(soil bacteria not isolated from AMF spores) were used as controls to compare the bacterial growth and attachment on fungal mycelium. Sampling was conducted in the Mirabel–Lachute area (Québec, Canada) on June 2006 in a natural
stand colonized with herbaceous and deciduous shrubs growing on a sandy soil. The sampling site was chosen because a G. irregulare isolate [formerly Glomus intraradices (Sokolski et al., 2010)] has been identified previously from AM fungal spores harvested from A. stolonifera L. roots collected from the same site in 1991 (Y. Dalpé, pers. commun.). Six A. stolonifera plants with roots and approximately 1 L of the surrounding soil were collected. GPS coordinates of the six sampling sites were between 45°41′36.35″N 74°08′33.32″W and 45°41′33.52″N 74°08′31.50″W. Dinaciclib solubility dmso There was a distance of about 10 m between each plant collected. Samples were kept at 4 °C and processed a few days
after sampling. To reduce the bias due to variation in the local composition of the soil, a composite sample was prepared by mixing six 100-g rhizosphere soil samples from each sampling site. This composite soil sample was CDK activation used for spore extraction. AM fungal spores were isolated by wet soil sieving (45 μm pore size) and sucrose density gradient centrifugation (Vilarino & Arines, 1990). Collected spores were kept in sterile water at 4 °C until use. The identity of collected spores was determined by PCR amplification and sequencing of 18S rRNA genes, as described in Yergeau
et al. (2006). Glomus irregulare isolate DAOM 197198 was grown on root-inducing (Ri T-DNA) transformed carrot roots in two-compartment Petri dishes, as described in St-Arnaud et al. (1995). The first compartment containing the transformed carrot roots was filled with 20 mL M medium, while the second compartment received 20 mL sterile water solidified with 0.4% (w/v) gellan gum (Sigma), containing 0.74 g L−1 MgSO4, but lacking nutrients. Plates were incubated for approximately 6 weeks at 25 °C in the dark until hyphae crossed the central wall and colonized unless the second compartment. Glomus irregulare spores harvested from the field were cleaned with autoclaved MilliQ water and cleaned spores were placed directly in contact with hyphae growing in the second compartment. An additional incubation period of 4 weeks at 25 °C in the dark was required to allow bacterial growth. Mixed bacterial colonies growing around hyphae were isolated and purified by successive inoculations on 10% tryptic-soy agar medium (TSA, QueLab Laboratories, Canada). To test whether these bacterial colonies were growing on organic residues contained in the gellifying agent, we also inoculated all isolated bacteria on sterile water solidified with 0.4% gellan gum as described above. After 1 week of incubation, no bacteria growth was observed, while bacterial colonies were clearly visible on 10% TSA medium. The complete 16S rRNA gene was used to identify bacterial isolates.
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