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 spacer regions of phytoplasmas associated with lethal decline diseases of coconut palm (Cocos nucifera), were amplified from infected plants from Florida and the Yucatan region in Mexico and from east and west Africa. Following sequencing of the rDNA products, phylogenetic analysis confirmed that these coconut phytoplasmas form a separate cluster within the phytoplasma clade and that the pathogen causing diseases in west Africa formed a new sub-clade within this cluster. Analysis of the 16S-23S intergenic spacer regions confirmed 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, produced PCR amplification products from healthy coconut DNA, infected coconut DNA from the Caribbean or DNA from a variety of periwinkle (Catharanthus roseus)-maintained phytoplasmas. These specific primers can serve as effective tools for identifying particular coconut phytoplasmas in field samples.