Characterization of Isolates (PGPR Capabilities)

In order to evaluate the capability of inorganic phosphate solubilization, Pikovskayas Agar plates were used (Gupta et al., 1994). Liquid precultures were grown to an OD₆₀₀ of 0.2–0.3 in MAG medium, and droplets of 10μl were applied to agar plates, 4 per plate, two experiments. Incubation was conducted for a minimum of 1week at 28°C with Bacillus subtilis DSM 10 as positive control. The capacity for phosphate solubilization was indicated in this assay by the formation of a white to translucent halo formation around the bacterial colony, rated only weakly positive when it took 8weeks to reach the reaction.

A colorimetric assay was used to indicate the capability for synthesis of indole-3-acetic acid (IAA) from L-tryptophan (Gordon and Weber, 1951). Precultures were grown in 20ml of liquid MAG medium cultures containing 6mM L-tryptophan for 2–5days to an optical density of approximately 1 at 28°C, depending on the growth speed of the strains. Five tenths milliliter of preculture was inoculated to the main culture (medium as above) and grown overnight. After removing cells by centrifugation for 10min, 1ml of supernatant was incubated with Salkowski reagent [0.5M iron (III) chloride in 35% perchloric acid] for 45min in the dark. An absorption peak at 530nm involving a color change from yellow to red indicated the formation of IAA. An absorbance at 530nm of 0.1–0.2 was rated as weak reaction, of around 0.6 as positive reaction. Azospirillum brasilense SP7 was used as an IAA-producing positive reference (Ona et al., 2003), and Azoarcus olearius BH72 as a negative control. Assays were done in duplicate.

For the detection of siderophore production, a universal assay based on chrome azurol (CAS) and hexadecyltrimethylammonium bromide (HDTMA) bound to iron as indicating complex (Schwyn and Neilands, 1987) was used. Since compounds of the CAS/HDTMA complex may be toxic to cells, the agar plates were overlaid with MAG agar. Liquid precultures were grown to an OD₆₀₀ of 0.2–0.3 in MAG medium, pelleted, and resuspended in SM medium without nitrogen (SM-N, Reinhold et al., 1986) to an optical density of 2; 10μl of the cell suspension were dropped on CAS Agar (four droplets replicated, two experiments), and incubated at 28°C 1–4weeks. The production of siderophores as iron chelators results in the removal of iron from the dye complex and a color change from green to orange as a halo around the colony. Azospirillum brasilense Sp7 was used as positive control.

Tests for temperature tolerance were carried out as previously described (Grönemeyer et al., 2014), with maximum growth temperature above 35°C (i.e., growth, at least, at 36°C) regarded as temperature tolerant (Grönemeyer and Reinhold-Hurek, 2018). Fresh overnight precultures in MAG medium grown to OD₆₀₀ of 0.2–0.5 at 28°C were used to inoculate the main cultures with a starting OD₆₀₀ of 0.001 in 20ml of MAG medium. Incubation at 28, 32, 36°C or higher temperature was maintained in a water bath with reciprocal shaking, with three replicate cultures. Cultures were inspected for visible growth within 7days of incubation. A similar set-up was used to analyze NaCl tolerance in 5ml of MAG broth at 28°C with rotary shaking, with 0.5 or 0.75% NaCl added. Moderate salt tolerance was assumed for strains growing with 0.5% NaCl, tolerant strains growing at 0.75% NaCl (Gao et al., 1994; Yao et al., 2015).