Progress 12/15/98 to 12/14/03
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? Postharvest losses due to decay of fruits during storage, shipping, marketing, and home consumption are significant and represent a financial burden to both growers and consumers. Furthermore, public concern over chemical residues, environmental concerns, and pathogen populations that are resistant to chemical fungicides, have created a critical need for safe and effective alternatives. Biological approaches that utilize microbial antagonists and natural compounds are being developed to address these issues. A basic understanding of the mode of action of biocontrol agents will lead to the selection of more effective antagonists. Fire blight is one of the most serious and destructive diseases of apple and pear. To combat this disease there is a need to understand the basis of host resistance, and the
epidemiology of the disease, and to develop non-chemical orchard and nursery management practices that will dramatically reduce the incidence of fire blight. Freeze damage, other types of environmental stress, and micronutrient deficiency in fruit trees often result in a significant loss in yield, fruit quality, or tree longevity. To address this problem, there is a need to develop a basic understanding of the biochemistry, physiology, and molecular biology of stress tolerance and micronutrient metabolism in fruit trees. Several key synthetic fungicides have been targeted for withdrawal from the market because of their potential impact on human health and/or the costs involved in registering these products and demonstrating their safety. Some examples are ipridione, used on stone fruit, and benomyl compounds used on pome fruit. Additionally, the development of fungicide resistance has made disease management more difficult. In the near future, some commodities may have no effective
synthetic fungicides registered for postharvest application. Fire blight infections in newly planted orchards (1 to 5 years) can result in up to 80% tree loss causing devastating financial loss for growers. A single fire blight epidemic that occurred in southwest Michigan in 2000 was estimated to have caused the death of 350,000 to 450,000 trees and the removal of 1,500 to 2,300 acres of apple orchards with a total economic loss of 42 million dollars. The phytotoxicity of copper and its limited efficacy, and resistance to streptomycin, (copper and streptomycin are presently the two main methods of controlling fire blight), make the need to find new ways to manage fire blight critically important. Freezing temperatures are the prime factor affecting the production of perennial fruit and nut crops nationally. Estimates of annual losses in the U.S. range from millions of dollars for the citrus industry to nearly one billion dollars for all crops. A basic understanding of cold
hardiness and stress tolerance can lead to the development of varieties that are resistant to extreme weather conditions and lead to new management tools that alleviate the impact of environmental stress on the performance of plants. Zinc deficiency in fruit crops is widespread throughout the United States. It is associated with poor tree growth, reduced yields, and inferior fruit quality. The inefficiency and subsequent repeated use of zinc foliar sprays have led to concern over heavy metal contamination of orchard soils. More effective methods of managing zinc nutrition are needed to assure healthy trees, good fruit quality, and reduce the risk of soil contamination. The goal of this research is to develop information on the identity and mode of action of biocontrol agents in order to make this strategy more effective and contributes to National Program 303: Plant Diseases (70%). This project also addresses the need to improve stress tolerance and micronutrient absorption in fruit
crops and contributes to National Program 302: Improving Plant Biological and Molecular Processes (30%). It is recognized that this is an important need given the impact of environmental stress and micronutrient deficiency on fruit production. 2. List the milestones (indicators of progress) from your Project Plan. Previous CRIS Project Plans and Annual Reports did not necessitate a list of specific milestones. Milestones achieved during the life of this project are described in previous annual reports and in a later section of this annual report. This project was terminated in December, 2003 and therefore only operational for two months since the last annual report (FY 03). The research is being continued in two new CRIS projects: 1931- 21220-013-00D, Management of Pre- and Postharvest Diseases of Fruit Crops; and 1931-21220-014-00D, Management of Abiotic Stress in Fruit Crops, that have been approved through OSQR. Further information on this research can be obtained by referencing
the annual reports for these new CRIS projects. Project Plan Objectives: 1) to develop new, environmentally-friendly methods for managing pre- and postharvest diseases of fruit trees, and 2) to increase environmental stress tolerance in fruit trees through genetic enhancement and cultural management. An integrated approach will be taken utilizing the combined expertise of plant pathologists, physiologists, and molecular biologists. Research on Objective 1 will focus on: 1) development of new biologically-based products fro controlling postharvest disease; 2) identification of genes involved in biocontrol efficacy and genetic enhancement of microbial antagonists; 3) development of biologically-based technologies for controlling major foliar diseases, e.g. apple scab, of fruit trees; 4) development and evaluation of biological approaches for managing fire blight; 5) use of natural volatile compounds as alternatives to methyl bromide fumigants; and 6) improvement of disease resistance
in fruit trees through the identification and manipulation of disease-resistance genes in apple and peach, as well as candidate genes from other species; Research on Objective 2 will focus on: 1) improvement of stress tolerance in fruit trees through the identification and evaluation of stress tolerance genes; and 2) evaluation and development of novel methods of frost protection. 3. Milestones: This project was terminated in December 2003 and therefore was only operational for two months since the last annual report (FY 03). The research is being continued in two new CRIS projects: 1931-21220-013-00D, Management of Pre- and Postharvest Diseases of Fruit Crops; 1931-21220- 014-00D Management of Abiotic Stress in Fruit Crops, that have been approved through OSQR. Further information on this research can be obtained by referencing the annual reports for these new CRIS projects. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment:
This project was terminated in December, 2003 and was therefore only operational for two months since the last annual report (FY 03). The research is being continued in two new CRIS projects: 1931-21220-013-00D, Management of Pre- and Postharvest Diseases of Fruit Crops; 1931-21220- 014-00D, Management of Abiotic Stress in Fruit Crops that have been approved through OSQR. Further information on this research can be obtained by referencing the annual reports for these new CRIS projects. B. Other accomplishments: None C. Significant activities that support special target populations: None D. Progress Report: None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A natural compound, hinokitiol, derived from cedar was identified that controls apple scab, a serious disease that lowers the productivity of apple trees and fruit quality. There is a critical need to develop new, safe and environmentally-friendly, approaches to
controlling diseases of apple. The natural compound was effective in greenhouse and field trials. This research will provide a new tool for managing apple scab that does not rely on the use of synthetic fungicides. A method to manage fire blight in young apple orchards with prohexadione- calcium (Phd-Ca). Fire blight, a disease of apples and pears, can be particularly devastating in newly planted orchards (1-5 years old) because infections of young trees often result in complete tree death. ARS scientists determined that fewer high-dose applications of Phd-Ca provided good blight control, without unduly restricting young tree growth or impairing young tree establishment, as opposed to multiple low- dose applications which did not effectively control fire blight. These results improve the grower's ability to manage this destructive disease in young orchards A vector was constructed for gene expression that can be used to evaluate gene expression by providing an internal control. Due to
public concerns about the safety of genetically-modified organisms (GMOs) there is a need to develop new, tissue-specific or inducible promoters and better understand how these new promoters compare with constitutive promoters such as the commonly-used 35S promoter. We constructed a vector which carries both a control gene (GUS) under the regulation of the (35S) promoter, and a gene encoding a green fluorescent protein (gfp) optimally modified for plants under control of the tissue-specific cab promoter (developed by an ARS scientist at Kearneysville, WV). This use of this vector will greatly improve of tissue-specific promoters and will result in better promoter selection for targeted genetic engineering. Two biocontrol agents were disovered, patented, and later commercialized as the products, Aspire and Bio-Save, through CRADAs with industry. Subsequently, ARS scientists have found that the efficacy of yeast antagonists can be greatly enhanced by the addition of natural, bioactive
compounds. Due to changes in pesticide regulations and increased demand by consumers for a reduction in the use of pesticides there is an urgent need to develop new methods of controlling postharvest diseases of fruit. This technology has been patented and is being commercialized via a CRADA with industry as a bioactive coating that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. The antagonist P. syringae, previously identified and commercialized to control postharvest diseases, can reduce the risk of potential contamination with E. coli O157:H7 on apples handled in water after harvest. Food safety is of paramount importance to consumers, as well as growers and processors. This work not only revealed an unforeseen benefit of this postharvest biocontrol agent, but also opened a new area of research for reducing risk from foodborne pathogens on intact and fresh cut fruits. We demonstrated that
benzaldehyde and acetic acid are effective fumigants against four major soilborne plant pathogens: Sclerotia minor; Rhizoctonia solan; Pythium aphanidermatum; and, Fusarium oxysporum. Methyl bromide is targeted for de-registration as a soil fumigant making it critical to find effective alternatives. We demonstrated that blighted summer prunings left on the orchard floor can serve as an active inoculum source for up to two months after pruning. Fire blight is the most serious disease of apple and pear in the United States. We verified the accuracy of the fire blight prediction model, MARYBLYT, to predict the occurrence of the blossom blight stage of the disease. These findings can be utilized as part of an integrated approach to manage fire blight. A patented technology for frost protection was developed in collaboration with a CRADA partner. Loss of horticultural crops due to late spring frosts is a serious problem that can result in severe economic losses for growers and higher
prices for consumers. The technology consists of the use of a hydrophobic clay material that is applied to the plant surface and blocks externally-induced freezing caused by the formation of ice on the plant surface. This technology represents a new approach to frost protection for crops. 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? A Specific Cooperative Agreement with a commodity group was extended to assess the ability of genetic enhancement to improve stress tolerance in apple trees using anti-oxidant genes. The results of this research should be known in a few years and demonstrate to fruit tree growers the utility of using biotechnology in conjunction with traditional breeding. A Specific Cooperative Agreement with a commodity group was
initiated to examine the effect of overexpression of zinc transporter genes in apple on zinc absorption and translocation. If successful, this technology could be used to develop apple rootstocks with enhanced ability to acquire zinc from the soil, thus eliminating the need for application of zinc fertilizers. The acceptance of genetically-modified plants is considered the major constraint. However, since the transgene will be in a rootstock, consumers will not be exposed to transgenic tissues and there will be no risk of spreading the transgene to non-target species. These factors are expected to greatly mitigate the constraints for adopting this technology. A CRADA with industry was extended to explore the use of natural compounds in consumer packaging to prolong storage life of purchased produce. A CRADA was extended with industry to assess the ability of technology developed by USDA-ARS scientists to control postharvest diseases to also control apple scab and fire blight. The
use of a methylation-minus E. coli strain as a source of plasmid to increase transformation efficiency in Pseudomonas syringae (L-59-66) was published in February 2003, and since then, numerous requests from microbiologists/molecular biologists in academia for reprints and the E. coli strain have been received. The technology was immediately available to the end users. No constraints are apparent in the application of this technology for dealing with similar problems in transformation of other bacterial strains and species. ARS scientists at Kearneysville, WV met with commodity groups, attended regional project workshops, presented invited seminars both nationally and internationally, and presented research at national meetings of various professional societies on topics of biological control and stress tolerance in fruit crops. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. This project was
terminated in December 2003 and was therefore only operational for two months since the last annual report (FY 03). All publications and presentations have been previously reported in previous annual reports. The research is being continued in two new CRIS projects: 1931-21220-013-00D Management of Pre- and Postharvest Diseases of Fruit Crops; 1931-21220-014-00D,Management of Abiotic Stress in Fruit Crops, that have been approved through OSQR. Further information on this research can be obtained by referencing the annual reports for these new CRIS projects.
Impacts
(N/A)
Publications
- BASSETT, C.L., CALLAHAN, A.M. CHARACTERIZATION OF A TYPE II CHLOROPHYLL A/B-BINDING PROTEIN GENE(LHCB2*PP1) IN PEACH:II. MRNA ABUNDANCE IN DEVELOPING LEAVES EXPOSED TO SUNOR SHADE IN 'LORING'. TREE PHYSIOLOGY. Vol. 23, pgs. 473-480 April 2003.
- Bassett, C.L., Janisiewicz, W.J. Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae. Biotechnology Letters. 2003. v.25(3). p.199-203.
- El Ghaouth, A., Wilson, C.L., Callahan, A.M. 2003. Induction of chitinase b-1,3-glucanase and phenylalanine ammonia lyase in peach fruit by uv-c treatment. Phytopathology, Vol. 93:349-355.
- El Ghaouth, A., Wilson, C.L., Wisniewski, M. 2003. Control of postharvest decay of apple fruit with Candida saitoana and induction of defense responses. Phytopathology 93: 344-348.
- Huang, T., Nicodemus, J., Zarka, D.G., Thomashow, M.F., Wisniewski, M., Duman, J.G. 2002. Expression of an insect (Dendroides cnadadensis) antifreeze protein in Arabidopis thaliana results in a decrease in plant freezing temperature. Plant Molecular Biology 50:333-344.
- Janisiewicz, W.J. Postharvest biocontrol products: development and commercialization. Phytopathologia Brasiliera. 2002. v.27 (Suplemento). p. S10-11.
- Janisiewicz, W.J. Advantages and limitations of postharvest biological control: toward the next generation products. 1st FEMS Congress of European Microbiologists. 2003. Abstract p.99.
- Janisiewicz, W.J. Peterson, D.L. Susceptibility of stem pull area of mechanically harvested apples to blue mold decay and its control with a biocontrol agent. 2003. Phytopathology. v.93. Abstract p.S40.
- Janisiewicz, W.J. Korsten, L. Biological Control of Postharvest Diseases of Fruits. Annual Review of Phytopathology. 2002. v.40. p.411-441.
- 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., 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. 2003. Phytopathology. v.93. Abstract p. S50.
- Norelli, J.L., Holleran, H.T., Johnson, W.C., Robinson, T.L., Aldwinckle, H.S. Resistance of Geneva and other apple rootstocks to Erwinia amylovora. Plant Disease. 2003. v.87. p.26-32.
- Norelli, J.L., Jones, A.L., Aldwinckle, H.S. Fire blight management in the 21st century: using new technologies that enhance host resistance in apple. Plant Disease. 2003. v.87. p.756-765.
- Segal, E., Yehuda, H., Droby, S., Wisniewski, M., Goldway, M. 2002. Cloning and analysis of CoEXGI, a secreted 1,3-b-glucanase of the yeast biocontrol agent Candida oleophila. Yeast 19: 1171-1182.
- Stier, J.C., Filiault, D.L., Wisniewski, M., Palta, J.P. 2003. Visualization of freezing progression in turfgrass using infrared thermography. Crop Science 43: 415-420.
- Wisniewski, M., Fuller, M., Glenn, D.M., Gusta, L., Duman, J., and M. Griffith. 2002. Extrinsic Ice Nucleation in Plants: What are the factors involved and can they be manipulated. In: Plant Cold Hardiness: Gene Regulation and Genetic Engineering. Eds. P.H. Li and E.T. Palva. Kluwer Academic, New York. Pp.211-221.
