Source: OKLAHOMA STATE UNIVERSITY submitted to
DEVELOPMENT AND ASSESSMENT OF DIAGNOSTICS FOR AGRICULTURAL BIOSECURITY AND FORENSIC PLANT PATHOLOGY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0221481
Grant No.
(N/A)
Project No.
OKL02773
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Project Director
Ochoa Corona, F.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Entomology And Plant Pathology
Non Technical Summary
Increasing global trade, passenger traffic and natural atmospheric disturbances associated with climate change favor the continental and intercontinental movement of insects, microbes and pathogens. New introductions of unwanted organisms associated with these pathways can eventually lead to complex incursions of emerging pests and diseases costly to control and/or eradicate. Moreover, during the last 7 years it has become evident that changes in world-geopolitical scenarios have also increased the risk of terrorism interventions in general and also bioterrorism against relevant agricultural commodities and the associated industries in particular. This new scenario poses risks to agriculture due to inadvertent, illegal or criminal introductions of exotic pathogens, giving rise to serious threats to the economy, environment, human health and cultural heritage. This project is expected to contribute solutions the agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for plant pathogen collection, detection, diagnosis and discrimination in plant tissue or associated material suspected of harboring microorganisms of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7122499116050%
2122499116050%
Goals / Objectives
This project is expected to contribute solutions the agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for plant pathogen collection, detection, diagnosis and discrimination in plant tissue or associated material suspected of harboring microorganisms of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. Both microbial forensics and agricultural biosecurity approaches are required to avoid and/or minimize regional, national or global movement of unwanted pathogens. Goal or objectives are: To develop and/or validate methods for plant pathogen detection, discrimination, and disease diagnosis. The new methods will be target-specific and broad detection to include variants within the target. The new methods can include diagnostic keys or diagnostics pathways if required. Methods will be developed for detection and diagnostic of specimens from active or passive surveillance activities, farm fields, borders or transitional sites, valuable imported or exported plant genetic material, including suspected bio-crime scenes. To develop and validate sampling devices and/or sampling procedures, for rapid screening. It will include the assessment of new biomaterial to streamline microbial forensic investigations, diagnostics and screening of plant material potentially carrying unwanted plant pathogens of agricultural biosecurity relevance. Attention will be given to requirements of the three described biosecurity zones: global, borders and pathways, and within Oklahoma and the U.S. The duration of this project is anticipated to be five years and the project activities including objectives time management are disclosed in Table 1. Table 1. Project core activities including objectives time management. 1.Development of artificial positive controls 2010,2011,2012,2013 2.Development of specific primers for Oomycetes 2009,2010 3.Development of multiplex RT-PCR for three wheat viruses.2010,2011,2012,2013 4.Assessment forward osmosis devises 2009,2010 5.Assessment of soluble membranes and 2009,2010,2011 6.development of a sampling devise 2011,2012,2013 7.Writing of standard operational procedures 2010,2011,2012,2013
Project Methods
This project will assess the potential for sensitive detection of viruses, oomycetes and bacteria, cost-effectiveness, and ease of use of forward osmosis (FO) hydration bag or filtration devices developed first for military, recreational, and emergency relief situations in which clean sources of drinking water are not available. Sampling irrigation water for detection of microorganisms is expected to work by immersion of the bags in tanks, ponds, rivers or irrigation channels. The sample to be analyzed will consist of the extracted membrane and the residual material captured on its outer surface during the FO of a standard volume of irrigation water. Currently available FO devices (HTI) filter up to 3 L/day over a 3 day period (overall filtration 9 L (~ 20 lbs). Sample volumes of cultured waterborne pathogens E. coli, Tomato bushy stunt virus and Pythium aphanidermatum will be optimized by assaying serially diluted preparations, and the limits of threshold sensitivity of current DNA-based molecular diagnostic methods will be determined. Testing will be repeated under stringent field conditions using irrigation water as a model in eastern, western and two central sites of the Oklahoma State. The information obtained as described above is expected to facilitate the design, standardization and validation of new water sampling devices and protocols, to be cost-effective and easy to use by growers for capturing targeted microorganisms within the threshold of sensitivity of current DNA-based molecular diagnostic methods and new high resolution melt (HRM) technology. Regarding primer design and molecular diagnostics, sequences of interest from the NCBI Genbank will be retrieved and aligned and subsequently analyzed for primer design using validated thermodynamic parameters and the Web-interface pathway Primer3-mFOLD-BLASTn as reported. The information obtained during the development of multiplex RT-PCR detection method will facilitate stepping toward the development of a multi-pathogen, diagnostic-detection platform by using solid phase, heat stable polymer surfaces to which primers or alternatively PCR products are immobilized. Procedures will be followed according to preliminary results and from experience documented at the OSU Microarray Core facility, including a variety of procedures and strategies reported on this regard. For development of artificial positive controls, which will contribute minimizing the risk of using true pathogens as positive controls, and in downstream routine or large scale diagnostic analysis. Constructions will be made by (a) ligation of a number of PCR amplifiable sequences which are targeted by optimized sets of diagnostics oligonucleotide primers. These constructions will be inserted in commercially available plasmids through standardized procedures as show in figure. This part of the research will require all constructs or synthetic primer strings to be validated by extensive PCR assays including reference positive and blank controls, cloning and sequencing verification.

