Suppressive soils have been identified as soils that can suppress naturally occurring soil-borne diseases. These soils contain microbial communities that are capable of suppressing or controlling disease-causing organisms, including fungi (e.g. Fusarium virguliforme (cause of sudden death syndrome, SDS), Macrophomina phaseolina (cause of charcoal rot), Phytophthora root rot (Phytophthora sojae) and nematodes (e.g., soybean cyst nematode, Heterodera glycines). How the native soil microbial communities reduce disease is not known. Knowledge of factors that contribute to and support these beneficial microbial communities is also unknown.
One example of this natural improvement in soil microbial community reducing disease was demonstrated in our previous research (sponsored by the Kansas Soybean Commission) that demonstrated that a high-glucosinolate mustard (Brassica juncea) reduced fungal populations that caused charcoal rot in soil and in soybean plants. The research proposed here builds on those results by exploring the interaction between soil health and disease pressure. Management practices will be tested in field studies to determine the impact on soil health, fungal pathogen presence, and soybean growth and yield. Impact of soil health on soybean disease.
Crop plants that are disease hosts increase the number of disease-causing organisms in the soil. We have previously shown the increase in colony forming units (CFU) of M. phaseolina in the soil after soybean production (Sassenrath et al., 2019). Other factors reduce soil-borne disease, include high-glucosinolate mustard as a cover crop (Sassenrath et al., 2017, 2019) and increasing the soil temperature (e.g., "solarization"). Use of animal manures greatly increases the diversity of the soil microbial community, and beneficial microorganisms in particular. In addition to improving the soil nutrient balance, manure may contribute to reduced disease pressure (Graham et al., 2014) and soybean cyst nematode populations (Bao et al., 2013).

Identify agronomic practices to control soil diseases.

Assessment of soil “health.”
Current standard practices of assessing soil health include phospholipid fatty acid analysis (PLFA), the Haney test, and soil respiration rates. The PLFA reports total community structure of bacteria (gram-negative and -positive), fungi (arbuscular mycorrhizal and saprophytes), protozoa, and undifferentiated organisms. While potentially useful, these tests give no information on the relative abundance of disease organisms in relation to beneficial organisms. The potential impact of these tests on productive capacity or return on investment are also difficult to interpret. The quantitative reported measurements of “soil health” are not readily interpretable to bushels per acre. Current methods of assessing soil community structure and disease organisms is limited to time-consuming lab-based analysis. A simpler method of assessing disease-suppressive potential of soils would be beneficial to better determine soil community structure and disease status.

Measuring the impact of soybean health on soybean diseases.
Management practices to alter the soil community structure will be implemented in replicated research plots at the Southeast Research and Extension Center fields in Parsons, KS. Treatments will include two treatments that are likely to increase disease (soybeans and corn stubble, both hosts of disease organisms), three treatments that are likely to decrease disease (brassica cover crop, animal manure, and solarization), and a fallow control.
Soils will be sampled at three times during the season to determine: soil nutrients, soil community structure, and pathogen populations. Soils will be sampled prior to implementation of treatments (early March), mid-season (late June) and after harvesting soybeans (October). Final soybean yield will be collected. Standard soil nutrient analysis will be performed at the K-State Soil Testing Lab. Soil community structure will be determined with PLFA. Because of the cost of this measurement, we will use funds from additional sources to complete this analysis. Disease presence will be measured using standard plating analysis of soils in the Department of Plant Pathology on campus.
M. phaseolina populations will be determined by counting the number of colony forming units (CFUs). Charcoal rot disease severity will be measured by randomly selecting ten plants per plot at the R7-R8 growth stage for root and stem severity ratings. The plants will be scored by splitting the stem and taproot of each plant and rating the degree of gray discoloration and microsclerotia in the vascular and cortical tissues on a scale of 1-5. M. phaseolina root populations will be estimated by grinding the split roots after the severity evaluation. The ground plant tissue and soil samples will be plated on microbiological media and incubated. CFUs of M. phaseolina will be counted and transformed to CFUs per gram of root tissue or gram of soil.

