Detection of the Apple Proliferation and Pear Decline Phytoplasmas by PCR Amplification of Ribosomal and Nonribosomal DNA

Abstract

Ribosomal and nonribosomal sequences were analyzed to design polymerase chain reaction (PCR) primers for detection and identification of the phytoplasmas that cause apple proliferation (AP) and pear decline (PD). A ribosomal primer pair (fU5/rU3) was developed that initiated amplification of the target DNA from all 42 samples from PD-infected pear trees and 36 samples from AP-infected apple trees. These primers also amplified rDNA in all other taxonomically different phytoplasma strains that were tested. A pair of group-specific primers (fO1/rO1) derived from the 16S rRNA gene was identified for detection of the closely related phytoplasmas associated with AP, PD, and European stone fruit yellows. PCR detection of the PD agent with primer pairs fU5/rU3 and fO1/rO1 was considerably more sensitive than microscopic detection using the 4'-6-diamidino-2-phenylindole fluorescence method. A more specific ribosomal primer pair (fPD/rO1) amplified phytoplasma rDNA in all samples from infected pear trees but showed some cross-amplification of AP rDNA. Restriction enzyme analysis of the PCR products obtained with primer pairs fO1/rO1 and fPD/rO1 distinguished the AP and PD phytoplasmas. One pair of ribosomal (fPD/rPDS) primers specifically amplified DNA of the PD phytoplasma but from only about 80% of the infected pear trees. Three pairs of nonribosomal primers amplified phytoplasmal DNA from AP- or PD-infected trees or from both phytoplasmas but failed to detect all strains of either of the two pathogens. These results show that is not possible to detect all strains of the AP and PD phytoplasmas, respectively, with pathogen-specific primers.