Progress 02/25/03 to 05/16/07
Outputs
Progress Report Objectives (from AD-416) Identify and characterize pathogen virulence factors important in postharvest decay. Optimize combined control strategies to reduce postharvest decay and maintain fruit quality. Approach (from AD-416) Determine virulence factors important in pathogenicity by comparing various characteristics of two pathogens of the same species, one of which is significantly less virulent than the other. Determine how environmental conditions involved in host-parasite interactions affect the virulence of the pathogen. Determine the combination of alternatives to chemical control that will equal the efficacy of fungicides in controlling postharvest plant pathogens. Significant Activities that Support Special Target Populations New treatment helps reduce postharvest fruit decay. It is necessary to find alternatives to chemical control to reduce losses due to postharvest decay. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists at Beltsville, Maryland, and scientists at Virginia Polytechnic Institute and State University the second year of a pilot test was completed at a commercial packing facility in Winchester, Virginia by combining controlled atmosphere storage, sodium bicarbonate, and two biocontrol agents, alone or combined, to reduce postharvest fungal decay of apples. The antagonists alone reduced blue mold decay but tended to be more effective when combined. The most effective treatment was the combination of the two antagonists and sodium bicarbonate on fruit stored under controlled atmosphere conditions. This control strategy could potentially be used commercially by the postharvest storage industry to reduce our dependency on fungicides. NP 303, component 4a. Accomplishments Virulence factors associated with Penicillium expansum and P. solitum during decay of apple fruit. Penicillium expansum and P. solitum are blue mold fungi that cause major losses of apples in storage due to decay. A comparison of the virulence factors produced by these fungi and the resulting decay of various varieties of apples will help us understand why P. expansum is more efficient at causing disease than P. solitum. We have purified and partially characterized the polygalacturonases produced by P. expansum and P. solitum in infected apple fruit as well as in various liquid media. It was found that both pathogens produce multiple but different forms (isozymes) of a pectin degrading enzyme known as polygalacturonase in a medium dependent fashion. In apple fruit P. expansum and P. solitum appear to produce two isoforms of the enzyme. In addition, both P. expansum and P. solitum produce cellulase activity in apple and pear fruit. Roles for the cellulase and specific polygalacturonase isozymes in the disease process are yet to be determined and are objectives of our upcoming project. This information will be of use to researchers as well as members of the apple storage industry that are exploring methods of blue mold decay control. NP 303, component 2a. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 3
Impacts
(N/A)
Publications
- Conway, W.S., Janisiewicz, W.J., Leverentz, B., Saftner, R.A., Camp, M.J. 2007. Control of blue mold of apple by combining controlled atmosphere, antagonist mixtures and sodium bicarbonate. Postharvest Biology and Technology. 45:326-332.
|
Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Economic losses caused by postharvest pathogens are greater than is often realized, and the avoidable losses between the farm gate and the consumer are cause for concern. Fresh fruits and vegetables, because of the added cost of harvesting and handling, increase several times in value as they are moved from the field to the consumer. Annual postharvest losses range from 10 to 50% depending upon the sophistication of available storage facilities. There is increasing concern among consumers and regulatory agencies concerning the health hazards of synthetic chemical residues. For some pathogens, no practical chemical control has been developed and many pathogens have developed resistance to commonly used chemicals, of which only a very few are still registered for postharvest treatment.
To decrease our dependency on chemical control to ensure quality of harvested produce, alternative, more natural methods of postharvest decay control are being developed. It is highly unlikely that any one of these alternative methods alone will be as effective as fungicides; however, the development of a strategy combining several of these methods may be successful. We are currently investigating such promising alternatives as biocontrol, natural antimicrobial volatiles and heat treatment alone and in various combinations to develop a successful postharvest decay control strategy. If chemical control is no longer an option due to pathogen resistance or legislative restriction due to health and environmental concerns, a successful strategy combining various alternatives will be available to the grower to protect fruit in storage. This research falls within National Program 303, Plant Diseases (60 percent) and National Program 306, Quality and Utilization of Agricultural Products
(40 percent) We are seeking biocontrol agents to block pathogens from causing disease and to manipulate the conditions under which these agents are used in order to optimize their effectiveness. In addition, our research is aimed at the identification, characterization, and manipulation of genes for resistance in crop species, which will allow us to seek natural mechanisms of resistance to postharvest decay control. As promising methods of resistance are identified, they will be used in various combinations to develop an integrated pest management strategy to reduce our dependency on synthetic chemical control. This research also aims to prevent spoilage by managing or eliminating postharvest pathogens. Specifically, this approach includes the development of new treatments to eliminate pathogens and biological control of pathogens and will allow scientists to seek natural mechanisms of resistance to control postharvest decay. 2. List by year the currently approved milestones
(indicators of research progress) Year 1 (FY 2004) Complete optimization of P. solitum transformation procedure. Complete comparisons of wild type virulence of P. solitum and P. expansum. Complete optimization of UV mutagenesis and mutant screening procedures. Establish quantitative relationship between inoculum level and development of C. acutatum. Year 2 (FY 2005) Complete crude purification of PGs. Complete hygromycin-tagged plasmid construction and transformation of P. solitum. Complete isolation of pH altered mutants of P. expansum and P. solitum. Complete isolation and sequencing of wild type glutamate dehydrogenase and pacC genes. Determine effect of MCP on various apple decays. Determine the effect of heat on the survival and growth of C. acutatum. Year 3 (FY 2006) Complete purification and biochemical characterization of PGs. Complete comparisons of PG production and virulence by P. solitum containing control vector DNA and P. solitum containing the P. expansum PG gene.
