Progress 10/01/12 to 09/30/17
Outputs
Target Audience:US Department of Homeland Security, US Federal Bureau of Investigation, US Department of State, US Department of Defense, USDA Animal and Plant Health Inspection Service, National Plant Diagnostic Network, National Plant Disease Recovery System, US Defense Threat Reduction Agency, Oklahoma Office of Homeland Security, European Union 7th Framework - Security, citizens of Oklahoma. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students and postdocs presented oral papers and posters at national and international professional meetings Students and postdocs wrote and submitted their manuscripts to peer reviewed journals At the request of the Federal Bureau of Investigation's Laboratory Unit, FBI scientists were hosted for a 2-day, hands-on, field and classroom Crop Biosecurity Field Training How have the results been disseminated to communities of interest? Research talks and posters at scientific meetings, both national and international Invited talks at a variety of national and international venues Submission of research and review papers to refereed journals Application as STEM activity for 4-H youth Field training for FBI scientists What do you plan to do during the next reporting period to accomplish the goals? Continue to work towards research goals Prepare and submit additional manuscripts to refereed journal articles
Impacts
What was accomplished under these goals?
1. Develop a novel approach for handling of next generation sequencing data for diagnostics using "E-probe Diagnostic Nucleic acid Analysis" (EDNA). A. EDNA. This USDA NIFA- funded project addresses the vulnerability of U.S. agriculture to intentional contamination with human pathogens by adapting massively parallel sequencing (MPS) for detection. The method is rapid, identifies any pathogen in a complex sample, and can detect hallmarks of genetic engineering. MPS strategies and the EDNA pipeline were optimized and validated with sequencing data sets of the human pathogens E. coli and Salmonella spp. In 2015 the assay was optimized for a collection of E. coli O157H7 as proof of concept for applications to foodborne human pathogens. A manuscript describing this work was submitted, in 2015, to a refereed journal (Blagden et al.) Significance: This bioinformatics approach to microbial detection has the potential for simultaneous detection of all foodborne pathogens present in a food sample. B. Whole Genome Sequencing for Applications in Microbial Forensics . The Department of Homeland Security established this program with the objectives of building an infrastructure to exploit genomic information of high-threat pathogens of humans, animals and plants for microbial forensic applications, identifying priorities for sequencing, and establishing strategies for using sequence information in the investigation of biocrimes. In Phase I (2013-2014), we prepared literature reviews, nucleic acid sequence gap analyses, and sequencing plans for 12 selected plant pathogens. We established relationships with potential pathogen strain providers. In collaboration with USDA ARS, we prepared and sequenced the genomes of two strains of the plant pathogen Rathayabacter toxicus. In 2015, in collaboration with scientists at the University of California at Riverside, we obtained and Illumina-sequenced several strains of Xylella fastidiosa; in collaboration with a research group at the University of Wisconsin we Illumina-sequenced several strains of Ralstonia solanacearum Race 3 Biovar 2; in collaboration with scientists at the University of Colorado and at INRA, France, we sequenced several strains of Xanthomonas oryzae pvs. oryzae and oryzicola. We established cooperation with scientists at the University of California and the USDA for obtaining genomic DNA for strains of Peronosclerospora raysiiae collected in the Philippines, and with scientists in India to obtain Sclerophthora raysiiae. DNA from one Xanthomonas isolate was submitted to DHS for PacBio sequencing. Significance: As the only U.S. entity focused specifically on plant pathogen forensics, OSU's contributions are filling a need within the U.S. security and defense communities. 2. Develop a specific forensic investigation strategy for a model plant disease and validate it in vivo. As part of a larger project on Crop and Food Biosecurity, funded by the European Union's 7th Framework Program, this project highlights a single plant pathosystem as a focus for multiple objectives relating to biosecurity and forensics. A forensically valid real time PCR assay specific for Fusarium proliferatum (Fp), cause of salmon blotch of onion, was developed and validated. Comparison of amplified DNA of 3 housekeeping genes from Fp from the US, Germany and Israel showed significant strain variability. Fp was isolated from onion sets and bulbs, weeds, soil, and surrounding plants and soil in Israel. Simple sequence repeats (SSRs) of genomic DNA were used to develop primers for discrimination among strain groups. Over 300 Fp isolates from a variety of locations, host plants and other matrices, were SSR typed and the data analyzed by population genetic software programs. Fp populations clustered into groups that suggest ecological niches and between-strain relationships. A manuscript presenting the adaptation of SSR technology to the typing of Fp isolates was submitted, in 2015, for journal publication. Significance: SSRs were shown to be useful in typing strains of Fp and in characterizing the fungus's population structure in different countries and hosts as well as within a single country and field. This work was the first comprehensive, real world test of a combination of field and laboratory methods for the forensic investigation of a plant disease outbreak. The methodologies can be applied similarly to other fungal pathogens.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Moncrief, I., C. Garzon, S. Marek, J. Stack, A. Gamliel, P. Garrido, F. Proano, M. Gard, H. Dehne, and J. Fletcher. 2016. Development of simple sequence repeat (SSR) markers for discrimination among isolates of Fusarium proliferatum. J. Molec. Methods
