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
Studies have continued with Pantoea agglomerans strain E325, a fire blight biocontrol agent that originated from our research program and became available commercially for apple and pear beginning in 2007. Strain E325 is antagonistic to Erwinia amylovora on flower stigmas, the primary site of pathogen establishment. In an artificial medium partially formulated based on chemical analyses of exudates from flower stigmas, E325 was shown to produce a compound inhibitory to the pathogen. Characteristics of the compound purified by RP-HPLC distinguished it from antibiotics of other strains of P. agglomerans. The antibacterial compound was active under acidic conditions, sensitive to basic pH and to high phosphate levels conventionally used in buffered media to test for antibiosis, and unaffected by amino acids or proteolytic and other enzymes. Further, the E325 compound exhibited a high specificity to E. amylovora, which may be advantageous in efforts to maximize biological control by integrating E325 with other microbial antagonists with complementary mechanisms or environmental adaptations. Since our research indicates that acidification by E325 is possibly related to antibiosis and may also directly contribute to the antagonism of E. amylovora, screening assays are being done to identify acid-tolerant bacteria or yeast that could potentially complement strain E325 in antagonist mixtures. Efforts are underway to develop a non-antibiotic mutant of E325 to evaluate the role of antibiosis by this antagonist in biological control. Our laboratory has continued to cooperate with the private company marketing E325 by recently evaluating the purity and stability of samples during production scale-up, generating DNA-based identification information required by regulatory agencies in the US and Canada, and sharing research information that may be useful for improving fermentation, formulation and efficacy of the product. As a follow up to our previous biochemical characterization of apple and pear stigmas, a field study was initiated in 2008 to study chemical changes on stigmatic surfaces and in flower tissues in response to colonization by E. amylovora and the antagonist strain E325. Stigma exudates are being evaluated for several chemical components, including free sugars, organic acids, and phenolic compounds, and flower tissues will be evaluated for PR protein gene expression. It is hoped that this data will lead to a better understanding of microbial interactions on apple and pear flowers and improved strategies for fire blight management. This research is part of NP 303, Component 4.
1. Title: Suppression of fire blight of apple by new biocontrol agent likely involves multiple mechanisms Fire blight, a serious disease of apple and pear caused by Erwinia amylovora has been controlled in the past mainly with antibiotics, but bacterial resistance to these chemicals has led to alternative strategies such as biological control. One biocontrol agent originating from our laboratory and recently made available commercially is Pantoea agglomerans strain E325. An explanation of why strain E325 is a superior antagonist of E. amylovora on flower stigmas, where the two organisms compete for establishment, has been elusive. Recent studies conducted by scientists in the Physiology and Pathology and Tree Fruits Research Unit in WEnatchee, WA indicate that in addition to competing for nutrients, strain E325 may also suppress E. amylovora by lowering the pH to levels less conducive for pathogen growth and produce a unique antibacterial compound highly specific to the pathogen and active under acidic conditions. This information and follow-up studies will likely lead to improvements in fire management with biological agents. The research is part of National Program 303 (Plant Diseases) and supports Component 4 (Biological and Cultural Strategies for Sustainable Disease Management) of the NP 303 Action Plan
5. Significant Activities that Support Special Target Populations
“Responded to farm managers and cooperative extension personnel seeking advice relating to pome fruit diseases, particularly fire blight (this sometimes involved receiving plant specimens for diagnosis). Responded to inquiries from the fruit industry regarding new fire blight biocontrol agent that originated in our lab and is now commercially available. Advised and cooperated with private company in meeting regulatory requirements and scaling up production of the biocontrol agent for use against fire blight in 2008.”