Source: ZYMTRONIX CATALYTIC SYSTEMS, INC. submitted to
A SEED-COATING MIXED ENZYME FORMULA FOR THE CONTROL OF FUNGAL AND BACTERIAL PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1012666
Grant No.
2017-33610-26746
Project No.
NY.W-2017-00113
Proposal No.
2017-00113
Multistate No.
(N/A)
Program Code
8.2
Project Start Date
Aug 1, 2017
Project End Date
Mar 31, 2019
Grant Year
2017
Project Director
Donnelly, M.
Recipient Organization
ZYMTRONIX CATALYTIC SYSTEMS, INC.
405 WEILL HALL, CORNELL UNIVERSITY
ITHACA,NY 14853
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
0%
Research Effort Categories
Basic
33%
Applied
33%
Developmental
34%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21214601160100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1160 - Pathology;
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.

Progress 08/01/17 to 03/31/18

Outputs
Target Audience:The Target Audience reached during this project includes seed producing companies, including HM Clause, Bejo Zaden, and Sakata;and small scale farmers in upstate NY who are concerned about pathogen problems we have addressed through this project. Efforts to reach the target audience included outreach via email and introductions at meetings by our collaborators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?These results have been presented to seed companies via direct outreach, resulting in potential new partnerships. Additionally, these results were used in a proposal submitted to the New York Farm Viability Institute's Farm Viability Grant program. In writing this proposal, farmers in NY state were consulted and presented these results. Their interest and support lead to the grant being successfully funded. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Modern agriculture relies on agrochemicals to control crop diseases, however, many products are losing their efficacy due resistance development.Novel crop protection productsare needed to protect against disease. Coating seeds with antimicrobials, such as fungicides, isone of the most efficient ways to ensure that seedlings are protected frompathogens. The ultimate goal of this project is to provide seed producers and farmers with a novel and sustainable seed coating product, which will increase yields and food production by protecting seeds and seedlings from disease and by protecting the longevity of existing antimicrobials by reducing the rate of resistance development. For this Phase 1 project, we targeted thediseases damping off caused by Pythium species (Py)and bacterial speck caused by Pseudomonas syringae pv tomato (Pst) as models for soil and seed borne plant diseases. We determined the efficacy of our stabilized enzymes against thesepathogens, both alone and in co-formulations with commercial fungicides and antibiotics in laboratory tests, demonstrating good control of the pathogens. This information informed the development of the seed coating, which was successfully applied to tomato seeds, with no inhibition of germination. These coated seeds were then used in greenhouse tests. A batch of coated seeds were planted in Py infested soil, and another batch was infected with Pst prior to coating. The results of the greenhouse test demonstrate that although the formula is effective in laboratory tests against the pathogens, more work is required to develop a seed coating that is sustained in the immediate seed environment for sufficient time to protect against the diseases. The combined results of this project are being used to develop an effective seed coating product for tomato seeds, and have been extended into new a new project in cabbage seeds. Our first objective was to evaluate theinteraction between the stabilized enzymes andcommercial antimicrobials, and to determineif these interactions were enhancing or antagonizing the activity of the commercial antimicrobials. We tested the stabilized enzymesin co-formulations with twofungicides, thiram and captan.In our experiments, the stabilized enzyme formula was placed on a petri dish containing growth media for Py. A 7 mm filter paper disc was impregnated with the commercial fungicide and placed on top of the enzyme formula on the petri dish. A 7 mm plug of actively growing Pyculture was placed on top and allowed to incubate at room temperature for two days. The diameter of the Pyculture that grew out from the initial plug in the presence of the fungicide co-formulations was measured and compared toa culture grown withoutfungicides. There was an average of 50% reduction in culture diameter from the enzyme formula alone. There was no difference in Pyculture diameter between thiram applied at the label rate alone and in thiram applied at the label rate in a co-formulation with the stabilized enzymes, in each case we observed80% reduction in diameter. Interestingly, when thiram was applied at a reduced