Source: Agricultural Research Service submitted to
MOLECULAR APPROACHES TO UNDERSTANDING HOST RESISTANCE AND PATHOGEN VARIABILITY FOR IMPROVING POTATO DISEASE MANAGEMENT
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0407105
Grant No.
(N/A)
Project No.
1275-21220-187-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 25, 2003
Project End Date
Feb 26, 2007
Grant Year
(N/A)
Project Director
JONES R W
Recipient Organization
Agricultural Research Service
BLDG 003 BARC W RM 331
BELTSVILLE,MD 20705-2351
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21213101160100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1310 - Potato;

Field Of Science
1160 - Pathology;
Goals / Objectives
Investigate strategies for protecting potato quality and yields against late blight and common scab diseases caused by Phytophthora infestans and Streptomyces scabies. Characterize aspects of resistance or susceptibility to P. infestans and S. scabies, including assessment of the role of specific potato genes in quantitative late blight resistance and in disease resistance responses, and responses of different potato varieties to scab. Utilize an alternative method for producing disease-resistant potatoes by expressing antimicrobial peptides in transgenic plants. Develop rapid protocols to aid in identification of new more virulent pathogen isolates, monitor the origins and spread of disease, and characterize pathogen variability. Determine underlying disease resistance mechanisms in potato, and infection mechanisms and genetic variability in pathogens that can be used in developing and selecting potato germplasm resistant to P. infestans and S. scabies.
Project Methods
(a) Characterize the diversity in alleles of 4 defense-related gene families encoding WRKY motif-containing proteins, osmotin, phenylalanine ammonia lyase, and chalcone isomerase that map to quantitative trait loci for late blight resistance, and relate this diversity to differences in late blight resistance among wild Solanum species and cultivated potato germplasm. Compare cDNA libraries generated from potato leaves infected with a virulent and a less virulent Phytophthora infestans isolate by sequencing at least 1000 ESTs from each library. Examine scab susceptibility in potato breeding material using a reliable assay with well-characterized, pathogenic Streptomyces scabies isolates. (b) Identify antimicrobial proteins and peptides inhibitory to P. infestans by screening peptide libraries. Develop methods for efficient and effective production of these inhibitory molecules in potato plants, utilizing gene promoter analysis and fusion protein studies. (c) Characterize molecular features used in P. infestans genotyping studies, primarily the gene encoding glucose-6-phosphoisomerase, and RG 57 fingerprint bands from Southern blots. Characterization will involve cDNA and genomic DNA cloning and sequencing. Proteins secreted from P. infestans will be investigated to assess their role in pathogenicity and/or virulence. Extracellular proteins will be analyzed in acrylamide protein gels and by cDNA cloning. Find molecular features useful in rapid simple identification of pathogenic Streptomyces scabies strains. BSL-1; recertified 8/16/01.

