LOCALIZATION OF PATHOGENIC MYCOPLASMA-LIKE ORGANISMS IN PLANT-TISSUE USING INSITU HYBRIDIZATION

A cloned chromosomal DNA fragment of clover proliferation mycoplasma-like like organism (MLO) was sequenced and used to synthesize biotinylated single-stranded DNA probes by asymmetric polymerase chain reaction. The biotinylated single-stranded DNA was identified by temperature gradient gel electrophoresis. Hand sections were prepared from tissues of periwinkle plants (Catharanthus roseus (L.) G. Don) individually inoculated with clover proliferation MLO or potato witches'-broom MLO. In situ hybridization of the sections with the biotinylated probes showed that clover proliferation MLO and potato witches'-broom MLO were located mainly in sieve tube elements. Western aster yellows MLO was not detected by in situ hybridization with the same biotinylated probes. MLO was detected first in the external phloem tissue and then in the internal phloem tissue as plants grew older. This investigation provides the first example of developing PCR-generated small biotinylated single-stranded DNA to trace the colonization of MLOs in plant tissues.