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

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

Production of Extracellular Polysaccharide by a Rhizobium Species from Root Nodules of the Leguminous Tree Dalbergia lanceolaria

The ability of the Rhizobium D1 10 species, which was isolated from the root nodules of the leguminous forest tree Dalbergia lanceolaria, for the production of extracellular polysaccharides (EPS) was investigated. High amounts of EPS (765 μg/mL) were produced by the bacteria (Rhizobium D1 10) in yeast extract mannitol medium. Both growth and EPS production started simultaneously, but the EPS production was at its maximum in the stationary phase of growth at 32 h. The EPS production was maximal when the medium was supplemented with mannitol (2 %), thiamine hydrochloride (1 μg/mL) and KNO₃ (0.1 %), which was accompanied by a great increase in the production compared to the control. The EPS contained xylose, rhamnose, glucose, galactose and arabinose. The possible role of rhizobial EPS production in root nodule 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.     

The extracellular polysaccharide produced by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. isolated from the root nodules of <Emphasis Type="Italic">Phaseolus mungo</Emphasis>

The symbiont isolated from root nodules of Phaseolus mungo L., a widely grown legume in India was identified as a Rhizobium sp. a Rhizobium sp. close to R. multihospitium based on a biochemical and 16S rRNA gene-based phylogenetic approach. This Rhizobium sp. was able to produce large amounts of extracellular polysaccharides (EPS) in a yeast extract mannitol (YEM) broth medium. Both growth and EPS production started simultaneously though each had different stationary phases. EPS production increased enormously with supplementation by the preferred carbon, nitrogen and vitamin sources. Attempts were made to optimize the cultural requirements for maximum growth and maximum EPS production. The EPS produced by the symbiont contained large amount of mannose together with small amounts of arabinose and xylose. The possible role of EPS production on the Rhizobium—root nodule symbiosis is briefly discussed.     

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

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

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.     

Production and composition of extracellular polysaccharide synthesized by a Rhizobium isolate of Vigna mungo (L.) Hepper

An extracellular polysaccharide (EPS) was produced by a Rhizobium sp. isolated from the root nodules of Vigna mungo (L.) Hepper. Maximum EPS production (346 mg l⁻¹) was when the yeast extract basal medium was supplemented with mannitol (1%), biotin (1.5 mg l⁻¹) and asparagine (0.3%). Ribose (53%) and mannose (47%) were the principle monomers of the EPS. Chemical, chromatographic and spectroscopic analysis showed that this polymer, which has Man₄Rib₁ as an oligomeric subunit, has an apparent molecular mass of 750 kDa.     

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

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

Chemotaxis of Rhizobium SP.S2 Towards Cajanus cajan Root Exudate and Its Major Components

The chemotactic response of Rhizobium sp. S2, a slow-growing Cajanus cajan isolate, towards its host root exudate was examined. Two classes of mutants, one nonchemotactic towards nutrients (amino acids and sugars) and signal compounds like flavonoids and the other, nonchemotactic towards amino acids and sugars but positive towards naringenin, the flavonoid present in Cajanus cajan root exudate, were obtained. The plasmid-cured derivative of the parent showed positive response towards amino acids and sugars but was nonchemotactic towards naringenin. A possible presence of dual chemotaxis pathways, one towards nutrients and the other for sensing signal compounds, was thus demonstrated. The possible involvement of naringenin as a chemoattractant in the preliminary stages of this Rhizobium-legume interaction was also established. Received: 18 September 1998 / Accepted: 22 October 1998     

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.     

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.     

News & Notes Indole Acetic Acid and Its Metabolism in Root Nodules of a Monocotyledonous Tree Roystonea regia

A monocotyledonous tree, Roystonea regia, was found to bear root nodules. The root nodules contained a high amount (16.9 μg/g fresh mass) of indole acetic acid (IAA). A big tryptophan pool (1555.1 μg/g fresh mass) was found in the root nodules, which might serve as a source of IAA production. The presence of IAA-metabolizing enzymes IAA oxidase and peroxidase indicated metabolism of IAA in the root nodules. The symbiont isolated from the root nodules of R. regia, a Rhizobium sp., produced high amount of IAA in culture when supplemented with tryptophan. The possible role of this IAA production in the monocotyledonous tree–Rhizobium symbiosis is discussed. Received: 31 December 1997 / Accepted: 5 February 1998     

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 fast and slow growing Rhizobium spp. nodulating Cqjanus cajan and Cicer arietinum

