Its. Eighteen chosen strains were assessed for siderophore production in accordance with
Its. Eighteen chosen strains have been assessed for siderophore production as outlined by the O-CAS technique [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 )two to each medium as insoluble P source. In each assays, Pseudomonas fluorescens2. Components and Methods2.1. Soil Sampling, Bacterial Isolation, and Azotobacter Reference Strains. In total, 74 bulk soil samples (00 cm) were collected from agricultural (53 samples) and non-agricultural web sites (21 samples) during spring 2006. Samples belonged to 38 unique areas of Northwest, Pampas, and Patagonia regions of Argentina (see Supplementary Material readily available on the net at dx.doi.org/10.1155/2013/519603). Soil aggregates (two 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 5 days at 28 C, slimy and glistening Azotobacter-like ErbB3/HER3 MedChemExpress colonies growing about soil particles had been chosen and additional purified in N-free LG with bromothymol blue agar medium [1]. Motility, pigment production, and encystment have been determined as previously described [1].The Scientific Globe Journal BNM233 (Banco Nacional de Microorganismos, Buenos Aires, Argentina) was used as a optimistic control. Auxin production was determined utilizing a colorimetric assay [20], with measurements just after 1, two, 3, and five days of development in modified LG (LGSP) liquid medium containing 1 sucrose and 0.five soymeal CXCR3 supplier 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], employing a Hewlett Packard Series II 5890 equipped having a flame ionization detector (FID) and a stainless-steel Porapak N column (3.2 mm 2 m; 80/100 mesh). The injector, oven, and detector temperatures had been 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 method 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 had been determined for six chosen Azotobacter spp. strains grown in LGSP liquid medium at 28 C for eight days. Z was identified and quantified by HPLC-UV, whereas IAA and GA3 had 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) 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 placed inside 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 8 days (108 cfu mL-1 ). Fifteen pregerminated seeds were inoculated with 100 L of bacterial culture (107 cells) per seed and grown for eight days as described ab.