Azospirillum Lipoferum Strain Research Articles

Two-stage enrichment of hydrogen-oxidizing bacteria as biofertilizers

Biofertilizers can replace chemical fertilizers to promote the plant growth without causing any pollution. The study of hydrogen-oxidizing bacteria (HOB) enrichment as biofertilizers from mixed culture is scarce. Our recent study shows that biofertilizing HOB are successfully enriched in a short hydraulic retention time of 10 h. While, the mechanism is unknown. This study intentionally used a two-stage method to enrich biofertilizing HOB specifically with nitrate as nitrogen source in Stage 1 and then 1-aminocyclopropane-1-carboxylate (ACC) as nitrogen source in Stage 2. It was found Pseudomonas (34.46%, reported HOB) predominated in Stage 1, while Azospirillum (59.35%), Azoarcus (36%) were dominant genera and Azospirillum lipoferum strain DSM 1691 (50%), Azoarcus olearius strain DQS-4 (50%) were dominant species in Stage 2. The enriched HOB of Stage 2 showed ACC deaminase activity. Furthermore, they could also fix N2 and consume Ca3(PO4)2. Thus, the two-stage method can be used as a specific enrichment for HOB as biofertilizers, which extends the application of HOB in agriculture.

Azospirillum lipoferum strain AL-3 reduces early blight disease of potato and enhance yield

In sustainable agricultural systems, plant disease management with biological agents is a cost-effective and eco-friendly strategy. This study was performed to screen the biocontrol efficacy of rhizobacterial strains against early blight disease of potatoes (Solanum tuberosum). Rhizobacterial antagonists that are capable of suppressing the growth of Alternaria solani up to 50% under in-vitro conditions were selected. Next, early blight susceptible potato cultivar Sadaf plants were pre-treated with a foliar application of selected antagonist rhizobacteria and challenged 1 d later with Alternaria solani. The results showed that foliar treatment with Azospirillum lipoferum AL-3 significantly reduced early blight disease severity by 68.1%. The results of 2-year studies revealed 52.7 and 39.23% reductions in disease index of A. lipoferum AL-3 -treated plants compared with the control group along with the significant increase of 11.3–17.9% and 16.1–22.7% in tuber yield both in the presence and absence of early blight pathogen, respectively. Azospirillum lipoferum AL-3 also helped potato plants fight early blight disease through induced systemic resistance. The quantity of total phenolics, defense-related enzyme (peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase) activities, and PR-gene transcriptomic levels were significantly increased by Azospirillum lipoferum AL-3. Furthermore, application of Azospirillum lipoferum AL-3 followed by pathogen challenge increased salicylic acid and H2O2 levels up to 1.9 and 3.1 fold, respectively and reduced cell death up to 1.73-fold in potato plants to fight the early blight pathogen. The current investigation highlighted the dual role of Azospirillum lipoferum AL-3 as an antagonist of the early blight pathogen that also induced resistance. Incorporation of this microbe in conventional agricultural systems could reduce management costs and environmental pollution.

Enrichment of hydrogen-oxidizing bacteria with nitrate recovery as biofertilizers in the mixed culture

Hydrogen-oxidizing bacteria (HOB) can utilize hydrogen and oxygen to produce valuable products in biomass, including polyhydroxyalkanoates, microbial proteins, and biofertilizers. However, the method of enriching HOB as biofertilizers from mixed culture remains unknown. In this study HOB were enriched with nitrate as nitrogen source at a hydraulic retention time of 10 h. The nitrate consumption rate was 120 mgN/L/d or 16 mg N/g VSS/h, which was comparable to those of denitrification using organic carbon or hydrogen. The percentage of Azospirillum (dominated genus, reported biofertilizing HOB) was 84.89% and the dominated species was Azospirillum lipoferum strain DSM 1691. Furthermore, the enriched HOB had the abilities of 1-aminocyclopropane-1-carboxylate conversion and phosphate solubilization, the functions of biofertilizers. This is the first report on the enrichment of biofertilizing HOB from mixed culture. Meanwhile, the enriched HOB can recover nitrate from wastewater without any secondary nitrogen pollution, extending HOB application for resource recovery from wastewater.

