Final report for GS22-266
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.
- 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.
- 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.
Research
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.
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Table 1. ASD amendment trials: the effect of protein to carbohydrate ratio and soil texture on VFA balance and disinfestation efficiency |
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Factor |
Levels |
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Amendment |
5 |
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1. 100% soybean protein isolate |
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2. 75% soybean protein isolate, 25% dried molasses |
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3. 50% soybean protein isolate, 50% dried molasses |
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4. 25% soybean protein isolate, 75% dried molasses |
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5. 100% dried molasses |
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Soil Texture |
3 |
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1. Silty clay |
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2. Sandy loam |
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3. Sand |
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Liming |
2 |
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1. no lime |
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2. 0.1% w/w dolomitic lime |
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Factorial combinations |
30 |
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# of experimental units total (2 trials) |
240 |
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.
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Table 2. ASD amendment greenhouse trials: the effect of ASD amendment type and soil texture on VFA balance and disinfestation efficiency |
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Factor |
Level |
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Amendment |
5 |
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1. Control |
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2. Dried molasses |
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3. Wheat bran |
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4. Azolla meal |
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5. Spent brewer's yeast |
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Soil Texture |
3 | |
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1. Sand |
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2. Sandy loam |
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3. Silty clay |
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Liming |
2 | |
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1. No lime |
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2. 0.1% w/w calcium carbonate lime |
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Factorial combinations |
30 | |
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# of replications total (3 reps x 2 trials) |
180 | |
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.
Our results demonstrate the importance of both soil texture and root-associated fungal communities for safeguarding the health of strawberry crops in heavily-infested soils even if ASD fails to kill a meaningful proportion of pathogens. Even though higher soil clay content was associated with greater Fo CFU viability, higher soil clay content was also associated with healthier plants. This may be due to a wide variety of reasons. Clay adsorbs not just the VFAs produced by ASD but also the extracellular enzymes and effector molecules utilized by soilborne plant pathogens to infect crop roots in addition to adsorbing root exudates that trigger the germination of pathogen resting spores, therefore restricting pathogen germination (Smith 1977; Alabouvette 1993; Li et al. 2018). Additionally, clay-enriched soils are also enriched in key micronutrients essential for crop health such as Fe, Mn, Mo, B, and Si compared to sandy soils (Siddiqui et al. 2016; Orr and Nelson 2018). Likewise, even though suppression of Fo by ASD was greater in sandy soils, overall crop health as demonstrated by vastly reduced root and shoot biomass was also the worst in sandy soils. This may be in part because none of the strawberries were fertilized beyond the original ASD amendment and application of aqueous nutrients to the pots upon transplantation so the nutrients within the sandy soils was leached out when the pots were irrigated.
The presence of beneficial fungi has also proven far more important than Fo suppression by VFA for safeguarding the health of strawberry plants grown in heavily infested soils. For instance, while Fo CFU viability levels were among the highest in all unlimed silty-clay soils, the strawberry plants growing in them were healthier than those grown in sandy soils, where the lack of nutrients may have stunted the growth of strawberry plants and likely made them more susceptible to fusarium wilt. In fact, even though both Serendipitaceae (ectomycorrhizae) and Glomeromycetes (arbuscular mycorrhizae) were present in the strawberry root microbiomes analyzed by amplicon sequencing, Serendipitaceae but not Glomeromycetes had significant positive impact on crop growth. This may be because of the greater versatility of members of the family Serendipitaceae, which can live as saprophytes when they are unable to form mycorrhizal partnerships. This may explain why Serendipitaceae were favored in pots amended with dried molasses or Azolla meal, which are carbohydrate-dominated amendments but not the protein-rich spent brewer’s yeast treatments.