Complete characterization of pH altered mutants for pectic enzyme expression and virulence. Complete construction of in vitro mutation of glutamate dehydrogenase and pacC genes. Identify biocontrol agents which are successful antagonists to C. acutatum. Year 4 (FY 2007) Complete in vitro construction of the PG gene knockout and subsequent transformations. Complete construction and isolation of a glutamate dehydrogenase and pacC mutants. Confirm causality of mutant phenotypes by restoration to a wild type phenotype via complementation. Optimize integrated strategy to control B. cinerea, C. Acutatum, and P. expansum. Year 5 (FY 2008) Complete mutant screens and mutant characterizations for PG expression and virulence. Complement mutants with the wild type PG gene to prove causality. Complete characterization of glutamate dehydrogenase and pacC mutants for pectic enzyme expression, virulence, and pH phenotype. Complete studies on the integrated strategy to control B. cinerea, C.
Acutatum, and P. expansum. 4a List the single most significant research accomplishment during FY 2006. Combined treatment reduces postharvest fruit decay. It is necessary to find alternatives to chemical control to reduce losses due to postharvest decay. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists at Beltsville, Maryland, for a second year of study, as well as in a pilot test in a commercial packing facility in Pennsylvania, combined controlled atmosphere storage, sodium bicarbonate, and two biocontrol agents, alone or combined, at Beltsville to reduce postharvest fungal decay of apples. The antagonists alone reduced blue mold decay but tended to be more effective when combined. The only treatment that completely eliminated decay caused by this pathogen was the combination of the two antagonists and sodium bicarbonate on fruit stored under controlled atmosphere conditions. This control strategy could
potentially be used commercially by the postharvest storage industry to reduce our dependency on fungicides. NP 303, component 4a. 4b List other significant research accomplishment(s), if any. Virulence factors associated with Penicillium expansum and P. solitum during decay of apple fruit. Penicillium expansum and P. solitum are blue mold fungi that cause major losses of apples in storage due to decay. A comparison of the virulence factors produced by these fungi and the resulting decay of various varieties of apples will help us understand why P. expansum is more efficient at causing disease than P. solitum. We have shown that P. expansum and P. solitum produce multiple but different forms (isozymes) of a pectin degrading enzyme known as polygalacturonase in a medium dependent fashion. Unlike P. expansum, P. solitum also produces a cellulase on a cellulose containing medium. Roles for the cellulase and specific polygalacturonase isozymes in the disease process are yet to be
determined. This information will be of use to researchers as well as members of the apple storage industry that are exploring methods of blue mold decay control. NP 303, component 2a. 5. Describe the major accomplishments to date and their predicted or actual impact. Bitter rot and blue mold decays were effectively controlled when combining one of several antagonists heat treatment. Combining several antagonists, sodium bicarbonate and heat treatment was also effective, as were antagonist combinations, sodium bicarbonate and controlled atmosphere storage. These strategies could be useful to growers who wish to reduce their reliance on chemical control. The bitter rot fungus produces two types of a certain protein that helps it attack and grow on apple fruit. This information could help explain the virulence mechanism in this fungus and aid in developing control methods useful to other scientists and eventually fruit growers. It is necessary to investigate as many alternatives to
chemical control as possible to reduce decay due to postharvest pathogens. In cooperation with scientists at the University of Tennessee, ARS scientists at Beltsville, Maryland, tested chitosan extracted from various fungi, including mushrooms, as well as commercial sources to determine the effectiveness of chitosan in reducing decay of apple caused by postharvest pathogens. In studying another alternative to chemical control, chitosan extracted from fungi was as effective as the commercial sources in reducing postharvest decay. Chitosan extracted from portions of mushrooms not sold commercially for consumption may give mushroom growers an additional source of revenue. NP 303, components 2a and 4a. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
The product to be delivered at the termination of this project will be a strategy combining several alternatives to chemical control which will equal the efficacy of chemical control. This strategy will be of importance to the apple growing industry, especially if postharvest fungicides are no longer effective or their use is no longer permitted.
