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 blossoms. Disease control historically depended on chemical control measures, but pathogen resistance and environmental and safety concerns prompted a search for alternative control strategies. ARS scientists in Wenatchee, WA, who developed what is now a commercially available biocontrol agent that suppresses the fire blight organism, conducted studies in 2010 to examine how the beneficial microorganism prevents fire blight. This was partly accomplished by comparing the performance of the biocontrol strain with mutated derivative strains lacking the ability to produce a unique antibiotic compound highly specific to the disease organism. The derivative strains were much less effective than the parent strain in suppressing the disease organism on plant floral surfaces. The role of antibiotic activity was additionally evidenced by the fact that the purified antibiotic alone inhibited the disease organism on blossoms. Collaboration with scientists at Washington State University, Pullman, have provided insight as to the identity of the antibiotic compound. In other research aimed at improving the commercial biocontrol agent, it was demonstrated that increasing salt levels in the production medium increased the survival of the organism during freeze drying and subsequent storage prior to application. The new procedure also improved the establishment of the beneficial organism in flower nectaries where infection usually occurs. This work led to modifications in the commercial production and formulation of the biocontrol agent in 2010. Progress is being made in efforts to further enhance biological control of fire blight through the use of microbial mixtures with complementary modes of action and ecological niches. DNA-based identifications of hundreds of microorganisms indigenous to blossoms were recently evaluated in conjunction with blossom assays indicating the biocontrol potential of these organisms. This information is being used in the selection of taxonomic groups and specific strains most suited for use in microbial mixtures effective for disease control. A successful fire blight management program in apple and pear orchards not only includes the use of protective agents, but an accurate weather-based predictive system for determining the risk of infection. In collaboration with Washington State University, Wenatchee, laboratory data relating temperature and growth of the fire blight organism on blossoms was recently incorporated in the Cougarblight model, a disease risk assessment system widely used in the Pacific Northwest.
1. Biocontrol agent for fire blight improved by increasing salt levels in production medium. Biological control of fire blight of apple and pear with beneficial microorganisms is a viable alternative to the use of antibiotics, which have become less effective due to resistance in the causal organism. One effective biocontrol agent, the bacterium Pantoea agglomerans strain E325, was originally discovered by ARS scientists at Wenatchee, WA, and is now commercially available. In efforts to improve the production and tolerance of this organism to dehydration and related stress factors, salt levels were increased in the growth medium. As a result, the organism survived at significantly higher levels during freeze drying and subsequent storage prior to application, and in some cases, established better on floral surfaces under dry conditions. The research led to commercial modifications and an improved biological product for fire blight management.