Progress 02/25/03 to 03/13/07
Outputs
Progress Report Objectives (from AD-416) The goal of this research is to identify natural, safe strategies for enhancing the protection of sugarbeet from fungi that cause root and leaf diseases in the field with an emphasis on the pathogens Aphanomyces cochlioides and Cercospora beticola. Specific objectives toward this goal are to identify genes and/or biochemical factors produces by A. cochlioides anc C. beticola that are crucial for sugarbeet plant infection, to identify microbes or compunds of sugarbeet, wild beet, plant, microbial, or synthetic origin capable of preventing or reducing the infection of sugarbeet by A. cochlioides, and to obtain molecular genetic markers associated with genes for resistance to diseases that limit sugarbeet production. Approach (from AD-416) Mutants of A. cochlioides and C. beticola will be generated using current gene-transfer technologies. Mutants will be tested for loss or gain of virulence on inoculated sugarbeet plants. Sites of insertion of the transferred DNA in the pathogen genome will be located by cloning out chromosomal regions flanking the inserted DNA. Pathogen genes crucial for pathogenicity or virulence will be discovered using this method. Biocontrol bacteria and plant species suppresive to sugarbeet diseases will be analyzed for compounds involved in controlling pathogen damage. This will involve biochemical fractionation of bioactive compounds and the determination of their structure. Natural genes in sugarbeet that condition resistance to common and important diseases of the crop will be tagged with molecular genetic markers. A combination of AFLP, RAPD, and SSR marker technologies will be employed in the search for markers linked to disease resistance genes. The markers will be used to facilitate the introgression of disease resistance into commerical hybrids. BSL-1 Recertified May 6, 2002. Accomplishments Potential Biocontrol for Sugarbeet Aphanomyces Root Rot Identified. Root rot diseases of sugarbeet such as Aphanomyces cause significant losses that cost the industry millions of dollars annually. In research conducted by the Sugarbeet and Potato Research Unit, Northern Crop Science Laboratory, Fargo ND over a two-year period, the efficacy of several biocontrol agents was determined in greenhouse and field trials. Research demonstrated that the biocontrol bacteria tested were ineffective but an inducer of systemic host plant resistance provided partial protection to Aphanomyces root rot in the field. These results suggest that biocontrol of this costly disease may provide a useful partner to chemical fungicides and genetic resistance. This project addresses the goals of National Program 303, Plant Disease, Problem Areas: 1. Identification of pathogens, 3. Pathogen biology, and 4. Host plant resistance to pathogens.
Impacts
(N/A)
Publications
- Weiland, J.J., Anderson, J.V., Bigger, B. 2006. Inexpensive chemifluorescent detection of antibody - alkaline phosphatase conjugates on Western blots using 4-methylumbelliferyl phosphate. Analytical Biochemistry. 361:140-142.
- Lai, Z., Faris, J.D., Weiland, J.J., Steffenson, B.J., Friesen, T.L. 2007. Genetic mapping of Pyreonphora teres f. teres genes conferring avirulence on barley. Fungal Genetics and Biology. 44:323-329.
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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? Fungal pathogens continue to cause significant disease problems in sugarbeet grown in the U.S. and world-wide. Leaf spot caused by C. beticola, root rot, crown rot and seedling blight caused by R. solani, and post-emergence damping off caused by A. cochlioides are of special concern to growers in the north central region. Although some resistance to these pathogens has been identified in Beta germplasm, cultivars grown for sugar production remain susceptible to disease. Chemical control of A. cochlioides and C. beticola currently exists, but is limited, and is of decreasing effectiveness for C. beticola; no chemical control of R. solani is currently available. Known cultural practices that might alleviate disease severity are not compatible with climactic constraints that effect the
north central growing region. With the recent discovery of Rhizomania disease caused by beet necrotic yellow vein virus in production fields of the region, a post-doctoral scientist has been added to our project to investigate novel means for the control of this devastating sugarbeet pathogen. The objectives of the project are to examine the nature of the interaction of fungal pathogens with their sugarbeet host and to determine genetic or biochemical factors that condition resistance in the plant and virulence in the fungus. Additional objectives are the evaluation of genetic diversity in the fungal pathogens of sugarbeet and the extent to which this genetic diversity is correlated with virulence traits, as well as the development of biological control measures aimed at reducing the use of environmentally-unfriendly fungicides. We are using molecular genetic and biochemical approaches to examine (a) the genetic variation in the pathogens described above and how this variation
relates to pathogen virulence, (b) the production of enzymes by both the plant and the fungus at the interface of these two entities, (c) the efficacy of gene silencing in sugarbeet and how it might be used to combat Rhizomania disease, and (d) the identification of DNA markers that are associated with genes in sugarbeet that confer resistance to the above and other biotic diseases. Reduction of disease by the application of beneficial bacteria and natural plant elicitor compounds are also being examined. The above illustrates the major disease problems that impact sugarbeet production in the U.S. as well as illuminates the approaches being taken in our laboratory in order to reach the stated objectives. Although these technologies are beginning to be applied to the improvement of sugarbeet, the techniques are used more intensively in other crops. Studies on the infection process of sugarbeet by fungal pathogens are acutely lacking in molecular genetic approaches. For this reason,
the focus of the project is on the interaction between sugarbeet and its fungal pathogens using molecular genetic techniques. This project addresses the goals of National Program 303, Plant Diseases (100%), Problem Areas: 1. Identification of pathogens, 3. Pathogen biology, and 4. Host plant resistance to pathogens. Greater than 80% of U.S. sugarbeet production is located north of the 40th parallel. Root rot in the field caused by R. solani and A. cochlioides and leaf spot caused by C. beticola constitute the most serious disease problems in sugarbeet in the northern sugarbeet production regions of the U.S. Yields can be reduced by 40% to 50% in fields subjected to severe epidemics of root rot or leaf spot. Although relatively new to the central plains states, Rhizomania has severely impacted sugarbeet yields in the western U.S. Post-harvest integrity of stored sugarbeet is compromised severely by previous infection of the beet in the field by all of the above organisms. These
