Cloning of the spoT gene of "Candidatus Phlomobacter fragariae" and development of a PCR-restriction fragment length polymorphism assay for detectionof the bacterium in insects

Marginal chlorosis is a new disease of strawberry in which the uncultured p hloem-restricted proteobacterium "Candidatus Phlomobacter fragariae" is inv olved. In order to identify the insect(s) vector(s) of this bacterium, homo pteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between "P . fragariae" and other insect-associated proteobacteria, isolation of "P, f ragariae" genes other than 16S rDNA was undertaken. Using comparative rando mly amplified polymorphic DNAs, an amplicon was specifically amplified from "P, fragariae"-infected strawberry plants. It encodes part of a "P. fragar iae" open reading frame sharing appreciable homology with the spoT gene fro m other proteobacteria, A spoT-based PCR test combined with restriction fra gment length polymorphisms was developed and was able to distinguish ''P. f ragariae" from other insect bacteria. None of the many leafhoppers and psyl lids captured during several years in and around infected strawberry fields was found to carry "P, fragariae." Interestingly however, the "P, fragaria e" spoT sequence could be easily detected in whiteflies proliferating on "P , fragariae"-infected strawberry plants under confined greenhouse condition s hut not on control whiteflies, indicating that these insects can become i nfected with the bacterium.