Phylogenetic relationships of coconut phytoplasmas and the development of specific oligonucleotide PCR primers

Using the polymerase chain reaction the 16S rRNA genes and the 16S-23S spac er regions of phytoplasmas associated with lethal decline diseases of cocon ut palm (Cocos nucifera), were amplified from infected plants from Florida and the Yucatan region in Mexico and from east and west Africa. Following s equencing of the rDNA products, phylogenetic analysis confirmed that these coconut phytoplasmas form a separate cluster within the phytoplasma clade a nd that the pathogen causing diseases in west Africa formed a new sub-clade within this cluster. Analysis of the 16S-23S intergenic spacer regions con firmed the sequence diversity of this region and enabled two primers to be designed which were specific for the diseases found in east and west Africa . None of these specific primers, when paired with a universal primer, prod uced PCR amplification products from healthy coconut DNA, infected coconut DNA from the Caribbean or DNA from a variety of periwinkle (Catharanthus ro seus)-maintained phytoplasmas. These specific primers can serve as effectiv e tools for identifying particular coconut phytoplasmas in field samples.