Molecular Diagnosis of Phytoplasmas

Abstract

Phytoplasmas are wall-less prokaryotes associated with diseases in numerous plant species worldwide.
In nature they are transmitted by phloem-sucking insects. Yellowing, decline, witches’ broom, leaf curl, floral virescence
and phyllody are the most conspicuous symptoms associated with phytoplasmas, although infections are sometimes
asymptomatic. Since phytoplasmas cannot be cultured in vitro, molecular techniques are needed for their diagnosis
and characterization. The titer of phytoplasma cells in the phloem of infected plants may vary according to the
season and the plant species, and it is often very low in woody hosts. Different DNA extraction procedures have
therefore been tried out to obtain phytoplasma DNA at a concentration and purity high enough for effective diagnosis.
DNA/DNA hybridization methods were reported in the nineties to be appropriate for the detection of phytoplasmas,
but at present PCR is considered the most suitable. Universal and group-specific primers have been designed on the
rRNA operon of the phytoplasma genome and on plasmid sequences. RFLP analysis of the obtained amplicons has
classified these pathogens into major 16Sr RNA groups. Group-specific primers have also been designed on other
genomic sequences. PCR is a very sensitive technique, but due to the low titre of phytoplasmas a further increase in
sensitivity may be required for accurate diagnosis. This is routinely obtained with a second round of PCR (nested
PCR). The drawback of nested PCR is that there is a greater chance of obtaining false positives due to contamination.
Many authors have therefore developed protocols based on hybridization (PCR/dot blot) or serological approaches
(PCR/ELISA) to increase the sensitivity and specificity of the direct PCR, reducing the risks due to nested PCR. Real
time PCR protocols may also improve the sensitivity and specificity of the direct PCR assay.