Fast and slow growing Rhizobium spp. isolated from Cajanus cajan and Cicer arietinum were compared in terms of colony characteristics, utilisation of carbon sources, acid production, symbiotic effectiveness and nodulating competitiveness. Fast growing isolates from C. cajan and C. arietinum formed 3–6 mm diameter colonies on yeast-extract mannitol agar after 4 days and were unlike the slow growers which produced colonies of c. 1 mm diameter after 7–10 days at 28 °C. The fast growing Rhizobium spp. from C. cajan utilised a wider range of carbon sources than the slow growing isolates from this host. Fast and slow growing strains from C. arietinum were able to utilise most of the carbon sources tested suggesting that the slow growers possessed glycolytic pathways similar to those in other fast growing species of Rhizobium. In culture, slow growing isolates from C. cajan produced a near-neutral to alkaline reaction (pH 66·7-5) whereas the fast growers from this host and both fast and slow growing isolates from C. arietinum produced an acidic reaction (pH 4·4–5·6). These data are discussed in the context of Norris' (1965) evolutionary concept of the Leguminosae. Under glassshouse conditions, fast and slow growing strains isolated from C. cajan and C. arietinum were equally effective on their respective hosts. In competition with slow growing rhizobia, half of the fast growers formed more than 70% of the nodules on C. cajan grown in sand. In all but one instance similar results were obtained when plants were grown in soil. With C. arietinum grown in sand or soil, all fast growing isolates from this host formed more than 85% of the nodules in competition with slow growing strains.     

Estimation of root nodule development by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. Using hydroponic culture

A nitrogen-fixing bacterium isolated from the root nodules of a cultivated leguminous plant, soybean (Glycine max L.), was cultivable and was identified as Rhizobium sp. Bacterial species isolated from root nodules of wild leguminous plants including -bush clover, white dutch clover, wisteria, and false acacia were identified as Burkholderia cepacia, Pseudomonas migulae, Pseudomonas putida, and Flavobacterium sp, respectively, all of which are heterotrophic bacteria that grow in the rhizosphere. Temperature gradient gel electrophoresis (TGGE) 16S-rDNA bands extracted directly from the bacterial population within the root nodules of the wild leguminous plants were identified as Rhizobium sp, Mesorhizobium sp, and Bradyrhizobium sp. none were cultivable. Rhizobium sp. isolated from soybean root nodule generated approximately 48 and 19 mg/L of ammonium in glucose- and starch-defined medium, respectively, during 8 days of growth. The growth rate of Rhizobium sp. was increased by the addition of yeast extract but not by the addition of ammonium. K _m and V _max for starch saccharification measured with the extracellular crude enzyme of Rhizobium sp. were 0.7556 mg/L and 0.1785 mg/L/min, respectively. The inoculation of Rhizobium sp. culture into a hydroponic soybean plant culture activated root nodule development and soybean plant growth. The inoculated Rhizobium sp. survived for at least 4 weeks, based on the TGGE pattern of 16S-rDNA. The 16S-rDNA of Rhizobium sp. isolated from newly developed root nodules was homologous with the inoculated species.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.     

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

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

Nodule haustoria and microbial features of Cajanus and Pongamia parasitized by sandal (sandal wood)

A field observation that roots of sandal wood tree (Santalum album (L.)) formed direct haustorial connections with root nodules of nodulating legumes was confirmed by pot culture studies on interaction between sandal wood plants and pigeonpea (Cajanus cajan (L.) Millsp.) or Pongamia glabra Vent. plants. The number of nodules and nitrogen content of plants decreased in parasitized nodulating species with corresponding increase in the nitrogen content of sandal plants. The root region of sandal had more of nitrogen-fixing bacteria and VAM fungi than those of pigeonpea.     

Activity of antioxidant enzymes in response to cadmium in Crotalaria juncea

The aromatic amine, -phenethylamine, was identified in various field-grown leguminous plants by analyses with HPLC, GC, GC-MS and ¹H-NMR. High concentration of -phenethylamine was generally detected only in mature root nodules, but not in other plant organs such as root, stem, leaf, pod and grain. Occurrence was specific to the root nodules formed by Bradyrhizobium infection. Ten of eleven legume crops including soybean [Glycine max (L.) Merr.], pigeon pea [Cajanus cajan (L.) Millsp.], adzuki bean (Vigna angularis), mung bean [V. radiata (L.) Wilczek] and cowpea (V. unguiculata) contained this aromatic amine, but groundnut (Arachis hypogaea L.) also nodulated by Bradyrhizobium sp. did not. Root nodules collected from garden pea (Pisum sativum L.), broad bean (Vicia fava L.), kidney bean (Phaseolus vulgaris L.) and various other herbaceous legumes nodulated by Rhizobium sp., Mesorhizobium sp., Sinorhizobium sp. or Azorhizobium caulinodans, and root-nodulated, woody non-legumes, nodulated by Frankia spp., contained little -phenethylamine.The amount of -phenethylamine in Bradyrhizobium-infected nodules varied with the legume species and their cultivars, and most significantly, with nodule age. In field-grown soybean plants, nodule -phenethylamine attained maximum concentration at the flowering stage and far exceeded that of the major polyamines of soybean nodules, putrescine and spermidine.