Effect of grazer regulation and algal inoculation on photodependent nitrogen fixation in a wetland rice field

Summary A dry season field experiment conducted for two consecutive years highlighted problems of achieving increased populations of N₂-fixing blue-green algae (BGA) in wetland rice fields. Inoculation of non-indigenous BGA strains, either dried or as fresh viable inocula even at high levels of application, was unsuccessful. A limiting effect of grazing invertebrate populations on BGA establishment was evident, but other factors were involved. Reducing grazer pressure did not permit establishment of inoculated BGA; interspecific competition and environmental factors may explain the inoculation failure. Grazer regulation permitted the establishment of a fast-growing indigenous N₂-fixing Anabaena and the doubling of N₂-fixing activity over a control. Neither inoculation nor grazer control affected grain yields significantly.     

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.     

Physiological enhancement of early growth of rice seedlings (Oryza sativa L.) by production of phytohormone of N2-fixing methylotrophic isolates

Three plant-growth promoting, N₂-fixing methylotrophic strains isolated from rice cultivars (Oryza sativa L.), viz, Methylobacterium sp. CBMB20, Enterobacter sp. CBMB30, Burkholderia sp. CBMB40, were selected, and their activities in promoting the early growth of rice were studied. Seeds treated with the methylotrophic strains improved seed germination, seedling vigor index (SVI), and biomass of rice seedlings. The methylotrophic population in the treated seedlings increased in the vegetative stages when compared to seeding stages. Treated seedlings showed a higher accumulation of plant hormones viz trans-zeatin riboside, isopentenyladenosine, and indole-3-acetic acid than untreated seedlings. Plant hormones were detected immunologically using the phytodetek kit. Conformational evidence suggested that cytokinins were produced by the epiphytic bacteria colonizing the plants rather than by the plants themselves. In addition, the inoculated early stage rice seedlings also exhibited a wide range of acetylene reduction activity. The results suggest the potential use of these bacteria to stimulate germination, SVI, and biomass production, which is mediated by production of plant hormone accumulation and nitrogen fixation.     

Dynamics of algal populations and acetylene-reducing activity in five rice soils inoculated with blue-green algae

Summary The dynamics of five inoculated strains of heterocystous blue-green algae (BGA) and indigenous algae were studied for 1 month in 1-m² microplots of five soils previously air-dried or oven-dried. The same soils were then dried and resubmerged for another 2 months to study the effect of controlling algal grazers with neem (Azardirachta indica) seeds on the revival and dynamics of indigenous and inoculated algae. During the month following inoculation, inoculated BGA multiplied to some extend in all soils but never dominated the total algal flora. They rarely dominated the indigeneous heterocystous BGA, and did so only when the growth of N₂-fixing BGA was poor or after the decline of blooms of indigenous strains. Once the soils were dried, two of the five inoculated strains did not reappear. During the 1st month following rewetting, the remaining inoculated strains again exhibited poor growth; however, after 2 months of submergence, inoculated Aulosira fertilissima developed an agronomically significant bloom in neem-treated plots of two soils. Correlations between acetylene-reducing activity and heterocystous BGA populations indicated a major contribution by indigenous BGA and a minor contribution by inoculated BGA to the N₂-fixing activity of the soils during the first experiment and the 1st month of the second experiment. The establishment of inoculated BGA exhibited clear differences among strains but was less affected by the nature of the soil and heat treatment. Neem application might have had a delayed positive effect on the late establishment of inoculated A. fertilissima and favored BGA growth and N₂ fixation by the total algal population.Visiting Scientist at IRRI     

Diversity of diazotroph populations in the rhizosphere of maize (Zea mays L.) growing on different French soils

Summary We studied the dominant diazotrophs associated with maize roots and rhizosphere soil originating from three different locations in France. An aseptically grown maize plantlet, the spermosphere model, was used to isolate N₂-fixing (acetylene-reducing) bacteria. Bacillus circulans was the dominant N₂-fixing bacterium in the rhizosphere of maize-growing soils from Ramonville and Trogny, but was not found in maize-growing sandy soil from Pissos. In the latter soil, Enterobacter cloacae, Klebsiella terrigena, and Pseudomonas sp. were the most abundant diazotrophs. Azospirillum sp., which has been frequently reported as an important diazotroph accociated with the maize rhizosphere, was not isolated from any of these soils. The strains were compared for their acetylene-reducing activity in the spermosphere model. The Bacillus circulans strains, which were more frequently isolated, also exhibited significantly greater acetylene-reducing activity (3100 nmol ethylene day^-1 plant^-1) than the Enterobacteriaceae strains (180 nmol ethylene day^-1 plant^-1). This work indicates for the first time that Bacillus circulans is an important maizerhizosphere-associated bacterium and a potential plant growth-promoting rhizobacterium.     

