N2-fixing bacteria in the rhizosphere: Quantification and hormonal effects on root development

The paper summarizes the results of a series of experiments on enumeration of N₂-fixing bacteria (diazotrophs) and hormonal effects of Azospirillum on root development. Numbers of N₂-fixing and N-heterotrophic bacteria were determined on the root (rhizoplane plus “inner” root surface) and in the rhizosphere soil (0–3 mm from the root surface) of Arrhenatherum elatius, other forage grasses and some herbaceous plant species. Pot experiments involved freshly collected soil from an unfertilized grassland area containing its natural population of N₂-fixing bacteria. The MPN (most probable number) of diazotrophs in relation to the MPN of the total bacterial population was always lower on the root than in the rhizosphere soil, suggesting that diazotrophs were not selectively advantaged at the root surface. Supply of mineral nitrogen (NH₄NO₃) decreased the proportion of N₂-fixing bacteria at the rhizoplane as well as in the rhizosphere soil. Similar results were obtained when N was supplied via the leaves. The data suggest that N₂-fixing bacteria in the rhizosphere are poor competitors once they loose their competitive advantage of binding dinitrogen. Correspondingly, the increase in the MPN of the diazotrophs found during plant development was interpreted as a result of decreased available combined N in the rhizosphere. The proportion of N₂-fixing bacteria relative to the total number of bacteria was generally below 1%. Considering the potential amount of substrate released from the roots and the substrate requirement of the bacterial population, N₂-fixation was considered insignificant for plant growth under the given conditions. For the investigations on possible beneficial effects on plant development by bacterial hormones, Azospirillum brasilense was chosen because evidence suggests that amongst the soil bacteria releasing hormones, especially IAA, certain strains of this species are more important than other bacteria. Application of A. brasilense Cd (ATCC 29710) onto the roots of young wheat plants grown in soil increased the number of lateral roots, the total root length and the number of root hairs. Similar results were obtained after application of IAA. This suggests that IAA is an important factor responsible for the effects observed after inoculation with A. brasilense. The increase in root surface may improve acquisition of nutrients and enhance growth of plants. Another hormonal effect of A. brasilense was an increase in nodulation of Medicago sativa grown on agar. Again pure IAA resulted in a similar increase in nodule number. Increases in nodule number were only in part associated with a change in root morphology. Therefore an effect of IAA on the plant immanent regulation system for nodulation is likely.     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

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     

Using the spermosphere model technique to describe the dominant nitrogen-fixing microflora associated with wetland rice in two Egyptian soils

Summary This study is an attempt to describe the dominant N₂-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 × 10^–6 mol C₂H₄ h^–1 g^–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 10⁸ bacteria g^–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.     

Occurrence and characterization of nitrogen-fixing Azospirilla in some soils of Egypt

The occurrence and characterization of N₂-fixing azospirilla in some Egyptian soils has been investigated. Seven soils, representing a wide range in texture and properties were selected from different localities in Egypt. The highest nitrogenase activity reported for soil samples under investigation were related to numbers of N₂-fixing microorganisms (Azotobacter spp., Azospirillum spp., and Clostridium spp.). Seven strains of azospirilla were isolated and purified. Based on morphological characteristics, three types of cell morphology were distinguished. Cultural and physiological characteristics as well as nitrogenase activity of representative isolates in presence of different concentrations of NaCl were determined. According to the physiological properties studied, all isolates were classified as members of Azospirillum brasilense.     

Fluorescent antibody technique to identify Azospirillum brasilense associated with roots of grasses

Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N₂-fixing ability was demonstrated by C₂H₂ reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum). These N₂-fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N₂-fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N₂-fixing bacteria associated with grass roots.     

Ammonium-excreting Azospirillum sp. become intracellularly established in maize (Zea mays) para-nodules

Maize seedlings develop nodule-like tumour knots (para-nodules) along primary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Inoculated NH ₄ ⁺ -excreting Azospirillum brasilense cells were shown to colonize these tumours, mostly intracellularly, promoting a high level of N₂ fixation when microaerophilic conditions were imposed. The nitrogenase activity inside the para-nodules was less sensitive to free O₂ than in non-para-nodulating roots. Both light and electron microscopy showed a dense bacterial population inside intact tumour cells, with the major part of the cell infection along a central tumour tissue. The bacteria colonized the cytoplasm with a close attachment to inner cell membranes. In an auxin-free growth medium, young 2,4-D-induced para-nodules grew further to become mature differentiated root organs in which introduced bacteria survived with a stable population. These results provide evidence that gramineous plants are potentially able to create a symbiosis with diazotrophic bacteria in which the NH ₄ ⁺ -excreting symbiont will colonize para-nodule tissue intracellularly, thus becoming well protected.     