Lastly, it is very important to keep in mind the limits of ASD for treating soilborne crop diseases such as black root rot of strawberry caused by Fo. Past studies demonstrating the effectiveness of ASD against Fo typically utilized fresh hyphal biomass and/or conidia/bud cells, or infested field soil, which can be expected to contain a mixture of macro- and microconidia, and chlamydospores. However, the resistance of conidia and hyphal biomass to environmental stresses, including VFAs is significantly lower than that of chlamydospores and in turn, Fo chlamydospores have higher inoculum potential than conidia and hyphae. This may explain the relative lack of success of ASD in suppressing Fo, particularly in sandy loam and silty clay soils compared to past ASD experiments. Likewise, the fact that ASD, particularly with amendments rich in protein in addition to carbohydrates actually led to reduced strawberry crop health compared to inoculated controls, as shown by root and shoot biomass may be because the amendments actually enhanced the germination of chlamydospores that survived ASD treatment. This is because Fo chlamydospores need to be induced to germinate by the presence of a combination of glucose, amino acids (i.e., asparagine), and dissolved Fe. Therefore, while ASD is often effective in treating Fo-infested soils, other disease management techniques may be necessary to prevent the buildup of inoculant, particularly chlamydospores. For example, infected plants can be rouged when found and residues of infected crops should be removed from the field post-harvest to minimize the chance of overwintering pathogens infecting future crops. Crop rotation can be used to prevent the buildup of the inoculum of Fo forma speciales specialized for a particular crop. Additionally, ensuring high root colonization levels by beneficial fungi such as members of the order Agaricales or family Serendipitaceae as was the case when dried molasses was used as the ASD amendment can protect crops from infection and help enable their healthy growth even in soils with high pathogen population densities.
Educational & Outreach Activities
Participation Summary:
Published Abstracts from Presentations at Professional Conferences:
Littrell, J., Ownley, B.H., and Butler, D.M. Examining the Biogeochemical Mechanisms of Fusarium oxysporum Suppression in Anaerobic Soil Disinfestation. Proceedings of the ASA – CSSA – SSSA International Annual Meeting, October 29 - November 1, St. Louis Mo. 2023
Littrell, J., Shrestha, U., Ownley, B.H., and Butler, D.M. Soil Texture, Soil pH, and amendment protein: carbohydrate ratio affect suppression of Fusarium oxysporum under anaerobic soil conditions by volatile fatty acids and reduced metal cations. Proceedings of the ASA – CSSA – SSSA International Annual Meeting, November 6-9, Baltimore MD. 2022
Littrell, J., Ownley, B.H., Shrestha, U., Rice, J.H., and Butler, D.M. Optimizing anaerobic soil disinfestation through soil fermentation experiments. Proceedings of the American Phytopathological Society Southern Division, March 7-10, Chattanooga TN. 2022.
Refereed Journal Articles:
Littrell, J.J., B.H. Ownley, and D.M. Butler. 2024. Unraveling the interplay: Soil biogeochemical factors shaping the efficacy of anaerobic soil disinfestation in suppressing Fusarium root rot of strawberry. Phytopathology 114:1782-1790, doi:10.1094/PHYTO-09-23-0323-R (recognized as Editor’s pick manuscript, August 2024 issue).
Littrell, J., B. H. Ownley, Z. R. Hansen, K. D. Gwinn, and D. M. Butler. Role of organic amendment composition and soil texture in modulating volatile fatty acids, Fe/Mn reduction, and Fusarium oxysporum suppression during anaerobic soil disinfestation. (in review)
Project Outcomes
While ASD can generate pathogen-suppressing volatile fatty acids and reduced metal cations, these may not necessarily be produced in the amounts necessary to suppress some of the more resistant pathogen resting spores such as Fusarium oxysporum chlamydospores. However, one can still potentially safeguard the health of crops grown in heavily infested soils with the help of beneficial root-associated fungi such as members of the genus Trichoderma, family Serendipitaceae, or order Agaricales. Therefore, further research in developing methods to use different amendments to encourage the growth of beneficial root-associated fungi following ASD treatments and/or inoculation of crop roots with beneficial fungi would help further the goal of developing safe low-cost alternatives to chemical soil fumigation.
