Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
DETECTION AND DIAGNOSTIC METHODS FOR AGRICULTURAL BIOSECURITY AND FORENSIC PLANT PATHOLOGY APPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1004354
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Dec 17, 2014
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Entomology And Plant Pathology
Non Technical Summary
This project develops solutions for agricultural biosecurity and microbial forensics needs developing, and 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. Especial emphasis is given to the study of waterborne plant viruses and other water phytopathogens as well as arthropod transmitted viruses.Goal or objectives are: A) To develop and/or validate methods for plant pathogen detection, discrimination, and disease diagnosis. Emphasis will be given to methods for non-skilled operators and friendly to use. B) The new methods will be target-specific and/or broad detection to include variants within the target. The new methods can include diagnostic keys or diagnostics pathways if required. C)Methods will be developed for detection and diagnostic of specimens from active or passive surveillance activities, farm fields, borders or transitional sites, high value crops, specialty crops, imported or exported plant genetic material, including samples from suspected bio-crime scenes. D)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.
Animal Health Component
70%
Research Effort Categories
Basic
10%
Applied
70%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2120210110125%
2121549110125%
2122199110125%
2121599110125%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other; 2199 - Ornamentals and turf, general/other; 0210 - Water resources; 1599 - Grain crops, general/other;

Field Of Science
1101 - Virology;
Goals / Objectives
Objectives of this project are:To develop and/or validate target-specific and broad detection methods for plant pathogens for improved detection, discrimination, and disease diagnosis 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 diagnosis of specimens applicable to active or passive surveillance activities, farm fields, borders or transitional sites, and valuable imported or exported plant genetic material, including those from suspected bio-crime scenes.To develop and validate sampling devices and/or sampling procedures, for dependable rapid screening. This activity will include the assessment of new biomaterials that can streamline microbial forensic investigations, diagnostics and screening of plant material potentially carrying unwanted plant pathogens of agricultural biosecurity relevance.Specific objectives of objective 1:Development of specific single target and/or multiplex RT-PCR methods for waterborne plant viruses, viruses and phytopathogens of ornamentals and high value and specialty crops to include insect/nematode vectors. Priority will be given to waterborne plant viruses in the genera Tombusvirus, Tobamovirus and Potexvirus.Development of artificial positive controls for new and existing pathogen detection assays to downstream routine and/or large scale diagnostic analysis for high value crops, i.e. potato and cereals to replace and minimize the risk of using true pathogens and /or insects and nematodes as positive controls.Specific objectives of objective 2:2.1. Validating and further characterizing the use of soluble membranes that can extend and facilitate sampling, microorganism recovery, and management and storage of microbes and/or nucleic acid compared to classic methods currently used such as blotting plant sap or natural fluids preparations.2.2. Assessment of isothermal DNA amplification methods seeking the development or non-skilled detection kits for field use by growers of high value and specialty crops.
