Synergistic effects of inoculating arbuscular mycorrhizal fungi and Methylobacterium oryzae strains on growth and nutrient uptake of red pepper (Capsicum annuum L.)

A greenhouse experiment was conducted to examine the effects of inoculation with two Methylobacterium oryzae strains (CBMB20 and CBMB110) and a consortium of three arbuscular mycorrhizal (AM) fungi on the growth of red pepper (Capsicum annum L.). Inoculation of red pepper plants with the M. oryzae strains resulted in a significant increase in root length and root fresh weight compared to untreated control plants. The combined inoculation of M. oryzae strains and AM fungi significantly increased various plant growth parameters and chlorophyll content compared to uninoculated controls. Mycorrhizal colonisation and the number of AM fungal spores were higher in co-inoculation treatments. In addition, the combined inoculation of M. oryzae strains and AM fungi resulted in significantly higher nitrogen (N) accumulation in the roots and shoots of red pepper plants compared to uninoculated controls. The combined inoculation of M. oryzae strain CBMB110 and AM fungi increased the phosphorus (P) content by 23.3% compared to untreated controls. The micronutrient content of the red pepper plants also increased in most of the inoculation treatments. A perfect mutualism among CBMB100-AMF was found which was attributed to the improved macro- and micronutrient uptake along with higher chlorophyll content in red pepper. Further research on in-depth understanding of the co-operative microbial interactions will facilitate the successful application of Methylobacterium-AM fungi products in biotechnology.     

Effect of congo red on the motility of the bacterium Azospirillum brasilense

In semiliquid laboratory media, the bacterium Azospirillum brasilense migrates with the formation of swarming rings. It is demonstrated that adsorption of the sulfonated azodye Congo Red confers on A. brasilense the ability to consistently spread in a semiliquid agar with formation of microcolonies. Spontaneous variants of A. brasilense with increased swarming rate are described, as well as variants that swarm in the presence of Congo Red. It is assumed that at least two types of compounds are formed, which are necessary for swarming and/or spreading with the formation of microcolonies and are capable of interacting with Congo Red.     

Effect of Congo red on the motility of the bacterium Azospirillum brasilense

In semiliquid laboratory media, the bacterium Azospirillum brasilense migrates with the formation of swarming rings. It is demonstrated that adsorption of the sulfonated azodye Congo Red confers on A. brasilense the ability to consistently spread in a semiliquid agar and form microcolonies. Spontaneous variants of A. brasilense with rapid swarming are described, as well as variants that swarm in the presence of Congo Red. It is assumed that at least two types of compounds are formed, which are (a) necessary for swarming and/or spreading with the formation of microcolonies and (b) capable of interacting with Congo Red.     

Genetics of Azospirillum brasilense with respect to ammonium transport,sugar uptake,and chemotaxis

This paper describes molecular aspects of Azospirillum-plant root association with respect to nitrogen flux and carbon utilization. In the first part, biochemical and genetic data are reported on the transport of ammonium and methylammonium in A. brasilense cells. Ammonium excreting A. brasilense mutants reported so far appear to result from alterations in genes encoding for enzymes involved in ammonium assimilation. Solid genetic evidence is given on the occurrence of a postulated ammonium transporter in A. brasilense. In the second part, biochemical and genetic evidence is likewise given for the occurrence of a high-affinity uptake system for D-galactose in A. brasilense. A sugar- binding protein that is part of this uptake system is required for chemotaxis of A. brasilense towards particular sugars, including D-galactose.     

Influence of Azospirillum inoculation on the mineral uptake and growth of rice under hydroponic conditions

Seedlings of rice (Oryza sativa L. var. IR42) were inoculated with nitrogen-fixingAzospirillum lipoferum (strain 34H) by immersing the roots in the inoculum for 6 h. The plants were grown in the prescence of NH₄ ⁺-N for 47 days in a hydroponic system under greenhouse conditions. Inoculation significantly enhanced PO₄-ion uptake of the plants in 4 of the 7 samplings tested while the uptake of NH₄-ion was significantly increased in two samplings and was decreased in one sampling. Inoculation reduced root length significantly and caused significant increases in shoot fresh and dry weights. Root surface area was not affected by inoculation. Bacterial population counts suggested thatA. lipoferum survived on the roots till the end of the experiment.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Effects of plant growth regulators on acetylene-reducing associations between Azospirillum brasilense and wheat

Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge. The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02–0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-d treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-d. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-d. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N₂-fixing system in cereals.     