Nitrogenase activity associated with leaf,root, and rhizosphere soils in an Indian tropical forest

Summary N₂ fixation (acetylene reduction assay) by phylloplane microorganisms was measured in dominant and co-dominant plant species growing in a tropical rain forest. No significant acetylene reduction was recorded with intact leaf samples. Azotobacter sp., Beijerinckia sp., Derxia sp., and Klebsiella pneumoniae were isolated as phylloplane N₂-fixing bacteria. Azospirillum lipoferum was only isolated from soil samples containing the roots of Poaceae. Nitrogenase activity was recorded in culture derived from the roots and rhizosphere soil samples, although low acetylene reduction activity indicates that these associations did not provide large amounts of N to the systems studied.     

Analysis of factors limiting associative N2-Fixation (C2H2 reduction) with two cultivars of Sorghum nutans

A 5–10 times larger nitrogenase activity per plant (max. 8 μmol C₂H₄ h⁻¹ plant⁻¹) was found in Sorghum nutans cultivar CSV 5 compared to the cultivar IS 5218 (max. 0.9 μmol C₂H₄ h⁻¹ plant⁻¹). This significant difference was reproduced for all water regimes from 8 to 21% soil moisture. The number of chemoorganotrophic bacteria (“total cell number”) on a medium with six carbon sources was very similar for both cultivars (3−4 × 10⁸ cells g⁻¹ rhizosphere soil). On the other hand, in the “high fixing” variety CSV 5 the number of N₂-fixing bacteria was greater than in IS 5218 by a factor of 3, the number of actinomycetes was reduced ten-fold and Arthrobacter strains to one-third or one-quarter. The number of aerobic N₂-fixing bacteria growing in an O₂ gradient system was increased 100-fold. Cultivar CSV 5 has a reduced photosynthetic area (20–30%), a reduced root weight (up to 50%, depending on the water regime) and a reduced transpiration rate (30–40%) compared to cv. IS 5218. Grain production however is 10–20% greater. At all soil moisture regimes the pH in the rhizosphere of cv. CSV 5 is 0.5–1.5 pH units below the values for cv. IS 5218.     

15N2还原法测定胡萝卜离体类根瘤的固氮作用

用低浓度的2,4-D处理胡萝卜离体根诱导结瘤,并接种固氮螺菌(Azospirillum.brasilense),用15N2还原法测定结果证明,胡萝卜离体类根瘤能固定15N2。电镜切片观察发现,类根瘤内有菌体存在,菌体进入细胞间隙中比进入细胞内多。     

Free-living nitrogen-fixing bacteria in temperate cropping systems: Influence of nitrogen source

Sustainable cropping systems rely on a minimum of external inputs. In these systems N is largely acquired in animal manures and leguminous green manures. Little is known of how these organic forms of N fertilizer influence the presence and activity of free-living N₂-fixing bacteria. High concentrations of inorganic N in soil inhibit N₂-fixation in cyanobacteria and Azotobacter spp. It is likely that manure and fertilizer applications would result in concentrations of inorganic N capable of inhibiting N₂ fixation and, ultimately, the presence of these organisms. We investigated the effect of synthetic and organic N fertilizer sources on the populations and N₂-fixation potential of free-living N₂-fixing bacteria in the Farming Systems Trial at the Rodale Research Institute. Field plots received the following N treatments prior to corn (Zea mays L.) production: (1) Legume rotations and green manures supplying about 165 kg N ha^-1; (2) beef cattle manure applied at a rate of 220 kg N ha^-1 (plus 60 kg N ha^-1 from 1994 hay plow-down); or (3) fertilizer N (urea and NH₄NO₃) applied at a rate of 145 kg N ha^-1. Soil samples were collected at two depths from corn plots four times during the growing season, and analyzed for soil moisture, soil pH, numbers of N₂-fixing cyanobacteria and Azotobacter spp., extractable NH _inf4 ^sup+ and NO _inf3 ^sup- , and potentially mineralizable N. Soil samples collected in mid-July were analyzed for nitrogenase activity (by C₂H₂ reduction) and total C and N. Populations of Azotobacter spp. and cyanobacteria were influenced only slightly by treatment; however, cyanobacteria species composition was notably influenced by treatment. Nitrogenase activity in surface soils was greatest in legume-N plots and in subsurface plots levels were greatest in fertilizer-N plots. Populations and activity of free-living N-fixing bacteria appeared to be somewhat reduced in all plots as a result of low soil pH levels and high concentrations of inorganic N across all treatments. Annual applications of N to all plots resulted in high levels of potentially mineralizable N that in turn may have reduced non-symbiotic N₂-fixation in all plots.     

