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? Due to the potential loss of pesticide registrations resulting from the Food Quality Plant Protection Act, worker safety issues, and public concerns about pesticide usage, methods for more efficient use of pesticides, and alternatives to synthetic chemical pesticides are needed. The long-term goal of this CRIS is the development of holistic production systems for horticultural crops where biological control and cultural methods are the dominant practices for managing multiple pests of horticulture crops. A combination of laboratory and field experiments combined with modern molecular techniques will be used to address these objectives. Knowledge and technologies gained from this research will enhance a grower's ability to utilize biocontrol and traditional control measures within their
production system. By understanding how microorganisms interact on foliar and fruit surfaces and how these interactions are impacted by the plant and environmental conditions, better management systems can be developed that deploy biocontrol and traditional control measures at appropriate times to enhance their survival and efficacy. The project has 3 specific goals: 1) Advance understanding of the biology of foliar and fruit pathogens and develop regional disease forecasting systems; 2) Determine the role of biofilms in the establishment and efficacy of a biocontrol agent; 3) Develop and implement strategies for enhancing the establishment and efficacy of biocontrol agents on aerial plant surfaces. This research falls under the National Program 303, Plant Diseases (100%) and addresses Components 2 (Biological Control) and 4 (Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationships with and Vectors) . We are addressing Biological Control Component 2 by developing
alternative systems for achieving biological control than the traditional application of individually formulated microorganisms and increasing our understanding of factors that influence the survival of microorganisms on aerial plant surfaces. We are addressing the Pathogen Biology Component 4 by focusing on powdery mildews of numerous crops and Botrytis cinerea on strawberry, caneberry and geraniums. We are developing techniques to more rapidly and accurately quantify disease levels in the field (particularly low levels). These methods will then be used to develop economic thresholds for action. We are increasing our understanding of how diurnal temperature fluctuation impacts powdery mildew development with emphasis on how brief exposure to nonconducive temperatures impact infection of powdery mildews. This data is being used to develop disease forecasting models that can be utilized for most powdery mildews across most crops. We are developing a better understanding of how and
when B. cinerea infects plants. 2. List by year the currently approved milestones (indicators of research progress) FY2004: Objective 1: 1. Forecasting Powdery Mildew Infection Risk; 2. Effects of fluctuating temperature; 3. Immediate exposure to supra-optimal temperatures. 4. Delayed exposure to supra-optimal temperatures. Objective 2: A: 1. Development of FP62 derivatives expressing GFPuv. 2. Microscopic Observation. 3. Spatial distribution of biofilms. B: 1. Cloning of restriction fragments with mini-Tn5-lacZ1 from FP62 mutants 2. Marker-exchange mutagenesis. 3. Sequence analysis of gene(s) disrupted by mini-Tn5-lacZ1 insertion. Objective 3: 1. Disease Assay. 2. Field Validation. FY2005: Objective 1: 1. Plant Characters effecting Etiology/Epidemiology of B. cinerea. 2. Forecasting Powdery Mildew Infection Risk; 3. Effects of fluctuating temperature; 4. Delayed exposure to supra-optimal temperatures. 5. Exposure to simulated field temperatures. Objective 2: A: 1. Development of
FP62 derivatives expressing GFP; 2. Microscopic Observation; 3. Spatial distribution of biofilms. B: 1. Marker-exchange mutagenesis; 2. Biofilm and biocontrol assessment of mutants. Objective 3: 1. Disease Assay; 2. Field Validation. FY2006: Objective 1: 1. Plant Characters effecting Etiology/Epidemiology of B. cinerea; 2. Forecasting Powdery Mildew Infection Risk; 3. Exposure to simulated field temperatures; 4. Field Validation. Objective 2: A: 1. Spatial distribution of biofilms; 2. Structure of FP62 Biofilms on the leaf surface; 3. FP62 biofilms and control of B. cinerea. B: 1. Biofilm and biocontrol assessment of mutants; 2. Functional characterization of biofilm genes; 3. Monitoring biofilm gene expression in the phyllosphere. Objective 3: 1. Disease Assay; 2. Field Validation. FY2007: Objective 1: 1. Forecasting Powdery Mildew Infection Risk; 2. Field Validation. Objective 2: A: 1. Structure of FP62 Biofilms on the leaf surface; 2. FP62 biofilms and control of B. cinerea. B: 1.