Assessment of soil “health”
Phospholipid fatty acid analysis (PLFA) is a standard method of determining microbial community composition. However, the price for this test has recently increased to $80 per sample, making it cost-prohibitive. To improve assessment of soil community structure, we will develop an assay soil community analysis using BioLog EcoPlates to measure the effects of management practices on microbial community changes in the soil based on their global metabolite utilization profiles.

Update:
Soil samples were collected prior to implementation of treatments for determination of soil microbial activity and disease organisms. Six treatments were implemented into replicated field trials in early June, 2022. Temperature sensors were installed 2" into the soil for continuous recording of soil temperatures in 1 replication.

Update:
Control of Soil-Borne Disease of Soybean

G.F. Sassenrath, C. Little, X. Lin, and S. Moraes

Keywords: charcoal rot; soybean; Macrophomina phaseolina; suppressive soils

Summary
Soil-borne diseases are a significant cause of reduction in crop yield. Alternative management of soils can enhance the natural disease-controlling organisms in the soil. This study explores the impact of alternative production methods on a primary soybean disease, charcoal rot, caused by the fungus Macrophomina phaseolina. Treatments that could potentially enhance or control the disease were implemented, and soil tests were conducted for nutrient and disease presence. Manure increased the nutrient levels in the soil, as expected, but did not impact the disease control. Solarization increased the temperature within the plots, and increased the number of colony forming units of M. phaseolina. Environmental conditions during the 2022 growing season were much hotter and drier than normal, leading to reduced soybean yields.

Introduction
Suppressive soils have been defined as soils that can inhibit the growth of naturally occurring soil-borne diseases. These soils are capable of suppressing or controlling disease-causing organisms, including fungi (e.g. Fusarium virguliforme (cause of sudden death syndrome, SDS), Macrophomina phaseolina (cause of charcoal rot), Phytophthora root rot (Phytophthora sojae), and nematodes (e.g., soybean cyst nematode, Heterodera glycines). How the native soil microbial communities reduce disease is not known. Knowledge of factors that contribute to and support these beneficial microbial communities is also unknown.

One example of this natural improvement in soil microbial community reducing disease was demonstrated in our previous research (Sassenrath et al., 2017, 2019) that demonstrated that a high-glucosinolate mustard (Brassica juncea) reduced fungal populations that caused charcoal rot in soil and in soybean plants. Here, we expand on those results by exploring the interaction between soil health and disease pressure. Management practices that increase, decrease, or maintain disease pressure were tested in field studies to determine the impact on soil health, fungal pathogen presence, and soybean growth and yield.

Impact of Soil Health on Soybean Disease
Crop plants that are disease hosts increase the number of disease-causing organisms in the soil. We have previously shown the increase in colony forming units (CFU) of M. phaseolina in the soil after soybean production (Sassenrath et al., 2019). Other factors reduce soil-borne diseases include high-glucosinolate mustard as a cover crop (Sassenrath et al., 2017, 2019) and increasing the soil temperature (e.g., "solarization"). Use of animal manures greatly increases the diversity of the soil microbial community, and beneficial microorganisms in particular. In addition to improving the soil nutrient balance, manure may contribute to reduced disease pressure (Graham et al., 2014) and soybean cyst nematode populations (Bao et al., 2013).

Diseases are primary factors that reduce the yield and quality of soybeans in Kansas and throughout the world. Soil-borne diseases are prevalent in eastern Kansas crop fields. Certain plants have been shown to produce chemicals that act as biofumigants that control or reduce harmful soil fungi. Animal manures have also been used to alter the soil microbiome to improve the control of disease organisms. Our working hypothesis is that improving the overall soil health by supporting healthy soil microbial communities can reduce disease pressure. Creating suppressive soils by altering management practices will reduce disease pressure. This research explores the relationship between soil health and disease pressure. The research tests the ability of cover crops, animal manure, and solarization to control or reduce charcoal rot in soybean production through improved soil microbial communities.