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Blagden, T., W. Schneider, U. Melcher, J. Daniels, and J. Fletcher. 2015. Adaptation and validation of E-proble diagnostic nucleic acid analysis for detection of Escherichia coli O157:H7 in metagenomic data of complex food matrices. J. Food Prot.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2016
Citation:
Fletcher, J., A. Gamliel, J. Stack, H. Dehne, and I. Moncrief. 2016. Applications and assessment of microbial forensics in a field outbreak of salmon blotch of onion in Israel. Pp. ____ in: Gullino, M.L., J.P. Stack, J. Fletcher and J. Mumford, eds. Practical Tools for Plant and Food Biosecurity - Results From a European Network of Excellence. Springer International Publishing AG, Gewerbestrasse 11, 6330 Cham, Switzerland.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2016
Citation:
Fletcher, J. and R.M. Hunger. 2016. Bioethics and agriculture. Pp. ___ in: S. Morse and R. Murch, eds. Bioethics and the Life Sciences: Challenges in a Globalized World. Wiley and Sons, Inc. Submitted April 2014.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2016
Citation:
Wayadande, A., J. Fletcher, & B. Bruton. 2016. Cucurbit yellow vine disease: A model for true bug-bacteria relationships. in: J. Brown, Ed. Microbe-Arthropod Vector Interactions. APS Press.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:US Department of Homeland Security, US Federal Bureau of Investigation, US Department of State, US Department of Defense, USDA Animal and Plant Health Inspection Service, USDA National Plant Diagnostic Network, USDA National Plant Disease Recovery System, US Defense Threat Reduction Agency, Oklahoma Office of Homeland Security, European Union 7th Framework in Security, citizens of Oklahoma. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Student and PI presented oral papers at national and international professional meetings. Students and PI submitted manuscripts to peer reviewed journals and included findings in book chapters. How have the results been disseminated to communities of interest? Research talks and posters at scientific meetings, both national and international. Invited talks at national and international venues. Submission of papers to refereed journals. What do you plan to do during the next reporting period to accomplish the goals?Continue to support NIMFFAB, the Department of Entomology and Plant Pathology, the Division of Agricultural Sciences and Natural Resources, and Oklahoma State University Participate in NIMFFAB strategic planning, including attendance and presentation at a planning retreat in Stillwater (December 2016) Present invited talks and posters that disseminate our research and education programs, with acknowledgement to OSU Submit several invited scientific review articles and chapters to peer-reviewed journals, with acknowledgement to OSU Continue to contribute to the USDA National Needs Fellowship Project, on which I am a principle investigator Continue to serve on Federal Agency panels Volunteer with the American Phytopathological Society through service on the APS Foundation Board, the Microbial Forensics Interest Group, the Food Safety Interest Group, and the International Congress of Plant Pathology Planning Committee Teach PLP 5304, Phytobacteriology, for graduate students
Impacts
What was accomplished under these goals?
Objective: Develop a novel approach for handling of next-generation sequencing data for diagnostics using "E-probe Diagnostic Nucleic acid Analysis (EDNA). Accomplishments: Research projects on which I was involved were completed and manuscripts submitted for publication. Significance: This bioinformatics approach to microbial detection has the potential for simultaneous detection of all pathogens present in a complex sample, such as a plant, animal, food, or soil. Objective: Whole genome sequencing for applications in microbial forensics (Department of Homeland Security/MRIGlobal project). Accomplishments: Sequencing of all plant pathogens on the DHS-approved work program was completed, sequences were annotated and submitted via a MRIGlobal pipeline to DHS as well as deposited at the National Genome Repository. Pathogens included were several strains each of Rathayabacter toxicus, Xanthomonas oryzae pv. oryzae and pv. oryzicola, Ralstonia solanacearum Race 3 Biovar 2, Xylella fastidiosa, and the fungus Coniothyrium. Significance: As the only U.S. entity focused specifically on plant pathogen forensics, NIMFFAB/OSU's contributions are filling a need within the U.S. security and defense communities. Objective: Develop a specific forensic investigation strategy for a model plant disease and validate it in vivo. Accomplishments: Final analysis of data establishing and validating the use of simple sequence repeat (SSR) primers for discrimination among strains of the plant pathogenic fungus Fusarium proliferatum (Fp) were performed and the research was submitted for publication. Significance: SSRs were shown to be useful in typing strains of Fp and in characterizing the fungus's population structure in different countries and hosts as well as within a single country and field. This methodology can be applied similarly to other fungal pathogens.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Wayadande, A., J. Fletcher, & B. Bruton. 2016. Cucurbit yellow vine disease: A model for true bug-bacteria relationships. in: J. Brown, Ed. Microbe-Arthropod Vector Interactions. APS Press.