Biofilm and biocontrol assessment of mutants; 2. Functional characterization of biofilm genes; 3. Monitoring biofilm gene expression in the phyllosphere. Objective 3: 1. Disease Assay; 2. Field Validation. 4a List the single most significant research accomplishment during FY 2006. New approaches to collection and quantification of aerobiota. We have developed a quantitative PCR protocol based on TaqMan probes that can consistently enumerate field samples containing >1000 of Podosphaera macularis (hop Powdery Mildew) and inexpensive solar power spore traps. Our ability to manage hop powdery mildew is limited by our understanding of factors influencing inoculum availability and ability to detect low levels of disease; this research allows for the rapid and accurate detection of P. macularis from field samples. This research was done by the Horticultural Crops Research Unit in Corvallis, Oregon and cooperators at National Forage Seed and Cereal Research Center in Corvallis and
Washington State University in Prosser, Washington. The qPCR technique will allow for detailed studies on factors influencing inoculum availability that will be used to enhance current disease forecasting models; coupled with an inexpensive solar powered spore trap that we developed, this technique appears to be useful in estimating disease levels in fields. These outcomes will enhance our ability to effectively and economically manage hop powdery mildew by more accurately timing and reducing fungicide applications, and aith development of other primers, these techniques will also be useful for other powdery mildews or other plant diseases. (NP 303, Component 2). 4b List other significant research accomplishment(s), if any. The distribution of blackberry rust and factors influencing disease development. Blackberry rust (Phragmidium violaceum) was first reported in the US near Coos Bay, Oregon in April 2006, and was later found to be devastating Evergreen blackberry fields in the
Willamette Valley. Blackberry rust was found in wild Himalaya blackberry west of the Cascade Mountains from northern California to the Canadian border and appears to be limited to elevations below 2500 ft. Temperature and leaf wetness conditions suitable for disease development from asexual spores were determined. Blackberry cultivars important to growers in the Pacific Northwest, other than Evergreen types, were determined to be resistant to two isolates of Phragmidium violaceum. This work was done at the Horticulture Crops Research Unit in Corvallis, Oregon and a cooperator at Oregon State University. The information developed can be used to develop disease forecasting models to help predict disease development of blackberry rust and assist growers in determining when to apply fungicides. (NP 303, Component 1 and 4). 5. Describe the major accomplishments to date and their predicted or actual impact. We developed methods for the trapping and identification of aerial borne spores
of hop and grape powdery mildew that are suitable for PCR and QPCR analysis. Using DNA sequence specific to individual species of powdery mildews and an inexpensive custom spore trap, we were able to detect spore movement 10-14 days prior to visually detecting disease in fields. These new methods will allow for highly accurate assessments of when an epidemic begins in a specific field and timing of fungicide applications. Preliminary results indicate that 2 or more applications could be eliminated in most years. It could also result in a more accurate determination of the environmental conditions favorable to spore movement occurs and could lead to more accurate assessment of disease incidence. (NP 303 Component 1, Problem Statement a; Component 2, Problem Statement c). Indentified GTPase and Hemoagglutinin likely genes that are associated with biofilm formation and biocontrol activity of the biocontrol agent Burkholderia pyrrocinia FP62. Some of the genetic determinants were
identified to be associated with biofilm formation and biological control of Botrytis cinerea by FP62. The alleles that were disrupted by transposon insertion in two mutants deficient in biofilm formation and biocontrol activity were identified and cloned. Sequence analysis tentatively indicates that they encode for a GTPase involved with ribosome binding and another unidentified activity and hemoagglutinin formation. Complementation analysis is still needed to confirm these relationships. Impact: Understanding the importance and mechanisms of biofilm formation in the efficacy of foliar biocontrol agents will lead to the development of improve delivery technologies to enhance the efficacy of biological control agents. (Dr. Walter. Mahaffee, Dr. Caroline Press). (NP 303 Component 4, Problem Statement a). Determined that the Gubler/Thomas model and previous literature do not accurately reflect the temperatures that are conducive for infection of Vitis vinifera by Erysphae necator
