Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.     

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.     

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 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.     

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.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.     

Real time PCR detection targeting nifA gene of plant growth promoting bacteria Azospirillum brasilense strain FP2 in maize roots

The plant growth promoting bacteria (PGPB) Azospirillum brasilense has been recommended for use in commercial inoculants in Brazil. Effective methods are necessary to monitor PGPB strains in the rhizosphere. Our purpose was to develop a real time PCR method for detection of A. brasilense strain FP2 in maize seedlings targeting nifA. Primer pairs were designed and their specificity was verified using DNA from 12 different bacterial species. Standard curves were prepared for DNA quantification using serial dilution of A. brasilense DNA extracts. PCR efficiencies and correlation coefficient presented values within the acceptable range for qPCR, mean PCR efficiency was 95 % and correlation coefficient was 0.98, indicating that nifA gene was suitable for the quantitative analysis of the target bacterial genome. Inoculated maize seedlings were grown in vitro or in pots, bacterial DNA copy number per gram of fresh root was quantified 1, 4, 7 and 10 days after inoculation. The developed primers targeting nifA will be useful for monitoring Azospirillum brasilense strain FP2 in crops.     

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.     

Ability of intestinal lactic bacteria to bind or/and metabolise phenol and p-cresol

Intestinal microflora can contribute to colon cancer by the production of substances playing a role in carcinogenesis. Metabolites of protein fermentation in the colon, such as ammonia, H₂S, indole, phenol, skatole are toxic. Lactic bacteria existing in the colon may exert an anti-carcinogenic action, but the mechanism is poorly understood. In the present study the ability of intestin|al lactobacilli to bind or metabolise phenol and p-cresolin vitro was determined.Lactobacillus strains were cultivated in MRS and in a modified MRS broth with reduced concentrations of carbon source. Phenol and p-cresol content in the media were from 2 to 10 μg/ml. In MRS medium lactobacilli could decrease the concentration of phenol and p-cresol and it was 0.2-5.8 μg/ml for phenol and 0.2-1.4 μg/ml for p-cresol. After cultivation in a modified MRS broth, the decrease was 0.5-2.0 μg/ml for phenol and 0.5-2.4 μg/ml for p-cresol. The binding capacity of bacterial cells was rather low. After incubation of non-growing bacteria the decrease of phenol concentration was 0.1-0.5 μg/ml and p-cresol 0.1-2.8 μg/ml. But the ability of growing lactobacilli to metabolise the compounds cannot be excluded. After interaction of lactobacilli with 10 μg/ml of phenol they displayed a lower genotoxicity, as evaluated by the alkaline comet assay. The phenomenon not always depended on the decrease of phenol concentration, but on the medium, the strain of bacteria and for phenol it ranged from 32 to 48%.Lactobacillus strains tested did not lower the genotoxicity of p-cresol.     

Screening and Optimization of Indole-3-Acetic Acid Production and Phosphate Solubilization from Rhizobacteria Aimed at Improving Plant Growth

A total of 216 bacterial strains were isolated from rice rhizospheric soils in Northern Thailand. The bacterial strains were initially tested for solubilization of inorganic phosphate, indole acetic acid (IAA) production, selected strains were then tested for optimized conditions for IAA production and whether these caused stimulatory effects on bean and maize seedling growth. It was found that all strains had solubilized inorganic phosphate (P), but only 18.05% produced IAA. The best IAA producer was identified by biochemical testing and 16S rDNA sequence analysis as Klebsiella SN 1.1. In addition to being the best IAA producer, this strain was a high P-solubilizer and produced the highest amount of IAA (291.97 ± 0.19 ppm) in culture media supplemented with l-tryptophan. The maximum production of IAA was achieved after 9 days of incubation. The culture requirements were optimized for maximum IAA production. The tested of IAA production by selected isolates was studied in a medium with 0, 0.1, 0.2, 0.5, 0.7, and 0.9% (v/v) l-tryptophan. Low levels (12.6 ppm) of IAA production was recorded without tryptophan addition. Production of IAA in Klebsiella SN 1.1 increased with an increase to 0.2% (v/v) tryptophan concentration. The production of IAA was further confirmed by extraction of crude IAA from this isolate and subsequent Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same R _f value. The Klebsiella strain SN 1.1 also demonstrated stimulatory effects on bean seedlings in vivo.     