problems, therefore, affect the sugarbeet grower by reducing yields in the field, as well as beet sugar processors, as fungal infection causes increased rotting of stored beets and leads to increases in impurities in sugarbeet extract. 2. List by year the currently approved milestones (indicators of research progress) Year 2003 Mutants of C. beticola deficient in cercosporin production in culture will be generated. The purification of an esterase enzyme from this same organism will commence. Protease production by A. cochlioides will be subject to initial biochemical analysis prior to purification. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. Preparations of DNA will be made for the initiation of marker generation tagging genes for resistance to beet mosaic virus. Year 2004 Mutants of C. beticola deficient in cercosporin production in culture will be generated. The purification of an esterase enzyme from
this same organism will continue and will be tested for the ability to damage sugarbeet leaf cells. Protease production by A. cochlioides will be subject to initial biochemical analysis prior to purification. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. Gene for beet mosaic virus resistance in sugarbeet will be tagged using RAPD markers. Year 2005 Mutants of C. beticola deficient in cercosporin production in culture will be generated and evaluated for virulence on sugarbeet plants. The purification of an esterase enzyme from this same organism will continue and will be tested for the ability to process various substrates. Purification of a major 49K protease found in A. cochlioides-infected seedlings will commence. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. A scar marker will be generated for robust detection of the resistance
gene to beet mosaic virus. Sugarbeet populations segregating for resistance to powdery mildew will be screened and DNA will be prepared from these plants. Year 2006 The effect of the loss of cercosporin production by C. beticola will be determined. The purification of an esterase enzyme from C. beticola will continue and purified protein will be used for the generation of polyclonal antiserum. Purification of a major 49K protease found in A. cochlioides-infected seedlings will continue and culminate with the generation of polyclonal antiserum to this protein. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. RAPD marker analysis will be applied to sugarbeet populations segregating for resistance to powdery mildew. Year 2007 Polyclonal antiserum will be used to confirm expression of the esterase at the site of leaf damage by C. beticola. Polyclonal antiserum to the A. cochlioides 49K protease will be used to
confirm production of this enzyme in infected sugarbeet and will aid in immunolocalization of the protein in beet tissue. Field assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. SCAR markers will be generated for resistance to powdery mildew based on the RAPD markers obtained in this study. 4a List the single most significant research accomplishment during FY 2006. Identification of beet black scorch virus (BBSV) in sugar beet in the U.S. for the first time; BBSV was discovered, purified, characterized, sequenced, and immunological reagents made for its diagnosis. Although sugar beet production nationwide is under stress from a number of root- infecting pathogens including Rhizomania, BBSV may play a role in the unusual severity that Rhizomania is exhibiting in some regions of the U.S. After the initial isolation of BBSV by USDA-ARS at Fort Collins, CO, and electron microscopic characterization by NDSU-Fargo, ND, the staff at
USDA-ARS, Fargo, ND, completed purification of the virus and production of antisera. The discovery reveals another in a series of emerging pathogens in U.S. agriculture and highlights the responsiveness of ARS scientists in the development of tools to monitor new diseases. This project addresses the goals of National Program 303, Plant Diseases (100%) , Problem Areas: 1. Identification of pathogens, 3. Pathogen biology, and 4. Host plant resistance to pathogens. 4b List other significant research accomplishment(s), if any. Candidate molecular genetic markers for resistance to sugarbeet cyst nematode (SBCN) on sugarbeet were obtained. A molecular tag for resistance to SBCN would aid breeding programs in the introgression of this resistance into sugarbeet varieties. The RAPD technique was used to screen a sugarbeet population segregating for resistance to this organism. DNA sequencing of the candidate markers will be completed by the end of 2006. Rating of the population was
performed by a research geneticist at USDA-ARS, Salinas CA, and marker generation was performed in the laboratories of USDA-ARS-NPA-RRVARC-NCSL Sugarbeet and Potato Research Unit, Fargo, North Dakota. The marker technology will be transferred to breeding programs for use in variety improvement. This project addresses the goals of National Program 303, Plant Diseases (100%), Problem Areas: 1. Identification of pathogens, 3. Pathogen biology, and 4. Host plant resistance to pathogens. 5. Describe the major accomplishments to date and their predicted or actual impact. Discovery of beet black scorch virus in the U.S. for the first time. This indicates a novel agent infecting sugar beet which must be addressed when implementing disease control strategies. High levels of protease activity were found to be associated with infections of beet seedlings by A. cochlioides. This identifies a potential new biochemical target for the control of this disease. A protocol for the reproducible
generation of stable oospores from A. cochlioides and P. aphanidermatum was developed. These will be useful to sugarbeet breeding concerns in the selection for germplasm or hybrid parents that exhibit superior resistance to these pathogens. Genetic diversity of C. beticola in the U.S. was determined, which will permit a more thorough testing panel of isolates for the development of fungicides and resistant sugarbeet germplasm. The molecular tagging of genes in sugarbeet that confer resistance to the economically-important disease powdery mildew and pest root-knot nematode was accomplished. This will reduce costs in the screening of elite sugarbeet germplasm containing these genes. Gene transfer to a Pythium member of the Oomycota was successful. Determination of pathogen genes important for disease production will result from this work. An esterase biochemical activity from C. beticola has been found in our laboratory to be light-inducible. This may prove to be a virulence factor
for the fungus. If so, characterization of this enzyme could shed light on new ways to control leaf spot disease caused by this fungus. This project addresses the goals of National Program 303, Plant Diseases (100%), Problem Areas: 1. Identification of pathogens, 3. Pathogen biology, and 4. Host plant resistance to pathogens. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A PCR marker and enzyme-linked immunosorbant assay (ELISA) reagents for beet black scorch virus diagnosis were distributed to private company researchers in Sweden (Syngenta Seeds). 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). Weiland, J.J.
