1Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; 2Danish Institute of Agricultural Sciences, Department of Crop Protection, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark
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Accepted 12 July 2004.
Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.
© 2004 The American Phytopathological Society