Grain filling and fatty acid composition of safflower fertilized with integrated nitrogen fertilizer and biofertilizers

Abstract: The objective of this work was to evaluate the effects of the integrated application of nitrogen fertilizer and biofertilizers on the yield, grain filling period, and composition of fatty acids of safflower (Carthamus tinctorius). Split-plot experiments were carried out during the 2011 and 2012 crop seasons. The treatments consisted of seed inoculation with plant growth-promoting rhizobacteria (Azotobacter chroococcum strain 5, Azospirillum lipoferum strain F, and Pseudomonas putida strain 186) in the subplots, including a control without seed inoculation; and of the application of N fertilizer at different rates (60, 120, and 180 kg ha-1 urea) in the main plots, including a control without N. The highest grain yield, grain filling period, and effective grain filling period were obtained by the application of 180 kg ha-1 urea and by seed inoculation with P. putida. The application of high N rates and P. putida inoculation resulted in 25.66% increase of the potential rate of grain filling. Biofertilizer inoculation in seed reduced the contents of saturated fatty acids (palmetic and stearic acids) and increased the contents of unsaturated fatty acids (linoleic, linolenic, and oleic acids). The suitable amount of N fertilizer (between 120 and 180 kg ha-1 urea) can improve plant growth, and the quantity and quality of oil in seeds treated with P. putida in safflower plants.

Genomic insights into metabolic versatility of a lithotrophic sulfur-oxidizing diazotrophic Alphaproteobacterium Azospirillum thiophilum.

Diazotrophic Alphaproteobacteria of the genus Azospirillum are usually organotrophs, although some strains of Azospirillum lipoferum are capable of hydrogen-dependent autotrophic growth. Azospirillum thiophilum strain was isolated from a mineral sulfide spring, a biotope highly unusual for azospirilla. Here, the metabolic pathways utilized by A. thiophilum were revealed based on comprehensive analysis of its genomic organization, together with physiological and biochemical approaches. The A. thiophilum genome contained all the genes encoding the enzymes of carbon metabolism via glycolysis, tricarboxylic acid cycle and glyoxylate cycle. Genes for a complete set of enzymes responsible for autotrophic growth, with an active Calvin-Benson-Bassham cycle, were also revealed, and activity of the key enzymes was determined. Microaerobic chemolithoautotrophic growth of A. thiophilum was detected in the presence of thiosulfate and molecular hydrogen, being in line with the discovery of the genes encoding the two enzymes involved in dissimilatory thiosulfate oxidation, the Sox-complex and thiosulfate dehydrogenase and Ni-Fe hydrogenases. Azospirillum thiophilum utilizes methanol and formate, producing CO2 that can further be metabolized via the Calvin cycle. Finally, it is capable of anaerobic respiration, using tetrathionate as a terminal electron acceptor. Such metabolic versatility is of great importance for adaptation of A. thiophilum to constantly changing physicochemical environment.

Foliar application of nitrogen fixing bacteria increases growth and yield of canola grown under different nitrogen regimes

A field study was conducted to evaluate the effects of foliar application of nitrogen fixing bacteria on canola growth and yield. Azotobacter chroococcum Strain 5 and Azospirillum lipoferum Strain 21 as well as mixture of them were used on aerial parts of two canola cultivars under different nitrogen regimes. The experiment was laid out in a randomized complete block design with factorial arrangement in three replications. The results indicated that foliar application of bacteria significantly promoted almost all agronomic traits. These positive effects amplified when both species of bacteria were applied simultaneously on the plants.

Effects of seed inoculation with plant growth promoting rhizobacteria and foliar application with nano-zinc oxide on yield and rate and period of grain filling of triticale

In order to study the effects of seed inoculation with plant growth promoting rhizobacteria (PGPR) and foliar application of nano-zinc oxide (NZO) on yield and rate and period of grain filling of triticale, a factorial experiment was conducted based on randomized complete blocks design with three replications, in Research Greenhouse of Faculty of Agriculture, University of Mohaghegh Ardabili, Iran, in 2013. Treatments consisted of foliar application of NZO at five levels [0 (as control), 0.25, 0.5, 0.75 and 1 g/L] and seed inoculation with PGPR at four levels (no inoculation as control, seed inoculation with Azotobacter chrocococum strain 5, Azospirillum lipoferum strain OF, and Psedomunas putida strain 9). Comparison of means showed that maximum yield, yield components and rate and period of grain filling was obtained by application of 1 g/L NZO and seed inoculation with Azotobacter, and minimum of these parameters were recorded for application of no NZO and no seed inoculation with PGPR. It seems that utilizing biological fertilizers and zinc micronutrient is a proper and cheap method for increasing triticale yield. Therefore, it can be suggested that inoculation of triticale seeds with Azotobacter and foliar application of 1 g/L NZO be performed in order to increase its yield, yield components and grain filling period.

