Inoculation of field-grown wheat (Triticum aestivum) with Azospirillum spp. in Brazil

Summary Three field experiments with wheat were conducted in 1983, 1984, and 1985 in Terra Roxa soil in Paraná, the major Brazilian wheat-growing region, to study inoculation effects of various strains of Azospirillum brasilense and A. amazonense. In all three experiments inoculation with A. brasilense Sp 245 isolated from surface-sterilized wheat roots in Paraná produced the highest plant dry weights and highest N% in plant tops and grain. Grain yield increases with this strain were up to 31 % but were not significant. The application of 60 or 100 kg N ha^–1 to the controls increased N accumulation and produced yields less than inoculation with this strain. Another A. brasilense strain from surface-sterilized wheat roots (Sp 107st) also produced increased N assimilation at the lower N fertilizer level but reduced dry weights at the high N level, while strain Sp 7 + Cd reduced dry weights and N% in the straw at both N levels. The A. amazonense strain isolated from washed roots and a nitrate reductase negative mutant of strain Sp 245 were ineffective. Strains Sp 245 and Sp 107st showed the best establishment within roots while strain Cd established only in the soil.     

Peculiar effect of Azospirillum inoculation on growth and nitrogen balance of winter wheat (Triticum aestivum)

Summary Wheat plants (Triticum aestivum) grown in pots and in the field under the Mediterranean climate of the south of France were inoculated with a strain of Azospirillum brasilense. Comparisons with non-inoculated plants grown under the same conditions showed significant responses to inoculation with an increase in the number of fertile tillers, shoot and root dry weight, and root to shoot biomass ratio. The roots of inoculated plants attracted relatively more assimilates than those of the control plants until a late stage of growth (heading stage) but the rhizosphere respiration expressed per unit of root growth was not increased by inoculation. Nitrogen yield, both total and in grains, was also enhanced; however, N percentages of all aerial parts of the plants grown in pots were always statistically lower after inoculation than in the control. At maturity, the N % in seeds was 1.81 and 2.45, respectively. The possible mechanisms of this effect of inoculation under the experimental conditions of this study are discussed.     

Association of Azospirillum brasilense with pearl millet (Pennisetum americanum (L.) Leeke)

Summary Microscopic observations of the root system of pearl millet (Pennisetum americanum (L.) Leeke) var. BJ 104 after surface sterilization and incubation in phosphate malate triphenyl tetrazolium chloride (TTC) revealed extensive colonization by Azospirillum spp. when plants were grown in sterile, partially sterile and field conditions as evidenced by the TTC-reducing property of active cells of the bacterium. Quantitative studies showed the need to standardize the techniques further to ensure more precise monitoring of the bacteria in the rhizosphere, as large numbers of soil bacteria were found capable of growth on specific media, thus interfering with the plate counts. Seed inoculation with A. brasilense increased the mean grain yield of pearl millet under different agroclimatic conditions in India. The mean increase in grain yield due to inoculation over uninoculated controls was also noticed with graded levels of fertilizer nitrogen (urea). Inoculation alone contributed to increased nitrogen uptake of plants with varying levels of fertilizer nitrogen application under sandy loam soil conditions (pH 7.3). The effects of inoculation were more prominent under lower levels of nitrogen than at the higher levels. The root biomass under field conditions was increased with Azospirillum spp. inoculation at 10 and 20 kg N/ha than their corresponding uninoculated controls.     

Yield response of spring wheat cultivars (Triticum aestivum and T. turgidum) to inoculation with Azospirillum brasilense under field conditions

Summary Eight commercial Israeli spring wheat cultivars (six Triticum aestivum and two T. turgidum) grown with 40 and 120 kg N/ha were tested for responses to inoculation with Azospirillum brasilense. At the low level of N fertilization (40 kg/ha), five cultivars showed significant increases in plant dry weight measured at the milky ripe stage; however, by maturation only the cultivar Miriam showed a significant increase in grain yield. Two cultivars, which had shown a positive inoculation effect at the earlier stages, had a significant decrease in grain yield. No significant effect of inoculation was found at the high N level. To confirm those results, four wheat (T. aestivum) cultivars were tested separately over 4 years in 4 different locations under varying N levels. Only Miriam showed a consistently positive effect of Azospirillum inoculation on grain yield. Inoculation increased the number of roots per plant on Miriam compared with uninoculated plants. This effect was found at all N levels. Nutrient (N, P and K) accumulation and number of fertile tillers per unit area were also enhanced by Azospirillum, but these parameters were greatly affected by the level of applied N. It is suggested that the positive response of the spring wheat cultivar Miriam to Azospirillum inoculation is due to its capacity to escape water stresses at the end of the growth season.     