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: Scientific community including students, Stakeholders (to include industry) Growers of high value crops Non-skilled operators within the field of agriculture, diagnostic networks, law enforcement, military, and forensics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four workshops about “Primer design for PCR based diagnostics” were organized and taught, providing new opportunities of training scientists, graduate students and lab personnel. How have the results been disseminated to communities of interest? Through four scientific articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A method for sensitive detection and discrimination and studying multiple infections of five major plant viruses infecting ornamental plants in nursery environments was developed and published. A method for highly sensitive end-point PCR and SYBR green qPCR detection of Phymatotrichopsis omnivora, causal fungus of cotton root rot was developed and published. A method for primer modification that improves rapid and sensitive in vitro and field deployable assays for detection of High plains virus variants was developed and published. A phylodynamic study about the migration of turnip mosaic potyvirus from Europe to Australia and New Zealand was completed and published.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Ryosuke Yasaka, Kiho Ohba, Mark W. Schwinghamer, John Fletcher , Francisco M. Ochoa Corona, John E. Thomas, Simon Y. W. Ho, Adrian J. Gibbs and Kazusato Ohshima. 2014. Phylodynamic evidence of the migration of turnip mosaic potyvirus from Europe to Australia and New Zealand. Journal of General Virology. Accepted. 10/26/2014.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Shefali Dobhal, Mohammad Arif, Jen Olsen, Abby Mendoza-Yerbafr�a, Stefanny Aguilar-Moreno, Marcos Perez-Garcia and Francisco M. Ochoa-Corona. Sensitive detection and discrimination method for studying multiple infections of five major plant viruses infecting ornamental plants in nursery environments. Annals of Applied Biology. Accepted: October 16, 2014.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Arif, M., Dobhal, S., Garrido, P. A., Orquera, G. K., Esp�ndola, A. S., Young, C. A., Ochoa-Corona, F. M., Marek, S. M., and Garz�n, C. D. 2014. Highly sensitive end-point PCR and SYBR green qPCR detection of Phymatotrichopsis omnivora, causal fungus of cotton root rot. Plant Dis. 98:1205-1212.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Arif, M., G. S. Aguilar-Moreno, A. Wayadande, J. Fletcher and F. M. Ochoa-Corona. 2014. Primer modification improves rapid and sensitive in vitro and field deployable assays for detection of High plains virus variants. Applied and Environmental Microbiology. Vol. 80 (1):32-327. doi:10.1128/AEM.02340-13.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Jacqueline Fletcher, Francisco M. Ochoa Corona, Mark Payton. Plant Disease Diagnostics for Forensic Applications, Chapter 7. In: Detection and Diagnostics of Plant Pathogens. Series: Plant Pathology in the 21st Century, Vol. 5. Gullino, Maria Lodovica, Bonants, Peter J. M. (Eds.). Springer. 2014, Pages 103-115.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Dobhal , M. Arif , J. Olson , A. Mendoza-Yerbafr�a , S. Aguilar-Moreno , M. Perez-Garcia , F. M. Ochoa-Corona. 2014. Screening for plant viruses including TMV, HVX, CMV, TSWV and INSV in ornamental and nursery crops from four states of the United States. Phytopathology 104(Suppl. 3):S3.34
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Dobhal , J. Olson , M. Arif , J. A. Garcia Suarez , F. M. Ochoa-Corona. 2014. Development of endpoint RT-PCR for Rose Rosette virus and screening of Rosa spp. from eight states of the United States. Phytopathology 104(Suppl. 3):S3.34
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Dobhal , M. Arif , J. Olson , F. M. Ochoa-Corona. 2014 Comparison of end point RT-PCR and RT-HDA using primer pairs for five viruses frequently infecting ornamental and nursery crops. Phytopathology 104(Suppl. 3):S3.34
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Donna Caasi, Mohammad Arif, Mark Payton, Ulrich Melcher, Louise Winder, Francisco Ochoa-Corona. 2014. A multi-target, non-infectious and clonable artificial positive control for PCR-based assays. International Union of Microbiological Societies Congresses (IUMS) Abstracts 2014.Montreal, Canada. VIR-PM2057. Pag 215.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Donna Caasi, Mohammad Arif, Denise Altenbach, Francisco Ochoa Corona. Rapid sampling of microorganisms and nucleic acids for PCR assays using a novel elution independent collection device. International Union of Microbiological Societies Congresses (IUMS) Abstracts 2014.Montreal, Canada. VIR-PM2075. Pag.224.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Robin MacDiarmid, Brendan Rodoni, Francisco Ochoa-Corona, Marilyn Roossinck, Ulrich Melcher, Fiona Constable. Biosecurity implications of new technology and discovery in plant virus research International Union of Microbiological Societies Congresses (IUMS) Abstracts 2014.Montreal, Canada. VIR-WK201.01. Pag 354.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Results from research activities were communicated to scientists, students and stakeholders related to agricultural biosecurity and microbial forensics in OK, the U.S. and other countries including Australia, India, New Zealand, Italy, Turkey, Colombia, Chile and Mexico. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I organized and taught one workshops on primer design for detection/diagnostics PCR based experiments. The workshop was sponsored by AGDIA Inc. and held in Elkhart, Indiana and addressed scientists of Agdia (November, 2013). I taught one workshops Bioterrorism: Primer design for pathogen diagnostics. The workshop was sponsored by USDA’s NIFA-HSI grant at the University of Puerto Rico (UPR-Mayaguez) (June, 2013). How have the results been disseminated to communities of interest? Through scientific publications At the international symposium ‘ Potato Harmful Organisms’ held in Ankara, Turkey from the 04-07 of November 2013. I addressed the audience with a talk over ‘new detection/diagnostic strategies to monitor health status of crops and/or spreading of regulated diseases’. At the primer design workshop sponsored by AGDIA, Inc. held in Elkhart, Indiana, from the 9-21 of November, 2013. During the attendance to one national scientific meetings, such as American Phytopathological Society (APS). Through my OSU web page. Addressing growers on Oklahoma during the Access Tour the 10 and 11 of May, 2013. What do you plan to do during the next reporting period to accomplish the goals? I will continue with my Hatch project (OKL02773) within NIMFFAB and the Division of Agricultural Sciences and Natural Resources. I will continue research related to the simultaneous detection of three genera of waterborne plant viruses and Ralstonia solanacearum R3B2 in irrigation water. USDA-APHIS FY12 Farm Bill section 10201. I will continue mentoring graduate students and postdoctoral associates as well as visiting students and scientists. I will be focusing on securing extramural funding for novel research in plant pathogen/virology/ and insects that are important to US National Security and development of new inventions generated in the lab. I will continue cooperative research activities with other scientists within the department, Oklahoma State University, and other institutions not in Oklahoma. I will continue writing scientific articles with data generated since 2009. Several manuscripts are currently under preparation. The data of these articles was entirely generated at OSU, Department of Entomology and Plant Pathology and NIMFFAB.