Experimental Procedures
Replicated plots were established at the Southeast Research and Extension Center in Parsons during the spring of 2022. Plots included: fallow, mustard cover crop, soybean, corn stubble, cow manure, and plastic sheets. Temperature sensors (Hobo, Onset, Inc., Bourne, MA) were installed at 2-in. depth in the soil in plots of one replication and temperatures were recorded continuously. Plastic sheets provide a “solarization” treatment, increasing soil temperature and potentially reducing soil microbes. Corn stubble was spread to about a 2-in. layer; corn stubble provides more carbon for soil microbes, increasing their abundance, but may also act as a host for M. phaseolina. Animal manure provides an additional food source for the microbes and adds additional microbes to the soil; manure has been shown to reduce some pathogens in soil. Mustard cover crop has been shown to reduce the number of CFUs of M. phaseolina, while soybeans are a host and increase the CFUs of M. phaseolina. The fallow treatment was left unplanted and served as a control.

Soil samples were collected in spring prior to implemented treatments, mid-season, and after harvest. Soils were analyzed for nutrients at the K-State Soil Testing Lab, for microbial activity and “Soil Health Score” at Ward Labs (Lincoln, NE), and for the number of CFUs of M. phaseolina in the Department of Plant Physiology at Kansas State University.

Results and Discussion
Nutrients changed in response to treatments (Table 1). As anticipated, the cow manure treatment had the highest levels of organic matter (OM), potassium (K), and phosphorus (P). This treatment also led to the highest Soil Health Score. Surprisingly, the solarization treatment (plastic film) had the highest total nitrogen (N) and microbiologically active carbon.

Interesting differences were observed in the environment within the soil in the different treatments (Figure 1). The plastic sheets (yellow line) raised the temperature more than 10 degrees above the fallow treatment (blue line), and temperatures remained elevated at night. Animal manure (grey line) also raised the day-time temperature, but temperatures decreased at night to that of fallow. Temperatures under corn stubble (orange line) were much lower throughout the day compared with the soil temperatures under fallow.

The only statistically significant difference in the number of CFUs was recorded in the solarization treatment (Figure 2), which showed a buildup in CFUs of M. phaseolina during the growing season. The corn stubble, cow manure, and soybean treatments had very similar levels of CFUs. The number of CFUs decreased slightly in the fallow and mustard seed treatments.

The summer growing season of 2022 was challenging for crop production due to high temperatures and low rainfall (Sassenrath et al., 2023). Crop establishment in the plots (mustard seed and soybeans) was impaired due to insufficient rainfall. Average yields for plots were very low (~5 bu/a) and well below the 12-year average at the station (Figure 3). Overall, statewide soybean yields were below average.

Conclusions
The soil microbiome controls many of the functions of the soil. It is possible, through alternative management practices, to alter the soil microbiome to support helpful organisms, such as arbuscular mycorrhizae, while controlling disease-causing organisms. Preliminary evidence from this study showed a minor change in microbial composition and activity with treatments. However, the unusually hot and dry weather may have compromised the results, as soybean yields were greatly reduced.


Acknowledgements
This research is supported by funding from the Kansas Soybean Commission and the U.S. Department of Agriculture National Institute of Food and Agriculture, Hatch project 1018005.


References
Bao, y., Chen, S., Vetsch, J., Randall, G. 2013. Soybean yield and Heterodera glycines responses to liquid swine manure in nematode suppressive soil and conducive soil. J. Nematology. 45(1):21-29.

Graham, E., Grandy, S., Thelen, M. 2014. Manure effects on soil organisms and soil quality. Michigan State Extension. https://www.canr.msu.edu

Sassenrath, G.F., Lingenfelser, J., Lin, X. 2023. Corn and soybean production – 2022 summary. Kansas Agricultural Experiment Station Research Reports: Vol. 9: Iss. 2.