- Leverentz, B., Janisiewicz, W.J., Conway, W.S. Biological control on minimally processed fruits and vegetables. Novak, J.S., Sapers, G.M., Juneja, V.K., editors. CRC Press, Boca Raton, FL. Microbial Safety of Minimally Processed Foods. 2003. Chapter 15. p.319-332.
- Saftner, R., Conway, W., Abbott, J. Leverentz, B., Janisiewicz, W. Physical and chemical control strategies to reduce postharvest decays of apple and other fresh fruit while maintaining quality; reducing dependence on pesticides. XXVI International Horticultural Congress. 2002. Abstract p. 273-274.
- Droby, S., Wisniewski, M., El Ghaouth, A., Wilson, C. 2003. Influence of food additives on the control of postharvest rots of apple and peach and efficacy of the yeast-based biocontrol product Aspire. Postharvest Biology and Technology 27: 127-135.
- Duke, S.O., Baerson, S.R., Dayan, Franck E., Rimando, Agnes, M., Scheffler, B.E., Tellez, M.R., Wedge, D.E., Schrader, K.K., Akey, D.H., Arthur, F.H., De Lucca, A.J., Gibson, D.M., Harrison, H.F., Peterson, J.K., Gealy, D.R., Tworkoski, T., Wilson, C.L., Morris, J.B. 2003. United States of Agriculture - Agricultural Research Service research on natural products for pest management. Pest Management Science 59:708-717.
- Wisniewski, M., Bassett, C., Farrell, R., Artlip, T. Two dehydrin genes in peach bark tissues: Transcript Accumulation in response to environmental stress and comparative analysis of their promoters. Proceedings Plant and Microbe Adaptations to Cold, Quebec, Canada, Abstract 46:p.67.
- 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.
|
Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Postharvest losses due to decay of fruits during storage, shipping, marketing, and home consumption are significant and represent a financial burden to both growers and consumers. Furthermore, public concern over chemical residues, environmental concerns, and pathogen populations that are resistant to chemical fungicides, have created a critical need for safe and effective alternatives. Biological approaches that utilize microbial antagonists and natural compounds are being developed to address these issues. A basic understanding of the mode of action of biocontrol agents will lead to the selection of more effective antagonists. Fire blight is one of the most serious and destructive diseases of apple and pear. To combat this disease there is a need to understand the basis of host resistance, the epidemiology of the disease, and develop non-chemical orchard and nursery management
practices that will dramatically reduce the incidence of fire blight. Freeze damage, other types of environmental stress, and micronutrient deficiency in fruit trees often results in a significant loss in yield, fruit quality, or tree longevity. To address this problem, there is a need to develop a basic understanding of the biochemistry, physiology, and molecular biology of stress tolerance and micronutrient metabolism in fruit trees. 2. How serious is the problem? Why does it matter? Several key synthetic fungicides have been targeted for withdrawal from the market because of their potential impact on human health and/or the costs involved in registering these products and demonstrating their safety. Some examples are ipridione used on stone fruit and benomyl compounds on pome fruit. Additionally, the development of fungicide resistance has made disease management more difficult. In the near future, some commodities may have no effective synthetic fungicides registered for
postharvest application. Fire blight infections in newly planted orchards (1 to 5 years) can result in up to 80% tree loss causing devastating financial loss for growers. A single fire blight epidemic that occurred in southwest Michigan in 2000 was estimated to have caused the death of 350,000 to 450,000 trees and the removal of 1,500 to 2,300 acres of apple orchards with a total economic loss of 42 million dollars. The phytoxicity of copper and its limited efficacy, and resistance to streptomycin, the two main methods of controlling fire blight, make the need to find new ways to manage fire blight critically important. Freezing temperatures are the prime factor affecting the production of perennial fruit and nut crops nationally. Estimates of annual losses in the U.S. range from millions of dollars for the citrus industry to nearly one billion dollars for all crops. A basic understanding of cold hardiness and stress tolerance can lead to the development of varieties that are
resistant to extreme weather conditions and lead to new management tools that alleviate the impact of environmental stress on the performance of plants. Zinc deficiency in fruit crops is widespread throughout the United States. It is associated with poor tree growth, reduced yields, and inferior fruit quality. The inefficiency and subsequent repeated use of zinc foliar sprays has led to concern over heavy metal contamination of orchard soils. More effective methods of managing zinc nutrition are needed to assure healthy trees, good fruit quality, and reduce the risk of soil contamination. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The goal of this research is to develop information on the identity and mode of action of biocontrol agents in order to make this strategy more effective and contributes to National Program 303: Plant Diseases (70%). This project also addresses the need to improve stress tolerance and
micronutrient absorption in fruit crops and contributes to National Program 302: Improving Plant Biological and Molecular Processes (30%). It is recognized that this is an important need given the impact of environmental stress and micronutrient deficiency on fruit production. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment: Due to public concern about pesticide residues, there is a critical need to improve the efficacy of postharvest biocontrol agents, understand their mode of action, and improve disease resistance in fruit crops. USDA- ARS scientists at the Appalachian Fruit Research Station, Kearneysville, WV, in collaboration with scientists in Israel, isolated an antimicrobial peptide gene from peach trees and expressed it in a yeast. The transformed yeast exhibited antimicrobial activity against Botrytis cinerea and Penicillium exapansum. This research demonstrated that biocontrol agents can be genetically enhanced to
improve their efficacy and also identified a compound responsible for innate resistance in peach trees. B. Other Significant Accomplishments: Fire blight, a disease of apples and pears, can be particularly devastating in newly planted orchards (1-5 years old) because infections of young trees often result in complete tree death. ARS scientists at the Appalachian Fruit Research Station, Kearneysville, WV, have conducted research to determine the effect of various prohexadione-calcium (Phd-Ca) dose levels on shoot growth and fire blight resistance in young apple trees. It was determined that fewer high-dose applications of Phd-Ca provided good blight control, without unduly restricting young tree growth or impairing young tree establishment, as opposed to multiple low- dose applications which did not effectively control fire blight. These results improve the grower's ability to manage this destructive disease in young orchards. There is a critical need to develop new, safe and
environmentally- friendly, approaches to controlling diseases of apple. A USDA-ARS scientist in Kearneysville, WV has shown that a natural compound, hinokitiol, derived from cedar, is effective against apple scab in greenhouse screens and in preliminary field trials. New formulations of hinokitiol have been developed by the ARS scientist which enhances its effectiveness. This research has commercial potential and may lead to a new product that could be used by growers to control apple diseases in the orchard. Due to significant losses in the quality of produce after harvest, there is an urgent need to develop safe and effective methods of maintaining the quality of post-consumer-purchase produce. A USDA-ARS scientist in Kearneysville, WV has developed "Active Packaging" for the storage of fruits and vegetables which extends their freshness and shelf-life. Moisture activated anti-microbial compounds are incorporated into the package which are released by water originating from the
stored commodity. This technology is being developed in collaboration with private industry and may result in a product that greatly benefits consumers. Current approaches to the development of new, mechanical approaches to apple harvesting, necessitated by a critical shortage in manual labor, have led to increased potential of rots in the stem area of apples due to stem pulls. A USDA-ARS scientist in Kearneysville, WV has identified microbial antagonists that are very effective in controlling infections that develop from stem pulls. This research demonstrates how biocontrol can be adapted to solve new problems in postharvest disease control and how an integrated approach (mechanical harvesting and postharvest biological control) can be used to solve critical problems for the fruit industry. Understanding mechanisms responsible for differential expression of genes in fruit could lead to improvement in fruit traits such as, quality, storage potential, disease resistance, etc. To
approach this issue experimentally, scientists at the AFRS, Kearneysville, WV in collaboration with a researcher from Pennsylvania State University at York, PA used a genetic protocol (RLM-5' RACE) technique to identify differential processing of a dehydrin mRNA from ripe peach fruit that alternatively targets the dehydrin protein for transport to either the cytosol or specifically to mitochondria. This observation is the first report of a possible role for dehydrins in mitochondria and provides a greater understanding of the complex regulation of stress genes in fruit crops. Due to public concerns about the safety of genetically-modified organisms (GMOs) there is a need to develop new, tissue-specific or inducible promoters and better understand how these new promoters compare with constitutive promoters such as the commonly-used 35S promoter. Scientists at the AFRS, Kearneysville, WV have constructed a vector for gene expression which carries both a control gene (GUS) under the
regulation of the (35S) promoter, and a gene encoding a green fluorescent protein (gfp) optimally modified for plants under control of the tissue- specific cab promoter (developed by an ARS scientist at Kearneysville, WV) . This vector can now be used to compare, in the same plant, gene expression driven by the cab promoter, or any other newly-identified promoter, with gene expression driven by the 35S promoter. Improved testing of tissue-specific promoters will result in better promoter selection for targeted genetic engineering. There is a need to develop new ways to improve disease resistance in fruit crops and a potential target for manipulation is a class of transcription factors (TFs) known as WRKY factors which have been shown in other plants to control response to a variety of biotic and abiotic stresses. Scientists at the AFRS, Kearneysville, WV have succeeded in isolating a gene encoding a WRKY TF from apple. Preliminary results that this gene is involved in
pathogen-induced responses in fruit. Manipulation of this gene could lead to enhanced resistance of fruit to diseases commonly seen during storage. There is a significant need to develop automated technologies at the packing house for detecting poor quality and diseased fruit. A USDA-ARS scientist in Kearneysville, WV has shown that with the use of an electronic nose (Cyranose 320) it was possible to distinguish three degrees of severity of watercore in 'Red Delicious' apples. This is the first time that an electronic nose has been used successfully to detect internal defects in fruit. Research is continuing to see whether this technology can be utilized for the on-line detection of watercore during processing and for the detection of other internal defects. Due to widespread deficiencies, there is a critical need to improve zinc absorption, transport, and utilization in fruit trees in order to reduce the need for continuous foliar sprays or root drenches that may accumulate and
contaminate soils with a heavy metal. USDA-ARS scientists in Kearneysville, WV in collaboration with a USDA-ARS scientist in Ithaca, NY, have made two gene constructs containing different zinc transporter genes and transformed M-26 apple rootstocks with these genes. If the resulting explants have enhanced zinc absorption, these genetically- enhanced genotypes would be available for commercial production providing a rootstock that would improve zinc nutrition and decrease or eliminate the need for zinc foliar sprays or root drenches. The resulting rootstock could be used with any apple scion cultivar. C. Significant Activities that Support Special Target Populations: None D. Progress Report: (Significant Milestone) There is a need to develop safer plant transformation constructs which do not express foreign genes in edible plant parts such as fruit. A plasmid construct was developed by a USDA-ARS scientist at the Appalachian Fruit Research Station, Kearneysville, WV containing the
regulatory (promoter) region of a photosynthetic gene (cab) and a reporter gene (GUS). The construct was transformed into a model plant system (tomato) and shown to produce high levels of GUS expression in leaves and significantly lower and decreasing expression in ripe fruit, flowers and roots, respectively. These results indicate that the cab promoter can be used to provide high levels of expression of a foreign gene in leaves without causing significant expression of the gene in fruits or roots, thus potentially yielding a safer, more acceptable transgenic plant. There is a critical need to improve stress tolerance in fruit trees. USDA-ARS scientists in Kearneysville, WV, in collaboration with Oregon State University, and the Washington State Tree Fruit Commission, have generated genetically-enhanced lines of Gala apple that overexpress a gene for the antioxidant enzyme, ascorbate peroxidase (APX). The resulting lines have been demonstrated to have increased resistance to heat,
cold, and UV-B stress in laboratory tests. The best of these lines are now being prepared for field testing. Developing new management tools for fire blight is a critical need for apple growers. USDA-ARS scientists in Kearneysville, WV have demonstrated how multiple, low-dose applications of a growth regulator, prohexadione-calcium, can be used to control fire blight in young orchards. Developing methods to improve the efficacy of postharvest biocontrol agents is essential so that they will work in a uniform and predictable manner under the wide array of conditions present in commercial packing houses. Scientists in the USDA-ARS, Kearneysville, WV, in collaboration with scientists in Israel, have transformed a yeast with a naturally- occurring, antimicrobial peptide gene responsible for innate resistance in peach and have demonstrated activity against two major postharvest pathogens of apple, Botrytis cinerea and Penicillium expansum. This research illustrates that genetic
enhancement of biocontrol agent is a feasible approach to improving their efficacy. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Due to changes in pesticide regulations and increased demand by consumers for a reduction in the use of pesticides there is an urgent need to develop new methods of controlling postharvest diseases of fruit. ARS scientists at Kearneysville, WV discovered and patented two biocontrol agents that were commercialized as the products, Aspire and Bio-Save, through a CRADA with industry. Subsequently, ARS scientists have found that the efficacy of yeast antagonists can be greatly enhanced by the addition of natural, bioactive compounds. This technology has been patented and is being commercialized via a CRADA with industry as a Abioactive coating@ that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. Food safety is of
