DETECTION AND DIAGNOSTIC METHODS FOR BIOSECURITY AND MICROBIAL FORENSICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019496
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2024
• 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 will focus on aspects of forensic plant pathology relevant to agricultural biosecurity in Oklahoma, the southern plains and the United States. It will include adaptation and development of methods for pathogen detection and discrimination with special focus on waterborne plant pathogens. Also, validation of methods for plant pathogen collection, detection, diagnosis and discrimination in plant tissue or associated material such as water, organic fluids, soil, and insect vectors of diseases suspected of harboring microorganisms of relevance for agricultural biosecurity in Oklahoma, the southern plains and the United States. This project will also consider developing detection discrimination methods for insect vectors of plant pathogens including mites and other phytopathogens such as bacteria, fungi, viroids, and phytoplasma. Specifically basic topics of this research within the outlined objectives are: Seeking to define what is a waterborne plant virus, characterizing the genomic organization of relevant genomes, molecular landmarks, conserved and divergent genomic sequences, and the implication of this variability on taxonomical relationships, morphology, host-pathogen associations and the dynamics of the plant pathogen bio-geographic distribution and global dispersal routes. The biodiversity of regulated-exotic naturalized and indigenous microorganisms and diseases of relevance to agricultural biosecurity will be also considered.

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
212	2410	1101	20%
212	4030	1101	70%
212	4040	1101	10%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4040 - Viroids, mycoplasmas, spiroplasmas, etc.; 2410 - Cross-commodity research--multiple crops; 4030 - Viruses;

Field Of Science
1101 - Virology;

Keywords

biosecurity

microbial forensics

detection

diagnostics

e-probe diagnostic nucleic acid analysis (edna)

waterborne plant viruses

plant viruses

viroids

phytopathogens

Goals / Objectives
The objectives of this project will directly address the priority, directions, and goals of the OSU Division of Agriculture Sciences and Natural Resources. This willinclude research drivers such as a) climate variability and b) land use and natural resources, which are seeking the transformation and better preparation of Oklahoma.To develop and/or validate target-specific and broad detection methods for waterborne plant viruses, and phytopathogens infecting high value and specialty crops, ornamentals to include insect/nematode vectors 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 multiplex assays for waterborne plant viruses, and phytopathogens infecting high value and specialty crops, ornamentals to include insect/nematode vectors, including waterborne plant viruses in the genera Tombusvirus, Tobamovirus and Potexvirus.Developing artificial positive controls for new and existing pathogen detection assays for routine and/or large scale diagnostic analysis for high value and specialty crops, and ornamentals to include insect/nematode vectors, 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 of methods or kits field detection of phytopathogens of high value and specialty crops to be used by growers or non-skilled operators.2.3 Development and validation of detection methods based on EDNA specific for phytopathogens infecting crops and grasslands.

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. 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.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 to 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 Electronic Diagnostics Nucleic-acid Analysis (EDNA) for staple crops.The main scope of this project is to develop an E-probe Diagnostic Nucleic acid Analysis (EDNA) to avoid the introduction, spreading and establishment of quarantined or high impact viruses or microorganisms infecting staple crops. EDNA combines High through put or Next Generation Sequencing (NGS) of nucleic acids in a given sample with bioinformatics for screening multiple viruses and/or microorganisms infecting staple crops at once and in a single plant sample. This technology is executed in an easy to use server portal (Mi-Fi), which operates in a Windows environment, and is easy to use by non bioinformaticians or non-highly skilled operators. EDNA/Mi-Fi can be transferred for virus-free propagation of virus-free plant stocks.

Progress 10/01/19 to 09/30/20

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, Chrysanthemums, and ornamentals, also 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?Two international 'Primer Design Workshops' were organized and taught in Colombia and Brazil. How have the results been disseminated to communities of interest?Through eight scientific articles, Three theses, and one book chapter. Also four international presentations and five national presentations in professional meetings. For a total of nine presentations. What do you plan to do during the next reporting period to accomplish the goals? Data generated during 2018-2020 will continue to be analyzed. The effort will be done to publish 2 to 3 articles during 2021. Efforts 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.