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Allen, C., C. Burge, P. Daszak, P. Genovesi, J. Fletcher, P. Formenty, D. Harvell, W.B. Karesh, R. Kock, E.H. Loh, J. Lubroth, C. Machalaba, K. M. Smith, P. Stoett, and H. S. Young. 2015. Infectious Diseases. in: State of Knowledge Review: Connecting Global Priorities: Biodiversity and Human Health. Convention on Biological Diversity and World Health Organization. Montreal.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Phytobiomes Writing Team. 2016. Phytobiomes: A Roadmap for Research and Translation. American Phytopathological Society, St. Paul, MN. www.phytobiomes.org/roadmap
- Type:
Other
Status:
Accepted
Year Published:
2016
Citation:
Fletcher, J. 2016. Cotton anthocyanosis virus. USDA APHIS Datasheet. Invited.
- Type:
Other
Status:
Accepted
Year Published:
2016
Citation:
Fletcher, J. 2016. Cotton virescence agent. USDA APHIS Datasheet. Invited.
- Type:
Other
Status:
Accepted
Year Published:
2016
Citation:
Fletcher, J. 2016. Cassava mosaic virus. USDA APHIS Datasheet. Invited.
- Type:
Other
Status:
Accepted
Year Published:
2016
Citation:
Fletcher, J. 2016. Cassava brown streak virus. USDA APHIS Datasheet. Invited.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Fletcher, J., T. Blagden, U. Melcher, R. Winegar, B. Campos, K. Hari, and D. Lin. 2016. Capturing global biodiversity of plant pathogens by whole genome sequencing. Sequencing, Finishing, and Analysis in the Future Meeting, Santa Fe, NM
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Ma, L.M., U. Melcher, F. Ochoa-Corona, C. Garzon, W. Schneider, A. Wayadande, R. Allen and J. Fletcher. 2016. Forensic plant pathology: Enhancing U.S. crop biosecurity through multidisciplinary graduate education, experience and research. North American Colleges and Teachers of Agriculture Annual Meeting, Honolulu, HI.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:US Department of Homeland Security, US Federal Bureau of Investigation, US Department of State, US Department of Defense, USDA Animal and Plant Health Inspection Service, National Plant Diagnostic Network, National Plant Disease Recovery System, US Defense Threat Reduction Agency, Oklahoma Office of Homeland Security, European Union 7th Framework - Security, citizens of Oklahoma. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students and postdocs presented oral papers and posters at national and international professional meetings Students and postdocs wrote and submitted their manuscripts to peer reviewed journals At the request of the Federal Bureau of Investigation's Laboratory Unit, FBI scientists were hosted for a 2-day, hands-on, field and classroom Crop Biosecurity Field Training How have the results been disseminated to communities of interest? Research talks and posters at scientific meetings, both national and international Invited talks at a variety of national and international venues Submission of research and review papers to refereed journals Application as STEM activity for 4-H youth Field training for FBI scientists What do you plan to do during the next reporting period to accomplish the goals? Continue to work towards research goals Prepare and submit additional manuscripts to refereed journal articles
Impacts
What was accomplished under these goals?
1. Develop a novel approach for handling of next generation sequencing data for diagnostics using "E-probe Diagnostic Nucleic acid Analysis" (EDNA). A. EDNA. This USDA NIFA- funded project addresses the vulnerability of U.S. agriculture to intentional contamination with human pathogens by adapting massively parallel sequencing (MPS) for detection. The method is rapid, identifies any pathogen in a complex sample, and can detect hallmarks of genetic engineering. MPS strategies and the EDNA pipeline were optimized and validated with sequencing data sets of the human pathogens E. coli and Salmonella spp. In 2015 the assay was optimized for a collection of E. coli O157H7 as proof of concept for applications to foodborne human pathogens. A manuscript describing this work was submitted, in 2015, to a refereed journal (Blagden et al.) Significance: This bioinformatics approach to microbial detection has the potential for simultaneous detection of all foodborne pathogens present in a food sample. B. Whole Genome Sequencing for Applications in Microbial Forensics . The Department of Homeland Security established this program with the objectives of building an infrastructure to exploit genomic information of high-threat pathogens of humans, animals and plants for microbial forensic applications, identifying priorities for sequencing, and establishing strategies for using sequence information in the investigation of biocrimes. In Phase I (2013-2014), we prepared literature reviews, nucleic acid sequence gap analyses, and sequencing plans for 12 selected plant pathogens. We established relationships with potential pathogen strain providers. In collaboration with USDA ARS, we prepared and sequenced the genomes of two strains of the plant pathogen Rathayabacter toxicus. In 2015, in collaboration with scientists at the University of California at Riverside, we obtained and Illumina-sequenced several strains of Xylella fastidiosa; in collaboration with a research group at the University of Wisconsin we Illumina-sequenced several strains of Ralstonia solanacearum Race 3 Biovar 2; in collaboration with scientists at the University of Colorado and at INRA, France, we sequenced several strains of Xanthomonas oryzae pvs. oryzae and oryzicola. We established cooperation with scientists at the University of California and the USDA for obtaining genomic DNA for strains of Peronosclerospora raysiiae collected in the Philippines, and with scientists in India to obtain Sclerophthora raysiiae. DNA from one Xanthomonas isolate was submitted to DHS for PacBio sequencing. Significance: As the only U.S. entity focused specifically on plant pathogen forensics, OSU's contributions are filling a need within the U.S. security and defense communities. 