Impacts
(N/A)
Publications
- Park, E., Mcevoy, J.L., Conway, W.S., Sams, C.E., Solomos, T. 2006. The effect of calcium on the virulence and polygalacturonase activity of colletotrichum acutatum on apple fruit. Plant Pathology Journal. 5:183-190.
- Mcevoy, J.L., Conway, W.S., Janisiewicz, W.J. 2006. Virulence factors associated with penicillium expansum and p. solitum during decay of apple fruit. Phytopathology. 96:S75
- Hong, Y., Leverentz, B., Conway, W.S., Janisiewicz, W.J., Abadias, M., Camp, M.J. 2006. Biocontrol of listeria monocytogenes on fresh-cut honeydew melons using a bacterial antagonist and bacteriophage. Phytopathology. 96:S51.
|
Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Economic losses caused by postharvest pathogens are greater than is often realized, and the avoidable losses between the farm gate and the consumer are cause for concern. Fresh fruits and vegetables, because of the added cost of harvesting and handling, increase several times in value as they are moved from the field to the consumer. Annual postharvest losses range from 10 to 50% depending upon the sophistication of available storage facilities. There is increasing concern among consumers and regulatory agencies concerning the health hazards of synthetic chemical residues. For some pathogens, no practical chemical control has been developed and many pathogens have developed resistance to commonly used chemicals, of which only a very few are still registered for postharvest treatment. To decrease
our dependency on chemical control to ensure quality of harvested produce, alternative, more natural methods of postharvest decay control are being developed. It is highly unlikely that any one of these alternative methods alone will be as effective as fungicides; however, the development of a strategy combining several of these methods may be successful. We are currently investigating such promising alternatives as biocontrol, natural antimicrobial volatiles and heat treatment alone and in various combinations to develop a successful postharvest decay control strategy. If chemical control is no longer an option due to pathogen resistance or legislative restriction due to health and environmental concerns, a successful strategy combining various alternatives will be available to the grower to protect fruit in storage. This research falls within National Program 303, Plant Diseases (60 percent) and National Program 306, Quality and Utilization of Agricultural Products (40 percent).
We are seeking biocontrol agents to block pathogens from causing disease and to manipulate the conditions under which these agents are used in order to optimize their effectiveness. In addition, our research is aimed at the identification, characterization, and manipulation of genes for resistance in crop species, which will allow us to seek natural mechanisms of resistance to postharvest decay control. As promising methods of resistance are identified, they will be used in various combinations to develop an integrated pest management strategy to reduce our dependency on synthetic chemical control. This research also aims to prevent spoilage by managing or eliminating postharvest pathogens. Specifically, this approach includes the development of new treatments to eliminate pathogens and biological control of pathogens and will allow scientists to seek natural mechanisms of resistance to control postharvest decay. 2. List the milestones (indicators of progress) from your Project
Plan. Year 1 (FY 2004) Complete optimization of P. solitum transformation procedure. Complete comparisons of wild type virulence of P. solitum and P. expansum. Complete optimization of UV mutagenesis and mutant screening procedures. Establish quantitative relationship between inoculum level and development of C. acutatum. Year 2 (FY 2005) Complete crude purification of PGs Complete hygromycin-tagged plasmid construction and transformation of P. solitum. Complete isolation of pH altered mutants of P. expans um and P. solitum. Complete isolation and sequencing of wild type glutamate dehydrogenase and pacC genes. Determine effect of MCP on various apple decays. Determine the effect of heat on the survival and growth of C. acutatum. Year 3 (FY 2006) Complete purification and biochemical characterization of PGs Complete comparisons of PG production and virulence by P. solitum containing control vector DNA and P. solitum containing the P. expansum PG gene. Complete characterization of pH
altered mutants for pectic enzyme expression and virulence. Complete construction of in vitro mutation of glutamate dehydrogenase and pacC genes. Identify biocontrol agents which are successful antagonists to C. acutatum. Year 4 (FY 2007) Complete in vitro construction of the PG gene knockout and subsequent transformations. Complete construction and isolation of a glutamate dehydrogenase and pacC mutants. Confirm causality of mutant phenotypes by restoration to a wild type phenotype via complementation. Optimize integrated strategy to control B. cinerea, C. Acutatum, and P. expansum Year 5 (FY 2008) Complete mutant screens and mutant characterizations for PG expression and virulence. Complement mutants with the wild type PG gene to prove causality. Complete characterization of glutamate dehydrogenase and pacC mutants for pectic enzyme expression, virulence, and pH phenotype. Complete studies on the integrated strategy to control B. cinerea, C. Acutatum, and P. expansum. 3a List the
milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Effect of MCP on various apple decays has been determined. Milestone Fully Met 2. Effect of heat on the survival of C. acutatum has been determined. Milestone Fully Met 3. Complete crude purification of PGs. Purification of crude PGs is in progress but not yet complete due to malfunction of necessary instrumentation. This equipment will be replaced and milestone completed in FY 2006. Milestone Substantially Met 4. Complete hygromycin-tagged plasmid construction and transformation of P. solitum. Optimization of the P. solitum growing conditions is still underway prior to performing the transformation procedure which will be addressed in FY 2006. Milestone Substantially Met 5. Complete isolation of pH altered mutants of P. expansum and P. solitum. pH altered mutants of P. expansum have been isolated, but once again,
optimization of the P. solitum growing conditions is still underway prior to performing the transformation procedure which will be addressed in FY 2006. Milestone Substantially Met 6. Complete isolation and sequencing of wild type glutamate dehydrogenase and pacC genes. Due to the large amount of time involved in the complete isolation and sequencing of wild type glutamate dehydrogenase and pacC genes, we do not feel we can address this milestone and complete the other studies as well. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 Complete purification and biochemical characterization of PGs. Compare PG production and virulence by P. solitum containing control vector DNA and P. solitum containing the P. expansum gene. Complete isolation of pH altered mutants of P. expansum and C. acutatum. Identify
biocontrol agents which are successful antagonists to C. acutatum. FY 2007 Perform PG gene knockout and subsequent transformations. Complete characterization of pH altered mutants for pectic enzyme expression and virulence. Confirm causality of mutant phenotypes by restoration to a wild type phenotype via complementation. FY 2008 Complete mutant screens and mutant characterizations for PG expression and virulence. Complement mutants with the wild type PG gene to prove causality. Complete characterization of glutamate dehydrogenase and pacC mutants for pectic enzyme expression, virulence, and pH phenotype. Complete studies on the integrated strategy to control B. cinerea, C. acutatum, and P. expansum. Optimize integrated strategy to control B. cinerea, C. acutatum, and P. expansum. 4a What was the single most significant accomplishment this past year? It is necessary to find alternatives to chemical control to reduce losses due to postharvest decay. In cooperation with an ARS
scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists at Beltsville, Maryland, combined controlled atmosphere storage, sodium bicarbonate, and two biocontrol agents, alone or combined, at Beltsville to reduce postharvest fungal decay of apples. The antagonists alone reduced blue mold decay but tended to be more effective when combined. The only treatment that completely eliminated decay caused by this pathogen was the combination of the two antagonists and sodium bicarbonate on fruit stored under controlled atmosphere conditions. This control strategy could potentially be used commercially to reduce our dependency on fungicides. 4b List other significant accomplishments, if any. Naturally occurring aromatic volatiles in blueberry fruit may be associated with postharvest disease resistance and contribute to reduced fruit losses caused by pathogen-induced postharvest decays. ARS scientists at Beltsville, Maryland, compared volatile
concentrations in headspace extracts of C. acutatum-inoculated and non-inoculated blueberry fruit stored 0 to 5 days from 10 cultivars having a wide range of resistance to C. acutatum. The results indicated that none of the aromatic volatiles in blueberry fruit were inducible by C. acutatum infection and the volatiles did not appear to contribute to disease resistance against Anthracnose Ripe Rot. Aromatic volatiles cannot be used by themselves as an alternative to fungicides to reduce postharvest decay. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Bitter rot and blue mold decays were effectively controlled when combining one of several antagonists and heat treatment. Combining several antagonists, sodium bicarbonate and heat treatment was also effective. These strategies could be useful to growers who wish to reduce their reliance on chemical control. The bitter rot fungus produces two types of a certain protein
that helps it attack and grow on apple fruit. This information could help explain the virulence mechanism in this fungus and aid in developing control methods useful to other scientists and eventually fruit growers. It is necessary to investigate as many alternatives to chemical control as possible to reduce decay due to postharvest pathogens. In cooperation with scientists at the University of Tennessee, ARS scientists at Beltsville, Maryland, tested chitosan extracted from various fungi, including mushrooms, as well as commercial sources to determine the effectiveness of chitosan in reducing decay of apple caused by postharvest pathogens. In studying another alternative to chemical control, chitosan extracted from fungi was as effective as the commercial sources in reducing postharvest decay. Chitosan extracted from portions of mushrooms not sold commercially for consumption may give mushroom growers an additional source of revenue. 6. What science and/or technologies have been
transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The product to be delivered at the termination of this project will be a strategy combining several alternatives to chemical control which will equal the efficacy of chemical control. This strategy will be of importance to the apple growing industry, especially if postharvest fungicides are no longer effective or their use is not longer permitted.