Progress 04/25/03 to 02/26/07

Outputs
Progress Report Objectives (from AD-416) Investigate strategies for protecting potato quality and yields against late blight and common scab diseases caused by Phytophthora infestans and Streptomyces scabies. Characterize aspects of resistance or susceptibility to P. infestans and S. scabies, including assessment of the role of specific potato genes in quantitative late blight resistance and in disease resistance responses, and responses of different potato varieties to scab. Utilize an alternative method for producing disease- resistant potatoes by expressing antimicrobial peptides in transgenic plants. Develop rapid protocols to aid in identification of new more virulent pathogen isolates, monitor the origins and spread of disease, and characterize pathogen variability. Determine underlying disease resistance mechanisms in potato, and infection mechanisms and genetic variability in pathogens that can be used in developing and selecting potato germplasm resistant to P. infestans and S. scabies. Approach (from AD-416) (a) Characterize the diversity in alleles of 4 defense-related gene families encoding WRKY motif-containing proteins, osmotin, phenylalanine ammonia lyase, and chalcone isomerase that map to quantitative trait loci for late blight resistance, and relate this diversity to differences in late blight resistance among wild Solanum species and cultivated potato germplasm. Compare cDNA libraries generated from potato leaves infected with a virulent and a less virulent Phytophthora infestans isolate by sequencing at least 1000 ESTs from each library. Examine scab susceptibility in potato breeding material using a reliable assay with well-characterized, pathogenic Streptomyces scabies isolates. (b) Identify antimicrobial proteins and peptides inhibitory to P. infestans by screening peptide libraries. Develop methods for efficient and effective production of these inhibitory molecules in potato plants, utilizing gene promoter analysis and fusion protein studies. (c) Characterize molecular features used in P. infestans genotyping studies, primarily the gene encoding glucose-6-phosphoisomerase, and RG 57 fingerprint bands from Southern blots. Characterization will involve cDNA and genomic DNA cloning and sequencing. Proteins secreted from P. infestans will be investigated to assess their role in pathogenicity and/or virulence. Extracellular proteins will be analyzed in acrylamide protein gels and by cDNA cloning. Find molecular features useful in rapid simple identification of pathogenic Streptomyces scabies strains. Accomplishments Genome analysis of Phytophthora. Advancement in our understanding of genes used by Phytophthora to incite plant disease relies on molecular analysis of the genome. A draft of the complete genomic DNA sequence of Phytophthora sojae and P. ramorum has been finalized. This information provides the basis for future studies on the location and sequence of genes in Phytophthora. Expression of these genes can be monitored to determine their use in plant infections. Close similarity in gene number and location was found between the two species studied. This genome template can be extended to all other Phytophthora species, helping to alleviate the billions of dollars of lost agricultural productivity due to these pathogens. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. Identification of novel secreted protein families in Phytophthora. A genomic map was used as a template for identification of a unique type of endoglucanase. The endoglucanase causes damage to the host plant cell wall, helping the fungus to invade. A duplicated cluster of genes were identified in each of three species of Phytophthora. These endoglucanases belong to family 12 glycosyl hydrolases, and represent the first report and characterization of this type of enzyme in any plant pathogen. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. Streptomyces species that cause common scab in the USA Potato common scab is found world-wide, but disease severity varies in different locations. Little information is available on the species and distribution of species and strains of the soil bacteria that cause potato common scab. To determine whether different species or strains of Streptomyces may characterize locations with different common scab disease pressure, molecular markers have been developed that are specific to individual species of Streptomyces. These markers have been used to demonstrate that several different species of Streptomyces that cause common scab are present in the U.S., including a species/strain from Idaho not previously described. Furthermore, different species are prevalent in different geographic regions. Some species appear to be world-wide in distribution or are found on other continents such as Europe, while other species that have been described from Europe and Asia have not been found in the US. Further work is underway to determine the significance of differences in pathogen populations to the incidence and severity of common scab in different geographic regions. This addresses the National Program 303 Component Identification and Classification of Pathogens. Influence of pathogen load on common scab disease In the laboratory, potato common scab symptoms are not seen at low pathogen densities, and symptom severity increases as the pathogen inoculum increases over several orders of magnitude. It is possible that severity of common scab disease in the field is related to the amount of the bacteria causing the disease found in soil, and the amount or ¿load¿ of the pathogen may change rapidly in soil in response to weather or other factors. Confirmation of this hypothesis depends on the availability of rapid inexpensive and high through-put techniques for specifically measuring the numbers of pathogenic bacteria in soil and on the plant surface. Specific identification of pathogenic species is complicated by their general genetic and morphological similarity to abundant non-pathogens also found in soil. We developed a quantitative PCR technique based on a gene that is found only in pathogenic species, but is found in all known pathogenic Streptomyces species. This technique will be applied to measurements of pathogen load in soil, and could eventually be applied to assaying the likelihood of a serious scab problem causing economic losses in specific field locations and years. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. Characterization of the late blight populations in the Pacific Northwest and Alaska. Tomato and potato late blight has become an increasingly important problem to agriculture in the United States in the past decade as more aggressive, fungicide-resistant, and specialized pathogenic strains have appeared. Epidemics occurred in 2004-2006 when over 50% of the commercial crop was destroyed in the Pacific Northwest and Alaska. Most growers reported that no available fungicides would control the rapid spread of the epidemic. Phenotype characterization of the isolates included mating type; sensitivity to fungicides; virulence on potato host differentials; and pathogenic fitness. Genotypes were also assessed with several molecular methods. The diverse phenotypic and genotypic structure of the current field populations suggests that this maybe due to local processes like host preference and selection rather than through long-distance migration. These research findings are beneficial because the US blight population structure is monitored and data on the distribution and modification of strains are available. Most predominant strain information allows breeders and geneticists to compare or exploit sources of resistance in their breeding programs. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. An international study on late blight of potato. Phenotypic and genetic characterization of several pathogenic fungal strains of the organism that causes late blight of potato and tomato crops in Europe clearly demonstrated an alarming shift in new and very dynamic strain diversity especially in Great Britain. The appearance of new genotypes in Wales, presumably due to migration, was first detected in 2001 with the identification of the rare mating type and a number of DNA fingerprints that had not been detected previously. This migration resulted in dramatic changes. Gene and genotype diversity and the frequency of uniquely detected genotypes all increased beginning in 2003, and reached a peak in 2005. A new clonal genotype, first detected in 2003, increased in frequency and made up almost 50% of all isolates collected in 2005. In contrast, the old clonal lineage that dominated populations from 1985 to 2002 was not detected after 2003. An international study of the diversity of the population in Europe has significantly increased knowledge of this pathogen, which still represents a threat to the health of potato crops in the entire area. These findings are vital to those concerned with the control of the pathogen with the application of fungicides or by exploiting new cultivars with resistance to late blight. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. Technology Transfer Number of Patent Applications filed: 1 Number of U.S. Patents granted: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 5 Number of Newspaper Articles,Presentations for NonScience Audiences: 7

Impacts
(N/A)

Publications

  • Costanzo, S., Ospina-Giraldo, M., Deahl, K.L., Baker, C.J., Jones, R.W. 2006. Gene duplication event in family 12 glycosyl hydrolase from phytophthora spp. Fungal Genetics and Biology. 43:707-714.
  • Deahl, K.L., Jones, R.W., Perez, F.G., Shaw, D.S., Cooke, L.R. 2006. Characterization of isolates of phytophthora infestans from four solanaceous hosts growing in association with late-blighted potatoes. American Journal of Potato Research. 41:1635-1639.
  • Tyler, B.M., Tripathy, S., Xuemin, Z., Dehal, P., Jiang, R.H., Aerts, A., Arredondo, F., Baxter, L., Benasson, D., Beynon, J.L., Damasceno, C.M., Dickerman, A., Dorrance, A.E., Dou, D., Dubchak, I., Garbelotto, M., Gijzen, M., Gordon, S., Govers, F., Grunwald, N., Huang, W., Ivors, K., Jones, R.W., Kamoun, S., Konstantinos, K., Lamour, K., Lee, M., Mcdonald, W.H., Medina, M., Meijer, H.J., Nordberg, E., Maclean, D.J., Ospina- Giraldo, M.D., Morris, P., Phuntumart, V., Putnam, N., Rash, S., Rose, J.K. , Sakihama, Y., Salamov, A., Savidor, A., Scheuring, C., Smith, B., Sobral, B.W., Terry, A., Torto-Alalibo, T., Win, J., Xu, Z., Zhang, H., Grigoriev, I., Rokhsar, D., Boore, J. 2006. Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science. 313:1261- 1266.
  • Wanner, L.A. 2006. A survey of genetic variation in streptomyces isolates causing potato common scab in the united states. Phytopathology. 96(12) :1363-1371.
  • Wanner, L.A. 2007. A new strain of streptomyces causing potato common scab. Plant Disease. 91:352-359.


Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Control strategies for the potato diseases late blight and common scab, caused by Phytophthora infestans and Streptomyces sp., are inadequate to prevent significant crop loss in the U.S. New variations within pathogen populations rather than changes in cropping systems or environmental conditions are responsible for these losses. Chemical control possibilities for both diseases are limited, as the prevalent Phytophthora strains have become increasingly resistant to traditional fungicides, and few chemical controls exist for common scab. Little information is available on variation within the scab pathogen populations in the field or variation in scab resistance in potato germplasm. Effective disease control relies on understanding how an organism causes disease and how plants resist infection. Strategic and sustainable deployment of durable disease resistance requires knowledge of resistance mechanisms in potatoes, and of infection mechanisms and genetic variability in pathogens. We are investigating several strategies for protecting potato quality and yields against late blight and common scab. The goals of the project are: (I) Molecular characterization of potato resistance/susceptibility responses to Phytophthora infestans and Streptomyces sp., (II) Engineering potatoes to express antimicrobial peptides effective against Phytophthora diseases, and (III) Development of new molecular tools for following the evolution, spread and disease- causing potential of the pathogens that cause these two diseases. Specific sub-objectives (milestones) for the project are outlined in section 2. Information on pathogen and plant variability is essential for identifying potential disease resistance markers that can be used in breeding more resistant potato varieties, and for developing disease management strategies to minimize losses to growers. This addresses the goals of National Program 303 Plant Diseases. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2004) Objective IB. Construct cDNA library from virulent P. infestans isolateSequence ESTs Objective IC. Develop scab assay Objective IIIA2. Clone and characterize glucose phosphoisomerase (GPI) -encoding enzymes from P. infestans Year 2 (FY 2005) Objective 1C. Confirm scab resistance/susceptibility in different potato varieties. Objective IIB. Construct and test peptide delivery systems Objective IIIA1. Develop new markers based on P. infestans RG57 marker Year 3 (FY 2006) Objective IA. Clone phenylalanine ammmonia lyase (PAL) and chalcone synthase (CHS) gene families (from potato) Objective 2A Screen peptide libraries. Objective IIIA3. Analyze secreted proteins from P. infestans. Year 4 (FY 2007) Objective IA. Clone transcription motifs (WRKY) and chitinase (CHI) gene families (from potato) Objective IC. Confirm scab resistance/susceptibility in different potato varieties Objective IIIA3. Analyze secreted proteins from P. infestans. Year 5 (FY 2008) Objective IA. Study expression of PAL, CHI, WRKY and CHS gene families in susceptible and resistant potato varieties. Objective IIC. Screen promoters for temporal and spatial differences in antimicrobial protein expression. Objective IIIB. Characterize gene regions associated with pathogenicity in Streptomyces. 4a List the single most significant research accomplishment during FY 2006. Discovery and characterization of a unique family of endoglucanases from Phytophthora. Enzymes are required for a pathogen to destroy plant tissues. A new group of enzymes that attack the cell walls of plants was identified from Phytophthora. Many copies of the gene for the enzyme were found, suggesting a source of variation for the pathogen. The genes were found in different Phytophthora species and were organized in the same manner, indicating that study of a single Phytophthora species is relevant to many species of Phytophthora. This information will be useful in selecting resistance to Phytophthora in a wide range of plants. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. 4b List other significant research accomplishment(s), if any. Development of molecular markers that specifically recognize different species of plant pathogenic Streptomyces. These species-specific markers have been used to demonstrate that there are several species of strains of common scab-causing Streptomyces found in potato growing regions of the northern U.S., including a previously undescribed species/strain from Idaho. Furthermore, different species are prevalent in different geographic regions. Further work is underway to determine the significance of differences in pathogen populations to the incidence and severity of common scab in different geographic regions. This addresses the National Program 303 Component Identification and Classification of Pathogens. Monitoring Phytophthora in alternate hosts. In 2006, we reported the isolation and characterization of P. infestans strains from other Solanaceous hosts, growing near fields of blighted potatoes in several states and in one location in the UK. It is recommended that when Phytophthora spp. is found in Solanaceous plants growing in close proximity of potato crops, the pathogen species should be identified and tested for variation in strain occurrence before disease control measures are implemented. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. 5. Describe the major accomplishments to date and their predicted or actual impact. Cloning of a marker gene for Phytophthora identification. Genes encoding glucose-6-phosphate isomerase (GPI), proteins produced by P. infestans, were cloned and characterized. This enzyme has been used worldwide as a tool for aiding in characterization of isolates. Sequence analysis and genome comparisons identified molecular variations that correlate with virulence profiles. The outcome of this was application of the information to improving our understanding of the origins of P. infestans isolates that may occur by worldwide transport or interspecies hybridization. Tracking of isolate origins can now be enhanced and provide a useful tool to scientists worldwide. This addresses the National Program 303 Component Identification and Classification of Pathogens. Construction of a novel method for delivery of antimicrobial peptides in transgenic plants. Very small antimicrobial peptides can be very potent inhibitors of pathogenic organisms, however their size precludes efficient expression in plants. To overcome this limitation a fusion protein delivery system was developed that mimicked the plants natural mechanism for producing very small peptides. Transgenic plants expressing a prosystemin-antimicrobial peptide fusion provided at least a 50% reduction in the rate of P. infestans lesion expansion. This information provides growers with new options for disease control. This addresses the National Program 303 Component Host Plant Resistance to Disease. Novel potential reservoir of the late blight pathogen in weed species. We have discovered the natural occurrence of P. infestans on black nightshade (a common weed in the UK and the United States) in Wales, and in northern Maine. This discovery is important, since these weed hosts may serve as reservoirs of inoculum originating from potato or tomato crops, and the existence of inoculum reservoirs affects disease management strategies. However, these infections have only been found when blight is already widespread in potato fields and there is no evidence to suggest that black nightshade acts as an over-wintering host. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. Demonstration that potato (and radish) common scab disease severity is a function of pathogen load. Scab disease symptoms and severity vary from year to year in the same field locations and cultivars, but the basis for this variation has not been well understood. We demonstrated that scab symptoms are not seen at low pathogen densities, and that symptom severity increases as the pathogen inoculum increases over several orders of magnitude. We conclude that fluctuation in pathogen population density is an important component of variation in disease severity. Therefore, it is imperative to (re-) analyze scab resistance in potato varieties and breeding material at carefully controlled inoculum levels to distinguish true genetic differences in pathogen susceptibility from the effects of isolate-specific variation in pathogenicity and pathogen load. Measurements of pathogen load could eventually be applied to assaying the likelihood of a serious scab problem causing economic losses in specific field locations and years. This addresses the National Program 303 Component Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors. 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? Preliminary information on the scab susceptibility of potato varieties and breeding lines grown in different geographic areas has been made available to breeders and growers by presentation at the annual potato association meeting. Genetic sequences were deposited in GenBank, making them available to the global research community free of charge: Consulted with scientists at APHIS about monitoring the changing regulations regarding permits and the movement of plant pathogens in the U.S. during which concerns regarding permit regulations and the need for risk-based assessment of pathogens were discussed. Markers for Verticillium resistance have been developed for use by breeders in modernizing selection methods.