2. Develop a specific forensic investigation strategy for a model plant disease and validate it in vivo. As part of a larger project on Crop and Food Biosecurity, funded by the European Union's 7th Framework Program, this project highlights a single plant pathosystem as a focus for multiple objectives relating to biosecurity and forensics. A forensically valid real time PCR assay specific for Fusarium proliferatum (Fp), cause of salmon blotch of onion, was developed and validated. Comparison of amplified DNA of 3 housekeeping genes from Fp from the US, Germany and Israel showed significant strain variability. Fp was isolated from onion sets and bulbs, weeds, soil, and surrounding plants and soil in Israel. Simple sequence repeats (SSRs) of genomic DNA were used to develop primers for discrimination among strain groups. Over 300 Fp isolates from a variety of locations, host plants and other matrices, were SSR typed and the data analyzed by population genetic software programs. Fp populations clustered into groups that suggest ecological niches and between-strain relationships. A manuscript presenting the adaptation of SSR technology to the typing of Fp isolates was submitted, in 2015, for journal publication. Significance: SSRs were shown to be useful in typing strains of Fp and in characterizing the fungus's population structure in different countries and hosts as well as within a single country and field. This work was the first comprehensive, real world test of a combination of field and laboratory methods for the forensic investigation of a plant disease outbreak. The methodologies can be applied similarly to other fungal pathogens.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Blagden, T., W. Schneider, U. Melcher, J. Daniels, and J. Fletcher. 2015. Adaptation and validation of E-proble diagnostic nucleic acid analysis for detection of Escherichia coli O157:H7 in metagenomic data of complex food matrices. J. Food Prot. (Submitted 10/1/15).
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: US Department of Homeland Security, US Federal Bureau of Investigation, US Department of State, US Department of Defense, USDA Animal and Plant Health Inspection Service, National Plant Diagnostic Network, National Plant Disease Recovery System, US Defense Threat Reduction Agency, Oklahoma Office of Homeland Security, European Union 7th Framework - Security, citizens of Oklahoma. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Students and postdocs presented oral papers and posters at national and international professional meetings. Students and postdocs wrote and submitted their manuscripts to peer reviewed journals. A Ph.D. student defended his thesis and graduated. How have the results been disseminated to communities of interest? Research talks and posters at several scientific meetings, both national and international Invited talks at a variety of national and international venues Submission of papers to refereed journal articles Application as STEM activity for 4-H youth What do you plan to do during the next reporting period to accomplish the goals? Continue to work towards research goals Prepare and submit manuscripts to refereed journal articles
Impacts
What was accomplished under these goals?
1. Develop a novel approach for handling of next generation sequencing data for diagnostics using "E-probe Diagnostic Nucleic acid Analysis" (EDNA). A. EDNA. This USDA NIFA- funded project addresses the vulnerability of U.S. agriculture to intentional contamination with human pathogens by adapting massively parallel sequencing (MPS) for detection. The method is rapid, identifies any pathogen in a complex sample, and can detect hallmarks of genetic engineering. MPS strategies and the EDNA pipeline were optimized and validated with sequencing data sets of several plant pathogenic bacteria, fungi and viruses. Unique e-probes were generated for human pathogens E. coli and Salmonella spp. Optimum E-probe length was established by calculating precision, and the assay was validated using a range of pathogen:matrix ratios. T-test statistical confidences were limited by the total number of e-probes available, despite 100% precision. Significance: This bioinformatics approach to microbial detection has the potential for simultaneous detection of all foodborne pathogens present in a food sample. B. Whole Genome Sequencing for Applications in Microbial ForensicsThe Department of Homeland Security established this program with the objectives of building an infrastructure to exploit genomic information of high-threat pathogens of humans, animals and plants for microbial forensic applications, identifying priorities for sequencing, and establishing strategies for using sequence information in the investigation of biocrimes. In Phase I (2013-2014), we prepared literature reviews, nucleic acid sequence gap analyses, and sequencing plans for 12 selected plant pathogens. We established relationships with potential pathogen strain providers. In collaboration with USDA ARS, we prepared and sequenced the genomes of two strains of the plant pathogen Rathayabacter toxicus. Significance: As the only U.S. entity focused specifically on plant pathogen forensics, OSU's contributions are filling a need within the U.S. security and defense communities. 