2006. Managing fungicide resistance in Cercospora leaf spot of sugar beet. Presentation to the California Sugar Beet Growers Annual Meeting, Brawley CA
Impacts
(N/A)
Publications
- Friesen, T.L., Weiland, J.J., Aasheim, M., Hunger, S., Borchardt, D.C., Lewellen, R.T. 2006. Identification of a SCAR marker associated with Bm, the beet mosaic virus resistance gene, on chromosome 1 of sugar beet. Plant Breeding. 125:167-172.
- Weiland, J.J., Mcgrath, J.M. 2006. An EST database of the sugarbeet pathogen, Aphanomyces Cochlioides [abstract]. Plant & Animal Genome XIV Conference. Abstract No. W181:48.
- Weiland, J.J., Larson, R.L., Freeman, T.P., Edwards, M.C. 2006. First report of Beet black scorch virus in the United States. Plant Disease. 90(6):828.
- Weiland, J.J. 2006. Presence and distribution of BNYVV and BSBMV in the Glyndon rhizomania research site in 2005. 2005 Sugarbeet Research and Extension Reports, Cooperative Extension Service, North Dakota State University. 36:315-322.
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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? Fungal pathogens continue to cause significant disease problems in sugarbeet grown in the U.S. and world-wide. Leaf spot caused by C. beticola, root rot, crown rot and seedling blight caused by R. solani, and post-emergence damping off caused by A. cochlioides are of special concern to growers in the north central region. Although some resistance to these pathogens has been identified in Beta germplasm, cultivars grown for sugar production remain susceptible to disease. Chemical control of A. cochlioides and C. beticola currently exists, but is limited, and is of decreasing effectiveness for C. beticola; no chemical control of R. solani is currently available. Known cultural practices that might alleviate disease severity are not compatible with climactic constraints that effect the north central
growing region. With the recent discovery of Rhizomania disease caused by beet necrotic yellow vein virus in production fields of the region, a post-doctoral scientist has been added to our project to investigate novel means for the control of this devastating sugarbeet pathogen. The objectives of the project are to examine the nature of the interaction of fungal pathogens with their sugarbeet host and to determine genetic or biochemical factors that condition resistance in the plant and virulence in the fungus. Additional objectives are the evaluation of genetic diversity in the fungal pathogens of sugarbeet and the extent to which this genetic diversity is correlated with virulence traits, as well as the development of biological control measures aimed at reducing the use of environmentally-unfriendly fungicides. We are using molecular genetic and biochemical approaches to examine (a) the genetic variation in the pathogens described above and how this variation relates to pathogen
virulence, (b) the production of enzymes by both the plant and the fungus at the interface of these two entities, (c) the efficacy of gene silencing in sugarbeet and how it might be used to combat Rhizomania disease, and (d) the identification of DNA markers that are associated with genes in sugarbeet that confer resistance to the above and other biotic diseases. Reduction of disease by the application of beneficial bacteria and natural plant elicitor compounds are also being examined. The above illustrates the major disease problems that impact sugarbeet production in the U.S. as well as illuminates the approaches being taken in our laboratory in order to reach the stated objectives. Although these technologies are beginning to be applied to the improvement of sugarbeet, the techniques are used more intensively in other crops. Studies on the infection process of sugarbeet by fungal pathogens are acutely lacking in molecular genetic approaches. For this reason, the focus of the
project is on the interaction between sugarbeet and its fungal pathogens using molecular genetic techniques. This project is consistent with the goals of National Program 303, Plant Diseases (100%) which has four principal thrusts: 1) identification and classification of sugarbeet fungal pathogens, 2) identification of non-pathogenic microorganism for use as biocontrol agents against sugarbeet fungal pathogens, 3) develop gene-transfer methodsfor use in determining sugarbeet fungal virulence loci, and 4) discover and exploit natural mechanism of host-plant resistance against fungal pathogens in sugarbeet. Greater than 80% of U.S. sugarbeet production is located north of the 40th parallel. Root rot in the field caused by R. solani and A. cochlioides and leaf spot caused by C. beticola constitute the most serious disease problems in sugarbeet in the northern sugarbeet production regions of the U.S. Yields can be reduced by 40% to 50% in fields subjected to severe epidemics of root rot
or leaf spot. Although relatively new to the central plains states, Rhizomania has severely impacted sugarbeet yields in the western U.S. Post-harvest integrity of stored sugarbeet is compromised severely by previous infection of the beet in the field by all of the above organisms. These problems, therefore, affect the sugarbeet grower by reducing yields in the field, as well as beet sugar processors, as fungal infection causes increased rotting of stored beets and leads to increases in impurities in sugarbeet extract. 2. List the milestones (indicators of progress) from your Project Plan. Year 2003 Mutants of C. beticola deficient in cercosporin production in culture will be generated. The purification of an esterase enzyme from this same organism will commence. Protease production by A. cochlioides will be subject to initial biochemical analysis prior to purification. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet
seedlings. Preparations of DNA will be made for the initiation of marker generation tagging genes for resistance to beet mosaic virus. Year 2004 Mutants of C. beticola deficient in cercosporin production in culture will be generated. The purification of an esterase enzyme from this same organism will continue and will be tested for the ability to damage sugarbeet leaf cells. Protease production by A. cochlioides will be subject to initial biochemical analysis prior to purification. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. Gene for beet mosaic virus resistance in sugarbeet will be tagged using RAPD markers. Year 2005 Mutants of C. beticola deficient in cercosporin production in culture will be generated and evaluated for virulence on sugarbeet plants. The purification of an esterase enzyme from this same organism will continue and will be tested for the ability to process various substrates.