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.     

Evidence for enhanced N availability during switchgrass establishment and seeding year production following inoculation with rhizosphere endophytes

Improvement in sustainable production of switchgrass (SG, Panicum virgatum L), as a purpose-grown biomass feedstock crop, could be realized through investigation of plant–microbe interactions associated with plant growth promoting rhizobacteria (PGPR), capable of biological nitrogen fixation (BNF). The objective of this study is to increase establishment year production of SG biofuels by inoculation with a mixed PGPR inoculum. We isolated pure strains of N₂-fixing, and other PGPR, from SG rhizomes. The bacteria were identified as Paenibacillus polymyxa, an N₂-fixing bacterium, and other PGPR capable of solubilizing phosphate and/or producing auxins. Field trials utilizing these strains in a mixed PGPR inoculum showed that inoculated plants contained more N in tillers during anthesis but not at senescence, suggesting that more N could be cycled to belowground roots and rhizomes for winter storage. The amount of N removal in biomass and recovery of fertilizer N were also greater for inoculated than uninoculated plants. PGPR inoculation also resulted in positive N balances, suggesting improved access to N from non-fertilizer N sources, possibly through BNF and improved soil N uptake. Overall, inoculation of SG with PGPR enhanced N acquisition and could be an effective strategy to increase the establishment year production of this crop.     

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.     

Nitrogen fixation in paddy soils

Several important features of the N. fixation in paddy fields which were reported previously were confirmed and some new additional results regarding the evaluation of the N₂ fixation in the rhizosphere were obtained by reinvestigation in the fields. In addition, rice plants were cultivated in the submerged soil in pots and various parts of the soil were analyzed for the N₂-fixing activity as well as several other properties. The results of the pot experiments were found to be fairly similar to those observed in the field investigations, indicating the validity of the submerged soil in a pot as a rather simulated model for the actual paddy field. By using this model system, the following facts were ascertained: (1) Water-percolation had almost no effect on the N₂-fixing activities of both the rhizosphere and the non-rhizosphere soils. (2) Suppressing effect of washing the root of rice plant on the N₂-fixing activity was slight in the seedling stage and marked in the tillering and flowering stages. (3) The N₂-fixing activity of a single rice root varied from tip to base.     

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

 Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N₂ fixation (BNF) using an agar growth medium containing 5 mg N l^–1of ¹⁵N-labelled (NH₄)₂SO₄. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with ¹⁵N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N₂-fixing bacteria and plants. Received: 4 May 1999     

Effect of plant growth-promoting bacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties and rhizosphere microflora

Inoculants are of great importance in sustainable and/or organic agriculture. In the present study, plant growth of barley (Hordeum vulgare) has been studied in sterile soil inoculated with four plant growth-promoting bacteria and mineral fertilizers at three different soil bulk densities and in three harvests of plants. Three bacterial species were isolated from the rhizosphere of barley and wheat. These bacteria fixed N₂, dissolved P and significantly increased growth of barley seedlings. Available phosphate in soil was significantly increased by seed inoculation of Bacillus M-13 and Bacillus RC01. Total culturable bacteria, fungi and P-solubilizing bacteria count increased with time. Data suggest that seed inoculation of barley with Bacillus RC01, Bacillus RC02, Bacillus RC03 and Bacillus M-13 increased root weight by 16.7, 12.5, 8.9 and 12.5% as compared to the control (without bacteria inoculation and mineral fertilizers) and shoot weight by 34.7, 34.7, 28.6 and 32.7%, respectively. Bacterial inoculation gave increases of 20.3–25.7% over the control as compared with 18.9 and 35.1% total biomass weight increases by P and NP application. The concentration of N and P in soil was decreased by increasing soil compaction. In contrast to macronutrients, the concentration of Fe, Cu and Mn was lower in plants grown in the loosest soil. Soil compaction induced a limitation in root and shoot growth that was reflected by a decrease in the microbial population and activity. Our results show that bacterial population was stimulated by the decrease in soil bulk density. The results suggest that the N₂-fixing and P-solubilizing bacterial strains tested have a potential on plant growth activity of barley.     