Impacts
(N/A)
Publications
- Conway, W.S., Leverentz, B., Janisiewicz, W.J., Blodgett, A.B., Saftner, R. A., Camp, M.J. 2004. Integrating heat treatment, biocontrol and sodium bicarbonate to reduce postharvest decay of apple caused by colletotrichum acutatum and penicillium expansum. Postharvest Biology and Technology. 34:11-20.
- Conway, W.S., Leverentz, B., Janisiewicz, W.J., Saftner, R.A., Camp, M.J. 2005. Improving biocontrol using antagonist mixtures with heat and/or sodium bicarbonate to control postharvest decay of apple fruit. Postharvest Biology and Technology. 36:235-244
- Conway, W.S., Janisiewicz, W.J., Leverentz, B., Saftner, R.A., Camp, M.J. 2005. Control of blue mold of apple by combining controlled atmosphere, antagonist mixtures, and sodium bicarbonate [abstract]. Phytopathology. 34:11-20.
- Leverentz, B., Conway, W.S., Janisiewicz, W.J., Kurtzman, C.P., Abadias, M. L., Camp, M.J. 2005. Biocontrol of foodborne pathogens on fresh-cut apples using naturally occurring bacterial and yeast antagonists [abstract]. Phytopathology 95. (Sppl.):59.
- Polashock, J.J., Saftner, R.A. 2005. Blueberry volatile analysis as a potential marker for disease resistance. Proceedings of the 2005 New Jersey Annual Vegetable Meeting. p. 121-122.
- Wu, T., Zivanovic, S., Draughon, A., Conway, W.S., Sams, C.E. 2005. Physiochemical properties and bioactivity of fungal chitin and chitosan. Journal of Agricultural and Food Chemistry. 53:3888-3894.
|
Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Economic losses caused by postharvest pathogens are greater than is often realized, and the avoidable losses between the farm gate and the consumer are cause for concern. Fresh fruits and vegetables, because of the added cost of harvesting and handling, increase several times in value as they are moved from the field to the consumer. Annual postharvest losses range from 10 to 50% depending upon the sophistication of available storage facilities. There is increasing concern among consumers and regulatory agencies concerning the health hazards of synthetic chemical residues. For some pathogens, no practical chemical control has been developed and many pathogens have developed resistance to commonly used chemicals, of which only a very few are still registered for postharvest treatment. To decrease
our dependency on chemical control to ensure quality of harvested produce, alternative, more natural methods of postharvest decay control are being developed. It is highly unlikely that any one of these alternative methods alone will be as effective as fungicides; however, the development of a strategy combining several of these methods may be successful. We are currently investigating such promising alternatives as biocontrol, natural antimicrobial volatiles and heat treatment in various combinations to develop a successful postharvest decay control strategy. If chemical control is no longer an option due to pathogen resistance or legislative restriction due to health and environmental concerns, a successful strategy combining various alternatives will be available to the grower to protect fruit in storage. This research falls within National Program 303, Plant Diseases (60 percent) and National Program 306, Quality and Utilization of Agricultural Products (40 percent). It mainly
addresses Plant Diseases (NP 303) and falls within component V (Host Resistance) of the Action Plan and addresses Research Need #3 under Pome Fruits (Development of Safe and Effective Alternatives to Synthetic Fungicides for Control of Blue Mold). It also addresses Research Need #4 under Pome Fruits (Elucidation and Manipulation of Internal Mechanisms of Resistance to Reduce Postharvest Decay). We are seeking biocontrol agents to block pathogens from causing disease and to manipulate the conditions under which these agents are used in order to optimize their effectiveness. In addition, our research is aimed at the identification, characterization, and manipulation of genes for resistance in crop species, which will allow us to seek natural mechanisms of resistance to postharvest decay control. As promising methods of resistance are identified, they will be used in various combinations to develop an integrated pest management strategy to reduce our dependency on synthetic chemical
control. This research also aims to prevent spoilage by managing or eliminating postharvest pathogens. Specifically, this approach includes the development of new treatments to eliminate pathogens and biological control of pathogens and will allow scientists to seek natural mechanisms of resistance to control postharvest decay. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Complete optimization of P. solitum transformation procedure. Complete comparisons of wild type virulence of P. solitum and P. expansum. Complete optimization of UV mutagenesis and mutant screening procedures. Establish quantitative relationship between inoculum level and development of C. acutatum. Year 2 (FY 2005) Complete crude purification of PGs. Complete hygromycin-tagged plasmid construction and transformation of P. solitum. Complete isolation of pH altered mutants of P. expansum and P. solitum. Complete isolation and sequencing of wild type glutamate dehydrogenase
