Title
Real-time detection of Phytophthora nicotianae and P. citrophthora
in citrus roots and soil
Abstract
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 ll)1 for P. nicotianae and 100 pg ll)1 for P. citrophthora, whereas
in separate reaction DNA up to 1 pg ll)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
Authors
Antonio Ippolito, Leonardo Schena, Franco Nigro, Vincenza Soleti Ligorio and Thaer Yaseen
Journal
European Journal of Plant Pathology, 2004 : 110(0), 833~843
