| Molecular |
|
Molecular Detection of Phytophthora spp.
P. ramorum, P. nemorosa, and P. pseudosyringae
The described PCR detection system is based on amplification of the spacer region between the mitochondrially-encoded cox I and II genes of Phytophthora using genus-specific primers developed from conserved regions of the flanking genes. A two-step multiplex amplification procedure is used for determining if a Phytophthora sp. was present in symptomatic plant tissue and clarifying if it was P. ramorum or P.nemorosa or P. pseudosyringae, two additional species commonly encountered in samples from forest ecosystems in California. The first round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for PCR amplification and a second primer pair specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in all the plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa or P. pseudosyringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum this included 24 isolates from California, Germany and the Netherlands. Using purified pathogen DNA the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure the sensitivity of detection was reduced by 100 to 1000 fold depending on the plant species. Using a dilution series of purified DNA from P. ramorum the nested mitochondrial marker system was found to have a level of sensitivity comparable to the ITS marker system. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions, and for P. ramorum, amplification with a previously published rDNA ITS species-specific primer pair. Results were compared and validated with three different brands of thermalcyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests they will have utility for pathogen detection in other Phytophthora pathosystems and the variability encountered in the spacer regions should be useful for constructing additional species-specific primers.
A real-time fluorescent PCR detection method for the sudden oak death pathogen P. ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System (Tooley et al. 2006). Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a 3-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae and plant DNA in a single tube. P. ramorum was detected down to a 10 -5 dilution of extracted tissue of artificially infected Rhododendron 'Cunningham's White' and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.
- References
- Martin, F.N, Tooley, P.W. and Blomquist, C. (2004) Molecular detection of Phytophthora ramorum, the causal agent ofsudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology 94: 621-631 (.pdf)
- Tooley, P.W., Martin, F.N., Carras, M.M. and Frederick, R.D. (2006) Real-time Fluorescent PCR Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions. Phytopatholgy 96: In Press (draft .pdf)
•• P. ramorum amplification procedure - conventional PCR 3/26/04
? Modifications to amplification procedure
•• Potential alternative plant primers 5/12/04
•• Detection of P. ramorum in a single amplification 5/12/04
•• Real-time PCR 3/14/06
•• Primers, TaqMan probes and PCR conditions
•• P. ramorum-specific primers and probe
•• P. pseudosyringae-specific primers and probe
•• Plant primers and probe
•• Testing primers and probes with plant DNA
•• Sensitivity of detection of real time PCR assay with infected tissue
•• Evaluating field samples from California
•• Multiplex amplification
Additional details
? Plant primers 5/1/04
? Phytophthora genus-specific primer pair 5/1/04
? P. ramorum species-specific primer pair 5/1/04
•• Multiplex amplification
•• Limit of detection 5/1/04
•• Sensitivity compared to the ITS marker system 5/12/04
•• Amplification from symptomatic plant tissue 5/1/04
? Additional species-specific primers nested in the genus-specific amplicon
•• P. nemorosa species-specific primer pair 5/1/04
•• P. pseudosyringae species-specific primer pair 5/1/04
•• Additional species-specific primers 5/1/04
•• Sequence alignments of the Phytophthora genus-specific amplicons
•• Additional references on phylogeny of P. ramorum and Phytophthora spp.
•• Martin, F.N and Tooley, P.W. (2003) Phylogenetic relationships of Phytophthora ramorum,P. nemorosa, and P. pseudosyringae, three speciesrecovered from areas in California with sudden oak death. Mycological Research 107:1379-1391.
•• Martin, F.N. and Tooley, P.W. (2003) Phylogenetic relationships among Phytophthora species inferred from sequence analysis of the mitochondrially-encoded cytochrome oxidase I and II genes. Mycologia 95:269-284.
•• Links to related web sites
- USDA-APHIS SOD National Survey: [NAPIS] Phytophthora ramorum Sudden Oak Death PILOT National Survey Plan 2002/2003
