Growth behaviour and indole acetic acid (IAA) production by a Rhizobium isolated from root nodules of Alysicarpus vaginalis DC

From the root nodules of Alysicarpus vaginalis DC, the symbiont was isolated and identified as a Rhizobium sp. The bacteria produced a high amount (107 microg/ml) of indole acetic acid (IAA) in culture from tryptophan supplemented yeast extract mannitol medium. The isolate preferred L-isomer of tryptophan for maximum IAA production. The production was maximum when the bacteria reached its stationary phase of growth. The production of IAA could be increased up to 70% over yeast extract glucose medium by supplementing ZnSO4, 7H2O (0.5 microg/ml). L-asparagine (0.2%) and sodium dodecyl sulfate (1.0 microg/ml). The possible relationship between the rhizobial IAA production and legume-rhizobia symbiosis is discussed.     

Indole acetic acid production by a Rhizobium species from root nodules of a leguminous shrub, Cajanus cajan

A Rhizobium sp. isolated from the root nodules of a leguminous pulse-yielding shrub, Cajanus cajan, was found to produce high amounts (99.7 microg/ml) of indole acetic acid (IAA) during growth in basal medium supplemented with L-tryptophan. The Rhizobium sp. was a fast growing species which reached its stationary phase at 28 h. The IAA production could be increased upto 653.3% over control by supplementing the carbon-free incubation medium with glucose (5 g/l), NiCl2 (10 microg/ml) and glutamic acid (0.5 g/l). The possible role of rhizobial IAA on the rhizobia-legume symbiosis is discussed.     

Studies on the root nodules of leguminous plants: The production of indole acetic acid in culture by a Rhizobium sp. from a leguminous climbing shrub Derris scandens benth

The Rhizobium sp. When isolated form the root nodules of a leguminous climbing shrub Derris scandens produced a high amount of indole acetic acid (IAA) (135.2 μg/ml) from the tryptophan-supple-mented basal medium. Growth and IAA production started simultaneously, and the maximum amount of IAA was produced as a secondary metabolite in the stationary phase of growth. The IAA production by the Rhizobium sp. was increased by 503% when the medium was supplemented with mannitol (2%), KNO₃ (0.2%), nicotinic acid (0.1 μg/ml) and MnSO₄ (1 μg/ml) in addition to tryptophan (4 mg/ml)/ The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is also discussed.     

Production of 3-indolylacetic acid in root nodules and culture by a <Emphasis Type="Italic">Rhizobium</Emphasis> species isolated from root nodules of the leguminous pulse <Emphasis Type="Italic">Phaseolus mungo</Emphasis>

The root nodules of Phaseolus mungo (a herbaceous leguminous pulse) contained a high amount of 3-indolylacetic acid (IAA). A tryptophan pool present in the nodule might play the role of precursor for IAA production. From the root nodule a Rhizobium sp. was isolated. The symbiont produced a large amount of IAA (142 mug/mL) from L-tryptophan-supplemented basal medium. The production of IAA by the symbiont was much increased over the control when a L: -tryptophan (2 mg/mL) supplemented C-free mineral medium was enriched with mannitol (1 %), L: -asparagine (0.3 %) and thiamine hydrochloride (1 mug/mL). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Studies on root nodules of leguminous plants bioproduction of indole acetic acid by a Rhizobium sp. from a twiner Clitoria ternatea L.

The Rhizobium sp. isolated from the root nodules of Clitoria ternatea L., a leguminous twiner, produced a high amount of IAA (16.4 μg/ml) from tryptophan in an unsupplemented basal medium. The production of IAA started simultaneously with the growth and had no different growth and production phase. The growth and production were parallel and increased up to 45–50 h. The IAA production by the Rhizobium sp. was increased by 520% when the medium was supplemented with fructose (1.5%), MnSO₄ (1.0 μg/ml), riboflavin (0.10 μg/ml) and Triton X-100 (0.01%). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Auxin production by bacteria associated with orchid roots

