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.     

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.     

Stimulation of indoleacetic acid production in a <Emphasis Type="Italic">Rhizobium</Emphasis> isolate of <Emphasis Type="Italic">Vigna mungo</Emphasis> by root nodule phenolic acids

The influence of endogenous root nodules phenolic acids on indoleacetic acid (IAA) production by its symbiont (Rhizobium) was examined. The root nodules contain higher amount of IAA and phenolic acids than non-nodulated roots. Presence of IAA metabolizing enzymes, IAA oxidase, peroxidase, and polyphenol oxidase indicate the metabolism of IAA in the nodules and roots. Three most abundant endogenous root nodule phenolic acids (protocatechuic acid, 4-hydroxybenzaldehyde and p-coumaric acid) have been identified and their effects on IAA production by the symbiont have been studied in l-tryptophan supplemented yeast extract basal medium. Protocatechuic acid (1.5 μg ml⁻¹) showed maximum stimulation (2.15-fold over control) of IAA production in rhizobial culture. These results indicate that the phenolic acids present in the nodule might serve as a stimulator for IAA production by the symbiont (Rhizobium). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

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.     

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.     

Content of indoleacetic acid and its metabolism in root nodules ofMelilotus alba

The root nodules ofMelilotus alba, a leguminous fodder herb, contain a high amount of indoleacetic acid (IAA). The tryptophan pool present in the nodule might serve as a source for the IAA production. Metabolism of IAA in the nodules was evidenced by the presence of IAA-metabolizing enzymes, IAA oxidase and peroxidase. A high amount of IAA was produced by the symbiont isolated from the nodules in culture, when supplemented with tryptophan. For IAA production, the bacteria preferred thel-isomer over thedl- ord-isomer of tryptophan. The possible role of nodular IAA production on the legume-Rhizobium symbiosis is discussed.     

Indoleacetic acid production byRhizobium sp. from a leguminous tree,Erythrina indica

Rhizobium sp. isolated from the root nodulesof Erythrina indica Lamk., a leguminous tree, produced large amounts of indoleacetic acid (IAA) from tryptophan. L-Tryptophan (2 mg/ mL) was preferred to DL-tryptophan for IAA synthesis. Attempts were made to optimize the cultural requirements for an accelerated IAA production. An enrichment of the carbon-free incubation medium with maltose (1 %), MnSO₄.H₂O (5 μg/mL), and Triton X-100 (100 ppm) promoted the synthesized IAA content of the medium by 480 %.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

Bioproduction of Ascorbic Acid in Root Nodule and Root of the Legume Pulse Phaseolus mungo

The root nodules of Phaseolus mungo (L.), a herbaceous leguminous pulse, contain high amounts of ascorbic acid (AsA). A glucose pool present in the nodule might serve as precursor for AsA production. From root nodule, a Rhizobium sp. was isolated. The symbiont produced a large amount of AsA (290.5 μg/ml) from glucose-supplemented basal medium. The production of AsA by the symbiont was much greater than that of the control when the glucose (0.5%)-supplemented mineral medium was enriched with thiamine hydrochloride (20 μg/100 ml), biotin (20 μg/100 ml), and L-asparagine (0.2%). The possible role of the rhizobial production of AsA on rhizobia–legume symbiosis is discussed.     

Roystonea regia a monocotyledonous tree, bears rhizobial root nodules

The first instance of a monocotyledonous treeRoystonea regia (royal palm) bearing root nodules is reported. They are formed by a Gram-negative bacterium which was tentatively identified asRhizobium sp. The nodules are formed on younger roots of the tree mostly in the rainy season. Younger plants also become nodulated. Mature nodules are oval to cylindrical in shape, unbranched and loosely attached to the roots which show ability to fix nitrogen.     