(Grape Powdery Mildew). We have determined that short exposures to temperatures greater than 36 degrees C are conducive for infection contrary to the previously report upper limit of 35 degrees C. Impact: This new information will result in a modified infection risk model of grape powdery mildew that will more accurately reflect the risk of infection based on how diurnal temperature fluctuations impact disease development. (Dr. Walter Mahaffee, Dr. Steve Scheuerell, Oregon State University, and Dr. Gary Grove, Washington State University). (NP 303 Component 2, Problem Statements a and c). 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Grove, G.G., Mahaffee, W. F., Gent, D.H., Peetz, A.B., and Falacy, J. S. Detection and quantification of powdery mildew aerobiota. Second International Workshop on Powdery Mildews Mahaffee, W.M., Turechek,
W.W., Thomas, C.S., Nelson, M., Fox, A.D., Gent, D.H., and Grove, G.G. 2006. Forecasting infection risk of hop powdery mildew. Second International Workshop on Powdery Mildews Gent, D.H., Mahaffee, W.M., and Turechek, W.W. 2006, Sequential estimation and classification of the incidence of hop powdery mildew on leaves and cones. Second International Workshop on Powdery Mildews Johnson, K.B. and Mahaffee, W.F. 2006. Understanding impacts of blackberry rust in the Pacific Northwest. Special Session on Introduced pathogens at the annual meeting of the Pacific Division of the American Phytopathological society. Peetz, A.B., Mahaffee, W.M., Turechek, W.W., Thomas, C.S., Nelson, M., Gent, D.H., Ocamb, C.A., and Grove, G.G. Hop powdery mildew in the Pacific Northwest. Special Session on Introduced pathogens at the annual meeting of the Pacific Division of the American Phytopathological society. Mahaffee, W.M. 2006. Response to an introduced disease: Hop powdery mildew in the Pacific
Northwest. Department of Plant Pathology at Ohio State University. Gent, D.H., Mahaffee, W.M., and Turechek, W.W. 2006. Spatial heterogenetity and sampling of hop powdery mildew in cones. Annual meeting of the American Phytopathological society. Mahaffee, W.M. 2006. Blackberry Rust. Annual Meeting Oregon/Washington Cane berry Commission Peetz, A.B., Mahaffee, W.M., Gent, D.H., and Grove, G.G. 2006. New tools for disease management. Annual meeting Hop research Council. Bassil, N.V., R. Botta, and S.A. Mehlenbacher. 2005. Additional microsatellite markers of the European hazelnut. Acta Horticulturae 686:105-110. Bassil, N.V., R. Botta and S. Mehlenbacher. 2005. Microsatellite markers in hazelnut: Isolation, characterization and cross-species amplification. J. Amer. Soc. Hort. Sci. 130:543-549. Boccacci, P., A. Akkak, N.V. Bassil, S.A. Mehlenbacher and R. Botta. 2005. Characterization and evaluation of microsatellite loci in European hazelnut (Corylus avellana L.) and
their transferability to other Corylus species. Mol. Ecol. Notes 5:934-937. Chen, H., S.A. Mehlenbacher and D.C. Smith. 2005. AFLP markers linked to eastern filbert blight resistance from OSU 408.040 hazelnut. J. Amer. Soc. Hort. Sci. 130:412-417. Chen , H., S.A. Mehlenbacher and D.C. Smith. 2006. Hazelnut accessions provide new sources of resistance to eastern filbert blight. HortScience (in press). Gokirmak, T. 2005. Characterization of European hazelnut (Corylus avellana L.) cultivars using SSR markers. M.S. thesis, Oregon State University. 170 p. Gokirmak, T., S.A. Mehlenbacher and N.V. Bassil. 2005. Investigation of genetic diversity among European hazelnut (Corylus avellana L.) cultivars using SSR markers. Acta Horticulturae 686:141-147. Lunde, C.F., S.A. Mehlenbacher and D.C. Smith. 2006. Segregation for resistance to eastern filbert blight in progeny of 'Zimmerman' hazelnut. J. Amer. Soc. Hort. Sci. (in press). McCluskey, R.L., A.N. Azarenko, S.A. Mehlenbacher,
and D.C. Smith. 2005. Advanced selection and cultivar performance of hazelnut trials planted in 1994 and 1998 at Oregon State University. Acta Horticulturae 686:71-78. Mehlenbacher, S.A. 2005. Release of the first commercially acceptable EFB-resistant hazelnut variety. Ann. Rep. Nut Growers Soc. OR, WA & BC 90:33-36. Mehlenbacher, S.A. 2005. Turkey trip. Ann. Rep. Nut Growers Soc. OR, WA & BC 90:36-40. Mehlenbacher, S.A. 2006. Evaluation of OSU hazelnut breeding program selections. Ann. Rep. Nut Growers Soc. OR, WA & BC 91 (in press). Mehlenbacher, S.A., R.N. Brown, E.R. Nouhra, N.V. Bassil and T.L. Kubisiak. 2005. A linkage map for hazelnut. Acta Horticulturae 686:135- 140. Mehlenbacher, S.A., R.N. Brown, E.R. Nouhra, T. Gokirmak, N.V. Bassil and T.L. Kubisiak. 2006. A genetic linkage map for hazelnut (Corylus avellana L.) based on RAPD and SSR markers. Genome 49:122-133. Grove, G.G., Mahaffee, W., Falacy, J., Galloway, H., and Peetz, A. 2005. Detection of Erysiphe
necator in the air of grape vineyards in Eastern Washington. Phytopathology 95:S36.
Impacts
(N/A)
Publications
- Scheuerell, S., Mahaffee, W.F. 2006. Variability associated with suppression of gray mold (Botrytis cinerea) on geranium (Pelargonium x hortorum) by foliar applications of non-aerated and aerated compost teas. Plant Disease. 90:1201-1208.