and pacC genes. Determine effect of MCP on various apple decays. Determine the effect of heat on the survival and growth of C. acutatum. Year 3 (FY 2006) Complete purification and biochemical characterization of PGs Complete comparisons of PG production and virulence by P. solitum containing control vector DNA and P. solitum containing the P. expansum PG gene. Complete characterization of pH altered mutants for pectic enzyme expression and virulence. Complete construction of in vitro mutation of glutamate dehydrogenase and pacC genes. Identify biocontrol agents which are successful antagonists to C. acutatum. Year 4 (FY 2007) Complete in vitro construction of the PG gene knockout and subsequent transformations. Conplete construction and isolation of a glutamate dehydrogenase and pacC mutants. Confirm causality of mutant phenotypes by restoration to a wild type phenotype via complementation. Optimize integrated strategy to control B. cinerea, C. acutatum, and P. expansum Year 5 (FY
2008) Complete mutant screens and mutant characterizations for PG expression and virulence. Complement mutants with the wild type PG gene to prove causality. Complete charactization of glutamate dehydrogenase and pacC mutants for pectic enzyme expression, virulence, and pH phenotype. Complete studies on the integrated strategy to control B. cinerea, C. Acutatum, and P. expansum. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones listed below were scheduled to be completed under year 1. The following milestones were completed. Complete comparisons of wild type virulence of P. solitum and P. expansum. Establish the quantitative relationship between inoculum level and development of C. acutatum. The following milestone was initiated and will be completed in FY
2005: Complete optimization of UV mutagenesis and mutant screening procedures. The following milestone was not done due to time constraints but will be initiated in FY 2005. Complete optimization of P. solitum transformation procedure. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006 and 2007). What do you expect to accomplish, year by year, over the next three years under each milestone? FY 2005 Complete crude purification of PGs. Optimize P. solitum transformation procedure Complete optimization of UV mutagenesis and mutant screening procedures. Determine effect of MCP on various apple decays. Determine the effect of heat on the survival of C. acutatum. FY 2006 Complete purification and biochemical characterization of PGs. Compare PG production and virulence by P. solitum containing control vector DNA and P. solitum containing the P. expansum gene. Complete isolation of pH altered mutants of P. expansum and C. acutatum. Identify biocontrol
agents which are successful antagonists to C. acutatum. FY 2007 Perform PG gene knockout and subsequent transformations. Complete characterization of pH altered mutants for pectic enzyme expression and virulence. Confirm causality of mutant phenotypes by restoration to a wild type phenotype via complementation. Optimize integrated strategy to control B. cinerea, C. acutatum, and P. expansum. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004 (one per Research Project): It is necessary to find alternatives to chemical control to reduce losses due to postharvest decay. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists at Beltsville, Maryland, combined heat treatment, sodium bicarbonate, and two biocontrol agents, alone or combined at Beltsville to reduce postharvest fungal decay of apples. Both bitter rot and blue mold decays were
effectively controlled by a combination of either of the antagonists and heat treatment. In nonheated fruit, the combination of the two biocontrol agents plus sodium bicarbonate resulted in the smallest lesions on fruit decayed by the blue mold fungus. This control strategy could potentially be used commercially to reduce our dependency on fungicides. B. Other Significant Accomplishments: None C. Significant Accomplishments/Activities that Support Special Target Populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ('D') projects and the projects listed in Apendix A; mandatory for all other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Bitter rot and blue mold decays were more effectively controlled when combining one of several antagonists and heat treatment than any of the treatments alone. This
strategy will be useful for apple growers who may need to reduce their dependence on synthetic fungicides. Action Plan Component V; Pome Fruits Research Need #3. The bitter rot fungus produces two types of a certain protein that helps it attack and grow on apple fruit. This information will help elucidate the mechanism of virulence involved in postharvest decay caused by this pathogen. Action Plan Component V; Pome Fruits Research Need #4. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. None Scientific Publications:
Impacts
(N/A)
Publications
- Conway, W.S., Leverentz, B., Janisiewicz, W.J., Saftner, R.A., Camp, M.J. 2004. Improving biocontrol using antagonist mixtures with heat and/or sodium bicarbonate to control postharvest decay of apple fruit. Phytopathology. 94(Suppl.):Abstract. P. S20.
