Performing Department
Plant Pathology
Non Technical Summary
This project seeks to improve food security through the development of a novel crop disease management tool. Modern agriculture relies on the heavy use of pesticides to control plant diseases and protect crops from significant losses, and many of the most important disease control products are quickly losing efficacy due to resistance development. Consequences of pesticide use include potentially negative effects on human health and the environment and selection for pesticide resistance, and the increased use of pesticides since 1960 has not resulted in a significant decrease in crop losses. Novel crop protection solutions will ensure crops are protected against diseases amid global trade and a changing climate which threaten to introduce or increase the severity of diseases in areas where they were previously insignificant. More sustainable practices are needed to protect arable lands from deterioration against the backdrop of intensive agricultural production as well as to protect effective pesticides from becoming obsolete through the selection of resistant pests and pathogens. From 2001 through 2003, actual crop loss in eleven major crops (including tomato) due to pathogens was estimated at 68% of the theoretical potential loss, compared to 61% and 26% for animal pests and weeds, respectively. This indicates a significant opportunity to improve the efficiency of plant disease management. The goal of this project is to provide seed producers with a novel and sustainable seed coating technology which will increase crop yields and food production by protecting seeds and seedlings against a diversity of diseases, and by protecting the longevity of important antimicrobials by reducing the rate of resistance development.Coating seeds with antimicrobials, mostly fungicides, to protect against disease has been practiced for centuries, and remains one of the most efficient ways of ensuring that seedlings are protected from soilborne and seedborne pathogens. Early seed coating technologies included the use of arsenic, mercury and copper, but concerns over acute toxicity, mishandling accidents such as the mass poisoning by methylmercury of 1971 in Iraq, and environmental impacts have given rise to more targeted, or site-specific, compounds. A drawback to site-specific products is an increased likelihood of resistance development compared to multi-site compounds, due to the fact that a single mutation can confer resistance. Zymtronix has developed a solution to this problem through the use of its enzyme immobilization system, which uses stabilized antimicrobial enzymes delivered as a seed coating to protect against a broad spectrum of plant pathogens. Zymtronix's enzyme system relies on the production of non-site-specific free radicals and reactive oxygen species, thereby reducing the likelihood of resistance development. Additionally, we have demonstrated compatibility with a commercial fungicide and antibiotic and shown potential for reducing pesticide application rates and reducing the probability of resistance development without compromising control. This novel method will reduce reliance on existing agrochemicals like fungicides and antibiotics, thereby decreasing the likelihood of resistance development while simultaneously providing an alternative, effective method for managing a broad spectrum of major crop diseases.For this Phase I project, we are initially targeting the devastating tomato seedling disease damping-off, which can affect nearly all crops, as well bacterial speck, an important bacterial disease of tomato that can serve as a model for many other seed-borne and soil-borne bacterial plant diseases. We will determine the efficacy of, and potential synergy between, our stabilized enzyme formula and several commercial fungicides and antibiotics in laboratory tests. We will then use that information to develop several unique seed-coating formulas. Tomato seeds treated with those formulas will be exposed to damping-off and bacterial speck pathogens in greenhouse tests, and disease incidence data will be collected. These data will serve as the foundation for expansion into other crop-disease systems. The ultimate goal is to introduce a novel disease management product into the agricultural market which will reduce losses due to disease, increase profitability for seedling producers and farmers, reduce the occurrence of pesticide resistance, and reduce reliance on synthetic agricultural chemicals.
