Sudden death syndrome (SDS) of soybean is responsible for important yield losses in Iowa and the Midwest. Annual yield losses due to SDS have reached 1.9 million metric tons in the US, with cumulative economic losses over 2.5 billion dollars for 1996-2016 in Iowa alone (Bandara et al. 2020). The disease is caused by Fusarium virguliforme, a soil-borne fungus that causes root rot and premature defoliation.

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 in years when weather conditions are not conducive to SDS development. 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. Nano-emulsion is widely used to encapsulate biopesticides such as essential oils. 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. Due to these benefits, nano-emulsions have been widely applied as vehicles for delivery of active compounds in several fields, including drugs, food, and agriculture. Unfortunately, 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.

Biopesticides are materials with pesticidal properties that originate from plants, animals, and microorganisms. They are promising crop protection options because they are safer and more environmentally friendly than their chemical counterparts. Plant essential oils are a type of biopesticide that has shown effectiveness against several plant pathogens and pests. For example, lemongrass oil reduced Fusarium solani growth in vitro, reduced root rot incidence by up to 45% in petri dish assays, and doubled soybean root length and shoot length in greenhouse assays (Eke et al. 2020). Thymol essential oil was also effective in inhibiting F. solani in petri dish assays (Kong et al. 2021). Although we are not aware of studies on the effectiveness of these essential oils specifically against SDS, these are promising results since F. solani is a closely related species to F. virguliforme. In addition, lemongrass and thymol essential oils have been effective in reducing 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), respectively.

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 for natural antimicrobials, drugs, and bioactive compounds. Due to the 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.

Goal: 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.

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:
Since the start of this project we have made progress on Objectives 1 and 2, as planned.

For Objective 1, Dr. Liu’s lab has prepared nanocellulose from soybean residues which is planned for use in the characterization and optimization of nano-encapsulated biopesticides. The prepared nanocellulose from soybean stover has gel-like structure. More information will be available after the characterization of the as-prepared nanocellulose. Dr. Liu’s lab started with 50 lb of soybean stover and currently is still in the process of massive production of nanocellulose for the encapsulation work of the project.

For Objective 2, Dr. Leandro's lab has conducted in-vitro assays to test the suppressiveness of essential oils on mycelial growth and spore germination of Fusarium virguliforme (Fv). Three essential oils were tested to date: lemongrass, thyme and clove. Initial experiments focused on Lemongrass oil to develop protocols for the growth and germination assays. In these preliminary experiments, lemongrass oil completely suppressed Fv mycelial growth at concentrations ranging from 0.5% to 5%, but effects on spore germination we inconclusive. We then conducted three experiments with each of the essential oils using lower concentrations of the oils to determine the lowest dose that was still inhibitory to Fv. We tested concentrations of 0%, 0.005%, 0.010%, 0.05%, 0.10%, 0.25%, 0.50% and 1% in the first experiment and concentrations of 0.000%, 0.001%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08% in the second and third experiment.

We have found that lemongrass oil suppressed mycelial growth at concentrations as low as 0.05%, and both thyme and clove leaf oil suppressed mycelial growth at a concentration of 0.10%. Lemongrass oil was ineffective at preventing spore germination at all concentrations tested, while thyme oil showed limited effectiveness with <65% germination at 0.08%. Clove leaf oil was the most effective inhibitor of spore germination, with <2% germination at a concentration of 0.08% and <20% germination at 0.07%.

An abstract was submitted to the Americal Phytopathological Society to present results in the form of a poster at the national conference in August 2022.

In the next two quarters, Dr. Leandro will continue to test different essential oils and Dr. Liu will characterize the nanocellulose produced in her lab and attempt nanoencapsulation of the most suppressive oil.

View uploaded report

Update:

Objective 1
Dr. Liu’s lab has prepared nanocellulose from soybean residues which is planned for use in the formulation of nano-encapsulated essential oils. The prepared nanocellulose from soybean stover has gel-like structure and it is semi-transparent. The average particle size and zeta potential of the nanocellulose was found to be 292.2 nm and -63.8 mV.
Nanoencapsulation of lemongrass essential oil was achieved by mixing certain concentrations of nanocellulose, Tween 80, and lemongrass essential oil together and forming nanoemulsion. The fresh nanoemulsion had an initial pale white and translucent appearance. All the formulated nanoemulsion (0.1%~1% nanocellulose, 5% Tween 80, and 5% lemongrass essential oil) were stable against centrifugation test and storage at room temperature (up to 10 days), but unstable against freeze-thaw cycle tests. Specifically, the creaming indices of the formulations as shown in Table 1 ranged from 8.8% to 13.1% after the first freeze-thaw cycle. Nanoemulsion containing 0.5% CNC had the lowest creaming index among all formulations. Table 2 shows the transmittance of the nanoemulsion on the first day and on the 10th day. For the fresh nanoemulsion, the transmittance of the nanoemulsion increased with the increase of nanocellulose concentration except at the 1% nanocellulose concentration. After storage for 10 days, the transmittance of the nanoemulsion decreased for all formulations.
As this project continues, more formulations will be prepared and characterization of the properties of the nano-encapsulated essential oil will be studied by Dr. Liu’s lab. The optimal conditions for the nano-encapsulation of lemongrass essential oil will be determined later.