Cloning, Sequencing, and Characterization of the Azospirillum brasilense fhuE Gene

The fhuE gene of Escherichia coli encodes the FhuE protein, which is a receptor protein in the coprogen-mediated siderophore iron-transport system. A fhuE gene homologue from Azospirillum brasilense, a nitrogen-fixing soil bacterium that lives in association with the roots of cereal grasses, was cloned, sequenced, and characterized. The A. brasilense fhuE encodes a protein of 802 amino acids with a predicted molecular weight of approximately 87 kDa. The deduced amino-acid sequence showed a high level of homology to the sequences of all the known fhuE gene products. The fhuE mutant was sensitive to iron starvation and defective in coprogen-mediated iron uptake. The mutant failed to express one membrane protein of approximately 78 kDa that was induced by iron starvation in the wild type. Complementation studies showed that the A. brasilense fhuE gene, when present on a low-copy number plasmid, could restore the functions of the mutant. Mutation in fhuE gene did not affect nitrogen fixation.     

Effect of silicon on the growth and phosphorus uptake of rice

A pot experiment was conducted to measure the effect of silicon on phosphorus uptake and on the growth of rice at different P levels. Rice (Oryza sativa L. cv. Akebono) was cultured in Kimura B nutrient solution without and with silicon (1.66 mM Si) and with three phosphorus levels (0.014 mM P, low; 0.21 mM, medium; and 0.70 mM, high).Shoot dry weight with Si (+Si) in solution increased with increasing P level, while shoot weight without Si (–Si) was maximum at 0.21 mM P, suggesting that +Si raised the optimum P level for rice. +Si increased shoot weight more when P was low or high than when P was medium.The concentration and amount of inorganic P in shoots increased with increasing P level. +Si did not significantly decrease P uptake by rice at 0.014 mM P, however, uptake at 0.21 and 0.70 mM P was 27 and 30 percent less than uptake with –Si, respectively. In –Si with 0.21 and 0.70 mM P, inorganic P in shoots was more than double the concentration in shoots grown in +Si solutions.The Si concentration in shoots decreased slightly with increasing P level, although Si uptake was not significantly affected by P. +Si decreased the uptake of Fe and Mn by an average of 20 and 50 percent, respectively, thus P/Mn and P/Fe ratios increased in the shoot when P was low.From the results above, the beneficial effect of Si on the growth of rice was clearly shown when P was low or high. This effect may have resulted from decreased Mn and Fe uptake, and thus increased P availability within P deficient plants, or from reduced P uptake when P was high.     

Effect of salinity, gibberellic acid and Azospirillum inoculation on growth and nitrogen uptake of Zea mays

Pot experiments were conducted to evaluate the possible interaction of salinity (osmotic potential -0.3, -0.9 and -1.2 MPa) and occurrence of Azospirillum lipoferum or exogenous gibberellic acid (GA₃) (100 μg g^-1) on growth and some physiological parameters of maize. ¹⁵N-uptake as well as the percentage of nitrogen derived from ¹⁵N-fertilizer were decreased by increasing the NaCl concentrations and completely inhibited at concentrations corresponding to osmotic potentials -0.9 and -1.2 MPa. The percentage of nitrogen originating from N₂ fixation was significantly correlated to the total counts of Azospirillum cells that colonized the histosphere. At high NaCl concentrations although no significant changes in N % in shoot dry mass either in inoculated or uninoculated plants were observed, the total N-yield [mg(N) pot^-1] was decreased. Fresh and dry shoot mass significantly increased by Azospirillum inoculation. Azospirillum and GA₃ treatments were positively correlated with most of the parameters analysed. Azospirillum inoculation or GA3 application at NaCl concentrations up to -1.2 MPa significantly increased the chlorophyll, K, Ca, soluble saccharides and protein contents as compared with control plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Azospirillum brasilense and Azospirillum lipoferum hydrolyze conjugates of GA20 and metabolize the resultant aglycones to GA1 in seedlings of rice dwarf mutants

