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Definition of vertical transmission of Candidatus Glomeribacter gigasporarum through generations of Gigaspora margarita

Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria. Based on their ribosomal sequences these endobacteria have been recently identified as a new taxon, Candidatus Glomeribacter gigasporarum. In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungi reproduction (asexual), a system using transformed carrot roots and single spore inocula of Gigaspora margarita was used. Under these in vitro, sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored. Parallel multisporal inocula (50 spores per pot) were used in clover pot cultures.

MORPHOLOGICAL ANALYSIS:
Endobacterial cells were constantly observed in all sporal generations produced in both Petri dish and pot cultures by using the Bacteria Counting Kit. In monosporal-inoculated Petri dish cultures, endobacteria were observed throughout all the mycelial structures produced during root colonisation and spore production: i.e. germination hyphae, extraradical mycelium, auxiliary cells and new spores.

TRANSMISSION OF THE ENDOBACTERIA:
In order to verify the identity of endobacteria contained in all spore generations (SGs), PCR experiments with specific primers for 16S and 23S rDNA were performed on single SG0, SG1, SG2, SG3, SG4 spores, harvested from both pot cultures and Petri dishes. A fragment of the expected size (565bp) was obtained from spore generation SG0, SG1, SG2, SG3 and SG4 multiplied in Petri dish cultures, by using the specific primers GlomGIGf-GlomGIGr (23S rDNA) The direct sequencing of the PCR fragments confirmed the presence of Candidatus G. gigasporarum. As in vitro SG4 generation included spores where bacterial DNA could not be amplified (also with16S rDNA specific primer, BLOf-BLOr), the reliability of DNA extraction was checked. PCR amplifications of SG4 spores that appeared lacking endobacteria were performed with the eukaryotic primers NS1-NS2. They systematically gave positive response. Fragments of the expected size were constantly amplified, from all spore generations (SG0 to SG4) obtained from pot cultures. DNA amplification with the non specific 16S rDNA bacterial primers 27f and 1495r from the five spore generations (SG0, SG1, SG2, SG3 and SG4) consistently produced a single 1500pb fragment. Restriction analyses performed with a total of 5 restriction enzymes led to identical profiles across the SGs, confirming the presence of the same bacterial population along fungal generations. The size of the digested DNA fragments rightly matched the profiles expected after in silico analysis for the Candidatus G. gigasporum 16S rDNA (X89727). No difference between SG0 spores and the four in vitro produced spore generations were observed. The results show that, at least in the four spore generations here investigated, only one bacterial population is present, and it corresponds to Candidatus G. gigasporarum.

BACTERIAL CELL QUANTIFICATION:
The repeated observation of sporal cytoplasm from successive generations revealed that the spores produced by monosporal-inoculated Petri dish cultures apparently harboured a progressively smaller population of endobacteria (eventually leading to some bacteria-free SG4 spores), while no decrease was evident through the spore generations produced by the multisporal-inoculated pot cultures. In order to better investigate this aspect, a quantification method was set up, exploiting the capabilities of confocal microscopy. An automatic quantification of the bacterial cells (based on particle size and counting algorithms) was discarded, as a human control of overestimates (due to double counting) turned out to be necessary. In addition, the counting of bacteria in each optical section rather than in a projected image of the whole volume was introduced in order to allow the discrimination of each bacterium, since in many cases their high density resulted in confluent fluorescent areas in the projected image. The average numbers of bacteria present in 100µm-sided cubes of sporal cytoplasm was calculated for each spore. Though a wide range of variability was a general decrease occurred from SG0 to SG4 spores in monosporal-inoculated Petri dish cultures. In conclusion, a method of confocal microscopy for quantifying the density of endobacteria in sporal cytoplasm was designed and applied; endobacteria were constantly found in all the SGs, although their number decreased progressively from SG0 to SG4.

In conclusion, our experiments demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus and that these bacteria are stable endocellular component of their AM fungal host.

More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/

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