Progress 05/17/07 to 05/16/12
Outputs
Progress Report Objectives (from AD-416): Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development. Approach (from AD-416): To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species. Maintaining pome fruit quality by eliminating decay in long-term storage is critical to prevent postharvest losses. Penicillium expansum and P. solitum are the most destructive pome fruit pathogens causing blue mold decay and they also produce carcinogens which contaminate processed fruit products. Investigations in the Food Quality Laboratory in Beltsville, Maryland have shown that Penicillium solitum modifies its host during apple fruit decay by lowering pH, production of organic acids and secretion of polygalacturonase (PG). This information will be used to design new disease control strategies that alter fruit surface pH or inhibit the production of PG. Research efforts have uncovered resistance in wild apple fruit to Penicillium species from the germplasm collection located in Geneva, New York. Nineteen of 452 accessions of wild apple fruit were resistant to P. expansum. Of these nineteen, four accessions performed consistently from year to year with respect to resistance. This information is beneficial to plant breeders and molecular geneticists interested in characterizing or introgressing resistance traits in to commercial apple cultivars. Fundamental studies involving P. expansum have shown that the fungus can grow and produce PG on a wide variety of carbon and nitrogen sources and that a single PG enzyme is produced when grown on apple pectin. These studies can provide information to design new postharvest decay control strategies. The role of P. solitum polygalacturonases (PGs) in predisposing wounds to infection by P. expansum have been conducted and a method has been developed to measure tissue maceration caused by PGs to determine their impact on fruit decay. Using this new method, purified PGs from P. solitum have been found to macerate intact apple and pear fruit tissue. This information will be used by other scientists in developing specific control measures targeting PGs produced by both P. expansum and P. solitum. Two new Penicillium species have been identified for the first time from decayed apple fruit obtained from a packinghouse in Pennsylvania and their detailed characterization has been carried out. Information concerning these two emerging pathogens will be used by other scientists to develop rapid detection techniques to ensure timely application of disease control methods to preserve apple fruit quality during storage. Progress contained in this report directly addresses the objectives contained in the terminating project plan that focused on identifying and characterizing virulence mechanisms to develop new control measures for postharvest decay of apple and pear fruit. Accomplishments 01 A polygalacturonase produced by the blue mold fungus is important for tissue maceration during postharvest decay of pear fruit. Polygalacturonases (PGs) are cell wall degrading enzymes that are involv in fungal decay. We have extracted and purified a PG from P. solitum- decayed pear fruit tissue and performed a full characterization. PG enzy activity at various pHs, temperature, and enzyme cleavage pattern have been completed. Ultimately, this knowledge may be applied either through manipulation of the host (pear) or by identifying PG inhibitors that may be applied to the fruit surface to block decay. 02 A new method of screening and testing microorganisms to control brown ro on plum fruit. Brown rot is the most destructive disease of plums that causes decay both in the field and during storage. In cooperation with a ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, we have developed a novel method for screening and testin the efficacy of microorganisms obtained from the surface of plum fruit t inhibit brown rot caused by Monolinia fructicola. Microbes have been isolated, identified, and were successful at stopping brown rot decay. This technique may be used to discover and test the efficacy of addition microorganisms which can be implemented as biocontrol agents in place of synthetic fungicides for control. 03 Polygalacturonase is regulated by many nutritional and environmental factors in the blue mold fungus Penicillium expansum. P. expansum is amo the most destructive apple and pear fruit pathogens causing blue mold an also produces toxins which contaminate processed fruit products like jui and butters. It was demonstrated that pH, carbon and nitrogen sources regulate the production and activity of a polygalacturonase. Understandi the factors that regulate polygalacturonase production in this fungus ha revealed new regulatory mechanisms involved in the production of this important virulence factor. This information can be used to target polygalacturonase in this fungus which could be used to control decay. 04 A new rapid, accurate and non-toxic method to measure cellulase enzyme activity. Cellulase is an important cell wall degrading enzyme that is produced by microorganisms to break down cellulose. Cellulases have been studied for their role in fungal decay and have also been used to produc ethanol from plant debris. However, the standard method of detecting cellulase activity involves the use of carcinogens that require special handling and disposal. Therefore, a non-toxic method to detect cellulase enzyme activity was developed and shown to be more accurate than the standard method cited in the scientific literature. The new assay can be used by scientists to accurately assay cellulase enzyme activity without the use of harmful chemicals.