Bacteria associated with the roots of greenhouse tropical orchids were shown to produce indole-3-acetic acid (IAA) and to excrete it into the culture liquid. The presence and activity of IAA were demonstrated colorimetrically, by thin-layer chromatography, and by biotests. The associated bacteria varied in their ability to excrete indole compounds (1-28 microg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced phytohormone production. Upon addition of 200 microg/ml tryptophan, the bacteria isolated from Dendrobium moschatum roots (Sphingomonas sp. 18, Microbacterium sp. 23, Mycobacterium sp. 1, Bacillus sp. 3, and Rhizobium sp. 5) produced 50.2, 53.1, 92.9, 37.6, and 60.4 microg IAA/ml respectively, while the bacteria isolated from Acampe papillosa roots (Sphingomonas sp. 42, Rhodococcus sp. 37, Cellulomonas sp. 23, Pseudomonas sp. 24, and Micrococcus luteus) produced 69.4, 49.6, 53.9, 31.0, and 39.2 microg IAA/ml. Auxin production depended on cultivation conditions and on the growth phase of the bacterial cultures. Treatment of kidney bean cuttings with bacterial culture liquid promoted formation of a "root brush" with location height 7.4- to 13.4-fold greater than the one in the control samples. The ability of IAA-producing associated bacteria to act as stimulants of the host plant root development is discussed.     

Production of extracellular polysaccharides by a Rhizobium species from root nodules of Cajanus cajan

The ability of the Rhizobium sp., isolated from the root nodules of the leguminous pulse yielding shrub Cajanus cajan, to produce extracellular polysaccharides (EPS) was checked. A large amount of EPS (1, 128 μg/ml) was produced by the bacteria in yeast extract mannitol medium. Growth and EPS production started simultaneously, but the production reached its maximum level in the stationary phase of growth at 28 h. The EPS production by this Rhizobium sp. was much higher than by many other strains from nodules of Cajanus cajan which took a much longer time to reach maximum EPS production than this strain. The maximum EPS production (2,561 μg/ml) was obtained when the medium was supplemented with mannitol (1%), cetyl pyridinium chloride (2 μg/ml) and KNO₃ (0.2%), in which the production was increased by 276% compared to the control. The EPS production rose in the period up to 65 h with increased mannitol concentration. The EPS contained arabinose, xylose and rhamnose monomers. The possible role of rhizobial EPS production in root nodule symbiosis is discussed.     

Production of indole acetic acid in root nodules and culture by a Rhizobium species from root nodules of the fodder legume Melilotus alba DESR.

The root nodules of Melilotus alba DESR ., a fodder legume, contained high amounts of IAA. A tryptophan pool present in the nodule might serve as a source of IAA production. Presence of IAA oxidase and peroxidase in the nodules indicated the metabolism of IAA, at least in part, in the nodules. The Rhizobium species isolated from the root nodules produced a high amount of IAA (190 μg/ml) from L-tryptophan supplemented basal medium. IAA production and microbial growth were coincident. The production of IAA by the Rhizobium sp. was increased by 315% when the medium was supplemented with lactose (1%), NiCl₂ (10 μg/ml), cetyl pyridinium chloride (0.5 μg/ml) and glutamic acid (0.4%), in addition to L-tryptophan (3 mg/ml). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.     

Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027

A low-cost medium, MGRS, has been developed for growth and lipase production from Bacillus THL027 at 65 degrees C and pH 7.0. MGRS was composed of 2% (v/v) buffer solution (7.3% (w/v) Na(2)HPO(4), 3.2% (w/v) KH(2)PO(4), pH 7.2), 40 microg ml(-1) FeSO(4) and 40 microg ml(-1) MgSO(4), 0.1% (w/v) (NH(4))(2)SO(4) supplemented with 3% NaCl, 0.1% glucose, 1.0% rice bran oil and 0.5% (w/v) rice bran. The lipase was purified 2.6-fold to apparent homogeneity by ultrafiltration and gel filtration chromatography. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties.     