The applications of plant growth promoting rhizobacteria (PGPRs) as biofertilizers for cold area rice variety JP-5

The aim of present study was to evaluate the effect of bacterial inoculation on different growth parameters of rice variety JP 5. A total of 18 bacterial strains were isolated from roots and rhizosphere of rice. Three bacterial strains (Azospirillum brasilense strain R1,Azospirillum lipoferum strain RSWT1 and Pseudomonas strain Ky1) were used to inoculate rice variety JP 5 at two experimental sites in Swat (Agriculture Research Institute (N) Mingora and Udigram). Plant growth promotion was observed in all inoculated treatments, which was evident from increase in number of tillers, straw and grain yields and total weight of the plants. A. brasilense strain R1 was more effective in plant growth promotion than other strains. At ARIN Mingora, Swat, inoculation of rice variety JP 5 withA. brasilense strain R1 showed 19% increase in the straw weight and 39.5% increase in the grain weight. At this experimental site, inoculation with A. lipoferum strain RSWT1 andPseudomonas strain Ky1 increased grain weight by 18. and 13.8% respectively. At Udigram, Swat, any significant beneficial effect of inoculation with A. lipoferum strain RSWT1 and Pseudomonas strain Ky1 was not observed whereas inoculation with A. brasilense strain R1 showed positive results of 15.5 and 27.4% increase over control in straw weight and grain weight, respectively. The study reveals that beneficial strains of PGPR can be used as biofertilizer for rice. Key words: Azospirillum, Pseudomonas, inoculation of rice, effects of inoculation.

Azospirillum humicireducens sp. nov., a nitrogen-fixing bacterium isolated from a microbial fuel cell

A Gram-negative, facultative anaerobic, motile, spiral, straight-to-slightly curved rod-shaped and nitrogen-fixing strain, designated SgZ-5(T), was isolated from a microbial fuel cell (MFC) and was characterized by means of a polyphasic approach. Growth occurred with 0-1 % (w/v) NaCl (optimum 1 %) and at pH 5.5-8.5 (optimum pH 7.2) and at 25-37 °C (optimum 30 °C) in nutrient broth (NB). The strain had the ability to grow under anaerobic conditions via the oxidation of various organic compounds coupled to the reduction of anthraquione-2,6-disulfonate (AQDS). Chemotaxonomic characteristics (main ubiquinone Q-10, major fatty acid C18 : 1ω7c/C18 : 1ω6c and DNA G+C content 67.7 mol%) were similar to those of members of the genus Azospirillum. According to the results of phylogenetic analyses, strain SgZ-5(T) belonged to the genus Azospirillum within the family Rhodospirillaceae of the class Alphaproteobacteria, and was related most closely to the type strains of Azospirillum lipoferum, Azospirillum thiophilum and Azospirillum oryzae (98.0, 97.6 and 97.1 % 16S rRNA gene sequence similarity, respectively). DNA-DNA pairing studies showed that the unidentified organism displayed reassociation values of 36.7 ± 3.7, 24.1 ± 2.2 and 22.3 ± 2.4 % to the type strains of A. lipoferum, A. thiophilum and A. oryzae, respectively. Similarities between nifH gene sequences of strain SgZ-5(T) and members of the genus Azospirillum ranged from 94.0 to 97.0 %. A combination of phenotypic, chemotaxonomic, phylogenetic and genotypic data clearly indicated that strain SgZ-5(T) represents a novel species, for which the name Azospirillum humicireducens sp. nov. is proposed. The type strain is SgZ-5(T) ( = CCTCC AB 2012021(T) = KACC 16605(T)).

