Sudden death syndrome (SDS) of soybean is responsible for important yield losses in Iowa and the Midwest. SDS management strategies rely primarily on the use of resistant soybean varieties and fungicide seed treatments. Neither approach is completely effective at suppressing SDS. Two fungicide seed treatments are currently available to manage SDS: ILeVO (fluopyram) and Saltro (pydiflumetofen). However, these treatments are expensive and may not result in economic profit to growers when weather conditions are not conducive to SDS.

In addition, ILeVO treatments are known to cause phytotoxicity in soybean seedlings, which can negatively impact root growth and sometimes reduce yield (Budi, 2020). Phytotoxicity problems are also known to occur with other fungicides, such as Topguard (flutriafol). Although this fungicide is labeled for SDS, it is not used by growers due to the lack of a safe application method that prevents plant stand loss from phytotoxicity. In this proposal, we hypothesize that treating seeds with a reduced rate of fungicide, in combination with another antimicrobial treatment, would reduce the risk of phytotoxicity without compromising SDS control.

Nanotechnology is being explored as a pesticide delivery system that can enhance the efficacy of pesticide and reduce pesticide use. With this technology, pesticides are encapsulated in nano-emulsions which have droplet size within ranges of 1~100 nanometers. These nano-emulsions provide many advantages, including increased stability, controlled release and higher absorption rates of the encapsulated active compounds. Not all compounds can be nano-encapsulated because of their chemical structure or properties. The fungicides in ILeVO, Saltro, and Topguard cannot be nano-encapsulated due to their extremely low water or oil solubility. For that reason, we are proposing to use nano-encapsulated biopesticides as the antimicrobial treatment to combine with the reduced rate of commercially fungicides.

Plant essential oils are a type of biopesticide that has shown effectiveness against several plant pathogens and pests. For example, lemongrass oil and thymol oil effectively inhibited Fusarium solani growth in vitro (Eke et al. 2020, Kong et al. 2021). In addition, lemongrass and thymol reduced disease caused by several other plant pathogens, including Phytophthora root rot in curcubits (Amini et al. 2016) and bacterial pustule in soybean (Kumari et al., 2018). Plant essential oils are promising crop protection options because they are safer and more environmentally friendly than their chemical counterparts.

Nano-encapsulation of biopesticides can increase their efficacy by up to ~20% (Kah et al., 2018), reduce their cost, protect them from adverse environmental conditions (Blanco-Padilla et al., 2014) and allow better control of their release (Mossa et al., 2018). Nanocellulose is an organic nanomaterial that is nontoxic, biodegradable, and an effective nanoencapsulation agent. Due to increasing demand for soy-based products, large amounts of soybean residues are generated every year (Costa et al., 2015; Li et al., 2019). Soybean residues have a cellulose content of up to 50% and are therefore great, low-cost sources of nanocellulose. We propose to use nanocellulose derived from soybean residues to encapsulate the biopesticides in this study.

The overall goal of this project is to enhance soybean productivity, profitability and environmental sustainability by combining fungicide seed treatments with nano-encapsulated biopesticides using soybean residue-derived nanocellulose as carriers. Our project goal fits into ISA’s research focus of ‘Soybean disease, nematode, insect pest and abiotic stress biology, management and yield loss mitigation’.

1. Develop nano-encapsulated essential oil seed treatments using nanocellulose derived from soybean residues
2. Test the effectiveness of the essential oils in suppressing growth of the SDS pathogen, F. virguliforme, in-vitro
3. Test the effectiveness of the nano-encapsulated essential oils, in combination with ILevo at reduced rates, against SDS development in soybean plants in greenhouse conditions

Protocols for optimized production of nanocellulose derived from soybean residues
Recommendations for future production of nano-encapsulated biopesticides, including storage conditions and effective dosage
New information about the antimicrobial activity of plant essential oils against F. virguliforme and the potential for using these compounds to help manage SDS
A highly effective biopesticide formulation that can be used to reduce fungicide phytotoxicity by combining it with reduced rates of seed treatments
Extension and research publications and presentations