Single and double inoculation with Azospirillum/Trichoderma: the effects on dry bean and wheat

A plant growth-promoting rhizobacterium (Azospirillum brasilense Sp7) and a bio-control fungus, which can solubilize insoluble phosphorus (Trichoderma harzianum Rifai 1295-22), were evaluated for their single and combined effects on dry bean (Phaseolus vulgaris) and wheat (Triticum aestivum L.) grown in soil. A pot experiment with bean and a field experiment with both bean and wheat were established. In contrast to single inoculation of Trichoderma, the single inoculation of Azospirillum and the double inoculation did not significantly (P >0.05) increase nodule numbers and nodule mass at 45 days after planting in pot grown beans. However, the Azospirillum inoculation with supplementary phosphorus significantly (P <0.05) increased nodule mass. There were no significant (P >0.05) differences among the inoculation treatments for plant dry weight, total plant nitrogen, and total plant phosphorus at 45 days after planting in both pot and field experiments with bean. However, the combined inoculation and rock phosphate application at 1 Mg ha^–1 significantly (P <0.05) increased bean seed yield, total seed nitrogen and phosphorus in the bean field trial. This treatment more than doubled the mentioned properties compared to the control. The microbial inoculations, with the exception of the combined inoculation, significantly (P <0.05) increased total seed nitrogen, but never affected seed yield in the wheat field trial (P >0.05). The combined inoculation improves many plant and yield parameters and, therefore, has some advantages over single inoculation provided that rock phosphate was supplied at an amount not exceeding 1 Mg ha^–1. Higher rock phosphate application rates decreased many plant and yield parameters in our study.This work was carried out at Gaziosmanpaa University, Tokat, Turkey.     

Influence of soluble phosphorus fertilizer on the interaction between a vesicular-arbuscular mycorrhizal fungus and Azospirillum brasilense in barley (Herdeum vulgare L.)

Summary Pot-culture studies were carried out to examine the response of barley (Hordeum vulgare L.) to inoculation with Azospirillum brasilense and Glomus versiforme, singly and/or in combination, under varying levels of nitrogenous [(¹⁵NH₄)₂SO₄] and soluble phosphatic (single superphosphate) fertilizers. The interaction between both the endophytes led to increased growth and nutrition of the barley plants. Roots from plants inoculated with Azospirillum brasilense and Glomus versiforme exhibited very low acetylene reduction activity. N₂ fixation in the plants increased with the increase in plant growth but the mycorrhiza alone gave a low level of N₂ fixation in the plants compared to combined inoculation with both the endophytes.     

The flavonoid naringenin enhances intercellular colonization of rice roots by<Emphasis Type="Italic"> Azorhizobium caulinodans</Emphasis>

The intercellular colonization of rice roots by Azorhizobium caulinodans and other diazotrophic bacteria has been studied using strains marked with the lacZ reporter gene. A. caulinodans were able to enter the roots of rice at emerging lateral roots (lateral root cracks) by crack entry and this was observed by light microscopy. After colonization of lateral roots, bacteria moved into intercellular space within the cortical cell layer of roots. Naringenin at 1×10^-5 and 5×10^-5 M concentration significantly enhanced root colonization. The role of nodABC and regulatory nodD genes was also studied; lateral root crack (LRC) colonization of rice was shown to be Nod factor and NodD independent. Lateral root crack colonization of rice was also observed with similar frequency following inoculation with Azospirillum brasilense and the colonization by A. brasilense was stimulated by naringenin and other flavonoid molecules.     

Effect of Azospirillum brasilense inoculation on rhizobacterial communities analyzed by denaturing gradient gel electrophoresis and automated ribosomal intergenic spacer analysis

Nucleic acid-based techniques allow the exploration of microbial communities in the environments such as the rhizosphere. Azospirillumbrasilense, a plant growth promoting rhizobacterium (PGPR), causes morphological changes in the plant root system. These changes in root physiology may indirectly affect the microbial diversity of the rhizosphere. In this study, the changes in the rhizobacterial structure following A. brasilense inoculation of maize (Zea mays) plants was examined by PCR-denaturating gradient gel electrophoresis (DGGE) and automated ribosomal intergenic spacer analysis (ARISA), using two universal primers sets for the 16S rRNA gene, and an intergenic 16S-23S rDNA primer set, respectively. Similar results were obtained when using either ARISA or DGGE performed with these different primer sets, and analyzed by different statistical methods: no prominent effect of A. brasilense inoculation was observed on the bacterial communities of plant roots grown in two different soils and in different growth systems. In contrast, plant age caused significant shifts in the bacterial populations.     

