Project Number: 5350-22000-015-00
Project Type: Appropriated

Start Date: Mar 12, 2007
End Date: Mar 11, 2012

Objective:
(1) Investigate microbe-induced chemical changes on flower surfaces, with particular attention to pH modification, as modes of antagonism towards the fire blight bacterium, Erwinia amylovora; (2) evaluate the contribution and possible relationship of different modes of microbial antagonism toward E. amylovora, including pH reduction, antibiotic production and competitive exclusion; and (3) develop mixtures of antagonists with complimentary mechanisms and ecological niches and integrate their use with other fire blight management strategies.

Approach:
To accomplish the first objective, we will focus primarily on Pantoea agglomerans strain E325, a patented biocontrol agent recently registered by EPA. This strain was shown in preliminary tests to increase the acidity of stigma exudates based on the pH of exudates extracted from inoculated flowers. Laboratory experiments will be performed with an artificial stigma-based medium (SBM) and detached crab apple flowers prior to conducting field studies. Work with SBM and variations of it will be used to evaluate the relationship between pathogen suppression and pH reduction, determine the pH range and optimum for both pathogen and antagonist bacteria, and evaluate changes in acid production under varying buffer capacities and oxygen conditions. In flower bioassays, pH on stigmatic surfaces will be directly measured using pH-sensitive fluorescent dyes and confocal laser microscopy or with fabricated microelectrodes. In addition, stigma exudates extracted from inoculated flowers will be analyzed for sugar consumption and bacteria-produced organic acids. In field experiments with apple, flowers will be sampled and their stigmas evaluated for bacterial population size, pH and specific organic acids. Similar methods will be used to assess whether biocontrol treatments can be enhanced through the addition of various soft agrochemicals (e.g., foliar nitrogen fertilizers and pH buffers) that may alter acid production by bacteria or directly affect pH on flower surfaces. For the second objective, a collection of antagonist strains, previously shown to be among the best performers in flower bioassays, will be evaluated for mode of action. This will involve a series of laboratory tests with SBM and flowers to determine the importance of acid production, antibiotic production and nutrient depletion as mechanisms of individual antagonist strains. Major extracellular compounds inhibitory to Erwinia amylovora will be identified or characterized. For the third objective, a strategy of enhancing biocontrol with antagonist mixtures will be largely dependent on results of the first two objectives. To fully exploit multiple antagonists and mechanisms, we will evaluate the compatibility of antagonists and avoid or eliminate incompatibilities. Further screening of microbial epiphytes from apple and pear may be necessary to develop the best complement of antagonists. Finally, to further improve the management of fire blight, we will test the integration of antagonist mixtures with other control approaches or agents.