Purification of a major 49K protease found in A. cochlioides-infected seedlings will commence. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. A scar marker will be generated for robust detection of the resistance gene to beet mosaic virus. Sugarbeet populations segregating for resistance to powdery mildew will be screened and DNA will be prepared from these plants. Year 2006 The effect of the loss of cercosporin production by C. beticola will be determined. The purification of an esterase enzyme from C. beticola will continue and purified protein will be used for the generation of polyclonal antiserum. Purification of a major 49K protease found in A. cochlioides-infected seedlings will continue and culminate with the generation of polyclonal antiserum to this protein. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. RAPD marker analysis
will be applied to sugarbeet populations segregating for resistance to powdery mildew. Year 2007 Polyclonal antiserum will be used to confirm expression of the esterase at the site of leaf damage by C. beticola. Polyclonal antiserum to the A. cochlioides 49K protease will be used to confirm production of this enzyme in infected sugarbeet and will aid in immunolocalization of the protein in beet tissue. Field assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. SCAR markers will be generated for resistance to powdery mildew based on the RAPD markers obtained in this study. 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. Cercosporin knock-outs confirmed and tested. Milestone Fully Met 2. Purification of 49K protease from A. cochlioides. Milestone Substantially Met 3. Investigatio of pathogen inhibiting
compounds. Milestone Substantially Met 4. Marker analysis of powdery mildew resistance. Milestone Fully Met 5. Purification of esterase enzyme. Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 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? In 2006, esterase production in C. beticola or protease production by A. cochlioides will be detected in infected plants using antisera developed to these proteins. Third year testing of biocontrol bacteria and inducers of plant resistance for field efficacy will occur. In 2007, markers obtained for resistance to A. cochlioides will be converted to the more robust SCAR marker format. Protease inhibitors will be used to determine which activities are important for the infection of sugarbeet seedlings by A. cochlioides. In 20008, the infection etiology of sugarbeet will
be determined using the GFP-transformed Cercospora beticola isolate. 4a What was the single most significant accomplishment this past year? Role of cercosporin toxin in sugarbeet leaf spot disease. Mutants of C. beticola deficient in cercosporin production were found to be severely reduced in their ability to cause leaf spot disease. The role of this toxin in damaging sugarbeet leaves has been debated for many decades. Chemical analysis of mutants with Dr. Jeff Suttle of the USDA- ARS in Fargo confirmed that cercosporin was not being produced in these mutants and mutants of C. beticola were produced by Dr. Kuang-Ren Chung at the University of FloridaLake Alfred with testing for virulence done by Dr. John Weiland at the USDA-ARS-NPA-RRVARC-NCSL Sugarbeet and Potato Research Unit, Fargo, North Dakota. The result indicates that targeting the detoxification of cercosporin, or breeding for cercosporin resistance, would be an effective means to limit leaf spot disease in this crop. 4b List
other significant accomplishments, if any. Candidate molecular genetic markers for resistance to powdery mildew disease on sugarbeet was obtained. A molecular tag for resistance to mildew would aid breeding programs in the introgression of this resistance into sugarbeet varieties. The RAPD technique was used to screen a sugarbeet population segregating for resistance to this virus. DNA sequencing of the candidate markers will be completed by the end of 2005. Rating of the population and marker generation was performed in the laboratory of Dr. John Weiland at the USDA-ARS-NPA-RRVARC-NCSL Sugarbeet and Potato Research Unit, Fargo, North Dakota. The marker technology will be transferred to breeding programs for use in variety improvement. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. High levels of protease activity were found to be associated with infections of beet seedlings by A. cochlioides. This identifies a
potential new biochemical target for the control of this disease. A protocol for the reproducible generation of stable oospores from A. cochlioides and P. aphanidermatum was developed. These will be useful to sugarbeet breeding concerns in the selection for germplasm or hybrid parents that exhibit superior resistance to these pathogens. Genetic diversity of C. beticola in the U.S. was determined, which will permit a more thorough testing panel of isolates for the development of fungicides and resistant sugarbeet germplasm. The molecular tagging of genes in sugarbeet that confer resistance to the economically-important disease powdery mildew and pest root-knot nematode was accomplished. This will reduce costs in the screening of elite sugarbeet germplasm containing these genes. Gene transfer to a Pythium member of the Oomycota was successful. Determination of pathogen genes important for disease production will result from this work. An esterase biochemical activity from C. beticola
has been found in our laboratory to be light-inducible. This may prove to be a virulence factor for the fungus. If so, characterization of this enzyme could shed light on new ways to control leaf spot disease caused by this fungus. The research reported is conducted under National Program 303, Plant Diseases contributes to research component IV, Pathogen Biology, Genetics, Population Dynamics, Spread, and Relationship With Hosts and Vectors. Research activities are further related to ARS Strategic Plan Goal 3, Enhance Protection and Safety of the Nations Agriculture and Food Supply; Objective 3.2, Provide fundamental and applied scientific information and technology to protect agriculturally important plants from pests and disease. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and
durability of the technology products? A molecular marker for a gene conferring resistance to beet mosaic virus was made public for use by the sugarbeet breeding industry. See Genbank ID# DQ022571 at http://www.ncbi.nlm.nih.gov/ 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). Weiland, J.J. 2005. Old and New Sources of Resistance to Rhizomania. The Sugarbeet Grower. 44:12-14.
