Soil Microcosms and Experimental Setup

For all experiments, soil from an agricultural field in Buinen, the Netherlands, was used (denoted B soil). The B soil was characterized as a loamy sand soil, with pH 5.3 and an organic matter content of 3.8% [3]. For some experimental treatments, the soil was adjusted to reduced pH values (about 4.6, 4.2, and 3.8) by adding different amounts of 0.2 M H₂SO₄ and subsequent mixing. Then, soils were autoclaved (121 °C, 27 min) for three times, with intermittent incubation at room temperature. The maintenance of the different pH values was confirmed following the last autoclaving before incubation. Soil pH was measured in water (soil/water = 1:5, w/v). Following these soil pH modulations, soil microcosms were prepared, using three-compartment petri dishes in accordance with previous studies [2, 4, 5]. Briefly, one of the compartments was filled with OFA, and the other two with either one of the pretreated B soil portions (adjusting soil moisture content to 12 or 17%, corresponding to 42 or 60% of water holding capacity [WHC], measured before the soil microcosm preparation to make the experimental conditions consistent). Using the soil microcosms with differently set pH (5.3; 4.6; 4.2; 3.8) and moisture content (12, 17%) values, OFA plugs containing fungal growth were placed in the OFA compartments, and the systems were incubated at 28 °C. Control systems received no fungus. Following incubation, with fungal growth reaching up to 20 (or 30) mm into the soil compartments, bacterial cells were introduced either at the tip of fungal growth or in the middle of the hyphal growth area. Specifically, in experimental set 1, the bacterial cells were introduced at the tip of fungal growth when this reached 20 mm into the soil. In experimental set 2, the bacterial cells were introduced 10 mm away from the tip of fungal growth when this tip reached 30 mm into the soil. Specifically, the bacterial strains (separate introductions for all strains used) were introduced at 5 × 10⁵ cells per soil compartment, establishing a 45 × 3 mm (length x width) inoculated soil zone, and systems were incubated at 28 °C. At set times (4, 7, and 15 days after bacterial inoculation), small samples were recovered, by punching out, from the introduction, “backward” (just behind the barrier) and “forward” (hyphal migration front) sites. All samples were suspended in water, shaken intensely (1 min, three times, with 30-s intervals), serially diluted, and spread on R2A agar plates. Following incubation of the plates, colonies were enumerated and CFU numbers per gram soil were calculated. For each experimental treatment, three replicate microcosms were used. The non-fungal controls, examined similarly, consistently revealed the absence of CFUs on the plates.

To assess the P. terrae BS001 population dynamics in the B soil, fresh washed suspensions of all strains (BS001 wild type, BS001ΔsctD, BS001ΔpilN, and BS001ΔfliF) were introduced separately into the different microcosms in the absence of fungal hyphae, and population densities were monitored over time.