1a. Objectives (from AD-416)
(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.
1b. Approach (from AD-416)
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.
3. Progress Report
Progress was made in understanding mechanisms of the bacterial antagonist Pantoea agglomerans strain E325, which originated from our research program and is now available commercially for use on apple and pear blossoms to reduce fire blight. Studies involving a synthetic medium, which included sugars and amino acids detected in previous chemical analyses of flower stigma exudates, showed a correlation between acidity in the medium and suppression of the disease bacterium (Erwinia amylovora). This led us to investigate pH modification on flower stigmas as a mode of antagonism. In an experimental apple orchard, blossoms were inoculated with bacteria, and the pH was determined in stigma exudates extracted from pooled samples of flowers. Results indicated a trend consistent with our hypothesis that strain E325 decreases pH to levels negatively affecting growth of E. amylovora, but were not statistically conclusive. To investigate further, we explored methods of directly measuring the pH on flower stigmas using pH-sensitive fluorescent dyes and laser microscopy, but were not successful with this approach. We are making progress, however, with an alternative method involving the fabrication of pH microelectrodes small enough to probe stigmatic surfaces. Meanwhile, we discovered that strain E325 produces an extracellular substance inhibitory to E. amylovora. The active compound is produced in the stigma-based medium when the phosphate buffer concentration is low and pH decreases during bacterial growth to levels between 3 and 5. It is stable when pH is neutralized, but inactivated under basic conditions. The compound has been partially characterized and was recently purified with high-performance liquid chromatography. As part of our effort to enhance biological control by finding antagonists that are compatible and complementary to strain E325, we collected 450 strains of Pseudomonas spp. and screened them for tolerance to acidic conditions. Selected acid-tolerant strains will be tested with E325 in antagonist mixtures applied to blossoms. In other experiments, E325 was evaluated in combination with certain agrochemicals, such as nitrogen fertilizers and pH buffers, but none of the additives significantly improved antagonist performance on flowers. In the past year we also cooperated with private company holding the patent license for strain E325, to improve production and formulation.
Phytosanitary concerns about fire blight prohibit export of US-grown pears to some countries without this disease. These concerns were evaluated in a 4-year regional project by scientists with Oregon State University and USDA-ARS, Wenatchee, Washington. We evaluated the potential for co-occurrence of the fire blight bacterium, Erwinia amylovora, with mature symptomless winter pear fruit by inoculation experiments and by survey of commercial orchards. It was concluded that E. amylovora shows similar survival characteristics on both pear and apple fruit, the disease organism does not exist inside mature symptomless pear fruit, its presence is exceptionally rare on commercially-produced fruit, and survival of E. amylovora on fruit surfaces is unlikely during postharvest chilling given the unrealistically high population size required for persistence. The information will be of value in efforts to expand foreign markets for US pears. Research is part of NP 303 (Plant Diseases), supporting Component 2(Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors) and Problem Statement 2C(Population Dynamics, Spread, and Epidemiology of Pathogens).
5. Significant Activities that Support Special Target Populations