran


4. Azolla meal


5. Spent brewer's yeast


Soil Texture


1. Sand


2. Sandy loam


3. Silty clay




1. No lime


2. 0.1% w/w calcium carbonate lime


Factorial combinations


# of replications total (3 reps x 2 trials)




VFA and Reduced Metal Extraction and Measurement

Volatile fatty acids (i.e., acetic, n-butyric, and isovaleric) produced during ASD will be extracted from soil and measured by HPLC (Agilent 1260, USA) using methods as described by Shrestha et al. (2020). Additionally, water-extractable reduced metals will be measured by ICP-AES analysis similarly to Momma et al. (2011).


Assessment of Fusarium oxysporum Populations and Plant Health

Fusarium oxysporum survival following ASD treatments will be quantified using serial dilution of 1 g of soil from each pot resuspended in 9 mL sterile deionized H2O and diluted further to make 10-1 and 10-2 dilutions. A 50-µL aliquot of each dilution for each sample will be plated on Fo selective medium (Komada’s) and incubated for 5 days. Identification of Fo will be confirmed as needed with compound microscopy at 100X magnification to identify microconidia, monophialids, and white colony color with purple pigments in agar, the defining features of Fo. Eight weeks after transplanting the strawberry plants, they will be removed, and roots assessed for infection symptoms. Plant health will be assessed using root and shoot biomass measurements and strawberry fruit counts and weights from fruits collected weekly throughout the trial.


Assessment of Soil Abundance of Trichoderma and Mycorrhizal Fungi

DNA will be isolated from soil from each pot using the DNAEasy PowerSoil Pro Kit (Qiagen) according to the manufacturer’s protocol. We will evaluate the abundance of Trichoderma spp. and arbuscular mycorrhizal fungi in rhizosphere soil through soil DNA analysis using primer sets specific for AMF as described by Bodenhausen et al. (2021) and Trichoderma spp. as described by Kim and Knudsen (2008). We will perform qPCR as described by Shrestha et al. (2020) to determine abundance of AMF or Trichoderma spp.


Data Analysis

The Fo population data will be log10 transformed before statistical analyses. Differences in soil physicochemical (VFAs, Mn2+ and Fe2+) and microbial properties among treatments will be compared using an F-protected LSD test at P ≤ 0.05 after a one-way ANOVA to test for significant differences in reduced metal and VFA balance and Fo suppression efficacy between the treatments. Two trials will be performed for both the soil fermentation and greenhouse studies. A randomized complete block design model blocked by replicate will be used and statistical analyses will be performed using SAS 9.4 software.

Participation Summary

Educational & Outreach Activities

2 Consultations
1 Curricula, factsheets or educational tools

Participation Summary:

Education/outreach description:

We are currently working on a manuscript with anticipated submission in Summer 2023 related to Obj. 1 results.  Other outreach activities will occur as the project progresses.

Project Outcomes

Knowledge Gained:

For objective 1, our preliminary results indicate that suppression of Fusarium oxysporum (Fo) was significantly influenced by both amendment composition and soil texture (P < 0.05). The suppressive effects of ASD were strongest in sandy soils, in which amendments containing 25-100% soybean protein isolate (SPI) with the rest being dried molasses (DM) reduced viable Fo CFUs by over 2 log units with 50% SPI and 50% DM being the most effective. On the other hand, 100% DM amendment only reduced the number of viable Fo CFUs by a single log unit. As the soil gets finer-textured, the suppressive effect of ASD became less pronounced. In sandy loam soils, amended soils exhibited reductions of viable CFUs by 1.2-1.8 log units compared to unamended soils with the greatest reductions seen in sandy loam soils amended with 100% DM. The effectiveness of ASD was lowest in silty clay soils, with reductions in Fo CFU viability by 0.4 to 0.7 log units with little difference between amendments.

Amendment composition and soil texture both significantly influenced the total amount of VFAs as well as the concentration of n-butyric and isovaleric acids, the two most Fo-suppressive VFAs, in the soil solution (P < 0.05). Higher protein amendments were associated with higher total VFA concentrations as soils amended with 100% SPI had the highest total VFA concentration (~30 mmol L-1) followed by 75% SPI/25% DM (~22.4 mmol L-1) while soils amended with 50% or less SPI combined with 50% or more DM had ~16-17 mmol L-1 total VFAs in the soil solution. Likewise, higher protein content in amendments were also associated with higher n-butyric + isovaleric acid concentrations, as soils amended with 100% SPI had ~2.7 mmol L-1 n-butyric + isovaleric acids followed by 75% SPI/25% DM and 50% SPI/50% DM (~1.5 mmol L-1), 25% SPI/75% DM (~1.2 mmol L-1) and 100% DM (~0.8 mmol L-1). Additionally, total n-butyric and isovaleric acid concentrations were the highest in sandy soils (~1.8 mmol L-1) followed by sandy loam (~1.3 mmol L-1) with the lowest concentrations in silty clay.


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.