Associative nitrogenase activity by some Hawaiian grass roots

Summary Nitrogenase activity by the roots of some tropical grasses was detected by the acetylene reduction technique. In some cases particular plant species, evaluated at different times from different sites, displayed marked variations in nitrogenase activity.Acetylene reduction by root samples which had undergone preincubation under low O₂ displayed an increase of approximately 5-fold when compared to plant-containing soil cores. These increases in nitrogenase activity correspond to an increase in the number of nitrogen-fixing bacteria associated with the roots.Journal Series No. 2066 of the Hawaii Agricultural Experiment Station, Honolulu, Hawaii 96822Journal Series No. 2066 of the Hawaii Agricultural Experiment Station, Honolulu, Hawaii 96822     

Nitrogen-fixing pseudomonads isolated from roots of plants grown in the canadian high arctic

Root-associated bacteria capable of reducing acetylene to ethylene (biological nitrogen fixation) were isolated from various native plants grown in the Canadian High Arctic. All the strains belonged to the genus Pseudomonas but varied in several physiological characteristics. The rates of acetylene reduction at 14 or 20 degrees C were higher than at 25 or 9 degrees C. Six strains reduced acetylene at 4 degrees C. All the strains exhibited chemotaxis to l-asparagine in semisolid agar at 4 to 25 degrees C. Eleven strains colonized roots of canola (Brassica campestris cv. Tobin) in field soil at population densities of log 4.3 to log 5.1 CFU/g of fresh root at 14 degrees C and log 4.0 to log 5.2 CFU/g of fresh root at 25 degrees C. Some of these nitrogen-fixing pseudomonad strains demonstrated a competitive advantage for root colonization over other rhizosphere bacteria at low temperatures. The combined capabilities of nitrogen fixation and root colonization by diazotrophic pseudomonads may be useful for the development of a biofertilizer inoculant for temperate and cold regions.     

Nitrogen Fixation by Marine Photosynthetic Bacteria

S ummary . Potential nitrogen-fixing marine photosynthetic bacteria isolated from the coastal waters and sea-bed sediments of Cardigan Bay and adjoining mud flats, were investigated, using the acetylene reduction technique, to ascertain the magnitude of their contribution of fixed nitrogen to the coastal ecosystem. Aberystwyth harbour and Iceland fjord mud readily yielded Athiorhodaceae, the majority of which consistently reduced acetylene when grown as pure cultures on marine media. Only one such strain was isolated from local seawater, but they were more common in Iceland fjord water. No marine Thiorhodaceae were isolated. It was essential to monitor crude and pure culture systems for ethylene production in the absence of acetylene.     

Enumeration of acetylene-reducing bacteria in strip-mined reclamation sites in a temperate grassland

Summary From 36 to 71% of bacteria, depending on the sampling site, that were isolated from the soil or rhizosphere of undisturbed prairie soil or reclamation sites of strip-mined grassland areas in western North Dakota were capable of reducing acetylene. These bacteria generally could be divided into two populations; one capable of acetylene reduction under aerobic conditions and another capable of acetylene reduction under anaerobic conditions. The reclamation site to which no topsoil had been applied, pH 8.5, had a bacterial population which generally was capable of higher levels of acetylene reduction than individual bacteria isolated from other sites.     

Nitrogen-fixing bacteria associated with bark beetles

Nitrogen-fixingEnterobacter agglomerans andEnterobacter spp. were consistently isolated from the bark beetleDendroctonus terebrans. Large populations of nitrogen-fixing bacteria were found with the beetle, although no in situ acetylene reduction was demonstrated. The constant occurrence of nitrogenfixing bacteria withD. terebrans suggests a symbiotic relationship. Nitrogen-fixing bacteria were also isolated from the bark beetlesDendroctonus frontalis andIps avulsus.Mention of trade names is not an endorsement by the U.S. Department of Agriculture.     

Invalidity of the acetylene reduction assay in alkane-utilizing, nitrogen-fixing bacteria.

