ADAPTATION OF REAL-TIME PCR ASSAY FOR SPECIFIC DETECTION OF APPLE PROLIFERATION PHYTOPLASMA

Abstract

Phytoplasmas are cell wall-less bacteria. Apple proliferation phytoplasma causes an important apple tree disease occurring in many apple-growing areas. Infected trees present symptoms such as witches’ brooms, enlarged stipules and small sized fruits with incomplete coloration. The goal of this study was to develop a real-time PCR assay for specific detection of AP phytoplasma. In the literature, the detection of phytoplasmas by real-time PCR has already been done through the use of SYBR Green, TaqMan probes and TaqMan MGB probes. To ensure a double level of specificity (primers and probe), the use of probes was privileged. A key factor of real-time PCR is the selection of an appropriate threshold. Two methods are commonly used to set the threshold: (i) threshold = 10 x SD, (ii) point of inflexion. A previously designed MGB probe (qAP-16S) was tested on our phytoplasmas collection (mainly AP, PD and ESFY). Using this probe, late fluorescent curves were obtained from ESFY isolates. These curves crossed the threshold calculated as 10 x SD. So, a sequence alignment was made using database sequences and our sequencing results. A new MGB probe was designed in a region presenting a single nucleotide polymorphism (SNP) between AP and ESFY isolates. Using this probe, a specific detection of AP was obtained whatever the method of threshold calculation. No late amplification was observed with other phytoplasmas. Moreover, the amplification of serial dilutions of initial template DNA showed that this method is at least 16 and 8 times more sensitive than conventional PCR with specific (AP5/AP4) and polyvalent (qAP-16S-F/R) primers, respectively. In addition, the phytoplasma infection on inoculated apple tress was sooner detectable using this new probe in real-time PCR than by conventional PCR or biological indexing. So, the optimization of an existing method led to a quick, specific and sensitive method for detection of AP.