2. Develop a specific forensic investigation strategy for a model plant disease and validate it in vivo.As part of a larger project on Crop and Food Biosecurity, funded by the European Union's 7th Framework Program, this project highlights a single plant pathosystem as a focus for multiple objectives relating to biosecurity and forensics. A forensically valid real time PCR assay specific for Fusarium proliferatum (Fp), cause of salmon blotch of onion, was developed and validated. Comparison of amplified DNA of 3 housekeeping genes from Fp from the US, Germany and Israel showed significant strain variability. Fp was isolated from onion sets and bulbs, weeds, soil, and surrounding plants and soil in Israel. Simple sequence repeats (SSRs) of genomic DNA were used to develop primers for discrimination among strain groups. Over 300 Fp isolates from a variety of locations, host plants and other matrices, were SSR typed and the data analysed by population genetic software programs. Fp populations clustered into groups that suggest ecological niches and between-strain relationships. Significance: SSRs were shown to be useful in typing strains of Fp and in characterizing the fungus's population structure in different countries and hosts as well as within a single country and field. The methodology can be applied similarly to other fungal pathogens.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Blagden, T., W. Schneider, U. Melcher, J. Daniels, and J. Fletcher. 2015. Adaptation and validation of E-probe diagnostic nucleic acid analysis for detection of Escherichia coli O157:H7 in metagenomics data of complex food matrices. J. Food Sci. Submitted January 16, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Dobhal, S., G. Zhang, D. Gautam, J. Fletcher and L.M. Ma. 2015. Uneven distribution of microorganisms on the surface of field-grown cantaloupes. Food Control pp. 185-189 DOI: 10.1016/j.foodcont.2014.07.002.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
James, M., U. Melcher, and J. Fletcher. 2014. Evaluating the impacts of stressors of Pseudomonas syringae pathovar tomato on the effectiveness of multi-locus variable number tandem repeat analysis and multilocus sequence typing in microbial forensics investigations. Investig. Genet. 5:10. doi:10.1186/2041-2223-5-10.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Budowle, B., S. Minot, N. D. Connell, A. Bielecka, R. R. Colwell, C. R. Corbett, M. Forsman, D. R. Kadavy, A. Markotic, S.A. Morse, R. S. Murch, S.E. Schmedes, K. Ternus, S. Turner, and J. Fletcher. 2014. Validation of high throughput sequencing and microbial forensics applications. Investig. Genet. 5:9. doi:10.1186/2041-2223-5-9.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Moncrief, I., C. Garzon, S. Marek, A. Gamliel, J. Stack, Y. Issac, and J. Fletcher. 2014. Adapting microbial forensics approaches for crop biosecurity: Validating the use of simple sequence repeats (SSRs) for typing populations of Fusarium proliferatum associated with salmon blotch of onion in southern Israel. APS Annual Meeting, Minneapolis, MN.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Gautam, D., S. Dobhal, M.E. Payton, J. Fletcher and L.M. Ma. 2014. Surface survival and internalization of Salmonella enterica through natural cracks on developing cantaloupe fruits, alone or in the presence of the melon wilt pathogen Erwinia tracheiphila. PLoS ONE 9 (8): e105248. doi:10.1371/journal.pone.0105248.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2014
Citation:
Fletcher, J. and R.M. Hunger. 2014. Bioethics and agriculture. Pp. ___ in: S. Morse and R. Murch, eds. Bioethics and the Life Sciences: Challenges in a Globalized World. Wiley and Sons, Inc. Submitted April 2014; publication expected August 2015.
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Allen, C., C. Burge, P. Daszak, P. Genovesi, J. Fletcher, P. Formenty, D. Harvell, W.B. Karesh, R.Kock, E.H. Loh, J. Lubroth, C. Machalaba, K.M. Smith, P. Stoett, H.S. Young (Coordinating authors: W.B. Karesh and P. Formenty). 2015. Infectious Diseases. In: State of Knowledge Review: Connecting Global Priorities: Biodiversity and Human Health. Convention on Biological Diversity and World Health Organization. Montreal.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2013
Citation:
Wayadande, A., J. Fletcher, and B. Bruton. 2013. Cucurbit yellow vine disease: A model for true bug-bacteria relationships. Pp. ___ in: J. Brown, Ed. Microbe-Arthropod Vector Interactions. APS Press. Accepted, in press.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Ma, L.M., J. Fletcher, and G. Zhang. 2014. Detection of human pathogens on plants. Pp. 87-102 in: Gullino, M.L. and P. Bonants, eds. Detection and Diagnostics of Plant Pathogens. Springer Press, Dordrecht, The Netherlands. 199 p.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Fletcher, J., F.M. Ochoa Corona, and M. Payton. 2014. Plant disease diagnostics for forensic applications. Pp. 103-118 in: Gullino, M.L. and P. Bonants, eds. Detection and Diagnostics of Plant Pathogens. Springer Press, Dordrecht, The Netherlands. 199 p.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Gelhaus, C., P. Aguilar, T. Blagden, K. Brown, A. Bukreyev, R. Chen, J. Fletcher, N. Forrester, K. Hari, R.Jain, T. Ksiazek, D. Lin, U. Melcher, D. Roberts, R.. Tesh, E. Wang, S. Weaver, R. Winegar. 2014. Advancing microbial forensics through genome sequencing. Defense Threat Reduction Agency Research Meeting, St. Louis, MO.
