DETECTION OF PHYTOPLASMAS BY DIENES' STAIN, DAPI AND ELECTRON MICROSCOPY
Potato Research Program, Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, Charlottetown, PEI, C1A 4N6, Canada
Background and objectives
The objective of this study was to compare three detection methods, Dienes' stain, DAPI and electron microscopy, used for detection of phytoplasmas in the phloem tissues of yellows-infected carrot and lettuce collected in the fields in Oklahoma and aster and periwinkle grown in the greenhouse.
Results and conclusions
Detection of phytoplasmas with DAPI: DAPI-stained plant material was observed under fluorescence microscope. Extracellular DNA in the phloem tissues of all of the 50 yellows-infected plants indicated the presence of phytoplasmas, whereas healthy plants had no extracellular DNA. Although the DAPI could detect phytoplasmas in the infected tissues, it is not specific for phytoplasmas. In addition, the detection procedure took 25-30 min per sample.
Detection of phytoplasmas with electron microscopy: sections of 20 infected and eight healthy control plant samples were observed under transmission electron microscope. Pleiomorphic, 70-800 nm diameter, unit membrane-bound phytoplasmas were observed at x7200 in the phloem regions of the infected plants. No phytoplasmas were seen in sections of healthy control samples. Although electron microscopy is the only convincing diagnostic method, it is time-consuming and requires expensive reagents and equipment.
In conclusion, of the three detection methods used, Dienes' stain, which was more rapid than DAPI and required less expensive equipment and reagents than electron microscopy, can be used as a preliminary diagnostic method to detect phytoplasmas in infected plants. Electron microscopy, however, can be used as a confirmatory test for visualization of phytoplasmas in infected tissues.