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Bacterial Diseases of Tree Fruit Crops and Their Control

Investigators
Sundin, George
Institutions
Michigan State University
Start date
2008
End date
2013
Objective
  1. Integrate growth regulation, biological control, and induced resistance strategies with antibiotics for management of fire blight in apple orchards. Screen native microflora for the occurrence of kasugamycin resistance; determine if kasugamycin resistance is associated with cross resistance to other aminoglycoside antibiotics.
  2. Perform surveys and genetic analyses to understand the evolution and spread of streptomycin resistance in the fire blight pathogen Erwinia amylovora.
  3. Examine the biofilm mode of growth of E. amylovora and determine the importance of biofilms to fire blight disease.
  4. 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.

The expected outputs of this project are novel disease management strategies that can be directly utilized by growers for fire blight disease management. These strategies may involve the use of new antibiotics or biological control materials integrated in programs emphasizing effective control and antibiotic resistance management. This information is transmitted to growers through individual consulting visits, talks and handouts at grower meetings, newsletter articles, and information provided in the annually updated Michigan Fruit Management Guide. Additional outputs include further knowledge and impacts of streptomycin resistance in the pathogen E. amylovora to apple growers in Michigan and surrounding states.

Longer term basic research knowledge generated in this project will include a more detailed understanding of the role of two pathogenicity factors, biofilm growth and the type III effector DspE, in the fire blight disease system and host-pathogen interactions. This work will be disseminated through refereed journal publications.

More information
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). Cherries are second in importance, with tart and sweet cherries grown on 37,800 acres with a value of $55.5 million. Peaches, plum, and pears are of less importance, and, combined, are grown on 6,200 acres with a value of $14.9 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. Fire blight is an exceedingly difficult disease to manage because of several interacting factors: 1) chemical management options such as antibiotics are limited, and are impacted by antibiotic resistance in the pathogen population 2) after the initial blossom phase of the disease, the pathogen is located internally within trees while antibiotic options are surface-associated 3) infection can occur on the scion, reducing yield, and in the rootstock, resulting in tree death 4) most of the popular apple varieties grown in Michigan are highly susceptible to the disease.

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.

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: ACTIVITIES: Experiments conducted included work on short-term control methods for fire blight disease of apple and pear. We examined the efficacy of experimental antibiotics and biological control agents for blossom blight control. Additional experiments included analyses of the distribution of streptomycin resistant strains of the pathogen Erwinia amylovora in Michigan and characterization of the mechanism of streptomycin resistance in these strains. We also continued work examining the role of the ubiquitous plasmid pEA29 in the virulence of E. amylovora, studied the mechanism of action of the growth inhibitor prohexadione-calcium on shoot blight disease control, and began studies examining the role of biofilms in fire blight disease.
EVENTS: Research presentations at scientific meetings included four presentations at the American Phytopathological national meeting in 2007 and six presentations at the International Fire Blight Working Group meeting in 2007. A total of 10 grower training sessions were given on fire blight disease control.
DISSEMINATION: Outreach activities included oral presentations given to growers and other clientele at grower meetings, newsletter type articles published through Michigan State University IPM CAT Alert, and informal one-on-one meetings with individual growers through on-farm visits.
PARTICIPANTS: George Sundin -- PI -- oversees all research projects, makes oral presentations at grower meetings, writes extension/newsletter articles, co-writes research publications. Gail Ehret -- laboratory research technician -- responsible for field projects relating to fire blight disease control. Gayle McGhee -- laboratory research assistant -- responsible for field projects relating to fire blight disease control and studies of streptomycin resistance in Erwinia amylovora in Michigan. Molly McGrath -- postdoctoral research associate -- responsible for experiments aimed at determining the mechanism of action of prohexadione-calcium for fire blight control. Jessica Koczan -- graduate student -- responsible for work on the role of biofilms in fire blight disease.
TARGET AUDIENCES: The target audience for this work is the grower community (apple, cherry, peach), extension agents, representatives for chemical companies and disease scouting consultants, mainly in Michigan but in other states as well. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit.
PROJECT MODIFICATIONS: None.