Wheat (Triticum aestivum L.) growth enhancement by Azospirillum sp. under drought stress

Plant growth promoting rhizobacteria (PGPR) can enhance plant growth by alleviating soil stresses. Although previously investigated, some new interesting details are presented regarding the alleviating affects of Azospirillum sp. on wheat growth under drought stress in this research work. We hypothesized that the isolated strains of Azospirillum sp. may alleviate the adverse effects of drought stress on wheat (Triticum aestivum L.) growth. Three different strains of Azospirillumlipoferum (B1, B2 and B3) were used to inoculate wheat seedlings under drought. During the flowering stage the seedlings were subjected to three drought levels with five different time longevity, including control. Pots were water stressed at 80% (S0), 50% (S1) and 25% (S2) of field capacity moisture in a 25 day-period. Soil and plant water properties including water potential and water content, along with their effects on bacterial inoculum and wheat growth, were completely monitored during the experiment. While stress intensity significantly affected bacterial population and wheat growth, stress longevity only affected wheat water potential and water content. Compared to uninoculated treatments strain B3 (fixing and producing the highest amounts of N and auxin, respectively, with P solubilizing and ACC-deaminase activities) increased wheat yield at S1 and S2 by 43 and 109%, respectively. However, strain B2 (producing siderophore) was the most resistant strain under drought stress. The results of this experiment may elucidate the more efficient strains of Azospirillum sp. for wheat inoculation under drought stress and the mechanisms by which they alleviate the stress.

Revised structure of the repeating unit of the O-specific polysaccharide from Azospirillum lipoferum strain SpBr17

A neutral O-polysaccharide liberated by mild acid hydrolysis of a lipopolysaccharide isolated from Azospirillum lipoferum SpBr17 was investigated using 1D and 2D 1H and 13C NMR spectroscopy, including HSQC, HMBC, and NOESY as well as SDS–PAGE electrophoresis along with sugar and methylation analyses. The structure of the O-specific polysaccharide repeating unit was established as follows: [Display omitted] The presented structure is a revised version of the formula that was published earlier in the Abstracts of the 9th International Congress on Nitrogen Fixation in Cancun (Mexico, 1992).

The effect of combined and separate inoculation of alfalfa plants with Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N2O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N2O (11 micromol N2O/pot x 24 h) with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C2H4 nmol/flask x 24 h) and low denitrification activity (1.8 x micromol N2O/flask x 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.

Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.

Evaluation of effect of inoculation of Azospirillum on the yield of Setaria italica (L.)

Inoculation of foxtail millet (variety Lepakshi) with three strains of Azospirillum lipoferum either alone or in combination with nitrogen fertilizer (40 kg N ha-1) increased the plant height, dry weight of shoot and root, and total N content of shoot, root and grain. The grain yield of the inoculated plants was significantly higher compared to the control plants. Both the panicle and the seed weight increased due to inoculation. Among the inocula of A. lipoferum tested, Inoculum-2 (In-2) was found superior to the other two inocula. Further, seasonal (Kharif and Rabi) variations in growth and yield of the plants were also observed.

Construction of a recA mutant of Azospirillum lipoferum and involvement of recA in phase variation

The plant-growth promoting rhizobacterium Azospirillum lipoferum strain 4B generates in vitro a stable phase variant designated 4V I at frequencies of 10 −4 to 10 −3 per cell per generation. Variant 4V I displays pleitropic modifications, such as the loss of swimming motility and the inability to assimilate certain sugars compared to the wild type. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are involved in phase variation, the recA gene of A. lipoferum 4B was cloned and sequenced and a recA mutant (termed 4B recA) was constructed by allelic exchange. Strain 4B recA showed increased sensitivity to UV and MMS compared with 4B and impaired recombinase activity. The ability to generate variants in vitro was not altered; the variants from 4B recA exhibited all morphological and biochemical features characteristic of the variant generated by strain 4B. However, the frequency of variants generated by 4B recA was increased by up to 10-fold. So, in contrast with many studies showing the abolition or a large reduction of the frequency of phase variation in recA mutants, this study describes an enhancement of phase variation in the absence of a functional recA.

