The goals of this project are to: <OL> <LI> Determine the efficacy of inducers of host resistance, the antibiotic kasugamycin and novel biological control agents for management of fire blight in apple orchards. 1a.) Screen native microflora for the occurrence of kasugamycin resistance; determine if kasugamycin resistance is associated with cross resistance to other aminoglycoside antibiotics. <LI> Perform surveys and genetic analyses to understand the evolution and spread of streptomycin resistance in the fire blight pathogen Erwinia amylovora. <LI> Develop a model of the effector-chaperone interaction of DspE and DspF, mapping areas and specific residues of both proteins required for effector-chaperone interaction and secretion. <LI> Examine the biofilm mode of growth of E. amylovora and determine the importance of biofilms to fire blight disease. </ol> Expected outputs include: <OL> <LI> We expect to identify efficacious materials for the control of the blossom blight phase of fire blight including the novel agricultural antibiotic kasugamycin. This will be the third year of assessment of kasugamycin in field trials. We are also screening native bacterial flora in apple orchards (flowers, leaves, soil) for the occurrence of resistance to kasugamycin. Since this antibiotic is naturally produced by Streptomyces sp. in soil, we expect that some level of resistance will be present in native microvlora. It is critical to get a handle of this for risk assessment for the potential for kasugamycin resistance development in the fire blight pathogen.<LI> We also expect to develop a structural model of the interaction of the major effector DspE with its chaperone DspF which is necessary for the secretion and translocation of DspE into apple tissues. This knowledge could help us target methods to inhibit the secretion of this effector, which is required for pathogenesis.<LI> The critical nature of biofilms for fire blight disease development and the importance of biofilms to a variety of bacterial pathogens suggests that biofilm development is an excellent target for future disease control strategies. We expect to begin to understand the role of biofilms in fire blight infection and colonization and movement through the plant of the fire blight pathogen.
NON-TECHNICAL SUMMARY: Tree fruit crops represent an important agricultural commodity in Michigan, with apple leading in importance, both in terms of acreage grown (44,000 A) and value received by growers ($81.6 million). The apple acreage in Michigan was reduced approximately 20% between 1997 and 2001 and one of the major diseases responsible for this reduction has been fire blight caused by bacterial pathogen Erwinia amylovora. A severe fire blight epidemic in southwest Michigan in 2000 caused losses exceeding $42 million including losses of approximately 350,000 to 450,000 trees. The introduction of a wide range of new apple cultivars grafted onto susceptible dwarfing rootstocks has resulted in the establishment of high value orchards vulnerable to catastrophic losses. Streptomycin resistance in the fire blight pathogen is a national problem resulting in the loss of an important disease control option. As such, it is critical that this work be performed in FY09-10; timely results are necessary for successful management of the disease. In addition, Michigan State University is an ideal location for this research work because of the importance of the disease problem to Michigan apple growers and because these results are used directly in Dr. Sundin's Extension program providing management recommendations to Michigan apple growers. The research proposed in this project is aimed at developing new short-term and long-term disease management measures for fire blight. As such, this work is relevant to three of the five research target areas in the MAES mission. Research on plant disease management contributes directly to Secure Food and Fiber Systems and to Enhancing Profitability in Agriculture and Natural Resources. Furthermore, our research on biological control of fire blight contributes to the Food and Health target area through increasing microbial and chemical food safety.
APPROACH: The focus of our field research for 2008-2013 is to continue to optimize timing and rates of the novel antibiotic kasugamycin for blossom blight control, and to increase the effectiveness of biological control agents for fire blight management. We have shown that kasugamycin when used singly is efficacious in blossom blight disease control in 2007 and 2008. We now need to integrate this material in with other control measures to formulate control strategies that would be appropriate for grower use. Although biological control agents such as Serenade MAX and Bloomtime E325 represent an excellent disease control technology, we have not observed consistent performance from these materials in previous field research. We are planning more monitoring experiments to understand the colonization parameters of the biological control organisms such that their performance can become more predictable and tuned with environmental conditions. In addition, we will integrate kasugamycin and streptomycin with the biological control agents and the growth regulator prohexadione-calcium (ProCa) in hopes of generating successful season-long disease control programs. Evaluation experiments will be performed on the fire blight-susceptible variety Jonathan. Trees will be inoculated with a virulent marked strain (E. amylovora Ea110 [RifR] for tracking purposes. Trees will be evaluated for % blossom blight and % shoot blight and all treatments will be compared with a non-treated control. Surveys for streptomycin resistance will be conducted during bloom and during times when fresh fire blight infections are visible in orchards. We plan to sample approximately 20 new orchards to add to our knowledge from previous years of the distribution of SmR E. amylovora in Michigan. We will also continue to monitor the movement of resistant strains into Oceana county and do some pilot sampling in Leelanau and Grand Traverse counties and in eastern Michigan. We will initially use standard in vitro techniques to characterize biofilm formation by E. amylovora including a crystal violet staining method to examine biofilm formation on polystyrene surfaces and a flow cell technique in which biofilm formation is observed using confocal laser-scanning microscopy. All assays will be performed using wild-type E. amylovora Ea1189, an amylovoran biosynthesis mutant constructed in the lab, and fluorescently-labeled derivatives of these strains. We will compile three-dimensional structural images that will reveal the architecture and distribution of E. amylovora, E. amylovora mutants that have been determined to have a defect in biofilm formation, as well as mixed cultures. Finally, we will examine apple tissue infected with E. amylovora using scanning electron microscopy to visualize E. amylovora infection in planta. We will first determine the secretion signal for DspE and identify the chaperone-binding domain on this effector to accompany our mutational studies of the chaperone DspF. We will map the region of DspE required for translocation into plant cells using the adenylate cyclase domain of the Bordetella pertussis CyaA protein.