Molecular detection and identification of phytoplasmas and establishment of phytoplasma-free clonal plants.

摘要

Polymerase chain reaction (PCR) was found to be the most sensitive method for detection of phytoplasmas requiring as little as 16 pg of total nucleic acid extracted from woody host plants. The concentration of phytoplasmas in tissue cultures was significantly higher than that in plants grown in the greenhouse. Short-duration microwave heating did not affect the structure of DNA and was very useful for preserving phytoplasma DNA in tissues for molecular analyses and international exchange of research specimens. Paulownia witches'-broom (PaWB) phytoplasma isolates were very closely related to each other when collected from diverse geographic sites, but were different from jujube witches'-broom (JWB) phytoplasma isolates on the basis of restriction fragment length polymorphism and 16S rDNA sequence analyses. PaWB isolates from China, Japan, and South Korea share the same 16S rRNA gene sequences and JWB phytoplasmas from these countries also share the same 16S rRNA gene sequences. Monarda yellows was, for the first time, identified to be associated with a phytoplasma which belongs to the aster yellows group through molecular analyses. Phytoplasma-free plantlets were established by PCR-based screening of meristem cultures of PaWB phytoplasma-infected paulownia after subjecting them to heat treatment at 35