Impacts
What was accomplished under these goals? During 2013, my Hatch research program (OKL02773) contributed addressing solutions to agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of four methods for collection, detection, diagnosis and discrimination of plant pathogens of relevance for agricultural biosecurity in Oklahoma, the southern plains, and the United States. Solutions contributed by this project during 2013 are: 1) An enhanced, reliable and accurate deployable bioforensic field method for detection and discrimination of Xylella fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis using Razor Ex technology and TaqMan quantitative PCR (PLoS ONE, accepted 10/16/2013). 2) A multi-target, non-infectious and clonable arti?cial positive control for routine PCR-based assays (Journal of Microbiological Methods 95: 229-234). 3) A theoretical approach for handling of next generation sequencing data for diagnostics named E-probe Diagnostic Nucleic acid Analysis (EDNA) (Journal of Microbiological Methods 94:356-366). 4) The EDNA technique, which allows a plant sample to be simultaneously screened bioinformatically for prokaryotes of concern (Journal of Microbiological Methods 94:356-366).

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ouyang P., M. Arif, 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. 8(11): e81647.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Caasi, Donna Ria J., Arif Mohammad, Payton Mark, Melcher Ulrich, Winder Louise, Ochoa-Corona Francisco M. 2013. A multi-target, non-infectious and clonable arti?cial positive control for routine PCR-based assays, Journal of Microbiological Methods 95: 229-234.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Stobbe, A.H., J. Daniels, A.S. Espindola, R. Verma, U. Melcher, F. Ochoa-Corona, C. Garzon, J. Fletcher, William Schneider. 2013. E-probe Diagnostic Nucleic acid Analysis (EDNA): A theoretical approach for handling of next generation sequencing data for diagnostics. Journal of Microbiological Methods 94:356-366.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: MacDiarmid, R, Rodoni, B., Melcher, U., Ochoa-Corona, F., Roossinck, M. 2013. Biosecurity implications of new technology and discovery in plant virus research. PLoS Pathogen 9(8): e1003337. Digital Object Identifier, doi:10.1371/journal.ppat.1003337
  • 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 field deployable Razor Ex BioDetection System and qPCR assay for detection of Phymatotrichopsis omnivora using multiple gene targets. Appl. Environ. Microbiol. 79 (7): 2312-20.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Arif, Mohammad, Francisco M. Ochoa-Corona. 2013. Comparative assessment of 5' A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity. Molecular Biotechnology 55:1726.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Arif, M. S. Dobhal, G. K. Orquera, P. A. Garrido, A. S. Esp�ndola, C. A. Young, F. M.Ochoa-Corona, S. M. Marek and C. D. Garz�n. 2013. Highly sensitive end-point PCR and SYBR Green qPCR detection of Phymatotrichopsis omnivora, causal fungus of cotton root rot. Plant Disease. (Accepted).