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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? Due to the potential loss of pesticide registrations resulting from the Food Quality Plant Protection Act, worker safety issues, and public concerns about pesticide usage, methods for more efficient use of pesticides, and alternatives to synthetic chemical pesticides are needed. The long-term goal of this CRIS is the development of holistic production systems for horticultural crops where biological control and cultural methods are the dominant practices for managing multiple pests of horticulture crops. A combination of laboratory and field experiments combined with modern molecular techniques will be used to address these objectives. Knowledge and technologies gained from this research will enhance a grower's ability to utilize biocontrol and traditional control measures within their production
system. By understanding how microorganisms interact on foliar and fruit surfaces and how these interactions are impacted by the plant and environmental conditions, better management systems can be developed that deploy biocontrol and traditional control measures at appropriate times to enhance their survival and efficacy. The project has 3 specific goals: 1) Advance understanding of the biology of foliar and fruit pathogens and develop regional disease forecasting systems; 2) Determine the role of biofilms in the establishment and efficacy of a biocontrol agent; 3) Develop and implement strategies for enhancing the establishment and efficacy of biocontrol agents on aerial plant surfaces. This research falls under the National Program 303, Plant Diseases (100%) and addresses Components 2 (Biological Control) and 4 (Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationships with and Vectors) . We are addressing Biological Control Component 2 by developing alternative
systems for achieving biological control than the traditional application of individually formulated microorganisms and increasing our understanding of factors that influence the survival of microorganisms on aerial plant surfaces. We are addressing the Pathogen Biology Component by focusing on powdery mildews of numerous crops and Botrytis cinerea on strawberry, caneberry and geraniums. We are developing techniques to more rapidly and accurately quantify disease levels in the field (particularly low levels). These methods will then be used to develop economic thresholds for action. We are increasing our understanding of how diurnal temperature fluctuation impacts powdery mildew development with emphasis on how brief exposure to nonconducive temperatures impact infection of powdery mildews. This data is being used to develop disease forecasting models that can be utilized for most powdery mildews across most crops. We are developing a better understanding of how and when B. cinerea
infects plants. 2. List the milestones (indicators of progress) from your Project Plan. 1.1.1 Environmental factors effecting Etiology/Epidemiology of B. cinerea. 1.1.2 Plant Characters effecting Etiology/Epidemiology of B. cinerea. 1.1.3 Forecasting Powdery Mildew Infection Risk 1.1.3.1 Effects of fluctuating Temperature. 1.1.3.1.1 Immediate exposure to supra-optimal temperatures 1.1.3.1.2 Delayed exposure to supra-optimal temperatures. 1.1.3.2 Exposure to simulated field temperatures. 1.1.3.3 Field Validation. 2.1.1 Characterize biofilms established on leaf tissue by FP62 2.1.1.2 Development of FP62 derivatives expressing GFPuv. 2.1.1.3 Microscopic Observation. 2.1.1.4 Spatial distribution of biofilms 2.1.1.5 Structure of FP62 Biofilms on the leaf surface. 2.1.1.6 FP62 biofilms and control of B. cinerea. 2.1.2 Evaluate the role of biofilm-associated genes in biocontrol. 2.1.2.1 Cloning of restriction fragments with mini-Tn5-lacZ1 from FP62 mutants 2.1.2.2
Marker-exchange mutagenesis. 2.1.2.3 Sequence analysis of gene(s) disrupted by mini-Tn5-lacZ1 insertion. 2.1.2.4 Biofilm and biocontrol assessment of mutants. 2.1.2.5 Functional characterization of biofilm genes. 2.1.2.6 Monitoring biofilm gene expression in the phyllosphere. 3.1.1 Disease Assay. 3.1.3 Field Validation. 4.1 Detection and Quantification of airborne powdery mildew spores. 4.1.1 Development of species specific primers for several powdery mildew species 4.1.2 Develop quantitative PCR protocol for Podosphaera macularis and Erysiphe necator 4.1.2.1 Develop efficient extraction protocol 4.1.2.2 Determine detection limits 4.1.3 Develop a reliable and inexpensive trapping method for air borne fungal propagules. 4.1.4 Determine relationship of disease levels in the field with quantity of spores trapped 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. Environmental factors effecting Etiology/Epidemiology of B. cinerea. Milestone Not Met Redirection of Research focus due to change in priorities 2. Plant Characters effecting Etiology/Epidemiology of B. cinerea. Milestone Not Met Redirection of Research focus due to change in priorities 3. Forecasting Powdery Mildew Infection Risk; Effects of fluctuating Temperature. Milestone Fully Met 4. Forecasting Powdery Mildew Infection Risk; Exposure to simulated field temperatures. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 5. Characterize biofilms established on leaf tissue by FP62; Development of FP62 derivatives expressing GFP. Milestone Fully Met 6. Characterize biofilms established on leaf tissue by FP62; Microscopic observation. Milestone Substantially Met 7. Characterize biofilms established on leaf tissue by FP62; Spatial distribution of biofilms. Milestone Not Met Progress slowed by resource limitation