Soil and Amendment Properties and Fo Suppression
The suppression of Fo was significantly influenced by amendment composition, soil texture, and their interaction. The suppressive effects of ASD were strongest in sandy soils, in which amendments containing 25 to 100% soybean protein isolate (SPI) and the balance as dried molasses (DM) reduced viable Fo CFUs by 1.1 to 2.6 log10(CFU+1)/g soil compared to the unamended control. As soil clay content increased, the suppressive effect of ASD became less pronounced. In sandy loam soils, amended soils exhibited average reductions of viable CFU by 1.2 to 1.8 log10(CFU+1)/g soil compared to unamended soil, and the largest reductions were observed when 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 log10(CFU+1)/g soil compared to unamended silty clay soil with minimal differences among amendments.
The main effects of amendment composition and soil texture but not their interaction also significantly influenced total VFA concentrations. As with Fo suppression, there was no significant effect of liming on soil VFA concentrations. Overall, soils of all textures amended with 100% SPI had the highest total VFA concentration (~30 mmol/kg soil) followed by 75% SPI/25% DM (~22.4 mmol/liter soil solution) while soils amended with 50% or less SPI combined with 50% or more DM had ~16 to 17 mmol/kg soil total VFAs in soil solution. Sandy loam soils exhibited higher total VFA concentrations (20 to 25 mmol/kg soil) than sandy (14 to 16 mmol/kg soil) or silty clay (12 to 16 mmol/kg soil) soils. Additionally, acetic acid dominated the VFA balance for all treatments, though the degree of its dominance varied. Acetic acid dominance of VFA fractions ranged from 70% in treatments amended with 100% soybean protein isolate to 88% in treatments amended with 100% dried molasses and the degree of acetic acid dominance of the VFA balance increases as the proportion of protein in the amendment decreases. Additionally, the degree of acetic acid dominance is the lowest in sandy soils at 68% and greatest in silty clay soils at 80%. Of the other VFAs, isovaleric acid is the most commonly produced by organic amendment fermentation, followed by n-butyric acid and the other VFAs (i.e., propionic, isobutyric, and valeric acids) for a given treatment.
Soil solution Fe2+ and Mn2+ were significantly influenced by individual effects of amendment composition, soil texture, and liming) as well as the interactive effects of amendment composition and soil texture and amendment composition and lime. Treatments in sandy soils exhibited the lowest overall total combined Fe2+ and Mn2+ concentrations, ranging from 0.27 to 3.22 mg/kg soil for unamended treatments and amendments containing 0 to 50% dried molasses to sand amended with 100% dried molasses at 33.6 mg/ kg soil. Combined soil solution Fe2+ and Mn2+ concentrations were significantly higher overall in sandy loam and silty clay treatments. In both cases, unamended treatments exhibited the lowest combined Fe2+ and Mn2+ concentrations (19 mg/kg soil in sandy loam and 41 mg/kg soil in silty clay) while soils amended with 75% dried molasses exhibited the highest Fe2+ and Mn2+ concentrations in sandy loam (301 mg/kg soil) and 100% dried molasses in silty clay soils (297 mg/kg soil). Additionally, the reduced metal balance in soil solutions were overwhelmingly dominated by Mn2+, except for sandy soils amended with 100% dried molasses (0.9 mg/kg soil Mn2+ and 30 mg/kg soil Fe2+), 25% soybean protein isolate and 75% dried molasses (1.8 mg/kg soil Mn2+ and 9 mg/kg soil Fe2+) and 50% soybean protein isolate and 50% dried molasses (1.1 mg/kg soil Mn2+ and 2.1 mg/kg soil Fe2+). Additionally, Mn2+ represents 98% or more of the reduced metals in soil solution in all the sandy loam and silty clay treatments. Likewise, treatments amended with 100% dried molasses had the highest Fe2+ fraction in the reduced metals in soil solution, at 7% for unlimed and 5% for limed treatments. However, differences between amended treatments were greater in sandy loam than in silty clay soils. Additionally, unamended soils, both limed and unlimed had the lowest Fe2+ and Mn2+ concentrations (~16-24 mg/ kg soil) while unlimed soils amended with mixtures containing 75 to 100% dried molasses had the greatest combined Fe2+ and Mn2+ concentrations (235 to 243 mg/kg soil ) but there were no significant differences between the combined Fe2+ and Mn2+ concentrations in soils amended with mixtures containing 50% or more SPI and/or were limed (123 to 171 mg/ kg soil ).
