Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Plant Soil & Entomological Sci
Non Technical Summary
There is a critical need for natural pesticides that can be used in organic and high value agricultural and horticultural crops. Mustard plants (Brassica juncea and Sinapis alba) contain natural pesticides that kill weeds and nematodes, and inhibit the sprouting of stored potatoes. Highest concentrations of the natural pesticides occur in seed meal; solid materials remaining after oil for biodiesel is removed from the seed by crushing. The primary obstacle in using mustard seed meals as natural pesticides is the bulky nature of the materials. We propose to develop methods to extract, concentrate, dry, and formulate natural pesticide powders from mustard seed meals. The products will cost less to transport, can be applied more easily, and will be more efficacious than seed meals. In this project we will develop scale-up procedures, optimize pesticide formulations so they are effective, and test the products on various plant pests. Commercialized products will target markets for which the few alternative products available are expensive, ineffective, or potentially harmful to humans or the environment.
Animal Health Component
30%
Research Effort Categories
Basic
30%
Applied
30%
Developmental
40%
Goals / Objectives
Our overall goal is to develop biopesticides from the mustard seed meals of B. juncea, and S. alba. Currently, our most promising candidates are a bioherbicide, nematicide, and potato sprout inhibitor. We propose here to continue these efforts by working with business partners, scaling up our activities, and eventually commercializing biopesticidal products. We propose therefore, to extract glucosinolates from seed meals, utilize the separated glucosinolates as biopesticides, and increase the nutritional value of the residual seed meals for animal feed. Glucosinolate extracts possess inherent advantages as biopesticides compared to the seed meals including 1) greater ability to manipulate active ingredient additions to achieve efficacy, 2) improved transportability, and 3) ease of application.Our specific objectives include the following:1. Develop scaled-up extraction procedures for glucosinolates from Brassicaceae seed meals.2. Optimize biopesticidal formulations to maximize biopesticide release.3. Develop an economically viable bioherbicide, nematicide, and potato sprout inhibitor.
Project Methods
Mustard seeds of S. alba (IdaGold variety) and B. juncea (Pacific Gold) will be obtained locally. Oil contents of seeds and meals will be analyzed gravimetrically after extraction with hexane. Homogenous seed meal samples of S. alba and B. juncea will be extracted with ethanol concentrations appropriate for the specific seed meal. The meal-ethanol slurry will be incubated for various times and temperatures to determine the optimal conditions required for complete glucosinolate or active ingredient extraction. The mixture will be passed through a 2-mm sieve to remove large seed meal particles. Smaller particles will be removed by placing the remaining extract into a nylon mesh bag with an approximate pore size of 200 µm, and a mechanical or hydraulic press will be used to remove entrained liquid. We will determine the appropriate type and size of press based on the level of scale-up required to supply our commercial partners with sufficient extract for testing. Nutritional value of the residual meal will be determined by measuring crude protein, crude fiber, acid detergent fiber, neutral detergent fiber, and total digestible nutrients. Techniques for most efficiently concentrating the extract will be determined. For scaling up we will determine if vacuum extraction will improve throughput and sample processing. We will also develop a system for recovering ethanol so that it may be reused for extraction. Once the ethanol is removed and the sample is concentrated by one of the above techniques, we will dry the material using either a spray dryer or a freeze dryer.Extraction efficiency will be quantified by analyzing the resulting solution for glucosinolate content using procedures routinely practiced in our laboratory including enzymatic desulfation, separation by High Performance Liquid Chromatography (HPLC), and quantitative/qualitative analysis on an Agilent time-of-flight (TOF) mass spectrometer (MS). Formulation of powdered products requires consideration of two primary aspects: 1) ensuring that hydrolysis occurs at an acceptable rate (B. juncea extracts only) and 2) buffering the reaction such that any pH decrease does not inhibit the enzymatic reaction.We will evaluate different mustard meals for their potential use as a source of enzyme for B. juncea extracts. S. alba meal typically contains higher concentrations of myrosinase than B. juncea seed meal. Determinations will be made as to whether B. juncea or S. alba seed meal is the better enzyme source, also determining if substrate