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.

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.