(human,fiscal,equipment, etc. 8. Evaluate the role of biofilm-associated genes in biocontrol; Marker- exchange mutagenesis. Milestone Fully Met 9. Evaluate the role of biofilm-associated genes in biocontrol; Biofilm and biocontrol assessment of mutants. Milestone Substantially Met 10. Develop and implement strategies for enhancing the establishment and efficacy of BCAs; Disease Assay and Field Validation. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 1.1.3 Forecasting Powdery Mildew Infection Risk 1.1.3.2 Exposure to simulated field temperatures. 1.1.3.3 Field Validation. 2.1.1 Characterize biofilms established on leaf tissue by FP62 2.1.1.4 Spatial distribution of biofilms 2.1.2 Evaluate the role of biofilm-associated genes in biocontrol. 2.1.2.4 Biofilm and biocontrol assessment of mutants. 2.1.2.5
Functional characterization of biofilm genes. 4.1 Detection and Quantification of airborne powdery mildew spores. 4.1.1 Development of species specific primers for several powdery mildew species 4.1.2 Develop quantitative PCR protocol for Podosphaera macularis and Erysiphe necator 4.1.2.1 Develop efficient extraction protocol 4.1.2.2 Determine detection limits 4.1.3 Develop a reliable and inexpensive trapping method for air borne fungal propagules. FY 2007 1.1.3 Forecasting Powdery Mildew Infection Risk 1.1.3.3 Field Validation. 2.1.1 Characterize biofilms established on leaf tissue by FP62 2.1.1.5 Structure of FP62 Biofilms on the leaf surface. 2.1.1.6 FP62 biofilms and control of B. cinerea. 2.1.2 Evaluate the role of biofilm-associated genes in biocontrol. 2.1.2.5 Functional characterization of biofilm genes. 4.1 Detection and Quantification of airborne powdery mildew spores. 4.1.3 Develop a reliable and inexpensive trapping method for air borne fungal propagules.
4.1.4 Determine relationship of disease levels in the field with quantity of spores trapped FY 2008 1.1.3 Forecasting Powdery Mildew Infection Risk 1.1.3.1 Effects of fluctuating Temperature. 2.1.2 Evaluate the role of biofilm-associated genes in biocontrol. 2.1.2.4 Biofilm and biocontrol assessment of mutants. 2.1.2.5 Functional characterization of biofilm genes. 2.1.2.6 Monitoring biofilm gene expression in the phyllosphere. 3.1.1 Disease Assay. 4.1 Detection and Quantification of airborne powdery mildew spores. 4.1.3 Develop a reliable and inexpensive trapping method for air borne fungal propagules. Publish papers 4a What was the single most significant accomplishment this past year? We developed methods for the trapping and identification of aerial borne spores of hop and grape powdery mildew that are suitable for PCR and QPCR analysis. Using DNA sequence specific to individual species of powdery mildews and an inexpensive custom spore trap, we were able to detect
spore movement 10-14 days prior to visually detecting disease in fields. This was done by the Horticultural Crops Research Unit in Corvallis, Oregon and cooperators at Washington State University in Prosser, Washington. These new methods will allow for highly accurate assessments of when an epidemic begins in a specific field and timing of fungicide applications. Preliminary results indicate that 2 or more applications could be eliminated in most years. It could also result in a more accurate determination of the environmental conditions favorable to spore movement occurs and could lead to more accurate assessment of disease incidence. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Indentified GTPase and Hemoagglutinin likely genes that are associated with biofilm formation and biocontrol activity of the biocontrol agent Burkholderia pyrrocinia FP62. Some of the genetic determinants were identified to be associated
with biofilm formation and biological control of Botrytis cinerea by FP62. The alleles that were disrupted by transposon insertion in two mutants deficient in biofilm formation and biocontrol activity were identified and cloned. Sequence analysis tentatively indicates that they encode for a GTPase involved with ribosome binding and another unidentified activity and hemoagglutinin formation. Complementation analysis is still needed to confirm these relationships. Impact: Understanding the importance and mechanisms of biofilm formation in the efficacy of foliar biocontrol agents will lead to the development of improve delivery technologies to enhance the efficacy of biological control agents. (Dr. Walter. Mahaffee, Dr. Caroline Press) (5358-22000-030-00D). Determined that the Gubler/Thomas model and previous literature do not accurately reflect the temperatures that are conducive for infection of Vitis vinifera by Erysphae necator (Grape Powdery Mildew). We have determined that
short exposures to temperatures greater than 36 degrees C are conducive for infection contrary to the previously report upper limit of 35 degrees C. Impact: This new information will result in a modified infection risk model of grape powdery mildew that will more accurately reflect the risk of infection based on how diurnal temperature fluctuations impact disease development. (Dr. Walter Mahaffee, Dr. Steve Scheuerell, Oregon State Univeristy, and Dr. Gary Grove, Washington State University) 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Peetz, A.B., Mahaffee, W.F., and. Grove, G.G 2005. Quantification of airborne hop powdery mildew inoculum, a molecular approach. Phytopathology 95 Peetz, A.B., Mahaffee, W.F., Jackson, I. Grove, G.G., and Galloway, H. 2005 Rapid detection and quantification of airborne hop powdery mildew inoculum.
