Updated October 27, 2022:
The first three months of the project went well overall with surprising discoveries. Extract was obtained from oilseed meals successfully and early trials of soybean cyst nematode treatment were conducted. Soybeans were planted with control and 3 treatment levels of glucosinolate extract, all infected with 300 juvenile nematodes per plant. The early trials were repeated twice, and in both cases cyst nematodes were not significantly affected without adding glucosinolate hydrolysis enzyme. However, soybean root mass and plant height increased with increasing treatment dosage levels in both experiments. This is a surprising discovery, as the extract was only expected to treat the cyst nematodes and was not hypothesized to behave as a biofertilizer. The reason this is surprising is due to the fact that the meal was not used in any of the trials and that was believed to contain the nutrients that allowed oilseed meals to act as a “green manure” from the earlier reviewed literature. Since cyst nematodes were not significantly treated in the early trials, some changes will be made to the downstream processing of glucosinolate recovery to make the treatment more effective (i.e. concentration, purification, etc.). Hopefully, this can make the treatment effective at treating cyst nematodes while maintaining the biofertilizer effect.
The graduate research assistant position was not filled for the first three months and is not expected to be filled in the next three months for the FA2022 semester. Due to this, and the surprising discovery, it is requested that half of the funds for GRA salary and tuition remission be moved to laboratory supplies. The supplies that we plan to acquire will be for identifying and analyzing the nutrients that are coming with the glucosinolates causing the biofertilizer effect. The other half of the GRA funds is requested to be left as originally budgeted, so if a student can be recruited in time for the SP2023 semester the support is still available to them.
There is no request for assistance from the farmers at this time. Trials will continue to be conducted with increased attention to the biofertilizer properties of the proposed treatment.
Updated January 27, 2023:
The greenhouse had a pipe freeze and burst in December resulting in our room losing heat. Thus, our plants froze and died before conclusive data was gathered for our latest fall trials. We replanted 1.5x the plants that we normally do for the next trial to try and catch back up on the research. If all goes well, we should have a large report in the spring.
Updated May 1, 2023:
Currently analyzing data from soybean trials, more students will be assisting on both the FY23 and recently funded FY24 project (continuation of FY23). So far, developing the enzyme to function properly is the largest technical risk for nematicidal effect, and the biostimulant effects for improve growth, stress resistance, etc. is easier and carries a lower technical risk. The SDSU Chem. Dept. will be consulted to help analyze and identify the biocompounds in our treatment that are causing the surprising biostimulant effects for better development. We may be requesting the Final Report for FY23 have a later due date (~Sept. 30) so we can include data and reports from the summer student research fellows in the final report. This will make the research around the FY23 more robust and thorough. Kayla Christopherson, presented the continuation of this project, is currently putting an application proposal together for an NSF iCorps program. This program inspires entrepreneurship in scientists through customer discovery activities. The main technology proposed for her program will be a seed treatment from the technology being developed in this project. This will allow Kayla and the team to connect directly with farmers, seed dealers, ag-chemical dealers, etc. to develop a Business Model Canvas to allow smooth implementation of the technology from a minimum viable product. This will lower the overall risk of technology commercialization and development to directly address farmers needs (create a pull for the tech, rather than a push into the market). These activities will allow us to identify methods for application, that were discussed at the meeting, like dry box spreading, seed coating, no-till applications, etc.
Updated July 30, 2023:
As part of previous research, we simply tried spraying antimicrobial compounds on topsoil. However, this did not yield the results we were hoping for. We believe this is because the antimicrobial compounds become "lost" and separated when simply sprayed on the soil. For this reason, we are trying a new delivery technique: encapsulation. To encapsulate the antimicrobial compounds, we opted for a hydrogel approach. Hydrogels are composed of polymer chains that have been cross-linked to form a 3D network structure. This structure allows hydrogels to swell and absorb large amounts of water, or in our case, GSL and myrosinase. As previously discussed with the Soybean Council, glucosinolates from the Brassicaceae plant family are a form of metabolites that contain many nitrogen and sulfur groups. When hydrolyzed by myrosinase, a catalyst, the glucosinolates break down to form bioactive compounds such as isothiocyanates, nitriles, and thiocyanates. These bioactive compounds possess anti-inflammatory, antioxidant, and antimicrobial capabilities.
For the first few weeks of this summer, we searched for the most effective way to develop a hydrogel. Initially, we used starch as the main structural element and citric acid as the crosslinker. However, this method proved ineffective, as the crosslinker did not strengthen the hydrogel and instead left it as a pile of goop that was very sticky (Image 1). Our next hydrogel utilized pectin and chitosan, cross-linked with sodium hydroxide. Although it took a long time to cure, it eventually formed a nice thin sheet. However, an issue arose. The hydrogel would initially absorb water, but if left to dry a second time, the thin sheet would become brittle and crumble to the touch (Image 2). We attempted to compensate for this issue by adjusting the pectin-to-chitosan ratio, but ultimately, this led to a decrease in the mechanical properties, making it unsuitable for long storage periods. Finally, at the start of July, we used sodium alginate and cross-linked it with a calcium cation either through calcium chloride or calcium carbonate (Image 3). This hydrogel seemed to possess ideal stability.
As of today, we have begun conducting mechanical property testing on various concentrations of sodium alginate to determine the strongest concentration. We have currently sent the hydrogels to a team in the engineering department and have yet to receive the results. We have also initiated microbial inhibition testing, in which we are mixing ampicillin within the hydrogel before it is cured. Subsequently, we will use the Kirby-Bauer inhibition test, except instead of placing an antibiotic disk, we will place a small section of the hydrogel containing ampicillin (Image 4). This test will provide a baseline for when GSL and myrosinase are added, as we will have a better understanding of the diffusion rates of the various concentrations of sodium alginate.
Moving forward, we will perform biodegradability tests. However, due to limited space and time, this aspect of the project has been put on the back burner. Additionally, we will begin incorporating GSL and myrosinase into the hydrogels and conduct inhibition testing.