Proceedings of the Public Security S&T Summer Symposium 2009

CRTI 04-0045RD

Development of Collections, Reference/DNA Databases, and Detection Systems to Counter Bioterrorism against Agriculture and Forestry

Project Lead: Agriculture and Agri-food Canada

Federal Partners: Canadian Food Inspection Agency, Natural Resources Canada – Canadian Forest Service

Objectives

This three-year project enabled the project team to assemble a biological collection of critical fungal plant pathogens of high risk to Canadian crops, forests, and the food supply, and develop a multi-gene DNA sequence database and molecular diagnostics for these fungi. A related activity involved updating an online database documenting the occurrence of all known plant pathogens in Canada.

Agriculture and Agri-food Canada (AAFC) serves as the repository for cultures and specimens through the Canadian Collection of Fungal Cultures and the Canadian National Mycological Herbarium. The Canadian Food Inspection Agency (CFIA) secured import permits and addressed biosecurity issues related to these cultures. Researchers from the AAFC and the Canadian Forestry Service (CFS) developed DNA databases, and were involved in the design and preliminary validation of molecular assays. The CFIA, the Canadian Grain Commission (CGC), and international partners such as the United States Department of Agriculture (USDA) – Animal and Plant Health Inspection Service (APHIS) undertook final validation of assays.

Relevance

Accidental introductions of exotic agricultural pathogens can cause quarantine crises and place entire ecosystems at risk. Deliberate introductions, even on a small scale, could result in loss of consumer confidence in the food system and provoke international trade embargoes, with severe economic and social consequences to Canada. To maintain open agricultural and forestry trade with partners who have already established new plant biosecurity measures, including our closest trade partners who have already implemented research programs and early detection systems, vigilance and monitoring by Canada are critical. This project resulted in improvements to the documentation of plant pathogens that occur in Canada, including close relatives of high-risk pathogens, and the development of tools for recognizing suspicious outbreaks and establishing their source of origin.

Recent Progress and Results

This final year of the project focused on the validation of previously developed assays and completion of the basic objectives for the lower priority targets. The project team was able to complete specimen collection for 21 of its 23 target pathogens and each of their five taxonomically similar relatives. In a few cases, researchers were only able to acquire DNA for the foreign target organism or one or more of the relatives. The two remaining pathogens proved impossible to obtain as a result of material transfer restrictions imposed by foreign states.

Researchers completed the sequencing of three or more gene regions for all but one of the 21 species, and were able to identify regions that differentiated the target organism from the nearest relatives in all but two of the targets. These regions have been used to design real-time (RT) polymerase chain reaction (PCR) assays; protocols were transferred to the CFIA for validation. A DNA array based on the RT-PCR probes was developed to enable parallel detection of the target pathogens.

Impact

Although the purpose for developing these assays was to detect the deliberate introduction of exotic pathogens in Canada, the immediate deployment and use of some assays for accidental introductions is helping to protect the health of oak trees and soybean, potato, and corn crops in parts of Canada. For example, although mitigation of existing outbreaks of Phytophthora ramorum, which causes sudden oak death and attacks many plant species, is underway in California and parts of Europe, an international blind trial found the project team’s P. ramorum three-gene test to be the most accurate test in avoiding both false positives and false negatives. The CFIA has processed close to 58,000 putative P. ramorum samples since April 2007. Of these, over 16,000 were tested further by the species-specific RT-PCR assay, which detected 317 positives. A second assay based on single nucleotide polymorphisms (SNPs) to detect the origin of strains, has been implemented and a multiplex assay was developed to further increase speed, throughput and reliability. As a result of their outstanding work on P. ramorum detection to prevent the establishment of this disease in Canada, four team members from the AAFC, Natural Resources Canada (NRCan), CFIA, and CRTI received a Merit Award from the Deputy Minister of NRCan in June 2008. Asian soybean rust disease was first confirmed in southern North America in 2004. Rainfall and air sample collectors were deployed at 12 sites in Ontario, Saskatchewan, and Manitoba. In 2007, the first positive Canadian molecular detection of soybean rust spores occurred in samples obtained from mid-summer to early fall at multiple sites. In October of 2007, the first infected soybean plant in Canada was found in southwestern Ontario. The project team’s American collaborators provided computer prediction models of spore trajectories and spore detection data that correlated with this event. This was the first detection in Canadian history of a plant pathogen in air and rain samples before a diseased plant was found. In 2009, spores were detected in Ontario and Manitoba but no diseased plants were found. The project team is looking into maintaining and expanding this network and using it to track other anticipated airborne threats such as the new potentially devastating strains of wheat stem rust called Uganda 99.

The project team also developed three RT-PCR assays for the fungus Synchytrium endobioticum, an obligate potato pathogen, which cannot be grown in culture and is on the US Agriculture Bioterrorism Act Select Agent List. This pathogen is known to occur in Canada, and was found again in Prince Edward Island in 2007, creating an immediate demand for the team’s assays. The project team produced clean suspensions of sporangia in the 106/ml range to spike soil samples at various concentrations. Sufficient clean DNA was obtained from a single tuber for eventual sequencing of the whole genome. The PCR assays are being implemented at CFIA and material transfer agreements have been signed with other agencies, including the USDA–APHIS.

In 2006, the biggest epidemic in Ontario history of Fusarium graminearum on corn was recorded. F. graminearum is a mycotoxin-producing species complex of high genetic diversity, requiring molecular markers of the appropriate resolution. After screening 24 genes, it was found that the mating type (MAT) gene had the best features for the design of RT-PCR assays. This assay has since been transferred to the CGC for use in validating Canadian seed exports. The CGC, in turn, funded further development by the team’s post-doctoral researcher to develop the assay. The Fusarium headblight complex is now being resolved at a resolution higher than planned.

Authors:

C. André Lévesque, Agriculture and Agri-Food Canada, andre.levesque@agr.gc.ca

Carolyn Babcock, Agriculture and Agri-Food Canada, carolyn.babcock@agr.gc.ca

John Bissett, Agriculture and Agri-Food Canada, john.bissett@agr.gc.ca

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