Update:
A protocol to evaluate select biological control agents (BCAs) in the field was developed and shared among the collaborators. The protocol was based on experiments conducted in greenhouse and growth chamber assays. BCA inoculum was produced at SIU, soybean seeds were coated with different BCA formulations and the coated seeds were shipped to MSU and ISU to conduct field trials.

Field experiments were conducted in three locations in IL (Carbondale, Shawneetown and Valmeyer), one location in IA and one location in MI. Treated soybean seeds were exposed to Fusarium virgulifome (SDS) and Macrophomina phaseolina (charcoal rot) in IL, and to oomycetes in IA and MI. Five different BCA treatments are being assessed in the IL locations and four different BCA treatments are being tested in MI and IA.

Stand counts were collected as well as disease symptoms. Plant samples were also collected. The data is still being processed. The field data will be compared and contrasted with QPCR data. DNA has been extracted from plants collected from the experimental plots. PCR probes specific to the pathogens and to the different BCAs are being used to assess wether the BCAs were able to protect the treated plants. The QPCR data will allow us to determine wether the BCAs were able to colonize the treated soybean roots, and whether this resulted in a decrease in root colonization by the pathogens. It is to be noted here that disease incidence and severity was low to moderate in most locations, which may make it difficult to discern any dramatic effect of the BCAs on disease under these conditions.

One of the objectives of this research is to correlate different management practices (tillage, crop rotation, etc...) with the incidence of BCAs in production soybean fields. Soil samples were collected from soybean production fields in KY, IL, and IN. DNA was extracted from these fields and BCA-specific QPCR probes are being used to assess the effect of different management practices on the incidence of BCAs in soils.

Another objective of the research is to assess the ability of the BCAs to induce resistance in treated plants. In a series of experiments, the expression of soybean genes involved in resistance to pathogens was assessed after treating the plants with BCAs. Our data shows that several of the soybean genes that are associated with resistance of soybean to plant pathogens were induced upon treating the seeds with BCAs. Hence, the BCAs may have a dual mode of action in affecting disease; they directly inhibit the growth of potential pathogens, and they indirectly affect the ability of the pathogens to infect soybean plants by inducing resistance in those plants.

We tested the effect of a set of recently identified potential bio-control agents on soil-inhabiting fungal, oomycete and nematode pathogens of soybean. Previously, a multi-state survey that was funded by USB was done to collect and identify soil- inhabiting organisms associated with seedling diseases of soybean. In that survey, we detected the presence of several fungal species that could potentially affect the ability of soybean pathogens to cause disease. In this current project, we investigated the direct effect of these species on the growth of a select number of soybean pathogens. We have also developed protocols to apply these bio-control agents to soybean seeds and we have conducted greenhouse and field assays conducted lab assays to determine the effect of these organisms (9 isolates) on the growth of several fungal, oomycete, and nematode pathogens of soybean.

The in-vitro screening of the activity of fifty-eight potential bio-control agents against several soybean pathogens is complete. In many instances, several isolates of the BCA species were tested against several isolates of the pathogen. BCAs with consistent high activity against the tested pathogens have been identified. Greenhouse experiments have been conducted to test the effectiveness of the BCAs against the pathogens and the the best performing BCAs were used in field experiments. Field experiments were conducted in IA, MI, and IL during the 2016 growing season. The experiments included five separate treatments of BCAs (individually or in mixture) that were selected based on the performance of the corresponding BCAs in lab, greenhouse or growth chamber assays. Data from 2016 included yield, stand count, and quantification of pathogens and BCAs in treated soybean plants. Preparations are underway to repeat the field experiments during the 2017 season. The data collected in 2016 will be used to optimize the application of the BCAs to the seeds and to maximize the impact of the BCAs on disease occurrence.

Another objective of our proposed research was to "follow the effect of commonly used management practices on the distribution and activity of BCAs in the soil. Soil samples were collected from several soybean production fields in IL, KY, and IN with different histories of management practices (crop rotation, tillage, etc..). We have developed molecular assays to detect and quantify the BCAs in soils. These assays are currently being used to assess the effect of different management practices on the presence of the BCAs in the collected soils. The objective is to be able to recommend management practices to soybean producers that would enhance the presence and activity in their soil improving thus the "health" of these soils in terms of their ability to limit the activity of soybean pathogens.

Finally, we proposed to investigate the ability of the BCAs to induce resistance in treated soybean plants. We ran several experiments to monitor whether treatment with BCAs affects the expression of soybean genes previously reported to be associated with resistance to disease. In other words, when these genes are turned on, defense mechanism are activated in soybean and the plants become more tolerant to pathogens. Our data shows that several of the soybean "resistance genes" were activated when soybean seeds were treated with BCAs.This indicates that the BCAs may have a dual mode of action in affecting disease; they directly inhibit the growth of potential pathogens, and they indirectly affect the ability of the pathogens to infect soybean plants by inducing resistance in those plants.