paramount importance to consumers, as well as growers and processors. An ARS scientist at Kearneysville, WV found that the antagonist P. syringae, previously identified and commercialized to control postharvest diseases, can reduce the risk of potential contamination with E. coli O157:H7 on apples handled in water after harvest. This work not only revealed an unforeseen benefit of this postharvest biocontrol agent, but also opened a new area of research for reducing risk from foodborne pathogens on intact and fresh cut fruits. Methyl bromide is targeted for de-registration as a soil fumigant making it critical to find effective alternatives. ARS scientists at Kearneysville, WV determined that benzaldehyde and acetic acid are effective fumigants against four major soilborne plant pathogens: Sclerotia minor; Rhizoctonia solan; Pythium aphanidermatum; and, Fusarium oxysporum. Fire blight is the most serious disease of apple and pear in the United States. An ARS scientist at
Kearneysville, WV demonstrated that blighted summer prunings left on the orchard floor can serve as an active inoculum source for up to two months after pruning. An ARS scientist verified the accuracy of the fire blight prediction model, MARYBLYT, to predict the occurrence of the blossom blight stage of the disease. These findings can be utilized as part of an integrated approach to manage fire blight. Loss of horticultural crops due to late spring frosts is a serious problem that can result in severe economic losses for growers and higher prices for consumers. USDA-ARS scientists at Kearneysville, WV in collaboration with Engelhard, Inc. have developed patented technology for frost protection. The technology consists of the use of a hydrophobic clay material that is applied to the plant surface and blocks externally- induced freezing caused by the formation of ice on the plant surface. This technology represents a new approach to frost protection. 6. What do you expect to
accomplish, year by year, over the next 3 years? FY2004 Genes encoding the rate-limiting enzymes for the synthesis of sorbitol and starch in apple will be cloned and the expression of these genes will be down-regulated in apple using sense and/or antisense transgenic gene expression in order to determine the role of sorbitol and osmotic potential in the susceptibility of apple to fire blight. Orchard evaluation of transgenic apples overexpressing antioxidant genes will be conducted. Transcription factors involved in the regulation of stress tolerance genes in fruit crops will be identified. This research is directed at improving stress tolerance in fruit trees with the use of transgenic technologies. A biocontrol system for fruit processed in small packinghouses where the drenching suspension is kept for a long period to time, i.e., up to 7 days will be finalized. The ability of hinokitoil, a natural compound obtained from cedar, to effectively control apple scab and fire blight
will be evaluated. Transposon tagged Pseudomonas syringae will be constructed and evaluated in order to identify genes responsible for biocontrol activity. An apple genomic library will be constructed in order to identify and isolate genes relevant to disease resistance and stress tolerance. M-26 apple rootstocks transformed with ZNT1 and ZIP4 zinc transporter genes will be evaluated under greenhouse conditions for their ability to absorb and translocate zinc to various plant tissues. FY 2005 A pistil and/or flower specific promoter and a reporter gene will be constructed and its expression in tomato examined. If successful, this construct will be used to specifically express cold hardiness genes in flowers of fruit crops. The mode of penetration and infection of apple shoots by Erwinia amylovora will be determined using green fluorescent protein (gfp) as a visual tag and as a "biosensor" to monitor the growth, movement and metabolism of E. amylovora in apple. Biocontrol products,
based on mutually-compatible antagonist mixtures exhibiting superior biocontrol potential will be completed. This goal originally defined for FY 2004 has required additional time to accomplish. The limiting nutrients in the interaction between the pathogen, P. expansum, and an antagonistic yeast effective in biocontrol of apple blue mold will be identified. Genetically-enhanced lines of M-26 apple rootstock that overexpress zinc transporter genes will be evaluated for increased zinc absorption and translocation in greenhouse plants. Additionally, 'Gala' and 'Fuji' apple scions will be propagated on the transformed apple rootstock. The ability of these trees to absorb and translocate zinc will be evaluated under greenhouse conditions. The activity of transcription factors associated with stress tolerance in fruit crops will be characterized. FY 2006 It will be determined if graft transmissible gene silencing can be used to transfer desirable trait modifications from a transgenic
apple rootstock to a fruiting scion with no genetic alteration of the fruiting variety. Overexpression of transcription factors identified in fruit crops will be evaluated in transgenic apple in order to determine their effect on the global induction of stress tolerance genes and concomitant stress resistance. Using transposon-tagged lines of Pseudomonas syringae (L59-66), genes responsible for biocontrol potential. 'Gala' and 'Fuji' apple trees, propagated on genetically-modified M-26 rootstocks overexpressing zinc transporter genes, will be field-evaluated in West Virginia for their ability to acquire zinc from the soil. 7. 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? A Specific Cooperative Agreement with a commodity group was extended to assess
the ability of genetic enhancement to improve stress tolerance in apple trees using anti-oxidant genes. The results of this research should be known in a few years and demonstrate to fruit tree growers the utility of using biotechnology in conjunction with traditional breeding. A Specific Cooperative Agreement with a commodity group was initiated to examine the effect of overexpression of zinc transporter genes in apple on zinc absorption and translocation. If successful, this technology could be used to develop apple rootstocks with enhanced ability to acquire zinc from the soil, thus eliminating the need for application of zinc fertilizers. The acceptance of genetically-modified plants is considered the major constraint. However, since the transgene will be in a rootstock, consumers will not be exposed to transgenic tissues and there will be no risk of spreading the transgene to non-target species. These factors are expected to greatly mitigate the constraints for adopting this
technology. A CRADA with industry was extended to explore the use of natural compounds in consumer packaging to prolong storage life of purchased produce. A CRADA was extended with industry to assess the ability of technology developed by USDA-ARS scientists to control postharvest diseases to also control apple scab and fire blight. The use of a methylation-minus E. coli strain as a source of plasmid to increase transformation efficiency in Pseudomonas syringae (L-59-66) was published in February 2003, and since then, numerous requests from microbiologists/molecular biologists in academia for reprints and the E. coli strain have been received. The technology was immediately available to the end users. No constraints are apparent in the application of this technology for dealing with similar problems in transformation of other bacterial strains and species. ARS scientists at Kearneysville, WV met with commodity groups, attended regional project workshops, presented invited seminars
both nationally and internationally, and presented research at national meetings of various professional societies on topics of biological control and stress tolerance in fruit crops. 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). 1. Presentation: "Fire blight infections on apple rootstocks" and "Fire blight management in the 21st Century" at the 2003 Virginia Grown Conference and Trade Show, sponsored by the Virginia State Horticultural and the West Virginia State Horticultural Societies, Richmond, VA, January 27-29, 2003. 2. Presentation: "Fire blight management in the 21st century: using new technologies that enhance host resistance" and "The role of ephiphytic bacteria in the shoot blight phase of fire blight" at the 78th Cumberland- Shenandoah Fruit Workers Conference, Winchester, VA; November 15-16, 2002. 3.
Presentation: "Epiphytic bacteria in the shoot blight phase of fire blight" at the Washington Tree Fruit Research Commission, Penticton, BC, January 6, 2003. 4. Presentation: "The shoot blight phase of fire blight" at the Annual Meeting of the New York State Apple Research and Development Program, December 10, 2002. 5. Poster Presentation: "Survival and growth of Erwinia amylovora on apple shoots" at the American Phytopathological Society Annual Meeting Charlotte, NC, August 9-13, 2003. 6. Presentation: Invited Plenary Lecture at the XXXV Annual Meeting of Brazilian Phytopathological Society on "Postharvest biocontrol products: development and commercialization." Recife, Brazil, 2002. 7. Presentation: Invited Lecture at the 8th International Congress of Plant Pathology - Workshop on Successes and Problems of Postharvest Biological Control of Postharvest Diseases "What have we learnt from past mistakes and achievements in postharvest biocontrol?" , Christchurch, New Zealand, 2003.
8. Presentation: Lectures at the Institute for Horticultural Development "Biological control of postharvest diseases of pome fruits: research, commercialization, and future prospects" and "Biocontrol of foodborne pathogens", Knoxfield, Australia, 2003. 9. Presentation: Invited Lecture at the 1st FEMS Congress of European Microbiologists "Advantages and limitations of postharvest biological control: toward the next generation products", Ljubliana, Slovenia, 2003. 10. Presentations: Taught 12 hour graduate level course on "Biological Control of Postharvest Diseases" at the Federal Rural University of Pernambuco (FRUP), in Recife, Brazil (August 12-14, 2002). 11. Presentation: "A novel Tata box element controls expression of the peach dehydrin gene, Ppdhn2, in ripe fruit and potentially alters the intracellular distribution of the cognate polypeptide", to the Mid- Atlantic Society of Plant Molecular Biologists, Beltsville, MD, August, 2003. 12. Presentation: "Overexpression of
antioxidant genes in apple and tomato improves stress tolerance," at the Washington Tree Fruit Research Commission, Penticton, BC, January 6, 2003. 13. Presentation: "Overexpression of zinc transporter genes in apple to improve zinc uptake and absorption," at the Washington Tree Fruit Research Commission, Penticton, BC, January 6, 2003. 14. Poster Presentation: "Expression and characterization of two dehydrin genes in peach," Gordon Conference on Temperature Stress. January, 2003. 15. Poster Presentations: "Enhanced stress tolerance in apple overexpressing a cytosolic APX gene," and "Analysis of the promoters of two dehydrin genes from peach," Plant and Microbe Adaptations to Cold, Quebec, Canada, May, 2003. 16. Poster Presentation: "Novel regulation of a dehydrin gene from peach, " Int. Congress of Plant Molecular Biology, Barcelona, Spain. July, 2003. 17. Invited Seminar: "An overview of cold hardiness research in fruit crops," Department of Genetics and Biotechnology, Helsinki
University, Finland, August, 2003.
Impacts
(N/A)
Publications
- BASSETT, C.L., CALLAHAN, A.M. CHARACTERIZATION OF A TYPE II CHLOROPHYLL A/B-BINDING PROTEIN GENE(LHCB2*PP1) IN PEACH:II. MRNA ABUNDANCE IN DEVELOPING LEAVES EXPOSED TO SUNOR SHADE IN 'LORING'. TREE PHYSIOLOGY. Vol. 23, pgs. 473-480 April 2003.
- Huang, T., Nicodemus, J., Zarka, D.G., Thomashow, M.F., Wisniewski, M., Duman, J.G. 2002. Expression of an insect (Dendroides cnadadensis) antifreeze protein in Arabidopis thaliana results in a decrease in plant freezing temperature. Plant Molecular Biology 50:333-344.
- Janisiewicz, W.J. Postharvest biocontrol products: development and commercialization. Phytopathologia Brasiliera. 2002. v.27 (Suplemento). p. S10-11.
- Janisiewicz, W.J. Advantages and limitations of postharvest biological control: toward the next generation products. 1st FEMS Congress of European Microbiologists. 2003. Abstract p.99.
- Janisiewicz, W.J. Peterson, D.L. Susceptibility of stem pull area of mechanically harvested apples to blue mold decay and its control with a biocontrol agent. 2003. Phytopathology. v.93. Abstract p.S40.
- Janisiewicz, W.J. Korsten, L. Biological Control of Postharvest Diseases of Fruits. Annual Review of Phytopathology. 2002. v.40. p.411-441.
- 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., 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. 2003. Phytopathology. v.93. Abstract p. S50.
- Norelli, J.L., Holleran, H.T., Johnson, W.C., Robinson, T.L., Aldwinckle, H.S. Resistance of Geneva and other apple rootstocks to Erwinia amylovora. Plant Disease. 2003. v.87. p.26-32.
- Norelli, J.L., Jones, A.L., Aldwinckle, H.S. Fire blight management in the 21st century: using new technologies that enhance host resistance in apple. Plant Disease. 2003. v.87. p.756-765.
- Segal, E., Yehuda, H., Droby, S., Wisniewski, M., Goldway, M. 2002. Cloning and analysis of CoEXGI, a secreted 1,3-b-glucanase of the yeast biocontrol agent Candida oleophila. Yeast 19: 1171-1182.
- Stier, J.C., Filiault, D.L., Wisniewski, M., Palta, J.P. 2003. Visualization of freezing progression in turfgrass using infrared thermography. Crop Science 43: 415-420.
- Wisniewski, M., Fuller, M., Glenn, D.M., Gusta, L., Duman, J., and M. Griffith. 2002. Extrinsic Ice Nucleation in Plants: What are the factors involved and can they be manipulated. In: Plant Cold Hardiness: Gene Regulation and Genetic Engineering. Eds. P.H. Li and E.T. Palva. Kluwer Academic, New York. Pp.211-221.
- 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.
- Leverentz, B., Janisiewicz, W.J., Conway, W.S. Biological control on minimally processed fruits and vegetables. Novak, J.S., Sapers, G.M., Juneja, V.K., editors. CRC Press, Boca Raton, FL. Microbial Safety of Minimally Processed Foods. 2003. Chapter 15. p.319-332.
- Saftner, R., Conway, W., Abbott, J. Leverentz, B., Janisiewicz, W. Physical and chemical control strategies to reduce postharvest decays of apple and other fresh fruit while maintaining quality; reducing dependence on pesticides. XXVI International Horticultural Congress. 2002. Abstract p. 273-274.
- Droby, S., Wisniewski, M., El Ghaouth, A., Wilson, C. 2003. Influence of food additives on the control of postharvest rots of apple and peach and efficacy of the yeast-based biocontrol product Aspire. Postharvest Biology and Technology 27: 127-135.
- Duke, S.O., Baerson, S.R., Dayan, Franck E., Rimando, Agnes, M., Scheffler, B.E., Tellez, M.R., Wedge, D.E., Schrader, K.K., Akey, D.H., Arthur, F.H., De Lucca, A.J., Gibson, D.M., Harrison, H.F., Peterson, J.K., Gealy, D.R., Tworkoski, T., Wilson, C.L., Morris, J.B. 2003. United States of Agriculture - Agricultural Research Service research on natural products for pest management. Pest Management Science 59:708-717.
- Wisniewski, M., Bassett, C., Farrell, R., Artlip, T. Two dehydrin genes in peach bark tissues: Transcript Accumulation in response to environmental stress and comparative analysis of their promoters. Proceedings Plant and Microbe Adaptations to Cold, Quebec, Canada, Abstract 46:p.67.
- Bassett, C.L., Janisiewicz, W.J. Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae. Biotechnology Letters. 2003. v.25(3). p.199-203.
- El Ghaouth, A., Wilson, C.L., Callahan, A.M. 2003. Induction of chitinase b-1,3-glucanase and phenylalanine ammonia lyase in peach fruit by uv-c treatment. Phytopathology, Vol. 93:349-355.