Phytopathology 95:S81 Grove, G.G., Spotts, R.A., Mahaffee, W.F., Galloway, H., and Lunden, J. 2005. Detection of Podosphaera clandestine in the air of cherry orchards using PCR and species-specific primers. Phytopathology 95:S37 Grove, G.G., Mahaffee, W.F., Falacy, J, Galloway, H., and Peetz, A.B. 2005. Detection of Erysiphe necator in the air of grape vineyards in Eastern Washington. Phytopathology 95:S36 Mahaffee, W.M., Thomas, C.S.,Turechek, W.W., Nelson, M., and Grove, G.G. 2005. The Rise and Fall of Hop Powdery Mildew in the Pacific Northwest. Phytopathology 95 Jackson, J., Hummer, K.E., Mahaffee, W.F. 2004. High temperature dependent resistance to powdery mildew in hops. Acta Horticulturae. p. 26.
Impacts
(N/A)
Publications
- Scheuerell, S.J., Sullivan, D.M., Mahaffee, W.F., 2005. Suppression of seedling damping-off caused by pythium ultimum, pythium irregulare, and rhizoctonia solani in container media amended with a diverse range of pacific northwest compost sources. Phytopathology.95(3):306-315.
- Scheurell, S., Mahaffee, W.F. 2004. Compost tea as a container media drench for supressing seedling damping-off caused by pythium ultimum. Phytopathology. 94(11)1156-1163.
- Turechek, W., Mahaffee, W.F. 2004. Spatial pattern analysis of hop powdery mildew in the pacific northwest: implication for sampling. Phytopathology. 94(10):1116-1128.
- Scheuerell, S., Mahaffee, W.F. 2005. Microbial recolonization of compost after peak heating needed for the rapid development of damping-off suppression caused by pythium ultimum. Compost Science and Utilization. 13:65-71.
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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? Due to the potential loss of pesticide registrations resulting from the Food Quality Plant Protection Act, worker safety issues, and public concerns about pesticide usage, methods for more efficient use of pesticides, and alternatives to synthetic chemical pesticides are needed. The long-term goal of this CRIS is the development of holistic production systems for horticultural crops where biological control and cultural methods are the dominant practices for managing multiple pests of horticulture crops. A combination of laboratory and field experiments combined with modern molecular techniques will be used to address these objectives. Knowledge and technologies gained from this research will enhance a grower's ability to utilize biocontrol and traditional control measures within their production
system. By understanding how microorganisms interact on foliar and fruit surfaces and how these interactions are impacted by the plant and environmental conditions, better management systems can be developed that deploy biocontrol and traditional control measures at appropriate times to enhance their survival and efficacy. The project has 3 specific goals: 1) Advance understanding of the etiology and epidemiology of foliar and fruit pathogens and develop regional disease forecasting systems; 2) Determine the role of biofilms in the establishment and efficacy of a biocontrol agent; 3) Develop and implement strategies for enhancing the establishment and efficacy of biocontrol agents on aerial plant surfaces. This research falls under the National Program 303, Plant Diseases (100%) and addresses Components 2 (Biological Control) and 4 (Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationships with and Vectors) . We are addressing Biological Control Component 2 by
developing alternative systems for achieving biological control and increasing our understanding of factors that influence the survival of microorganisms on aerial plant surfaces. We are addressing the Pathogen Biology Component by focusing on powdery mildews of numerous crops and Botrytis cinerea on strawberry, caneberry and geraniums. We are developing techniques to more rapidly and accurately quantify disease levels in the field (particularly low levels). These methods will then be used to develop economic thresholds for action. We are increasing our understanding of how diurnal temperature fluctuation impacts powdery mildew development with emphasis on how brief exposure to nonconducive temperatures impact infection of powdery mildews. This data is being used to develop disease forecasting models that can be utilized for most powdery mildews across most crops. We are developing a better understanding of how and when B. cinerea infects plants. 2. List the milestones (indicators
