Role of Pseudomonas putida indoleacetic acid in development of the host plant root system

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.     

Effect of Wild-Type and Mutant Plant Growth-Promoting Rhizobacteria on the Rooting of Mung Bean Cuttings

Mung bean cuttings were dipped in solutions of wild type and mutant forms of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 and then incubated for several days until roots formed. The bacteria P. putida GR12-2 and P. putida GR12-2/aux1 mutant do not produce detectable levels of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, whereas P. putida GR12-2/acd36 is an ACC deaminase minus mutant. All bacteria produce the phytohormone indole-3-acetic acid (IAA), and P. putida GR12-2/aux1 overproduces it. Treatment of cuttings with the above-mentioned bacteria affected the rates of ethylene production in the cuttings in a way that can be explained by the combined effects of the activity of ACC deaminase localized in the bacteria and bacterial produced IAA. P. putida GR12-2 and P. putida GR12-2/acd36-treated cuttings had a significantly higher number of roots compared with cuttings rooted in water. In addition, the wild type influenced the development of longer roots. P. putida GR12-2/aux1 stimulated the highest rates of ethylene production but did not influence the number of roots. These results are consistent with the notion that ethylene is involved in the initiation and elongation of adventitious roots in mung bean cuttings. Received October 21, 1998; accepted January 3, 1999     

Isolation and Characterization of Mutants of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2 That Overproduce Indoleacetic Acid

Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that were able to grow in the presence of the tryptophan analog 5-fluorotryptophan were selected. Seven of the 50 5-fluorotryptophan-resistant mutants overproduced the phytohormone indoleacetic acid (IAA). Of these seven mutants, the highest level of IAA was observed with strain P. putida GR12-2/aux1, which produced four times the amount of indoleacetic acid synthesized by the wild-type strain. Strain P. putida GR12-2/aux1, in contrast to the wild type, lost the ability to stimulate the elongation of the roots of canola seedlings under gnotobiotic conditions. The growth rate, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity of mutant strain P. putida GR12-2/aux1 were identical to those of the wild-type strain. The role of IAA in the mechanism of plant growth stimulation by P. putida GR12-2 and other plant growth-promoting rhizobacteria is discussed.     

Utilization of the Plant Hormone Indole-3-Acetic Acid for Growth by Pseudomonas putida Strain 1290

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.     

Contribution of Indole-3-Acetic Acid Production to the Epiphytic Fitness of Erwinia herbicola

Erwinia herbicola 299R produces large quantities of indole-3-acetic acid (IAA) in culture media supplemented with l-tryptophan. To assess the contribution of IAA production to epiphytic fitness, the population dynamics of the wild-type strain and an IAA-deficient mutant of this strain on leaves were studied. Strain 299XYLE, an isogenic IAA-deficient mutant of strain 299R, was constructed by insertional interruption of the indolepyruvate decarboxylase gene of strain 299R with the xylE gene, which encodes a 2,3-catechol dioxygenase from Pseudomonas putida mt-2. The xylE gene provided a useful marker for monitoring populations of the IAA-deficient mutant strain in mixed populations with the parental strain in ecological studies. A root bioassay for IAA, in which strain 299XYLE inhibited significantly less root elongation than strain 299R, provided evidence that E. herbicola produces IAA on plant surfaces in amounts sufficient to affect the physiology of its host and that IAA production in strain 299R is not solely an in vitro phenomenon. The epiphytic fitness of strains 299R and 299XYLE was evaluated in greenhouse and field studies by analysis of changes in the ratio of the population sizes of these two strains after inoculation as mixtures onto plants. Populations of the parental strain increased to approximately twice those of the IAA-deficient mutant strain after coinoculation in a proportion of 1:1 onto bean plants in the greenhouse and onto pear flowers in field studies. In all experiments, the ratio of the population sizes of strain 299R and 299XYLE increased during periods of active growth on plant tissue but not when population sizes were not increasing with time.

