Molecular diagnosis of phytoplasmas

الملخص AR

-Phytoplasmas are non-culturable, wall-less and phloem-restricted pathogens transmitted in a persistent manner by leafhoppers and planthoppers (Homoptera: Auchenorrhyncha) and psyllids (Homoptera: Sternorrhyncha).

They are associated with diseases in many wild and cultivated plant species belonging to different families and cause economically important epidemics world-wide.

The colonization of the plant by the phytoplasmas depends on the season, organ, host and pathogen species, and results in different symptoms due to complex interference with the host physiology.

Sensitive and accurate diagnosis of these pathogens is crucial for the management of phytoplasma-associated diseases.

Phytoplasmas are difficult to detect due to their low concentration especially in woody hosts and their erratic distribution in the infected plants.

Their detection is now routinely done by nucleic acid-based techniques, mainly PCR.

Total DNA preparations of good quality and enriched in phytoplasma DNA are usually obtained by including a time-consuming phytoplasma enrichment step, although simpler protocols have been developed using commercially available microspin columns.

Successful phytoplasma detection in insect vectors may be attained with quicker total DNA extraction procedures, probably due to the high titre of the bacteria in the insect body.

Universal phytoplasma-specific PCR primers have been identified in different positions of the ribosomal RNA operon, and group-specific primers have also been designed following comparison of the phytoplasma-specific 16SrRNA and 16S-23S intergenic regions of phytoplasmas belonging to different strain clusters.

Ribosomal sequence-based primers are the most used for routine diagnosis of phytoplasmas.

Universal and group-specific primers have also been targeted to other gene sequences, to sequences with no obvious predicted function and to the sequence of plasmids hosted by phytoplasmas.

Routine diagnostic protocols usually involve the use of nested PCR.

More recently phytoplasma diagnostic assays based on RT-PCR, real time PCR, PCR-ELISA, PCR-dot blot, heteroduplex mobility assay, 16S-23S spacer length polymorphism, microarray and nanobiotransducer hybridization have also been proposed.