of progress) from your Project Plan. Objective 1: Environmental factors effecting Etiology/Epidemiology of B. cinerea. 1. Plant Characters effecting Etiology/Epidemiology of B. cinerea; 2. Forecasting Powdery Mildew Infection Risk; 3. Effects of fluctuating Temperature; 4. Immediate exposure to supra-optimal temperatures; 5. Delayed exposure to supra-optimal temperatures; 6. Exposure to simulated field temperatures; 7. Field Validation. Objective 2: A. Characterize biofilms established on leaf tissue by FP62. 1. Development of FP62 derivatives expressing GFPuv; 2. Microscopic Observation; 3. Spatial distribution of biofilms; 4. Structure of FP62 Biofilms on the leaf surface; 5. FP62 biofilms and control of B. cinerea. B. Evaluate the role of biofilm-associated genes in biocontrol. 1. Cloning of restriction fragments with mini-Tn5-lacZ1 from FP62 mutants; 2. Marker-exchange mutagenesis; 3. Sequence analysis of gene(s) disrupted by mini-Tn5-lacZ1 insertion; 4. Biofilm and biocontrol
assessment of mutants; 5. Functional characterization of biofilm genes; 6. Monitoring biofilm gene expression in the phyllosphere. Objective 3: Develop and implement strategies for enhancing the establishment and efficacy of BCAs. 1. Disease Assay; 2. Field Validation. 3. Milestones: A. FY2004: Objective 1: Environmental factors effecting Etiology/Epidemiology of B. cinerea. We demonstrated that humidity does not impact the epiphytic colonization of plant material. 1. Forecasting Powdery Mildew Infection Risk; 2. Effects of fluctuating temperature; 3. Immediate exposure to supra-optimal temperatures. We have determined that short exposures to temperatures greater than 36 C are conducive for infection contrary to the previously reported upper limit of 35 C. 4. Delayed exposure to supra-optimal temperatures. We have almost completed these experiments and will have done so by the end of October 2004. Objective 2: A. Characterize biofilms established on leaf tissue by FP62. 1. Development
of FP62 derivatives expressing GFPuv. We were unable to get GFPuv, or GFPdsRed to express in FP62. However, we have gotten GFPmut2 to express in FP62. We are attempting to create an expression vector using indigenous promoters from FP62 due to problems with stable expression with other promoters. 2. Microscopic Observation. We generated one mutant using a promoter probe to achieve constitutive expression of GPF mut2, however, this mutant was defective in biofilm formation 5-6 days post-inoculation. It failed to form organized 3-D structures. 3. Spatial distribution of biofilms. We have not begun to due to problems incountered in #1 B. Evaluate the role of biofilm-associated genes in biocontrol. 1. Cloning of restriction fragments with mini-Tn5-lacZ1 from FP62 mutants Two fragments were cloned and identified as homologus to GTPase and Hemoagglutinin. 2. Marker-exchange mutagenesis. We have not succeeded in numerous approaches and are looking to complementation. 3. Sequence analysis
of gene(s) disrupted by mini-Tn5-lacZ1 insertion. We have identified two genes that were disrupted as a GTPase and Hemoagglutinin. Their function has not been confirmed. Objective 3: Develop and implement strategies for enhancing the establishment and efficacy of BCAs. 1. Disease Assay. We identified several adjuvants that enhanced control efficacy of bacterial and fungal biological control agents 2. Field Validation. We conducted field trials of several adjuvants to enhance the efficacy of commercial biological control agents against powdery mildew and gray mold of grapes. B. FY2005 Objective 1: Environmental factors effecting Etiology/Epidemiology of B. cinerea. Further examine how temperature and humidity impact the epiphytic growth of B. cinerea 1. Plant Characters effecting Etiology/Epidemiology of B. cinerea. Will begin research into how trichomes impact the infection rate of B. cinerea 2. Forecasting Powdery Mildew Infection Risk; 3. Effects of fluctuating temperature; 4.
Delayed exposure to supra-optimal temperatures. Further examine how short exposures to non-conducive temperatures impact disease development. 5. Exposure to simulated field temperatures. Experiments will be conducted in growth chambers using 15 min temperature and humidity data to determine how diurnal temperature and humidity fluctuations impact disease development. Objective 2: A. Characterize biofilms established on leaf tissue by FP62. 1. Development of FP62 derivatives expressing GFP; 2. Microscopic Observation; 3. Spatial distribution of biofilms B. Evaluate the role of biofilm-associated genes in biocontrol. 1. Marker-exchange mutagenesis; 2. Biofilm and biocontrol assessment of mutants. Objective 3: Develop and implement strategies for enhancing the establishment and efficacy of BCAs. 1. Disease Assay; 2. Field Validation. FY2006 Objective 1: Environmental factors effecting Etiology/Epidemiology of B. cinerea. 1. Plant Characters effecting Etiology/Epidemiology of B.
