Real time detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soil

Two primers, specific for Phytophthora nicotianae ( Pn6) and P. citrophthora ( Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species of Phytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianae specific primers a delayed and lower fluorescence increase was also obtained from P. cactorum DNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg mul(-1) for P. nicotianae and 100 pg mul(-1) for P. citrophthora, whereas in separate reaction DNA up to 1 pg mul(-1) was detected for both pathogens. Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification ( nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 ( second round), it was possible to achieve real-time detection of P. nicotianae and P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.