Azospirillum species are plant growth-promotive bacteria whose beneficial effects have been postulated to be partially due to production of phytohormones, including gibberellins (GAs). In this work, Azospirillum brasilense strain Cd and Azospirillum lipoferum strain USA 5b promoted sheath elongation growth of two single gene GA-deficient dwarf rice (Oryza sativa) mutants, dy and dx, when the inoculated seedlings were supplied with [17,17-2H2]GA20-glucosyl ester or [17,17- 2H2]GA20-glucosyl ether. Results of capillary gas chromatography-mass spectrometry analysis show that this growth was due primarily to release of the aglycone [17,17-2H2]GA20 and its subsequent 3beta-hydroxylation to [17,17-2H2]GA1 by the microorganism for the dy mutant, and by both the rice plant and microorganism for the dx mutant.     

Effect of water-soluble vitamins on the production of indole-3-acetic acid by Azospirillum brasilense

The effects of six water-soluble vitamins on tryptophan-dependent synthesis of indole-3-acetic acid in Azospirillum brasilense were investigated. A multifactorial regression analysis was employed to produce models of indole-3-acetic acid synthesis versus concentrations of tryptophan and the vitamins added to the growth medium. Very low levels of the B-group vitamins added at 10 to 100 microg l(-1) affected production of indole-3-acetic acid in A. brasilense. The largest release of this phytohormone was observed after amendment with pyridoxine and nicotinic acid. Results of the study suggest a role these vitamins may fulfil in the regulation of indole-3-acetic acid synthesis in A. brasilense.     

Effect of replacing nutrient potassium by sodium on uptake and distribution of sodium in tomato plants

Summary Tomato plants (Lycopersicon esculentum cv. Amberley Cross) were grown in a series of nutrient solutions in which 0, 60, 90, 95, 98 and 99 per cent of the K was replaced by Na. The plants selectively absorbed K relative to Na from the nutrient solutions and transported K to the shoots in preference to Na. In the nutrient solution having the highest K/Na ration most of the Na taken up by the plants accumulated in the roots, but as the K was progressively replaced by Na an increasing proportion of the total Na absorbed was transported to the leaves. Sodium was present at up to 2.4 per cent of the dry wt of whole, fully-expanded leaves without there being any apparent visual signs of damage or reduction in the rate of growth of the plants. On closer examination it was found that most of the Na transported to the leaves was excluded from the laminar tissue and accumulated in the adjacent petioles. The ability of the roots and petioles to retain large amounts of Na depended on an adequate supply of K to the plants.     

Effect of mercury and gold on growth,nutrient uptake,and anatomical changes in Chilopsis linearis

Plants of Chilopsis linearis were grown with 0, 50, 100, and 200 μM Hg [as Hg(CH₃COO)₂] and 0 and 50 μM Au (as KAuCl₄) in hydroponics. The results showed that seedling grown with 50 μM Au + 50 μM Hg and 50 μM Au + 100 μM Hg had roots 25 and 55% shorter than control roots, respectively. The element uptake determination using ICP/OES demonstrated that Hg at 50 and 100 μM (with and without Au) significantly increased (p < 0.05) the S concentration in leaves. On the other hand, the concentration of Fe significantly increased in roots of plants treated with Au–Hg. In addition, the stems of plants treated with Hg at 100 μM, with and without Au, had 239 and 876 mg Hg/kg dry biomass (d wt), respectively. Also, at 50 μM Hg, with and without Au, stems accumulated 375 and 475 mg Hg/kg d wt. The Hg concentration in leaves (287 mg Hg/kg d wt) was higher (p < 0.05) for the treatment containing 50 μM Au + 100 μM Hg. Without Au, the Hg concentration in leaves decreased to 75 mg Hg/kg d wt. Toxicity symptoms induced by Hg in cortex cells and the vascular system were lower in plants exposed to 50 μM Au + 50 μM Hg compared to plants exposed to 50 μM Hg only. Further, the SEM micrographs revealed deposition of Au–Hg particles inside the root. Although the concentrations of Hg used in this study showed different degree of toxicity, the plants displayed good agronomic value.     