Bradyrhizobium japonicum subspecies (USDA 110 and 26) characterized by fixed-nitrogen uptake and symbiotic indoleacetic acid production

Two dissimilar subspecies ofBradyrhizobium japonicum (USDA 110 and 26) differ in ammonia (NH₃) assimilation and symbiotic indoleacetic acid (IAA) production. Free-living cultures of type-strain USDA 26 grow on NH₃ as a sole N source and take up an NH₃ analog, methylamine, whereas USDA strain 110 does neither. Although both strains nodulate soybean effectively, root nodules infected with symbiont 26 contain 0.3–1.1 g IAA per gram fresh weight. Nodules infected by tryptophan catabolic variants 4b and 20d, derived from strain 26, also elicit an increased IAA content, two- to fourfold (2.0–3.9 g · g^–1). In contrast, nodules infected with the dissimilar subspecies (strains 110 and 123) contain significantly less IAA.     

Studies of the root nodules of leguminous plants IV: Production of indole acetic acid by a bradyrhizobium sp. from the root nodules of a leguminous shrub,crotalaria retusa L

Bradyrhizobium sp. isolated from the root nodules of a leguminous shrub, Crotalaria retusa L., produced a high amount of indole acetic acid (IAA) from tryptophan in the culture. The bacteria preferred D-isomer to the DL- or L-isomer of tryptophan for the IAA production. The IAA production could be increased up to 153.6% over control by supplementing the medium with arabinose (0.5%), ZnSO₄(0.01 μg/ml), KNO₃ (0.1%), thiamine-HCl (0.01 μg/ml) and EDTA (5 μg/ml). The possible role of the rhizobial production of IAA with the rhizobia-legume symbiosis is discussed.     

The effect of aluminium on growth of and nitrogen fixation in vegetable soybean germplasm

Vegetable soybeam germplasm was screened for its tolerance to 0, 50 and 100 μM Al in solution culture. Plants were inoculated with prescreened acid-Al tolerantBradyrhizobium japonicum strain USDA 110 and a localRhizobium isolate SM867. Aluminum concentrations of 0, 50, and 100 μM affected the root lengths of all germplasm lines in the first few weeks of their growth. At 100 μM, the plants had severely stunted roots throughout the growing period of 35 days, but at 50 μM the initial stunting of the roots was overcome after the third week of growth, and there were no significant differences between the root lengths of these plants and of the controls. The appearance of the first nodule was delayed for 2–3 and 4–5 days at 50 μM and 100 μM Al, respectively. There was a significant reduction in nodule numbers and acetylene reduction activity (ARA) at 100 μM Al. At 50 μM Al, even though the number of nodules was decreased significantly, nodules were larger in size, so there was no significant reduction in nodule fresh weight and ARA. No significant differences in nitrogen fixing abilities of the soybean lines were observed between the twoRhizobium strains. Germplasm line Kahala showed the greatest tolerance to 50 μM Al, and Kahala, Kim and Wolverine tolerated 100 μM Al better than other germplasm lines.     

Root hair elongation is inhibited by hypaphorine, the indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius, and restored by indole-3-acetic acid

Hypaphorine, the major indolic compound isolated from the ectomycorrhizal fungus Pisolithus tinctorius, controls the elongation rate of root hairs. At inhibitory concentrations (100 μM), hypaphorine induced a transitory swelling of root hair tips of Eucalyptus globulus Labill. ssp. bicostata. When the polar tip growth resumed, a characteristic deformation was still visible on elongating hairs. At higher hypaphorine concentrations (500 μM and greater), root hair elongation stopped, only 15 min after application. However, root hair initiation from trichoblasts was not affected by hypaphorine. Hypaphorine activity could not be mimicked by related molecules such as indole-3-acetic acid (IAA) or tryptophan. While IAA had no activity on root hair elongation, IAA was able to restore the tip growth of root hairs following inhibition by hypaphorine. These results suggest that hypaphorine and endogenous IAA counteract in controlling root hair elongation. During ectomycorrhiza development, the absence of root hairs might be due in part to fungal release of molecules, such as hypaphorine, that inhibit the elongation of root hairs. Received: 27 October 1999 / Accepted: 14 March 2000     

Production of 3-indoleacetic acid by aRhizobium sp. fromMimosa pudica

ARhizobium species isolated from the root nodules of the sensitive plant,Mimosa pudica, produced 60 mg/L of 3-indoleacetic acid (IAA) froml-tryptophan in culture. The production of IAA started simultaneously with the growth and had no different growth or production phase. The stationary phase of growth was reached after 55 h, but the production of IAA increased gradually up to 80 h, and then remained constant. The IAA production could be promoted in the culture medium up to 365% by supplementing the medium with maltose, CuSO₄ and Triton X-100.     

Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.     

Indole-3-Acetic Acid (IAA) Production in Symbiotic and Non-Symbiotic Nitrogen-Fixing Bacteria and its Optimization by Taguchi Design

Production of Indole-3-acetic acid (IAA) in 35 different symbiotic and non-symbiotic nitrogen-fixing bacteria strains isolated from soil and plant roots was studied and assayed by chromatography and colorimetric methods. These bacteria included Agrobacterium, Paenibacillus, Rhizobium, Klebsiella oxytoca, and Azotobacter. The best general medium and synergism effects of isolates for IAA production were investigated. Effects of different variables containing physical parameters and key media components and optimization of condition for IAA production were performed using the Design of Experiments. Qualitek-4 (W32b) software for automatic design and analysis of the experiments, both based on Taguchi method was used. The results showed that Rhizobium strains, symbiotic, and Paenibacillus non-symbiotic bacteria yielded the highest concentrations of IAA (in the range of 5.23–0.27 and 4.90–0.19 ppm IAA/mg biomass, respectively) and IAA production was increased by synergism effect of them. Yeast Extract Mannitol medium supplemented with l-tryptophan was the best general medium for IAA production. The analysis of experimental data using Taguchi method indicated that nitrogen source is very prominent variable in affecting the yield and mannitol as carbon source, potassium nitrate (1%), and l-tryptophan (3 g/l) as nitrogen sources after 72-h incubation at 30°C were the optimum conditions for production of IAA. 5.89 ppm IAA/mg biomass was produced under these optimal conditions.     

Anthraquinones from in vitro root culture of <Emphasis Type="Italic">Morinda royoc</Emphasis> L.

Morinda royoc L. (Rubiaceae) root cultures were established for the production of anthraquinones (AQs). Three independent experiments were performed to evaluate the effects of different levels of indole-3-acetic acid (0–22.8 μM), culture duration (15–75 days) and subculture number (0–4). The following indicators were recorded: root fresh weight per Erlenmeyer and intracellular and extracellular AQ production. The experiments performed in this study allowed an increase of intracellular AQ content up to a maximum of 4.5 mg g⁻¹ of fresh mass, after 30 days of culture in a medium 5.7 μM of IAA. In addition, isolation and identification of seven AQs from M. royoc L. roots is described, one of them being reported for the first time for this species. The structures of isolated compounds were determined from ¹H-NMR data. To the best of our knowledge, this is the first report on AQ production from root culture of this plant.     

Effects of Salt and pH Stress on Temperature-Tolerant Rhizobium sp. NBRI330 Nodulating Prosopis juliflora

A study was conducted to examine the growth response of a rhizobial strain Rhizobium sp. NBRI330 isolated from root nodules of Prosopis juliflora growing in alkaline soil. The strain had the ability to nodulate P. juliflora. Nursery grown plants inoculated with Rhizobium sp. NBRI330 had 60.6% higher plant dry weight, as compared with uninoculated plants. The individual stress survival limit of a rhizobial strain Rhizobium sp. NBRI330 isolated from alkaline soil in a medium containing 32% (wt/vol) salt was 8 h, and at 55°C up to 3 h. The length of Rhizobium sp. NBRI330 in salt-stressed cells increased significantly to 3.04 μm from 1.75 μm of non-stressed control cells. On the contrary, the length of pH-stressed cells declined to 1.40 μm. Compared with non-stressed control rod-shaped cells, the shape of temperature-stressed cells changed to spherical, of 0.42 μm diameter. High temperature (45°C) was tolerated efficiently by Rhizobium sp. NBRI330 in the presence of salt at pH 12, as compared with pH 7. Received: 13 September 1999 / Accepted: 14 October 1999     

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.