Yield response of wheat and barley to inoculation of plant growth promoting rhizobacteria at various levels of nitrogen fertilization

The yield response of a wheat (Kirik) and a barley (Tokak 157/37) cultivar to inoculation with Azospirillum brasilense Sp246 and Bacillus sp. OSU‐142 was studied in relation to three levels of N fertilization (0, 40, and 80 kg ha^–1) under field conditions in Erzurum, Turkey, in 1999 and 2000. Seed inoculation with A. brasilense Sp246 significantly affected yield and yield components, both in wheat and barley. On average of years and N doses, inoculation with A. brasilense Sp246 increased spike number per m², grain number per spike, grain yield, and crude protein content by 7.2, 5.9, 14.7, and 4.1 % in wheat and by 6.6, 8.1, 17.5, and 5.1 % in barley, respectively, as compared to control. Inoculation with Bacillus sp. OSU‐142 significantly increased kernel number per spike in wheat, but no significant effect was determined in the other characteristics. Grain yields and yield components were also higher at all levels of nitrogen fertilizer in the inoculated plots as compared to the control. However, these increases diminished at high fertilizer levels. These results suggest that application of the growth promoting bacteria A. brasilense Sp246 may have the potential to be used as a biofertilizer for spring wheat and barley cultivation in organic and low‐N input agriculture.     

Agronomical Performance of Upland Rice Cultivars Inoculated with Azospirillum brasilense Depends on the Plant Genotype

Plant growth–promoting bacteria may play an important role on rice development; however, its interaction with different genotypes is still uncertain. This work aimed to assess the effect of inoculation of Azospirillum brasilense AbV5 strain on the agronomical performance of different rice cultivars under field conditions during two consecutive years and to determine the response of rice cultivars to indole-acetic acid (IAA) rates under in vitro conditions using for both conditions a randomized block design. Field experiments evaluated the productivity components and grain yield of inoculated and noninoculated cultivars and in vitro assays monitored the plantlet growth under different IAA rates. Field experiments showed that the general average grain yield of rice cultivars inoculated with A. brasilense AbV5 strain was significantly greater. Fifty percent of the evaluated cultivars had statistically significant increase in yield varying from 40 to 108 percent upon inoculation with A. brasilense AbV5 strain, while 35 percent showed no significant change in yield (?6 to ?28 percent). In vitro assays showed rate- and cultivar-dependent responses of upland rice to exogenous IAA. These results show a cultivar-dependent response of rice to inoculation with A. brasilense AbV5 strain, which can be related with IAA production by AbV5 strain.     

N2-fixation by ammonium-excreting Azospirillum brasilense in auxine-induced root tumours of wheat (Triticum aestivum L.)

Summary Wheat seedlings, treated with the auxine 2,4-dichlor-phenoxy acetic acid (2,4-D) during germination developed only a residual root system. Root elongation was extremely restricted and root tips were deformed to thick club-shaped tumours. When 2,4-D was added in a later stage of plant growth the plants developed additional nodule-like knots along primary roots. Root and shoot dry-matter production was slightly repressed in all 2,4-D treatments and N translocation from roots to shoots was repressed as well. When transferred to an auxine-free growth medium, the 2,4-D-affected roots were not capable of complete recovery. In plants inoculated gnotobiotically with Azospirillum brasilense, either with the wild type or with the NH ₄ ⁺ -excreting mutant strain C3, a 2,4-D addition increased rhizosphere acetylene-reduction activity at pO₂ 1.5 kPa. The O₂ sensitivity of root-associated nitrogenase activity tended to be reduced. The number of root-colonizing bacteria, at approximately 10⁸ colony-forming units (cfu) per g dry root, was similar in the 2,4-D treatments and untreated controls. Plant treatment with high concentrations of the chemical isomer 3,5-dichlor-phenoxy acetic acid (3,5-D) did not have comparable effects, either on plant development or on rhizosphere-associated nitrogenase activity. Root-tumour tissue inhabited by A. brasilense showed purple staining when subjected to a tetrazolium chloride solution, which may indicate intensive local nitrogenase activity in this tissue. Exposed to an ¹⁵N₂-enriched atmosphere, plants treated with 2,4-D and with A. brasilense incorporated significantly higher amounts of ¹⁵N than untreated controls. In all cases the highest values of ¹⁵N enrichment were found following inoculation with the NH ₄ ⁺ -excreting mutant strain C3. Present address: F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, Willem de Croylan 42, B-3001 Heverlee, Belgium     