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2013 Citation: Fletcher J., Ochoa Corona F.M. and Payton M. 2013. Chapter: Plant Disease Diagnostics For Forensic Applications. In: Detection and Diagnostics of Plant Pathogens. Springer. Submitted 09-30-13.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Andreason, S., M. Arif, J. K. Brown, F. M. Ochoa-Corona, J. Fletcher, A. Wayadande. 2013. Multiplex PCR identification of high consequence Bemisia tabaci biotypes and Trialeurodes vaporariorum. Phytopathology 103 (Suppl. 2):S 2.7
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Arif, M., M. Perez Garcia, S. Dobhal, F. M. Ochoa-Corona. 2013. A simple and rapid method to generate full sequence reads from small qPCR amplicons using direct sequencing. Phytopathology 103 (Suppl. 2):S2.8
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Arif, M., G. S. Aguilar Moreno, A. Wayadande, J. Fletcher, F. M. Ochoa-Corona. 2013. SYBR green and Taqman qRT-PCR, helicase dependent amplification, end-point RT-PCR and Razor Ex BioDetection System for etection of High plains virus. Phytopathology 103(Suppl. 2):S2.9
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Arif, M., A. Mendoza Yerbafria, G. S. Aguilar Moreno, M. Perez Garcia, J. Olson, D. Smith, F. M. Ochoa-Corona. 2013. Development of end-point multiplex RT-PCR and helicase dependent amplification for detection of CMV, HVX, INSV, TMV, and TSWV. Phytopathology 103(Suppl. 2):S2.9.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Schneider, W., R. Verma, A. Stobbe, J. Daniels, A.Espindola, T. Blagden, J. Fletcher, F. M. Ochoa-Corona, C. Garzon, U. Melcher. 2013. Bioinformatics strategies for microbial forensics. Phytopathology 103 (Suppl. 2):S2.176.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Research dissemination Results from research activities were communicated to scientists and stakeholders in agricultural biosecurity and microbial forensics through 3 refereed publications and thirteen scientific presentations delivered in two professional meetings, and three invited lectures during 2012. All refereed articles were published in 2012. One of the two attended meetings was an international invitation as a Keynote speaker. One of the three invited lectures was an international invitation. The two attended meetings were: American Phytopathological Society. August 4-8, 2012. Providence, Rhode Island. 2do Simposio Internacional Agroindustrial (2nd International Agro-Industrial Symposium) Universidad Autonoma de Chapingo. April, 19-21, 2012. Texcoco, Mexico. Inventions No inventions, disclosed or submitted to the OSU Office of Intellectual Property Management in 2012. Status of previously reported inventions OSU Ref No. 2010.26 disclosed and submitted on February 23, 2010, got granted a patent pending status and protection until February 2012. The patent application was posted in the web and the patenting process continues and is in progress. A first draft of a license agreement between OSU and BIOREBA (Switzerland) is in final stage. OSU Ref No. 2011.23 (Application of soluble plastic and/or starch films for collection, nucleic acid recovery and release of polymerization chain reaction (PCR) reagents in diagnostic and forensic applications) disclosed and submitted to the OSU Office of Intellectual Property Management (IPM) on February 8, 2011, was approved by the IPM Screening Committee on March 29, 2011. OSU has a proprietary interest in the invention and the IPM office is conducting an assessment of patentability and market potential. No filing for patenting has been done. Teaching One undergraduate course, PLP 2143 or ENTO 2143 Global issues in agricultural biosecurity and forensics. This course is offered yearly during spring semesters. The course has a 3 credit value and is open to all majors. The aim of this new course is to introduce undergraduate students to the field of biosecurity and microbial forensics, associated sciences and the fundamental components of an effective agricultural biosecurity system. PARTICIPANTS: Current participants This project have cooperative research with: a) Dr. Louise Winder from the Biosecurity Group, Biocontrol, Biosecurity and Bioprocessing Section of AgResearch Limited, Lincoln, New Zealand on the the development of artificial positive controls and assessment of of DNA melting analysis as a tool for species identification and discrimination. b) Drs. Anna Whitfield and Dallas Seifers from the Department of Plant Pathology, Manhattan, Kansas State University on the development of a sensitive detection and discrimination method for WSMV, TriMV and HPV using multiplex RT-PCR. c) Dr. George Opit, Assistant professor at the department of entomology and plant pathology Oklahoma State University on the development of a sensitive detection and discrimination method for stored-product pests of the genus Liposcelis (Psocoptera: Liposcelididae) and Lepinotus (Psocoptera: Trogiidae). d) Master Science graduate student Jon Daniels. e) Dr. Rosa La Rosa, Istituto di Patologia Vegetale Universita degli studi di Catania, Italy. f) Dr. Richard Lee, Supervisory Research Plant Pathologist. USDA-ARS National Clonal Germplasm Repository for Citrus & Dates. Riverside, California. Former participants This project had cooperative research with: a) Postdoctoral associates Postdoctoral associate Dr. Mohammad Arif, Dr. loannis Oikonomakos and Dr. Ouyan Ping who were recruited to form part of this project and are no longer active in this project. TARGET AUDIENCES: Scientists and stakeholders associated to agricultural biosecurity and microbial forensics in OK, the US and other countries including Australia, New Zealand, Italy, Colombia and Mexico. Students PLP 2143 or ENTO 2143 Global issues in agricultural biosecurity and forensics and students in three universities in Mexico, Colombia and Turkey. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project contributed addressing solutions to agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for collection, detection, diagnosis and discrimination of plant pathogens of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. Solutions contributed by this project are: (1) Development of a PCR and isothermal-based molecular identification of the stored-product psocid pest Lepinotus reticulatus (Psocoptera: Trogiidae). (2) A comparative assessment of 5 feet A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity. (3) A survey on the methods of primer design among plant pathologists. (4) The project is making progress developing molecular detection and discriminatory methods for High plain virus with loop-mediated isothermal amplification; detection of Bean pod mottle virus using RT-PCR, RT-qPCR and isothermal amplification; discrimination among Cherry leafroll virus, Grapevine fanleaf virus and Tomato ringspot virus using multiplex RT-PCR; and detection ; discrimination of Maize dwarf mosaic virus, Sugarcane mosaic virus and Sorghum mosaic viruses. This project also interacted with other project and is making progress collaborating in the development of a (5) Massively Parallel Sequencing (MPS) detection system. The MPS technique, allows a plant sample to be simultaneously (a) screened bioinformatically for organisms of concern, (b) strain-typed for forensic purposes, and (c) searched for signs of genetic engineering. We have been making progress developing a bioinformatic pipeline, created sample sequence datasets to test the pipeline and used the database for pathogen typing to support forensic microbiological attribution. This collaboration is contributing with a strong educational component that addresses a critical emerging national need for scientists trained and experienced in both traditional and modern areas of plant pathology, also knowledgeable and appreciative of new National initiatives in agricultural biosecurity and forensic capabilities. Teaching outcomes The research activities of this project assisted to consolidate the course PLP 2143 or ENTO 2143 Global issues in agricultural biosecurity and forensics. This course was offered for a third time in 2012. The aim of this new course is to introduce undergraduate students to the field of biosecurity and microbial forensics associated science and the fundamental components of an effective agricultural biosecurity system. This course received only good evaluations from students during the last three years. The number of undergraduate students that received biosecurity and microbial forensics education is as follow: three in 2010, 11 in 2011, thirteen in 2012 for a total of 27 students in the last three years. Teaching activities had expanded in 2012 to other countries by teaching on line and mentoring students in Colombia and Mexico. The project contributed one doctoral graduate student, Donna Caasi, who graduated in the summer of 2012.

