Its. Eighteen chosen strains had been assessed for siderophore production in accordance with
Its. Eighteen selected strains were assessed for siderophore production in accordance with the O-CAS method [17]. Phosphate-solubilizing activity was tested on Pikovskaya medium [18], NBRIP medium [19] and modified Burk’s agar medium [1], adding 0.5 of Ca3 (PO4 )two to every medium as insoluble P supply. In both assays, Pseudomonas fluorescens2. Components and Methods2.1. Soil Sampling, Bacterial Isolation, and Azotobacter Reference Strains. In total, 74 bulk soil samples (00 cm) have been collected from agricultural (53 samples) and non-agricultural internet sites (21 samples) through spring 2006. Samples belonged to 38 distinctive places of Northwest, Pampas, and Patagonia regions of Argentina (see Supplementary Material available on-line at dx.doi.org/10.1155/2013/519603). Soil aggregates (2 mm) were spread onto the surface of Petri dishes containing N-free Burk’s agar medium with mannitol as C-source [1]. Soon after 5 days at 28 C, slimy and glistening Azotobacter-like colonies increasing around soil particles were selected and additional purified in N-free LG with bromothymol blue agar medium [1]. Motility, pigment production, and encystment had been determined as previously described [1].The Scientific World Journal BNM233 (Banco Nacional de Microorganismos, Buenos Aires, Argentina) was employed as a positive control. Auxin production was determined employing a IRAK4 custom synthesis colorimetric assay [20], with measurements immediately after 1, two, 3, and 5 days of growth in modified LG (LGSP) liquid medium containing 1 sucrose and 0.5 soymeal ACAT2 list peptone. At each time interval, the amount of cells (cfu mL-1 ) was determined by plate counting on LG agar. Nitrogenase activity was estimated by the acetylene reduction assay. Bacterial cultures have been grown in N-free Burk’s agar medium at 28 C for 24 h and ethylene production was measured by gas chromatography [21], working with a Hewlett Packard Series II 5890 equipped having a flame ionization detector (FID) in addition to a stainless-steel Porapak N column (three.two mm 2 m; 80/100 mesh). The injector, oven, and detector temperatures were 110 C, 90 C, and 250 C, respectively. N2 was made use of as carrier gas (4.five cm s-1 linear gas velocity). Total protein concentration of bacterial cells was determined by the Lowry strategy with the DC Protein Assay kit (BioRad, USA). Nitrogenase activity was expressed as mmol ethylene produced per mg of protein in 24 h. Indole-3-acetic acid (IAA), gibberellic acid (GA3 ), and zeatin (Z) production have been determined for six selected Azotobacter spp. strains grown in LGSP liquid medium at 28 C for 8 days. Z was identified and quantified by HPLC-UV, whereas IAA and GA3 have been identified by gas chromatography-mass spectrometry with selective ion monitoring (GC-MS-SIM), as previously described [21]. 2.7. Effects of Azotobacter Inoculation and IAA Pure Solutions on the Number of Seminal Roots and Root Hairs of Wheat Seedlings. For plant tests, seeds of wheat (Triticum aestivum cv. Baguette Premium 13, Nidera, Buenos Aires, Argentina) have been surface-disinfected (1 NaClO for three minutes) and germinated in plastic containers (15 25 4 cm) on filter paper soaked with sterile distilled water. To sustain humidity, containers have been wrapped in transparent plastic bags and placed in a development chamber at 25 C with a 16 h light/8 h dark regime for 24 h. For inoculation, bacterial strains have been grown in LGSP liquid medium at 28 C for eight days (108 cfu mL-1 ). Fifteen pregerminated seeds had been inoculated with 100 L of bacterial culture (107 cells) per seed and grown for 8 days as described ab.
