Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees

Abstract

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 simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of ‘Candidatus Phytoplasma mali’

“…Serially diluted extracts were analysed for the presence of the template to be detected by TaqMan MGB real-time PCR (Aldaghi et al, 2007(Aldaghi et al, , 2008. The results of these experiments are summarized in Table 3.…”

“…Ct values for real-time PCR (Aldaghi et al, 2007(Aldaghi et al, , 2008 assays performed after serial dilution of crude versus purified extracts of leaf and bark samples. Although phytoplasma was detected sooner by purified extracts for dilutions 10 and 10 2 , detection limit of both extraction techniques for phytoplasma infection was similar (up to dilution 10 5 ).…”

“…The method used for extraction of purified DNA from phytoplasma-infected plants has been previously published (Aldaghi et al, 2007). Briefly, after homogenizing 200 mg plant materials (leaves or bark tissue) in a mortar with CTAB buffer containing 0.2% 2-mercaptoethanol, the samples were incubated at 60 • C for 30 min.…”

“…Three established real-time PCR protocols, a TaqMan MGB method (protocol 1) (Aldaghi et al, 2007(Aldaghi et al, , 2008 and two SYBR green methods (protocol 2 and 3) (respectively, Jarausch et al, 2004;Torres et al, 2005), were also used for phytoplasma detection from both DNA extract preparation methods. Reactions were performed in a total volume of 25 l containing 1× qPCR MasterMix for TaqMan or SYBR Green from Eurogentec (respectively for protocols 1 and 2), or Power SYBR Green ® PCR MasterMix from Applied Biosystems, Warrington, UK (for protocol 3), 400 nM of each primer (used in original protocols), 200 nM TaqMan MGB probe (if applicable), and 5 l purified or crude DNA extract.…”

“…The C t Table 1 Comparison of 'Ca. P. mali' detection by real-time PCR (Aldaghi et al, 2007(Aldaghi et al, , 2008 performed with crude and purified DNA extracts of different types of samples. The results of the two methods are in complete agreement.…”