Its. Eighteen chosen strains had been assessed for siderophore production in line with
Its. Eighteen chosen strains have been assessed for siderophore production according to 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.five of Ca3 (PO4 )2 to each and every medium as insoluble P supply. In both assays, Pseudomonas fluorescens2. Components and Methods2.1. Soil Sampling, Bacterial Isolation, and Azotobacter CB1 Storage & Stability Reference Strains. In total, 74 bulk soil samples (00 cm) have been collected from agricultural (53 samples) and non-agricultural websites (21 samples) through spring 2006. Samples belonged to 38 distinctive locations of Northwest, Pampas, and Patagonia regions of Argentina (see Supplementary Material accessible on line at dx.doi.org/10.1155/2013/519603). Soil aggregates (2 mm) had been spread onto the surface of Petri dishes containing N-free Burk’s agar medium with mannitol as C-source [1]. Soon after five days at 28 C, slimy and glistening Azotobacter-like colonies expanding about soil particles were selected and further 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 Planet Journal BNM233 (Banco Nacional de Microorganismos, Buenos Aires, Argentina) was employed as a constructive control. Auxin production was determined applying a colorimetric assay [20], with measurements following 1, two, 3, and 5 days of growth in modified LG (LGSP) liquid medium containing 1 sucrose and 0.5 soymeal peptone. At every 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], utilizing a Hewlett Packard Series II 5890 equipped with a flame ionization detector (FID) as well as a stainless-steel Porapak N column (three.two mm two m; 80/100 mesh). The injector, oven, and detector temperatures had been 110 C, 90 C, and 250 C, respectively. N2 was employed as carrier gas (4.5 cm s-1 linear gas velocity). Total protein concentration of bacterial cells was determined by the Lowry strategy together with the DC Protein Assay kit (BioRad, USA). Nitrogenase activity was expressed as mmol ethylene made per mg of protein in 24 h. Indole-3-acetic acid (IAA), gibberellic acid (GA3 ), and zeatin (Z) production were 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 were 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 Options around 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) had been surface-disinfected (1 NaClO for three minutes) and germinated in plastic containers (15 25 four cm) on filter paper soaked with sterile distilled water. To preserve humidity, containers were wrapped in transparent plastic bags and JNK Storage & Stability placed in a development chamber at 25 C having a 16 h light/8 h dark regime for 24 h. For inoculation, bacterial strains were 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 eight days as described ab.