Publications

  • Refereed articles 2011 Three refereed articles: Mohammad Arif, Francisco M. Ochoa-Corona. 2012. Comparative assessment of 5 feet A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity. Molecular Biotechnology: Published online first .DOI 10.1007/s12033-012-9617-5
  • Mohammad Arif, Francisco M. Ochoa-Corona, George P. Opit, Zhi-Hong Li, Zuzana Kucerova, Vaclav Stejskal, Qian-Qian Yang. 2012. PCR and isothermal-based molecular identification of the stored-product psocid pest Lepinotus reticulatus (Psocoptera: Trogiidae). J. Stored Prod. Res. 49: 184 to 188.
  • Francisco M. Ochoa-Corona, Brendan Rodoni and Joe Tang. 2012. A survey on the methods of primer design among plant pathologists in Australia and New Zealand. Journal of Life Sciences 6 454-459.
  • Abstracts and presentation in professional meetings Nine abstracts and scientific presentations: Arif, M., Fletcher, J. and Ochoa-Corona, F.M. (2012) Application of primer and probe modifications in detection, biosecurity and microbial forensics. Phytopathology, 102 (7), S4.6
  • Arif, M., Daniels, J., Chalam, C., Fletcher, J. and Ochoa-Corona, F.M. (2012) Detection of High plain virus with loop-mediated isothermal amplification. Phytopathology, 102 (7), S4.6
  • Chalam, C., Arif, M., Fletcher, J. and Ochoa-Corona, F.M. (2012) Detection of Bean pod mottle virus using RT-PCR, RT-qPCR and isothermal amplification. Phytopathology, 102 (7), S4.20
  • Chalam, C., Arif, M., Cassi, D. R., Fletcher, J. and Ochoa-Corona, F.M. (2012) Discrimination among Cherry leafroll virus, Grapevine fanleaf virus and Tomato ringspot virus using multiplex RT-PCR. Phytopathology, 102 (7), S4.20
  • Ozakman, Y., Arif, M., Cassi, D. R. and Ochoa-Corona, F.M. (2012) RT-PCR assays for the detection and discrimination of Maize dwarf mosaic virus, Sugarcane mosaic virus and Sorghum mosaic viruses. Phytopathology, 102 (7), S4.89
  • Andreason, S., Brown, J., Fletcher, J., Ochoa-Corona, F.M., Arif, M. and Wayadande, A. (2012) Development of mtCOI primers for the rapid identification of Bemisia tabaci biotypes and Trialeurodes vaporariorum. Phytopathology, 102 (7), S4.5
  • Schneider, W. L., A. Stobbe, J. Daniels, A. Espindola, R. Verma, T. Blagden, J. Fletcher, F. Ochoa-Corona, C. Garzon, P. Hoyt, and U. Melcher. 2012. Finding the diagnostic needle in a deep sequence data haystack: E-probe Diagnostic Nucleic acids Analysis (EDNA). APS Annual Meeting, Providence, RI.
  • 2nd International Agro-Industrial Symposium. Universidad Autonoma de Chapingo. (2012). Mexico. Title: Invencion vs. Innovacion. Instrumentos de bioseguridad agricola y microbiologia forense para minimizar los efectos de la globalizacion y comercio internacional. (Invention vs innovation. Agricultural biosecurity and mirocrobial forensics instruments to minimize the effects of globalization and international trade).
  • Daniels, J., A. Stobbe, A. Espindola, W. Schneider, J. Fletcher and F. Ochoa Corona. 2012. Next generation sequencing as a diagnostic tool for biosecurity agencies. ASM Biodefense Conference, Washington, D.C.
  • Jon Daniels, Bill Schneider, Jacqueline Fletcher and Francisco Ochoa Corona. A need for simple and user-friendly detection methods for waterborne microbes. (2012). Water Research Symposium. Tulsa, Oklahoma. Invited lectures and Simposia. Four invited lectures: Emerging infectious agents II. MICR 5153
  • Visiting students from two Mexican Universities (Chapingo and Chihuahua) visitors (2012). Entitled: Ejemplos de Innovacion & Creatividad (Examples of innovation and creativity) (Summer semester)
  • The Autonomous Popular University of Puebla, Tehuacan Campus (2012). Language week 2012. UPAEP lecture Entitled: The way I visualize and teach biosecurity. Fall semester.
