1. Biologie Des Microorganisms Pdf Viewer Software

. Conceptual drawing of the experimental design for the investigation of interactions between bacteria and Glomus irregulare hyphae using a two-compartment Petri dish. Results Isolation of bacteria from G. Irregulare spores AMF spores with morphological features such as color, size and shape that were typical to G. Irregulare were collected from the soil samples. We confirmed the identity of these spores by sequencing of the 18S rRNA gene amplified by PCR from single spores. The sequences obtained showed 100% homology with G.

Irregulare isolate DAOM197198 (accession number ). After 1 month of incubation of these spores on the G. Irregulare hyphae growing in vitro on water–gellan gum medium, bacterial growth was clearly visible around hyphae as shown in. Bacteria did not affect the growth of hyphae and spore development of G. These colonies were reinoculated repeatedly until single morphotypes were obtained on TSA medium. In total, 29 morphotypes were recovered.

PCR amplification and sequencing of the 16S rRNA gene allowed the grouping of these 29 morphotypes into seven different bacterial species. Blast nucleotide searches of the 16S rRNA gene showed sequence homologies 99% for all isolates, except Bacillus simplex (98.8%). Phylogenetic analysis revealed that three bacterial taxa clustered in Firmicutes in the Bacillus genus, two in Actinobacteria and one each in Alpha- and Betaproteobacteria. Bacterial taxa recovered from Glomus irregulare spores isolated from natural soil and identified using sequencing of a 16S rRNA gene No. Of colonies Strain Phylogenetic affiliation (strain, accession number)% identity (sequence length) 1 SE712 Bacillus cereus (ATCC 14579, ) 99.1 (1438 bp) 2 SE342 B. Megaterium (SB 3112, ) 99.7 (1445 bp) 3 SE713B B.

Simplex (N25, ) 98.8 (874 bp) 2 SE713J Kocuria rhizophila (TA68, ) 99.9 (1424 bp) 5 SE33c Microbacterium ginsengisoli (unknown, ) 99.9 (1412 bp) 3 SE31c Sphingomonas sp. (MUELAK1, ) 99.8 (1386 bp) 13 E1347c Variovorax paradoxus (rif200835, ) 99.8 (1430 bp).

Phylogenetic analysis of a partial sequence of the 16S rRNA gene inferred using the maximum likelihood method. Bootstrap values 96 are shown. Assessment of bacterial biodiversity DGGE patterns of 16S bacterial gene fragments amplified from field-collected G. Irregulare spores showed a total of 37 migration positions, with 17–24 bands per sample. The three individual spores showed different banding patterns, with only seven bands common to all spores and between five and nine bands unique to each spore, indicating that bacterial communities varied markedly among spores. The positive control E.

Coli showed one very bright band and a faint band that was probably a contaminant, while the negative control did not show any band. DGGE pattern of the V9 portion of the 16S rRNA gene. Lanes 1–3 show bacterial DNA from one individual Glomus irregulare spore taken from natural soil.

Lane 5 shows a positive control ( Escherichia coli) and lane 6 shows a negative control (sterile water). Lanes M show markers Positions in marker from top to bottom are Cellvibrio vulgaris NCIMB 8633, uncultured bacterium clone LO13.6, uncultured bacterium clone LO13.6, Pseudomonas sp. Gu5828, Bacillus sp.

TH64, uncultured bacterium O18F3 and Alteromonadaceae bacterium LA34A. Interactions between bacteria and G. Irregulare mycelium When inoculated on G. Irregulare mycelium grown in vitro, bacterial isolates grew exclusively along hyphae and around spores and showed different growth speed and patterns. Some bacterial isolates, such as B. Simplex and Pseudomonas sp.

, showed profuse development around hyphae after 15–30 days of incubation. Other isolates, such as Kocuria rhizophila, Variovorax paradoxus and Microbacterium ginsengisoli , showed very little growth after 30 days, but significant growth and attachment after 45 days of incubation. On the other hand, Bacillus cereus showed little if any growth on the fungal surface (data not shown). In contrast to the spore-collected bacteria, the non-soil control bacteria E. Coli showed no growth on the water media and little affinity to the fungal surface. As expected, negative controls using sterile water inoculated either on the mycelium or on the media without mycelium showed no growth (data not shown). The growth and attachment of the bacterial isolates on the hyphal surface are summarized in the Supporting Information.

Bacterial growth patterns on Glomus sp. Hyphae cultivated in vitro and observed with a DIC microscope using a × 63 objective. (a) Bacillus simplex; (b) Kocuria rhyzophila; (c) Bacillus megaterium; (d) Variovorax paradoxus; (e) Sphingomonas sp.; (f) Microbacterium ginsengisoli; (g) Pseudomonas sp.; and (h) Escherichia coli. (a) and (g) bacteria are shown after 15 days of growth; (b) and (c) bacteria after 30 days of growth while (d)–(f) and (h) bacteria are shown after 45 days of growth.

Images in (d), (e) and (h) were acquired using confocal microscopy. Scale bars=10 μm. Discussion This study aimed to isolate soil bacteria closely associated with the mycelium of the AMF G.

Irregulare grown in vitro. We developed an experimental Petri dish system to study the hyphal attachment of bacteria in the absence of nutrients other than those derived from the mycelium and monitored the interactions of bacteria inoculated at similar concentrations on the mycelium and incubated for 15, 30 or 45 days before observation.

It is well known in the literature that bacterial colonization of the rhizosphere is extremely important for plant–microorganism interactions including pathogenic and symbiotic interactions. In addition, research in microbial ecology has revealed that bacteria from soil adhere specifically to AMF hyphae. However, these interactions are quite complex and community structure changes are affected by many factors. In the present work, 29 bacterial morphotypes associated with the AMF G.