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.     

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.     

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.     

Influence of various soil factors on nitrogen fixation by Azospirillum spp

The influence of soil pH, redox potential and added organic matter on N₂-fixation by Azospirillum was studied. Application of rice straw to alluvial, laterite and acid-sulphate Pokkali soils under submerged conditions enhanced the population of N₂-fixing Azospirillum spp. An acid-sulphate saline soil of extremely low pH (3.2) harboured Azospirillum spp with appreciable N₂-fixing activity. Enrichment cultures originating from soils with low pH (<4.0) possessed lower N₂-fixing activity compared to cultures from soils with higher pH values (upto 6.6). Azospirillum cultures from soils that had undergone prolonged waterlogging showed lower N₂-fixing activity than cultures isolated from soils submerged for a few days. A relationship was shown between the in vitro N₂-fixing activity of Azospirillum cultures and the redox status of the soil samples; activity was high when the soil redox potential was between ?50 to ?150mV. The results show that the N₂-fixing activity of Azospirillum cultures is governed by fluctuations in soil redox potential, pH and organic matter.     

水稻根际和周围土壤中微生物功能基因丰度与碳、氮状况及其通量之间的关系

Rapid nitrogen（N） transformations and losses occur in the rice rhizosphere through root uptake and microbial activities. However,the relationships between rice roots and rhizosphere microbes for N utilization are still unclear. We analyzed different N forms（NH＋4,NO-3, and dissolved organic N）, microbial biomass N and C, dissolved organic C, CH4 and N2O emissions, and abundance of microbial functional genes in both rhizosphere and bulk soils after 37-d rice growth in a greenhouse pot experiment. Results showed that the dissolved organic C was significantly higher in the rhizosphere soil than in the non-rhizosphere bulk soil, but microbial biomass C showed no significant difference. The concentrations of NH＋4, dissolved organic N, and microbial biomass N in the rhizosphere soil were significantly lower than those of the bulk soil, whereas NO-3in the rhizosphere soil was comparable to that in the bulk soil. The CH4 and N2O fluxes from the rhizosphere soil were much higher than those from the bulk soil. Real-time polymerase chain reaction analysis showed that the abundance of seven selected genes, bacterial and archaeal 16 S rRNA genes, amoA genes of ammonia-oxidizing archaea and ammonia-oxidizing bacteria, nosZ gene, mcrA gene, and pmoA gene, was lower in the rhizosphere soil than in the bulk soil, which is contrary to the results of previous studies. The lower concentration of N in the rhizosphere soil indicated that the competition for N in the rhizosphere soil was very strong, thus having a negative effect on the numbers of microbes. We concluded that when N was limiting, the growth of rhizosphere microorganisms depended on their competitive abilities with rice roots for N.     

Inoculation of rice with nitrogen-fixing bacteria —problems and perspectives

Summary The present status and merits of inoculating rice with N₂-fixing bacteria are discussed in the light of recent advances. Bacterial inoculation improves plant growth and rice yield but not uniformly. The yield response to inoculation is more pronounced in the presence of moderate levels of fertilizer N. Evidence for the establishment and activity of the inoculated bacteria is limited, and the poor survival of the inoculum under field conditions further complicates the effects of inoculation. There is no clear evidence that improved growth and mineral content following inoculation are due to increased N₂ fixation. Beneficial effects of the inoculum on rice, such as plant growth promotion, N₂ fixation and antagonism effects against pathogens need to be further investigated under laboratory and field conditions. Improved management practices, such as organic amendments, suitable water and soil management, selection of efficient microbial strains, selection of effective breeding lines with high associative nitrogen fixation, and better management of agrochemicals are some of the measures suggested for deriving benefits from bacterial associations with rice.     

