Oligodeoxynucleotides as probes for in situ hybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells

Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They bel ong to several groups in a unique phylogenetic clade. Non-related phytoplas mas may infect the same plant species, often with similar symptoms. Hence m ethods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embedding i n situ hybridization (ISH) with transmission electron microscopy using olig odeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplas ma-infected periwinkle were submitted to ISH using digoxigenin or biotin-la belled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplific ation of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Tog ether with preliminary digestion with pepsin, different in situ denaturatio n conditions and formamide concentrations were tested. The grids were incub ated in the hybridization mixture at 37 degrees C overnight. Detection of h ybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A s pecific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleoti des may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy. (C) 1999 Academic Press.