- El Ghaouth, A., Wilson, C.L., Wisniewski, M. 2003. Control of postharvest decay of apple fruit with Candida saitoana and induction of defense responses. Phytopathology 93: 344-348.
|
Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Postharvest losses due to decay of fruits during storage, shipping, marketing, and home consumption are significant and represent a financial burden to both growers and consumers. Furthermore, public concern over chemical residues, environmental concerns, and pathogen populations that are resistant to chemical fungicides have created a critical need for safe and effective alternatives. Biological approaches that utilize microbial antagonists and natural compounds are being developed to address these issues. A basic understanding of the mode of action of biocontrol agents will lead to the selection of more effective antagonists. Fire blight is one of the most serious and destructive diseases of apple and pear. To combat this disease there is a need to understand the basis of host resistance, the epidemiology of the disease, and to develop non-chemical orchard and nursery management
practices that will dramatically reduce the incidence of fire blight. Freeze damage, and other forms of environmentally-induced injury to fruit trees often results in a significant loss in yield, fruit quality, or tree longevity. To address this problem, there is a need to develop a basic understanding of the biochemistry, physiology, and molecular biology of stress tolerance in fruit trees. 2. How serious is the problem? Why does it matter? Several key synthetic fungicides have been targeted for withdrawal from the market because of their potential impact on human health and/or due to the costs involved in registering these products and demonstrating their safety. Some examples are ipridione, used on stone fruit, and benomyl compounds used on pome fruit. Additionally, the development of fungicide resistance has made disease management more difficult. In the near future, some commodities may have no effective synthetic fungicides registered for postharvest application. Fire blight
infections in newly planted orchards (1 to 5 years) can result in up to 80% tree loss causing devastating financial loss for growers. A single fire blight epidemic that occurred in southwest Michigan in 2000 was estimated to have cuased the death of 350,000 to 450,000 trees and the removal of 1,500 to 2,300 acres of apple orchards with a total economic loss of 42 million dollars. The phytoxicity of copper and its limited efficacy, and resistance to streptomycin, the two main methods of controlling fire blight, make the need to find new ways to manage fire blight critically important. Freezing temperatures are the prime factors affecting the production of perennial fruit and nut crops nationally. Estimates of annual losses in the U.S. range from millions of dollars for the citrus industry to nearly one billion dollars for all crops. A basic understanding of cold hardiness and stress tolerance can lead to the development of varieties that are resistant to extreme weather conditions
and lead to new management tools that alleviate the impact of environmental stress on the performance of plants. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The goal of this research is to develop information on the identity and mode of action of biocontrol agents in order to make this strategy more effective and contributes to National Program 303: Plant Diseases (70%). This project also addresses the need to improve stress tolerance in plants and contributes to National Program 302: Improving Plant Biological and Molecular Processes (30%). It is recognized that this is an important need given the impact of environmental stress on crop production. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: There is a critical need to develop new approaches to disease control in order to reduce the use of synthetic pesticides in fruit production. ARS
scientists at the Appalachian Fruit Research Station, Kearneysville, WV, in collaboration with an industry partner, have developed a new biofungicide ('Biocure') based on a combination of a yeast antagonist and natural compounds. Extensive tests of this new biofungicide were conducted on apples in West Virginia and citrus in California, Israel, Italy, and Morocco and it proved as effective as the recommended synthetic fungicide for the control of postharvest decay. This new technology will provide a safe and environmentally-friendly alternative to synthetic fungicides for the control of postharvest decay of harvested commodities. B. Other Significant Accomplishments: Fire blight, a major disease of pome fruits, can have devastating effects on the health of fruit trees and has the potential to cause the loss of entire orchards. ARS scientists at the Appalachian Fruit Research Station, Kearneysville, WV have conducted research to identify the sources of fire blight bacteria that
initiate the shoot blight phase of the disease. It was determined that fire blight bacteria could not survive on the surface of apple leaves for more than 48 hours. These results will improve our ability to predict and prevent this destructive disease. The full potential use of biological control for postharvest decay of fruits is limited by the lack of eradicative activity and a relatively narrow spectrum of effective application. A scientist at the Appalachian Fruit Research Station, Kearneysville, WV, in collaboration with ARS scientists in Beltsville, MD, investigated combining the eradicative effects of heat treatment with the use of an ethylene inhibitor 1-MCP (which slows down maturation of fruit), in combination with a heat- tolerant, antagonistic yeast, on bitter rot and blue mold decay of 'Golden Delicious' apples. When used in combination with the heat treatment, the yeast antagonist provided effective control against bitter rot and blue mold. This research demonstrates
that integrated strategies of postharvest decay management can effectively address the limitations of biological control and can be used to accommodate new technologies such as 1-MCP. Sunscald or sunburn of apples results in a browning of the apple skin which in turn dramatically lowers the value of the fruit and affects overall fruit quality. ARS scientists at the Appalachian Fruit Research Station, Kearneysville, WV, in collaboration with researchers from Oregon State University, have produced transgenic apple plants (and tomato plants) overexpressing the antioxidant enzyme gene, ascorbate peroxidase (APX). The resulting transgenic lines of apple show significantly greater resistance to injury induced by high temperatures, prolonged exposure to UV-B, as well as freezing temperatures. This research demonstrates that genetic enhancement of fruit crops is a feasible approach to improving resistance to environmental stress. In order to select better antagonists for the biocontrol of
postharvest diseases of fruits or develop technologies to enhance the performance of existing biocontrol agents, a better understanding of antagonist and pathogen biology is needed. A scientist at the Appalachian Fruit Research Station, Kearneysville, WV examined sugars occurring in apple, and various microelements for their ability to stimulate germination of P. expansum conidia, a major apple decay organism causing "blue mold." Only one sugar, sucrose, in the presence of microelements, stimulated germination of the pathogen conidia within the first 24 hours, the time within which biocontrol must occur. This finding can improve selection of bacterial and yeast antagonists used for biocontrol of P. expansum, by selecting those that are efficient in utilizing sucrose. Although biocontrol agents are effective in reducing losses of fruit due to postharvest diseases, there is need to enhance their effectiveness in order to make their use commercially acceptable. A scientist at the
Appalachian Fruit Research Station, Kearneysville, WV developed a method for increasing the transformation efficiency of the biocontrol bacterium, Pseudomonas syringae. Growing the plasmid vectors in a methylation- deficient E. coli host significantly reduced DNA losses during subsequent transformation of the biocontrol bacterium. This method will greatly facilitate the testing of genes to enhance the biocontrol properties of this bacterial strain. C. Significant Activities that Support Special Target Populations: None. D. Progress Report: There is a need to develop safer plant transformation constructs which do not express foreign genes in edible plant parts such as fruit. A plasmid construct was developed by a scientist at the Appalachian Fruit Research Station, Kearneysville, WV containing the regulatory (promoter) region of a photosynthetic gene (cab) and a reporter gene (GUS). The construct was transformed into a model plant system (tomato) and shown to produce high levels
of GUS expression in leaves and significantly lower and decreasing expression in ripe fruit, flowers and roots, respectively. These results indicate that the cab promoter can be used to provide high levels of expression of a foreign gene in leaves without causing significant expression of the gene in fruits or roots, thus potentially yielding a safer, more acceptable transgenic plant. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Due to changes in pesticide regulations and increased demand by consumers for a reduction in the use of pesticides there is an urgent need to develop new methods of controlling postharvest diseases of fruit. ARS scientists at Kearneysville, WV discovered and patented two biocontrol agents that were commercialized as the products, 'Aspire' and 'Bio-Save', through a CRADA with industry. Subsequently, ARS scientists have found that the efficacy of yeast antagonists can be greatly enhanced by
the addition of natural, bioactive compounds. This technology has been patented and is being commercialized via a CRADA with industry as a bioactive coating that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. Food safety is of paramount importance to consumers, as well as growers and processors. An ARS scientist at Kearneysville, WV found that the antagonist P. syringae, previously identified and commercialized to control postharvest diseases, can reduce the risk of potential contamination with E. coli O157:H7 on apples handled in water after harvest. This work not only revealed an unforseen benefit of this postharvet biocontrol agent, but also opened a new area of research for reducing risk from foodborne pathogens on intact and fresh cut fruits. Methyl bromide is targeted for de-registration as a soil fumigant. So there is a critical need to find effective alternatives. ARS scientists at
Kearneysville, WV determined that benzaldehyde and acetic acid are effective fumigants against four major soilborne plant pathogens: Sclerotia minor; Rhizoctonia solan; Pythium aphanidermatum; and, Fusarium oxysporum. Fire blight is the most serious disease of apple and pear in the United States. An ARS scientist at Kearneysville, WV demonstrated that blighted summer prunings left on the orchard floor can serve as an active inoculum source for up to two months after pruning. An ARS scientist verified the accuracy of the fire blight prediction model, MARYBLYT, to predict the occurrence of the blossom blight stage of the disease. These findings can be utilized as part of an integrated approach to manage fire blight. Loss of horticultural crops due to late spring frosts is a serious problem that can result in severe economic losses for growers and higher prices for consumers. USDA-ARS scientists at Kearneysville, WV in collaboration with Engelhard, Inc. have developed patented
technology for frost protection. The technology consists of the use of a hydrophobic clay material that is applied to the plant surface and blocks externally- induced freezing caused by the formation of ice on the plant surface. This technology represents a new approach to frost protection. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2003: ARS scientists in Kearneysville, WV will determine if transgenic apple trees overexpressing stress tolerance genes such as ascorbate peroxidase, dehydrin, and superoxide dismutase have increased resistance to environmental stress. ARS scientists will develop a "second generation" of biocontrol agents based on combining biocontrol agents with natural compounds or using mixtures of different species of biocontrol organisms. The second generation products will have improved performance over the first generation of products developed by ARS in collaboration with industry partners. ARS scientists will develop a
biocontrol system for fruit processed in small packinghouses where the drenching suspension is kept for a long period to time, i.e., up to seven days. The role of chitinase genes in the mechanism of action of biocontrol agents will be determined. ARS scientists at Kearneysville, WV will improve the ability to predict and control fire blight on apple by determining what role populations of Erwinia amylovora (the agent that causes fire blight) growing on the surface of leaves and shoots play in initiating the shoot blight phase of the disease and determining how environmental factors affect fire blight bacteria populations on shoots. FY2004: An ARS scientist in Kearneysville, WV will clone the genes encoding the rate-limiting enzymes for the synthesis of sorbitol and starch in apple, down-regulate the expression of these genes in apple using sense and/or antisense transgenic gene expression in order to determine the role of sorbitol and osmotic potential in the susceptibility of apple
to fire blight. Further evaluation of transgenic apples overexpressing antioxidant genes will be made in the orchard. An ARS scientist in Kearneysville, WV will develop a transgenic strain of Pseudomonas sp. that expresses a green fluorescent protein (GFP). This strain will be utilized to study the microbial ecology of postharvest biocontrol agents. An ARS scientist at Kearneysville, WV will test another version of the cab promoter in tomato fruit to determine if further reduction in the expression of transgenes can be attained in ripe fruit. ARS scientists at Kearneysville, WV will identify transcription factors involved in the regulation of stress tolerance genes in fruit crops. This research is directed at improving stress tolerance in fruit trees with the use of transgenic technologies. An ARS scientist at Kearneysville, WV will complete the development of an approach to postharvest decay control based on the use of mutually compatible mixtures of antagonists. FY 2005:
Overexpression of transcription factors identified in fruit crops by ARS scientists in Kearneysville, WV will be evaluated in transgenic apple in order to determine their effect on the global induction of stress tolerance genes and concomitant stress resistance. An ARS scientist in Kearneysville, WV will construct a pistil and/or flower specific promoter and a reporter gene and examine its expression in tomato. If successful, this construct will be used to specifically express cold hardiness genes in flowers of fruit crops. An ARS scientist at Kearneysville, WV will determine the mode of penetration and infection of apple shoots by Erwinia amylovora using green fluorescent protein (gfp) as a visual tag and as a "biosensor" to monitor the growth, movement and metabolism of the E. amylovora in apple. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints,
if known, to the adoption durability of the technology? A patent (US 6,235,683) was issued to cover a, "Method for enhanced supercooling of plants to provide frost protection." This technology was developed by ARS scientists in Kearneysville, WV through a CRADA with industry. This technology provides a novel method of frost protection for herbaceous plants based on the use of a hydrophobic particle film. A specific cooperative agreement with a commodity group was extended to assess the ability of genetic enhancement to improve stress tolerance in apple trees using anti-oxidant genes. The results of this research should be known in a few years and demonstrate to fruit tree growers the utility of using biotechnology in conjuction with traditional breeding. A CRADA with industry was extended to explore the use of natural compounds in consumer packaging to prolong storage life of purchased produce. This technology is expected to be available to the public in 2004. ARS scientists at
Kearneysville, WV met with commodity groups, attended regional project workshops, presented invited seminars both nationally and internationally, and presented research at national meetings of various professional societies on topics of biological control and stress tolerance in fruit crops. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) 1. Presentation: Inaugural Lecture at the XXIV National Biological Control Congress entitled "Development of Biocontrol Product BioSave for Control of Postharvest Decays of Fruits", Chihuahua, Mexico, 2001. 2. Presentation: Resistance of Geneva apple rootstocks to Erwinia amylovora when grown as vegetative shoots and orchard trees. At 9th International Workshop on Fire Blight, Napier, New Zealand (October, 2001) . 3. Presentation: Effect of Apogee dose level on fire blight shoot infection in young 'Gala' and 'York' apple
trees. At 77th Cumberland- Shenandoah Fruit Workers Conference, Winchester, VA (November, 2001). 4. Presentation: "Overexpression of antioxidant genes in apple for enhanced resistance to environmental stress," to the Washington State Tree Fruit Research Commission, Wenatchee, WA (January, 2002). 5. Poster Presentation: "Seasonal differences in transcript initiation from promoters of two peach dehydrin genes," at the Mid-Atlantic Society of Plant Molecular Biologists, Beltsville, MD, August, 2002. 6. Popular article on studying ice nucleation using infrared thermography appeared in Finnish popular science magazine (similar to Discovery). Article, entitled, "Kun halla vie syy on usein bakteerin," appeared in the magazine, Tiede (July, 2001). 7. Article on the development of "Biocure" a new product for postharvest decay control developed by ARS scientists at Kearneysville, WV and industry appeared in Agricultural Research (July, 2002).
Impacts
(N/A)
Publications
- Artlip, T., Wisniewski, M. Induction of proteins in response to biotic and abiotic stresses. Pessarakli, M. Editor. Marcel Dekker, Inc., New York, NY. Handbook of Plant and Crop Physiology. 2001. Chapter 33. p. 657-679.
- Bar-Shimon, M., Yehuda, H., Goldway, M., Wisniewski, M., Droby, S. Characterization of extracellular lytic enzymes produced by the postharvest biocontrol agent, Candida oleophila. XXVI International Horticultural Congress. 2002. Abstract p. 269.
- Bassett, C.L., Artlip, T.S., Callahan, A.M. Characterization of the peach homologue of the ethylene receptor, PpETR1, reveals some unusual features regarding transcript processing. Available from: http://link.springer. de/link/service/journals/00425/contents/tfirst.htm Planta online publications, original article. [2002]. Planta. v. 215. p. 679-688, DOI: 10.1007/s00425-002-0797-2
- Bassett, C.L., Artlip, T.S., Wisniewski, M.E. Discrete TATA boxes within the promoters of two peach dehydrin genes control differential expression. Available from: http://abstracts.aspb.org/pb2002/public/P30/0177.html Abstracts of the American Society of Plant Biologists. [2002].
- Carter, J., Brennan, R., Wisniewski, M. Patterns of ice formation and movement in blackcurrant. HortScience. 2001. v. 36. p. 1027-1032.
- Droby, S., Wisniewski, M., El Ghaouth, A., Wilson, C. Biological control of postharvest diseases of fruits and vegetables: Current achievements and future challenges. XXVI International Horticultural Congress. 2002. Abstract p. 273.
- Wang, Y., Wisniewski, M., Meilan, R., Fuchigami, L. Stress tolerance in transgenic tomato (Lycopersicon esculentum) seedlings that overexpress Mn- superoxide dismutase.. XXVI International Horticultural Congress. 2002. Abstract p. 137-138.
