RAPID, SIMPLE AND INEXPENSIVE DETECTION OF POTYVIRUS IN SEED POTATOES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 0221854
• Grant No.: 2010-33610-20978
• Proposal No.: 2010-00472
• Project Start Date: Jul 1, 2010
• Project End Date: Aug 31, 2011
• Grant Year: 2010
• Program Code: [8.2]- Plant Production and Protection-Biology

Recipient Organization
RXOA BIOSCIENCES, LLC
1212 FOURIER DR., STE 200
MADISON,WI 53717

Performing Department
(N/A)

Non Technical Summary
Rxoa Biosciences (Rxoa) proposes to develop a fast, reliable, cost-effective assay for Potato Virus Y (PVY). The $3 billion per year US potato industry requires a reliable and healthy seed potato supply. PVY is one of the top disease management issues for the over 2 billion pounds of seed potatoes produced in the United States each year. Seed potato certification programs in the US require a post-harvest grow-out test to assess virus incidence in seed potato lots. Unfortunately, foliar symptoms are not always clear cut, especially with recently established tuber necrotic lesion strains of PVY. With no or limited foliar symptoms, there is a need for an inexpensive, rapid and dependable PVY assay for post-harvest grow-out testing. Rxoa will use established technologies combined in a unique way to develop a new PVY assay. Rxoa will design and have synthesized a proprietary reagent that reacts to PVY. Concurrently, a production system will be developed to increase the supply of the test reagent. Due to time and monetary parameters of the phase I program, there may be only limited testing with one potato variety and one PVY strain. Specifically, Rxoa will: Design and have synthesized a PVY specific reagent, design and make a reagent production system and conduct initial testing of reagent in a known PVY infected potato plant. At the end of the phase I portion of the project, we expect to have confirmation that our assay strategy shows promise. Phase II activities will concentrate on validating the assay using multiple potato varieties with various strains of PVY across multiple environments. Upon validation, we believe we will have a fast, powerful, cost-effective assay for one of the most damaging viruses affecting the seed potato industry. Rxoa will be in a position to market the assay reagent or assay kit to both state and private potato seed producers and commercial testing labs. The use of this testing strategy is not limited to PVY and potatoes and could have huge potential for virus indexing in the $45 billion/yr ornamental industry.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1310	1040	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1310 - Potato;

Field Of Science
1040 - Molecular biology;

Keywords

potato virus y

pvy

potato

plant disease diagnostics

virus index

assay

Goals / Objectives
Rxoa will design and have synthesized a PVY specific reagent. To assure sufficient supplies of reagent, we will design and make a reagent production system. Rxoa will then use the PVY specific reagent to conduct preliminary PVY detection assays using known PVY infected potato plants. At the end of the phase I portion of the project, we expect to have data confirming that our assay strategy shows promise. This will lead to phase II activities consisting of assay validation across multiple varieties of potato, strains of PVY and environments

Project Methods
Rxoa Biosciences LLC is taking existing technologies and combining them in a unique and proprietary way to develop an effective, fast and inexpensive assay for PVY infections in potatoes. Rxoa will design a PVY specific reagent paired with a colorimetric indicator. This new reagent, when in the presence of PVY, will cause a visually detectable color change. Key to the success of this project is the development of the reagent production system. The reagent production system will be designed, produced and confirmed to produce the required reagent component. The component will then be used in a limited test with PVY infected potato plants to confirm the basic assay strategy works. At the end of the phase I, we should have all assay components in place and preliminary data to support our application for phase II funding.

Progress 07/01/10 to 08/31/11

Outputs
OUTPUTS: The most significant outputs during the phase I portion of this project have been: 1) Activities of conducting experiments and analyzing results. 2) The products of the amassed data generated from the experiments, collaboration with a researcher at the University of Wisconsin and new skills developed for Rxoa Biosciences LLC personnel working on the project. Data, conclusions and potential next steps have been disseminated to the collaborator and letters of introduction sent to potential future collaborators for follow up research. PARTICIPANTS: PI: William L. Petersen, Managing Member, conducted all experiments, gathered and analyzed data. Mr. Petersen was responsible for project management, working with our informal collaborator and producing all reports. Dr. Steven C. Slater, Chief Scientific Officer, co-designed genetic constructs used in the project. Dr. Carol Foster, Senior Scientist, co-designed genetic constructs, provided molecular biology support, conducted protein immunoblot assays on plant samples and contributed to report writing. Informal collaborator Dr. Amy Charkowski of the University of Wisconsin provided advice, expert opinions and provided plant materials. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: All protocols, tasks and scientific approaches that were laid out in the project proposal, were carried out during this project. There were additional protocols and approaches tested in response to a fourteen week delay from a supply vendor. New protocols included: 1) Testing a transient expression method (Agro-infiltration). 2) The use of reagent created in bacteria instead of from plants. 3) Cloning plants for use in experiments while waiting for R1 generation plants. 4) Testing various light spectrum to improve visual detection of virus infection in plants. 5) Various methods of applying the reagent to plants, including the use of accelerated particles (Biolistics). These were in addition to the tasks proposed. The project was granted a no-cost extension from February 28, 2011 to the new end date of August 31, 2011 due to the delay from the vendor (promised in 10 weeks, received in 24 weeks).

Impacts
Outcomes include Change in Knowledge: 1) greater depth of knowledge of project subject matter. 2) Improved skills in protein analysis. Most significant was the Change In Action. To make up for delays in the project, several new approaches were found in the available literature and applied to this project in an attempt to show proof-of-concept in a timely manner. For example, the application of Agro-infiltration techniques, plant cloning and new nucleic acid precipitation techniques used for particle mediated plant genetic engineering.

Publications

• No publications reported this period