- Sams, C.E., Zivanovic, S., Charron, C.S., Wu, T., Blodgett, A.B., Conway, W.S. 2004. Fungal chitosan extracts are as effective in reducing decay caused by botrytis cinerea, penicillium expansum, and penicillium solitum as commercial seashell chitosan extracts. Phytopathology. 95 (Suppl.) :Abstract. P. S91.
- Janisiewicz, W.J., Bassett, C.L., Wisniewski, M.E., Conway, W.S., Roberts, D.P. Use of a luxgfp bioreporter system to determine metabolic activity of an antagonist in fruit wounds. American Phytopathological Society Abstracts.Abstract to be presented at Annual Meeting of the American Phytopathological Society, July 31 - August 4, 2004 in Anaheim, CA.
- LIAO, C., MCEVOY, J.L., SMITH, J.L. 2003. CONTROL OF BACTERIAL SOFT ROT AND FOODBORNE HUMAN PATHOGENS ON FRESH FRUITS AND VEGETABLES. IN: Research Signpost, Kerala, India. Advances in Plant Disease Management. P. 165-193.
- Prusky, D., Mcevoy, J.L., Saftner, R.A., Conway, W.S., Jones, R.W. 2003. Relationship between host acidification and virulence of penicillium spp. on apple and citrus fruit. Biochemistry and Cell Biology. Phytopathology Vol. 94, No. 1, 2004, page 44-51
|
Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Economic losses caused by postharvest pathogens are greater than is often realized, and the avoidable losses between the farm gate and the consumer are cause for concern. Fresh fruits and vegetables, because of the added cost of harvesting and handling, increase several times in value as they are moved from the field to the consumer. To decrease our dependency on chemical control to ensure quality of harvested produce, alternative methods of postharvest decay control are being developed. It is highly unlikely that any one of these alternative methods alone will be as effective as fungicides. However, the development of a strategy combining several of these methods may be successful. We are currently investigating promising alternatives, such as biocontrol; natural antimicrobial volatiles; and heat treatment alone and in various combinations; to develop a successful postharvest decay
control strategy. 2. How serious is the problem? Why does it matter? Annual postharvest losses range from 10 to 50% depending upon the sophistication of available storage facilities. There is also increasing concern among consumers and regulatory agencies concerning the health hazards of synthetic chemical residues. For some pathogens, no practical chemical control has been developed and many pathogens have developed resistance to commonly used chemicals, of which only a very few are still registered for postharvest treatment. We must therefore investigate more natural methods of resistance to pathogens to reduce our dependency on chemical treatments and continue to reduce postharvest losses. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 303, Plant Diseases (60 percent)and National Program 306, Quality and Utilization of Agricultural Products (40 percent) We are seeking biocontrol agents to block
pathogens from causing disease and to manipulate the conditions under which these agents are used in order to optimize their effectiveness. In addition, our research is aimed at the identification, characterization, and manipulation of genes for resistance in crop species. This will allow us to seek natural mechanisms of resistance to postharvest decay control. As promising methods of resistance are identified, they will be used in various combinations to develop an integrated pest management strategy to reduce our dependency on synthetic chemical control. This research also aims to prevent spoilage by managing or eliminating postharvest pathogens. Specifically, this approach includes the development of new treatments to eliminate pathogens and biological control of pathogens and will allow scientists to seek natural mechanisms of resistance to control postharvest decay. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment
during FY 2003 Year: It is necessary to find alternatives to chemical control to reduce losses due to postharvest decay. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists in the Produce Quality and Safety Laboratory, Beltsville MD, combined either one of two heat tolerant yeast biocontrol agents, heat treatment and sodium bicarbonate to reduce postharvest fungal decay of apples. Both bitter rot and blue mold decays were effectively controlled by a combination of either of the antagonists and heat treatment. This control strategy could potentially be used commercially to replace some commonly used fungicides. B. Other Significant Accomplishments: It is necessary to develop a better understanding of the mechanisms by which postharvest fungal pathogens decay fruit in order to develop better methods of control and reduce our dependency on fungicides. ARS scientists in the Produce Quality and Safety Laboratory,