Impacts
(N/A)
Publications
- Weiland, J.J., Shelver, W.L. 2005. Production and characterization of antiserum to aphanomyces cochlioides. Journal of Sugarbeet Research. 41(4) 179-190.
- Weiland, J.J. 2005. Presence and distribution of BNYVV and BSBMV in the Glyndon rhizomania research site. 2002 Sugarbeet Research and Extension Report, Cooperative Extension Service, North Dakota State University. 35:239-242.
- Weiland, J.J. 2004. Production of protease isozymes by Aphanomyces cochlioides and Aphanomyces euteiches. Physiological and Molecular Plant Pathology. 65:225-233.
- Weiland, J.J., Larson, R.L., Lewellen, R.T. 2005. Progress on resistance gene tagging and the development of functional genomics tools in sugarbeet. Plant & Animal Genomes XIII Conference. W336. Available: http://www.intl- pag.org/13/abstracts/PAG13_W336.html
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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? Fungal pathogens continue to cause significant disease problems in sugarbeet grown in the U.S. and world-wide. Leaf spot caused by C. beticola, root rot, crown rot and seedling blight caused by R. solani, and post-emergence damping off caused by A. cochlioides are of special concern to growers in the north central region. Although some resistance to these pathogens has been identified in Beta germplasm, cultivars grown for sugar production remain susceptible to disease. Chemical control of A. cochlioides and C. beticola currently exists, but is limited, and is of decreasing effectiveness for C. beticola; no chemical control of R. solani is currently available. Known cultural practices that might alleviate disease severity are not compatible with climactic constraints that effect the north central
growing region. With the recent discovery of Rhizomania disease caused by beet necrotic yellow vein virus in production fields of the region, a post-doctoral scientist has been added to our project to investigate novel means for the control of this devastating sugarbeet pathogen. The objectives of the project are to examine the nature of the interaction of fungal pathogens with their sugarbeet host and to determine genetic or biochemical factors that condition resistance in the plant and virulence in the fungus. Additional objectives are the evaluation of genetic diversity in the fungal pathogens of sugarbeet and the extent to which this genetic diversity is correlated with virulence traits, as well as the development of biological control measures aimed at reducing the use of environmentally-unfriendly fungicides. We are using molecular genetic and biochemical approaches to examine (a) the genetic variation in the pathogens described above and how this variation relates to pathogen
virulence, (b) the production of enzymes by both the plant and the fungus at the interface of these two entities, (c) the efficacy of gene silencing in sugarbeet and how it might be used to combat Rhizomania disease, and (d) the identification of DNA markers that are associated with genes in sugarbeet that confer resistance to the above and other biotic diseases. Reduction of disease by the application of beneficial bacteria and natural plant elicitor compounds are also being examined. The above illustrates the major disease problems that impact sugarbeet production in the U.S. as well as illuminates the approaches being taken in our laboratory in order to reach the stated objectives. Although these technologies are beginning to be applied to the improvement of sugarbeet, the techniques are used more intensively in other crops. Studies on the infection process of sugarbeet by fungal pathogens are acutely lacking in molecular genetic approaches. For this reason, the focus of the
project is on the interaction between sugarbeet and its fungal pathogens using molecular genetic techniques. This project directly addresses the goals of National Program 303, Plant Diseases (100%)which has four principal thrusts: 1) identification and classification of sugarbeet fungal pathogens, 2) identification of non- pathogenic microorganism for use as biocontrol agents against sugarbeet fungal pathogens, 3) develop gene-transfer methods for use in determining sugarbeet fungal virulence loci, and 4) discover and exploit natural mechanism of host-plant resistance against fungal pathogens in sugarbeet. Greater than 80% of U.S. sugarbeet production is located north of the 40th parallel. Root rot in the field caused by R. solani and A. cochlioides and leaf spot caused by C. beticola constitute the most serious disease problems in sugarbeet in the northern sugarbeet production regions of the U.S. Yields can be reduced by 40% to 50% in fields subjected to severe epidemics of root
rot or leaf spot. Although relatively new to the central plains states, Rhizomania has severely impacted sugarbeet yields in the western U.S.. Post-harvest integrity of stored sugarbeet is compromised severely by previous infection of the beet in the field by all of the above organisms. These problems, therefore, affect the sugarbeet grower by reducing yields in the field, as well as beet sugar processors, as fungal infection causes increased rotting of stored beets and leads to increases in impurities in sugarbeet extract. 2. List the milestones (indicators of progress) from your Project Plan. Year 2002 Mutants of C. beticola deficient in cercosporin production in culture will be generated. The purification of an esterase enzyme from this same organism will commence. Protease production by A. cochlioides will be subject to initial biochemical analysis prior to purification. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of
sugarbeet seedlings. Preparations of DNA will be made for the initiation of marker generation tagging genes for resistance to beet mosaic virus. Year 2003 Mutants of C. beticola deficient in cercosporin production in culture will be generated. The purification of an esterase enzyme from this same organism will continue and will be tested for the ability to damage sugarbeet leaf cells. Protease production by A. cochlioides will be subject to initial biochemical analysis prior to purification. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. Gene for beet mosaic virus resistance in sugarbeet will be tagged using RAPD markers. Year 2004 Mutants of C. beticola deficient in cercosporin production in culture will be generated and evaluated for virulence on sugarbeet plants. The purification of an esterase enzyme from this same organism will continue and will be tested for the ability to process various
substrates. Purification of a major 49K protease found in A. cochlioides-infected seedlings will commence. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. A scar marker will be generated for robust detection of the resistance gene to beet mosaic virus. Sugarbeet populations segregating for resistance to powdery mildew will be screened and DNA will be prepared from these plants. Year 2005 The effect of the loss of cercosporin production by C. beticola will be determined. The purification of an esterase enzyme from C. beticola will continue and purified protein will be used for the generation of polyclonal antiserum. Purification of a major 49K protease found in A. cochlioides-infected seedlings will continue and culminate with the generation of polyclonal antiserum to this protein. Growth chamber assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. RAPD
