We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA(3)), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA(3), ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA(3) were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 microg ml(-1)). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 microg ml(-1)). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus L-methionine (3.94 ng ml(-1) h(-1)) and Az39 cultured in NFb plus L-lysine (36.55 ng ml(-1) h(-1)). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.

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The aim of this work was to evaluate phytohormone biosynthesis, siderophores production, and phosphate solubilization in three strains (E109, USDA110, and SEMIA5080) of Bradyrhizobium japonicum, most commonly used for inoculation of soybean and nonlegumes in USA, Canada, and South America. Siderophore production and phosphate solubilization were evaluated in selective culture conditions, which had negative results. Indole-3-acetic acid (IAA), gibberellic acid (GA(3)), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS-flame ionization detection and high-performance liquid chromatography (HPLC-UV), respectively. IAA, zeatin, and GA(3) were found in all three strains; however, their levels were significantly higher (p < 0.01) in SEMIA5080 (3.8 microg ml(-1)), USDA110 (2.5 microg ml(-1)), and E109 (0.87 microg ml(-1)), respectively. ABA biosynthesis was detected only in USDA110 (0.019 microg ml(-1)). Ethylene was found in all three strains, with highest production rate (18.1 ng ml(-1) h(-1)) in E109 cultured in yeast extract mannitol medium plus L-methionine. This is the first report of IAA, GA(3), zeatin, ethylene, and ABA production by B. japonicum in pure cultures, using quantitative physicochemical methodology. The three strains have differential capability to produce the five major phytohormones and this fact may have an important technological implication for inoculant formulation.

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Soybean seeds are non-sterile and their bacterial population interferes with the enumeration of beneficial bacteria, making it difficult to assess survival under different conditions. Within this context, the principal aims of this work were: (1) to improve a selective media for the enumeration of B. japonicum recovered from inoculated soybean seeds; (2) to establish the most representative mathematical function for B. japonicum mortality on soybean seeds after inoculation; (3) to evaluate if environmental or physiological conditions modify B. japonicum mortality on soybean seeds; and (4) to create a new protocol for quality control of soybean inoculants. We successfully evaluated the combination of pentachloronitrobenzene and vancomycin added to the yeast-mannitol medium to inhibit most fungi and Gram-positive soybean microbiota, thus producing reliable counts of B. japonicum from inoculated soybean seeds. Percentages of recovery and survival factors were obtained and used to construct a two-phase exponential decay non-linear regression function. High temperature and desiccation decreased these parameters, while the optimization of temperature and the use of osmoprotective compounds with inoculants increased them. The use of this protocol minimized heterogeneity between experiments and may be considered more reliable than the simple expression of direct colony count of bacteria recovered from seeds.