  • The Autonomous Popular University of Puebla, Tehuacan Campus. Language week 2012. UPAEP lecture 11-12-12 Entitled: The way I visualize and teach biosecurity. Offered on line.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Research dissemination Results from research activities were communicated to scientists and stakeholders in agricultural biosecurity and microbial forensics through 4 refereed publications and eleven scientific presentations delivered through six professional meetings during 2011. Two of the four refereed articles were published in 2011 and the other two are articles not reported in 2010. One of the six attended meetings was an international invitation as a Keynote speaker. The six meetings attended were: 1.2011 Chemical & Biological Terrorism Defense Gordon Research Conference held in conjunction with the Chemical & Biological Terrorism Defense Gordon-Kenan Research Seminar. March 20-25, 2011. Ventura, California. 2.18th Biennial Australasian Plant Pathology Conference concurrent with the 4th Asian Conference on Plant Pathology. 26-29 April 2011. Darwin, NT, Australia. 3.American Phytopathological Society. August 6-10, 2011. Honolulu, Hawaii. 4.3rd National meeting of the National Plant Diagnostic Network (NPDN). November 6-9, 2011. Berkeley, California. 5.2011 Chemical and Biological Defense Science and Technology (CBD S&T) Conference. November 14-18, 2011. Las Vegas, Nevada. 6.59th Entomological Society of America (ESA) meeting. November 13-16, 2011. Reno, Nevada. Inventions 1.One invention, disclosed and submitted to the OSU Office of Intellectual Property Management (IPM) (OSU Ref No. 2011.23) on February 8, 2011. The invention is entitled: Application of soluble plastic and/or starch films for collection, nucleic acid recovery and release of polymerization chain reaction (PCR) reagents in diagnostic and forensic applications by Ochoa Corona F, Daniels J, Caasi D, Andreason S, Moncrief I, and Arif M. This disclosure was approved by the IPM Screening Committee on March 29, 2011. OSU has a proprietary interest in the invention and the IPM office is conducting an assessment of patentability and market potential. Status of previously reported inventions 1.One invention, disclosed and submitted to the OSU Office of Intellectual Property Management (IPM) (OSU Ref No. 2010.26) on February 23, 2010, got granted a patent pending status and protection until February 2012. The patenting process continues and is in progress. Teaching Research activities of this project assisted consolidating a new course ENT/PLP 2143 Global issues in agricultural biosecurity and forensics. PARTICIPANTS: Current participants This project have cooperative research with: a) Dr. Louise Winder from the Biosecurity Group, Biocontrol, Biosecurity and Bioprocessing Section of AgResearch Limited, Lincoln, New Zealand on the the development of artificial positive controls and assessment of of DNA melting analysis as a tool for species identification and discrimination. b) Drs. Anna Whitfield and Dallas Seifers from the Department of Plant Pathology, Manhattan, Kansas State University on the development of a sensitive detection and discrimination method for WSMV, TriMV and HPV using multiplex RT-PCR. c) Dr. George Opit, Assistant professor at the department of entomology and plant pathology Oklahoma State University on the development of a sensitive detection and discrimination method for stored-product pests of the genus Liposcelis (Psocoptera: Liposcelididae) and Lepinotus (Psocoptera: Trogiidae). d) Doctoral graduate student, Donna Caasi, Master Science graduate student Jon Daniels, and Postdoctoral associate Dr. Mohammad Arif were recruited to form part of this project and are still active. Former participants This project had cooperative research with: a) Postdoctoral associates Dr. loannis Oikonomakos and Dr. Ouyan Ping who were recruited to form part of this project and are no longer active in this project. TARGET AUDIENCES: Scientists, stakeholders, and students associated to agricultural biosecurity and microbial forensics. Teaching Research activities of this project assisted consolidating a new course ENT/PLP 2143 Global issues in agricultural biosecurity and forensics. This course was offered for a second time in 2011. The aim of this new course is to introduce undergraduate students to the field of biosecurity and microbial forensics associated science and the fundamental components of an effective agricultural biosecurity system. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project contributed addressing solutions to agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for collection, detection, diagnosis and discrimination of plant pathogens of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. Solutions contributed by this project are: (1) assessment of DNA melting analysis as a tool for species identification and discrimination, (2) development molecular detection and discriminatory methods for Apple stem grooving virus and Alternanthera mosaic virus, and for Broad bean wilt virus 1 and Verbena latent virus infecting Nandina domestica and Tropaeolum majus respectively. (3) Developing an elution-independent collection device (EIDC) for rapid sampling of microorganisms and nucleic acids for PCR assays (Patent pending OSU Ref No. 2010.26). EICD collects fluid specimens by contact and lateral flow. Minute pieces (1.2 mm diameter) of a built-in soluble element dissolve directly in commercial PCR mixtures without an intermediate elution step, thereby streamlining PCR based assays. (4) Disclosure of a new invention entitled: Application of soluble plastic and/or starch films for collection, nucleic acid recovery and release of polymerization chain reaction (PCR) reagents in diagnostic and forensic applications. (5) In silica validation of a Massively Parallel Sequencing (MPS) platform as a broad detection tool for bacterial plant pathogens. This technology merges bioinformatics and plant pathology for the means of addressing national security needs in the agriculture industry. MPS is rapid, flexible and can detect multiple targeted pathogens in a sample simultaneously, including pathogens that are genetically modified. Special emphasis was given communicating: (3) the prototyping of an elution-independent collection device (EIDC) for rapid sampling of microorganisms and nucleic acids for PCR assays, and (5) Massively Parallel Sequencing (MPS) because this technology has the means of addressing the national security needs in the agriculture industry.

