Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real‐time PCR

Abstract

SUMMARY New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.

Management of plant health risks associated with processing of plant-based wastes: A review

“…Baiting, serological and DNA based detection methods for P. ramorum and P. kernoviae are available but data on detection limits are scant (Schena et al, 2006).…”

“…Phytophthora kernoviae sporangia oospores similar to P. ramorum no data Schena et al (2006) Polymyxa spp cystosori in roots or soil 100-150 cystosori detection of spores from soil using Real Time PCR 1.39 × 10 3 cystosori g -1 soil Ward et al (2004) bait tests followed by glutathione-S-transferase ELISA or microscopy no data √ Kingsnorth et al (2003) MPN method bait test method 50-100 cystosori √ Tuitert (1990) 58 Roenhorst et al (2005) …”

Pathways to false positive diagnoses using molecular genetic detection methods; Phytophthora cinnamomi a case study

“…The numbers above the branches correspond to the bootstrap support for the branch. Notes: a Ypt = RAS related protein Ypt1; ITS = Internal transcribed spacer region; LPV = LPV gene which encodes putative storage protein; ATP = mitochondrial ATP synthase; NAD = mitochondrial NADH dehydrogenase (Hughes et al 2006 (Shen et al 2005) P. boehmeriae 10 14 6 0 a Number of species present within the same clade as the target species b Number of species tested from the same clade as target species (excluding target species) c includes undescribed taxa…”

“…Using conventional PCR, the primer Ypt (C2) could be used to detect P. cinnamomi from 150 fg of DNA. Using a nested PCR approach, where universal Phytophthora primers Yph1F -Yph2R (Schena et al 2006) were used in the first round, increased the sensitivity of the assay at least 100-fold, down to 15.0 fg of DNA. Similarly, ITS (N2) primers could be used to detect as little as 0.015 fg of DNA.…”

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

“…Although in multiplex real-time PCR assays several target DNAs can be simultaneously detected from different microorganisms by differences in emission wavelength or amplicon size, a major limitation of multiplexing is the competition between different primers and probes, resulting in lower specificity and sensitivity (Okubara et al 2005). However, the detection of more than two targets without reduction of sensitivity has also been reported (Schena et al 2006). In order to obtain the best results, primer and probe design, amplification conditions, and amplicon length should be optimised.…”