ENHANCED-THROUGHPUT QUANTIFICATION OF RHIZOCTONIA AND PYTHIUM DNA IN SOIL SAMPLES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 0221847
• Grant No.: 2010-33610-20868
• Cumulative Award Amt.: $73,598.00
• Proposal No.: 2010-00285
• Project Start Date: Jul 1, 2010
• Project End Date: Feb 29, 2012
• Grant Year: 2010
• Program Code: [8.2]- Plant Production and Protection-Biology

Recipient Organization
Western Laboratories
P. O. Box 1020
Parma,ID 83660

Performing Department
(N/A)

Non Technical Summary
Soilborne fungal pathogens Rhizoctonia and Pythium are major yield limiting factors in cereal production systems in the Pacific Northwest with an estimated $101 million loss per year due to th ese pathogens. The expected outcome is a more effective management system for these pathogens that provides risk models to the grower clientele. This will allow growers to choose disease resistant varieties according to the information through the growers risk advisory.

Animal Health Component

50%

Research Effort Categories

Basic

(N/A)

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	7299	1040	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
7299 - Research equipment and methods, general/other;

Field Of Science
1040 - Molecular biology;

Keywords

diagnostic kits

pythium quantification

rhizoctonia quantification

real time pcr

risk management

root rot

taqman

Goals / Objectives
The Phase I proposal seeks to examine the technical feasability of adapting quantitative real time PCR assays for enhanced throughput analysis of three types of Rhizoctonia and three species of Pythium that are most pathogenic to PNW cereals. The expected output will be commercialization of the real time PCR technology developed in the laboratories of Dr. Pat Okubara and Dr. Tim Paulitz at the USDA-ARS Root Disease and Biological Research Unit at Pullman, Washington.

Project Methods
A semi-automated DNA extraction platform designed for soil samples will be integrated with pressure cycling, a technology to improve the threshold of pathogen detection from soils. Exploration of TaqMan chemistry and assay multiplexing to further increase diagnostic throughput. This analytical system will provide fast information that will be used to construct growers advisory on the risk of loss from soil borne pathogens.

Progress 07/01/10 to 02/29/12

Outputs
OUTPUTS: This study met all of the objectives of the Phase I project. Parallel analysis of spiked and naturally infested soil samples at Western Laboratories and the USDA Root Disease and Biological Control Research Unit demonstrated that the high throughput system at Western Laboratories produced reliable quantification of Rhizoctonia solani AG-8, Rhizoctonia oryzae group II, Pythium ultimum, Pythium irregulare group I, and Pythium irregulare group IV. A set of proposed Taqman suites was developed. PARTICIPANTS: Harry Kreeft, Nematologist/Plant Pathologist at Western Laboratories supervised the project at Western Laboratories and coordinated cooperative work with the USDA Root Disease and Biological Control Research Unit at Pullman, WA. The primer pairs and probe suites were developed by Dr. Patricia Okubara and Dr. Tim Paulitz at the USDA. They also conducted a parallel extraction and analysis at Pullman of the spiked soil samples using the USDA primer pairs. The partner organizations involved in this project were the USDA Root Disease and Biological Control Research Unit and the Department of Plant Pathology at Washington State University. TARGET AUDIENCES: The target audiences for this project are grain growers, agricultural scientists and extension personnel involved in the management of soil-borne plant pathogens in small grains in the Pacific Northwest. These people, in turn, will interact with grain growers and agricultural professionals to integrate a growers advisory based on this analytical technology into crop management systems for small grains in the Pacific Northwest. The implementation of this technology will enable growers to realize the benefits of conservation tillage while minimizing losses from the soil-borne diseases that have limited adoption of improved tillage practices. As primer pairs become available for soil-borne diseases in other crops, this technology can be readily transferred to management of soil-borne diseases in numerous cropping systems. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project developed a high throughput system for quantification of Rhizoctonia and Pythium DNA in soil samples. This work paves the way for commercialization of PCR based analytical technology derived from research conducted by scientists at the USDA Root Disease and Biological Control Research Unit at Pullman, Washington. This technology will allow for the development of a grower's advisory for soil-borne disease of small grains based on soil analyses using these methods. The set of proposed Taqman suites will further enhance the efficiency of this analytical system. With the grower's advisory, PNW small grain growers will be able to develop management programs for the pathogens present in individual fields. This, in turn, will permit growers to realize the benefits of conservation tillage practices that are oftentimes limited by soil-borne plant pathogens. Impacts beyond the purview of this project will include the use of this technology for plant disease management in other cropping systems where soil-borne pathogens are a serious problem.

Publications

• No publications reported this period