Influence of Amendment Type and Soil Texture on Fo Suppression and Strawberry Health
Fo suppression in the strawberry pot studies was significantly influenced by the first-order effects of amendment type, soil texture, and liming; the second-order interactive effects of amendment and texture and amendment type and liming; and most importantly, the third-order interactive effects of amendment type, soil texture, and liming. Fo viability was generally higher in finer-textured sandy loam and silty clay soils than sandy soils, particularly unlimed sandy soils. In addition, differences in Fo suppression based on amendment type and whether or not the soil was limed were minimal in fine-textured sandy loam and silty clay soils. In both limed and unlimed silty clay soils, Fo CFUs ranged from 4.2 log10(CFU+1)/g to 4.5 log10(CFU+1)/g soil for soils amended with dried molasses, spent brewer’s yeast, and wheat bran while for soils amended with Azolla meal or controls, both inoculated and uninoculated, Fo CFU counts ranged from 4.1 – 4.3 log10(CFU+1)/g soil. However, the effects of liming were greater in course-textured than in finer-textured soil. For instance, liming in sandy soils was associated with an increase in Fo CFU counts by 0.9 log10(CFU+1)/g soil in sandy soil amended with wheat bran and by 2.1 log10(CFU+1)/g soil in soils amended with Azolla meal. On the other hand, liming was associated with a decrease in Fo CFUs by 0.7 log10(CFU+1)/g soil in sandy soils amended with spent yeast. Additionally, the number of viable Fo CFUs in inoculated sandy loam and silty clay soils exceeded that of sandy soils by 2.7 to 3.6 log10(CFU+1)/g soil while amended sandy soils, both limed and unlimed exhibited significantly higher rates of Fo viability compared to inoculated controls, ranging from an increase of 1.7 log10(CFU+1)/g soil for Azolla meal to 3.1 log10(CFU+1)/g soil for wheat bran.
Soil solution VFA concentrations were significantly influenced by amendment type, soil texture, and the interactive effects of amendment type and soil texture. In general, soil solution VFA concentrations were higher in soils with greater sand content than soils with greater clay content with sandy soils amended with spent brewer’s yeast (5.3 mmol/kg soil) or dried molasses (4.3 mmol/kg soil) along with sandy loam soils amended with spent brewer’s yeast (4.11 mmol/kg) having the highest VFA concentrations. In contrast, soil solution VFA concentrations were lowest in silty clay soils, where average soil solution VFA concentrations for all amendment types was <1 mmol/kg soil with the pots amended with Azolla meal having the lowest VFA concentrations (0.1 mmol/kg soil). Soil solution Fe and Mn concentrations were significantly influenced by amendment type and soil texture but not the interaction of these factors. Soil solution Fe and Mn concentrations were highest in soils amended with dried molasses (8.2 mmol/kg soil) and lowest in soils amended with spent brewer’s yeast (6.3 mmol/kg soil). Unlike with VFAs, greater soil clay content is associated with greater soil solution Fe and Mn concentrations as both silty clay (8.2 mg/kg soil) and sandy loam (8.7 mg/kg soil) had significantly higher soil solution Fe and Mn concentrations than sandy soils (5.2 mg/kg soil).