Sassenrath, G.F., Little, C., Roozeboom, K., Lin, X., Jardine, D. 2019. Controlling soil-borne disease in soybean with a mustard cover crop. Kansas Agricultural Experiment Station Research Reports: Vol. 5: Iss. 2. https://doi.org/10.4148/2378-5977.7740

Sassenrath, G.F., Little, C.R., Hsiao, C.-J., Shoup, D.E., Lin, X. 2017. Cover crop system to control charcoal rot in soybeans. Kansas Agricultural Experiment Station Research Reports: Vol. 3: Iss. 2. https://doi.org/10.4148/2378-5977.1383


Table 1. Changes in soil nutrients with treatment
Mid-season
Organic matter, % Potassium, ppm Mehlich P, ppm Total N,
ppm Soil health score Microbially active carbon, %
Corn stubble 1.9 74.5 23.0 13.4 9.6 47.6
Cow manure 2.4 150.5 47.3 16.1 13.4 54.3
Fallow 2.0 66.8 18.5 13.2 10.3 48.8
Mustard seed 2.1 78.5 23.0 15.6 12.0 42.8
Plastic film 2.0 62.3 19.5 21.2 13.9 64.9
Soybeans 2.0 73.8 20.3 12.9 11.7 53.1

At harvest
Organic matter, % Potassium, ppm Mehlich P, ppm Total N, ppm Soil health score Microbially active carbon, %
Corn stubble 2.0 80.3 27.3 19.6 10.4 36.7
Cow manure 2.2 160.3 53.0 27.8 13.2 43.3
Fallow 2.0 60.8 25.8 20.1 12.1 63.4
Mustard seed 2.0 74.5 29.0 21.8 15.3 73.4
Plastic film 2.0 60.3 29.0 33.3 12.1 91.0
Soybeans 2.0 71.8 25.5 19.2 14.1 68.4

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Soil-borne diseases are a significant cause of reduction in crop yield. Alternative
management of soils can enhance the natural disease-controlling organisms in the soil.
This study explores the impact of alternative production methods on a primary soybean
disease, charcoal rot, caused by the fungus Macrophomina phaseolina. Treatments that
could potentially enhance or control the disease were implemented, and soil tests were
conducted for nutrient and disease presence. Manure increased the nutrient levels in
the soil, as expected, but did not impact the disease control. Solarization increased the
temperature within the plots, and increased the number of colony forming units of
M. phaseolina. Environmental conditions during the 2022 growing season were much
hotter and drier than normal, leading to reduced soybean yields.

Diseases are primary factors reducing the yield and quality of soybeans in Kansas and throughout the world. Soil-borne diseases are highly prevalent in eastern Kansas crop fields. Certain plants have been shown to produce chemicals that act as biofumigants that control or reduce harmful soil fungi. Animal manures have also been used to alter the soil microbiome to improve control of disease organisms. Our working hypothesis is that improving the overall soil health by supporting healthy soil microbial communities can reduce disease pressure, i.e. creating suppressive soils by altering management practices will reduce disease pressure. This research will explore the relationship between soil health and disease pressure. The research outlined here will test the ability of cover crops, animal manure, and solarization to control charcoal rot in soybean production through improved soil microbial communities. The research will also work to develop a rapid, accurate test to assess the disease organisms in the soil microbiome.
This proposal builds on previous research demonstrating that mustard reduces the number of CFUs of the charcoal rot pathogen. Experiments in other cropping systems at Parsons have indicated that soil microbial communities are modified even by different varieties of the same crop. This research will complement additional research in progress. Proposals are being developed to USDA-NIFA to leverage KSC funds to explore the impacts of soil health and natural soil biocontrol methods on crop disease and productivity. The proposed research will be coordinated with on-going funded soil health studies to further leverage KSC funds.