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: State of Oklahoma, US Department of Homeland Security, US Federal Bureau of Investigation, USDA Animal and Plant Health Inspection Service, National Plant Diagnostic Network, National Plant Disease Recovery System, US Defense Threat Reduction Agency, Oklahoma Office of Homeland Security, European Union 7th Framework – Security Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? STEM training for 4-H youth and educators Teaching and mentoring experience for graduate students and postdocs teaching graduate laboratories and a 4-H summer workshop Students and postdocs presented oral papers and posters at national and international professional meetings Students and postdocs wrote and submitted their manuscripts to peer reviewed journals How have the results been disseminated to communities of interest? Research talks and posters at several scientific meetings, both national and international Invited talks at a variety of national and international venues Submission of papers to refereed journal articles Application as STEM activity for 4-H youth What do you plan to do during the next reporting period to accomplish the goals? Continue to work towards research goals in each project Prepare and submit manuscripts to refereed journal articles Prepare funding applications for follow-on research
Impacts
What was accomplished under these goals?
1. Develop a novel approach for handling of next generation sequencing data for diagnostics using “E-probe Diagnostic Nucleic acid Analysis” (EDNA). A. EDNA. This USDA NIFA- funded project addresses the vulnerability of U.S. agriculture to intentional contamination with human pathogens by adapting massively parallel sequencing (MPS) for detection. The method is rapid, identifies any pathogen in a complex sample, and can detect hallmarks of genetic engineering. MPS strategies and the EDNA pipeline were optimized and validated with sequencing data sets of several plant pathogenic bacteria, fungi and viruses. Unique e-probes were generated for human pathogens E. coli and Salmonella spp. Optimum E-probe length was established by calculating precision, and the assay was validated using a range of pathogen:matrix ratios. T-test statistical confidences were limited by the total number of e-probes available, despite 100% precision. Significance: This bioinformatics approach to microbial detection has the potential for simultaneous detection of all foodborne pathogens present in a food sample. B. Whole Genome Sequencing for Applications in Microbial Forensics The Department of Homeland Security has established this program with the objectives of building an infrastructure to exploit genomic information of high-threat pathogens of humans, animals and plants for microbial forensic applications, identifying priorities for sequencing, and establishing strategies for using sequence information in the investigation of biocrimes. In Phase I (2013-2014), we began to prepare reviews and nucleic acid sequence gap analyses for 12 selected plant pathogens. These analyses will be used to prepare a sequencing strategy and approach. Significance: As the only U.S. entity focused specifically on plant pathogen forensics, OSU’s contributions are filling a need within the U.S. security and defense communities. 2. Develop a specific forensic investigation strategy for a model plant disease and validate it in vivo. As part of a larger project on Crop and Food Biosecurity, funded by the European Union’s 7th Framework Program, this project highlights a single plant pathosystem as a focus for multiple objectives relating to biosecurity and forensics. A forensically valid real time PCR assay specific for Fusarium proliferatum (Fp), cause of salmon blotch of onion, was developed and validated. Comparison of amplified DNA of 3 housekeeping genes from Fp from the US, Germany and Israel showed significant strain variability. Fp was isolated from onion sets and bulbs, weeds, soil, and surrounding plants and soil in Israel. Simple sequence repeats (SSRs) of genomic DNA identified in one isolate were used to develop primers that allow discrimination among strain groups. Significance: SSRs were shown to be useful in typing strains of Fp and in characterizing the fungus’s population structure in different countries and hosts as well as within a single country and field. The methodology can be applied similarly to other fungal pathogens. 3 & 4: 3. Determine the impact of elapsed time on the validity of molecular typing of for forensic matching assays. 4. Assess several bacterial characterization methods for their ability to reveal a past history of laboratory culture. In an investigation of agricultural biocrime or bioterror events, it is important to discern whether an incident-associated pathogen has had a history of laboratory culture (human handling). Using multi-locus variable number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST) in microbial forensics may be problematic because periods between pathogen introduction and discovery may allow evolutionary changes in the pathogen genome; bacteria grown in a laboratory may exhibit unusual rates or types of genetic change. We tested the reliability of MLVA and MLST to type populations of the plant pathogen, Pseudomonas syringae pv. tomato (Pst) after its exposure to experimental treatments simulating environmental conditions to which it could be exposed during a biological release, while the bacterium was being subcultured sequentially for 1 year. Both MLVA and MLST