The growth of agricultural productivity in the past century can be partly attributed to an intense focus on pest management, including the development of fungicides and bactericides. Agricultural history contains many examples of fungi or bacteria causing complete crop loss. The use of fungicides and bactericides can prevent such catastrophic losses and maximize yield, however, these benefits come with some risks. The early fungicides, developed in the 1950's and 1960's, have been associated with health risks to people and the environment, resulting in increased regulatory restrictions. The newer fungicides introduced during and after the 1970's have an improved safety profile, however, fungi resistance is threatening their long term viability. A similar resistance threat faces bactericides used to prevent bacterial diseases. We have identified formulations that show strong fungicidal and bactericidal efficacy alone and in combination with existing conventional fungicides. The benefit of these combined formulations is three-fold: (1) the older fungicides with more prominent health risks can be used at much lower concentrations, (2) the newer, safer fungicides can be enhanced to perform against the resistant strains and (3) the overall level of fungicide application can be reduced. Therefore, these combined formulations can mitigate the risks of fungicide use while maintaining performance. OBJECTIVES: This Phase I proposal is designed to provide the foundation of the next stage in development. Our key objective is to characterize the synergy of our formulations with commercial fungicides and optimize the formulas that will be evaluated in greenhouse and field studies in Phase II. Specifically, the objectives of Phase I are (1) determine the minimum inhibitory concentration (MIC) for Agion, chlorothalonil and mancozeb against each of the selected organisms, (2) perform checkerboard experiments to produce an interaction map and calculate the Fractional Inhibitory Concentration (FIC), a metric of synergy, and (3) perform studies in the vicinity of the optimized formulation to confirm efficacy on plants. The MIC values will be used to determine the range of concentrations to be explored in the checkerboard experiments. In this procedure, dilutions of the formulations are incorporated in broth. Each dilution tube is inoculated with a standard inoculum of the test organism. After appropriate incubation, the lowest concentration showing macroscopic inhibition of growth is considered the minimum inhibitory concentration (MIC). The checkerboard method is a two dimensional MIC that combines two actives over a range of concentrations. It is commonly used in studies of combinations of antibacterial agents, especially when looking at synergy or at strategies to combat resistant strains. The method has also been used in the evaluation of antifungal agents. The combination of antifungal agents and species results in six separate checkerboard experiments, with five replicates at each position in the matrix. The objective of the plant studies is to confirm that the formulations optimized in the first part of the study are optimal for control on the plant. A matrix experiment similar to the checkerboard study will be performed using the plant samples. A three by three matrix of samples will be tested, each treated with a different formulation. The center of the 3x3 matrix will be the optimized formulation for each of the six active/organism combinations. The eight surrounding samples will be treated with the corresponding step up or down in dilution of Agion and the particular coactive. The samples will be sprayed to runoff with the formulation and allowed to dry before inoculation with the pathogens. The formulation with the lowest concentration of active ingredient, whether the target formulation from the previous experiment or an adjacent sample, will be selected for Phase II studies. APPROACH: The Proof of Concept program in Phase I is designed to demonstrate that the Agion formulation can enhance the performance of mancozeb and chlorothalonil, allowing a significant reduction in use concentration. Fungicides and bactericides perform a vital role in crop protection and therefore the livelihood of American farmers. However, these benefits come with some risks. Some fungicides pose a health risk for applicators and several common actives are on the Food Quality Protection Act (FQPA) Priority List for review, including the two discussed here, along with captan, maneb, etridiazole and iprodione. Alternate actives have better safety profiles but many are at high risk of driving the development of resistant organisms. These two factors, health concerns and resistance development, pose a challenge to future crop protection strategies. Through our expertise in the use of metal ions for bacteria and fungus control, we have discovered formulations that show high activity against crop pathogens at very low active ingredient levels. It is interesting to note that the fungicides cited as priority for review by the FQPA are the older pesticides developed between the 1950's and 1970's, which utilize multi-site modes of action. The newer pesticides with a more moderate safety profile, on the other hand, generally utilize a single-site mode of action. From a biological standpoint, and as noted in the classification scheme devised by the Fungicide Resistance Action Commettee (FRAC), the singles-site modes of action results in high resistance potential, while the multi-site modes correlate to low resistance potential. Taking a broad look at the issues of toxicity and resistance, a picture emerges of an inverse correlation between health concerns and resistance potential. The older fungicides have low resistance potential but pose health risks. The bactericides and newer fungicides are associated with lower health risk, but their single-mode action makes them susceptible to resistant strains. Our research suggests that by combining the Agion formulation with commercial fungicides we can maintain the benefits of high performance, while mitigating the health risk of some fungicides and the performance loss due to resistance development in others. In Phase I we will evaluate the performance of combinations of Agion and conventional fungicides through a "checkerboard" study, in which the actives are tested together at concentrations below which they would normally be used. This method will identify if the effect of combining the actives is additive or synergistic. In addition, it will provide target formulations for future greenhouse and field studies to be pursue in Phase II