Project Methods
Objective 1. Development methods for plant pathogen detection, discrimination, and disease diagnosis.Specific objective 1.1. Development of specific primers for phytopathogens infecting high value crops and specialty crops to include insect/nematode vectors requires computational intensive analysis of sequences of interest to be retrieved from the NCBI Genbank, aligned and subsequently analyzed for primer design using validated thermodynamic parameters through the Web-interface pathway Primer3-mFOLD-BLASTn as reported [24]. This part of the research will require validation by extensive PCR assays of all amplified products obtained and cloned. These assays will include reference positive and blank controls, specificity and sensitivity testing and sequencing verification.Specific objective 1.2. Development of multiplex RT-PCR for high value crops and specialty crops to include insect/nematode vectors.Similarly 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 [20]. The information obtained during the development of the multiplex RT-PCR detection method will facilitate stepping toward the development of novel multi-pathogen, diagnostic-detection platform based on E-probe Diagnostic Nucleic acid Analysis (EDNA). The output of this part of the project will contribute fulfilling a need for a multiplex detection to downstream the diagnostics of high value and specialty crops to include insect/nematode vectors, and waterborne phytopathogens.Specific objective 1.3. Development of artificial positive controls.New constructions will be made by (a) ligation of a number of PCR primers amplifiable sequences which are arranged as optimized sets of diagnostics oligonucleotide primers. These contructions will be inserted in commercially available plasmids through standardized procedures. The output of this part of the project will contribute minimizing the risk of using true pathogens as positive controls, and in downstream routine or large scale diagnostic analysis. Diseases of high value crops, such as potato, cereals, citrus, grapevine. and specialty crops like rose and ornamentals will be given special attention.Objective 2. Assessment of a new biomaterial to streamline microbial forensic investigations)New biomaterial will be assessed stressing its ability to capture and retain phytopathogens. Recovered nucleic acids or microorganisms will be assayed using PCR based experiments. The output of this part of the project will contribute to a better understanding of the nucleic acid binding properties, carry-on residues, buffer compatibility and solubility of new membranes or biomaterials when used for sampling and recovery of plant pathogen nucleic acids. This information will be used for designing new and more practical sampling devices, i.e. devises using soluble plastic, lateral flow type [9], or easy-to-implement sampling and extraction protocols for the detection and diagnosis of microorganisms.

Progress 12/17/14 to 09/30/19

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?Three 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 and presentations in scientific meetings. 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 brief description of a few amenable technologies developed for research in biomedical and agricultural diagnostics tested using plant pathogens as microbial surrogate. Plant pathogen surrogate microbes can assist in easing compliance with regulations and reducing costs, ethical and biosafety concerns in biomedical research. Adv Biotech & Micro 13(2): AIBM.MS.ID.555859 (2019) A contribution was made to a multidisciplinary approach to control Rose rosette disease (RRD). In the short term we developed Best Management Practices and educational materials based on host, virus, and vector biology to minimize the effects of RRD. Key to this effort were the development of efficient user-friendly diagnostic tools. In the long term, roses are being assessed for resistance to RRD using both replicated field trials and observational data from collaborators. Acta Hortic. 1232. ISHS 2019. DOI 10.17660/ActaHortic.2019.1232.30 Suggestions for long-term planning for microbial stocks were proposed, as well as inducements for long-term preservation. Collections of microorganisms are a crucial element of life science research infrastructure, but are vulnerable to loss and damage caused by natural or man-made disasters, the untimely death or retirement of personnel, or the loss of research funding. Preservation of biological collections has risen in priority due to a new appreciation for discoveries linked to preserved specimens, emerging hurdles to international collecting, and decreased funding for new collecting. Journal of Applied Microbiology https://doi.org/10.1111/jam.14525.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: D.H Byrne, P.E. Klein, C. Hall, M. Windham, F.M. Ochoa-Corona, J. Olson, M. Paret, B. Babu, G. Knox, R. Jordan, J. Hammond, K. Ong, R. Ochoa, G.B. Bauchan, T. Evans, A. Windham, F. Hale, M.A. Palma, L. Ribera, H.B. Pemberton. Combating Rose rosette disease US national project. Acta Hortic. 1232. ISHS 2019. DOI 10.17660/ActaHortic.2019.1232.30 Proc. VII International Symposium on Rose Research and Cultivation Ed.: F. Foucher.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Francisco M Ochoa-Corona, Kitty F Cardwell, Andres S Espindola. New Technologies from the Microbial World: Alternatives for Biomedical Surrogate Research. Adv Biotech & Micro. 2019; 13(2): 555859. DOI: 10.19080/AIBM.2019.13.555859