produced consistent profiles for all treatments throughout the experiment. Significance: We have shown that MLVA and MLST, using the specified primers and conditions, are reliable typing tools for forensics investigations involving Pst. Similar experiments should be conducted with other high consequence plant pathogens to ensure that the assays are reliable for pathogens infecting plants in natural environments and for organisms that display mutation rates faster than those of Pst. 5. Investigate the ability of Salmonella to colonize cantaloupe, either alone or in the presence of the cucurbit pathogen Erwinia tracheiphila. We investigated the interactions between S. enterica (Se) Poona, the plant pathogen Erwinia tracheiphila (Et), and cantaloupe fruit. Fruit rinds were inoculated at the natural cracking stage with Se and Et, independently or together, over a crack. As long as 24 DPI (fruit maturity) Se was detected on 14% and 40% of the fruit inoculated with Se alone and the two-pathogen mixture, respectively, but Se declined gradually. Et, alone or with Se, caused watersoaked lesions on cantaloupe fruit, and Se survival was higher near lesions. Of fruit inoculated with Et alone and sampled at 24 DPI, 61% had watersoaked lesions. In nearly half of those symptomatic fruits the watersoaking extended into the sub-rind mesocarp. Significance: Although Se was not detected in the fruit interior in our experiments, the fact that Et did so suggests that human pathogens that contaminate fresh produce might also do so. The possibility should be investigated under a wider range of conditions and produce types. 6. Effect of fumigant use in tree nut orchards on the number and types of microbial residents on nut shell surfaces. Nothing to report. 7. Development of enhanced detection technology suitable for forensic analysis of cases involving the foodborne human pathogens Salmonella and E. coli O157:H7 on fresh produce. A multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) assay was developed and validated for strain discrimination among 6 common non-O157 Shiga toxin-producing E. coli (STEC) serogroups in the United States. Previously, twelve VNTR loci were identified and used to type 65 clinical non-O157 STEC isolates. This year, 11 additional VNTR loci were identified and screened in the MLVA assay and 3 MLVA PCR primer sets were optimized. Pulsed-field gel electrophoresis (PFGE) was completed for 84 isolates and the results compared to those of MLVA. The most informative VNTR loci were selected. The final optimized MLVA assay revealed in 68 unique MLVA-types among 84 non-O157 STEC isolates. Significance: The discriminatory power and robustness of the MLVA assay was improved so that the method can augment PFGE in foodborne illness outbreak investigations. 8. Determine risk factors associated with filth fly transmission of Salmonella and E. coli O157:H7 to leafy greens Filth flies are mechanical vectors of pathogenic bacteria in hospital and restaurant settings, but their role as vectors for disseminating microbes to plants has not been demonstrated. E. coli O157:H7 deposition by flies onto spinach was studied using molecular, microbiological, and microscopy techniques. Flies acquired bacteria from contaminated cattle manure and deposited it on leaves, where it multiplied. The data are consistent with a hypothesis that fly regurgitation material supplies nutrients to bacteria on the spinach phylloplane. E. coli O157:H7 persisted on fly body surfaces up to 13 d after exposure, consistent with a hypothesis of bioenhanced transmission of human pathogens by house flies. Significance: The results suggest that filth flies may affect the microbial safety of fresh produce and highlight the need for additional research.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Arif, M., G.S. Aguilar-Moreno, A. Wayadande, J. Fletcher and F.M. Ochoa Corona. 2013. Primer modification improves rapid and sensitive in vitro and field deployable assays for detection of High plains virus variants. Appl. Env. Microbiol. 80:320-327. Published ahead of print 25 October 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Arif, M., G.S. Aguilar-Moreno, A. Wayadande, J. Fletcher, and F.M. Ochoa-Corona. 2013. Primer modification improves rapid and sensitive in vitro and field deployable assays for detection of High plains virus variants. Appl. Env. Microbiol. http://aem.asm.org/content/early/2013/10/21/AEM.02340-13.abstract
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Arif, M., J. Fletcher, S. M. Marek, U. Melcher and F. M. Ochoa-Corona. 2013. Development of a rapid, sensitive, and feld-deployable Razor Ex BioDetection System and quantitative PCR assay for detection of Phymatotrichopsis omnivora using multiple gene targets. Appl. Environ. Microbiol. 2013, 79(7):2312.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Arif, M., F.M. Ochoa Corona, S. Marek, U. Melcher, and J. Fletcher. 2013. Multi-gene based detection and identification of Phymatotrichopsis omnivora for enhanced reliability and accuracy. Appl. Env. Microbiol. Published ahead of print 25 January 2013, doi: 10.1128/AEM.03239-12 AEM.03239-12.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Carpane, P., Melcher, U., Wayadande, A., de la Paz Gimenez Pecci, M., Laguna, G., Dolezal, W., and Fletcher, J. 2013. An analysis of the genomic variability of the phytopathogenic mollicute Spiroplasma kunkelii. Phytopathology 103:129-134.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Fletcher, J., J. Leach, K. Eversole and R. Tauxe. 2013. Human pathogens on plants: Designing a multidisciplinary strategy for research. Phytopathology 103:306-315.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