Production of indole-3-acetic acid by Thai native orchid-associated fungi

Thirteen endophytic fungi were isolated from roots of three orchid species, Spathoglottis affinis, Paphiopedelum bellatulum and Phaius tankervilleae. Of these, three fungal isolates produced high levels of indole-3-acetic acid (IAA) in culture medium supplemented with 2 mg/ml of L-tryptophan, and were selected for further analysis. Morphological characteristics and a phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that the fungal isolates CMU-SLP 007 and CMU-NUT 013 belonged to family Tulasnellaceae, genus Tulasnella (the anamorphic genus Epulorhiza) and the fungal isolate CMU-AU 006 belonged to Colletotrichum gloeosporioides. These three fungal isolates produced maximum levels of IAA when grown in a culture medium supplemented with 4 mg/ml of L-tryptophan (C. gloeosporioides CMU-AU 006, 243.56 μg/ml and Tulasnella sp. CMU-SLP 007, 155.63 μg/ml) and 6 mg/ml of L-tryptophan (Tulasnella sp. CMU-NUT 013, 104.03 μg/ml). Thin layer chromatography revealed that all fungal IAA presented the same R_f value as the standard IAA. The biological activity of fungal IAA showed that it increased the length of stem forming roots and the number of roots of kidney bean (Phaseolus vulgaris), promoted seed germination, the length of roots and root to shoot ratio of corn (Zea mays) and increased the elongation of rice (Oryza sativa) coleoptiles when compared with all controls (water and culture medium treatments). In addition, the results of all biological activities using fungal IAA indicated that the quality of fungal IAA were similar to standard IAA.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Production of extracellular polysaccharides by a Rhizobium species from the root nodules of Melilotus alba

The Rhizobium sp., isolated from the root nodules of the leguminous fodder herb Melilotus alba, produced large amounts of extracellular polysaccharides (EPS) (963.5 μg/ml) in a yeast extract mannitol medium. Growth and EPS production started simultaneously, but EPS production reached its maximum during the stationary phase of growth of the bacteria, at 20 hours. EPS production was increased with all of the thirteen sugars tested. Different nitrogen sources, such as nitrates, glutamic acid, casamino acid and L-asparagine, increased the EPS production although it was inhibited by glycine, nitrite and ammonium salts. Among the vitamins and metal ions, only pyridoxal phosphate and ZnSO₄ promoted EPS production. Attempts were made to optimize the cultural requirements for growth and maximum EPS production. Maximum EPS production (1457.0 μg/ml) was obtained when the medium was supplemented with glucose (1%), pyridoxal phosphate (2 μ g/ml), ZnSO₄ × 7 H₂O (10 μg/ml) and glutamic acid (0.1%). Under these conditions, the production was increased by 254.3% compared to the control. The EPS contained arabinose, xylose and rhamnose monomers. The presence of arabinose and xylose in the EPS produced by a Rhizobium sp. was uncommon.     

Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth

A total of 40 endophytic bacterial isolates obtained from banana tree roots were characterized for their biotechnological potential for promoting banana tree growth. All isolates had at least one positive feature. Twenty isolates were likely diazotrophs and formed pellicles in nitrogen-free culture medium, and 67% of these isolates belonged to the genus Bacillus sp. The isolates EB-04, EB-169, EB-64, and EB-144 had N fixation abilities as measured by the Kjeldahl method and by an acetylene reduction activity assay. Among the 40 isolates, 37.5% were capable of solubilizing inorganic phosphate and the isolates EB-47 and EB-64 showed the highest solubilization capacity. The isolate EB-53 (Lysinibacillus sp.) had a high solubilization index, whereas 73% of the isolates had low solubilization indices. The synthesis of indole-3-acetic acid (IAA) in the presence of L-tryptophan was detected in 40% of the isolates. The isolate EB-40 (Bacillus sp.) produced the highest amount of IAA (47.88 μg/ml) in medium supplemented with L-tryptophan and was able to synthesize IAA in the absence of L-tryptophan. The isolates EB-126 (Bacillus subtilis) and EB-47 (Bacillus sp.) were able to simultaneously fix nitrogen, solubilize phosphate and produce IAA in vitro. The results of this study demonstrated that the isolates analyzed here had diverse abilities and all have the potential to be used as growth-promoting microbial inoculants for banana trees.     

Factors affecting lipase production in Syncephalastrum racemosum

Syncephalastrum racemosum grown as a static culture showed maximum lipase production at 30°C in 2d at pH 8.0. When the medium was supplemented with fructose, maximum production of lipase per unit of growth was achieved, followed by raffinose, sucrose, ribose, galactose, maltose, lactose, mannitol and glucose. Amongst the nitrogen sources tested, corn steep liquor at 8% (v/v) produced maximum enzyme; there was evidence of catabolite repression by glucose when groundnut protein, soybean meal, milk casein or wheat bran were the sources of nitrogen. Calcium, potassium and sodium citrates, each at 0.1% (w/v), increased the yield of lipase.     