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Kyria Boundy-Mills, Kevin McCluskey, Patrick Elia, Jessie A. Glaeser, Daniel L. Lindner, David R. Nobles, Jr., Jennifer Normanly, Francisco M. Ochoa-Corona, James A. Scott, Todd J. Ward, Kimberly M. Webb, Katie Webster, and John E. Wertz. Preserving U.S. Microbe Collections Sparks Future Discoveries. Journal of Applied Microbiology. Accepted manuscript. https://doi.org/10.1111/jam.14525
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Maday Galeana-Figueroa 2018. Master: Development of diagnostic platforms based on EDNA for Leukemia.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Lizbeth Pe�a-Z��iga, Andr�s Esp�ndola, Patricia Klein, Thomas Debener, David Byrne, and Francisco M. Ochoa-Corona. E-probe Diagnostic Nucleic-acid Analysis (EDNA-Rose) A High Throughput Sequencing (HTS) analysis & bioinformatics approach for complete Rose-virome detection. Intnal. Advances in Plant Virology 2019 October Rome Italy.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: F.M. Ochoa-Corona, A. Salazar-Aguirre, S. Molina-Cardenas, A. Olmedo-Velarde, Lizbeth Pe�a-Z��iga, Andr�s Esp�ndola1 S. Dobhal, J. Olson, M. Paret, B. Babu, R. Jordan3 J. Hammond, K. Ong, And D.H Byrne. Exploring the best diagnostic fit for Rose rosette virus. Intnal. Advances in Plant Virology 2019 October Rome Italy.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience: Scientific community including students, Biosecurity and Microbial forensics community Stakeholders (Ornamental plants, cereals, cucurbits, grapevine, water quality, Federal government USDA, State Government ODAFF) Growers of high value and specialty crops (Roses and ornamentals, Cucurbits, Cereals, Grapevine) 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?One "Primer design for PCR based diagnostics" workshops were organized and taught, providing new opportunities for training and recruiting scientists, graduate students and lab personnel. How have the results been disseminated to communities of interest?Through two scientific articles and 10 professional meetings. Three meetings were of national scope, three meetings were on campus, one was out of state (University of Arkansas, Plant Pathology Department) and two were international at the Secretary of Agriculture of Mexico (SAGARPA/SENASICA) and a total of 13 presentations. What do you plan to do during the next reporting period to accomplish the goals? Data generated during 2014, 2017 will continue to be analyzed. Effort will be done to publish 2 to 3 articles during 2018. Effort on securing extramural funding through NSF or USDA program to fund novel research in plant pathogen/virology/ and arthropods important to US, OK and National Security will continue. Cooperative research activities with other scientists/faculty within the EPP department, Oklahoma State University, and other institutions in Oklahoma and internationally will continue.

Impacts
What was accomplished under these goals? A review of studies conducted using RPA for detection/diagnosis of plant viruses. These detection methods have been developed with the advent of molecular techniques. Among them, Recombinase Polymerase Amplification (RPA) is becoming a very important technique for the rapid, sensitive and cost-effective detection of plant viruses. The RPA technology has the advantage to be implemented in field-based scenarios because the method requires a minimal sample preparation, and is performed at constant low temperature (37-42 °C), which circumvent the use of thermocyclers. The RPA technique is rapidly becoming a promising tool for use in rapid detection and further diagnostics in plant clinics and monitoring quarantine services. This review includes examples with either DNA genomes (Banana bunchy top virus, Bean golden yellow mosaic virus, Tomato mottle virus, Tomato yellow leaf curl virus) or RNA genomes (Little Cherry virus 2, Plum pox virus and Rose rosette virus). A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGRLAMP) An assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/μl (25μl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGRLAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Babu B, Ochoa-Corona FM, Paret ML. Recombinase polymerase amplification applied to plant virus detection and potential implications. Analytical Biochestry, 546 (2018) 7277.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Noden BH, Martin J, Carrillo Y, Talley JL, Ochoa-Corona FM. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia. PLoS ONE (2018) 13(2): e0192331.