The third-order interactive effects of amendment type, soil texture, and liming significantly influenced dry shoot biomass. Average dry shoot biomass was lowest for plants grown in sandy soils ranging from 0.13 g - 0.65 g/plant in limed sandy soils amended with dried molasses, spent yeast, or Azolla meal to 1.9 g/plant for limed inoculated controls. Average dried shoot biomass was highest in plants grown in unlimed silty clay soils amended with dried molasses and unamended, limed inoculated controls (5.2 g - 5.7 g/plant). Additionally, liming increased average dried shoot biomass by 1.2 g/plant in sandy loam and by 1.7 g/plant in silty clay soils and likewise, by 1.1 g/plant in both sandy loam and silty clay soils amended with wheat bran. However, liming was associated with reductions of average strawberry plant shoot biomass by 1.3 g/plant in silty clay soil amended with dried molasses, 2.0 g/plant for silty clay soils amended with spent yeast, and 2.3 g/plant for silty clay soils amended with Azolla meal.
Strawberry fresh root biomass was significantly influenced by amendment type and soil texture but not by lime application or any of the interactive effects. Average strawberry fresh root biomass was highest in unamended inoculated controls (~5.41 g/plant) and lowest in plants grown in soil amended with spent yeast (~2.58 g/plant). The average fresh root biomass of plants grown in soils amended with Azolla meal, wheat bran, and dried molasses did not differ significantly from one another and ranged from ~3.51 g to 4.11 g/plant. Additionally, strawberries grown in sandy loam soil had the highest average root masses (~5.08 g/plant) while those grown in sandy soils had the lowest (~2.53 g/plant). Those grown in silty clay soils had an intermediate average root mass of ~3.85 g/plant.
Analysis of Root-Associated Fungal Communities and Their Relationship to Strawberry Health
Strawberry plants grown in silty clay soil were healthier than the others, regardless of the amendment added or whether or not it was inoculated with Fo. In all these samples Fo represented only 0.25 to 0.64% of the total ITS2 counts associated with the genus Fusarium and 0.003 to 0.023% of the total root-associated fungal counts. Additionally, there was no significant correlation between the abundance of Fo and other members of the genus Fusarium and strawberry health, as indicated by root, shoot, and fruit biomass. On the other hand, the abundance of many beneficial fungal clades was positively associated with plant health. While there was no significant correlation between any strawberry health measure and the total abundance of beneficial fungi of the phylum Glomeromycota and the species Clonostachys rosea, higher total abundance of members of the order Agaricales and family Serendipitaceae and genus Trichoderma were correlated with enhanced crop health. The abundance of Trichoderma spp. was positively correlated with fresh root biomass (Pearson correlation coefficient (r) = 0.726, P < 0.0001) and dried shoot biomass (Pearson correlation coefficient (r) = 0.515, P < 0.05), Agaricales abundance was positively correlated with fresh root biomass (Pearson correlation coefficient (r) = 0.769, P <0.0001) and dried shoot biomass (Pearson correlation coefficient (r) = 0.515, P < 0.01), and Serendipitaceae abundance was positively correlated with fresh root biomass (Pearson correlation coefficient (r) = 0.423, P < 0.05) and fruit biomass (Pearson correlation coefficient (r) = 0.512, P < 0.05). Of these beneficial fungal clades found to be positively associated with strawberry health, only Serendipitaceae abundance was significantly influenced by differences in amendment. The highest abundance of Serendipitaceae counts was found in strawberry roots from pots amended with dried molasses (~7,300 ITS2 OUT counts/g root tissue) and Azolla meal (~5,700 ITS2 counts/g root tissue) while strawberry roots from pots amended with spent yeast had the least abundance of Serendipitaceae counts (~700 ITS2 counts/g root tissue).