Mass Spectrometric Quantification of Indole-3-Acetic Acid in Rhizobium Culture Supernatants: Relation to Root Hair Curling and Nodule Initiation

Indole-3-acetic acid (IAA) has been unequivocally identified in culture supernatants of Rhizobium strains by gas chromatography-mass spectrometry. A method for accurately quantitating IAA in bacterial culture supernatants, employing deuterium-labeled IAA as an internal standard, has been developed. Similar IAA concentrations were found in culture supernatants of chosen Rhizobium mutants (defective in nodule formation) and their corresponding parent strains. Since some of the mutants are known to adhere to root hairs, it can be concluded that root hair curling is not simply a consequence of IAA production by rhizobia.     

Streptomycin resistance in Rhizobium japonicum

Mutants resistant to varying concentrations of streptomycin were recovered from two streptomycin-sensitive, effective nitrogen-fixing strains of Rhizobium japonicum. To determine if there were an upper limit of resistible antibiotic concentration, 3 mutants which were resistant to 10000 μg/ml were challenged by higher concentrations of streptomycin. Only one grew well at 25000 μg/ml, and none grew at 50000 μg/ml. All mutants maintained a smooth colonial morphology, and none exhibited streptomycin-dependence. Streptomycin-resistant mutants of both strains were examined for properties of infectivity and effectiveness. All mutants tested retained the symbiotic properties of the parental strains. The retention of these parental properties by the streptomycin-resistant mutants of R. japonicum is different from the properties described for phenotypically similar mutants in certain other rhizobial species.     

Endophytic actinomycetes from spontaneous plants of Algerian Sahara: indole-3-acetic acid production and tomato plants growth promoting activity

Twenty-seven endophytic actinomycete strains were isolated from five spontaneous plants well adapted to the poor sandy soil and arid climatic conditions of the Algerian Sahara. Morphological and chemotaxonomical analysis indicated that twenty-two isolates belonged to the Streptomyces genus and the remaining five were non-Streptomyces. All endophytic strains were screened for their ability to produce indole-3-acetic acid (IAA) in vitro on a chemically defined medium. Eighteen strains were able to produce IAA and the maximum production occurred with the Streptomyces sp. PT2 strain. The IAA produced was further extracted, partially purified and confirmed by thin layer chromatography (TLC) analysis. The 16S rDNA sequence analysis and phylogenetic studies indicated that strain PT2 was closely related to Streptomyces enissocaecilis NRRL B 16365^T, Streptomyces rochei NBRC 12908^T and Streptomyces plicatus NBRC 13071^T, with 99.52 % similarity. The production of IAA was affected by cultural conditions such as temperature, pH, incubation period and l-tryptophan concentration. The highest level of IAA production (127 μg/ml) was obtained by cultivating the Streptomyces sp. PT2 strain in yeast extract-tryptone broth supplemented with 5 mg l-tryptophan/ml at pH 7 and incubated on a rotary shaker (200 rpm) at 30 °C for 5 days. Twenty-four-hour treatment of tomato cv. Marmande seeds with the supernatant culture of Streptomyces sp. PT2 that contained the crude IAA showed the maximum effect in promoting seed germination and root elongation.     

Production of alternarioi and alternariol monomethyl ether in natural substrates in comparison with semisynthetic culture medium

The comparison In toxins production and growth byAlternarla strains in liquid, solid culture media and natural substrates (rice and sunflower) was evaluated. Ground rice- corn steep liquor medium (GRCS) was the more suitable medium for production of alternariol (AOH) and alternariol monomethyl ether(AME). The maximum levels produced were 676 μg/50ml AOH and 1570/50ml AME. Rice was better than sunflower In supporting toxins production. Different ratios AOH/AME were found according to the substrate evaluated.     

Plant-growth-promoting compounds produced by two agronomically important strains of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>, and implications for inoculant formulation

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 (GA₃), 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, GA₃, ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA₃ were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 μg ml⁻¹). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 μg 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.     