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Scientific community including students, Stakeholders (Ornamental plants, cereals, cucurbits, Federal government USDA, State Government ODAFF) Growers of high value and specialty crops (Roses, Cucurbits, Cereals) 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?One "Primer design for PCR based diagnostics" workshops was organized and taught, providing new opportunities of training and recruiting scientists, graduate students and lab personnel. How have the results been disseminated to communities of interest?Through three scientific articles and ten presentations given in four scientific conferences. 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 single-target and multiplex endpoint PCR assays with the eight primer sets were developed to target genomic DNA extracted from individual adult whiteflies. Specific and discriminatory PCR assays are for B. tabaci MEAM1, MED, and NW, and T. vaporariorum. Sequencing and phylogenetic analysis confirmed primer-target amplification specificity. Using these primer sets in single-target or multiplex PCR allows a quick discrimination and specific identification discrimination of B. tabaci complex from members and T. vaporariorum. A novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay was developed using primer/probe designed based on the nucleocapsid gene of the Rose rosette virus (RRV). The assay is highly specific and does not cross react with other viruses infecting roses. The primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. Moreover, a rapid technique for the extraction of viral RNA (< 5 min) has been standardized for direct trapping of RRV into PCR tubes. RT-exoRPA analysis of the infected rose plants using primer and probe developed in this project allows detection from leaves, stems, petals, pollen, primary roots and secondary roots. The entire process, including the extraction can be completed in 25 min, with less sophisticated equipment. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Different methods of tissue homogenization (liquid nitrogen and lyophilization) combined with RNA extraction (Trizol, CTAB and RNeasy mini kit) were assessed with a native ascomycete, Xylaria sp. Higher RNA purity was obtained using CTAB and the higher yields using the RNeasy mini kit. Extracted RNA is amplifiable by RT-PCR regardless of the homogenization and extraction methodology used. However, it is recommended to homogenize the tissue with liquid nitrogen and to extract RNA with the RNeasy mini kit due the shortness and efficiency of these protocols.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Babu B, Washburn BK, Ertek TS, Miller SH, Riddle CB, Knox GW, Ochoa-Corona FM, Olson J, Kat?rc?o?lu YZ, Paret ML. A field based detection method for Rose rosette virus using isothermal probebased Reverse transcription-recombinase polymerase amplification assay. Journal of Virological Methods 247 (2017) 8190
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Andreason SA, Arif M, Brown JK, Ochoa-Corona FM, Fletcher J, Wayadande A. Single-Target and Multiplex Discrimination of Whiteflies (Hemiptera: Aleyrodidae) Bemisia tabaci and Trialeurodes vaporariorum With Modified Priming Oligonucleotide Thermodynamics. Journal of Economic Entomology, 2017, 110
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Sandoval-Pineda JF, Ochoa-Corona FM, Torres-Rojas E. Evaluation of different RNA extraction methods from the native fungus Xylaria sp. Rev. Colomb. Biotecnol. Vol. XIX No. 1 2017, 42-54
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Larrea-Sarmiento, A.E., Arif, M., Olmedo-Velarde, A., Jen Olson , Ochoa-Corona, F.M. 2016. Sensitive detection and discrimination of HPWMoV, WSMV and TriMV using multiplex RT-PCR (PART II): Discrimination and detection of populations by high resolution melting. CIBB III: 2016 International Congress of Biotechnology and Biodiversity. CIBB-FP-EO-012. Pag. 39.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Garc�a-Suarez, J. A., Dobhal, S., Ochoa-Corona. F.M. 2016.Virus detection of Tobamovirus with wide spectrum degenerate oligonucleotides by TD-RT-PCR high resolution melting. CIBB III: 2016 International Congress of Biotechnology and Biodiversity. CIBB-FP-EO-013. Pag. 40
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Olmedo-Velarde, A. Larrea-Sarmiento, A.E., Flores, F.J., Elbeaino, T., Ochoa-Corona, F.M. 2016. Toward a broad detection of Emaravirus species: qRT-PCR-HRM and endpoint RT-PCR. CIBB III: 2016 International Congress of Biotechnology and Biodiversity. CIBB-FP-EO-014. Pag.41
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ochoa-Corona, F.M., Daniels, J. Gallucci-Mazziero B., Carrillo-Tarazona Y., Cardozo-Burgos C., Ochoa F.M. 2016.Toward broad detection of plant waterborne viruses. CIBB III: 2016 International Congress of Biotechnology and Biodiversity. CIBB-FP-EO-015. Pag. 42
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Jon Daniels. Ph.D. thesis title: Capturing Waterborne Plant Pathogenic Viruses: A Novel Tool for Agricultural Diagnostics using Three Model Viruses.
