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

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 25^oC 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 25^oC.

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.

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.

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.