Update:
Objective 1:
Dr. Liu’s lab has worked on nanoencapsulation of lemongrass essential oil in a nanoemulsion format. Macroscopic images of the nanoemulsion were shown in Figure 1. With a loading of 5% lemongrass essential oil and 5% Tween 80, the nanoemulsion formulated with 0.1%~1% soybean nanocellulose had an encapsulation efficiency of 100%. The nanoemulsion samples formulated were stable against centrifugation. For instance, the nanoemulsion containing 0.7%~1% soybean nanocellulose had an average particle size of ~32 nm, and the average particle size did not change after centrifugation. The nanoemulsion samples were stored at room temperature and no phase separation was observed for the sample containing 0.1% soybean nanocellulose after 7 months’ storage. Stable formulations will later on be sent to Dr. Leonor’s group for antifungal tests.

Objective 2:
Dr. Leandro's lab has completed in-vitro screeing of 8 plant essential oils against Fusarium virguliforme. In initial experiments, fungal growth media was amanded with essential oils at concentrations ranging from 0.03 - 0.08% by volume and lemongrass oil was show to inhibit fungal growth an concentrations of 0.05 or higher. In subsequent experiments, screening focused on more oils and only at concentrations of 0.03 and 0.05% with the goal of identiifying oils that were are effective or more than lemongrass. In those screens, cinnamon bark oil, geraniol and palmarosa oil were found to be highly effective at suppressing F. virguliforme growth. We are currently planning to screen an additional 8 oils to determine the most effective oil against the SDS pathogen.

In addition, we have conducted in-vitro phytotoxicity assay with lemongrass oil. We found that this oil is phytotoxoc to soybean seeds but that this effect is dose dependent, ie. by reducing the oil concentration 0.01% the soybean seeds germinate normally. In the next quarter, we will conduct similar phytotoxocity screens on the most effective oils identified in the fungal assays.

Update:

For objective 1, Dr. Liu’s lab prepared nanoencapsulated essential oil with different formulations of essential oil, tween 80, soybean nanocellulose in the presence of varied concentrations of salt. The thermodynamic stability of the nanoencapsulated essential oil was tested, including freeze-thaw stability test, centrifugation test, heating-cooling test, and room temperature storage test. The nanoemulsion stabilized by tween 80 and 1 wt % soybean nanocellulose had a mean particle size of 76 nm and it was stable against centrifugation test and room temperature storage for at least 30 days. The presence of sodium chloride at 40 mM enhanced the encapsulation efficiency of the nanoemulsion stabilized by tween 80 and 1 wt% soybean nanocellulose. Further thermodynamic stability tests will be performed for this.

For Objective 2, Dr. Leandro's lab has conducted in-vitro assays to test the suppressiveness of essential oils on mycelial growth of Fusarium virguliforme (Fv). A set of 15 essential oils have been screened, in two experimental runs each, at concentrations of 300 and 500 ppm in PDA growth media. Several of the essential oils showed strong inhibitory effects on Fv, with lemongrass, cinnamon bark and cassia oils being the most effective. We have also started to test phytotoxicity of the essential oils on soybean seed germination using two assays: a agar plate assay where seeds are germinated on agar amended with the essential oils and a rolled towel assay where seeds are coated with an emulsion of essential oils in agar, then germinated in moist rolled towels. To date these assays have demonstrated that essential oils can significantly reduce seed germination but that this phytotoxicity can be minimized by reducing the dose and type of application. Our goal is to complete testing the phytotoxicity of the 15 essential oils to determine which has the most promise for effective pathogen suppression with minimal phytotoxicity.

Two abstracts were submitted to the American Phytopathological Society to present results in poster format at the national conference in August 2023. In addition, Dr. Leandro presented research results at the ISU Crops Team Spring In-Service meeting. Dr. Leandro is scheduled to record two extension video presentations in January 2024.

Update:
Objective 1. Develop nano-encapsulated essential oil seed treatments using nanocellulose derived from soybean residues
Dr. Liu’s lab prepared cellulose nanocrystals from soybean stover and characterized its properties, including particle size, zeta potential, and crystallinity values. Soybean nanocellulose was then used to encapsulate lemongrass essential oil (EO) with the aid of surfactant Tween 80. The EO loaded nanoemulsion stabilized by soybean nanocellulose, sodium chloride, and Tween 80, showed excellent thermodynamic stability as it was stable against centrifugation forces, freeze-thaw cycles, heating-cooling cycles and room temperature storage for at least 30 days. The nanoencapsulated essential oil was then provided to Dr. Leandro’s group for further tests.