Root-associated nitrogen-fixing bacteria and their role in the nitrogen nutrition of wheat estimated by 15N isotope dilution

The ability of two cultivars of spring wheat, Cadet and Rescue, and their reciprocal chromosome substitution lines, C-R5D and R-C5D, to obtain significant quantities of N from atmospheric N₂ was investigated in glasshouse experiments using ¹²⁵N dilution. The wheat was inoculated with N₂-fixing bacteria, including Azotohacter, Azospirillum, Klebsiella and Bacillus spp, in pure and mixed culture, at N concentrations ranging from 1 to 56 mg N plant^?1 (14–168 μg N ml^?1), in sand culture and in three soils of differing N content. Root-ussociutcd N₂-fixalion was negligible unless carbohydrate was added to the rooting medium. Atmospheric N₂ was incorporated into wheat roots and translocated to the tops, when plants inoculated with Azotobacter beijerinckii or Azospirillum brasilense sp. 107 were amended with glucose and malate respectively, under monoxenic conditions in sand culture.     

Enhancement of aerobic nitrogenase activity (acetylene reduction assay) by phenol in soils and the rhizosphere of cereals

By addition of phenol at concentrations between 0.1 and 10 mmol·l^?1, nitrogenase activity (acetylene reduction assay) is enhanced by a factor of 5 in the rhizosphere of Pennisetum glaucum (pearl millet) incubated under 20% O₂. No increase is found under microaerobic conditions. This enhancement effect is also noticed in a soil amended with a sucrose concentration of 20 mmol·l^?1. Under those conditions, however, an enhancement is found under aerobic as well as under microaerobic conditions and a further increase of the phenol added reduces the activity to almost zero. A 4-fold increase of N₂-fixation by phenol addition under aerobic conditions was determined with homogenous sediments from a fresh water lake while anaerobic N₂-fixation was already slightly reduced by the same concentration added. Excised roots of Sorghum nutans CSH 5 failed to show any phenol enhancement of nitrogenase activity. After a preincubation of 6h, inhibition of nitrogenase activity under air by addition of 1 mmol·l^?1 was much more pronounced than under microaerobic conditions.     

Biological nitrogen fixation at elevated temperature in different Azospirillum species and strains

Nitrogenase activity, nitrogenase synthesis, and the growth of four species of Azospirillum were examined at 30°C and 42°C. Azospirillum brasilense Sp 7 did not synthesize nitrogenase at 42°C nor was the enzyme stable at this temperature. In A. lipoferum 708 and A. brasilense 12S the nitrogenase activity was stable at 42°C but the synthesis of nitrogenase was reduced. In A. brasilense Sp 9 the nitrogenase activity was stable and showed the highest C₂H₂ reduction activity at 42°C.     

Interaction of the herbicides Bromoxynil and Afalon S with Azospirillum and growth of maize

The possible interaction of herbicides Bromoxynil and Afalon S with Azospirillumspp. and growth of maize was investigated in a greenhouse experiment. Neither inoculation nor herbicide application with or without inoculation had significant effect on the major groups of soil microflora (bacteria, actinomycetes, and fungi). The highest values of nitrogenase as well as dehydrogenase activity were recorded in treatment received only Azospirillum. Incorporation in soil of either Bromoxynil or Afalon S at the recommended field dose seemed to have no significant effect on the enzymatic activities, while application of these herbicides with Azospirillum had stimulatory effects in some cases. The application of either Bromoxynil or Afalon S significantly increased the dry weight of roots and shoots at 45 days period. The effect of herbicide on plant growth was more pronounced when applied with Azospirillum and the highest stimulatory effect was observed when Afalon S was applied with the N₂-fixing microorganism.     

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).     

Root-induced changes in the rhizosphere: Importance for the mineral nutrition of plants