Effect of Cultivation Conditions on the Composition of Extracellular Polysaccharide-Containing Substances in the Bacterium Azospirillum brasilense

Maintenance of pH 7.0 during the fermentation period favors accumulation of high molecular weight polysaccharide-containing components called lipopolysaccharide–protein and polysaccharide–lipid complexes in the capsules and culture medium. Increased pH of the culture medium to 8.0 reduced the period of exponential growth and the yield of polysaccharide-containing complexes as compared to optimal conditions. Maintenance of pH 5.5 suppressed the culture growth and polysaccharide production. The polysaccharide–lipid complexes obtained when pH was stabilized at the level of 7.0–8.0 had relatively low molecular weights and included only acidic polysaccharides. The use of potassium gluconate instead of sodium malate as a source of carbon in the culture medium changed the polysaccharide composition and increased the content of glucosamine, which increased the affinity of polysaccharides for wheat germ agglutinin. Prolongation of Azospirillum cultivation to five days introduced new glucose-containing polysaccharide components in the capsule.     

Plasmid P85 from Azospirillum brasilense SP245: study of the circle of possible hosts and incompatibility with plasmids from Azospirillum brasilense SP7]

The possibility of the stable inheritance of the plasmid p85 mobilized derivatives from Azospirillum brasilense Sp245 in the cells of the bacterial genera Rizobiaceae (Agrobacterium tumfaciens) and Pseudomonadaceae (Pseudomonas putida) has been shown. The plasmid p85 participates in coding for the physiologically active products (the plant hormones). It is not inherited by the Escherichia coli strains. For the first time the incompatibility of azospirillium plasmids has been demonstrated on the example of the plasmid p85 from Azospirillum brasilense Sp245 and the plasmid p115 from Azospirillum brasilense Sp7.     

Effect of iron plaque outside roots on nutrient uptake by rice (Oryza sativa L.). Zinc uptake by Fe-deficient rice

This solution culture study examined the effect of the deposition of iron plaque on zinc uptake by Fe-deficient rice plants. Different amounts of iron plaque were induced by adding Fe(OH)₃ at 0, 10, 20, 30, and 50 mg Fe/L in the nutrient solution. After 24 h of growth, the amount of iron plaque was correlated positively with the Fe(OH)₃ addition to the nutrient solution. Increasing iron plaque up to 12.1 g/kg root dry weight increased zinc concentration in shoots by 42% compared to that at 0.16 g/kg root dry weight. Increasing the amount of iron plaque further decreased zinc concentration. When the amounts of iron plaque reached 24.9 g/kg root dry weight, zinc concentration in shoots was lower than that in shoots without iron plaque, implying that the plaque became a barrier for zinc uptake. While rice plants were pre-cultured in –Fe and +Fe nutrient solution in order to produce the Fe-deficient and Fe-sufficient plants and then Fe(OH)₃ was added at 20, 30, and 50 mg Fe/L in nutrient solution, zinc concentrations in shoots of Fe-deficient plants were 54, 48, and 43 mg/kg, respectively, in contrast to 32, 35, and 40 mg/kg zinc in shoots of Fe-sufficient rice plants. Furthermore, Fe(OH)₃ addition at 20 mg Fe/L and increasing zinc concentration from 0.065 to 0.65 mg Zn/L in nutrient solution increased zinc uptake more in Fe-deficient plants than in Fe-sufficient plant. The results suggested that root exudates of Fe-deficient plants, especially phytosiderophores, could enhance zinc uptake by rice plants with iron plaque up to a particular amount of Fe.