marker analysis will be applied to sugarbeet populations segregating for resistance to powdery mildew. Year 2006 Polyclonal antiserum will be used to confirm expression of the esterase at the site of leaf damage by C. beticola. Polyclonal antiserum to the A. cochlioides 49K protease will be used to confirm production of this enzyme in infected sugarbeet and will aid in immunolocalization of the protein in beet tissue. Field assays will be used to investigate compounds able to control Aphanomyces root rot of sugarbeet seedlings. SCAR markers will be generated for resistance to powdery mildew based on the RAPD markers obtained in this study. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. 1. Evaluation of cercosporin-deficient mutants of C. beticola; virulence testing
and in planta cercosporin production studies. Met. 2. Analysis of esterase produced by C. beticola: antisera production and in planta esterase assays. Not met. Loss of non-ARS university cooperator resulted in temporary delay of esterase purification. Alternative strategies are being developed and new cooperators have been identified. 3. Analysis of protease secretion by A. cochlioides: purification of trypsin-like protease. Met. 4. Biocontrol of A. cochlioides: greenhouse testing of induced resistance, evaluation of bacteria as biocontrol agents, field testing of novel control methods. Partially met. Field testing of novel control methods has been temporarily suspended pending hiring of new post-doctoral scientist to fill vacancy created during FY 2004. 5. Molecular tagging of disease resistance genes. Met. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years
under each milestone? In 2005, efforts to tag resistance genes to Rhizomania and Aphanomyces root rot will continue. Populations segregating for resistance and susceptibility to A. cochlioides will be used for marker analysis. Protease from A. cochlioides will be purified and antibody production to this protein will commence, followed by a similar treatment for esterase from C. beticola. The green fluorescent protein (GFP) gene will be used to transform C. beticola so that this isolate can be used by our lab and that of our collaborators at USDA-Fort Collins and Montana State University for plant infection studies. Antisera raised against A. cochlioides esterase/protease protein will be used in conjunction with Real-Time PCR for the screening of populations segregating for resistance to A. cochlioides. Molecular markers for powdery mildew resistance will converted to the more robust SCAR marker format. Induced resistance will be examined in the greenhouse for the protection of
beet seedlings from the effect of A. cochlioides. In 2006, Esterase production in C. beticola or protease production by A. cochlioides will be detected in infected plants using antisera developed to these proteins. Third year testing of biocontrol bacteria and inducers of plant resistance for field efficacy will occur. In 2007, markers obtained for resistance to A. cochlioides will be converted to the more robust SCAR marker format. Protease inhibitors will be used to determine which activities are important for the infection of sugarbeet seedlings by A. cochlioides. The infection etiology of sugarbeet will be determined using the GFP-transformed Cercospora beticola isolate. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004. Fungal pathogens continue to cause significant disease problems in sugarbeet grown in the U.S. and world-wide. Mutants of C. beticola deficient in the production of the phytotoxin
cercosporin were produced and tested for virulence on sugarbeet plants. Cercosporin production by C. beticola has long been speculated for decades to be crucial for the infection of sugarbeet by this fungus, yet conclusive data was lacking. In collaborative research, cercosporin-deficient mutants of C. beticola were produced by Dr. Kuang-Ren Chung at the University of Florida-Lake Alfred and virulence assays and cercosporin production determinations were conducted by Dr. John Weiland at the USDA-ARS-NPA-RRVARC-NCSL Sugarbeet and Potato Research Unit, Fargo, North Dakota. . Mutants in CTB1 were found to be severely reduced in their ability to cause leaf spot disease. The result indicates that targeting the detoxification of cercosporin, or breeding for cercosporin resistance, would be an effective means to limit leaf spot disease in this crop. B. Other Significant Accomplishment(s), if any. A molecular genetic marker for resistance to beet mosaic virus (BMV) was obtained. A
molecular tag for resistance to BMV would aid breeding programs in the introgression of this resistance into sugarbeet varieties. The RAPD technique was used to screen a sugarbeet population segregating for resistance to this virus. A marker was identified that associated highly with resistance and this marker was converted to a SCAR marker. Rating of the population and marker generation was performed in the laboratory of Dr. John Weiland at the USDA-ARS-NPA-RRVARC-NCSL Sugarbeet and Potato Research Unit, Fargo, North Dakota. The marker technology will be transferred to breeding programs for use in variety improvement. C. Significant activities that support special target populations. None. D. Progress Report. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. High levels of protease activity were found to be associated with infections of beet seedlings by A. cochlioides. This identifies a potential new biochemical target
for the control of this disease. A protocol for the reproducible generation of stable oospores from A. cochlioides and P. aphanidermatum was developed. These will be useful to sugarbeet breeding concerns in the selection for germplasm or hybrid parents that exhibit superior resistance to these pathogens. Genetic diversity of C. beticola in the U.S. was determined, which will permit a more thorough testing panel of isolates for the development of fungicides and resistant sugarbeet germplasm. The molecular tagging of genes in sugarbeet that confer resistance to the economically-important disease powdery mildew and pest root-knot nematode was accomplished. This will reduce costs in the screening of elite sugarbeet germplasm containing these genes. Gene transfer to a Pythium member of the Oomycota was successful. Determination of pathogen genes important for disease production will result from this work. An esterase biochemical activity from C. beticola has been found in our laboratory
to be light-inducible. This may prove to be a virulence factor for the fungus. If so, characterization of this enzyme could shed light on new ways to control leaf spot disease caused by this fungus. 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? Information from research conducted under the Project Plan has been transferred to sugarbeet producers, processors and other scientists through presentations at industry and scientific meetings and publications. Dissemination of information will continue as research progresses. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Work referred to in: Agricultural Research Magazine, April 2004, 52:4-8, 'New breed of beet
geneticists shows how sweet it can be'. Weiland, J.J. Oral presentation: Presence and distribution of BNYVV and BSMV in the Glyndon Rhizomania Research Site. Sugarbeet Research and Extension Board of MN & ND Annual Meeting, Fargo, ND. January 6, 2004.