Impacts
(N/A)
Publications
- Jurick Ii, W.M., Vico, I., Gaskins, V.L., Whitaker, B.D., Garrett, W.M., Janisiewicz, W.J., Conway, W.S. 2012. Penicillium solitum produces a polygalacturonase isozyme in decayed �Anjou� pear fruit capable of macerating host tissue in vitro. Mycologia. DOI:10.3852/11.119.
- Janisiewicz, W.J., Pimenta, R.S., Jurick Ii, W.M. 2011. A novel method for selecting antagonists against postharvest fruit decays originating from latent infections. Biological Control. 59:348-389.
- Jurick Ii, W.M., Vico, I., Gaskins, V.L., Peter, K.A., Park, E., Janisiewicz, W.J. 2012. Carbon, nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum. Archives of Phytopathology and Plant Protection. DOI:10. 1080/03235408.2012.657893.
- Jurick Ii, W.M., Vico, I., Whitaker, B.D., Gaskins, V.L., Janisiewicz, W.J. 2012. Application of the 2-cyanoacetamide method for spectrophotometric assay of cellulase enzyme activity. Plant Pathology Journal. DOI:10. 3923/ppj.2012.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development. Approach (from AD-416) To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species. Maintaining the quality of apple and pear fruit by eliminating decay in long-term storage is critical to prevent postharvest losses. ARS scientists at Beltsville, MD, have determined that Penicillium solitum is capable of decaying apple fruit by making them more acidic and producing a protein (polygalacturonase) that is responsible for softening the fruit tissue. This information may be used by plant pathologists to design new disease control strategies that alter the fruit surface and or inhibit fruit decay. Resistance to blue mold has been evaluated in a collection of wild apple fruit trees located in Geneva, New York. We have identified 19 of 452 different wild apple fruit that are resistant to the fungus that causes blue mold (Penicillium expansum). Of these nineteen, four were resistant from year to year which is a valuable characteristic as these same fruit can then be used for further study. This information will be beneficial to plant breeders and molecular geneticists interested in characterizing and/or incorporating resistance into commercial apple cultivars. Understanding the basic mechanisms that the blue mold fungus (P. expansum) uses to cause apple and pear fruit decay are important for uncovering new ways to halt postharvest decay. We have determined that P. expansum can grow and produce an enzyme responsible for tissue maceration (polygalacturonase) on a wide variety of nutrient sources and that a single enzyme is produced in the presence of apple pectin. This fundamental information on enzyme production may aid other scientists in designing new postharvest decay control strategies. ARS scientists at Beltsville, MD, have kept in close communication with the apple and pear fruit growers and packers. We have sampled decayed fruit from storage bins in their state-of-the-art storage facility. These fungi will be used to characterize their sensitivity to commonly used postharvest fungicides. It is important to conduct fungicide sensitivity tests to determine if they are developing resistance to the chemicals used to control decay in storage. This information will directly benefit other scientists and the packing industry as it may lead to fungicide management implications. Accomplishments 01 Low temperature storage of apple fruit delays blue mold decay. We demonstrated that storing fruit at 0�C after harvest delays decay by affecting fungal growth compared to other temperatures (5, 10, and 20�C) used for storing intact and fresh cut fruit. It was also determined that PG enzyme activity was affected by temperature which explains why tissue maceration of inoculated apple fruit was deterred. The impact of this accomplishment is that this information may then be used by other scientists to target specific biological processes in this fungus which could be used to control decay. 02 Purification of Polygalacturonase (PG) from P. expansum and P. solitum- decayed apple and pear fruit. We extracted and purified PGs from P. expansum and P. solitum-decayed pear and apple fruit tissue. We found th both fungi produced a single enzyme in decayed tissue that differ in the abilities to function at various pHs and also at various incubation temperatures. We also demonstrated that the purified PGs are capable of macerating host tissue. The impact of this accomplishment is that this knowledge may be applied either through genetic manipulation of the host (apple) or by identifying PG inhibitors that may be applied to the fruit surface to block decay.