Irregulare spores were recovered successfully. 16S rRNA gene sequencing showed that they belong to only seven different bacterial species: B. Cereus, Bacillus megaterium, B.

Rhizophila, M. Ginsengisoli, Sphingomonas sp.

Paradoxus. Interestingly, V.

Paradoxus was the most frequent bacterial taxon isolated from spores (13 morphotypes out of 29). All these bacterial taxa are commonly found in soil. The results supported the hypothesis that some bacteria adhering onto the spore surface and likely living on the AMF mycelium surface were able to grow in vitro with AMF hyphae as the sole energy source.

We tested the growth of the isolated bacteria on sterile water solidified with gellan gum lacking nutrients and we did not observe any bacterial growth. This test shows that these bacteria are not able to grow on gellan gum alone. It is likely that the bacterial taxa having grown around G. Irregulare hyphae in vitro were mostly taxa adapted to metabolize the molecules released by the hypha because no other nutrients were available. However, it is also likely that this method would isolate only the most competitive and fast-growing taxa whose portion of the total bacterial biodiversity is unknown.

Estimated that standard microbiological techniques may allow the growth of only about 1% of the soil bacterial taxa from environmental samples. Some bacterial taxa possibly present in the samples could have been obligatory biotrophs because such associations have been found previously in AMF spores. A higher number of replicates could have allowed the isolation of numerous other bacterial taxa as suggested by DGGE that explored the biodiversity of bacterial communities present on AMF spores collected from a field. Moreover, reported that bacteria from the Oxalobacteraceae family are abundant and adhere to AMF hyphae.

Other reports hypothesized that Oxalobacteraceae may specifically interact with mycorrhizal fungi. Have found different Pseudomonas spp., Herbaspirilium sp., Acidobacterium sp., Bacillus spp. And Verrucomicrobium sp. Specifically associated with G.

Irregulare or Glomus mosseae. However, although we isolated three members of the Bacillus genus in our study, they were less abundant and represent only six morphotypes in contrast to V. Paradoxus, which represents 13 morphotypes. Using DGGE, we assessed the bacterial biodiversity of washed spores of G. Irregulare isolated from soil.

DGGE patterns from three field-collected spores were markedly different in the number of bands formed, but mainly in their migration positions, indicating a widely different community structure between spores. The number of bands ranged from 17 to 24, and although 29–41% of the band positions were common to all spores, 28–38% were unique to each one. The markedly variable banding pattern seen on the DGGE clearly shows that a much higher number of bacterial taxa were associated with the spores than the number suggested by isolation. Soils may contain noncultivable taxa or taxa with specific nutritional needs that were not met with the isolation protocol used in this study. Some of the bacterial isolates recovered from the spores were also analyzed in DGGE. However, our DGGE analyses confirmed that we isolated only a very small proportion of bacterial taxa living on the surface of AMF spores.

In addition, bacterial community structure varies considerably among spores. It should be remembered that we cannot be certain whether these spores originate from the same location because we mixed six samples taken from six sites spread along a 50-m distance to reduce the bias due to variation in the local composition of the soil.

Soil biotic and abiotic conditions where each AM spore used was taken could then differ markedly and change the bacterial pattern associated with AMF spore. When we monitored the interactions between the isolated bacteria and the mycelium of G. Irregulare based on morphological growth and adherence, we found that all bacterial taxa except for B. Cereus grew and adhered on the surface of hyphae and spores (Table S1 and ). The growth rates and patterns were, however, different between taxa. For example, B. Cereus was rarely detected and grew very slowly.

In contrast, B. Simplex grew quickly and strongly adhered on the hyphal surface. Megaterium shows significant growth, but no adherence on the G.

Biologie Des Microorganisms Pdf Viewer Software

Irregulare mycelium, V. Paradoxus was fast growing and formed a dense colony around hyphae after only 45 days of incubation. This member of Bulkhoderiales was reported previously as a frequently isolated species in the Glomus intraradices hyphosphere and was also recovered from the hyphosphere of G. The taxon was shown to promote plant growth. The second most often isolated species was M. This strain also showed adherence after 45 days of incubation. Kocuria rhizophila, a soil actinomycete, showed abundant growth and adherence after 30 days of incubation, while the Sphingomonas sp.

Isolate showed slow growth and little adherence on hyphae. Microbacterium and Sphingomonas genera were shown to have a potential for bioremediation by degrading hydrocarbon. The Pseudomonas isolate used here as a control soil bacteria was not isolated from AMF spores, but was rather recovered from a black spruce rhizosphere, an ectomycorrhizal tree species not forming associations with AMF.

Coli strain was used as a non-soil bacterial control and did not show any adherence to the fungal surface. The bacterial isolates growing in close to loose association with the AMF mycelium may play important roles in association with the mycorrhizal symbiosis. For example, certain bacterial strains could improve mineral availability for AMF and the plant or could be antagonistic to certain opportunistic pathogenic organisms and improve the stability of the plant–AMF association. However, the data presented in this study cannot be extrapolated to the natural soil because we isolated and studied only the bacteria that can grow with hyphal exudates as the only nutrient source, but those existing in the soil and associated with AMF that may use additional nutrient sources were not included in this study. Understanding the interactions between AMF and bacteria and their biodiversity will advance our knowledge on microbial ecology in soil and therefore could have the potential to sustain modern agriculture systems with the use of AMF and associated bacterial as biofertilizers or in bioremediation. Acknowledgements This work was supported by NSERC discovery grants to both M.S.-A. We thank the Canada Foundation for Innovation (CFI) for microscopy facility support to M.H.

We also thank Maureen Marie-Joseph for technical assistance, Dr David Morse for comments and English editing and Dr G.V. Blomberg for kindly providing fluorescent protein plasmid vectors.

Ancillary Article Information.