|Chen, Wen -|
|Robleh-Djama, Zeinab -|
|Coffey, Michael -|
|Denton, Geoffrey -|
|Lévesque, C. -|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 24, 2012
Publication Date: January 1, 2013
Citation: Chen, W., Robleh-Djama, Z., Coffey, M.D., Martin, F.N., Bilodeau, G.J., Radmer, L.E., Denton, G., Lévesque, C.A. 2013. Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species. Phytopathology. 103(1):43-54. Interpretive Summary: This manuscript reports the development of a molecular detection system capable of identifying Phytophthora species. DNA sequences were generated for three different locations for 97 species of Phytophthora and unique regions for each species used to design markers that can be used to identify each species were attached to a nylon membrane. The three regions are amplified by PCR from environmental samples and exposed to the membrane. Where the DNA from the environmental sample bound to the marker DNA on the membrane identifies which species are present. The array was able to identify 84 out of the 97 species that were tested.
Technical Abstract: Many Phytophthora species are destructive plant pathogens imposing severe threats to both natural and agricultural vegetation. Effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of such diseases. DNA array hybridization technique is a sensitive, reliable, fast and high-throughput diagnostic molecular tool for the detection and identification of microbial organisms from environmental samples. In the current study, membrane-based oligonucleotide arrays were developed from three DNA regions that can be used for separating Phytophthora species reliably, namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 (cox1), and the intergenic region between cytochrome c oxidase 2 (cox2) and cox1 (cox2-1 spacer, CS). Each sequence dataset contained approximately 250 sequences representing 97 species of Phytophthora with 7 Pythium isolates used as outgroup. Arrays were validated with 141 pure cultures covering all available Phytophthora species and a few Pythium isolates. Cross reactions were widely observed on the cox1 array, indicating this genome region is not suitable for DNA array development. Together, non-rejected oligonucleotides from all three arrays have the ability to reliably detect 84 Phytophthora species, whilst the detection of the rest of the species can be facilitated using additional oligonucleotides specific for particular groups. Validation tests were done with 35 field samples collected from the USA, UK and Canada, in which the presence of target species had been validated using alternative molecular and/or conventional approaches. Our results showed that signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for detection and differentiation by the DNA array hybridization technique. The combination of selected oligonucleotides from all three regions has the potential to be used as a routine diagnostic tool for Phytophthora species from complex environmental samples without the need for culturing.