Phytoplasma detection by LNA probe-based real time-PCR in Solanum tuberosum

Phytoplasmas are unculturable, wall-less prokaryotes that cause disease in many plant species World-wide. Different phytoplasmas have been associated with diseases of potatoes, including virescence, witches' broom and the two quarantine diseases: purple top wilt and stolbur. Following classification based on RFLP analysis of the 16SrRNA gene sequence, potato-associated phytoplasma were found to belong to 16SrI, 16SrII, 16SrVI, 16SrXII groups and to the proposed specie 'Candidatus Phytoplasma americanum' (Lee et al., 2006). More recently, a 16SrIII group Phytoplasma in Montana (Lee et al., 2009) and the 'Candidatus Phytoplasma australiense' in New Zealand (Liefting et al., 2009), have been reported. Due to the wide diversity found in phytoplasmas affecting this host a detection method which is specific, yet sensitive and reliable is required. Phytoplasma detection using the available universal primers designed from the 16SrRNA gene, produced many false positives resulting from the presence of other bacteria naturally present in the potato samples analyzed. Once sequenced these bacteria were found to be close relatives of phytoplasmas, on the basis of their 16SrRNA gene. A similar approach based on nested-PCR improved the specificity of this diagnostic test but with inconsistent results using different primer combinations. As a consequence, an alternative approach based on the use of locked nucleic acid (LNA) probes and real-time PCR was investigated. The chemistry of LNA probes offers advantages of improved specificity and sensitivity over conventional DNA probes (Costa et al., 2004; Josefsen et al., 2009). The detection assay developed using this approach has been trialled with 100 potato samples and improvements in specificity, repeatability, and sensitivity were all evident when compared against results obtained using conventional PCR. This is the first report of use of LNA probe in Real Time PCR as diagnostic tool for phytoplasmas.