Plantlet Regeneration via Multiple Shoot Induction in Indian Cultivars of Lucerne (Medicago sativa L)

An efficient protocol has been developed for in vitro plant regeneration via multiple shoot induction in lucerne (Medicago sativa L). Shoot tips from in vitro grown 5–6 days old seedlings of 3 cultivars, LLC-3, Chetak and RL-88 were used as explants for multiple shoot induction on MS medium supplemented with cytokinins. Maximum of 14 shoots per apical meristem were observed in case of cv Chetak on MS medium supplemented with BAP (12.6 μM) and KN (9.3 μM). Shoot elongation on MS medium supplemented with GA (5.8 μM), while root induction was achieved on MS medium supplemented with IAA (11.4 μM) and activated charcoal (2.0 g l^?1). Tissue raised plants showed 75% survival after transfer to soil under field conditions.     

Production of indole-3-acetic acid by mutant strains of Ustilago maydis (maize smut / huitlacoche)

Production of indole-3-acetic acid (IAA) by four strains of the maize pathogen Ustilago maydis was analyzed. The fungus induces gall formation on its host plant and IAA production by  U. maydis may be required as a pathogenicity or virulence factor. The study included the FB2 wild-type strain and the 103, 130FZ and 130FT mutants. Results show that treatment with clofibric acid, alone or in combination with UV light, can be used to obtain  U. maydis strains with defective IAA production in vitro, as quantified with the Salkowski reagent and by HPLC. The strain with the lowest production was 130FT, and its peak IAA level represented only 16% of the highest value obtained for the FB2 wild-type strain (124 μg/ml). Received: 11 April 1996 / Received last revision: 5 September 1997 / Accepted: 11 September 1997     

The production of auxins by the endophytic bacteria of winter rye

The ability of the actinomycetes and coryneform bacteria isolated from the root tissues of winter rye to produce auxin in a liquid culture was studied. The isolates of coryneform bacteria produced indolyl-3-acetic acid (IAA) into the medium in the amount of 9.0–95.0 μg/ml and the isolates of actinomycetes in the amount of 39.5–83.0 μg/ml. The maximal IAA accumulation in culture liquid of actinomycetes coincided, in general, with the beginning of the stationary growth phase. The dependences of IAA synthesis by actino-mycetes on the composition and pH of nutrient medium, tryptophan concentration, and aeration conditions were determined. Biological activity of the bacterial IAA was assessed. Treatment of winter rye seeds with coryneform auxin-producing bacteria increased the germination capacity and enhanced an intensive seedling growth in vitro.     

Inoculation with selected strains of Azospirillum brasilense and A. lipoferum improves yields of maize and wheat in Brazil

Interest in the use of inoculants containing bacteria that promote plant growth is likely to increase in the coming years, due to higher costs of fertilizers, concerns over pollution and emphasis on sustainable agriculture. Although Brazil has a long tradition in research on nitrogen fixation in Azospirillum-grass associations, it has not led to recommendations of strains for use in commercial inoculants. In this study, we report the selection and evaluation of Azospirillum strains for the maize (Zea mays L.) and wheat (Triticum aestivum L.) crops, following protocols established by the Brazilian legislature, i.e. field experiments have to be performed in at least two different localities representing the crop growing regions, and for at least two seasons. In a first set of nine trials performed at Londrina and Ponta Grossa, southern Brazil, nine Azospirillum strains were evaluated after application to seeds as peat-based inoculants. A. brasilense strains Ab-V4, Ab-V5, Ab-V6 and Ab-V7 increased grain yields of maize by 662–823 kg ha^?1, or 24–30%, in relation to non-inoculated controls. Two A. lipoferum strains were tested in two of these experiments and promising results were also obtained. With wheat, A. brasilense strains Ab-V1, Ab-V5, Ab-V6 and Ab-V8 were the most effective, increasing yields by 312–423 kg ha^?1, or 13–18%. In a second trial set with eight field experiments at Londrina an Ponta Grossa, liquid and peat-based inoculants carrying a combination of A. brasilense strains Ab-V5 and Ab-V6 increased maize and wheat yields by 27% and 31%, respectively. Effects of inoculation were attributed to general increases in uptake of several macro and micronutrients and not specifically to biological nitrogen fixation. All experiments received only a low N-fertilizer starter at sowing (24 kg and 20 kg of N ha^?1 for the maize and wheat, respectively) and although yields can be globally considered low, they were compatible with Brazilian mean yields. This study resulted in the identification of the first Azospirillum strains authorized for the production of commercial inoculants in Brazil.