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Beatriz Gallucci Mazziero. M.Sc. thesis title: Assessment of a virus sampling and detection method for water testing using Pepino mosaic virus.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Salazar, A., Molina, S., Ochoa-Corona, F.M., Olson, J. 2016. Detecci�n de Rose rosette virus mediante el m�todo de amplificaci�n isot�rmica de �cidos nucleicos LAMP (LOOP-MEDIATED ISOTHERMAL AMPLIFICATION). CIBB III: 2016 International Congress of Biotechnology and Biodiversity. CIBB-FP-EO-016. Pag. 43
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Olmedo-Velarde, A., Larrea-Sarmiento, A. E., Ochoa-Corona, F. M. 2016. Discriminating Potexvirus species by qRT-PCR coupled to high resolution melting. CIBB III: 2016 International Congress of Biotechnology and Biodiversity. CIBB-FP-CA-002. Pag. 63
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: J. Fletcher, L.M. Ma, Francisco Ochoa-Corona, U. Melcher, C. Garzon, W. Schneider, A. Wayadande, & R. Allen. Forensic Plant Pathology: Enhancing U.S. Crop Biosecurity Through Multidisciplinary Graduate Education, Experience and Research. Poster 243. NACTA Conference. June 21-24, 2016. Honolulu, Hawaii
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jon Daniels, Beatriz Gallucci, Patrick Rydzak, and Francisco Ochoa Corona. Water as a vehicle for waterborne plant pathogens and the global impact. 2016 Water Research symposium October 11-12, in conjunction with the 2016 Oklahoma Governor's Water Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Beatriz Gallucci, Jon Daniels, Patrick Rydzak, and Francisco Ochoa Corona. Waterborne plant virus sampling and detection in aqueous environment. 2016 Water Research symposium October 11-12, in conjunction with the 2016 Oklahoma Governor's Water Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Francisco Ochoa-Corona. Title of seminar: Exploring detection-diagnostic methods for Rose rosette virus.Combating Rose rosette Disease (RRD). USDA-Specialty Crop Research Initiative (USDA-SCRI) project meeting. McKinney, TX. November 11-10, 2016.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Scientific community including students. Stakeholders (to include industry). Growers of high value and specialty 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?Two "Primer design for PCR based diagnostics" workshops were organized and taught, providing new opportunities of training and recruiting scientists, graduate students and lab personnel. How have the results been disseminated to communities of interest?Through two 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 simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries. This study generated a single primer-set based detection method for Rose rosette virus (RRV) and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses (HRM). The developed assay is sensitive with a detection limit of 1 fg from infected plant tissue. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. A second and highly reliable, specific and sensitive detection assay was developed to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays showed all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Babu B, Jeyaprakash A, Jones D, Schubert T S, Baker C, Washburn B K, Miller S H , Poduch K, Knox G W, Ochoa-Corona F M, Paret M L. Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets. Journal of Virological Methods. 235 (2016) 41-50.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shefali Dobhal, Jennifer D Olson; Mohammad Arif, Johnny A Garcia Suarez; Francisco M Ochoa-Corona. A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries. Journal of Virological Methods. 232 (2016) 4756.