2. Test the effectiveness of the essential oils in suppressing growth of the SDS pathogen, F. virguliforme, in-vitro
Dr. Leandro's lab completed testing of 16 plant essential oils (EO) for suppressiveness of Fusarium virguliforme (Fv) mycelial growth. Each oil was tested at concentrations of 300 and 500 ppm in PDA growth media in two separate experimental runs, each with 3 replicate plates. When inconsistent results were obtained, the experiment was repeated a third time. Growth was measured every 2 days on the underside of the plate. We analyzed the data and summarized the effectiveness of each EO at suppressing. The three essential oils with strongest inhibitory effects on Fv were cassia, cinnamon bark, and geraniol, with cassia inhibiting Fv growth by more than 95% at both concentrations.
We also compared three methods of testing phytotoxicity of the essential oils on soybean seed germination: 1) an agar plate assay where seeds are germinated on agar amended with the essential oils at different concentrations, 2) a rolled towel assay where seeds were coated with an emulsion of essential oils, then germinated in moist rolled towels, and 3) a pot assay where seeds treated with the emulsified essential oils were planted into pasteurized soil.
In the plate assay, lemongrass oil at rates of 0.05% to 1% inhibited soybean seed germination but germination was not affected at a rate of 0.01%. However, the plate method was considered to not be very applicable to the seed coating delivery method we are aiming for.
In the rolled towel assay, seeds were coated with an emulsion of agar with 5% lemongrass oil. This concentration is five times higher than the concentration used in the plates as the volume of emulsion that attaches to the seeds is very small. The results from this assay also showed that lemongrass oil can reduce seed germination and seedling growth rate. However, with this assay we also encountered frequent problems with seed rot and fungal contaminants, probably due to rich seed exudates being readily available for contaminant growth. We therefore decided to change to a soil assay, which is more representative of the end use of the seed treatment we are trying to develop.
For the pot assay, seeds were treated with 5% lemongrass oil, then planted in pasteurized soil and grown in the greenhouse for 3 weeks. Seed germination and root length were measured. With this assay, the lemongrass seed treatment did not affect germination compared to untreated seeds, although some of the seedlings showed abnormal root curling.
To date the phytotoxicity assays have demonstrated that essential oils can reduce seed germination but that this phytotoxicity can be minimized by reducing the dose and modifying the type of application to the seed. In the new funding cycle, we are planning to use the nanoemulsions produced by Dr. Liu to encapsulate the essential oils with the goal of reducing phytotoxicity to seed and enhance the inhibitory activity against F. virguliforme.

3. Test the effectiveness of the nano-encapsulated essential oils, in combination with ILevo at reduced rates, against SDS development in soybean plants in greenhouse conditions
For this objective, we made progress towards developing an assay to compare seed treatment with nanoencapsulated essential oils with the commercial seed treatment Ilevo. We treated seeds with the nanoencapsulated lemongrass oil prepared by Dr. Liu’s lab. The seeds were planted in pasteurized soil infested with Fv using sorghum grain inoculum. Foliar SDS symptoms were assessed every 2 days for approximately three weeks, and root rot severity and shoot/shoot dry weights were assessed at the end of the experiment.
For plants grown in pasteurized soil, seed treatment with lemongrass oil resulted in minor phytotoxicity. However, for plants grown in soil infested with F. virguliforme, plants treated with lemongrass oil showed more disease than the control plants. However, we consider these results inconclusive as SDS severity was very high in this experiment and the plants also suffered a fungal gnat infestation that may have caused additional stress on the plants. We are currently working on optimizing the greenhouse assay for testing new formulations of essential oil seed treatments. Preliminary tests are showing promising results with cassia oil applied at a seed treatment.