Impacts
What was accomplished under these goals? Most surrogate models for human medicine are vertebrates, such as rodents and primates, but even simple organisms such as yeast provide valuable insights and feedback by elucidating bioprocesses at the molecular and cellular levels. Similarly, research in food and agricultural microbiology uses surrogate organisms. We developed a few technologies for agricultural purposes that are amenable alternatives for biomedical diagnostics research using plant pathogens as microbial surrogates. They are: IV.1 An elution independent collection device (EICD), Patent US 9,423,398 B2, was conceived to streamline sample collection and microbial processing directly into detection assays such as PCR or ELISA. The compact, easy-to-use EICD collects fluid specimens by contact and lateral flow. After samples are collected onto the EICD, minute pieces (1.2 mm diameter) of a built-in soluble element are excised and dissolved directly in commercial PCR or ELISA mixtures without intermediate elution steps. IV.2 Artificial positive controls (APC), which consist of custom synthetic DNA inserts were designed de novo in tandems of forward and reverse complement primer sequences to be inserted in circularized plasmid vectors. IV.3 The Electronic-probe Diagnostic Nucleic-acid Analysis (EDNA), which includes: a) MiProbe© which houses all tools needed for building and validating E-probes, and b) MiDetect© the diagnostic side of the program rapidly identifies the genetic signatures of targeted pathogens in metagenomic datasets. IV.4 Several detection methods such as RT-PCR, the current standard, loop-mediated amplification of DNA (LAMP), helicase dependent amplification with self-quenched primers (HDA-SqP), and a genus-specific discriminatory Emaravirus RT-PCR coupled to high resolution melting analysis (RT-PCR+HRM) were studied. Methods were developed and validated. An improved real-time PCR is reliable and provides results in as little as 3 h reducing the time for analysis by 50% (Dobhal et al., 2016). Subsequent research work has emphasized developing isothermal DNA amplification technologies. Of these, the LAMP approach showed high potential as it uses isothermal amplification carried out at a constant temperature (60-65°C) which avoids the use of a thermal-cycler and the results can be measured by turbidity, change of color, or by fluorescence using intercalating dyes that allow visualization of the reaction by the naked eye.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Rydzak, P., Ochoa Corona F.M., Whitfield, A.E., Wayadande, AC. 2020. Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals. Journal of Virological Methods. 278:113823 https://doi.org/10.1016/j.jviromet.2020.113823
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Andreason S.A., Arif M., Brown J.K., Ochoa-Corona F., Wayadande A. Exploring the Use of High-Resolution Melting Analysis and Helicase-Dependent Amplification for Discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species and Trialeurodes vaporariorum. Journal of Economic Entomology, XX(XX), 2020, 110. doi: 10.1093/jee/toaa180
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Andrea Gentili, Sauro Simoni, Laura Tomassoli, Francisco M.Ochoa-Corona, Francesco Faggioli. Il virus del rosettamento � una minaccia. Colture Protette, n. 09 - ottobre 2020, 52-54.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ramos K., Sivaprasad Y., Guevara F., Ochoa-Corona F., Viera W., Flores F. Occurrence of potato yellowing virus in naranjilla (Solanum quitoense Lam.) in Ecuador. Journal of Plant Pathology (2020) 102:597 https://doi.org/10.1007/s42161-019-00479-0
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: EDNA-HOST: Detection of global plant viromes using High Throughput Sequencing. Lizbeth Daniela Pena-Zuniga 2020-04. F4N. Development of a rapid Anaplasma detection (rad) kit to identify three Anaplasma species infecting livestock. Andrea Mercedes Salazar Aguirre. 2020-06.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Vazquez-Iglesias I., Ochoa-Corona F.M., Tang J., Robinson R., Clover G.R.G., Fox A., Boonham N. Facing Rose rosette virus: A risk to European rose cultivation. Plant Pathology. 2020;00:115. DOI: 10.1111/ppa.13255