IMPACT: 2007/01 TO 2007/12
CHANGE IN KNOWLEDGE: Through this work, we learned of the spread of streptomycin resistance in E. amylovora into new areas of Michigan; we also learned that the spread of resistant strains in other areas was more limited than previously predicted. We found that new materials were efficacious for fire blight control under field conditions. We determined that the role of pEA29 in virulence of the fire blight pathogen was mainly due to the impact of the thiamin-biosynthetic genes of the plasmid on the expression of exopolysaccharide biosynthesis genes involved in biofilm formation.
CHANGE IN ACTIONS: By disseminating information on streptomycin resistance directly to growers, we enabled these growers to change fire blight management practices and thus not be exposed to the possibility of catastrophic losses due to disease control failures.

PROGRESS: 2006/01/01 TO 2006/12/31
Fire blight of apple and pear, bacterial canker of sweet and tart cherry, and bacterial spot of peach are the three most important tree fruit bacterial diseases in Michigan. My group is working with short and long-term applications for disease management. Regarding fire blight, we have focused on optimizing the management of this disease through control of the blossom blight phase. These are the compounds we have been testing for fire blight control: copper bactericides, the antibiotics streptomycin, oxytetracycline, kasugamycin, and gentamicin, the growth regulator prohexadione-calcium, the fungicide famoxate, the host resistance elicitor laminarin, and the biological control agents Serenade, BlightBan, Bloomtime, and Pantoea agglomerans C9-1. Streptomycin is the most effective fire blight control available; however, we have detected streptomycin-resistant strains of Erwinia amylovora in two important fruit-growing areas of Michigan. Oxytetracycline is less effective than streptomycin, but remains the best alternative in orchards where streptomycin resistance is a problem. Copper is an effective early season bactericide, but cannot be used later due to problems with fruit russeting. The biological control options were partially effective in fire blight control in our 2006 experiments. Prohexadione calcium was highly effective in reducing the shoot blight phase of the disease. The bactericide control programs must be combined with growing varieties that are less susceptible to fire blight in order to achieve high levels of control. In terms of genetic analyses, we have completed a study in which approximately 394 E. amylovora genes that are induced during infection were identified. We identified two new effector genes, avrRpt2EA and hopPtoCEA, and several other candidate virulence genes. We demonstrated that avrRpt2EA was similar to the homologous effector protein from Pseudomonas syringae and functioned similarly in eliciting the hypersensitive response in Arabidopsis plants containing the RPS2 resistance gene. We performed experiments aimed at identifying genes contributing to host specificity in E. amylovora by isolating genes unique to apple pathogenic strains and not present in raspberry pathogenic strains. We continued our work on the role of the ubiquitous plasmid pEA29 in virulence. This study has shown that the thiOGF genes are the critical genes on pEA29 in controlling the virulence effect of this plasmid. In addition, we continue to focus our efforts on determining the function of DspE, the critical effector for disease.

IMPACT: 2006/01/01 TO 2006/12/31
This research is aimed at determining sound management practices for controlling bacterial diseases of tree fruits. Effective management is difficult due to the lack of available compounds, the growth of highly disease-susceptible varieties, and bactericide resistance. We have developed optimized control programs for these diseases based on currently-available compounds.

