Intracellular structures very similar to bacteria and called Bacteria-like Organisms (BLOs) were first described in the 1970s (Mosse, 1970; Scannerini, Bonfante 1991 for a review). Ultrastructural observations clearly revealed their presence in many field-collected fungal isolates. Further investigation on these BLOs, including the demonstration of their prokaryotic nature, was long hampered because of their inability to grow on plate. Only a combination of morphological observations (electron and confocal microscopy) and molecular analyzes allowed us to identify BLOs as true bacteria and to start unraveling their symbiotic relationship with AM fungi (Bianciotto et al. 1996).
Isolate BEG 34 of Gigaspora margarita contains a large number of BLOs which can be easily detected by staining with fluorescent dyes specific for bacteria and capable of distinguishing between live and dead bacteria. About 250,000 live bacteria were counted in a single spore, which is a large structure of about 260-400 pm. Ultrastructural observations performed on high-pressure freezing/freeze-substituted samples revealed a large number of rod-shaped BLOs in the vacuoles of germinating spores, often associated to the abundant protein bodies. On the basis of the 16S rDNA sequences the bacterial endosymbionts living in the fungus Gigaspora margarita (BEG 34) were identified as belonging to the genus Burkholderia.
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