DNA-based methods for the detection and the identification of phytoplasmas in insect vector extracts

DNA extraction and storage methods have been evaluated using laboratory- reared leafhoppers and/or field-collected leafhoppers and psyllids. Detection of four different phytopathogenic phytoplasmas, belonging to three taxonomic groups, has been achieved by several direct or nested polymerase chain reaction methods with such DNA extracts. Reactions differed in both 16/23S ribosomal primer pairs used and specific assay and cycling conditions. Merits and possible hindrances of the various primer pairs, in relation to insect DNA extracts, are discussed. However, identification of the phytoplasma(s), necessarily relied on the comparison of the length polymorphism of the amplified DNA fragments obtained by restriction with appropriate endonucleases. Endonuclease digestion is crucial for determining the identity (=subgroup affiliation) of phytoplasmas of the same groups that can be carried by one individual vector.