Impacts
(N/A)

Publications

  • Jones, R.W., Ospina-Giraldo, M., Deahl, K. 2006. Gene silencing indicates a role for potato endoglucanase inhibitor protein in germplasm resistance to late blight. American Journal of Potato Research 83:41-46.


Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Control strategies for the potato diseases late blight and common scab, caused by Phytophthora infestans and Streptomyces sp., are inadequate to prevent significant crop loss in the US. New variations within pathogen populations rather than changes in cropping systems or environmental conditions are responsible for these losses. Chemical control possibilities for both diseases are limited, as the prevalent Phytophthora strains have become increasingly resistant to traditional fungicides, and few chemical controls exist for common scab. Little information is available on variation within the scab pathogen populations in the field or variation in scab resistance in potato germplasm. Effective disease control relies on understanding how an organism causes disease and how plants resist infection. Strategic and sustainable deployment of durable disease resistance requires knowledge of resistance mechanisms in potatoes, and of infection mechanisms and genetic variability in pathogens. We are investigating several strategies for protecting potato quality and yields against late blight and common scab. The goals of the project are: (I) Molecular characterization of potato resistance/susceptibility responses to Phytophthora infestans and Streptomyces sp., (II) Engineering potatoes to express antimicrobial peptides effective against Phytophthora diseases, and (III) Development of new molecular tools for following the evolution, spread and disease- causing potential of the pathogens that cause these two diseases. Specific sub-objectives (milestones) for the project are outlined in section 2. Information on pathogen and plant variability is essential for identifying potential disease resistance markers that can be used in breeding more resistant potato varieties, and for developing disease management strategies to minimize losses to growers. This work addresses NP 303 Plant Diseases components (4) pathogen biology, genetics, populations dynamics, spread, and relationship with host vectors, and (5) host plant resistance to disease. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Objective IB. Construct cDNA library from virulent P. infestans isolateSequence ESTs Objective IC. Develop scab assay Objective IIIA2. Clone and characterize glucose phosphoisomerase (GPI)- encoding enzymes from P. infestans Year 2 (FY 2005) Objective 1C. Confirm scab resistance/susceptibility in different potato varieties. Objective IIB. Construct and test peptide delivery systems Objective IIIA1. Develop new markers based on P. infestans RG57 marker Year 3 (FY 2006) Objective IA. Clone phenylalanine ammmonia lyase (PAL) and chalcone synthase (CHS) gene families (from potato) Objective 2A Screen peptide libraries. Objective IIIA3. Analyze secreted proteins from P. infestans. Year 4 (FY 2007) Objective IA. Clone transcription motifs (WRKY) and chitinase (CHI) gene families (from potato) Objective IC. Confirm scab resistance/susceptibility in different potato varieties Objective IIIA3. Analyze secreted proteins from P. infestans. Year 5 (FY 2008) Objective IA. Study expression of PAL, CHI, WRKY and CHS gene families in susceptible and resistant potato varieties. Objective IIC. Screen promoters for temporal and spatial differences in antimicrobial protein expression. Objective IIIB. Characterize gene regions associated with pathogenicity in Streptomyces. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Objective 1C. Confirm scab resistance/susceptibility in different potato varieties. Initial scab resistance data have been obtained for 32 potato lines, 27 commercial or breeding tetraploid varieties and 5 wild Solanum species. No variety is completely scab-resistant, though there is variation. Some wild species showed no scab, although tuber set was limited in these plants, and more data are needed to confirm these observations. Finding true scab resistance in any compatible Solanum species or variety provides the potential for introducing it into commercial varieties. Milestone Substantially Met 2. Objective IIB. Construct and test peptide delivery systems. Very small antimicrobial peptides can be very potent inhibitors of pathogenic organisms, however their size precludes efficient expression in plants. To overcome this limitation, a fusion protein delivery system was developed that mimicked the plant's natural mechanism for producing very small peptides. Transgenic plants expressing a prosystemin-antimicrobial peptide fusion provided at least a 50% reduction in the rate of P. infestans lesion expansion (Molecular Breeding 14:83-89). Milestone Fully Met 3. Objective IIIA1. Develop new markers based on P. infestans RG57 marker. Recent studies indicate that single sequence repeat (SSR) markers may prove more valuable than current RG 57 markers, so emphasis has shifted to SSR development and application. Milestone Not Met Other 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The milestones to be addressed over the next 3 years (FY 2006, 2007, 2008) are listed below, with descriptions of what we expect to accomplish specified under each milestone. Year 3 (FY 2006) Objective IA. Clone PAL and CHS gene families (from potato). In FY 2004, sequence data available in the public domain for these two gene families from potato and from other Solanaceous relatives has been obtained, aligned and used to identify conserved and non-conserved regions for primer design. In FY 2005, primers will be used to clone additional gene family members from potato. These will be sequenced and the sequence information will be used to design gene-specific probes for each family member in FY 2005 and 2006. This will allow for better understanding of these defense related genes, and their role in disease resistance. Objective IIA Screen peptide libraries. Selected peptides will be screened to identify new candidates for delivery in transgenic plants. This will support ongoing research on application of antimicrobial peptides for pathogen control. Objective IIIA3. Analyze secreted proteins from P. infestans. The genes encoding the extracellular enzymes cutinase and endoglucanase will be cloned, sequenced and over-expressed, allowing us to determine their substrate specificity and role in pathogenicity. These enzymes may also find use in industrial processing applications. A manuscript will be published reporting on these sequences and enzyme activities. Year 4 (FY 2007) Objective IA. Clone WRKY and CHI gene families (from potato). In FY 2005, sequence data available in the public domain for these two gene families from potato and from other Solanaceous relatives will be obtained, aligned and used to identify conserved and non-conserved regions for primer design. Objective IC. Confirm scab resistance/susceptibility in different potato varieties. Cultivated potato varieties, genetic breeding material, and wild Solanum species will be tested for scab susceptibility using both field tests and controlled-environment tests with well-defined Streptomyces strains. Results are being made available to growers and breeders as they are obtained. Objective IIIA3. Analyze secreted proteins from P. infestans. The genes encoding the extracellular enzymes, based on signal sequence analysis, will be cloned, sequenced and over-expressed. A manuscript will be published reporting on these sequences and enzyme activities. Year 5 (FY 2008) Objective IA. Study expression of PAL, CHI, WRKY and CHS gene families in susceptible and resistant potato varieties. Emphasis of these studies will be to correlate either specific alleles or gene expression patterns with resistance to disease. Objective IIC. Screen promoters for temporal and spatial differences in antimicrobial protein expression. Methods for expressing antimicrobial peptides and other inhibitory compounds, useful in disease resistance, will be optimized through selection of tissue specific promoters. Objective IIIB. Characterize gene regions associated with pathogenicity in Streptomyces. These gene regions will be used to design strain or isolate-specific primers to be used in monitoring the origins and spread of pathogenic Streptomyces isolates. 4a What was the single most significant accomplishment this past year? Single most significant accomplishment during FY 2005 (one per Research (OOD) Project) Construction of a novel method for delivery of antimicrobial peptides in transgenic plants. Very small proteins can be very potent inhibitors of pathogenic organisms, however their size precludes efficient expression in plants. To overcome this limitation a fusion protein delivery system was developed that mimicked the plant's natural mechanism for producing very small peptides. Transgenic plants expressing a prosystemin- antimicrobial peptide fusion provided at least a 50% reduction in the rate of P. infestans lesion expansion. This information provides growers with new options for disease control. 4b List other significant accomplishments, if any. Development of molecular assessment strategy for selection of Verticillium resistant potato. Verticillium wilt is a major disease problem in potato, and is controlled through resistant varieties. These varieties vary in levels of resistance and heritability of resistance. Analysis of specific regions of the Verticillium resistance gene (Ve) revealed a method for determining levels of Verticillium resistance and allow for tracking of heritability. This will greatly accelerate the proper selection of parental crosses for progeny resistance, a benefit to both breeders and growers. Discovery of alternate hosts for Phytophthora infestans. In 2005, we reported the isolation and characterization of P.infestans strains from another Solanaceous host, hairy nightshade, growing within and around fields of blighted potatoes in Maine. It is recommended that when Phytophthora spp. are found in Solanaceous plants growing within or near potato crops, the pathogen species should be identified and tested for fungicide resistance before disease control strategies are implemented. Monitoring of pathogen populations in bridging hosts is important, since these may play a major role in the introduction and spread of damaging pathogens to new locations. Demonstration of the significance of variation in virulence and pathogen load in potato common scab symptom development. To understand the contribution of the type(s) and variation in scab-causing Streptomyces strains and their abundance to scab incidence and severity, a rapid radish bioassay has been developed. Using this assay in carefully controlled laboratory condition, we tested more than 50 Streptomyces isolates for pathogenicity and virulence, and found differences in virulence between Streptomyces isolates. This will aid growers in determining if they have highly virulent isolates in their soil and may allow for proper potato variety selection. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. These accomplishments address aspects of NP 303 Plant Diseases. Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors Cloning of a marker gene for Phytophthora identification. Genes encoding glucose-6-phosphate isomerase (GPI), proteins produced by P. infestans, were cloned and characterized, This enzyme has been used worldwide as a tool for aiding in characterization of isolates. Sequence analysis and genome comparisons identified molecular variations that corre late with virulence profiles. The outcome of this was application of the information to improving our understanding of the origins of P. infestans isolates that may occur by worldwide transport or interspecies hybridization. Tracking of isolate origins can now be enhanced and provide a useful tool to scientists worldwide. Construction of a novel method for delivery of antimicrobial peptides in transgenic plants. Very small antimicrobial peptides can be very potent inhibitors of pathogenic organisms, however their size precludes efficient expression in plants. To overcome this limitation a fusion protein delivery system was developed that mimicked the plant's natural mechanism for producing very small peptides. Transgenic plants expressing a prosystemin-antimicrobial peptide fusion provided at least a 50% reduction in the rate of P. infestans lesion expansion. This information provides growers with new options for disease control. 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? Preliminary information on the scab susceptibility of 11 potato varieties and 6 breeding lines grown in different geographic areas has been made available to breeders and growers by presentation at the annual potato association meeting. Genetic sequences were deposited in GenBank, making them available to the global research community free of charge: Consulted with scientists at APHIS about monitoring the changing regulations regarding permits and the movement of plant pathogens in the U.S. during which concerns regarding permit regulations and the need for risk-based assessment of pathogens were discussed.