specificity plays a role. Additional studies will be performed to determine the amount of seed meal required for glucosinolate hydrolysis and how the rate of the reaction varies with enzyme concentration. Our ultimate goal is to formulate B. juncea extracts with a seed meal concentration that results in complete sinigrin hydrolysis within a relatively short time period. We will thus determine the optimal ratio of extract to seed meal required for a formulated product.Solution pH is the second significant parameter affecting the efficiency of glucosinolate hydrolysis. Our preliminary hydrolysis studies with sinalbin concentrations four times higher than endogenous concentrations resulted in a pH of 2.5 and consequently myrosinase inactivation. Correspondingly, the pH of a sinigrin hydrolysis mixture decreased to 4.6. To prevent inactivation of myrosinase by increased acidity, we will test different buffers. We will determine the viability and buffering requirement, we will test the potential to use carbonate and bicarbonate as alternative buffering agents. In addition to cost, another advantage of using a carbonate buffer for pH adjustment is that it can be obtained from natural sources, thus making the final product eligible for organic certification.The formulated glucosinolate/myrosinase products obtained from the two different species of oilseeds will be tested for pest control efficacy using bioassay procedures. We will focus our efforts on pests of critical importance and those for which we have business partners willing to pursue commercialization. Our efforts with S. alba meal biopesticide development will be focused exclusively on bioherbicide development. The target markets are organic and high value agricultural and horticultural crops for which the few alternative products available are expensive, ineffective, or harmful to humans or the environment. We will conduct greenhouse studies to determine if tolerance to the extract varies among species, the rates required for control, and whether the extracts function as contact or systemic bioherbicides. Greenhouse weed bioassays will be conducted using a Palouse silt loam soil (20% sand, 20% clay, pH 5.9) with six replications for each treatment. Both pre- and post-planting applications of the potential bioherbicides will be tested using glucosinolate rates equivalent to that found in field applications ranging from 0.5 to 4 t ha-1. Weed seed germination and plant growth will be monitored at regular time intervals during the bioassay. Total dry weight of root and shoots will be determined at experiment termination. Once we have established efficacy, plant tolerance, and use rates, a protocol for application will be developed.Hydrolysis products from B. juncea show broad spectrum biological activity and thus will be tested as biopesticides against nematodes. Our objective is to use concentrated extracts to formulate soil-applied biopesticides that will more consistently control nematodes. Greenhouse bioassays of cysts exposed to formulated extracts for 2 weeks will be conducted using bioassays to determine egg viability. Hatching and viability of initial populations of eggs will be assessed and reproduction of nematodes will be determined by enumerating extracted cysts. Treatments will be organized in a completely randomized design and reduction of cyst content as well as reduction in reproduction will be measured. Treatment effect on numbers of hatched juveniles and reduction in cyst content will be analyzed using SAS.The final market for a product relevant to Idaho agriculture includes that of a sprout inhibitor for stored potatoes to maintain quality and marketability. Initial tests in our laboratory and storage facilities maintained by our commercial collaborator indicate that B. juncea extracts are effective in preventing sprouting in stored potatoes. In cooperation with our commercial partner, we propose to first scale up testing to 900 kg of stored potatoes and then to potato storage facilities of a commercial scale. Standard protocols for sprout inhibitor dispersal within the storage facilities will be utilized. Dispersal of active ingredient produced by B. juncea extracts will be facilitated by the volatility of 2-propenyl isothiocyanate. Rate tests have shown that 5 µmol isothiocyanate/g potato has been effective. Future testing will focus on rates 0.5 to 3 times this rate based on headspace volumes of the storage facilities. Enzyme activation requires only water addition. Future tests will first involve 1,000 kg of potatoes, followed by 3,000 kg, and finally storages of 500,000 kg and above.The most promising formulations will be selected for further testing in laboratory, greenhouse, and field bioassays. Assistance from commercial partners will be used to conduct large scale testing and to pursue registration of commercially viable biopesticides with EPA. Our ultimate goal during the reporting period is product commercialization.