Discussion and Conclusions:
Our results demonstrate the importance of both soil texture and root-associated fungal communities for safeguarding the health of strawberry crops in heavily-infested soils even if ASD fails to kill a meaningful proportion of pathogens. Even though higher soil clay content was associated with greater Fo CFU viability, higher soil clay content was also associated with healthier plants. This may be due to a wide variety of reasons. Clay adsorbs not just the VFAs produced by ASD but also the extracellular enzymes and effector molecules utilized by soilborne plant pathogens to infect crop roots in addition to adsorbing root exudates that trigger the germination of pathogen resting spores, therefore restricting pathogen germination (Smith 1977; Alabouvette 1993; Li et al. 2018). Additionally, clay-enriched soils are also enriched in key micronutrients essential for crop health such as Fe, Mn, Mo, B, and Si compared to sandy soils (Siddiqui et al. 2016; Orr and Nelson 2018). Likewise, even though suppression of Fo by ASD was greater in sandy soils, overall crop health as demonstrated by vastly reduced root and shoot biomass was also the worst in sandy soils. This may be in part because none of the strawberries were fertilized beyond the original ASD amendment and application of aqueous nutrients to the pots upon transplantation so the nutrients within the sandy soils was leached out when the pots were irrigated.
The presence of beneficial fungi has also proven far more important than Fo suppression by VFA for safeguarding the health of strawberry plants grown in heavily infested soils. For instance, while Fo CFU viability levels were among the highest in all unlimed silty-clay soils, the strawberry plants growing in them were healthier than those grown in sandy soils, where the lack of nutrients may have stunted the growth of strawberry plants and likely made them more susceptible to fusarium wilt. In fact, even though both Serendipitaceae (ectomycorrhizae) and Glomeromycetes (arbuscular mycorrhizae) were present in the strawberry root microbiomes analyzed by amplicon sequencing, Serendipitaceae but not Glomeromycetes had significant positive impact on crop growth. This may be because of the greater versatility of members of the family Serendipitaceae, which can live as saprophytes when they are unable to form mycorrhizal partnerships. This may explain why Serendipitaceae were favored in pots amended with dried molasses or Azolla meal, which are carbohydrate-dominated amendments but not the protein-rich spent brewer’s yeast treatments.
Lastly, it is very important to keep in mind the limits of ASD for treating soilborne crop diseases such as black root rot of strawberry caused by Fo. Past studies demonstrating the effectiveness of ASD against Fo typically utilized fresh hyphal biomass and/or conidia/bud cells, or infested field soil, which can be expected to contain a mixture of macro- and microconidia, and chlamydospores. However, the resistance of conidia and hyphal biomass to environmental stresses, including VFAs is significantly lower than that of chlamydospores and in turn, Fo chlamydospores have higher inoculum potential than conidia and hyphae. This may explain the relative lack of success of ASD in suppressing Fo, particularly in sandy loam and silty clay soils compared to past ASD experiments. Likewise, the fact that ASD, particularly with amendments rich in protein in addition to carbohydrates actually led to reduced strawberry crop health compared to inoculated controls, as shown by root and shoot biomass may be because the amendments actually enhanced the germination of chlamydospores that survived ASD treatment. This is because Fo chlamydospores need to be induced to germinate by the presence of a combination of glucose, amino acids (i.e., asparagine), and dissolved Fe. Therefore, while ASD is often effective in treating Fo-infested soils, other disease management techniques may be necessary to prevent the buildup of inoculant, particularly chlamydospores. For example, infected plants can be rouged when found and residues of infected crops should be removed from the field post-harvest to minimize the chance of overwintering pathogens infecting future crops. Crop rotation can be used to prevent the buildup of the inoculum of Fo forma speciales specialized for a particular crop. Additionally, ensuring high root colonization levels by beneficial fungi such as members of the order Agaricales or family Serendipitaceae as was the case when dried molasses was used as the ASD amendment can protect crops from infection and help enable their healthy growth even in soils with high pathogen population densities.