Diversity of phytoplasmas isolated from insects, determined by a DNA heteroduplex mobility assay and a length polymorphism of the 16S-23S rDNA spacerregion analysis

Two techniques were developed for the analysis of non-cultivable mollicutes in insects. The first was aimed at detecting organisms belonging to undisc overed groups within the phytoplasma clade. After prescreening by polymeras e chain reaction with phytoplasma-specific primers, nucleic acids from 54 p ositive samples were amplified using phytoplasma-specific fluorescein-label led primers flanking the 16S-23S rDNA spacer region, which is variable in l ength among the phytoplasmas. The sizes of all the detected products were o nly those expected for already-described phytoplasma subclades. It was also shown that a single leafhopper might carry different phytoplasmas, at simi lar or very different relative concentrations. The second technique, based on the heteroduplex mobility assay, was designed for the detection of organ isms phylogenetically similar to phytoplasmas but not recognized by the spe cific primer pair. As a result, signals generated by ribosomal DNA of organ isms which appear to be closely related but not identical to phytoplasmas w ere detected.