- Wang, Y., Wisniewski, M., Webb, R., Meilan, R., Fuchigami, L. Improved tolerance to salinity and high temperature in transgenic tomato overexpressing ascorbate peroxidase (APX). XXVI International Horticultural Congress. 2002. Abstract p. 139.
- Wilson, C. L. and El Ghaouth, A. Biological coating with a protective and curative effect for the control of postharvest decay. 2002. U. S. Patent 6, 423,310, B1.
- Wisniewski, M., Fuchigami, L., Wang, Y., Srinivasan, C., Norelli, J. Overexpression of a cytosolic ascorbate peroxidase gene in apple improves resistance to heat stress. XXVI International Horticultural Congress. 2002. Abstract p. 147.
- Wisniewski, M., Fuller, M., Glenn, D.M., Duman, J. Using infrared thermography to study ice nucleation and propagation in plants. XXVI International Horticultural Congress. 2002. Abstract p. 149.
- Wisniewski, M., Fuller, M., Glenn, D.M., Palta, J., Carter, J., Gusta, L., Griffith, M., Duman, J. Factors involved in ice nucleation in plants: An overview based on new insights gained from the use of infrared thermography. Buvisindi Icelandic Agricultural Science 2001. v. 14. p. 41- 47.
- Wisniewski, M., Glenn, D.M., Fuller, M.P. Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants. Journal of the American Society of Horticultural Science. 2002. v. 127. p. 358-364.
- Yehuda, H., Droby, S., Wisniewski, M., Goldway, M. A transformation system for the biocontrol yeast, Candida oleophila, based on hygromycin B resistance. Current Genetics. 2001. v. 40. p. 282-287.
- El Ghaouth, A. and Wilson, C.L. Protective and curative methods and compositions for biocontrol of plant postharvest decay. 2002. U.S. Patent 6,419,922. B1.
- El Ghaouth, A. Wilson, C.L., Wisniewski, M., Droby, S., Smilanick, J.L., Korsten, L. Biological control of postharvest diseases of citrus fruits. Gnanamanicham, S. S., editor. Marcel Dekker, Inc., New York . Biological Control of Crop Diseases. 2002. p. 289-312.
- Glenn, D.M., Wisniewski, M., Puterka, G.J., Sekutowski, D.G. Method for enhanced supercooling of plants to provide frost protection. 2001. U.S. Patent 6,235,683.
- Janisiewicz, W.J., Tworkoski, T., Kurtzman, C.P. Biological control potential of Metchnikowia pulcherrima strains against blue mold of apple. Phytopathology. 2001. v. 91. p. 1098-1108.
- Janisiewicz, W.J., Korsten, L. Microbial Control of Postharvest Diseases and Spoilage. Bredt, J., Bartz, J., editors. Marcel Decker, New York, NY. Postharvest Physiology of Vegetables. 2002. p. 543-562.
- Janisiewicz, W.J., Tworkoski, T. Role of sugars and the nutrients in germination of Penicillium expansum conidia with implication to biological control of postharvest decays on apple. Phytopathology. 2002. v. 92 (Suppl. 1). Abstract p. S-39.
- Ko, K., Norelli, J.L., Reynoird, J.P., Brown, S.K., Aldwinckle, H.S. T4 lysozyme and attacin genes enhance resistance of transgenic 'Galaxy' apple against Erwinia amylovora. Journal American Society for Horticultural Science. 2002. v. 127. p. 515-519.
- Leverentz, B., Janisiewicz, W.J., Conway, W.S., Saftner, R.A., Reed, A.N. Integrated strategy to control blue mold and bitter rot of 'Golden Delicious' apples. 2002. Phytopathology. v. 92 (Suppl. 1). Abstract p. S- 46.
- Vero, S., Mondino, P., Burgueno, J., Soubes, M., Wisniewski, M. Characterization of biocontrol activity of two yeast strains from Uruguay against blue mold of apple. Postharvest Biology and Technology. 2002. v. 26. p. 91-98.
|
Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
Postharvest losses due to decay of fruits during storage, shipping, marketing, and home consumption are significant and represent a financial burden to both growers and consumers. Furthermore, public concern over chemical residues, environmental concerns, and pathogen populations that are resistant to chemical fungicides have created a critical need for safe and effective alternatives. Biological approaches that utilize microbial antagonists and natural compounds are being developed to address these issues. A basic understanding of the mode of action of biocontrol agents will lead to the selection of more effective antagonists. Fire blight is one of the most serious and destructive diseases of apple and pear. To combat this disease there is a need to understand the basis of host resistance, the epidemiology of the disease, and develop non-chemical orchard and nursery management practices that will dramatically reduce the incidence of fire blight. Freeze damage to fruit and nut
trees often results in a significant loss in yield, fruit quality, or tree longevity. To address this problem, there is a need to develop a basic understanding of the biochemistry, physiology, and molecular biology of stress tolerance in fruit trees.
2. How serious is the problem? Why does it matter?
Several key synthetic fungicides have been targeted for withdrawal from the market because of their potential impact on human health and/or the costs involved in registering these products and demonstrating their safety. Some examples are ipridione used on stone fruit and benomyl compounds on pome fruit. Additionally, the development of fungicide resistance has made disease management more difficult. In the near future, some commodities may have no effective synthetic fungicides registered for postharvest application. Fire blight infections in newly planted orchards (1 to 5 years) can result in up to 80% tree loss causing devastating financial loss for growers. A single fire blight epidemic that occurred in southwest Michigan in 2000 was estimated to have caused the death of 350,000 to 450,000 trees and the removal of 1,500 to 2,300 acres of apple orchards with a total economic loss of 42 million dollars. The phytoxicity of copper and its limited efficacy, and resistance to
streptomycin, the two main methods of controlling fire blight, make the need to find new ways to manage fire blight critically important. Freezing temperatures are the prime factor affecting the production of perennial fruit and nut crops nationally. Estimates of annual losses in the U.S. range from millions of dollars for the citrus industry to nearly one billion dollars for all crops. A basic understanding of cold hardiness and stress tolerance can lead to the development of varieties that are resistant to extreme weather conditions and lead to new management tools that alleviate the impact of environmental stress on the performance of plants.
3. How does it relate to the National Program(s) and National Component(s)?
The goal of this research is to develop information on the identity and mode of action of biocontrol agents in order to make this strategy more effective and contributes to National Program 303: Plant Diseases (70%). This project also addresses the need to improve stress tolerance in plants and contributes to National Program 302: Improving Plant Biological and Molecular Processes (30%). It is recognized that this is an important need given the impact of environmental stress on crop production.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2001: Developing new approaches to disease control is essential in order to reduce the use of synthetic pesticides in fruit production. A naturally occurring antimicrobial peptide was identified in peach and apple bark tissues. The gene encoding this protein was cloned and placed in a yeast-expression system which secretes the protein into the culture medium and allows for the collection of enough protein to characterize its antimicrobial activity. The result of this research will lead to a more basic understanding of disease resistance in fruit crops and provide a method to genetically enhance disease resistance in commercial fruit varieties. B. Other Significant Accomplishments: 1. There is a need to develop safer methods of plant transformation that prevent the expression of foreign genes in plant parts consumed by people, which may greatly improve the willingness of the public to accept and consume improved horticultural crops
resulting from transgenic technology. Previous research discovered that the CAB (chlorophyll A-B binding protein) gene was expressed in vegetative tissues but absent in fruit tissue. Using this knowledge, researchers isolated the promoter responsible for CAB expression and joined it with a reporter gene, GUS, to verify whether the expression pattern of the CAB promoter/GUS gene follows the same pattern of expression in nature, i.e., only in vegetative tissues. This technology would reduce or eliminate the expression of the foreign genes in fruit tissue, ensuring that proteins produced by transgenic technology are not ingested by consumers. 2. Biological control, using naturally-occurring yeasts, is an alternative approach to the use of synthetic fungicides for the control of postharvest diseases of fruit. Several strains of the yeast, Metschnikowia pucherrima were identified and evaluated. Some of these strains provide excellent control of blue mold disease on apple. These strains
differ in a number of characteristics which should make it possible to select a strain that can be used commercially to control blue mold disease of apple under both fresh market and storage conditions. 3. Environmental stress limits the productivity of crop plants and often results in severe economic losses for fruit growers. In collaboration with Oregon State University, Corvallis, the antioxidant genes, SOD and APX (that scavenge free radicals), were overexpressed in tomato and apple plants. This research demonstrated increased resistance to several types of environmental stress, such as: cold and high temperature; UV-B exposure; and, salt. If further evaluation of the transgenics demonstrate that the overexpression of the APX and SOD genes are non-deleterious to other aspects of plant metabolism and productivity, these genes can be used to genetically enhance commercial varieties of horticultural crops for increased tolerance to environmental stress. 4. Reduction in the risk of
rot pathogens and human pathogens on harvested commodities is important for consumer confidence and safety. The use of combinations of natural products was evaluated for the control of microbial pathogens. Synergistic combinations of natural compounds were identified that could greatly reduce or eliminate plant and human microbial pathogens from the surface of fruits. This technology (patent pending) could potentially be used as a wash by commercial packing houses or consumers to improve the quality and safety of harvested commodities. 5. In order to identify effective microbial biocontrol agents, a better understanding of the traits that are responsible for bicontrol efficacy is needed. A chitinase gene obtained from the bacterium, Serratia marcescens, was successfully cloned into E. coli under a constitutive promoter and the resultant transformants secreted chitinase into the medium. Cloning of this gene into the biocontrol agent, Pseudomonas syringae, may enhance the biocontrol
activity and help to elucidate the role this gene plays in biocontrol activity. 6. Developing a basic understanding of the genetic and biochemical basis for stress tolerance is essential for producing plants with improved resistance to environmental stress. Several "stress tolerance" genes and their promoters, were cloned from peach and transferred into a model plant system. Their expression and function are now being studied. The results of this research should lead to a better understanding of how to improve stress tolerance in fruit crops through biotechnology. C. Significant Accomplishments/Activities that Support Special Target Populations: The accomplishments in biological control research especially impacts organic farms and sustainable agricultural systems.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
1. Due to changes in pesticide regulations and increased demand by consumers for a reduction in the use of pesticides, there is an urgent need to develop new methods of controlling postharvest diseases of fruit. ARS scientists at Kearneysville, WV discovered and patented two biocontrol agents that were commercialized as the products, 'Aspire' and 'Bio-Save', through a CRADA with industry. Subsequently, ARS scientists have found that the efficacy of yeast antagonists can be greatly enhanced by the addition of natural, bioactive compounds. This technology has been patented and is being commercialized via a CRADA with industry as a "bioactive coating" that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. 2. Food safety is of paramount importance to consumers, as well as growers and processors. An ARS scientist at Kearneysville, WV found that the antagonist P. syringae, previously identified and
commercialized to control postharvest diseases, can reduce the risk of potential contamination with E. coli O157:H7 on apples handled in water after harvest. This work not only revealed an unforeseen benefit of this postharvest biocontrol agent, but also opened a new area of research for reducing risk from food borne pathogens on intact and fresh cut fruits. 3. Methyl bromide is targeted for de-registration as a soil fumigant, so there is a critical need to find effective alternatives. ARS scientists at Kearneysville, WV determined that benzaldehyde and acetic acid are effective fumigants against four major soilborne plant pathogens: Sclerotia minor; Rhizoctonia solan; Pythium aphanidermatum; and, Fusarium oxysporum. 4. Fire blight is the most serious disease of apple and pear in the United States. An ARS scientist at Kearneysville, WV demonstrated that blighted summer prunings left on the orchard floor can serve as an active inoculum source for up to two months after pruning. An
ARS scientist verified the accuracy of the fire blight prediction model, MARYBLYT, to predict the occurrence of the blossom blight stage of the disease. These findings can be utilized as part of an integrated approach to manage fire blight. 5. Loss of horticultural crops due to late spring frosts is a serious problem that can result in severe economic losses for growers and higher prices for consumers. USDA-ARS scientists at Kearneysville, WV in collaboration with Engelhard, Inc. have developed patented technology for frost protection. The technology consists of the use of a hydrophobic clay material that is applied to the plant surface and blocks externally-induced freezing caused by the formation of ice on the plant surface. This technology represents a new approach to frost protection.
6. What do you expect to accomplish, year by year, over the next 3 years?
FY2002: 1. Determine the utility of using the CAB promoter as a promoter for regulating foreign gene expression in plants. If successful, this will provide a way of expressing foreign genes of interest in fruit trees without having expression of the gene product in fruit tissues eaten by the consumer. 2. Determine if increasing chitinase activity in a biocontrol agent will increase its effectiveness. A chitinase gene will be expressed in the biocontrol agent, Pseudomonas syringae, in order to determine if its effectiveness against postharvest pathogensis improved. The antimicrobial peptide, defensin, will be expressed in yeast cells in order to determine its ability to inhibit fruit tree pathogens. 3. Evaluate the use of natural compounds and biocontrol agents for the control of apple scab. 4. Determine the effect of combining biocontrol agents with the eradicative effect of heat treatments on controlling postharvest decay of pome fruits. 5. Determine the feasibility of a
plant growth regulator use strategy for newly planted apple orchards, whereby significant fire blight protection will be achieved in young trees without sacrificing tree growth. FY2003: 1. Determine if transgenic apple tree overexpressing stress tolerance genes such as ascorbate peroxidase, dehydrin, and superoxide dismutase have increased resistance to environmental stress. 2.) ARS scientists will have developed and commercialized a "second generation" of biocontrol agents based on combining biocontrol agents with natural compounds, or using mixtures of different species of biocontrol organisms. The second generation products will have improved performance over the first generation of products developed by ARS in collaboration with industry partners. 3. Develop a biocontrol system for decentralized fruit production in small packinghouses, where the drenching suspension is kept for a long period of time, i.e., up to seven days. Various modifications will be introduced in a
bacterial chitinase gene by ARS scientists in order to attempt to improve its ability to digest chitin (a major cell wall component of rot pathogens). The expression of the modified chitinase gene will be tested in a strain of Pseudomonas syringae used as a biocontrol agent. 4. Improve the ability to predict and control fire blight on apple by determining what role populations of Erwinia amylovora (the agent that causes fire blight) growing on the surface of leaves and shoots, play in initiating the shoot blight phase of the disease, and determining how environmental factors affect fire blight bacteria populations on shoots. FY2004: 1. Clone the genes encoding the rate-limiting enzymes for the synthesis of sorbitol and starch in apple, down-regulate the expression of these genes in apple using sense and/or antisense transgenic gene expression in order to determine the role of sorbitol and osmotic potential in the susceptibility of apple to fire blight. Further evaluation of
transgenic apples overexpressing antioxidant genes will be made in the orchard.