Animal Health Component
33%
Research Effort Categories
Basic
33%
Applied
33%
Developmental
34%
Goals / Objectives
The overall goal of this project is to demonstrate the proof-of-concept that our stabilized lactoperoxidase:glucose oxidase (LP:GOx) enzyme technology effectively controls Pythium spp. and Pseudomonas syrigae pv. tomato and is compatible with existing fungicides and antibiotics through in-vitro laboratory assays. Furthermore, we will demonstrate efficacy of the seed coating and mixed seed coating/fungicide and seed coating/antibiotic formulations on two important tomato diseases, damping-off and bacterial speck,that will serve as models for other important crop diseases.This will be achieved through the following objectives:(1) Determine if there is an interaction in-vitro between stabilized LP:GOx and two commercial fungicides and antibiotics, and whether that interaction enhances or antagonizes the activity of the commercial antimicrobials. This will be done by (i) testingtwo commercial fungicides, thiram and captan, which are commonly used as components of seed coatings to protect against damping-off caused by Pythium spp. and (ii) testing two antibiotics, streptomycin and oxytetracycline, which are registered for use in commercial agriculture, especially to protect greenhouse-grown seedlings from various bacterial diseases like bacterial speck.(2) Develop a seed coating formulation incorporating stabilized LP:GOx enzymes, with registered fungicides or antibiotics, at concentrations optimized during in-vitro experiments. This will be done by (i) optimizing a seed coating formulation using carboxymethyl cellulose (CMC), which is a safe and FDA-approved food adjuvant, to make a film-forming solution upon drying and (ii) using Cornell's rotary pan seed-coating technologies for application of liquid formulations developed by Zymtronix for a two-layer coating through a collaboration with the Professor Alan Taylor laboratory. Commercial fungicides and antibiotics will be incorporated into one of the two layers to produce 21 tomato seed lots coated with various combinations of LP:GOx and commercial antimicrobials.(3) Test treatment-coated tomato seeds in the greenhouse to determine whether seed coatings containing stabilized enzymes alone or together with commercial fungicides and antibiotics can reduce the incidence of damping-off and bacterial speck of tomato. Additionally, we will determine whether a reduced rate of fungicide or antibiotic in combination with stabilized enzymes can achieve disease suppression comparable to that of the recommended rate of each commercial antimicrobial alone. We will also determinethe effect of each treatment on disease incidence and seed germination and seeding vigor. This will be done by (i) planting treatment-coated seeds inPythium-infested soil and recording incidence of damping-off, (ii) planting treatment-coated seeds that have been inoculated withPseudomonas syringaepv.tomato (Pst) and recording incidence of bacterial speck, (iii) testing leaf samples from thePstexperiment forPstDNA using quantitative PCR, and (iv) recording germination and seedling vigor data on treatment-coated non-inoculated controls.
Project Methods
Preliminary in-vitro studies will be conducted to determine concentrations of each antimicrobial necessary to achieve measurable growth inhibition of P. ultimum (fungicides) and Pst (antibiotics) at a range of four concentrations. Once appropriate assay concentrations are determined for each antimicrobial, sterile filter paper discs measuring 7 mm in diameter will be impregnated with 5 µl of antimicrobials at each of the four pre-determined concentrations. Control filter paper discs will be impregnated with 5 µl sterile H2O. LP:GOx concentrations necessary to provide measurable growth inhibition of P. ultimum and Pst have been determined in previous studies. Experiments will be conducted by placing one enzyme disc in the center of a petri dish containing cornmeal agar (P. ultimum) or LB agar (Pst). Filter paper discs impregnated with antimicrobials will immediately be placed on top of enzyme discs. P. ultimum will be assayed by placing one 7 mm plug taken from the edge of an actively growing culture on top of each treatment. Pst will be assayed by depositing 106 CFU onto each plate of LB and spreading using sterile glass beads to create bacterial lawns. Treatments will then be applied to each plate as previously described. Controls will include one H2O-impregnated filter paper disc only. After two days P. ultimum colony diameters and zones of inhibition of Pst will be measured. All treatments will be triplicated and each experiment will be done twice. If it is determined that the stabilized enzymes are not compatible with one or more of the target commercial antimicrobials we will proceed by coating and testing the stabilized enzymes alone.A seed coating formulation will be developed by incorporating stabilized LP:GOx enzymeswith registered fungicides or antibioticsat concentrations optimized during in-vitro experiments. For seed coating, we will use carboxymethyl cellulose (CMC), which is a safe and FDA-approved food adjuvant, to make a film-forming solution upon drying. This is available with degrees of polymerization