Molecular detection of phytoplasmas in apple with rubbery wood symptoms

Rubbery wood is a severe disease which affects both apple and pear trees and may cause severe losses in some countries. The affected plants show a partial lack of lignification of young branches which gives the plant a typical prostrate appearance. In longitudinal sections of flower petioles and green prebursted shoots from grafts or diseased trees, strong fluorescent areas associated with DNA due to phytoplasmas or to other infectious agents were observed using fluorescent microscopy-DAPI technique. Round structures resembling phytoplasmas were also observed under the scanning electron microscope. Further analyses were performed to verify the presence of phytoplasma and allow their molecular identification. Samples from two source plants from the Czech Republic and one from Canada were employed in direct and nested polymerase chain reaction assays together with apple proliferation samples and group specific control phytoplasma strains as positive controls. General ribosomal primer pairs employed were R16F1/R0 and R16F2/R2, while group-specific primer pairs were R16(I)F1/R1, R16(III)F2/R1, R16(V)F1/R1 and R16(X)F1/R1. A DNA fragment of 1.2 kb was produced from some of the rubbery wood samples and from apple proliferation samples as well as from the positive controls when nested PCR was carried out with the primer pair R16F2/R2. The amplification with specific primers yielded a 1.1 kb fragment from the rubbery wood samples amplified using primer pairs R16(I)F1/R1. RFLP analysis confirmed that phytoplasmas that belong to the aster yellows group (16SrI) were present. PCR experiments with primers that amplify a chromosomal portion of aster yellows group phytoplasmas followed by Southern hybridization with the homologous DNA probe confirmed the presence, in some of the apple rubbery wood samples, of phytoplasma that belong to the aster yellows group.