7. 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 & durability of the technology product?
1. A specific cooperative agreement has been established with a commodity group to assess the ability of genetic enhancement to improve stress tolerance in apple trees using anti-oxidant genes. The results of this research should be known in a few years and demonstrate to fruit tree growers the utility of using biotechnology in conjunction with traditional breeding. 2. A trust agreement was established with a commodity group to determine the role of epiphytic bacteria in the shoot blight phase of fire blight. The results of this research should be known in approximately one year and be available for use by the commodity group and other fruit growers, extension personnel, and scientists. 3. One CRADA was extended with its industry partner to commercialize ARS patented technology for postharvest biocontrol based on the use of combining a yeast and bioactive compound (lysozyme and/or chitosan). The company is seeking EPA registration and a commercial product should be available in
2002-2003. 4. An ARS scientist developed a Material Transfer Agreement with industry to evaluate a biocontrol agent against postharvest decay of fruits. 5. A CRADA was developed with industry to explore the use of natural compounds in consumer packaging to prolong storage life of purchased produce. This technology is expected to be available to the public in 2004. 6. ARS scientists at Kearneysville, WV have met with commodity groups, attended regional project workshops, presented invited seminars both nationally and internationally, and presented research at national meetings of various professional societies on topics of biological control and stress tolerance in fruit crops. 7. An ARS scientist at Kearneysville, WV has also served as an Associate Editor for the Journal of the American Society for Horticultural Science, and Chair of the Binational Agricultural Research and Development grant panel for Fruit Crops.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
1. Presentation: Microbial and Decay Control in Packaging Using Natural Compounds. Given to representatives of Clorox, Inc. at USDA-ARS, Kearneysville, WV. March 2001. 2. Presentation: Historical Perspective in Developing Biocontrol of Postharvest Decays of Fruit and The Next Generation of Biocontrol Against Postharvest Fruit Decay. EcoScience, FL. June 2001. 3. Presentation: Fire Blight and Rootstocks - A Look at Resistance, and, Genetic Engineering to Solve Plant Pest Problems: Benzie-Mainistee Horticultural Show, Thompsonville, MI. March 2001.
Impacts
(N/A)
Publications
- Bassett, C.L., Janisiewicz, W. Electroporation and Stable Maintenance of a copy Plasmid in a Pseudomonas syringae Post-harvest Biocontrol Strain. Proceedings of the 76th Annual Cumberland-Shendandoah Fruit Workers Conference. 2001. p. 119-127.
- Bassett, C.L., Nickerson, M.L., Cohen, R.A., Rajeevan, M.S. Alternative Transcripts and Novel Post-transcriptional Regulation of a Gene Encoding a Leucine-rich Repeat Receptor-like Protein Kinase that Responds to Short Day Photoperiodic Floral Induction in Morning Glory (Ipomoea nil). Plant Molecular Biology. 2000. v. 43. p. 43-58.
- Bassett, C.L. The Molecular Biology of Plant Hormone Reception. Horticultural Reviews. 2001, v. 26. p. 49-84.
- Glenn, D.M., Scorza, R., Bassett, C.L. Physiological and Morphological Traits Associated with Increased Water Use Efficiency in the Willow-leaf Peach. HortScience. 2000. v. 35. p. 1241-1243.
- Janisiewicz, W.J., Tworkoski, T., Sharer C. Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients. Phytopathology. 2000. v. 90. p. 1196-1200.
- Wang, Y., Boyer, C., Cheng, L., Fuchigamil, L., Wisniewski, M., Meilan, R., Dandekar, A. Increased resistance to oxidative stress in transgenic tomatoes (Lycopersicon esculentum) that overexpress MN-superoxide dismutase from rubber tree (Hevea brasiliensis). HortScience. 2001. v. 36. Abstract p. 492.
- El Ghaouth, A., Smilanick, J., Brown, E., Wilson, C. Control of decay of apple and citrus fruit in pilot-plant with Canida saitoana and 2-deoxy-D-glucose. Biological Control 2001. v. 20. p. 96-101.
- Leverentz, B., Janisiewicz, W.J., Conway, W.S., Safner, R.A., Fuchs, Y., Sams, C.E., Camp, M.J. Combining yeasts or a bacterial biocontrol agent and heat treatment to reduce postharvest decay of 'Gala' apples. Postharvest Biology and Biotechnology. 2001. v. 21. p. 87-94.
- Leverentz, B., Conway, W.S., Alavidze, Z., Janisiewicz, W.J., Fuchs, Y., Camp, M.J. Sulakvelidze, A. Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruit: a model study. 2001. Journal of Food Protection. 2001. v. 64. p. 1116-1121.
- Ippolitto, A., El Ghaouth, A., Wisniewski, M., Wilson, C. Control of apple decay by Aureobasidium pullans and induction of resistance responses. Postharvest Biology and Technology. 2000. v. 19. p. 265-272.
- Sutinen, L., Arora, R., Wisniewski,M., Ashworth, E., Strimbeck, R., Palta., J. Mechanisms of frost survival and freeze damage in nature. Bigras, F.J., Colombo, S.J., editors., Kluwer Academic Press, Dordrecht, Netherlands. Conifer Cold Hardiness. 2001. p. 89-120.
- Droby, S., Wilson, C., Wisniewski, M., El Ghaouth, A. Biologically Based Technology for the Control of Postharvest Disease of Fruits and Vegetables. Wilson, C., Droby, S. editors. CRC Press, Boca Raton, FL. Microbial Food Contamination. 2001. p. 187-206.
- Janisiewicz, W.J. Next generation of biological bicontrol of postharvest diseases of fruits. Phytopathology. 2001. v. 91. p. 154.
- Janisiewicz, W.J., Tworkoski, T.J., Kurtzman, C.P. Phenotypic, genetic and functional characterizing of Metchnikowia pulcherrima strains that control blue mold of apple. Phytopathology. 2001. v. 91. p. 43.
- Leverentz, B., Janisiewicz, W.J., Conway, W.S., Saftner, R.A. Effect of combining biocontrol, heat treatment, and MCP-treatment on the reduction of postharvest decay of 'Delicious' apples. Phytopathology. 2001. v. 91. p. 55.
- Bassett, C.L., Janisiewicz, W.J. Stability of E. coli/Pseudomonas shuttle vector in a biocontrol strain of Pseudomonas syringae. Phytopathology. 2001. v. 91. p. 7.
- Wisniewski, M. Extrinsic ice nucleation in plants: What are the factors involved and can they be manipulated. Proceedings of the 6th International Plant Cold Hardiness Seminar. 2001. p. 8-9.
- Wang, Y., Boyer, C., Cheng, L., Fuchigamil, L., Wisniewski, M., Meilan, R., Dandekar, A. Overproduction of pea (Pisum sativum) cytosolic ascorbate peroxidase gene in tomato provides protection against oxidative stress. HortScience. 2001. v. 36. Abstract p. 562.
|
Progress 10/01/99 to 09/30/00
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
Postharvest losses due to decay of fruits during storage, shipping, marketing, and home consumption are significant and represent a financial burden to both growers and consumers. Furthermore, public concern over chemical residues, environmental concerns, and pathogen populations that are resistant to chemical fungicides have created a critical need for safe and effective alternatives. Biological approaches that utilize microbial antagonists and natural compounds are being developed to address these issues. A basic understanding of the mode of action of biocontrol agents will lead to the selection of more effective antagonists. Fire blight is one of the most serious and destructive diseases of apple and pear. To combat this disease there is a need to understand the epidemiology of the disease and develop non-chemical orchard and nursery management practices that will dramatically reduce the incidence of fire blight. Freeze damage to fruit and nut trees often results in a
significant loss in yield, fruit quality, or tree longevity. To address this problem, there is a need to develop a basic understanding of the biochemistry, physiology, and molecular biology of stress tolerance in fruit trees.
2. How serious is the problem? Why does it matter?
Several key synthetic fungicides have been targeted for withdrawal from the market because of their potential impact on human health and/or the costs involved in registering these products and demonstrating their safety. Some examples are ipridione used on stone fruit and benomyl compounds on pome fruit. Additionally, the development of fungicide resistance has made disease management more difficult. In the near future, some commodities may have no effective synthetic fungicides registered for postharvest application. In many years losses due to fire blight can range from 20 to 100%. The phytoxicity of copper and resistance to streptomycin, the two main methods of controlling fire blight, make the need to find new ways to manage fire blight critically important. Freezing temperatures are the prime factor affecting the production of perennial fruit and nut crops nationally. Estimates of annual losses in the U.S. range from millions of dollars for the citrus industry to nearly one
billion dollars for all crops. A basic understanding of cold hardiness and stress tolerance can lead to the development of varieties that are resistant to extreme weather conditions and lead to new management tools that alleviate the impact of environmental stress on the performance of plants.
3. How does it relate to the National Program(s) and National Component(s)?
The goal of this research is to develop information on the identity and mode of action of biocontrol agents in order to make this strategy more effective and contributes to National Program 303: Plant Diseases. This project also addresses the need to improve stress tolerance in plants and contributes to National Program 302: Improving Plant Biological and Molecular Processes. It is recognized that this is an important need given the impact of environmental stress on crop production.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2000 year: Better methods of protecting fruit blossoms and tender plants from killing frosts are needed to prevent the significant economic losses experienced by growers. ARS scientists in Kearneysville, WV, in collaboration with a CRADA partner, developed a method of blocking ice formation in plants that is induced by external agents (moisture, frost, bacteria, etc.). A patent was filed for a formulation of hydrophobic kaolin (particle film) that when applied to plant surfaces prevents moisture from collecting on the plant surface which would freeze and then cause the plant to freeze. This technology may provide a new method of frost protection that is both effective and economic. B. Other Significant Accomplishments: 1. Genetic enhancement of biocontrol agents with specific genes may enhance their performance. ARS scientists at Kearneysville, WV developed a reproducible and efficient transformation system for the wild strain of
a bacterium, Pseudomonas syringae [L59-66]) which is presently registered for controlling postharvest diseases of pome fruits. Using this method, the effectiveness of this biocontrol strain can be improved which will in turn reduce the use of chemical pesticides to control postharvest diseases of fruit. 2. Controlling expression of foreign genes in transgenic plants so that they are expressed only at certain times (e.g, during pathogen attack) or only in specific tissues (e.g., only in leaf tissue but not in fruit) is necessary to insure consumer safety regarding edible plant parts. An ARS scientist at Kearneysville, WV designed a gene promoter system based on the photosynthetic-specific promoter, chlorophyll a/b-binding protein (cab), to prevent expression of certain foreign genes in mature tree fruit. The cab promoter can be used to obtain genetically engineered plants which are safer for the consumer. 3. ARS scientists in Kearneysville, WV and ARO, Bet Dagan, Israel identified
novel, antifungal materials derived from cell walls from a variety of yeast species. The material was demonstrated to be antifungal against a wide range of plant pathogenic fungi. The compatibility of this material with the commercial biocontrol product, based on an antagonistic yeast, was demonstrated and improved the efficacy of the product. 4. Development of new approaches to controlling postharvest and foodborne diseases in harvested fruit is critical because of the continuing loss of key chemical fungicides due to regulatory scrutiny. ARS scientists at Kearneysville have discovered that combinations of certain essential oils of plants give synergistic control of postharvest decay organisms and foodborne human pathogens. These combinations of natural compounds may serve as an effective commercial alternative to synthetic fungicides. 5. Increasing the effectiveness of biocontrol agents is essential if this approach is going to become a commercially acceptable practice. ARS
scientists in Kearneysville, WV and Beltsville, MD found that treating wounded apples with hot air (38 C for 4 days) and biocontrol agents after harvest can reduce fruit decay originating from wounds without compromising storage life of fruit. Combining these two non-chemical methods further increased efficacy of the biocontrol agent and may make this approach more competitive with chemical control. 6. Developing a basic understanding of genetic and biochemical basis for stress tolerance is essential for producing plants with improved resistance to environmental stress and for the development of management strategies that alleviate stress. An ARS scientist in Kearneysville, WV and collaborating scientists at the University of Wisconsin, University of Minnesota, University of Saskatchewan, and Notre Dame University, have used infrared thermography to develop a detailed understanding of the freezing process of plants and the role of antifreeze proteins. This information can be used by
other scientists and for the development of new methods of frost protection. An aspect of this research was awarded the Outstanding Cross-Commodity Publication 2000, by the American Society for Horticultural Science.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
1. Due to changes in pesticide regulations and increased demand by consumers for a reduction in the use of pesticides there is an urgent need to develop new methods of controlling postharvest diseases of fruit. ARS scientists at Kearneysville, WV discovered and patented a bioactive coating that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. This technology is being commercialized and will serve as an alternative to chemical control for the management of postharvest diseases of fruit. 2. Food safety is of paramount importance to consumers, as well as growers and processors. An ARS scientist at Kearneysville, WV found that the antagonist P. syringae can reduce the risk of potential contamination with E. coli O157:H7 on apples handled in water after harvest. This work not only revealed an unforeseen benefit of this biocontrol agent, which was developed to control postharvest decays of fruits, but also
opened a new area of research for reducing risk from foodborne pathogens on intact and fresh cut fruits. 3. Methyl bromide is targeted for de-registration as a soil fumigant. So there is a critical need to find effective alternatives. ARS scientists at Kearneysville, WV determined that benzaldehyde and acetic acid are effective fumigants against four major soilborne plant pathogens (Sclerotia minor, Rhizoctonia solani, Pythium aphanidermatum, and Fusarium oxysporum). 4. Fire blight is the most serious disease of apple and pear in the United States. An ARS scientist at Kearneysville, WV demonstrated that blighted summer prunings left on the orchard floor can serve as an active inoculum source for up to two months after pruning. An ARS scientist verified the accuracy of the fire blight prediction model, MARYBLYT, to predict the occurrance of the blossom blight stage of the disease. These findings can be utilized as part of an integrated approach to manage fire blight. 5. There are
considerable reductions in fruit yield and quality, and tree longevity directly caused by environmental stresses and plant diseases. ARS scientists at Kearneysville, WV have isolated genes from apple and peach trees that are associated with stress tolerance and disease resistance in peach. These genes can be evaluated as potential candidates for the genetic enhancement of stress tolerance in fruit trees and reduce economic losses.