Many plant-associated bacteria have the ability to produce the plant growth regulator indole-3-acetic acid (IAA) (5, 9, 25, 33). IAA is involved in diseases caused by gall- and knot-forming bacterial species (33); however, its role in other bacteria remains undefined. It is unclear whether these bacteria produce IAA during colonization of plant surfaces and whether this metabolite is beneficial to the bacteria during their growth and survival in the phyllosphere. The production of IAA may enable bacteria to detoxify tryptophan analogues present on plant surfaces (15), to downregulate genes involved in plant defense responses (33), or to inhibit the development of the hypersensitive response by plants (26). We recently demonstrated that the ipdC gene, which encodes the indolepyruvate decarboxylase of Erwinia herbicola (Pantoea agglomerans) 299R and which is involved in the indolepyruvate pathway for IAA synthesis in this epiphytic strain (2), is osmoresponsive and plant inducible (3). We hypothesized that the secretion of IAA may modify the microhabitat of epiphytic bacteria by increasing nutrient leakage from plant cells; enhanced nutrient availability may better enable IAA-producing bacteria to colonize the phyllosphere and may contribute to their epiphytic fitness (1).Few studies have attempted to determine the ecological significance of IAA production in pathogenic bacteria. Varvaro and Martella (31) showed that IAA-deficient mutants of Pseudomonas syringae pv. savastanoi, obtained by selection for resistance to α-methyltryptophan, were reduced in their ability to colonize and survive on olive leaf surfaces. The survival of an α-methyltryptophan-resistant IAA-deficient mutant of P. syringae pv. savastanoi in knots also was affected, its population declining more rapidly than that of the parental strain when inoculated alone into oleander leaf tissue (28). The importance of IAA production in bacterial colonization of bean leaves was also tested with the brown spot pathogen P. syringae pv. syringae and an IAA-deficient mutant derived by insertional mutagenesis (21). Although no difference in the survival of the parental and mutant strains on bean leaves was observed in the greenhouse, a small difference in their behavior was apparent in experiments conducted in a mist chamber (21). There have been no studies of the role of IAA production in plant-associated bacteria that do not cause disease.IAA biosynthesis is not essential for bacterial growth and survival, since IAA-deficient mutants grow as well as their IAA-producing parental strain in vitro (2, 29). Large differences in the epiphytic behaviors of IAA-producing bacteria and isogenic IAA-deficient mutants consequently would not be expected. Even small contributions of IAA production to epiphytic fitness could account for the common presence of this phenotype in epiphytic bacteria (19). Measurements of changes in the ratio of two strains following coinoculation, a common approach in ecological studies, can allow the detection of even small differences in the competitive behaviors of two organisms. This approach can detect much smaller differences in behavior between closely related species than comparison of populations of these species when present singly in separate habitats (16). In this study, we tested the role of IAA in the epiphytic fitness of E. herbicola by comparing the relative changes in the population sizes of the parental and IAA-deficient mutant strains with time after their inoculation onto plants in both controlled and field environments.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Proofs of different kind are presented of the existence of highly active bacteria producing IAA from tryptophan on plant surfaces and in plant homogenates. Both homogenates and washing solutions of nonsterile pea plant parts are active in producing IAA from tryptophan. Activity is much enhanced by the addition of glucose or by preincubating the preparations; it is abolished by sterile filtration, by some bactericidic and bacteriostatic substances, by chloramphenicol, streptomycin, and albucid (penicillin being only partly effective). Preparations of sterile plants do not produce IAA from tryptophan within the detection limit of the Salkowski test. The bacteria are even present on seed surfaces, in the air, and in aceton or ammonium sulfate precipitations of homogenates. Main products of the bacterial tryptophan conversion, as demonstrated by paper chromatography, are indolepyruvic acid, indoleacetic acid, indolecarboxaldehyde, and indolecarboxylic acid. In presence of glucose indolepyruvic acid is by far dominating. Many hitherto known results about tryptophan conversion to IAA by higher plants are likely to be falsified by epiphytic bacteria.     

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT^T) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10⁻⁶. DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.     

Plant-microbial interaction under gnotobiotic conditions: A scanning electron microscope study

Inoculation of canola seeds withPseudomonas putida GR12-2 stimulates root elongation under gnotobiotic conditions. Transformation ofP. putida GR12-2 with the broad-host-range plasmid pGSS15 abolishes the enhancement of root elongation. With scanning electron microscopy it was found that both transformed and nontransformedP. putida GR12-2 are capable of binding to canola seed coats. In addition, it was observed that 4 days after the initial inoculation the roots of bothP. putida GR12-2- and GR12-2/pGSS15-treated seedlings were free of adhering bacteria despite the fact that it was subsequently shown that both bacterial strains are capable of binding to roots. Thus, adhesion to roots is not necessary for the initial phase of enhanced root elongation that is induced byP. putida GR12-2 under gnotobiotic conditions.     

Control of microorganisms in the rhizosphere of wheat by inoculation of seeds withPseudomonas putida and by foliar application of urea

After inoculation of wheat seeds with various bacterial strains germination of plants was usually inhibited at first but growth was stimulated later. After inoculation withPseudomonas putida K 11 producing physiologically active compounds the total number of bacteria increased together with the bacteria: fungi ratio in the rhizosphere. These characteristic were further increased after foliar application of urea due to increased root exudation. Dry mass of upper wheat parts was about 15 — 80 % higher in green-house experiments, in which the plants were treated in the two above ways. More reliable results, were usually obtained by bacterization ofP. putida and foliar application of urea as compared with the situation when the seeds were inoculated without the foliar application or, on the contrary, after foliar application without inoculation of the seeds. Only when urea was applied early and in a soil contaminated with the fungusGaeumannomyces graminis var.tritici (causing “take-all” of the wheat) no favourable results could be detected. In these cases the foliar application without inoculation of the seeds was more successful. Symptoms of the disease of wheat roots caused byG. graminis were less frequently observed after the inoculation of the seeds with the strainP. putida K 11 and after the foliar application of urea.     