Impacts
(N/A)
Publications
- Vico, I., Jurick Ii, W.M., Camp, M.J., Janisiewicz, W.J., Conway, W.S. 2010. Temperature suppresses decay on apple fruit by affecting Penicillium solitum conidial germination, mycelial growth and polygalacturonase activity. Plant Pathology Journal. 9(3):144-148.
- Jurick Ii, W.M., Janisiewicz, W.J., Saftner, R.A., Vico, I., Gaskins, V.L., Park, E., Forsline, P.L., Fazio, G., Conway, W.S. 2011. Identification of wild apple germplasm (Malus spp.) with resistance to the postharvest decay pathogens Penicillium expansum and Colletotrichum acutatum. Plant Breeding. 130:481-486.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development. Approach (from AD-416) To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species. Examining fungal virulence factors to develop control strategies for blue mold on pome fruit during postharvest storage. Maintaining fruit quality by preventing decay is critical to prevent postharvest losses. P. expansum and P. solitum are among the most destructive pome fruit pathogens causing blue mold. Polygalacturonases (PGs) are cell wall degrading enzymes that are involved in fungal virulence and facilitate decay. Isolation, purification and biochemical characterization of PGs from P. expansum and P. solitum-decayed pear and apple fruit have been completed. Biochemical characterization of these enzymes and determining their role in fruit decay may provide a foundation for development of novel disease control methods. Synthesis of a recombinant phage display library against P. expansum produced PG and isolation of cDNA clones encoding antibodies against PGs have been achieved. This work will ultimately lead to inhibition studies in the lab and eventually will be tested on apple and pear fruit surfaces. The use of recombinant antibodies to block PG activity may be an effective non- chemical method of preventing postharvest decay caused by Penicillium species on pome fruits. Accomplishments 01 Identification of wild apple germplasm accessions from Kazakhstan for resistance to Penicillium expansum and Colletotrichum acutatum. Maintaining fruit quality by inhibiting decay is crucial to maintain quality and prevent postharvest losses. P. expansum and C. acutatum are the most destructive causal agents of apple fruit decay. Sources of resistance to these fungi are lacking in commercial apple cultivars. However, we have identified several wild apple accessions that are resistant to one or both pathogens. These wild apple fruit will be used identify the underlying genetic and biochemical resistance mechanisms to fungal decay pathogens. This information will be beneficial to plant breeders and geneticists in transferring resistance to commercial apple cultivars. Ultimately, this knowledge may be applied through genetic manipulation in the host to prevent decay during storage. 02 Isolation and identification of cDNA clones encoding recombinant antibodies against P. expansum PGs. It is necessary to develop alternatives to chemical control for reducing postharvest losses. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists in Beltsville, Maryland have made significant progress toward construction of a phage library against polygalacturonase from P. expansum, the causal agent of blue mold of apples. We have successfully synthesized the recombinant phage display library, completed 2 rounds of panning, identified several binders, and obtained corresponding cDNA clones. Blocking polygalacturonase activity on the fruit surface using recombinant antibodies against polygalacturonase may lead to a novel control strateg to halt postharvest decay mediated by P. expansum on apple fruit. This control strategy could potentially benefit the postharvest industry by reducing dependency on synthetic fungicides.