Deliverables:
Two posters presented at the annual meeting of the American Phytopathological Society (APS) in Denver, CO, in August 2023.
• Starkey, A., and Leandro, L. 2023. Evaluating the efficacy of essential oils on inhibition of mycelial growth of Fusarium virguliforme and impacts on soybean seed germination. Phytopathology (in press) – Poster presented at the American Phytopathological Society (APS) national meeting in Denver, CO, in August 2023.
• Mulder, D. M., Silva, V. K., Starkey*, A., and Leandro, L. 2023. Screening essential oils for mycelial growth reduction of Sclerotinia sclerotiorum, Macrophomina phaseolina, and Pythium sylvaticum. Phytopathology (in press)
Poster presented at the World food prize event in Des Moines and at the ISU Borlaug Lecture, in Ames, IA, in October 2023. Graduate student Alexandra Starkey won the 2nd place for the Borlaug International Dialogue event and 1st place for her poster presentation at ISU.
Video recording on the topic of “Plant essential oils: Potential for soybean pathogen suppression”, for the Iowa Seed Treatment Continuing Instruction Course, ISU Pesticide Safety Education Program, Jan 12, 2024, available Feb-Dec 2024 via county offices throughout Iowa
Video recording of an episode of ISU CropsTV - Season 4 entitled “Plant essential oils: Potential for soybean pathogen suppression” video recorded Jan 11, 2024; available online @ https://www.aep.iastate.edu/cropstv/
Research highlighted in article entitled “Nano-encapsulation of Essential Oils: A Unique Approach to Soybean Disease Control” prepared for the Soybean Research & Information Network (SRIN) website. Posted Aug 14, 2023. https://soybeanresearchinfo.com/research-highlight/nano-encapsulation-of-essential-oils-a-unique-approach-to-soybean-disease-control/
Presentation entitled “Potential of biochar and plant essential oils to suppress soybean stem/seedling diseases”, at the ISU Agriculture and Natural Resources Crops Team Spring In-Service meeting, Boone, IA, March 22, 2023

View uploaded report

Plant essential oils are a type of biopesticide that has shown effectiveness against several plant pathogens and pests. We conducted laboratory research to identify plant essential oils with inhibitory activity against Fusarium virguliforme (Fv), the pathogen that caused soybean sudden death syndrome (SDS). We screened 16 plant essential oils for suppression of growth in Fv culture plates. Of the 16 oils, we identified 7 EO’s that inhibited growth by more than 50%, 3 of which inhibited Fv growth by more than 85%. The three essential oils with strongest inhibitory effects on Fv were cassia, cinnamon bark, and geraniol, with cassia oil completely preventing Fv growth at the low concentration of 0.05% (500ppm). These results showed the strong antifungal activity of plant essential oils against Fv.

Because essential oils contain powerful solvents and toxic chemical compounds, it is important to ensure they can be applied at rates that are not phytotoxic to seeds but are still suppressive to the pathogen. We developed assays and started screening the most effective essential oils for phytotoxicity on soybean seedlings. We have found that some of these oils can reduce germination of soybean seeds but that this phytotoxicity is highly dependent on the rate applied and seed delivery method.

Our goal is to encapsulate the essential oils in corn or soybean nanocellulose. We expect that nanoencapsulation will reduce phytotoxicity in two ways: 1) by providing a slow release delivery system and 2) by increasing the efficacy of the essential oils against the target pathogen, allowing us to reduce the rate applied to seeds to a level that is not phytotoxic but still effective against the pathogen. We were able to create nanoformulations of plant essential oils that were stable against centrifugation forces, freeze-thaw cycles, heating-cooling cycles and room temperature storage for at least 30 days.

The next step is to test the effectiveness of seed treatments with nanoencapsulated essential oils against SDS. We have conducted research to optimize the application method onto seeds, and developed a greenhouse protocol. We are currently preparing to conduct these greenhouse assays with the most effective essential oil formulation.

Soybean growers are faced not only with yield losses due to SDS but also loss of productivity and profitability due to the cost and phytotoxicity of some fungicides. The proposed work will determine the potential for growers to reduce risk of phytotoxicity by applying lower rates of existing fungicide seed treatment in combination with nano-encapsulated biopesticides, while maintaining effective SDS control. Growers will also have information about the effectiveness of essential oils as a safe alternative treatment option for SDS management. The development of a delivery system for biopesticides will offer organic farmers more treatments options against SDS.