A new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of 'Candidatus Phytoplasma mali'. The new probe can distinguish a single mismatch between 'Ca. P. mali' and 'Candidatus Phytoplasma prunorum', this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real-time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real-time PCR method, three other real-time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real-time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our realtime PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.
a b s t r a c tDifferent PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter-and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB-or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10 5 ). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.
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.
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