Publications

  • Refereed articles 2011 Ochoa-Corona Francisco M. 2011. Biosecurity, microbial forensics and plant pathology:education challenges, overlapping disciplines and research needs. Australasian Plant Pathol. 40:335 338
  • Winder, Louise; Phillips, Craig; Richards, Nicky; Ochoa-Corona, Francisco; Hardwick, Scott; Vink, Cor; Goldson, Stephen. 2011. Evaluation of DNA melting analysis as a tool for species identification. Methods in Ecology and Evolution. 2 (3):229-332.
  • Abstracts and presentation in professional meetings Ochoa-Corona Francisco M. 2011. Biosecurity, microbial forensics and plant pathology:education challenges, overlapping disciplines and research needs. Australasian Plant Pathol. 40:335 to 338
  • S. ANDREASON, J. Brown, J. Fletcher, F. Ochoa-Corona, A. Wayadande. 2011. Development of mtCOI PCR primers with 5 feet AT-rich flaps for rapid identification of high consequence Bemisia tabaci. Phytopathology. 101:S7.
  • F. OCHOA-CORONA. 2011. Microbial forensics: Investigative plant pathology. Phytopathology 01:S217.
  • C. R. Donna, F. OCHOA-CORONA. 2011. An elution-independent collection device for rapid sampling of microorganisms and nucleic acids for PCR assays. Phytopathology 101:S44.
  • M. ARIF, C. Garzon, F. Ochoa Corona. 2011. 2011 Meeting Detection and discrimination of Pythium aphanidermatum and P. deliense by single probe based real time PCR and multiplex end point PCR. Phytopathology 101:S9.
  • M. ARIF, F. Ochoa-Corona, S. Marek, J. Fletcher. 2011. Multi-gene based detection and identification of Phymatotrichopsis omnivora. Phytopathology 101:S9.
  • P. Ouyang, M. ARIF, F. Ochoa-Corona, U. Melcher, J. Fletcher. 2011. Sensitive detection and discrimination of Xyllela fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis. Phytopathology 101:S135.
  • J. DANIELS, T. Stobbe, A. Espindola, W. L. Schneider, J. Fletcher, F. O choa-Corona. 2011. In silico simulation of massively parallel sequencing as a diagnostic tool for bacterial phytopathogens. Phytopathology 101:S41.
  • M. ARIF, J. Olson, A. Whitfield, F. Ochoa-Corona. 2011. Sensitive detection and discrimination of WSMV, TriMV and HPV using multiplex RT-PCR. 2011.Phytopathology 101:S9.
  • Detection of Xylella fastidiosa in the insect vector, the glassy-winged sharpshooter, Homalodisca vitripennis. Mohammad Arif, Ping Ouyang, Francisco Ochoa-Corona, Astri Wayadande, and Jacqueline Fletcher 2011. Pag 125. D0282
  • Sensitive detection and discrimination of stored-product pests of genus Liposcelis (Psocoptera: Liposcelididae) and Lepinotus (Psocoptera: Trogiidae). Mohammad Arif, F. M. Ochoa Corona, George Opit, , Zhi-Hong Li, Zuzana Kucerova, Vaclav Stejskal and Qian Qian Yang. 2011. Pag 129. D0380


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Results from research activities were communicated to scientist within agricultural biosecurity and microbial forensics sciences through 17 publications and twelve scientific presentations that were delivered at eight professional meetings from 2008 to 2010. 8th Australasian Plant Virology Workshop, Lake Okataina, Rotorua, New Zealand, November 19-22, 2008. American Phytopahological Society, Portland, Oregon, August, 1-4, 2009 Union of Forest Research Organizations (IUFRO) conference, Rotorua, New Zealand, March 16-19, 2009 Plant Virus Noble foundation retreat. April 17-18, 2009 American Phytopahological Society, Charlotte, NC, August 7-11, 2010 FAPC Symposium, OSU Stillwater, OK, February 16, 2010 Plant Virus Noble foundation retreat. May 2-3, 2010 American Phytopahological Society North-Central division, Rapid City, SD, June 7-11, 2010 PARTICIPANTS: a)This project had cooperative research with Dr. Louise Winder from the Biosecurity Group, Biocontrol, Biosecurity and Bioprocessing Section of AgResearch Limited, Lincoln, New Zealand for the development of artificial positive controls. b)Also with Drs. Stanley Bellgard and Sarah Dodd from Landcare Research, Auckland New Zealand, on the biosecurity aspects of using viruses for the manufacture of Bioherbicides as an innovative, inundative biocontrol option to manage forest weeds. c)A doctoral graduate student (Donna Caasi), a laboratory technician (Dr. Ioannis Oikonomakos) and a Postdoctoral associate (Dr. Mohammad Arif) were recruited to form part of this project TARGET AUDIENCES: Scientists and students within agricultural biosecurity and microbial forensics sciences. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project contributed addressing solutions to agricultural biosecurity and microbial forensics needs by developing, and/or providing validation of methods for plant pathogen collection, detection, diagnosis and discrimination in plant tissue suspected of harboring microorganisms of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. One invention was disclosed and submitted to the Office of Intellectual Property Management (IPM) (OSU Ref No. 2010.26) on February 23, 2010. The invention is entitled: Rapid microbial collection and nucleic acid recovery device. This disclosure was approved by the IPM Screening Committee on March 11, 2010. OSU has a proprietary interest in the invention and the IPM office is conducting a complete assessment of patentability and market potential. The disclosure is currently at the office of Fellers, Snider, Blankenship, Bailey & Tippens, P.C. Attorneys and Counsellors at Law for patent writing and filing. The project also contributed establishing a new course ENT/PLP 2143 Global issues in agricultural biosecurity and forensics. The aim of this new course is to introduce undergraduate students to the field of microbial forensics and fundamental components of an effective agricultural biosecurity system.