Impacts
(N/A)

Publications

  • Wanner, L.A. 2003. Molecular and pathogenic variation in streptomycetes causing potato or radish scab [abstract and talk]. 20th annual meeting of the Mid-Atlantic Plant Molecular Biology Society, Beltsville, MD 8-9 Aug 2003.
  • Wanner, L.A. 2003. Molecular and pathogenic variation in streptomycetes causing common scab [abstract and talk]. Phytopathology 93 (6):S89 Supplement
  • Deahl, K. 2004. Population dynamics of Phytophthora infestans assessed by mitochondrial DNA.[abstract] American Journal of Potato Research 81(1):54.
  • Deahl, K.L., Shaw, D.S., Cooke, L.R. 2004. Natural Occurrence of Phytophthora infestans on Black Nightshade (Solanum nigrum) in Wales. Plant Disease. 88(7):771.
  • Wattier, R.A.M., Gathercole, L.L., Assinder, S.J., Gliddon, C.J., Deahl, K. L., Shaw, D.S., Mills, D.I. 2003. Sequence variation of intergenic mitochondrial DNA spacers (mtDNA-IGS) of Phytophthora infestans (Oomycetes) and related species. Molecular Ecology Notes 3:(1)136-138.
  • Ospina-Giraldo, M., Jones, R.W. 2003. Characterization of the glucose-6- phosphate isomerase gene in Phytophthora infestans reveals the presence of multiple alleles. Fungal Genetics and Biology 40:197-206.
  • Wanner, L.A. 2003. Molecular and pathogenic variation in streptomycetes causing potato or radish scab [poster] 8th International Congress of Plant Pathology, Christchurch, NZ, 2-7 Feb. 2003


Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Control strategies for the potato diseases late blight and common scab, caused by Phytophthora infestans and Streptomyces sp., are inadequate to prevent significant crop loss in the US. New variations within pathogen populations rather than changes in cropping systems or environmental conditions are responsible for these losses. Chemical control possibilities for both diseases are limited, as the prevalent Phytophthora strains have become increasingly resistant to traditional fungicides, and few chemical controls exist for common scab. Little information is available on variation within the scab pathogen populations in the field or variation in scab resistance in potato germplasm. Effective disease control relies on understanding how an organism causes disease and how plants resist infection. Strategic and sustainable deployment of disease resistance requires knowledge of resistance mechanisms in potatoes, and of infection mechanisms and genetic variability in pathogens. We are investigating several strategies for protecting potato quality and yields against late blight and common scab. The goals of the project are: (I) Molecular characterization of potato resistance/susceptibility responses to Phytophthora infestans and Streptomyces sp., (II) Engineering potatoes to express antimicrobial peptides effective against Phytophthora diseases, and (III) Development of new molecular tools for following the evolution, spread and disease- causing potential of the pathogens that cause these two diseases. Specific sub-objectives (milestones) for the project are outlined in section 2. Information on pathogen and plant variability is essential for identifying potential disease resistance markers that can be used in breeding more resistant potato varieties, and for developing disease management strategies to minimize losses to growers. This research fits into the objectives of National Program 303, Plant Disease to reduce crop and commodity losses due to pathogens, using effective and affordable technology that poses minimum risk to consumers and the environment. The project specifically addresses components 4 and 5 of National Program 303 Action Plan: (4) Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship with Hosts and Vectors, and (5) Host Plant Resistance to Disease, for the commodity, potato. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Construct cDNA library from virulent P. infestans isolate-Sequence ESTs Develop scab assay Clone and characterize GPI-encoding genes from P. infestans Year 2 (FY 2005) Construct cDNA library from less virulent P. infestans isolate-Sequence ESTs Develop new markers based on P. infestans RG57 marker Construct and test peptide delivery systems Year 3 (FY 2006) Clone PAL and CHS gene families (from potato)Screen peptide libraries Analyze secreted proteins from P. infestans. Year 4 (FY 2007) Clone WRKY and CHI gene families (from potato)Catalog and compare EST libraries Confirm scab resistance/susceptibility in different potato varieties Year 5 (FY 2008) Study expression of PAL, CHI, WRKY and CHS gene families in susceptible and resistant potato varieties Study expression of selected ESTs in infected potato plants. Screen promoters for temporal and spatial differences in antimicrobial protein expression. Characterize gene regions associated with pathogenicity in Streptomyces. 3. Milestones: List the milestones (from the list in Question #2) that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. The milestones listed below were scheduled to be completed in FY 2004 (Year 1) and were substantially or fully met:. Develop scab assay. Clone and characterize GPI-encoding genes from P. infestans. The following milestone was not met: Construct cDNA library from virulent P. infestans isolate-Sequence ESTs. We propose to eliminate it from future plans in this project, because substantially similar work was published in May of 2004 [Ros, et al. (2004) Analysis of differentially expressed genes in a susceptible and moderately resistant potato cultivar upon Phytophthora infestans infection. Mol Plant Pathol 5:191-201.] B. List the milestones (from the list in Question #2) that you expect to address of the next 3 years (FY 2005, 2006, 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 2 (FY 2005) Construct cDNA library from less virulent P. infestans isolate-Sequence ESTs. We propose to eliminate this objective on the grounds that substantially similar work has recently been published, as described above in FY 2004. Develop new markers based on the P. infestans RG57 molecular marker. Discrete bands arising from new molecular fingerprints will be cloned and sequenced to determine their relationship to previously known RG57 regions. The fragments will be tested to determine if they could be adapted to PCR-based screening of isolates. Construct and test peptide delivery systems. A novel prosystemin-based carrier protein will be designed and used in transgenic plants to test effectiveness in delivering antimicrobial peptides that are useful in pathogen control. A manuscript will be published describing the outcome. Year 3 (FY 2006) Clone PAL and CHS gene families (from potato). In FY 2004, sequence data available in the public domain for these two gene families from potato and from other Solanaceous relatives has been obtained, aligned and used to identify conserved and non-conserved regions for primer design. In FY 2005, primers will be used to clone additional gene family members from potato. These will be sequenced and the sequence information will be used to design gene-specific probes for each family member in FY 2005 and 2006. Screen peptide libraries. Selected peptides will be screened to identify new candidates for delivery in transgenic plants. This will support ongoing research on application of antimicrobial peptides for pathogen control. Analyze secreted proteins from P. infestans. The genes encoding the extracellular enzymes cutinase and endoglucanase will be cloned, sequenced and over-expressed. A manuscript will be published reporting on these sequences and enzyme activities. Year 4 (FY 2007) Clone WRKY and CHI gene families (from potato). In FY 2005, sequence data available in the public domain for these two gene families from potato and from other Solanaceous relatives will be obtained, aligned and used to identify conserved and non-conserved regions for primer design. Catalog and compare EST libraries. We propose to eliminate this objective (see above). Confirm scab resistance/susceptibility in different potato varieties. Cultivated potato varieties, genetic breeding material, and wild Solanum species will be tested for scab susceptibility using both field tests and controlled-environment tests with well-defined Streptomyces strains. Results are being made available to growers and breeders as they are obtained. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004 (one per Research (OOD) Project) New improved molecular markers identify pathogen origins, and suggest a mechanism of virulence enhancement. Genes encoding glucose-6-phosphate isomerase (GPI) proteins produced by P. infestans were cloned and characterized. This enzyme has been used worldwide as a tool for aiding in characterization of P. infestans isolates. Sequence analysis and genome comparisons identified molecular variations (gene duplications) that correlate with enhanced virulence. New P. infestans isolates appear to originate both by worldwide transport and by interspecies hybridization. Detailed characterization of GPI enhances tracking of isolate origins, providing a useful tool to scientists worldwide. B. Other significant accomplishments, if any. 1) Spread of fungicide-resistant new mating type of P. infestans into the northeastern US. On-going genetic change was assessed in P. infestans populations infecting potato and tomato across the US. Tomato and potato late blight, caused by the oomycete pathogen Phytophthora infestans, has become an increasingly important problem to agriculture in the United States in the past decade as more aggressive, fungicide (metalaxyl)- resistant, and host-specialized isolates have appeared. To obtain new information about the on-going evolution of this species, we extracted DNA from the mitochondria of P. infestans and determined the genetic sequence of this DNA. Comparing unique mitochondrial DNA sequences from 325 strains collected during a 2-year period from 2003-2004, we found that novel metalaxyl-resistant strains of the A2 mating type were introduced into northeastern states during this time. We are using this information to elucidate how populations of the pathogen are sub- structured, and to estimate the contribution of sexual reproduction, which has been possible in the US for 15+ years, in generating variation and in permitting over-wintering of P. infestans inoculum. 2) Novel potential reservoir of the late blight pathogen in a weed species in Wales. We have discovered the natural occurrence of P. infestans on black nightshade (a common weed in the UK and Ireland) in Wales. The strain isolated is a member of a recent immigrant population, and may infect a wider host-range than pathogens in the older P. infestans populations. This discovery is important, since these weed hosts may serve as reservoirs of inoculum originating from potato or tomato crops, and the existence of inoculum reservoirs affects disease management strategies. However, these infections have only been found when blight is already widespread in potato fields and there is no evidence to suggest that black nightshade acts as an over-wintering host in Wales. This research was a cooperative effort with the University of Wales, Bangor and Queens University, Belfast, Northern Ireland. 3) Demonstration that potato (and radish) common scab disease severity is a function of pathogen load. Scab disease symptoms and severity vary from year to year in the same field locations and cultivars, but the basis for this variation has not been well understood. We demonstrated that scab symptoms are not seen at low pathogen densities, and that symptom severity increases as the pathogen inoculum increases over several orders of magnitude. We conclude that fluctuations in pathogen population density are important components of variation in disease severity. Measurements of pathogen load could eventually be applied to assaying the likelihood of a serious scab problem causing economic losses in specific field locations and years. C. Significant activities that support special target populations. None D. Progress Report---opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ('D') projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This information is especially useful to NPS during the National Program assessment in the 4th and 5th years of the program cycle. This is the first year of this project; the accomplishments of the project are therefore the same as the accomplishments for the past year, listed above. 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? Preliminary information on the scab susceptibility of 11 potato varieties and 6 breeding lines grown in different geographic areas has been made available to breeders and growers. Genetic sequences were deposited in GenBank, making them available to the global research community free of charge: These included an extensive list of GPI clones submitted by Ospina-Giraldo and Jones. The occurrence of new strains P. infestans that have been isolated and identified has been reported to potato farmers, agriculture agents and crop consultants. Potato germplasm that exhibited significant reduction in late blight compared to known standards was selected for extensive field characterization studies and results were reported to interested plant bioengineers in California. Recent information about late blight disease movement via potato tubers was communicated to staff and graduate students conducting cooperative studies at Queens University in Belfast, Northern Ireland, Jersey, UK and Michigan State University. Information about late blight disease control was communicated to tomato growers in New Jersey, Tennessee and North Carolina and potato growers at the Potato Annual Meeting. Cooperative testing late blight resistant germplasm and newly developed transgenic clones was accomplished in an agreement with Mexican scientists. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. The research career of Dr. Deahl was the focus of an article entitled "Blight Fighter" in the April 2003 issue of "The Furrow" magazine.

