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ISHS Acta Horticulturae 551: IX International Symposium on Small Fruit Virus Diseases

DEVELOPMENT OF A SPOT-BASED PCR-RFLP ASSAY FOR THE DETECTION OF

Authors:   X. Foissac, J.L. Danet, L. Zreik, J. Gandar, J.M. Bové, M. Garnier, J.G. Nourrisseau
Keywords:   Phlomobacter fragariae, PCR detection, Marginal chlorosis of strawberry, Phytoplasma, Whiteflies, Proteobacteria
Abstract:
Marginal chlorosis is a new disease of strawberry in which an uncultured phloem-restricted proteobacterium “Candidatus Phlomobacter fragariae” is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on 16S rDNA applied to these insects was unable to discriminate between “P. fragariae” and insect endogenous proteobacteria, isolation of “P. fragariae” genes other than 16S rDNA was undertaken. Using comparative Randomly Amplified Polymorphic DNAs (RAPD) a 1.1 kbp amplicon was specifically amplified from “P. fragariae”-infected strawberry plants. It encodes part of a “P. fragariae” Open Reading Frame sharing appreciable homology with spoT gene from other proteobacteria. A spoT-based PCR test combined with Restriction Fragment Length Polymorphisms (RFLP) was developed and was able to distinguish “P. fragariae” from other insect bacteria. Indeed, different RFLP profiles of the spoT gene were also evidenced for various endogenous bacteria of insects. However, none of the many leafhoppers, psyllids and whiteflies captured during several years in and around infected strawberry fields were found to carry “P. fragariae”. Interestingly, however whiteflies proliferating on “P. fragariae”-infected strawberry plants under confined glasshouse conditions were shown to have become infected with the bacterium. In the absence of other insect candidate for the transmission of “P. fragariae” the possibility of a transmission by whiteflies has to be further studied.
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