4.2. Soil and Biological Materials

The soil used was a loam and was collected on 1 September 2021 at the grounds of IFAPA (Granada, Spain), sieved (2 mm), diluted with quartz sand (<1 mm) (1:1, soil:sand, v/v) and sterilized by steaming (100 °C for 1 h on 3 consecutive days). The soil pH was 8.1 (water) and contained 0.85% organic matter with the following nutrient concentrations (mg kg⁻¹): N, 1; P, 10 (NaHCO₃-extractable P); and K, 110. The soil texture was made of 38.3% sand, 47.1% silt and 14.6% clay.

Seeds of maize (Zea mays L.), cultivar PR34B39, were provided by Pioneer Hi-Bred, Spain (DuPont Pioneer Corporation, Johnston, IA, USA). Seeds were pre-germinated on moist sand for 5 days (from 7 to 12 September 2021) and then transferred to pots of 1.5 L capacity containing 1250 g of the soil/sand mixture described above on 12 September 2021. A total of 120 pots were prepared, each one containing one maize seedling.

Mycorrhizal inoculum was bulked in an open-pot culture of Z. mays L. and consisted of soil, spores, mycelia and infected root fragments. The AM fungus was Rhizophagus irregularis (Schenck and Smith), belonging to the Zaidin Experimental Station (EEZ) Collection, strain EEZ 58. Ten grams of inoculum with about 65 infective propagules per gram (according to the most probable number test) was added to the appropriate pots at the time of seedlings transplantation to pots (12 September 2021). Non-inoculated control plants received the same amount of autoclaved mycorrhizal inoculum together with a 3 mL aliquot of a filtrate (<20 µm) of the AM inoculum in order to provide a general microbial population free of AM propagules.

Appropriate pots were inoculated with a Bacillus megaterium strain isolated and tested in previous studies [36,38,83,84]. For that, two days before starting this experiment, B. megaterium strain was grown in nutrient broth medium for 48 h at 28 °C and then centrifuged at 4500× g for 5 min. The pellet was suspended in sterilized water. One milliliter of the suspension containing 10⁸ cfu mL^–1 was added to each pot at the time of the seedlings’ transplantation to pots (12 September 2021) and repeated seven days later (19 September 2021).