Table 1 Creaming index of the nanoemulsion after one freeze-thaw cycle
Creaming index
Nanoemulsion containing 0% nanocellulose 10.123%
Nanoemulsion containing 0.1% nanocellulose 9.492%
Nanoemulsion containing 0.5% nanocellulose 8.833%
Nanoemulsion containing 0.7% nanocellulose 10.863%
Nanoemulsion containing 1% nanocellulose 13.0517%

Table 2 Transmittance of nanoemulsion on the first day and on the 10th day
Transmittance (%)
Fresh (day 1) After storage (day 10)
Nanoemulsion containing 0% nanocellulose 23.574 6.7360
Nanoemulsion containing 0.1% nanocellulose 27.133 9.2352
Nanoemulsion containing 0.5% nanocellulose 41.297 2.8175
Nanoemulsion containing 0.7% nanocellulose 47.817 5.7545
Nanoemulsion containing 1% nanocellulose 19.014 0.6805


Objective 2
Dr. Leandro's lab conducted in-vitro assays to test the suppressiveness of essential oils on mycelial growth and spore germination of Fusarium virguliforme (Fv). Three essential oils were tested: lemongrass, thyme and clove. Potato dextrose agar was amended with lemongrass, thyme, and clove leaf oil at concentrations of 0.000%, 0.001%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08% (v/v). Mycelial growth inhibition was measured using a Petri dish assay and inhibition of Fv spore germination was tested on cavity slides observed under the microscope.

We found that lemongrass oil suppressed mycelial growth at concentrations as low as 0.05%, and both thyme and clove leaf oil suppressed mycelial growth at a concentration of 0.10%. Lemongrass oil was ineffective at preventing spore germination at all concentrations tested, while thyme oil showed limited effectiveness with <65% germination at 0.08%. Clove leaf oil was the most effective inhibitor of spore germination, with <2% germination at a concentration of 0.08% and <20% germination at 0.07%.

Our findings suggest that essential oils are promising candidates for a new era of biofungicides to combat SDS and the economic losses sustained by farmers each year, without the drawbacks posed by traditional fungicides. Current limitations to their use include active compound volatility, lack of an effective delivery system to plant roots and unknown impacts on beneficial microorganisms.
In the new year of funding, we have started to screen additional essential oils to determine the most effective against the SDS pathogen. The most effective oil will be nano-encapsulated in Dr. Liu’s lab and will be tested against SDS development in plants.

Results of this project have been shared at the annual plant pathology conference in poster format, at the board meeting of the Iowa Soybean Asspciation, during a ISA board member visit to the ISU campus and during an extension event in NE Iowa in the summer.

View uploaded report

Dr. Liu’s lab has prepared nanocellulose from soybean residues which is planned for use in the formulation of nano-encapsulated essential oils. The prepared nanocellulose from soybean stover has gel-like structure and is semi-transparent, with an average particle size of 292.2 nm.
Nanoencapsulation of lemongrass essential oil was achieved by mixing certain concentrations of nanocellulose, Tween 80, and lemongrass essential oil together and forming a nanoemulsion. The fresh nanoemulsion had an initial pale white and translucent appearance. All the formulated nanoemulsion (0.1%~1% nanocellulose, 5% Tween 80, and 5% lemongrass essential oil) were stable against centrifugation test and storage at room temperature (up to 10 days), but unstable against freeze-thaw cycle tests. Specifically, the creaming indices of the formulations as shown in Table 1 ranged from 8.8% to 13.1% after the first freeze-thaw cycle. Nanoemulsion containing 0.5% CNC had the lowest creaming index among all formulations.

As this project continues, more formulations will be prepared and characterization of the properties of the nano-encapsulated essential oil will be studied by Dr. Liu’s lab. The optimal conditions for the nano-encapsulation of lemongrass essential oil will be determined later.

Dr. Leandro's lab conducted in-vitro assays to test the suppressiveness of essential oils on mycelial growth and spore germination of Fusarium virguliforme (Fv). Three essential oils were tested: lemongrass, thyme and clove. Potato dextrose agar was amended with lemongrass, thyme, and clove leaf oil at concentrations ranging from 0% to 0.08% (v/v). Mycelial growth inhibition was measured using a Petri dish assay and inhibition of Fv spore germination was tested on cavity slides observed under the microscope.

We found that lemongrass oil suppressed mycelial growth at concentrations as low as 0.05%, and both thyme and clove leaf oil suppressed mycelial growth at a concentration of 0.10%. Lemongrass oil was ineffective at preventing spore germination at all concentrations tested, while thyme oil showed limited effectiveness with <65% germination at 0.08%. Clove leaf oil was the most effective inhibitor of spore germination, with <2% germination at a concentration of 0.08% and <20% germination at 0.07%.

Our findings suggest that essential oils are promising candidates for a new era of biofungicides to combat SDS and the economic losses sustained by farmers each year, without the drawbacks posed by traditional fungicides. Current limitations to their use include active compound volatility, lack of an effective delivery system to plant roots and unknown impacts on beneficial microorganisms.

In the new year of funding, we have started to screen additional essential oils to determine the most effective against the SDS pathogen. The most effective oil will be nano-encapsulated in Dr. Liu’s lab and will be tested against SDS development in plants.

Results of this project have been shared at the annual plant pathology conference in poster format, at the board meeting of the Iowa Soybean Asspciation, during a ISA board member visit to the ISU campus and during an extension event in NE Iowa in the summer.

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 fungicide treatment. The results from the proposed work, growers will be able 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 an alternative, safe and organic treatment option for SDS management. The development of a delivery system for biopesticides will offer organic farmers more treatments options against SDS.