Setting up and evaluation of new primer sets for the detection of Spiroplasma citri and further investigations on the genetic diversity among the Mediterranean S. citri isolates
Citrus stubborn disease caused by Spiroplasma citri is a limiting factor for citrus production in countries with desert or semi arid conditions in the Mediterranean basin. Several polymerase chain reaction (PCR) assays has been previously developed but most of them lacked specificity or sensitivity for Spiroplasma citri detection. In order to develop a PCR assay specific and sensitive for detecting Mediterranean S. citri strains, three PCR primers were designed on the basis of repetitive sequences annotated “Hypothetical proteins”; a BLAST of the selected genes on the GeneBank showed no homology with genes of other organisms, spiroplasmas plasmids, or known spiroplasma virus sequences. The primer pairs were annotated as ScX 6, ScX7, ScX8, ScX9 and ScX 10, their sensitivity was evaluated by PCR with serial dilutions of positive S. citri cultures and DNA extracted from S.
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citri infected plants, while the specificity was determined by PCR using the DNA extracted from cultures of other Spiroplasma species. The ScX7 was selected, owing to its high specificity and sensitivity when compared with all other primers. Furthermore, investigations on the genetic diversity among S. citri Mediterranean isolates, carried out on collected sources from Egypt, Turkey, Syria and Algeria, targeting the TraG and spiralin genes, showed a noticeable diversity among them.