Impacts
(N/A)
Publications
- WEILAND, J.J. PRESENCE AND DISTRIBUTION OF BNYVV AND BSBMV IN THE GLYNDON RHIZOMANIA RESEARCH SITE. 2004 SUGARBEET RESEARCH AND EXTENSION REPORTS. 2004.V. 34. P. 294-299.
- BARGABUS, R.L., WEILAND, J.J. APPLICATION OF RNA SILENCING FOR THE CONTROL OF BENYVIRUSES OF SUGARBEET. PLANT AND ANIMAL GENOME XII ABSTRACTS. 2004. Abstr. No. W257. p. 69.
- WEILAND, J.J., EDWARDS, M.C. APPROACHES FOR GENE SILENCING IN SUGARBEET. PLANT AND ANIMAL GENOME XII ABSTRACTS. 2004. Abstr. No. P880. p. 291.
- Weiland, J.J., Koch, G. 2004. Sugar-beet leaf spot disease (cercospora beticola sacc.). Molecular Plant Pathology. 5:(3)157-166.
- McGrath, J.M., Shaw, R.S., De Los Reyes, B., Weiland, J.J. 2004. Construction of a sugarbeet BAC library from a hybrid that combines diverse traits. Plant Molecular Biology Reporter. 22(1):23-28.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Fungal pathogens continue to cause significant disease problems in sugarbeet grown in the U.S. and world-wide. Leaf spot caused by C. beticola, root rot, crown rot and seedling blight caused by R. solani, and post-emergence damping off caused by A. cochlioides are of special concern to growers in the north central region. Although some resistance to these pathogens has been identified in Beta germplasm, cultivars grown for sugar production remain susceptible to disease. Chemical control of A. cochlioides and C. beticola currently exists, but is limited, and is of decreasing effectiveness for C. beticola; no chemical control of R. solani is currently available. Known cultural practices that might alleviate disease severity are not compatible with climactic constraints that effect the north central growing region. With the recent discovery of Rhizomania disease caused by beet necrotic
yellow vein virus in production fields of the region, a post-doctoral scientist has been added to our project to investigate novel means for the control of this devastating sugarbeet pathogen. The objectives of the project are to examine the nature of the interaction of fungal pathogens with their sugarbeet host and to determine genetic or biochemical factors that condition resistance in the plant and virulence in the fungus. Additional objectives are the evaluation of genetic diversity in the fungal pathogens of sugarbeet and the extent to which this genetic diversity is correlated with virulence traits, as well as the development of biological control measures aimed at reducing the use of environmentally-unfriendly fungicides. We are using molecular genetic and biochemical approaches to examine (a) the genetic variation in the pathogens described above and how this variation relates to pathogen virulence, (b) the production of enzymes by both the plant and the fungus at the
interface of these two entities, (c) the efficacy of gene silencing in sugarbeet and how it might be used to combat Rhizomania disease, and (d) the identification of DNA markers that are associated with genes in sugarbeet that confer resistance to the above and other biotic diseases. Reduction of disease by the application of beneficial bacteria and natural plant elicitor compounds are also being examined. The above illustrates the major disease problems that impact sugarbeet production in the U.S. as well as illuminates the approaches being taken in our laboratory in order to reach the stated objectives. Although these technologies are beginning to be applied to the improvement of sugarbeet, the techniques are used more intensively in other crops. Studies on the infection process of sugarbeet by fungal pathogens are acutely lacking in molecular genetic approaches. For this reason, the focus of the project is on the interaction between sugarbeet and its fungal pathogens using
molecular genetic techniques. 2. How serious is the problem? Why does it matter? Greater than 80% of U.S. sugarbeet production is located north of the 40th parallel. Root rot in the field caused by R. solani and A. cochlioides and leaf spot caused by C. beticola constitute the most serious disease problems in sugarbeet in the northern sugarbeet production regions of the U.S. Yields can be reduced by 40% to 50% in fields subjected to severe epidemics of root rot or leaf spot. Although relatively new to the central plains states, Rhizomania has severely impacted sugarbeet yields in the western U.S.. Post-harvest integrity of stored sugarbeet is compromised severely by previous infection of the beet in the field by all of the above organisms. These problems, therefore, affect the sugarbeet grower by reducing yields in the field, as well as beet sugar processors, as fungal infection causes increased rotting of stored beets and leads to increases in impurities in sugarbeet extract. 3.