Root-induced changes in the rhizosphere may affect mineral nutrition of plants in various ways. Examples for this are changes in rhizosphere pH in response to the source of nitrogen (NH₄-N versus NO₃-N), and iron and phosphorus deficiency. These pH changes can readily be demonstrated by infiltration of the soil with agar containing a pH indicator. The rhizosphere pH may be as much as 2 units higher or lower than the pH of the bulk soil. Also along the roots distinct differences in rhizosphere pH exist. In response to iron deficiency most plant species in their apical root zones increase the rate of H⁺ net excretion (acidification), the reducing capacity, the rate of Fe^III reduction and iron uptake. Also manganese reduction and uptake is increased several-fold, leading to high manganese concentrations in iron deficient plants. Low-molecular-weight root exudates may enhance mobilization of mineral nutrients in the rhizosphere. In response to iron deficiency, roots of grass species release non-proteinogenic amino acids (?phytosiderophores”?) which dissolve inorganic iron compounds by chelation of Fe^III and also mediate the plasma membrane transport of this chelated iron into the roots. A particular mechanism of mobilization of phosphorus in the rhizosphere exists in white lupin (Lupinus albus L.). In this species, phosphorus deficiency induces the formation of so-called proteoid roots. In these root zones sparingly soluble iron and aluminium phosphates are mobilized by the exudation of chelating substances (probably citrate), net excretion of H⁺ and increase in the reducing capacity. In mixed culture with white lupin, phosphorus uptake per unit root length of wheat (Triticum aestivum L.) plants from a soil low in available P is increased, indicating that wheat can take up phosphorus mobilized in the proteoid root zones of lupin. At the rhizoplane and in the root (root homogenates) of several plant species grown in different soils, of the total number of bacteria less than 1 % are N₂-fixing (diazotrophe) bacteria, mainly Enterobacter and Klebsiella. The proportion of the diazotroph bacteria is higher in the rhizosphere soil. This discrimination of diazotroph bacteria in the rhizosphere is increased with foliar application of combined nitrogen. Inoculation with the diazotroph bacteria Azospirillum increases root length and enhances formation of lateral roots and root hairs similarly as does application of auxin (IAA). Thus rhizosphere bacteria such as Azospirillum may affect mineral nutrition and plant growth indirectly rather than by supply of nitrogen.     

Biological nitrogen fixation associated with roots of field-grown barley (Hordeum vulgare L.)

Nitrogenase (C₂H₂) activity was measured in microbial media inoculated with barley root segments or corresponding rhizosphere soil. Three different media were used, Döbereiner's malate medium, a modified Ashby medium, and an acid nitrogen-free medium. Only Döbereiner's medium gave consistently positive results, and cultures inoculated with roots showed higher activity than cultures inoculated with corresponding rhizosphere soil. Similar experiments with roots and rhizosphere soil from wheat gave only negligible nitrogenase activity, whereas the tropical grass, Cynodon dactylon, gave higher activity than barley. Measurements on intact soil cores containing barley root systems showed an initial lag phase followed by a rather stable activity level over a period from 12 h to 48 h, and then the activity again decreased. The activity during the stable period corresponded to fixation of about 100 to 200 g N₂ ha^?1 24 h^?1. Measurements on isolated, washed barley roots showed only negligible nitrogenase activity.     

Maize growth and yield responses to seed‐inoculated N2‐fixing bacteria under Dryland production conditions

Maize (Zea Mays L.) seeds were inoculated with the N₂‐fixing bacteria, Azospirillum brasilense and Azorhizobium caulinodans. Shoot growth, shoot nitrogen (N) concentration, and grain yield was determined under dryland production conditions in a silt loam. Fertilizer N was applied according to soil test recommendations at either 0, 50, 75, or 100% of the recommended N requirements for a 7,500 kg ha^‐1 yield goal. Both A. brasilense and A. caulinodans increased shoot dry matter production, shoot N concentration, and grain yield somewhat at the lower N recommended rates. There was no agronomic benefit with either A. brasilense and A. caulinodans inoculations under dryland conditions for high N fertility soils under dryland production conditions in a subhumid or semi‐arid moisture regime.     

Effect of Azospirillum brasilense inoculation on root morphology and respiration in tomato seedlings

Summary The level of Azospirillum brasilense strain Cd colonization in the rhizosphere of some vegetables was 10⁴–10⁵ colony-forming units (CFU) per root of one plant in 2-week-old plants inoculated with 5 × 10⁸ Azospirillum cells. Significant increases in root length (35%) and in top (90%) and root (50%) dry weight and total leaf area (90%) were observed in 18-day-old inoculated tomato plants compared with non-inoculated controls. An inoculum concentration of 1 × 10⁸ to 5 × 10⁸ CFU/ml stimulated the appearance of root hairs. Large numbers of bacteria (1 × 10⁹ CFU/ml) caused asymmetrical growth of the root tip. In a petri dish system, Azospirillum (1 × 10⁸ CFU/ml) increased root dry weight (150%), protein content (20%), respiration rate per root (70%) and the specific activity of malate dehydrogenase (45%–65%) over non-inoculated controls. The specific respiration rate, expressed as micromol of O₂ per minute per milligram of dry weight of roots, was significantly lower in inoculated roots, suggesting that less energy was spent for accumulation of more dry material.