Effect of calcium carbonate, sand, and organic matter levels on mortality of five species of Azospirillum in natural and artificial bulk soils

 Five bacterial strains, one from each of the five known species of the plant growth-promoting bacteria (PGPB) Azospirillum (A. brasilense, A. lipoferum, A. amazonense, A. halopraeference, and A. irakense) were inoculated into two natural, semiarid soils (terra rosa and loessial sandy) from Israel, and two artificial soils constructed to simulate the native soils. Within 60 days, the populations of all five Azospirillum species declined significantly in a linear fashion, in both the native soils and in the homologous artificial soils. Increased levels of CaCO₃, and fine and rough sand, had significant detrimental effects on the survival of the five Azospirillum species, whereas increased organic matter content improved survival. In contrast, when the bacterial strains were incubated in the rhizosphere of tomato seedlings grown in the artificial soils, manipulation of these soil variables had only a marginal effect on bacterial survival; all Azospirillum species survived well in the tomato rhizosphere under conditions that are otherwise detrimental. This study indicates that most cells of the strains of five known species of Azospirillum died out linearly over time in two semiarid soils, and that only the major soil components affected Azospirillum survival in soil. Because mortality was similar in native soils and in artificial homologous soils, artificial soils can be used to study the soil behavior of Azospirillum. Received: 9 April 1999     

Bacterial composition and N2-fixation of some Egyptian soils cultivated with wheat (Triticum aestivum L.)

The composition of the microflora, N₂-fixing bacteria particularly, in different soils cultivated with wheat in Egypt was investigated in some samples collected from the fields after applying the agricultural practices recommended for wheat cultivation and just before sowing. The influence of carbon sources, mineral nitrogen and water regimes on potential dinitrogen fixation (acetylene reduction assay) in soils was investigated. The bacterial population densities including-N₂-fixing organisms were related to a number of environmental factors such as organic matter content. Among diazotrophs, Azotobacter spp. and Azospirillum spp. were encountered in higher densities in comparison with clostridia. Unamended soils showed a lower acetylene-reducing activity (0.5–61.5 nmoles C₂H₄ g^?1 h^?1). Addition of glucose (1% w/w) greatly enhanced such activity being the highest (86.9–2846.5 nmoles C₂H₄ g^?1 h^?1) in the clay soil with the highest organic carbon content (1.42%). Glucose amendment had no significant influence on acetylene reduction in the saline soil. N₂-fixation in barley straw-amended (1%) soils was not much higher than in unamended soils. Concentrations of up to 70 ppm ammonium-nitrogen depressed N₂-fixation in soils that received barley straw. Acetylene reduction in submerged soil increased after addition of cellulose. Non-flooded conditions favoured N₂-fixation in the fertile clay soil amended with sucrose.     

Heterotrophy-coordinated diazotrophy is associated with significant changes of rare taxa in soil microbiome

Soil heterotrophic respiration during decomposition of carbon (C)-rich organic matter plays a vital role in sustaining soil fertility. However, it remains poorly understood whether dinitrogen (N₂) fixation occurs in support of soil heterotrophic respiration. In this study, ¹⁵N₂-tracing indicated that strong N₂ fixation occurred during heterotrophic respiration of carbon-rich glucose. Soil organic ¹⁵N increased from 0.37 atom% to 2.50 atom% under aerobic conditions and to 4.23 atom% under anaerobic conditions, while the concomitant CO₂ flux increased by 12.0-fold under aerobic conditions and 5.18-fold under anaerobic conditions. Soil N₂ fixation was completely absent in soils replete with inorganic N, although soil N bioavailability did not alter soil respiration. High-throughput sequencing of the 16S rRNA gene further indicated that: i) under aerobic conditions, only 15.2% of soil microbiome responded positively to glucose addition, and these responses were significantly associated with soil respiration and N₂ fixation and ii) under anaerobic conditions, the percentage of responses was even lower at 5.70%. Intriguingly, more than 95% of these responses were originally rare with < 0.5% relative abundance in background soils, including typical N₂-fixing heterotrophs such as Azotobacter and Clostridium and well-recognized non-N₂-fixing heterotrophs such as Sporosarcina, Agromyces, and Sedimentibacter. These results suggest that only a small portion of the soil microbiome could respond quickly to the amendment of readily accessible organic C in a fluvo-aquic soil and highlighted that rare phylotypes might have played more important roles than previously appreciated in catalyzing soil C and nitrogen turnovers. Our study indicates that N₂ fixation could be closely associated with microbial turnover of soil organic C when available in excess.