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. In 2008, ARS scientists in Wenatchee, WA, who developed what is now a commercially available biocontrol agent that suppresses the fire blight organism, conducted studies to examine how the beneficial microorganism prevents fire blight. This has been partly accomplished by comparing the performance of the biocontrol strain with a mutated derivative strain lacking the ability to produce a unique antibiotic compound highly specific to the disease organism. The derivative strain was much less effective than the parent strain in suppressing the disease organism on plant floral surfaces. Studies also showed that the purified antibiotic alone can inhibit the disease organism on blossoms. Collaboration with scientists at Washington State University, Pullman, have provided insight as to the identity of the antibiotic compound. Additional research was performed to increase the survival of the biocontrol agent and other beneficial microorganisms under low relative humidity conditions typical of fruit production areas in the western U.S. Laboratory and field experiments done in cooperation with the commercial producer of the biocontrol agent showed that the addition of certain ingredients to production media, including the commercial fermentation medium, enhances the tolerance of the biocontrol agent to dry environments. This work, still in progress, likely will lead to an improved commercial formulation in 2010. As an additional focus, we revisited a previous survey of microorganisms occurring naturally on tissues of apple blossoms by supplementing earlier identifications with DNA sequence analyses. This information, evaluated in conjunction with prior blossom assays involving hundreds of the same microbes, has allowed us to better select taxonomic groups and specific strains that may enhance biological control through the use of microbial mixtures with complementary modes of action and ecological niches.
1. Naturally-occuring mirobial residents on apple blossom surfaces and their potential use in fire blight control. 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. Although some biocontrol agents have now been commercialized for this disease, information regarding population sizes, distributions, and diversities of natural microbial residents on floral surfaces of apple trees and their potential use in disease control was lacking. ARS scientists in Wentachee, WA detected a wide diversity of bacteria and yeasts on blossoms of apple and, through laboratory bioassays with detached flowers, identified microbial genera and specific strains highly effective in suppressing the disease organism. The information will be useful in developing effective microbial mixtures with complementary modes of action and ecological niches for enhancing biological control of fire blight.
Pusey, P.L., Stockwell, V.O., Rudell Jr, D.R. 2008. Antibiosis and acidification by Panoea agglomerans strain E325 may contribute to suppression of Erwinia amylovora. Phytopathology. 98:1136-1143.