Santos-Garcia, D. - Department of Entomology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel;
Naor, V. - Shamir Research Institute, Katzrin, Israel;

‘Candidatus Phytoplasma’ species are obligate plant parasites limited to the phloem tissue. The difficulties to grow phytoplasma in culture seriously impedes the study of these prokaryotes. In this study, a protocol to extract intact phytoplasmas from infected periwinkle plants using filtration, differential and density-gradient centrifugations was optimized. Confocal and transmission electron microscopy was used to visualize intact phytoplasmas in the preparation. Quantitative-PCR and DNA-seq analyses revealed that phytoplasma DNA was significantly enriched following the extraction procedure. Challenging periwinkle plants with phytoplasma preparation induced the expression of one defence-related gene. This study provides an optimized procedure for phytoplasma DNA enrichment and for extracting intact phytoplasmas that can be used to examine host responses to them.