The spermosphere refers to the soil environment in the zone around the soybean seed from the time it is planted until a root system is established. The spermosphere is chemically and biologically altered by the presence of the seed and anything applied to the seed, but there is very little information about the specific changes that take place as a result of using different seed treatments. In order to understand these seed treatment effects, we will conduct experiments in the growth chamber and in the field with different soybean cultivars treated with various chemical and biological seed treatments, with or without inoculation with pathogens such as Fusarium graminearum. Soil from the planting zone will be sampled just before planting. After planting, seeds and soil from the spermosphere will be carefully excavated at intervals and the microbiome will be characterized using amplicon sequencing to profile the bacterial and fungal microbial communities. This will allow us to measure changes in the entire microbial community, including both pathogenic and beneficial microorganisms in the spermosphere, providing insight into the mechanisms by which seed treatments affect crop performance.

Seed treatments are known to have benefits for crop performance, but there is little information about exactly how they affect detrimental and beneficial microbes’ colonization of the spermosphere as it germinates and how long these effects last. Presumably, there are long-term consequence on plant performance that are dictated by early colonization events.
We will learn about the reaction of soil microflora, including pathogens, to the presence of different seed treatment products on soybean seeds.

Work plan includes conducting growth chamber experiments at the SSC and field experiments in multiple locations with different soil types, using seed/soil inoculation with pathogens representing different fungal genera. Seed treatment active ingredients have not been determined, but the plan will include multiple chemical and biological active ingredients targeting fungi and Oomycetes.

Updated January 25, 2024:
In the first year of this project, our primary task was to identify a graduate student to work on the project and establish an experimental system that would allow reliable microbiome sampling of spermosphere, rhizosphere, and endosphere. We successfully recruited a first-year student in the Interdepartmental Microbiology Graduate program after she had completed a rotation in our lab, joining the project May 2023. Here we outline progress made in establishing our experimental system. First, we decided to use soil from a typically managed corn-soybean rotation at the Marsden Long-term cropping system experimental site. This site was selected because the management history is well documented and our prior experience using this site for studies examining how crop diversification influences the soybean rhizosphere and endosphere microbiome. Second, we selected Williams82 soybean as the genotype to be used in these studies given its history and its well-characterized genetics. Third, preliminary experiments were conducted to identify a reasonable growth temperature for these experiments. We selected growth at 20 °C in order to mimic cool soil conditions during the planting season, while still allowing for relatively rapid germination and uniform emergence, facilitating collection of microbiome samples from plants at similar developmental stages. From this finding we were able to establish a timeline for collecting spermosphere and then rhizosphere and endosphere microbiome samples. Frequency of sampling will decrease over time since we anticipate seed treatments to have the greatest influence on the spermosphere microbiome assembly and the influence of the seed treatment on the rhizosphere and endosphere microbiome will diminish as the root develops (See Figure 1). We will grow plants in containers packed to the same bulk density. Fourth, in ongoing experiments we are exploring two approaches (Figure 2) for inoculating soil with F. graminearum that would result in increased pathogen pressure without killing all the plants or making them too sickly. This will allow us to assess potential interactions between seed treatments and pathogen pressure on the soybean spermosphere and rhizosphere/endosphere microbiomes. Lastly, we are working to optimize DNA extraction from rhizosphere/spermosphere samples and plant endosphere for microbiome analyses and for assessing the extent of F. graminearum infection of plants.
Supporting photos, graphs, and graphics can be viewed in the attachment

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Updated June 30, 2025:
In the second year of the project, our main goal was to perform experiments to collect samples to assess the effects of a fungicide and a bacterial seed treatment on the soybean spermosphere, rhizosphere, and root endosphere microbiome. This experimental design includes two important controls; seeds that were not treated and those treated with the carrier and colorant to directly assess whether these components of the seed treatments could explain effects of the chemical and biological treatments. To have sufficient power to assess treatment effects, there were 5 biological replications where sampling occurred over 11 time points post-planting as outlined in the first-year progress report.
After contacting several seed treatment companies, DPH Biologicals agreed to provide a bacterial strain from their TerraTrove SP-1 Classic biofertilizer as the biological seed treatment. We designed qPCR primers that could detect the presence of the bacterial species to gain a better understanding of how the population of the bacterial species changed over time in comparison to the control treatments. The amount of culturable bacterial treatment was also accessed at each timepoint. To aid in this assessment, the bacterial treatment was tested for antibiotic resistance to limit the amount of other bacterial growth. After completing the completing experiments to collect the soybean spermosphere, rhizosphere, and root endosphere microbiome, the presence of culturable bacterial treatment was found to dissipate over time, with no colonies being found after 5 days post planting.
We also completed optimization of DNA isolation from different types of samples before proceeding with the microbiome experiments. In anticipation of the Fusarium graminearum soil inoculation experiments, a qPCR primer pair was evaluated for their ability to detect F. graminearum. While the primer pair could detect F. graminearum, it did not meet the recommended 90 – 110% efficiency during qPCR trials. We are currently exploring ways to optimize the efficiency of these primers and identifying alternative primers.
While successfully isolating DNA from seed and root samples, unfortunately, due to an equipment malfunction we lost a majority of the spermosphere and rhizosphere samples. Attempts to salvage the samples were unsuccessful due to potential contamination of DNA from neighboring wells of the microtiter plate system. This develop will require a repetition of the entire experiment, which is currently underway.
In addition to obtaining samples for isolating DNA for microbiome analyses, roots were scanned to determine seed treatment effects on plant development. After analyzing the root scan data, it was determined that the seed treatments had no statistically significant effect on the root architecture in comparison to the control treatments, in natural soil with no added pathogens.

These results will be valuable for making decisions about seed treatment usage in soybean