Impacts
(N/A)
Publications
- Jurick II, W.M., Vico, I., Gaskins, V.L., Garrett, W.M., Whitaker, B.D., Janisiewicz, W.J., Conway, W.S. 2010. Purification and biochemical characterization of polygalacturonase produced by Penicillium expansum during postharvest decay of �Anjou� pear. Phytopathology. 100(1):42-48.
- Janisiewicz, W.J., Conway, W.S. 2010. Combining biological control with physical and chemical treatments to control fruit decays after harvest. Stewart Postharvest Review. 6(1):1-16.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development. Approach (from AD-416) To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species. Significant Activities that Support Special Target Populations Targeting fungal virulence factors and development of novel control strategies for control of blue mold on pome fruit. Maintaining fruit quality by inhibiting decay is crucial to prevent postharvest losses. P. expansum and P. solitum are the most destructive pome fruit pathogens causing blue mold. Polygalacturonases (PGs) are cell wall degrading enzymes that are involved in fungal virulence and aid in pathogen ingress and decay. Isolation and purification of PGs from P. expansum and P. solitum-decayed pear and apple fruit has been completed. Biochemical characterization of these enzymes and determination of their role in fruit decay may provide a foundation for development of novel disease control methods. Synthesis of a recombinant phage display library against P. expansum produced PG has been achieved and will lead to inhibition studies in the lab and eventually on apple and pear fruit surfaces. The use of recombinant antibodies to block PG activity may provide an effective and non-chemical method of preventing postharvest decay caused by Penicillium species.
Impacts
(N/A)
Publications
- Janisiewicz, W.J., Saftner, R.A., Conway, W.S., Yoder, K.S. 2008. Control of blue mold decay of apple during commercial controlled atmosphere storage with yeast antagonists and sodium bicarbonate. Postharvest Biology and Technology. 49:374-378.
- Jurick II, W.M., Vico, I., Mcevoy, J.L., Whitaker, B.D., Janisiewicz, W.J., Conway, W.S. 2009. Isolation, purification, and characterization of a polygalacturonase produced in Penicillium solitum-decayed 'Golden Delicious' apple fruit. Journal of Phytopathology. 99:636-641.
- Trentham, R.W., Sams, C.E., Conway, W.S. 2008. Histological effects of calcium chloride in stored apples. Journal of the American Society for Horticultural Science. 133:487-491.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development. Approach (from AD-416) To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species. Significant Activities that Support Special Target Populations Production of polyclonal antisera and a recombinant phage library against polygalacturonase produced by P. expansum. It is necessary and ecologically responsible to develop alternatives to chemical control for reducing postharvest losses. In cooperation with and ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, and ARS scientists in Beltsville, MD, have made progress toward the production of polyclonal antisera and a phage library against polygalacturonase from P. expansum, the causal agent of blue mold of apples. Polygalacturonase from P. expansum-infected apple fruit was purified using gel filtration chromatography and injected in to mice. Spleens from immunized mice were harvested and polyclonal antisera were produced. Current progress provides a foundation for the construction of a recombinant phage library specific to polygalacturonase produced by P. expansum which will be used to produce recombinant antibodies. Blocking polygalacturonase activity on the fruit surface using recombinant antibodies against polygalacturonase may lead to a novel control strategy to halt postharvest decay mediated by P. expansum on apple fruit. This control strategy could potentially benefit the postharvest industry by reducing dependency on chemical fungicides. National Program 303, component IIa.
Impacts
(N/A)
Publications
- Janisiewicz, W.J., Saftner, R.A., Conway, W.S., Forsline, P.L. 2008. Preliminary evaluation of apple germplasm from Kazakhstan for resistance to blue mold decay caused by Penicillium expansum after harvest. HortScience.43(2):420-426.
- Polashock, J.J., Saftner, R.A. 2007. Blueberry fruit volatiles as a potential marker for resistance to anthracnose fruit rot. Journal of the American Society for Horticultural Science. 132:859-868.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development. Approach (from AD-416) To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species. Accomplishments None, this project is too early to have any accomplishments.
Impacts
(N/A)
Publications
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