Infectivity and acetylene reduction of diazotrophic rhizosphere bacteria in wheat (Triticum aestivum) seedlings under gnotobiotic conditions

Summary Wheat seedlings were inoculated with rhizosphere nitrogen-fixing bacteria and grown gnotobiotically for 15 days. The growth medium consisted of semisolid agar with or without plant nutrients. The bacteria, isolated from roots of field-grown wheat, were three unidentified Gram-negative rods (A1, A2, E1), one Enterobacter agglomerans (C1) and two Bacillus polymyxa (B1, B2). A strain of Azospirillum brasilense (USA 10) was included for comparison.Nitrogenase activity (acetylene reduction activity, ARA) was tested on intact plants after 8 and 15 days of growth. In semisolid agar without plant nutrients, five isolates showed ARA of 0.01–0.9 nmol C₂H₄ plant^–1 h^–1, while the two strains of B. polymyxa had higher ARA of 3.3–10.6 nmol C₂H₄ plant^–1 h^–1.Plant development was not affected by inoculation with bacteria, except that inoculation with B. polymyxa resulted in shorter shoots and lower root weight.Transmission electronmicroscopy of roots revealed different degrees of infection. A. brasilense, A1 and A2, occurred mainly in the mucilage on the root surface and between outer epidermal cells (low infectivity). B. polymyxa strains and E1 were found in and between epidermal cells (intermediate infectivity) while E. agglomerans invaded the cortex and was occasionally found within the stele (high infectivity).     

Effect of inoculation with different strains of Azospirillum brasilense on cotton (Gossipium barbadense)

Summary The response of the cotton plant to inoculation with six strains of Azospirillum brasilense was investigated under subtropical conditions in Egypt. Azospirilla populations and activities were increased as a result of root inoculation with liquid inoculum of Azospirillum sp. Highest C₂H₂ — reduction activities on roots were obtained with strains S631 and Sp Br 14 (means of 216.85 and 209.50 nmol C₂H₄g^–1 root h^–1 respectively) while strain M4 gave the lowest activity (mean of 100.8 nmol C₂H₄g^–1 root h^–1). Statistical analysis showed that Azospirillum strains 5631, Sp Br 14, E15 and SC22 significantly increased the plant dry weight and nitrogen uptake while inoculation with strains M4 and SE had no significant effect in that respect.     

Response of maize (Zea mays) to Azotobacter inoculation under fertilized and unfertilized conditions

Summary Inoculated seeds of maize (Zea mays) with 11 Azotobacter strains, sown in the fields receiving no fertilizer and fertilizers (N and P at the rate of 125 and 40 kg ha^–1 respectively) increased the grain yield by 19.63% and 15.89% respectively over the corresponding control. The effect was greater in unfertilized than in fertilized soil. The increase in yield due to fertilizers was 21.2% without inoculation and 37.09% with inoculation. The correlations between total yield, and N, P and K uptake were highly significant and comparable among themselves. This indicated that increase in yield due to inoculation was not due to N₂ fixation but that some other mechanisms like production of growth hormones by this bacterium may be responsible.     

Growth stimulation and nitrogen supply to wheat plants inoculated with Azospirillum brasilense

Twelve Azospirillum brasilense strains isolated from wheat (Triticum aestivum L.) roots were compared for root colonization, growth stimulation, and nitrogen (N) supply to young wheat plants cv. Klein Chamaco grown in sterile nutrient solutions without N. All the strains inoculated colonized both the root surface and interior, and most strains stimulated root and shoot growth, although the degree of stimulation was different for the different strains. Some strains increased the total N content of roots and tops at the end of the experiment, in one case up to 80% of the uninoculated plants, while others produced no effect on N content. No correlation could be found between growth stimulation or the amount of N supplied to the plant with the degree of root colonization. When the most efficient strain for N fixation was inoculated to different wheat cultivars, it stimulated growth and supplied N to the five cultivars tested, although the degree of root colonization, growth stimulation and N supply showed differences among the cultivars. Our results suggest that there exists the potential of A. brasilense to supply N to wheat plants in considerable amounts, although an adequate strain are still to be identified.     

