Phylogenetic Tree for the Endosymbiotic Burkholderia

To determine whether bacteria are also harbored by other members of Gigasporaceae, eleven fungal isolates collected from different geographic areas and belonging to six different species were analyzed by morphological and molecular approaches. A fluorescent dye was used to visualize the bacteria inside the spores, while 16S ribosomal genes were amplified by PCR using universal eubacterial primers and primers specific for the endobacteria identified in Gi. margarita BEG 34. With the exception of Gigaspora rosea, isolates from all other species harbored bacteria in their cytoplasm and gave an amplified fragment of the expected size with the universal primers (Bianciotto et al. 2000). Six out of seven isolates (belonging to five different species) could be also amplified with the specific primers. These specific primers were used as probes for in situ hybridization on Gi. margarita spores, where they successfully identified bacteria. The 16S rDNA amplified from isolates of Scutellospora persica, S.castanea and Gi. margarita was sequenced and aligned with the closer bacterial sequences available in databases. With neighbor-joining analysis a strongly supported branch containing all endosymbiotic bacteria so far sequenced in Gigasporaceae could be identified nested in the genus Burkholderia. These results demonstrate therefore that endobacteria are widespread in Gigasporaceae. In addition, preliminary experiments showing that they pass through one fungal generation to another with a vertical transmission mechanism (V. Bianciotto and G. Becard, in preparation) suggest that they represent a stable cytoplasmic component.

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