Progress 12/17/14 to 09/30/15

Outputs
Target Audience:Results from research activities 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.were communicated to scientists, students and stakeholders through seminars, invited conferences and journal publications (refereed scientific articles). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I organized and taught two workshops on primer design for detection/diagnostics using PCR based assays. The workshop was sponsored by Ege University, Izmir, Turkey and Ankara Plant Protection Central Research Institute, Ministry of Agriculture and Rural Affairs. Ankara, Turkey. How have the results been disseminated to communities of interest?Results have been disseminated through six scientific publications, two presentation in conferences and seven invited seminars. Results have been published through my OSU web page. What do you plan to do during the next reporting period to accomplish the goals?I will continue with my Hatch project (OKL02950) 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, and Rose rosette virus. 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 anddevelopment 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 2014-2015, my Hatch research program (OKL02950) contributed 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 2014-2015 are: 1) A sensitive detection and discrimination method for studying multiple infections of five major plant viruses infecting ornamental plants in nursery environments. (Ann Appl Biol.166:286-296). 2) An array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species. (PLoS ONE 10(6): e0129810). 3) Phylodynamic evidence of the migration of turnip mosaic potyvirus from Europe to Australia and New Zealand. (J Gen Virol. 2015 96:701-713). 4) A highly sensitive end-point PCR and SYBR green qPCR detection of Phymatotrichopsis omnivore which is the causal fungus of cotton root rot. 5) A clarification about the virus-free status of New Zealand regarding High Plains virus, and that the virus requires a name revision by the ICTV (J Virol 89:7439 -7440). 6) A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries (Journal of Virological Methods, submitted).

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ochoa-Corona FM, Lebas BSM, Ward LI. 2015. New Zealand stresses that it is High Plains virus free, and the virus struggles with an identity crisis. J Virol 89:7439 7440. doi:10.1128/JVI.00676-15.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Arif M, Opit G, Mendoza-Yerbafr�a A, Dobhal S, Li Z, Ku?erov� Z, et al. (2015) Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species. PLoS ONE 10(6): e0129810. doi:10.1371/journal.pone.0129810.
  • Type: Journal Articles Status: Published 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. J Gen Virol. 2015 96:701-713. doi: 10.1099/jgv.0.000007
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Dobhal Shefali, 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. Ann Appl Biol. 166:286-296; DOI: 10.1111/aab.12182
  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Dobhal Shefali, Jennifer D Olson; Mohammad Arif, Johnny A Garcia Suarez; Francisco M Ochoa-Corona. A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries. Journal of Virological Methods. Under review.
  • 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: 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. (previously reported as A5N)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Jordan R., J. Hammond, M. L. Paret, B. Babu, F. Ochoa-Corona, J. Olson, R. Ochoa, E. Roundey, D. Byrne. Combating Rose Rosette Disease: Development of rapid, efficient, easy-to-use virus diagnostic tools and studying virus-vector interactions. APS 2015 Annual Meeting, Pasadena, CA, Aug 1-5.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: W. SCHNEIDER, A. Stone, D. Sherman, M. Malapi-Wight, J. Crouch, S. Andreason, J. Daniels, A. Wayadande, F. Ochoa-Corona. Insect E-probe Diagnostic Nucleic acid Analysis (EDNA): a novel bioinformatic tool for detection of vectors and pathogens in insect trap metagenomes. APS 2015 Annual Meeting, Pasadena, CA, Aug 1-5.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. San diego. Agricultural challenges in microbial forensics and few solutions you can use. 2015 International symposium of microbial forensics. San Diego, CA, Feb 17, 2015. Invited speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. Invention vs. Innovation. New Diagnostic methods useful for GPDN. Great Plains diagnostic Network (GPDN) Webinar. Feb 04, 2015. Invited speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. Diagnostics 101, ornamental plants and biosecurity Master Gardeners Association. Aug 5, 2015. Invited speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. Reliable diagnostics design by design. University of Florida: North Florida Research & Education Center-Quincy. Oct 01, 2015. Invited speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. Reliable diagnostics design by design. University of Tulsa. Oct 23, 2015.Invited speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. Successful primer design by design. Ege University. Izmir, Turkey. Nov 02, 2015. Invited speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ochoa-Corona, F. Successful primer design by design. Ankara Plant protection Central Research Institute, Ministry of Agriculture and Rural Affairs. Ankara,Turkey. Nov 10, 2015. Invited speaker.