PROGRESS: 2005/01/01 TO 2005/12/31
Fire blight of apple and pear, bacterial canker of sweet and tart cherry, and bacterial spot of peach are the three most important tree fruit bacterial diseases in Michigan. My group is working with short and long-term applications for disease management. Regarding fire blight, we have focused on optimizing the management of this disease through control of the blossom blight phase. These are the compounds we have been testing for fire blight control: copper bactericides, the antibiotics streptomycin, oxytetracycline, and gentamicin, the growth regulator prohexadione-calcium, the fungicide famoxate, the host resistance elicitor laminarin, and the biological control agents Serenade, BlightBan, and Pantoea agglomerans C9-1. Streptomycin is the most effective fire blight control available; however, we have detected streptomycin-resistant strains of Erwinia amylovora in two important fruit-growing areas of Michigan. Oxytetracycline is less effective than streptomycin, but remains the best alternative in orchards where streptomycin resistance is a problem. Copper is an effective early season bactericide, but cannot be used later due to problems with fruit russeting. The compounds famoxate and laminarin show promise, but need to be tested again in 2006. The biological control options were partially effective in fire blight control. Prohexadione calcium was highly effective in reducing the shoot blight phase of the disease. The bactericide control programs must be combined with growing varieties that are less susceptible to fire blight in order to achieve high levels of control. We examined the efficacy of copper in a bacterial canker management program but found that, because copper cannot be sprayed during bloom, bacterial populations quickly recover on blossoms following early-season copper applications. A large-scale survey of Michigan sweet cherry orchards revealed that populations of the pathogen Pseudomoans syringae are quite large on blossoms throughout the state. Analyses of copper resistance in P. syringae and the bacterial spot pathogen Xanthomonas arbicola pv. pruni (Xap) revealed high levels of copper resistance in Xap only. In terms of genetic analyses, we have completed a study in which approximately 160 E. amylovora genes that are induced during infection were identified. We identified a new effector gene, hopPtoCEA, and several other candidate virulence genes. We continued our work on the role of the ubiquitous plasmid pEA29 in virulence. We have shown that expression of many of the promoters of pEA29 genes is induced during infection and are currently constructing knockout mutations in several of the genes for functional characterization. This study has shown that the thiOGF genes are the critical genes on pEA29 in controlling the virulence effect of this plasmid. In addition, we continue to focus our efforts on determining the function of DspE, the critical effector for disease.

IMPACT: 2005/01/01 TO 2005/12/31
This research is aimed at determining sound management practices for controlling bacterial diseases of tree fruits. Effective management is difficult due to the lack of available compounds, the growth of highly disease-susceptible varieties, and bactericide resistance. We have developed optimized control programs for these diseases based on currently-available compounds.

PROGRESS: 2004/01/01 TO 2004/12/31
Fire blight of apple and pear, bacterial canker of sweet and tart cherry, and bacterial spot of peach are the three most important tree fruit bacterial diseases in Michigan. My group is working with short and long-term applications for disease management. Regarding fire blight, we have focused on optimizing the management of this disease through control of the blossom blight phase. These are the compounds we have been testing for fire blight control: copper bactericides, the antibiotics streptomycin, oxytetracycline, and gentamicin, the growth regulator prohexadione-calcium, and the biological control agents Serenade, BlightBan, and Pantoea agglomerans C9-1. Streptomycin is the most effective fire blight control available; however, we have detected streptomycin-resistant strains of Erwinia amylovora in two important fruit-growing areas of Michigan. Oxytetracycline is less effective than streptomycin, but remains the best alternative in orchards where streptomycin resistance is a problem. Copper is an effective early season bactericide, but cannot be used later due to problems with fruit russeting. The biological control options were partially effective in fire blight control. Prohexadione calcium was highly effective in reducing the shoot blight phase of the disease. The bactericide control programs must be combined with growing varieties that are less susceptible to fire blight in order to achieve high levels of control. We examined the efficacy of copper in a bacterial canker management program but found that, because copper cannot be sprayed during bloom, bacterial populations quickly recover on blossoms following early-seaseon copper applications. A large-scale survey of Michigan sweet cherry orchards revealed that populations of the pathogen Pseudomoans syringae are quite large on blossoms throughout the state. Analyses of copper resistance in P. syringae and the bacterial spot pathogen Xanthomonas arbicola pv. pruni (Xap) revealed high levels of copper resistance in Xap only.

IMPACT: 2004/01/01 TO 2004/12/31
This research is aimed at determining sound management practices for controlling bacterial diseases of tree fruits. Effective management is difficult due to the lack of available compounds, the growth of highly disease-susceptible varieties, and bactericide resistance. We have developed optimized control programs for these diseases based on currently-available compounds.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
MICL02084
Accession number
199062
Categories
Chemical Contaminants
Natural Toxins
Bacterial Pathogens
Commodities
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