The Effect of Pseudomonas putida Colonization on Root Surface Peroxidase

Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.     

Pathways of IAA Production from Tryptophan by Plants and by Their Epiphytic Bacteria: A Comparison

Metabolites of tryptophan were investigated using 2 systems: a bacterial (Peastem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). The plant system produces: indolepyruvic acid (IPyA), indoleacetaldehyde (IAAld) indoleacetic acid (IAA), indoleethanol (tryptophol, IAAol), indolecarboxylie acid (ICA), indolecarboxaldehyde (ICAld). Bacteria produce additionally: indoleactic acid (ILA), tryptamine (TNH₂) and the unknown Xb and Yb, but IAAld was not detected. A nonacidic inhibitor extract from pea stems decreases the gain of IAA, IPyA, ILA, Yb. It increases the gain of IAAld, IAAol, TNH₂, Xb, and (only in the bacterial system) ICA and ICAld. Three sites of inhibitor action are suggested, namely the steps Try → IPyA, TNH₂→ IAAld, IAAld → IAA.     

Comparison of growth,exogenous auxin sensitivity,and endogenous indole-3-acetic acid content in roots ofHordeum vulgare L. and an agravitropic mutant

The chemically induced barley (Hordeum vulgare L.) mutation, agr, was found to be a simple recessive trait resulting in agravitropic roots and normal gravitropic shoots. The total seedling root growth was similar for mutant and wild-type roots, although the mutant had fewer roots per seed and greater elongation per root. Although the concentration of exogenous indole-3-acetic acid (IAA) required to reduce root growth by 50% (GR50) was 12 times greater for the agravitropic mutant, agravitropic and gravitropic roots were equally sensitive to exogenous applications of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA). Root IAA contents, determined by high-pressure liquid chromatography (HPLC), were not different for gravitropes and agravitropes. The greater root elongation rates, lack of sensitivity to exogenous IAA, and normal endogenous IAA levels indicate that auxin-controlled growth regulation may be altered in the mutant.     

Pathways of IAA production from tryptophan by plants and by their epiphytic bacteria: Metabolism of indolepyruvic acid and indolelactic acid

Metabolites of indolepyruvic acid and indolelactic acid were investigated using 2 systems: a bacterial (pea stem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). The products of spontaneous indolepyruvic acid decomposition in aqueous solution and during chromatography were investigated, too. Biological indolepyruvic acid conversion yields, besides those substance amounts which occur spontaneously, indoleacetic acid, indoleethanol (tryptophol) and (only in the sterile plant system) indoleacetaldehyde. An inhibitor extract from pea stems decreases the indoleacetic acid and increases the indoleethanol and indoleacetaldehyde gain. Indolelactic acid is not metabolized in the sterile plant sections. Indolelactic acid oxidation by the bacteria-containing homogenate yields indolepyruvic acid and is inhibited by the inhibitor extract.     

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 ( tdd1 ) , which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase β-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1 , indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1 , which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR , which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.     

Influence of 5-Methyltryptophan-Resistant Bradyrhizobium japonicum on Soybean Root Nodule Indole-3-Acetic Acid Content

Bradyrhizobium japonicum mutants resistant to 5-methyltryptophan were isolated. Some of these mutants were found to accumulate indole-3-acetic acid (IAA) and tryptophan in culture. In greenhouse studies, nodules from control plants inoculated with wild-type bradyrhizobia contained 0.04, 0.10, and 0.58 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. Nodules from plants inoculated with 5-methyltryptophan-resistant bradyrhizobia contained 0.94, 1.30, and 10.6 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. This manyfold increase in nodule IAA content indicates that the Bradyrhizobium inoculum can have a considerable influence on the endogenous IAA level of the nodule. Further, these data imply that much of the IAA that accumulated in the high-IAA-containing nodules was of bacterial rather than plant origin. These high-IAA-producing 5-methyltryptophan-resistant bacteria were poor symbiotic nitrogen fixers. Plants inoculated with these bacteria had a lower nodule mass and fixed less nitrogen per gram of nodule than did plants inoculated with wild-type bacteria.     

IAA production during germination of Orobanche spp. seeds

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.     

