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
Fire blight is a potentially devastating disease of apple and pear trees that is generally initiated in flowers. Disease control historically depended on chemical control measures, but pathogen resistance and safety-related concerns prompted a search for alternative strategies. ARS scientists at Wenatchee, who developed what is now a commercial biocontrol agent for fire blight, completed studies showing that antibiosis is an important mechanism of the biocontrol activity. This was accomplished by comparing the performance of the beneficial bacterium, Pantoea agglomerans strain E325, with mutated derivative strains lacking the ability to produce a unique antibiotic compound. The derivative strains were much less effective than the parental strain in suppressing the disease organism on floral surfaces. Through collaborations with other researchers, new information was obtained relating to the identity and structure of the E325 antibiotic, and the gene for its production was found located on a plasmid. Efforts to improve E325 through a process known as osmoadaptation continued from the previous year, with experiments demonstrating that high salt levels in growth media increase the survival of E325 during freeze drying and subsequent storage prior to application. These results led to modifications in the commercial production and formulation of the biocontrol agent. As another attempt to improve product formulation, collaboration with bioengineering scientists at the University of Illinois led to preliminary data indicating the potential of microencapsulation as a means of protecting biocontrol agents and controlling their release in orchard environments. Also, progress was made toward the goal of enhancing biological control of fire blight through the use of microbial mixtures with complementary modes of action and ecological niches. Based on previous evaluations of hundreds of microorganisms screened for biocontrol potential on flowers, effective yeast strains that possibly could complement E325 were selected and field tested. Further research is necessary to assess whether E325 combined with a yeast strain is more effective than either type of biocontrol agent alone. The above activities relate to Objective 1A (“Optimize…production and product formulation”), Objective 2 (“Evaluate the contribution and possible relationship of different modes of microbial antagonism toward E. amylovora, including...antibiotic production…”), and Objective 3 (“Develop mixtures of antagonists…”).
1. Fire blight biocontrol agent produces novel antibiotic encoded by gene on plasmid. Biological control of fire blight of apple and pear is a viable alternative to the use of broad-spectrum antibiotics, which have diminished as an option due to resistance in the causal bacterium and concerns over the possibility of such resistance being transferred to organisms involved in human diseases. ARS scientists at Wenatchee, WA previously discovered biocontrol strain Pantoea agglomerans E325, which is now available commercially, and found that it produces an unique antibiotic highly specific to the fire blight organism. Recent collaboration with scientists in Switzerland revealed that the gene encoding the antibiotic compound is located on a plasmid, i.e., DNA independent of the chromosomal DNA. The discovery is significant because it opens up new possibilities for studying or enhancing biological control of fire blight involving microbial production of a novel antibiotic.