Publications

  • Arif, M., Dobhal, S., Flores, F.J., Ochoa-Corona, F.M, and Garzon C.D. Endpoint and real-time PCR protocols for detection and discrimination of Pythium aphanidermatum and P. deliense. (2010). Molecular ecology resource. Status: Submitted, under review.
  • Arif, M., Oikonomakos, I, Caasi, D. and Ochoa-Corona, F. (2010) Increased PCR amplification incorporating primer flap sequences and free energy values near equilibrium. Phytopathology, 100 (6), S7
  • Caasi, D., Arif, M. and Ochoa-Corona, F. (2010) Assessment of new biomaterials for sample collection and nucleic acid recovery. Phytopathology 100 (6), S19
  • Arif, M., Marek, S., Ochoa-Corona, F. Young, C. and Garzon, C.D. (2010) PCR detection and identification of Phymatotrichopsis mnivore. Phytopathology 100 (6), S7.
  • D. R. Elliott, B. S. M. Lebas, F. M. Ochoa‐Corona, J. Tang, B. J. R. Alexander. 2009. Investigation of Impatiens necrotic spot virus outbreaks in New Zealand. Australasian Plant Pathology. 38 (5):490 to 495.
  • B. S. M. Lebas, F.M. Ochoa‐Corona, D.R. Elliott, J. Tang, A.G. Blouin, O. E. Timudo, S. Ganev and B. J. R. Alexander. 2009. Investigation of an outbreak of Soil‐borne wheat mosaic virus in New Zealand. Australasian Plant Pathology 38, 85 to 9
  • Lebas B.S.M., F.M. Ochoa-Corona, B.J.R. Alexander, R.A. Lister, J.D.F. Fletcher, S.L. Bithell, and G.M. Burnip. 2009. First report of Wheat streak mosaic virus on wheat in New Zealand. Plant Disease 93(4), 430;
  • Ochoa Corona F.M. and Ward Lisa. Citrus (Citrus), Fortunella (Kumquat) & Poncirus (Trifoliate orange) Post-Entry Quarantine Testing Manual. Biosecurity New Zealand. July 2009. 58 pp http://www.biosecurity.govt.nz/regs/imports/plants/high-value-crops
  • F. M. Ochoa Corona, E. C. Locali-Fabris, J. Freitas-Astua. 2009. An RT-PCR procedure for detection and surveillance of Citrus mnivore virus C (CiLV-C) in post entry quarantine stocks of citrus. Phytopathology 99:S96
  • Stanley Bellgard, Sarah Dodd, and Francisco Ochoa Corona. Bioherbicides innovative, inundative biocontrol options to manage forest weeds. International Union of Forest Research Organizations (IUFRO) Forest Biosecurity Conference. Pag. 122. Rotorua, New Zealand, 16 to 19 of March, 2009.
  • Arif, M., Flores, F., Ochoa-Corona, F. and Garzon, C.D. (2010) Highly sensitive molecular detection of five Pythium species. Phytopathology 100 (6), S7.
  • Dobhal, S., Blazheva, D., Arif, M., Garrido, P., Ochoa-Corona, F. M., Opit, G. and Garzon, C. (2010) PCR detection of aflatoxin producing strains of Aspergillus spp. From corn and red flour beetle. Phytopathology 100 (6), S30.
  • Jacqueline Fletcher, Neel G. Barnaby, James P. Burans, Ulrich Melcher, Forrest W. Nutter Jr., Carla Thomas, and Francisco M. Ochoa Corona. Chapter 7. Forensic Plant Pathology. In: Microbial Forensics, 2nd Edition. B. Budowle, S.E. Schutzer, R.G. Breeze, P.S. Keim and S.A. Morse Editors. Academic Press-Elsevier. 2010. Pp. 89 to 105.
  • Committee on Scientific Milestones for the Development of a Gene-Sequence-Based Classification System for the Oversight of Select Agents: James W. LeDuc, Ralph Baric, Roger G. Breeze, R. Mark Buller, Sean Eddy, StanleyFalkow, Rachel E. Levinson, John Mulligan, Alison D. OBrien, Francisco Ochoa-Corona, Jane S. Richardson, Margaret Riley, Sequence-Based Classification of Select Agents: A Brighter Line. National Research Council. ISBN: 0-309-15905-9, 234 pages, (2010). http://www.nap.edu/catalog/12970.html
  • Francisco M. Ochoa Corona, Brendan Rodoni, Joe Z. Tang. Primer design. How we learn and do it in Australia and New Zealand. 8th Australasian Plant Virology Workshop, Lake Okataina, Rotorua, New Zealand, 19 to 22 of November, 2008. Pag. 3.
  • J. Tang, J. D. Olson, F. M. Ochoa-Corona and G. R. G. Clover. 2010. Nandina domestica, a new host of Apple stem grooving virus and Alternanthera mosaic virus. Australasian Plant Disease Notes, 2010, 5, 1 to 3.
  • F. M. Ochoa-Corona, J. Z. Tang, B. S. M. Lebas, L. Rubio, A. Gera and B. J. R. Alexander. Diagnosis of Broad bean wilt virus 1 and Verbena latent virus in Tropaeolum majus in New Zealand. 2010. Australasian Plant Pathology, Vol. 39 (2): 120 to 124.