rate, 50% of the label rate, we observed only a 40% reduction in culture diameterwith the fungicide alone, but when the reduced rate thiram was applied in a co-formulation with the stabilized enzymes, there was a 75% reduction in diameter, indicating an enhanced effect fromthe combination. Captan applied by itself at label rate and at areduced rate showed only a 25% and 10% reduction in diameter respectively, but when combined with the stabilized enzymes, showed a 70% and 65% reduction in diameter for label rate and reduced rate respectively. These data are a change in knowledge, demonstrating that a reducedfungicide dose can be usedfor the same level of Pycontrol when the fungicides are applied in a co-formulation with the stabilized enzymes. A similar set of experiments was conducted using the bacterial plant pathogen Pst, measuring the diameter of the cleared area resulting from the antimicrobial formulations in a lawn of bacteria growing on a petri dish. Streptomycin and oxytetracycline were applied to a lawn of Pstat both label and reduced rates, alone and in co-formulations with the stabilized enzymes. While no antagonistic effects were observed, there was no benefit to co-formulating the antibiotics with the stabilized enzymes observed either.These data also represent a change in knowledge, that lead to a change inaction. The results of the antibiotic in vitro assays indicatethere will be no benefit from developing a seed coatingthat combines the stabilized enzymes withantibiotics. The key outcomes from this objective aredata showing an additive effect from combining the stabilized enzymes with fungicides and no benefit from combining the stabilized enzymes with antibiotics. Both of these results show that the active ingredient in the seed coatingis effective at controlling the plant pathogens Pyand Pst. Our second objective was to produce a seed coating formula using the stabilized enzymes as the active ingredient, andto apply the formula to batches of tomato seeds both alone and in co-formulations with commercial antimicrobials. The conclusions from objective 1 informed the batches of coated seeds produced.Based on these conclusions, we decided not to pursue the co-formulation with antibiotics, and to produce batches of seeds coated with the stabilized enzymes alone, and batches of seeds coated with a co-formulation of the stabilized enzymes andthiram and captan. The stabilized enzymes seed coating formula was produced successfully, and then applied using a rotary seed coater. The coated seeds had a germination rate that was the same as the germination rate observed in the uncoated seeds, and in seeds coating in the inert ingredients only. Coated seeds were used in more in vitro studies against Pyand Pstas described in objective 1. The observed enzyme activity was lower than in the in vitro formulations previously used, requiring us to increase the concentration on the active ingredients and to change the drying agent used to adhere the formulas to the seeds. The seed coating formulation development was an iterative process, which resulted in changes in our knowledge of the problem and the materials themselves, and consequently changes in our actions. The original drying agent was replaced with a cellulose polymer, resulting in improved enzyme activity and more available substrate for the stabilized enzymes. Eventually, three batches of seeds, uncoated, inert ingredients only, and stabilized enzyme coated, were produced. These three sets of seeds were then used in the greenhouse trials described in objective 3. Our third objective was to demonstrate the effectiveness of the stabilized enzyme formula against Pyand Pstin soil and on seeds planted in greenhouse trials. Potting soil was artificially infested with Pyand seeds from each of the three batches described in objective 2 were planted in the infested soil. Seedling germination, shoot height, and root and shoot weight were measured after 4 weeks. There was no difference observed between the seedlings grown from the stabilized enzyme coated seeds and the uncoated seeds. Similarly, seeds were artificially infected with Pstbefore coating and planting in the greenhouse. Seedling shoots and leaves were harvested and evaluated for Pstinfection using qPCR. There was no observed difference between the coated seeds and the uncoated seeds. These results, combined with the results from both objective 1 and objective 2 indicate that though the stabilized enzymes are effective against pathogens, the current formula is insufficient in the more complex soil environment. This result reflects both a change of knowledge and a change of action. Future work will be dedicated to improving the formula to deliver the stabilized enzymes to the seed surface in soil and retaining it at the seed surface throughout the possible period of infection.

Publications