Alternative mechanism for the evaluation of indole-3-acetic acid (IAA) production by Azospirillum brasilense strains and its effects on the germination and growth of maize seedlings

We evaluated the production of indole-3-acetic acid (IAA) by Azospirillum brasilense strains in vitro (cell culture supernatants) and in vivo (stems and roots of maize seedlings) to clarify the role of this phytohormone as a signaling and effector molecule in the symbiotic interaction between maize and A. brasilense. The three strains all showed IAA production when cultured in NFb medium supplemented with 100 μg/ml L-tryptophan. The level of IAA production was 41.5 μg/ml for Yu62, 12.9 μg/ml for Az39, and 0.15 μg/ml for ipdC-. The release of IAA into culture medium by the bacteria appeared to be the main activator of the early growth promotion observed in the inoculated maize seedlings. The application of supernatants with different IAA contents caused significant differences in the seedling growth. This observation provides the basis for novel technological tools for effective quality control procedures on inoculants. The approach described can be incorporated into different inoculation methods, including line sowing, downspout, and foliar techniques, and increase the sustainability of symbiotic plant-bacteria systems.     

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree,Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L‐tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L‐tryptophan. The preferred carbon and nitrogen sources were glucose and KNO₃ and the optimum concentrations 1% and 0.02%, respectively. FeSO₄ × 7 H₂O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO₄ × 7 H₂O (10 μg/ml), KNO₃ (0.02%) as well as EDTA (5 μg/ml) and L‐tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree‐Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.     

Gas chromatography-mass spectrometry analysis of indoleacetic acid and tryptophan following aqueous chloroformate derivatisation of Rhizobium exudates.

A new method for preparing alkyl esters of indole-3-acetic acid (IAA) in aqueous solution is adapted from the chloroformate method originally described by Husek for the analysis of amino acids. This method has the significant advantage of avoiding the generation and use of diazomethane, and is done in aqueous solution without the need to dry the sample with concomitant non-specific losses of IAA. The effectiveness of this method is demonstrated by its use in an isotope dilution gas chromatography-mass spectrometry (GC-MS) assay of IAA and L-tryptophan (Trp) in the culture supernatant of a series of Sinorhizobium meliloti and Rhizobium leguminosarum bv. trifolii strains that can interact with rice to either enhance or inhibit rice plant growth. We were testing the hypothesis that the rice growth inhibition was related to the biosynthesis of IAA. It was found that S. meliloti and Rhizobium strains produced high amounts of IAA in Trp supplemented BIII minimal medium compared to BIII media. All the strains produced more than the minimum amount of IAA required to inhibit rice growth and thus IAA is not the major inhibitory factor of rice seedling growth from S. meliloti and Rhizobium strains.     

Morphogenesis in hypocotyl cultures of Digitalis obscura: Influence of carbohydrate levels and sources

Hypocotyl explants of Digitalis obscura L. were grown on Murashige and Skoog medium supplemented with 0.57 μM IAA and 4.40 μM BA. The effects of sucrose, maltose, glucose, galactose or mannitol on their growth and bud formation were investigated. None of the carbohydrate sources tested was superior to sucrose, and best results were obtained with 2.0% (w/v) of this disaccharide. Although mannitol did not support morphogenesis, it had a promotive effect on bud formation when added to 1.0 or 1.5% sucrose-supplied media to give the molar sucrose equivalent to 2.0%. The inhibitory effect of high sucrose concentrations could be duplicated by substituting mannitol for sucrose on a molar basis. Our data suggest a dual role of sucrose as osmotic and energy source in D. obscura hypocotyl cultures.     

Extracellular polysaccharide production by aRhzobium sp. from root nodules ofDerris scandens

TheRhizobium sp. isolated from the root nodules of the leguminous climbing shrubDerris scandens produced a large amount of extracellular polysaccharides in a yeast extract—mannitol medium in the stationary phase of growth. The production was maximum when the medium was supplemented with mannitol (3%), (+)-biotin (3 mg/L) and KNO₃ (0.3%). The extracellular polysaccharides contained glucose, galactose and mannose. The possible role of the rhizobial extracellular polysaccharide is discussed.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.