Stimulation of Plant Growth and Drought Tolerance by Native Microorganisms (AM Fungi and Bacteria) from Dry Environments: Mechanisms Related to Bacterial Effectiveness

In this study we tested whether rhizosphere microorganisms can increase drought tolerance to plants growing under water-limitation conditions. Three indigenous bacterial strains isolated from droughted soil and identified as Pseudomonas putida, Pseudomonas sp., and Bacillus megaterium were able to stimulate plant growth under dry conditions. When the bacteria were grown in axenic culture at increasing osmotic stress caused by polyethylene glycol (PEG) levels (from 0 to 60%) they showed osmotic tolerance and only Pseudomonas sp. decreased indol acetic acid (IAA) production concomitantly with an increase of osmotic stress (PEG) in the medium. P. putida and B. megaterium exhibited the highest osmotic tolerance and both strains also showed increased proline content, involved in osmotic cellular adaptation, as much as increased osmotic stress caused by NaCl supply. These bacteria seem to have developed mechanisms to cope with drought stress. The increase in IAA production by P. putida and B. megaterium at a PEG concentration of 60% is an indication of bacterial resistance to drought. Their inoculation increased shoot and root biomass and water content under drought conditions. Bacterial IAA production under stressed conditions may explain their effectiveness in promoting plant growth and shoot water content increasing plant drought tolerance. B. megaterium was the most efficient bacteria under drought (in successive harvests) either applied alone or associated with the autochthonous arbuscular mycorrhizal fungi Glomus coronatum, Glomus constrictum or Glomus claroideum. B. megaterium colonized the rhizosphere and endorhizosphere zone. We can say, therefore, that microbial activities of adapted strains represent a positive effect on plant development under drought conditions.     

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.)

Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml^?1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml^?1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.     

Host-specificity mutants of Rhizobium meliloti have additive effects in situ on initiation of alfalfa nodules

Pairs of Rhizobium meliloti nod mutants were co-inoculated onto alfalfa (Medicago saliva L.) roots to determine whether one nod mutant could correct, in situ, for defects in nodule initiation of another nod mutant. None of the Tn5 or nod deletion mutants were able to help each other form nodules when co-inoculated together in the absence of the wild-type. However, as previously observed, individual nod mutants significantly increased nodule initiation by low dosages of co-inoculated wild-type cells. Thus, nod mutants do produce certain signal substances or other factors which overcome limits to nodule initiation by the wild-type. When pairs of nod mutants were co-inoculated together with the wild-type, the stimulation of nodulation provided by individual nodABC mutants was not additive. However, clearly additive or synergistic stimulation was observed between pairs of mutants with a defective host-specificity gene (nodE, nodF, or nodH). Each pair of host-specificity mutants stimulated first nodule formation to nearly the maximum levels obtainable with high dosages of the wild-type. Mutant bacteria were recovered from only about 10% of these nodules, whereas the co-inoculated wild-type was present in all these nodules and substantially outnumbered mutant bacteria in nodules occupied by both. Thus, these mutant co-inoculants appeared to help their parent in situ even though they could not help each other. Sterile culture filtrates from wild-type cells stimulated nodule initiation by low dosages of the wild-type, but only when a host-specificity mutant was also present. The results from our studies seem consistent with the possibility that pairs of host-specificity mutants are able to help the wild-type initiate nodule formation by sustained production of complementary signals required for induction of symbiotic host responses.     

The hisC1 gene, encoding aromatic amino acid aminotransferase-1 in Azospirillum brasilense Sp7, expressed in wheat

Background and aims

Production of indole-3-acetic acid (IAA) by Azospirillum brasilense is one of the most important mechanisms underlying the beneficial effects observed in plants after inoculation with this bacterium. This study determined the contribution of the hisC1 gene, which encodes aromatic amino acid aminotransferase-1 (AAT1), to IAA production, and analyzed its expression in the free-living state and in association with the roots of wheat.

Methods

We determined production of IAA and AAT activity in the mutant hisC::gusA-sm ^R . To study the expression of hisC1, a chromosomal gene fusion was analyzed by following β-glucuronidase (GUS) activity in vitro, in the presence of root exudates, and in association with roots.

Results

IAA production in the hisC mutant was not reduced significantly compared to the activity of the wild-type strain. AAT1 activity was reduced by 50% when tyrosine was used as the amino acid donor, whereas there was a 30% reduction when tryptophan was used, compared to the activity of the wild-type strain. Expression of the fusion protein was up-regulated in both logarithmic and stationary phases by several compounds, including IAA, tryptophan, tyrosine, and phenyl acetic acid. We observed the expression of hisC1 in bacteria associated with wheat roots. Root exudates of wheat and maize were able to stimulate hisC1 expression.

Conclusions

The expression data indicate that hisC1 is under a positive feedback control in the presence of root exudates and on plants, suggesting that AAT1 activity plays a role in Azospirillum–plant interactions.