Phytoplasma molecular detection and classification

Following their discovery, phytoplasmas have been difficult to detect due to their low concentration in hosts and their erratic distribution in the sieve tubes of the infected plants. The establishment of electron microscopy-based techniques represented an alternative approach to the indexing procedure based on graft transmission to healthy indicator plants. Lately, protocols for the production of enriched phytoplasma-specific antigens have been developed, thus introducing serological-based detection techniques for the study of these pathogens in plants or insect vectors, however the antisera production is quite limited. Phytoplasma identification is currently performed by PCR/RFLP analyses of 16Sr DNA gene. The first comprehensive phytoplasma classification scheme was in fact based on restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S rRNA, that provides a reliable means for the differentiation of a broad array of phytoplasmas. This technique allowed first the classification of phytoplasmas in 19 groups and more than 40 subgroups and has become the most comprehensive and widely accepted phytoplasma classification system. Further classification allow to differentiate phytoplasmas in 32 groups and several subgroups. Several primers designed on the 16Sr DNA sequence universal for phytoplasmas or group specific were developed; they can be used in different combinations in direct, nested or semi-nested systems for routine phytoplasma detection. This procedure allows the identification of the majority of phytoplasmas at the 16Sr group/subgroup level for both epidemiology and quarantine purposes. A universal DNA barcoding based tool was also recently developed. Two sets of primers amplifying short fragments (500-600 bp) of the Tuf and of the 16S rDNA genes provide successful amplification and sequencing of more than 150 phytoplasma strains. Their ability to separate various phytoplasma strains to ‘Candidatus species’ level, 16S ribosomal group and sub-group level indicate this as a fast, efficient and reliable phytoplasma identification tool. On the other hand, the ‘Candidatus Phytoplasma’ species description, recently adopted, refers to 16S rRNA gene sequence with a threshold <97.5% similarity to that of any previously described ‘Ca. Phytoplasma’ species. Several phytoplasmas were already described as ‘Candidatus’ however gaps are still present since several of them were not yet validly published and other were only proposed based on sequences available in the Genbank.