续断菊与玉米间作的铅累积及根系低分子量有机酸分泌特征研究

为探明间作作物根系分泌低分子量有机酸对土壤重金属生物有效性的影响,采用矿区周边农田土壤进行室内盆栽试验,研究了云南本土超累积植物续断菊(Sonchus asper L. Hill)和玉米(Zea mays L.)间作下,植物生长、根系低分子量有机酸分泌量、根际土壤Pb提取形态以及植物Pb积累特点。结果表明:与单作相比,间作续断菊地上部和根部生物量、根长、根内径和根系体积均显著增加(P0.05);间作玉米根部生物量、根长、根内径和根系体积显著增加(P0.05)。柠檬酸、草酸是续断菊和玉米根系分泌的主要低分子量有机酸,间作导致续断菊根系低分子量有机酸的分泌量增加,玉米根系低分子量有机酸的分泌量降低。续断菊根际土壤生物有效态Pb含量增加85.2%(P0.05),而玉米根际土壤生物有效态Pb含量降低26.1%(P0.05)。续断菊体内Pb含量显著增加18.0%～43.2%(P0.05),富集系数提高26.0%,而转运系数降低42.0%;玉米地上部Pb含量显著降低24.3%(P0.05),转运系数降低43.1%。续断菊根系分泌的柠檬酸和草酸数量,均与土壤生物有效态Pb含量呈显著正相关,且土壤有效态Pb含量分别与续断菊地上部和根部的Pb含量呈显著正相关。表明间作增加了续断菊对Pb的吸收积累量,与间作体系植物根系分泌的低分子有机酸介导下的土壤有效态Pb含量增加密切相关。     

Effect of inoculation with Azotobacter chroococcum on nitrogenase activity of Zea mays roots grown in agricultural soils under aseptic and non-sterile conditions

Summary Inoculated and non-inoculated seedlings of Zea mays were grown in agricultural soils under aseptic and non-sterile conditions. Acetylene reduction activity and microbial counts were determined after 7 and 30 days of growth. Irrespective of the soil type Azotobacter spp. were commonly isolated under maize cultivation. Inoculation of agricultural soils with a suspension of A. chroococcum led to an increase in Azotobacter numbers, although this effect diminished with time. Nitrogenase activity was detected on maize roots and increased in response to the inoculation with A. chroococcum, showing that this associative growth could be of primary importance for the plant. The results of assays for acetylene reduction activity indicated that the nitrogenase activity was associated only with the root systems.     

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.     

Macronutrient accumulation in wheat crop (Triticum aestivum L.) with Azospirillum brasilense associated with nitrogen doses and sources

Abstract

New studies are needed to optimize the nitrogen (N) amount that can be applied to utilize the Azospirillum brasilense benefits. In addition, information regarding the interaction between the urease inhibitor and biological nitrogen fixation (BNF) and how they affect the macronutrients accumulation are also needed. We evaluate the effect of N sources and doses associated with A. brasilense regarding the macronutrients accumulation in straw and grains and wheat grain yield in tropical conditions. A randomized block experimental design was used with four replications in a 2?×?5?×?2 factorial arrangement as follows: two N sources (urea and urea with urease enzyme inhibitor NBPT; five N doses (0, 50, 100, 150, and 200?kg ha^?1) applied in topdressing; with and without A. brasilense inoculation. We found that an increase in N doses positively influenced the accumulation of macronutrients in straw and grains and the wheat grain yield. N sources have similar effects. Inoculation with A. brasilense increased accumulation of Mg and S in straw and P, Ca, and Mg in grains, regardless of the N dose. The inoculation with A. brasilense associated with 140?kg ha^?1 of N increased wheat grain yield. The inoculation can contribute in a more sustainable way to wheat nutrition and optimizing N fertilization.     

Yield and N assimilation of winter wheat (Triticum aestivum L., var. Norstar) inoculated with rhizobacteria

The effects of bacterial inoculants on the growth of winter wheat were studied in a growth chamber. Azospirillum brasilense, Azotobacter chroococcum, Bacillus polymyxa, Enterobacter cloacae, or a mixture of the four rhizobacteria were the inoculants tested. Inoculation effects on yield, yield components, and N-derived from fertilizer (Ndff) were assessed. The response of plants inoculated with individual bacteria was inconsistent and varied with treatment. At the first harvest (58 days after planting-DAP) plants inoculated with the mixture exhibited increases in plant dry weight, total-N and Ndff. At the second harvest (105 DAP), plants inoculated with A. brasilense and the mixture exhibited increases in shoot biomass, whereas at maturity (170 DAP), the inoculated plants showed no differences in total-N or shoot dry matter yield, as compared to the uninoculated controls. Inoculation with A. brasilense, however, increased the Ndff in the shoots, and B. polymyxa tended to enhance grain yield. Practical use of these rhizobacteria as inoculants for winter wheat may have limited value until such time as we better understand factors which influence rhizosphere competence of bacterial inoculants.