Adsorption and anchoring of Azospirillum strains to roots of wheat seedlings

Recent microscopic evidence acquired using strain-specific monoclonal antibodies and specific gene probes confirms earlier claims that some strains of Azospirillum lipoferum and A. brasilense, but not others, are capable of infecting the interior of wheat roots. The present study was performed to determine whether this strain specificity in the infection of the interior of wheat roots was apparent in the first 24 h of adsorption (`anchoring') of Azospirillum cells to the root surface. Strains of A. brasilense, originally isolated from surface-sterilised wheat roots (Sp 245, Sp 107) or with a proven ability to infect the interior of wheat roots (Sp 245), showed no greater ability to anchor to the roots than other Azospirillum strains isolated from the wheat rhizosphere (Sp 246) or from the rhizosphere or rhizosphere soil of other gramineae (Sp 7, Cd, S 82). The SEM images showed that at the root tip the Azospirillum cells were principally located in cracks between epidermal cells. In the root hair zone the bacteria were more numerous but again principally located in the depressions between epidermal cells. In all zones of the roots mucilage was present, and near the tip this appeared to have been partially digested, forming `halos' around the bacteria and revealing fibril-like strands attached to the bacteria. Subsequent studies were conducted using a technique originally developed for investigating competition of rhizobia for adsorption sites on legume roots. In the adaptation of this technique it was found that the presence of any significant concentration of Ca++ in the incubation medium reduced bacterial adsorption, as did concentrations of (PO4)3- above 50 mM. The influence of the pH of the incubation medium on the adsorption of ten different strains of Azospirillum showed, that with one exception, strains isolated from the roots or rhizosphere of wheat showed optimum adsorption at pH 6.0, and all other strains pH 7.0. Apart from this effect of pH no differences in adsorption were detected between strains with a proven capacity to infect wheat roots and those unable to do so. However, strains varied in their capability to compete for adsorption sites, there being a tendency for strains with a proven capacity to invade the internal tissues of wheat roots to be more competitive for adsorption sites.

Effect of some granular insecticides currently used for the treatment of maize crops (Zea mays) on the survival of inoculated Azospirillum lipoferum.

Four insecticides, carbofuran, chlormephos, terbufos and benfuracarb, currently used on maize (Zea mays) at sowing, were tested for their compatibility with Azospirillum lipoferum strain CRT1 used as an inoculant to improve maize growth and yield. The growth or survival of A lipoferum was studied in the presence of the insecticides: (1) in liquid and solid cultures of the bacteria, (2) when a commercial inoculant (Azogreen-m, Liphatech, Meyzieu, France) was inoculated directly on insecticide granules, (3) when inoculated Azogreen-m granules were mixed with insecticide granules and (4) when inoculated Azogreen-m granules were delivered separately to the seed bed. Of the four insecticides tested, only terbufos had a slight effect on growth of A lipoferum in solid cultures. All the insecticides decreased the survival of A lipoferum when the bacteria were inoculated directly on to the granules, or when inoculated Azogreen-m granules were mixed with an insecticide. We hypothesize that the discrepancies between bacterial culture tests and survival studies might be explained by the conditions of desiccation encountered during inoculation of the granules. Desiccation stress could increase the toxic effect of the insecticides. We therefore suggest including desiccation stress in the biotest used to assess inoculant-pesticide compatibility.

Emergence of a laccase-positive variant of Azospirillum lipoferum occurs via a two-step phenotypic switching process

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V I, spontaneously emerged from the wild-type at frequencies in the order of 10 −4 to 10 −3 per cell generation. Compared to the wild-type, the 4V I variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V I variant, a second atypical stable form, variant 4V II, which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.

Emergence of a laccase-positive variant of Azospirillum lipoferum occurs via a two-step phenotypic switching process.

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V(I), spontaneously emerged from the wild-type at frequencies in the order of 10(-4) to 10(-3) per cell generation. Compared to the wild-type, the 4V(I) variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V(I) variant, a second atypical stable form, variant 4V(II), which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.

Hydrolysis of [17,17-2H2]gibberellin A20-glucoside and [17,17-2H2]gibberellin A20-glucosyl ester by Azospirillum lipoferum cultured in a nitrogen-free biotin-Based chemically-defined medium

Azospirillum lipoferum strain USA 5b, a gibberellin producing bacterium, was cultured in a nitrogen-free biotin-based chemically-defined medium in the presence of the glucosyl ester or the 13-O-glucoside of [17,17-2H2]-gibberellin A20. The [17,17-2H2]-gibberellin A20 conjugates were added at both the stationary phase of the cultures and at the beginning of the growth curve. Metabolism of the conjugates was examined after 72 h of incubation using capillary gas chromatography-mass spectrometry, with identification by full scan mass spectra. Metabolites identified were [17,17-2H2]-gibberellin A20, [17,17-2H2]-gibberellin A1 and [17,17-2H2]-gibberellin A3. Also, in the Azospirillum cultures fed at the beginning of the growth curve, gibberellin A5 and gibberellin A20 were characterized as endogenous by mass spectrometry/full spectrum. These results support the concept that the growth promotion in plants that is induced by Azospirillum infection may occur by a combination of both gibberellin production and gibberellin-glucoside/glucosyl ester deconjugation by the bacterium.