6. What do you expect to accomplish, year by year, over the next 3 years?
FY2001. ARS scientists at Kearneysville, WV will determine the utility of using the cab promoter as a promoter for regulating foreign gene expression in plants. If successful, this will provide a way of expressing foreign genes of interest in fruit trees without having expression of the gene product in fruit tissues eaten by the consumer. ARS scientists will determine if increasing chitinase activity in a biocontrol agent will increase its effectiveness. FY2002. Various antimicrobial genes will be expressed in the biocontrol agent, Pseudomonas syringae, in order to improve its effectiveness against postharvest pathogens. The antimicrobial peptide, defensin, will be expressed in yeast cells and plants in order to determine its ability to inhibit fruit tree pathogens and increase disease resistance, respectively. ARS scientists will evaluate the use of natural compounds and biocontrol agents for the control of apple scab. FY2003. ARS scientists in Kearneysville, WV will determine if
transgenic apple trees overexpressing stress tolerance genes such as ascorbate peroxidase, dehydrin, and superoxide dismutase have increased resistance to environmental stress. ARS scientists will have developed and commercialized a second generation of biocontrol agents based on combining biocontrol agents with natural compounds or using mixtures of different species of biocontrol organisms. The second generation products will have improved performance over the first generation of products developed by ARS in collaboration with industry partners. ARS scientists will develop a biocontrol system for decentralized fruit production in small packinghouses where the drenching suspension is kept for up to seven days
7. 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 & durability of the technology product?
Two CRADAs have been established to either improve currently licensed ARS biocontrol technology or commercialize new ARS biocontrol technology. A Specific Cooperative Agreement has been established with a commodity group to assess the ability of genetic enhancement to improve stress tolerance in apple trees using anti-oxidant genes. Meetings were held with industry to assess the potential of new CRADAs to commercialize ARS-developed technologies based on the use of natural compounds to control postharvest diseases of fruit. ARS scientists at Kearneysville, WV have met with commodity groups, attended regional project workshops, presented invited seminars both nationally and internationally, and presented research at national meetings of various professional societies on topics of biological control and stress tolerance in fruit crops. An ARS scientist at Kearneysville, WV has also served as an Associate Editor for the Journal of the American Society for Horticultural Science, and
Chair of the Binational Agricultural Research and Development grant panel for Fruit Crops.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
1. Bio-Save. EcoScience Product System Division Technical Update December 1999. v.1 Issue 6. Industry and producers. 2. Workmaster, B., Palta, J., Wisniewski, M. Observing How Cranberry Plants Freeze. Cranberries. 2000. v.64(1). p.14-19. Trade journal. 3. Presentation: Stress Tolerance in Fruit Trees: Washington State Tree Fruit Commission. Wenatchee, WA. December 1999. 4. Presentation: Microbial and Decay Control in Packaging Using Natural Compounds: Kraft Foods and DuPont, Chicago, IL. November 1999.
Impacts
(N/A)
Publications
- Stevens, C., Kahn, V.A., Lu, J.Y., Wilson, C.L., Chalutz, E., Droby, S., Kabwe, M.K., Hung, Z., Adeyeye, O., Pusy, L.P., Tang, A.Y. A. Induced resistance of sweetpotato to Fusarium root rot by UV-C hormesis. Crop Protection. 1999. v. 18. p. 463-470.
- El Ghaouth, A., Smilanick, J.L., Wilson, C.L. Enhancement of the performance of Candida saitoana by the addition of glycochitosan for the control of postharvest decay of apple and citrus fruit. Postharvest Biology and Technology. 2000. v. 19. p. 103-110.
- Janisiewicz, W.J., Conway, W.S., Leverentz, B. Biological control of apple decay can prevent growth of Escherichia coli O157:H7 in apple wounds. Journal of Food Protection. 1999. v. 62. p. 372-1375.
- Conway, S W., Leverentz, B., Janisiewicz, W. J., Saftner, R.A., Sams, C.E. Survival and growth of Listeria monocytogenes on fresh-cut apple slices and its interaction with Glomerella cingulata and Penicillium expansum. Plant Disease. 2000. v. 84. p. 177-181.
- Wisniewski, M., Fuller, M. Ice nucleation and deep supercooling in plants: new insights using infrared thermography. In: Cold-Adapted Organisms: Ecology, Physiology, Enzymology, and Molecular Biology. R. Margesin and F. Schinner, editors. Springer-Verlag. 2000. p. 105-118.
- Wisniewski, M., Arora, R. Structural and biochemical aspects of cold hardiness in woody plants. In: Molecular Biology of Woody Plants, Vol. 2. S. Mohan Jain and S. Minocha, editors. Kluwer Academic Press. 2000. p. 419-438.
- Workmaster, B., Palta, J., Wisniewski, M.. Ice nucleation and propagation in cranberry uprights and fruit using infrared video thermography. Journal of American Society of Horticultural Science. 1999. v. 124. p. 619-625.
- Carter, J., Brennan, R., Wisniewski, M. Low-temperature tolerance of blackcurrant flowers. HortScience 1999. v. 34. p. 855-859.
- Janisiewicz, W. J. Biological Control of Blue-mold and Gray-mold of Pome Fruits with Natural Antagonists: Framework of a Successful Approach. p.147-150 in 10th International Symposium on Biocontrol, April 21-23, 1999, Skierniewice, Poland. Bull. Polish Academy of Sciences, Biological Sciences. 1999. v. 47. p. 2-4.
- Janisiewicz, W. J. Biological control of fungal and bacterial pathogens with Pseudomonas syringae and challenges for the future in the field of postharvest diseases. 9th European Conference on Biotechnology, July 11-15, 1999, Brussels, Belgium. 2000. ISBN 805215-1-5 (C) 1999-2000 Branche Belge de la Societe de Chimie Industrielle M164.
- Janisiewicz, W.J., Conway, W.S., Leverentz, B. Reducing the risk of Escherichia coli O157:H7 contamination of apple by an antagonist used to control postharvest decay of pome fruits. Phytopathology. 1999. v. 89. p. S36.
- Conway, W.S., Janisiewicz, W.J., Sams, C.E., Leverentz, B., Klein J.D. Integration of postharvest control strategies to reduce decay of 'Gala' apples. Phytopathology. 1999. v. 89. p. S18.
- Janisiewicz, W.J., and Tworkoski. T.J. A novel plate insert method for studying mechanisms of biological control of postharvest diseases of fruits. Phytopathology. 2000. v. 90. p. S39.
- Janisiewicz, W. J., Conway, W.S., Tworkoski, T.J., Leverentz, B., Sams, C.E. 2000. Improving Control of Postharvest Decays of Pome Fruits by Manipulating a Biocontrol System and Combining it with Non-fungicidal Methods. Abstracts of 4th International Conference on Postharvest Science: 4. Jerusalem, Israel. March 26-31, 2000. p. 4.
- Leverentz, B., Conway, W.S., Janisiewicz, W.J., Saftner, R.A., Fuchs, Y., Sams, C.E. Effect of combining biocontrol and heat treatment to reduce postharvest decay of Gala apples. Phytopathology. 2000. v. 90. p. S46.
- Wisniewski, M. Non-chemical approaches to postharvest disease control. Abstracts of 4th International Conference on Postharvest Science: 4. Jerusalem, Israel. March 26-31, 2000. p. 19.
- Segal, E., Yehuda, H., Droby, S., Goldway, M., Wisniewski, M. Cloning of B-1,3- glucanase of the yeast biocontrol agent, Candida oleophila. Abstracts of 4th International Conference on Postharvest Science: 4. Jerusalem, Israel. March 26-31, 2000. p. 62.
- Wisniewski, M., Fuchigami, L. Identification and partial characterization of seasonally-regulated proteins in apple bark tissues. HortScience. 2000. v. 35. p. 402.
- Wisniewski, M, Glenn, M., Fuller, M. The use of hydrophobic clay films as a barrier to ice nucleation in plants. HortScience. 2000. v. 35. p. 423.
|
Progress 01/01/99 to 09/30/99
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Postharvest losses due to decay of fruits during storage, shipping, marketing, and home consumption are significant and represent a financial burden to both growers and consumers. Synthetic fungicides are currently the major method of dealing with this problem. Public concern over safety, environmental concerns, and pathogen populations that are resistant to chemical fungicides, however, have created a critical need for safer alternatives. A major objective of this project is to produce safe, viable, and effective methods of controlling postharvest diseases through the use of biocontrol agents and natural compounds. While two successful biocontrol products (BioSave 110 - based on the bacterium, Pseudomonas syringae, and Aspire - based on the yeast, Candida oleophila) have been developed in this lab, current research is focused on developing "second generation" products utilizing
antagonistic mixtures, combining biocontrol agents with natural compounds and other additives, and developing a systems approach to managing postharvest diseases. Identification of genes involved in biocontrol activity and their utilization to genetically enhance microbial antagonists is another approach. The ability to use natural compounds as alternatives to methyl bromide is also being investigated. Fire blight is one of the most serious and destructive diseases of apple and pear. The major method of controlling this disease is through the use of copper compounds and streptomycin. The excessive use of the latter has resulted in resistance. The recent acceptance of cultivars that are highly susceptible to fire blight has also exacerbated the problem. Studies are designed to focus on the epidemiology of the disease in order to develop non-chemical orchard and nursery management practices that will dramatically reduce the incidence of fire blight. Although agricultural crops
have the genetic potential for high yield, there is typically a many-fold shortfall between potential and actual yields as a direct result of environmental stress. In particular, freeze damage to fruit and nut trees often results in a significant loss in yield, quality, or tree longevity. To address this problem we are identifying genes and proteins that are associated with stress tolerance. The objective of this approach is to determine the function of these proteins and genes so that they can be used as selection markers in breeding programs and for mapping stress tolerance in genome-mapping projects. Development of transgenic plants with increased stress and disease tolerance is also a goal of this project. Therefore, identification of tissue-specific promoters is also being pursued. Lastly, infrared thermography is being used to study ice nucleation in plants in order to develop new approaches to frost protection. 2. How serious is the problem? Why does it matter? Synthetic
fungicides have provided a major means of controlling postharvest decay of fruits and vegetables, however, many of these fungicides are being withdrawn from the market due to concerns over health and environmental impact. Public demand has created a need for safe and effective alternatives. Additionally, the development of fungicide resistance in pathogens, such as Penicillium and Botrytis, has made disease management more difficult. Chemical companies are reluctant to expend the capital necessary to develop and register new fungicides. In the near future, some commodities may have no effective synthetic fungicides registered for postharvest application. The situation is particularly acute in the case of apple and pear where only a few compounds are registered for postharvest use and their effectiveness is questionable. Recently, it was stated that: Overwhelmingly, the most significant problem for the pear industry was decay in stored fruits. The situation in apple packing houses
is also critical. All the above factors emphasize the need to find safe and effective methods of managing postharvest diseases. In many years losses due to fire blight can range from 20 to 100%. The phytoxicity of copper and resistance to streptomycin, the two main methods of controlling fire blight, make the need to find new ways to manage fire blight critically important. Freezing temperatures are the prime factor affecting the production of perennial fruit and nut crops nationally. Cold hardiness is a highly dynamic plant condition and air temperature is one of the least predictable climatic variables. Thus freezing temperatures often cause devastating losses. Estimates of annual losses in the U.S. range from millions of dollars for the citrus industry to nearly one billion dollars for all crops. Interactions between various plant factors and environmental conditions produce many different kinds of freezing stress. This emphasizes the need for continuing basic research on
the biochemical and genetic regulation of stress tolerance in fruit trees. Concern over the consumption of genetically-enhanced food products, makes the identification of tissue-specific promoters essential if transgenic fruit trees with enhanced disease and stress tolerance are to be acceptable to consumers. 3. How does it relate to the National Program(s) and National Component(s) to which it has been assigned? This research is embodied in Crop Protection, Product Value, and Safety, Section 303 - Plant Diseases, which states: The overall goal of the Plant Disease National Program is to develop and improve ways to reduce losses caused by plant diseases. In the subsection Problems to be Addressed, it states: There is a need for information on the identity and mode of action of biocontrol agents in order to make this strategy more effective and reliable. This project also directly addresses the need to improve stress tolerance in plants (National Program 302, Improving Plant
Biological and Molecular Processes). It is recognized that this is an important need given the impact of environmental stress on crop production. 4. What were the most significant accomplishments this past year? Antagonist mixture with superior biocontrol potential compared to the use of individual antagonist have been identified by an ARS scientist in Kearneysville, WV. The procedure includes isolation of the antagonists colonizing fruit tissue in the orchard before harvest, determining nutritional profile of the antagonists, and pairing antagonists, preferably from the same isolation site. Additional factors under consideration in refining the procedure include production of the killer toxins and vitamin requirements of the yeasts. An ARS scientist in Kearneysville, WV found that the beneficial bacterium P. syringae developed commercially for control of postharvest decay of apple also prevents growth of foodborne pathogen E. coli O157:H7 on wounded apple tissue. This bacterium
was effective when co-inoculated or inoculated 24 and 48 hours prior to inoculation with E. coli O157:H7. We have a strong indication that some of our beneficial yeasts are also effective. ARS scientists in Kearneysville, WV and Beltsville, MD, have developed & integrated strategy to control blue mold decay of Gala' apples after harvest which combines heat, calcium and beneficial bacterium Pseudomonas syringae treatments. The recommended procedure is to apply heat (38 C for four days), followed by calcium infiltration (CaCl2 2%) and beneficial bacterium application at commercial rate. An ARS scientist developed and tested a "bioactive coating" for fruits & vegetables that reduced decay and had an "eradicant action." Previous biofungicides did not have an eradicant action. ARS scientists at Kearneysville, WV and Beltsville, MD made the discovery that acetic acid fumes are effective in killing major soilborne pathogens (Sclerotinia minor, Rhizoctonia solani, Pythium aphanidermatum,
and Fusarium oxysporum). An ARS scientist in Kearneysville, WV recognized two new symptoms in fire blight epidemiology: an extremely late season shoot blight, termed autumnal shoot blight, which was associated with wet weather conditions that enhanced vegetative growth, the other a delayed expression of canker blight, termed latent canker blight, which is caused by endophytic E . amylovora bacteria traveling from a canker source to the succulent shoots along the periphery of the tree. This latter finding may explain the shoot blight outbreaks often observe by growers during the mid growing season. ARS scientists in Kearneysville, WV identified bacterial isolates that produce and have chitinase activity at low temperatures (1 - 4C). These scientists also obtained chitinase genes and laid the groundwork for transformation of Pseudomonas spp. An ARS scientist at Kearneysville, WV purified plasmids containing two versions of a chlorophyll a/b-binding protein promoter that will eventually
