Detection of DNA of Phytoplasmas Associated with Phyllody Disease of Sesame in Thailand.

Abstract

Sesame phyllody (SP) caused by phytoplasmas occurs in many countries in the tropics. DNA extracted from sesame plants with phyllody in Thailand, purified for the phyllody-specific fraction with repeated bisbenzimide-CsCl equilibrium density gradient centrifugation and fragmented by restriction enzyme HindIII was cloned into a plasmid-Escherichia coli system. Recombinant plasmids which reacted with the peroxidase-labeled DNA of SP-affected sesame plants but not with the labeled DNA of healthy sesame plants were selected. Southern analysis revealed that the inserts of some recombinant plasmids consisted of fragments of chromosomal DNA of the phytoplasma, whereas the inserts of the other recombinant plasmids consisted of fragments of two types of extrachromosomal DNAs of the phytoplasma. Hybridization using the cloned chromosomal and extrachromosomal DNA fragments associated with the SP phytoplasmas as probes as well as the PCR procedure to amplify the 16S rDNA of phytoplasmas was effective in assaying SP phytoplasmas in the field. Cloned DNA probes hybridized strongly with SP specimens whereas most of them hybridized weakly with rice yellow dwarf, sugarcane white leaf, gentian witches' broom, and tsuwabuki witches' broom specimens. Preferential dot hybridization and restriction enzyme cutting pattern analysis of the 16S rDNA of several phytoplasma isolates found in Northeast Thailand indicated that the SP phytoplasmas are more closely related to some phytoplasmas associated with phyllody and witches' broom type symptoms than to phytoplasmas associated with white leaf symptoms including sugarcane white leaf phytoplasma.