How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 303, Plant Diseases (100%) Research conducted in this project has four principal thrusts: 1) identification and classification of sugarbeet fungal pathogens, 2) identification of non-pathogenic microorganism for use as biocontrol agents against sugarbeet fungal pathogens, 3) develop gene-transfer methodsfor use in determining sugarbeet fungal virulence loci, and 4) discover and exploit natural mechanism of host-plant resistance against fungal pathogens in sugarbeet. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003: Since protease activity secreted by A. cochlioides might be a significant virulence factor in the infection of sugarbeet, we sought to determine whether protease production indeed occurred in plants. All investigation at this level was performed in the laboratory of Project
Leader Dr. John Weiland at the USDA-ARS-NPA-RRVARC-NCSL Sugarbeet and Potato Research Unit, Fargo, North Dakota. It was determined that protease activity produced in culture by A. cochlioides is indeed produced in high levels in infected sugarbeet tissue, a result not previous reported for any plant-infecting Aphanomyces. This could impact crop protection through the potential use of protease inhibitors or through the selection of germplasm exhibiting increased inhibition of protease activity produced by A. cochlioides. This is a new project initiated on 2-24-2003 after completing the peer review process for CRIS projects. B. Other Significant Accomplishments: C: Significant Accomplishments/Activities that Support Special Target Populations: 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A protocol for the reproducible generation of stable oospores from A. cochlioides and P. aphanidermatum was developed. These will
be useful to sugarbeet breeding concerns in the selection for germplasm or hybrid parents that exhibit superior resistance to these pathogens. Genetic diversity of C. beticola in the U.S. was determined, which will permit a more thorough testing panel of isolates for the development of fungicides and resistant sugarbeet germplasm. The molecular tagging of genes in sugarbeet that confer resistance to the economically-important disease powdery mildew and pest root-knot nematode was accomplished. This will reduce costs in the screening of elite sugarbeet germplasm containing these genes. Gene transfer to a Pythium member of the Oomycota was successful. Determination of pathogen genes important for disease production will result from this work. A PCR protocol has been developed that permits identification of the major fungal pathogens of sugarbeet without need for pathogen culture. The technology will expedite the identification of pathogens in a diseased sugarbeet crop leading to more
rapid deployment of control measures. A technique for the detection of resistance to Rhizoctonia root rot has been refined within this period as well, which will permit rating of members of mapping populations for resistance and selection of individuals with superior disease resistance. An esterase biochemical activity from C. beticola has been found in our laboratory to be light-inducible. This may prove to be a virulence factor for the fungus. If so, characterization of this enzyme could shed light on new ways to control leaf spot disease caused by this fungus. 6. What do you expect to accomplish, year by year, over the next 3 years? In 2004 research into the use of gene silencing in sugarbeet will commence. In addition, experiments on the control of Aphanomyces root rot by pre-cropping with oat, of storage rot and in-field root rot using biological control will be initiated: this will extend the biocontrol projects on-going in our lab. Cercosporin-deficient mutants of C.
beticola will be characterized for virulence in plant inoculations. Laboratory staff will continue efforts to tag resistance genes to Rhizomania, beet mosaic virus, and Aphanomyces root rot. Populations segregating for resistance and susceptibility to A. cochlioides will be used for marker analysis. Esterase from C. beticola or protease from A. cochlioides will be purified and antibody production to these proteins will commence. The green fluorescent protein (GFP) gene will be used to transform C. beticola so that this isolate can be used by our lab and that of our collaborators at USDA-Fort Collins and Montana State University for plant infection studies. In 2005, Antisera raised against A. cochlioides esterase/protease protein will be used in conjunction with Real-Time PCR for the screening of populations segregating for resistance to A. cochlioides. Molecular markers for beet mosaic virus and powdery mildew resistance will converted to the more robust SCAR marker format.
Esterase production in C. beticola or protease production by A. cochlioides will be detected in infected plants using antisera developed to these proteins. Mutants of C. beticola generated by transformation will be determined in inoculations of sugarbeet plants in the greenhouse. Third year testing of biocontrol bacteria and inducers of plant resistance for field efficacy will occur. In 2006, markers obtained for resistance to A. cochlioides will be converted to the more robust SCAR marker format. Protease inhibitors will be used to determine which activities are important for the infection of sugarbeet seedlings by A. cochlioides. The infection etiology of sugarbeet will be determined using the GFP-transformed Cercospora beticola isolate.
Impacts
(N/A)
Publications
- WEILAND, J.J. A SURVEY FOR THE PREVALENCE AND DISTRITUBTION OF CERCOSPORA BETICOLA TOLERANT TO TRIPHENYLTIN HYDROXIDE AND RESISTANCE TO THIOPHANATE METHYL IN 2002. SUGARBEET RESEARCH AND EXTENSION REPORT,. COOPERATIVE EXTENSION SERVICE, NORTH DAKOTA STATE UNIVERSITY. 2003. V. 33. P. 241-246.
- WEILAND, J.J. TRANSFORMATION OF PYTHIUM APHANIDERMATUM TO GENETICIN RESISTANCE. CURRENT GENETICS. 2003. V. 42 P. 344-352.
- Lartey, R.T., Weiland, J.J., Caesar-TonThat, T.C., Bucklin-Comiskey, S. A PCR protocol for rapid detection of Cercospora beticola in sugarbeet tissues. Journal of Sugarbeet Research. 2003. v. 40. p. 1-10.
- METZGER, M.S., WEILAND, J.J. FIELD BIOCONTROL OF APHANOMYCES COCHLIOIDES. ABSTRACTS OF JOINT MEETING OF INTERNATIONAL INSTITUTE FOR BET RESEARCH AND THE AMERICAN SOCIETY OF SUGARBEET TECHNOLOGISTS. 2003. ABSTRACT P. 53.
- WEILAND, J.J., FRIESEN, T.L. FUNCTIONAL GENOMICS OF CERCOSPORA BETICOLA. ABSTRACTS OF JOINT MEETING OF INTERNATIONAL INSTITUTE FOR BEET RESEARCH AND THE AMERICAN SOCIETY OF SUGARBEET TECHNOLOGISTS. 2003. ABSTRACT P. 56.
- Weiland, J.J., Protease Secretion in Aphanomyces Cochlioides. Proceedings of the 1st Joint International Institute for Beet Research and the American Society of Sugar Beet Technologists Congress. 2003. p. 415-417.
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