be tested as a tissue-specific promoter. An ARS Scientist at Kearneysville, WV identified two defense-related genes in peach and apple and characterized their seasonal expression. These genes will be used in transgenic apple plants to attempt to increase disease resistance. This scientist also identified and partially cloned two genes in peach that are seasonally-regulated and may be involved in the regulation of dormancy and/or cold hardiness. ARS scientists in Kearneysville, WV filed for a patent demonstrating the ability of using novel film-forming materials as a method of blocking ice nucleation and hence providing frost protection. An ARS scientist in Kearneysville, WV transformed "Royal Gala" apple with a peach dehydrin gene and ascorbic peroxidase gene for increased resistance to environmental stresses. Putative transgenic plants are now growing in antibiotic selection, tissue culture media. 5. Describe the major accomplishments over the life of the project including their
predicted or actual impact. ARS scientists at Kearneysville, WV discovered and patented a "bioactive coating" that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. It is anticipated that this technology will be transferred to a private company, Micro Flo, which plans to have a products on the market within 2 years. ARS scientists at Kearneysville, WV determined that benzaldehyde and acetic acid are effective fumigants against four major soilborne plant pathogens (Sclerotinia minor, Rhizoctonia solani, Pythium aphanidermatum, and Fusarium oxysporum). An ARS scientist at Kearneysville, WV found that the antagonist P. syringae can reduce the risk of potential contamination with E.coli O157:H7 on apples handled in water after harvest. This work revealed an unforeseen benefit of this biocontrol agent, which was developed to control postharvest decays of fruits. An ARS scientist at Kearneysville, WV demonstrated
that blighted summer prunings left on the orchard floor can serve as an active inoculum source for up to two months after pruning. An ARS scientist verified the accuracy of the fire blight prediction model, MARYBLYT, to predict the occurrence of the blossom blight stage of the disease. These findings can be utilized in an integrated program to manage fire blight. ARS scientists at Kearneysville, WV have demonstrated the ability to use "novel film-forming materials" as a means of blocking ice nucleation in plants thereby providing frost protection. Genes involved in stress tolerance and disease resistance were isolated from peach and apple. ARS scientists are now producing transgenics that overexpress these genes. These accomplishments may lead to transgenic fruit trees with improved stress tolerance and also provide a new method of frost protection. 6. What do you expect to accomplish, year by year, over the next 3 years? During FY2000, ARS will develop and patent a "bioactive
coating" that can serve as an effective alternative to synthetic fungicides for the control of postharvest diseases of pome and citrus fruit. It is anticipated that this technology will be developed and commercialized in cooperation with a private company, Micro Flo, during FY 2000 and FY 2001. It is anticipated that a product will be on the market by 2002. During FY99, ARS determined that benzaldehyde & acetic acid are effective fumigants against four major soilborne plant pathogens (Sclerotinia minor, Rhizoctonia solani, Pythium aphanidermatum, and Fusarium oxysporum). During FY2000, research will be conducted to determine an effective means of applying these natural soil fumigants to soil and testing their efficacy. It is expected that large field plot testing will be conducted during FY2001 and FY2002. During FY2000, ARS will screen a number of natural compounds and biocontrol agents for control of apple scab. During FY2000, ARS will identify the most effective natural compounds
and microbial agents and begin field testing of natural fungicides against apple scab Venturia inaequalis). During FY2000, ARS will continue work on developing second generation biocontrol products based on the use of mutually compatible antagonist mixtures with superior biocontrol potential. During FY2001 and FY2002, these mixtures will be tested under packinghouse conditions and their performance on specific commodities and specific packinghouse conditions will be determined. During FY 2000, ARS will also conduct work on combining biocontrol with other non-fungicidal methods, such as heat treatment, which by themselves do not provide adequate control but when combined with biocontrol may increase total performance of the system. Developing an integrated approach to postharvest disease control will continue into FY2001 and FY2002. During FY 2000, ARS will continue work on developing a model system for testing importance of various biocontrol traits in our postharvest fruit system
by developing a transformation system for the biocontrol bacterium Pseudomonas. During FY2001, and FY 2002, various genes will be inserted into Pseudomonas in order to determine their effect on biocontrol activity. During FY2000, ARS will use a portable drench unit to develop a biocontrol system for decentralized fruit production in small packing houses where the drenching suspension is kept for up to 7 days. This system will be further tested in FY2001 and recommendations made at the end of FY2002. In FY2000 & FY2001, ARS will establish cycle of blooming crabapple trees in the greenhouse to perform various tests with clay particles and bacterial antagonists to test their efficacy in reducing fire blight. ARS will also continue verification of latent canker blight symptomatology and in order to include this symptom of fire blight into the Maryblyte prediction system. In FY2002, ARS will integrate all the positive methods of fire blight reduction or control in order to make a
recommendation on a comprehensive management program for the control of fire blight. In FY2000, ARS will produce transgenic apple trees overexpressing two stress tolerance genes, dehydrin and ascorbate peroxidase. Laboratory testing of these transgenics will be conducted during FY2001, and field testing in FY2002. In FY2000, ARS will continue to isolate stress tolerance and disease resistance genes from apple and continue to work on decoding expressed sequence tags in peach and apple. In FY2001, genes of interest will be inserted into apple and the resulting transgenics will be evaluated for increased resistance to abiotic and biotic stress in FY2002. In FY2000, ARS will construct two versions of a tissue-specific gene promoter (chlorophyll a/b binding protein gene promoter) and test their expression. In FY2000, ARS will continue to evaluate the ability of "novel film-forming materials" to block ice nucleation in plants and therefore provide a means of frost protection. 7. 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 & durability of the technology product? The first yeast-based postharvest biocontrol product (Aspire) developed by ARS scientists in Kearneysville, WV is presently being marketed by Ecogen. Limitation of this product is that it does not exhibit eradicant activity, i.e., it cannot control existing infections, it can only protect the fruit from new infections. Presently, negotiations are underway to transfer ARS patents on a "bioactive coating" to Micro Flo (subsidiary of BASF). A CRADA is being developed with Micro Flo to develop effective formulations of the "bioactive coating" and test them in large-scale commercial tests in Florida and California on citrus and on apples in West Virginia. This product exhibits both protective and eradicant activity and
therefore represents a significant improvement over the first generation of yeast- based postharvest biocontrol products. The first bacterial-based, postharvest biocontrol product (BioSave) based on the bacterium, Pseudomonas syringae, developed by an ARS scientist at Kearneysville, WV and licensed and marketed by EcoScience Corp. has been used in fruit packing houses for the past three seasons and the use of this product is increasing. Although considered a commercial success, this product has its limitations. The performance margin for acceptable biological control is generally lower than that for fungicides. For example, higher concentrations of the antagonist must be used to achieve the same control of decay as fruit mature. Thus the performance margin for biocontrol must be increased. Plasticity of the biocontrol system must also be improved to make biocontrol effective under a wider range of conditions (e.g. more cultivars) and against a greater number of pathogens at a
reduced cost. ARS research is geared to develop more second generation biocontrol products which address those issues and are part of an integrated approach to disease management. The basic information developed by ARS scientists at Kearneysville, WV regarding the biochemical and genetic regulation of stress tolerance and disease resistance has been presented to other scientists and professionals at national and international meetings, as well as through invited seminars and peer-reviewed publications. ARS scientists are also submitting a patent application for the use of "novel film-forming materials" as a means of providing frost protection. If the utility of this technology can be demonstrated on a large scale, it is expected that this technology would be available to growers in two years. At present, a limitation is obtaining adequate coverage of the material and various formulations are being tested to resolve this problem. The genes and expressed tag sequences are being
made available for academic and commercial use as they are being characterized. Genes discovered by ARS scientists, such as the peach dehydrin gene, and the ethylene receptor gene have been requested and sent to other academic institutions. The goal of producing transgenic fruit trees with improved stress tolerance and/or disease resistance is a long- range goal (5-10 years). ARS scientists at Kearneysville, WV have met with commodity groups, participated in grower workshops, attended regional project workshops, such as the W-130 Western Regional Project on Freeze Damage to Fruit and Nut Trees, presented invited seminars both nationally and internationally, and presented papers at national meetings of professional societies, such as the American Phytopathological Society, American Society for Horticultural Science, and the American Society of Plant Physiologists. An ARS scientist at Kearneysville, WV has also served as an Associate Editor for the Journal of the American Society of
Horticultural Science, and Chair of the Binational Agricultural Research and Development grant panel for horticultural crops. An ARS scientists also manage a newsletter on fire blight and postharvest biological control through the USDA-ARS Appalachian Fruit Research Station web page. 8. List your most important non-peer reviewed publications and presentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which are listed below). 1. Anonymous. 1999. E. coli can take flight. Science News 155 (4): 63. 2. Anonymous. 1999. Fruit fly seen as cause of contamination. UniSci, UniScience News Net, Inc.http://unisci.com/stories/19991/0113993.htm 3. Anonymous. 1999. Yeast may prevent fresh fruit contamination. Food Technology Alert. John Wiley&Sons, Inc., New York, NY 10158. 4. Dr. Dean. 1999. Fruit flies transfer E. coli in apple tests. Dr. Dean Digest.
http://healthcentral.com/DrDean/Deanfulltexttopics.cfm?ID=8923 5. Naegely, S. 1999. Bio Controls to the rescue. American Vegetable Grower. August, 1999, Western Edition: 2* - 4*. 6. Naegely, S. 1999. Biocontrols cut postharvest losses. American Fruit Grower. June, 1999 : 2*-4* 7. Rose, D. 1999. The fruit fly's hidden danger. Science, Daily inScight. Posted 22, January, 1999, http://www.academicpress.com/inscight/01221999/graphb.htm 8. Applications of Genetic Engineering to Agriculture" to the Damascus Garden Society (March 5, 1999), Damascus, Maryland. 9. A poster entitled "Characterization of an ETR homologue from peach (Prunus persica)" was presented at the 1999 meeting of the American Society for Plant Physiology 10. Organized a symposium at the XIVth International Plant Pathology Congress Jerusalem, Israel, July 25-30, 1999 titled "Advances in Alternative Control Methods of Post-harvest Diseases of Fruits." Organized and chaired a symposium at the Society of Industrial
Microbiology 50th Anniversary Annual Meeting, August 3-7, 1999 titled, "Commercializing Microbes for Pest Control Products of the Future." Organized a BARD-sponsored workshop on "Microbial Contamination of Food", May 4-8, 1999, Shepherdstown, WV. Articles about our research on methyl bromide alternatives appeared in the New York Times and other major newspapers. Presented an invited lecture to the US EPA Office of Pesticide Programs, Alexandria, VA on "Natural Alternatives to Synthetic Pesticides" , July 20, 1999. Presented invited symposium lecture at an international symposium, in Wales, UK, August 24-28, 1999 titled, "The Next Generation of Biofungicides for the Control of Fruit and Vegetable Postharvest Diseases." Presented seminar on, "The use of infrared thermography to study the freezing process in plants," to the Department of Plant Science, University of Saskatchewan, Saskatoon, CA - May 1999. Presented a poster at the Gordon Conference of Temperature Stress in Plants,
January 1999. Presented an invited lecture on, "Postharvest Biological Control" at the First Argentine Conference on Biological Control, Buenos Aires, Argentina, October 1998. Gave an invited presentation on, "The use of infrared thermography in horticultural research," at the Annual Meeting of the American Society for Horticultural Science, Minneapolis, MN, August 1999. Presented a poster on the cloning and expression of a defensin gene in peach at the annual meeting of the American Society for Horticultural Science, Minneapolis, MN, August 1999
Impacts
(N/A)
Publications
- JANISIEWICZ, W.J., CONWAY,W.S., BROWN, M.W., SAPERS, G.M., FRATAMICO, P. and BUCHANAN, R.L. 1999. Fate of Escherichia coli O157:H7 on fresh-cut apple tissue...by fruit flies. APL. Environ. Mcrobiol. 65:1-5.
- CONWAY, W.S., JANISIEWICZ, W.J.,KLAIN, J.D. and SAMS, C.E. 1999. Strategy for combining heat treatment, calcium infiltration and biological control to reduce postharvest decay of Gala" apples. HortScience 34:700-704.
- WILSON, C.L., SOLAR, J.M., EL GHAOUTH, A. and FRAVEL, D. 1999. Benzaldehyde as a soil fumigant, and an apparatus for rapid fumigant evaluation 1999. HortScience 34: 681-685.
- VAN DER ZWET, T. and BEER, S.V. 1999. Fire Blight Its Nature, Prevention and Control; a practical guide to integrated disease management. USDA, ARS, Agric. Inform. Bul. 631, pp 97.
- CARTER, J., BRENNAN, R. and WISNIEWSKI, M. 1999. Low-temperature tolerance of blackcurrant flowers. HortScience 34: 855-860.
- WISNIEWSKI, M. and ARORA, R. 1999. Structural and biochemical aspects...plants. IN: Molecular Biology of Woody Plants. Eds. M. Jain and S. Minocha. Kluwer, Copenhagen.
- SAUTER, J., WESTPHAL, S. and WISNIEWSKI, M. 1999. Immunological identification of dehydrin-related proteins in the wood of five species...and in Salix caprea. J. Plant Physiol. 154:781-788.
- JANISIEWICZ, W.J. 1998. Development of a biocontrol product for postharvest application on apple and pear. Phytopathology 88:S130.
- CONWAY, W.S., JANISIEWICZ, W.J., WATADA, A.E. and SAMS, C.E. 1998. Survival and growth of Listeria monocytogenes on fresh-cut apple slices. Phytopathology 88:S18.
- CONWAY, W.S., JANISIEWICZ, W.J., KLAIN, J.D. and SAMS, C.E. 1998. Effect of heat treatment, calcium infiltration and biological control on postharvest decay of Gala'. Phytopathology 88:S18.
- BASSETT, C.L. and NICKERSON, M. 1999. Differential Processing Could Lead...Gene Expression, In: Current Topics in Plant. Food for the 21st Cent Abs. pg. 70.
- BASSETT, C.L., ARTLIP, T.S. and NICKERSON, M. 1999. A Gene Encoding an ETR1...IN: Current Topics in Plant Biochemistry. Food for the 21st Cent Abs. pg.71.
- BASSETT, C.L. and ARTLIP, T.S. 1999. Isolation of an ETR1 Ethylene Receptor Homologue from Peach Prunus persica (L.) Batsch]. HortScience 34:542.
- BASSETT, C.L. 1999. Bioinformatic tools on the world wide web: Programs for Gene Analysis. HortScience 34:430.
- WISNIEWSKI, M., ARTLIP. T., WEBB, R., BASSETT, C.L. and CALLAHAN, A. 1999. Cloning of a defensin-like gene and its expression during dormancy and fruit development in peach. HortScience 34: 451.
- WISNIEWSKI, M., FULLER, M. and FUCHIGAMI, L. 1999. The use of infrared thermography in horticulture. HortScience 34: 430.
|
|