Beltsville MD, studied various types of proteins produced by Colletotrichum acutatum, an important postharvest apple pathogen. We showed that the fungus produced two major types of a certain protein (polygalacturonase) that helps it attack and grow on apple fruit by breaking down the apple tissue, and that increasing the calcium content of apple fruit reduces decay and postharvest losses. Apple packing and storage facilites may use calcium treatment to reduce the amount of fungicides used to maintain fruit quality during cold storage. C. Significant Accomplishments/Activities that Support Special Target Populations. None. D. Progress Report 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. We have identified a number of alternatives to synthetic chemicals for the control of postharvest diseases. A number of yeasts and bacteria have been identified which are wound-site competitors of several important postharvest
pathogens. Natural, internal mechanisms of resistance involving polygalacturonase-inhibiting protein from apple fruit, methyl jasmonate, and MCP have also been shown to have antimicrobial activity. The shelf-life of fresh-cut honeydew melon slices can be extended by hot water treatment of the intact fruit. A dip in chlorine water supplemented with calcium propionate maintained the quality and more than doubled the shelf life of fresh-cut melon. The integrated strategy of heat-treating fruit, followed by calcium infiltration and then treatment with an antagonist or the use of heat treatment and an antagonist may be a useful alternative to controlling postharvest decay with fungicides. The foodborne human pathogen Listeria monocytogenes can grow on apple fruit if fruit are not properly refrigerated. Also, pre-inoculation of apples with plant pathogens can affect the growth of this human pathogen. The mechanisms by which pathogenic fungi alter the host microenvironment to enhance
decay are being studied. Decay by Colletotrichum species of avocado, apple, and tomato resulted in ammonia accumulation in the infected area where the pH increased to 7.4, which is optimal for the activity of cell wall degrading enzymes produced by these fungi as an attack mechanism. Penicillium species were found to use two mechanisms of acidification to lower the host pH to that which is optimal for the activity of the cell wall degrading enzymes produced by these fungi. Colletotrichum acutatum produced two distinct proteins which are thought to be involved in pathogenesis. 6. What do you expect to accomplish, year by year, over the next 3 years? During FY 2004, we will integrate methods of enhancing resistance and reducing decay to develop more effective decay control strategies, ie., better than is offered by any of these methods alone. Also, we will complete a comparison of the virulence of Penicillium solinum with that of Penicillium expansum. During FY 2005, the most
promising strategy observed from previous research in 2004 will be tested, and we will determine it's efficacy under simulated commercial conditions. In addition, we will complete the crude purification of Penicillium solitum polygalacturonase. During FY 2006, we will use this strategy with further refinements to determine it's effectiveness during a commercial pilot test. Also, the purification and characterization of P. solitum polygalacturonases will be done for future studies on its role in fungal decay and rotting of fruit after harvest. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Saftner, R., Conway, W. Optimizing 1-MCP effects on fresh market apples. Apple Postharvest Review of Washington Tree Fruit Research Commission. 2002. p. 273.
Impacts
(N/A)
Publications
- Saftner, R.A., Abbott, J.A., Conway, W.S., Barden, C.L. 2002. Effects of 1- methylcyclopropene and heat treatments on ripening and postharvest decay development in 'golden delicious' apples. Journal of the American Society for Horticultural Science 128(1):120-127, 2003
- Saftner, R.A., Conway, W.S., Abbott, J.A., Leverentz, B., Janisiewicz, W. Physical and chemical strategies to reduce postharvest decay of apples and other fresh fruit while maintaining quality: reducing dependence on fungicides. Proceedings XXVIth International Horticultural Congress. 2002. Abstract p. 273.
- Leverentz, B., Janisiewicz, W.J., Conway, W.S., Blodgett, A.B., Saftner, R. A. Control of postharvest decay of apple by combining heat treatment, biocontrol and sodium bicarbonate. Phytopathology. 2003. v. 93(Suppl.) :Abstract p. S50.
- Park, E., McEvoy, J.L., Conway, W.S. Cloning and molecular characterization of Colletotrichum acutatum endopolygalacturonases. Phytopathology. 2003. v. 93(Suppl.):Abstract p. S69.
- Chardonnet, C.O., Charron, C.S., Sams, C.E., Conway, W.S. Chemical changes in the cortical tissue and cell walls of calcium-infiltrated 'Golden Delicious' apples during storage. Postharvest Biology and Technology. 2003. v. 28. p. 97-111.
- Janisiewicz, W.J., Leverentz, B., Conway, W.S., Saftner, R.A., Reed, A.N., Camp, M.J. Control of bitter rot and blue mold of apples by integrating heat and antagonist treatments on 1-MCP treated fruit stored under controlled atmosphere conditions. Postharvest Biology and Technology. 2003. v. 29. p. 129-143.
- Leverentz, B., Conway, W.S., Janisiewicz, W.J., Saftner, R.A., Camp, M.J. Effect of combining MCP treatment, heat treatment, and biocontrol on the reduction of postharvest decay of 'Golden Delicious' apples. Postharvest Biology and Technology. 2003. v. 27. p. 221-233.
- Sams, C.E., Conway, W.S. Preharvest nutritional factors affecting postharvest physiology. Bartz, J.A., Brecht, J.K., editors. Marcel Dekker, New York, NY. Postharvest Physiology and Pathology of Vegetables. 2003. p. 161-176.
|
|