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United States Department of Agriculture

Agricultural Research Service

Research Project: Detection and Management of Pathogens in Strawberry and Vegetable Production Systems Title: Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order

Authors
item Bilodeau, Guillaume -
item Martin, Frank
item Coffey, Mike -
item Blomquist, Cheryl -

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 28, 2014
Publication Date: July 1, 2014
Citation: Bilodeau, G., Martin, F.N., Coffey, M.D., Blomquist, C. 2014. Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order. Phytopathology. 104:733-748.

Interpretive Summary: The availability of a molecular diagnostic assay for Phytophthora that is specific, sensitive, has both genus and species specific detection capabilities multiplexed and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need a new real time PCR marker system was developed that was based on the high copy sequences of the mitochondrial DNA. To reduce the importance of highly controlled annealing temperatures for maintaining amplification specificity the marker system was developed based on gene orders that were highly conserved in Phytophthora but different when compared to plants and the related genus Pythium. Two different markers were developed, one that had both a genus and species specific capability (14 species specific markers were validated; based on sequence differences detection should be able to be done for 84 species) while the other was primarily for genus specific detection. These markers were validated with 130 samples collected from the environment and should provide an additional tool for diagnosticians and regulatory personnel to use for detecting this important genera of plant pathogens.

Technical Abstract: The availability of a molecular diagnostic assay for Phytophthora that is specific, sensitive, has both genus and species specific detection capabilities multiplexed and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need a new marker system was developed that was based on the high copy sequences of the mitochondrial DNA. To reduce the importance of highly controlled annealing temperatures for maintaining amplification specificity the marker system was developed based on gene orders that were highly conserved in Phytophthora but different when compared to plants and the related genus Pythium. A single amplification primer pair was designed from highly conserved regions that generated an amplicon of approximately 340 bp specific for the Phytophthora genus. The amplicon contained a highly conserved portion of a gene that was used for designing the Phytophthora genus specific TaqMan probe as well as variable spacer sequences that were used for designing species specific TaqMan probes. Species specific probes were developed for 13 species and the P. citricola species complex, including the quarantine pathogens P. kernoviae and P. ramorum. In silico analysis suggests species specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes will expand this list. Due to its smaller size (approximately 206 bp) and ability to detect all Phytophthora spp. a second locus was also evaluated for genus specific detection capabilities (it can be used for species specific detection but due to less sequence variation it is not as effective for systematic development of a broad range of species-specific probes). All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 24 Pythium spp. and 39 plant species. Species specific probes were further validated against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level as well as with other species having high levels of sequence similarity to ensure specificity. Markers were also validated against 130 environmental samples from a range of hosts that were tested blind to confirm accuracy of detection. Both genus specific markers were highly specific and nonspecific background detection from plant, Pythium or nontarget DNAs in the environmental sample was not observed. The only limitation was the primers for the 340 bp amplicon did not amplify template from P. alticola, P. bisheria or P. frigida, so these species were not detected by the genus specific TaqMan probe. All 14 species specific TaqMan probes were highly specific and effective for detecting pathogen in environmental samples. Due to the genus specific nature of amplified template, the identification of species present in a sample can be determined without the need for culturing by sequencing the amplicon and BLAST analysis of results against the sequence database.

Last Modified: 11/27/2014
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