James, M., T. Blagden, I. Moncrief, J. Burans, K. Schneider, and J. Fletcher. 2013. Validation of real-time PCR assays for bioforensic detection of model plant pathogens. J. Forensic Sci. DOI:10.1111/1556-4029.12321.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kamenidou, S., R. Jain, K. Hari, J.M. Robertson, and J. Fletcher. 2013. Microbial Rosetta Stone Central Agricultural Database: An information resource on high consequence plant pathogens. Plant Dis. 97:1097-1102.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ouyang, P., Arif, M., J. Fletcher, U. Melcher and F.M. Ochoa-Corona. 2013. Enhanced reliability and accuracy for field deployable bioforensic detection and discrimination of Xylella fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis using Razor Ex technology and TaqMan quantitative PCR. PLOS-ONE DOI:10.1371/journal.pone.0081647.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Stobbe, T., J. Daniels, A. Espindola, U. Melcher, F. Ochoa Corona, C. Garzon, R. Verma, J. Fletcher and W. Schneider. 2013. Electronic diagnostic nucleic acid analysis (EDNA): A theoretical approach for improved handling of massively parallel sequencing data for diagnostics. J. Micr. Meth. http://dx.doi.org/10.1016/j.mimet.2013.07.002
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Timmons, C., S. Dobhal, J. Fletcher and L.M. Ma. 2013. Primers with 5 flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7. J. Food Prot. 76:668-673. doi:10.4315/0362-028X.JFP-12-428
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wasala, L., J. Talley, U. Desilva, J. Fletcher and A. Wayadande. 2013. Transfer of Escherichia coli O157:H7 to spinach by house flies (Musca domestica). Phytopathology 103:373-380
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Fletcher, J., F.M. Ochoa Corona, and M. Payton. 2013. Plant disease diagnostics for forensic applications. In: Proceedings of the International Congress of Plant Pathology.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Stack, J.P., J. Fletcher and M.L. Gullino. 2013. Climate change and plant biosecurity: A new world disorder? Pp. 161-181 In: Balajz, B., C. Burnley, I. Comardicea, A. Maas, and R. Roffey, Eds. Global Environmental Change: New Drivers for Resistance, Crime and Terrorism? Adelphi Press, Baden-Baden, Germany
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Andreason, S., M. Arif, J. Brown, F. Ochoa-Corona, J. Fletcher, and A. Wayadande. 2013. Multiplex PCR identification of high consequence Bemisia tabaci biotypes and Trialurodes vaporarium. Phytopathology 103: S2.7 (abstract).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Andreason, S., M. Arif, J.K. Brown, J. Fletcher, F. Ochoa-Corona, and A. Wayadande. 2013. Multiplex PCR identification of high consequence Bemisia tabaci biotypes and Trialeurodes vaporariorum. APS Annual Meeting, Austin, TX, August 2013.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Blagden, T.D., W. Schneider, U. Melcher, and J. Fletcher. 2013. In silico Adaptation of EDNA (E-probe Diagnostic Nucleic Acid Analysis) for Detection of Foodborne Pathogens. International Association of Food Protection Annual Meeting. Charlotte, North Carolina.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Daniels, J., W. Schneider, J. Fletcher and F. Ochoa Corona. 2013. Next generation sequencing and its application as a biosecurity tool. Gordon Rsch Conf. Chem. & Biolog. Terrorism Defense, Ventura, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Daniels, J., T. Stobbe, A. Espindola, W. Schneider, J. Sallee, T. Blagden, F. Ochoa Corona, C. Garzon, and J. Fletcher. 2013. CSI in a tomato disease plot: Engaging 4-H youth and educators in STEM through investigative plant pathology. APS Annual Meeting, Austin, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Gamliel, A., J. P. Stack, J. Fletcher, Y. Issac, I. Moncrief, H. Dehne, and M. L. Gullino. 2013. Fusarium proliferatum - Allium cepa pathosystem: a model encompassing crop protection, agricultural biosecurity and animal/human health. Intl Congr. Plant Pathology, Beijing, China, August 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Moncrief, I., C. Garzon, S. Marek, J. Stack, A. Gamliel, Y. Issac, H. Dehne, and J. Fletcher. 2013. Specific discrimination of Fusarium proliferatum using Inter-simple sequence repeats (ISSRs) and simple sequence repeats (SSRs). APS Annual Meeting, Austin, TX, August 2013.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Moncrief, I., Garzon, C., Marek, S., Stack, J., Gamliel, A., Issac, Y.m Dehne, H., Fletcher, J. 2013. Specific discrimination of Fusarium proliferatum using inter-simple sequence (ISSRs) and simple sequence repeats (SSRs). Phytopathology 103: S99.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Moncrief, I., Garzon, C., Marek, S., Stack, J., Gamliel, A., Issac, Y.m Dehne, H., Fletcher, J. 2013. Specific discrimination of Fusarium proliferatum using inter-simple sequence (ISSRs) and simple sequence repeats (SSRs). Biochemistry and Molecular Biology Graduate Student Research Symposium.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Schneider, W.S., R. Verma, A. Stobbe, J. Daniels, A. Espindola, T. Blagden, J. Fletcher, F. Ochoa-Corona, C. Garzon, and U. Melcher. 2013. Bioinformatics strategies for microbial forensics. APS Annual Meeting, Austin, TX.
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