Impacts
(N/A)

Publications

  • Wanner, L.A. 2003. Molecular and pathogenic variation in streptomycetes causing potato or radish scab [abstract and talk]. 20th annual meeting of the Mid-Atlantic Plant Molecular Biology Society, Beltsville, MD 8-9 Aug 2003.
  • Wanner, L.A. 2003. Molecular and pathogenic variation in streptomycetes causing common scab [abstract and talk]. Phytopathology 93 (6):S89 Supplement
  • Deahl, K. 2004. Population dynamics of Phytophthora infestans assessed by mitochondrial DNA.[abstract] American Journal of Potato Research 81(1):54.
  • Deahl, K.L., Shaw, D.S., Cooke, L.R. 2004. Natural Occurrence of Phytophthora infestans on Black Nightshade (Solanum nigrum) in Wales. Plant Disease. 88(7):771.
  • Wattier, R.A.M., Gathercole, L.L., Assinder, S.J., Gliddon, C.J., Deahl, K. L., Shaw, D.S., Mills, D.I. 2003. Sequence variation of intergenic mitochondrial DNA spacers (mtDNA-IGS) of Phytophthora infestans (Oomycetes) and related species. Molecular Ecology Notes 3:(1)136-138.
  • Ospina-Giraldo, M., Jones, R.W. 2003. Characterization of the glucose-6- phosphate isomerase gene in Phytophthora infestans reveals the presence of multiple alleles. Fungal Genetics and Biology 40:197-206.
  • Wanner, L.A. 2003. Molecular and pathogenic variation in streptomycetes causing potato or radish scab [poster] 8th International Congress of Plant Pathology, Christchurch, NZ, 2-7 Feb. 2003


Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Disease control strategies for Phytophthora and other pathogens are inadequate to prevent significant crop loss in the US. Effective disease control relies on understanding how an organism causes disease and how plants resist infection. Strategic and sustainable deployment of durable disease resistance requires knowledge of resistance mechanisms in potatoes, and of infection mechanisms and genetic variability in pathogens. We are investigating several strategies for protecting potato quality and yields against late blight and common scab. These include development of new molecular tools for following the evolution, spread and disease causing potential of the pathogens that cause these two diseases. We will conduct molecular studies of potato resistance responses, investigate potential molecular markers for disease resistance that could be used in potato breeding programs, and engineer potatoes to express antimicrobial peptides [effective against Phytophthora diseases, and potentially against other potato diseases]. 2. How serious is the problem? Why does it matter? In the past 10 years, potato production has suffered substantial losses due to dramatic increases in foliar and tuber diseases, especially those caused by the Phytophthora pathogens. New variations within pathogen populations rather than changes in cropping systems or environmental conditions are responsible for these losses. Chemical control possibilities have also become more limited as the prevalent Phytophthora strains have become increasingly resistant to traditional fungicides. Potato common scab is the fourth most important potato disease in the US, and few chemical controls exist. Little information is available on variation within pathogen populations in the field and variation in potato resistance. Information on pathogen and plant variability will be essential in breeding for more resistant potato varieties, and for developing disease management strategies to minimize losses to growers. Potato breeders will benefit by increased understanding of variations in pathogen populations as they can use this information to design more robust screens for disease resistance. Growers will also benefit, as they can choose potato cultivars better suited to their growing conditions, based on the prevalence of specific pathogen varieties and resistance characteristics. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research falls under National Program 303, Plant Diseases, with the goal to develop control strategies to reduce losses caused by plant diseases that are both effective and affordable while maintaining environmental quality, and reduce economic and environmental risks through improved management of agricultural production systems. The results of this research will allow scientists to devise new strategies to determine vulnerable sites in the life cycle of the pathogens, and to accurately determine the possible variation in pathogen population due to migration or mutation, thus enabling growers to employ the most effective integrated disease control methods with the least amount of fungicide. 4. What were the most significant accomplishments this past year? A. Chronicling of diversity in Phytophthora pathogen populations. Genetic variation was examined in a study evaluating over 320 strains of P. infestans that were collected in the Pacific Northwest during an eight- year period from 1992 to 1999. During this timeframe a fungicide resistant strain of the A2 mating type was introduced into the Columbia Basin (in 1993) and changes in the population were monitored in the ensuing years. DNA fingerprinting was used to characterize population diversity of this pathogen. By using unique DNA sequences as molecular markers, we were able to demonstrate greater levels of genotypic diversity in the new populations of P. infestans, and that population structures differ from one location to another. The current more diverse populations have pathogenic characteristics not previously found. DNA fingerprinting provides a mechanism for monitoring changes that occurred and continue to occur, either because of sexual recombination or through introduction of genetically different strains to potato stock. The major impact of understanding pathogen diversity will be to ensure that development of potato cultivars resistant to late blight or to scab is nonspecific (horizontal) and not limited to virulence genes present in the local populations of the pathogens. B. Discovery of additional alternate hosts for Phytophthora infestans. We have discovered the natural occurrence of P. infestans on woody nightshade (a common hedgerow plant in the UK and Ireland) in Northern Ireland. This isolated strain was a member of the recent immigrant population that may infect a wider host range than pathogens in the older populations. However, these infections have only been found when blight is already widespread in potato fields and there is no evidence to suggest that woody nightshade acts as an over-wintering host in Ireland. This discovery will have major impact if future results indicate that these plants are serving as reservoirs for the pathogen. C. In 2001 and 2002, a new disease was detected on petunia transplants at a glasshouse in central Maryland that provides flowers and bedding plants to local markets. This leaf blight on petunia is caused by P. infestans. This is the first time that this plant pathogen, P. infestans, was isolated and characterized on greenhouse grown petunias in the state of Maryland. The impact of this discovery is that floral and bedding plants could harbor this pathogen, presenting a new potential reservoir of the pathogen that could spread to potatoes, tomatoes, and other solanaceous crops. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A new, highly virulent race of P. infestans appears to have at least three copies of the gene coding for glucose 6 phosphate isomerase, suggesting that gene duplication may be leading to greater virulence. Scientists in the Vegetable Laboratory at Beltsville, MD, provided a molecular characterization of glucose 6 phosphate isomerase, and cloned the gene. The gene has now been cloned from P. infestans, P. erythroseptica and other Phytophthora species. This discovery allows for monitoring of populations to determine potential gene flow and possible virulence enhancement. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2004: Population diversity in P. infestans will be characterized by DNA fingerprinting and molecular markers. Cloning of families of genes in potatoes, as an initial step to determine whether specific allele combinations may be useful as selectable markers for late blight resistance. FY2005: Screening of peptide libraries for candidate antimicrobial peptides effective against late blight. Development of new markers for P. infestans genotypic diversity based on the RG57 restriction fragment length polymorphism. FY2006: Natural variation in aggressiveness of Streptomyces isolated from scabby potatoes documented, and molecular fingerprints for particularly aggressive strains developed. Pathogen load of Streptomyces scabies determined in potato production areas where scab occurs. cDNA libraries constructed from P. infestans-infected potato leaves, and ESTs sequenced. Antimicrobial peptide delivery systems constructed and tested. 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? Recent information about late blight disease movement in potato tubers was communicated to university staff and graduate students conducting cooperative studies Northern Ireland. Information about late blight disease control was communicated to tomato growers in Tennessee and North Carolina and potato growers at the Potato Annual Meeting in Spokane, WA. 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). Research on late blight was cited in a Science article entitled "Taking the Bite Out of Potato Blight" by Glenn Garelik, November 2002. The research career of Vegetable Lab scientist was the focus of an article entitled "Blight Fighters" in April issue of John Deere magazine.

Impacts
(N/A)

Publications

  • Deahl, K.L., Fravel, D.R., Occurrence of Leaf Blight on Petunia Caused by Phytophthora infestans in Maryland. Plant Disease. 2003. v.87. p.1004.
  • Deahl, K.L., Pagani, M.C., Vilaro, F.L., Perez, F.M., Moravec, B. Cooke, L. R. Characteristics of Phytophthora infestans isolates from Uruguay. European Journal of Plant Pathology. 2003. v. 109. p. 277-281.