3.3.1
DETECTION OF PHYTOPLASMAS BY DIENES' STAIN, DAPI AND ELECTRON MICROSCOPY

D ERRAMPALLI

Potato Research Program, Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, Charlottetown, PEI, C1A 4N6, Canada

Background and objectives
Phytoplasmas, which cause yellows diseases, are known to infect more than 350 plant species. Because of the non-culturable nature of these pathogens, phytoplasma-associated diseases have traditionally been differentiated by symptomatology, host range and plant-vector relationships. The presence of phytoplasmas in the phloem tissues of yellows-infected plants can be detected by Dienes' stain, 4-6-diamidino-2-phenyl indole-2 HCl (DAPI) and electron microscopy [1]. Although serological techniques and DNA-based probes are used to detect phytoplasmas in infected plants and insect vectors, electron microscopy is the only convincing detection method available for the phytoplasmas [2].

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 Dienes' stain: all of the 190 yellows symptomatic plants showed a positive reaction with Dienes' stain under bright-field microscope. Phloem tissue of stem, petiole, midrib and root infected with phytoplasma stained dark blue, while xylem was stained turquoise blue and cortex stained light blue. Phloem of healthy plants and plants infected with Sclerotinia sp., Sclerotium sp., S. rolfsii or Xanthomonas sp. remained unstained. This method is specific for phytoplasmas, requires inexpensive equipment, and the whole procedure took only 2-5 min per sample.

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.

References
1. Doi Y, Teranaka M, Yora K, Suyama H, 1967. Annals of the Phytopathological Society of Japan 33, 259-266.
2. Errampalli D, Fletcher J, Claypool PL, 1991. Plant Disease 75, 579-584.