cinerea; 2. Forecasting Powdery Mildew Infection Risk; 3. Exposure to simulated field temperatures; 4. Field Validation. Objective 2: A. Characterize biofilms established on leaf tissue by FP62. 1. Spatial distribution of biofilms; 2. Structure of FP62 Biofilms on the leaf surface; 3. FP62 biofilms and control of B. cinerea. B. Evaluate the role of biofilm-associated genes in biocontrol. 1. Biofilm and biocontrol assessment of mutants; 2. Functional characterization of biofilm genes; 3. Monitoring biofilm gene expression in the phyllosphere. Objective 3: Develop and implement strategies for enhancing the establishment and efficacy of BCAs. 1. Disease Assay; 2. Field Validation. FY2007 Objective 1: Plant Characters effecting Etiology/Epidemiology of B. cinerea. 1. Forecasting Powdery Mildew Infection Risk; 2. Field Validation. Objective 2: A. Characterize biofilms established on leaf tissue by FP62. 1. Structure of FP62 Biofilms on the leaf surface; 2. FP62 biofilms and control of B.
cinerea. B. Evaluate the role of biofilm-associated genes in biocontrol. 1. Biofilm and biocontrol assessment of mutants; 2. Functional characterization of biofilm genes; 3. Monitoring biofilm gene expression in the phyllosphere. Objective 3: Develop and implement strategies for enhancing the establishment and efficacy of BCAs. 1. Disease Assay; 2. Field Validation. 4. What were the most significant accomplishments this past year? A. A currently available model (Gubler/Thomas) and the literature do not accurately reflect the temperatures that are conducive for infection of Vitis vinifera by Erysiphae necator (Grape Powdery Mildew). Research conducted by scientists in the Horticulture Crops research Unit, Corvallis, OR. in collaboration with Dr. Steve Scheuerell of Oregon State University and Dr. Gary Grove, Washington State University found that short exposures to temperatures greater than 36 C are conducive for infection. These results are contrary to the previously reported upper
limit of 35 C. This new information will result in a modified infection risk model of grape powdery mildew that will more accurately reflect the risk of infection based on how diurnal temperature fluctuations impact disease development. B. None C. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Identified putative GTPase and Hemoagglutinin genes that are associated with biofilm formation and biocontrol activity of the biocontrol agent Burkholderia pyrrocinia FP62. Some of the genetic determinants were associated with biofilm formation and biological control of Botrytis cinerea by FP62. The alleles that were disrupted by transposon insertion in two mutants deficient in biofilm formation and biocontrol activity were identified and cloned. Sequence analysis tentatively indicates that they encode for a GTPase involved with ribosome binding and another unidentified activity and hemoagglutinin formation.
Complementation analysis is still needed to confirm these relationships. Impact: Understanding the importance and mechanisms of biofilm formation in the efficacy of foliar biocontrol agents will lead to the development of improve delivery technologies to enhance the efficacy of biological control agents. 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? Five years of research (previous CRIS 5358-22000-024-00D) has culminated in the public release of an infection risk forecasting model for hop powdery mildew. Once the infection risk model was created and validated (2000 and 2001) the utility of the risk index was increased by partnering with FieldWise, Inc and Fox Weather, LCC to develop a 5 day forecast. In 2002- 2003, growers reporting to use the
model to assist in fungicide applications reported using 1.5 fewer applications and 55% less incidence of cone infection. Approximately 75% hop acreage was being managed using the infection risk forecaster. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Mahaffee, W.F. 2003. Can we count? Estimating microbial populations on plant surfaces Phytopathology 93:S105 Pscheidt, J., Mahaffee, W.F., Martin, R.M. and Pinkerton, J. Diseases of Grapes. 2003 In: Oregon wine grape growers guide. Pp. 185-196. E. Hellman, Ed. OSU Press, Corvallis, OR.
Impacts
(N/A)
Publications
- Henning, J.A., Townsend, M.S., Mahaffee, W.F., Kenny, S., Haunold, A. 2004. Registration of newport hop. Crop Science.44:1018-1019.
- Mahaffe, W.F., Thomas, C.S., Turechek, W.W., Ocamb, C.M., Nelson, M.E., Fox, A., Gubler, W.D. Responding to an Introduced Pathogen: Podosphaera macularis (Hop Powdery Mildew) in the Pacific Northwest. Online. Plant Health Progress doi:10.1094/PHP-2003-1113-07-RV.
- Mahaffee, W.F., Turechek, W., Ocamb, C. Effect of variable temperature on the infection frequency of Podosphaera macularis on Humulus lupulus. Phytopathology. 2003. v93 p. 1587-1592.
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