Specific detection and quantification of the phytopathogenic agent 'Candidatus Phytoplasma prunorum'

'Candidatus Phytoplasma prunorum' is a wall-less bacterium associated with European stone fruit yellows (ESFY), a severe disease of Prunus spp. (mainly apricot and Japanese plum trees). It can be spread by one insect vector, Cacopsylla pruni, and by the trade of infected material. The availability of PCR-based methods allowing a sensitive and specific detection of 'Ca. P. prunorum' is crucial for this phytoplasma because, at present, it is uncultured and cannot be detected serologically. We developed a PCR test which, in contrast to the existing detection tools, provides a fast, specific and sensitive detection of 'Ca. P. prunorum' in plants and insects. For studies requiring an absolute quantification of the phytoplasma titer, the same primers were used to develop a real-time PCR assay, including a standard for C. pruni. The sensitivity of these molecular tools was compared by serial dilutions and their specificity was assessed both in silico and experimentally for reference strains and field samples of the closely related phytoplasma 'Ca. P. prunorum', 'Ca. P. pyri' (pear decline agent) and 'Ca. P. mali' (apple proliferation agent), as well as for representative strains of the 'Ca. Phytoplasma' genus.