and substitution that confer a broad range of viscosities even at low concentrations of the polymer. For our preliminary tests, we have assayed different Aqualon CMC batches (Ashland, USA) and retained a blend that maximizes the concentration of cellulose in the coating while slowly hydrating under high moisture content. Blending two types of CMC allows us to accurately control the quantity of film-forming material in the coating layer and also the activation of formulation. Another key advantage of CMC is that it is degraded to glucose in the presence of endo- and exocellulases, which are excreted by most plant pathogens to penetrate plant cell walls. This additional source of glucose can also serve as an oxidizing reagent for the GOx to produce H2O2 and prolong activity. We will adapt Cornell's rotary pan seed-coating technologies for application of liquid formulations developed by Zymtronix for a two-layer coating. Commercial fungicides and antibiotics will be incorporated into one of the two layers. This work will be conducted through a sub-contract with Cornell University in the laboratory of Dr. Taylor. Rotary pan coating is state-of-the-art seed coating technology that can achieve simultaneous liquid and solid particulates application. We will use a laboratory-scale rotary pan, R-6 (Universal Coating Systems, Independence, OR), which can treat samples as small as 25 g, reducing the need for large formulation application volumes. There are several seed coating technologies that may be employed; optimization is dependent on the amount of weight increase or build-up during the coating operation. For this project, as seeds are rapidly rotating in the coating pan, liquid components will be applied to the spinning pan to atomize the suspension onto the seeds. Solid filler materials will be applied as a dry formulation during the coating operation. A binder or adhesive is needed to adhere the filler to the seed surface, and binders are commonly applied as a liquid. The seed moisture will be measured with a non-destructive method with a water activity instrument (AquaLab 4TE, Decagon, Pullman, WA).We will also determine if seed coatings, composed of stabilized enzymes and reduced-rate antimicrobials, can achieve control of damping-off and bacterial speck of tomato comparable to or better than label-rate antimicrobials alone. If it is determined that the stabilized enzymes are not compatible with one or more of the target commercial antimicrobials we will proceed by testing the stabilized enzymes alone.For damping-off assays Pythium inoculum will be generated by soaking 20 g wheat seeds at room temperature in 25 ml DDI H2O in 250 ml flasks for 24 hours. Flasks will then be autoclaved twice daily for two consecutive days. Each flask will then be inoculated with five 5 mm disks of two-day-old Pythium ultimum cultures growing on corn meal agar. Control flasks will be inoculated with sterile CMA. Inoculated flasks will be incubated in the dark for 8 days at 25°C and shaken periodically to ensure uniform colonization of wheat seeds. Soil will be infested by adding 2.5 g infested wheat seeds per 150 g potting mix and mixed thoroughly in plastic bags. Treatments will include seeds coated with stabilized enzymes only, thiram only (label rate), thiram only (reduced rate), captan only (label rate), captan only (reduced rate), enzymes + each fungicide (label rate), enzymes + each fungicide (reduced rate), and enzymes coated with inert ingredients only (CMC). Coated seeds will be planted into 96-cell flats containing Pythium-infested soil and non-infested soil as controls. Treatments will be assigned to blocks of 16 cells, and each treatment will be replicated 4 times in a completely randomized design. Damping-off incidence will be recorded for each replicate, and analysis of variance will be done to determine if treatments differences are significant. To evaluate the treatment effect on plant vigor, seedlings grown in non-inoculated soil will be inspected for signs of phytotoxicity, and shoot and root biomass will be measured. For bacterial speck assays a preliminary study will be conducted to determine a concentration of Pst cells that, when used to inoculate tomato seeds, is sufficient to induce bacterial speck symptoms in tomato seedlings. Treatments will include seeds coated with stabilized enzymes only, streptomycin only (label rate), streptomycin only (reduced rate), oxytetracycline only (label rate), oxytetracycline only (reduced rate), enzymes + each antibiotic (label rate), enzymes + each antibiotic (reduced rate), and enzymes coated with inert ingredients only (CMC). Inoculated and non-inoculated seeds will be coated with each treatment in the laboratory of Dr. Taylor as previously described. The same experimental design as described for the damping-off assay will be used for the bacterial speck assay. After five weeks, incidence and severity of bacterial speck will be recorded. Leaf tissue samples will be collected and pooled from each seed-inoculated treatment replicate and a non-inoculated control treatment, and DNA will be extracted from each pooled sample in triplicate. DNA will then be tested for Pst using quantitative PCR and previously-described species-specific primers.