The cause of the failure of the C2H2-C2H4 assay for nitrogen-fixing bacteria growing on lower alkanes was studied. Acetylene was a strong competitive inhibitor of methane oxidation for methane-utilizing bacteria, as well as for the oxidation of lower alkanes by other bacteria, so that energy and reducing power were no longer available for the reduction of acetylene by nitrogenase. Nitrogen-fixing bacteria grown on alkanes may reduce acetylene when intermediates of alkane-breakdown or other substrates oxidizable in the presence of acetylene are supplied. Ethylene co-oxidation is not responsible for the failure of the test, because acetylene also inhibits this co-oxidation along with methane oxidation.     

Nitrogen-fixing chemo-organotrophic bacteria isolated from cyanobacteria-deprived lichens and their ability to solubilize phosphate and to release amino acids and phytohormones

AIMS: Cyanobacteria-deprived lichens of the species Canoparmelia caroliniana, Canoparmelia crozalsiana, Canoparmelia texana, Parmotrema sancti-angeli and Parmotrema tinctorum were screened for the presence of chemo-organotrophic nitrogen-fixing bacteria. METHODS AND RESULTS: Fifty-three lichen samples subjected to enrichment selection using a nitrogen-free minimal medium were positive for acetylene reduction. Seventeen isolates, able to fix nitrogen, belonged to Gamma-proteobacteria group and were identified as: Acinetobacter sp., Pantoea sp., Pseudomonas sp., Pseudomonas stutzeri, Serratia marcescens and Stenotrophomonas maltophilia, according to 16S rRNA gene sequences and biochemical tests. The excretion of amino acid and phytohormone and the ability of mineral phosphate solubilization were determined in 14 isolates. All isolates were able to release amino acids and 3-indoleacetic acid. About 64% of the isolates solubilized phosphates and 30% released ethylene. CONCLUSIONS: These data confirm sparse evidence from the literature on the occurrence of chemo-organotrophic nitrogen-fixing bacteria in cyanobacteria-deprived lichens; the isolates presented physiologic features which might benefit the host if they are expressed when the bacteria are harboured by lichens. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemo-organotrophic nitrogen-fixing bacteria were isolated from a high percentage (72.6%) of cyanobacteria-deprived lichens. All isolates presented important physiological characteristics, some of which are being described here for the first time.     

Field technique using the acetylene reduction method to assay nitrogenase activity and its association with the rice rhizosphere

Summary A plastic bag, used as a field assay chamber, assisted in the investigation of the acetylene reduction method as a technique to measure nitrogen-fixing activity in rice paddy fields. A study of the change in volume of the plastic bag and of the loss of acetylene and ethylene from the bag provided evidence that this plastic bag method was feasible in field assays. The field assay of rice at the grain ripening stage showed that the nitrogen-fixing activity increased linearly after a lag phase of 0 to 3 hours during a time-course experiment.Detachment of the aerial part of the rice plant from its root remaining in the field did not affect the nitrogen-fixing activity of the root.The markedly higher nitrogen-fixing activity in the planted areas of the field compared with the nonplanted areas between the plant rows indicated that the nitrogenase activity in the field is associated with the roots of the rice plant. re]19751204     

A new approach for the biolistic method: Bombardment of living nitrogen-fixing bacteria into plant tissues

Summary A new utilization of the biolistic gun was developed for the direct introduction of nitrogen-fixing bacteria (Azotobacter vinelandii) into strawberry (Fragaria x ananassa) tissues. This was the first case of using living bacteria as microprojectiles for the bombardment of plant tissues. Bacterial cells, adhered to tungsten particles, were accelerated by a nitrogen-powered device, and delivered into the target leaves and regenerating shoot meristems. The presence of bacteria in the developing strawberry callus tissues and regenerating plants was detected by microscopy, acetylene reduction assay, and selective polymerase chain reaction. Practically, the elaborated method proved to be suitable for the establishment of artificial intereellular, associations between nitrogen-fixing bacteria and higher plants.     

nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

ROLE OF THE NITROGEN-FIXING BACTERIAL MICROFLORA IN THE EPIPHYTISM OF TILLANDSIA (BROMELIACEAE)

Nitrogen-fixing activity in the phyllosphere of 12 species of Tillandsia from different Mexican habitats was evaluated by the acetylene reduction assay, and nitrogen-fixing microorganisms were isolated and characterized. The leaves from eight of the 12 Tillandsia species examined exhibited nitrogenase activity in enrichment cultures. Among the microorganisms implicated—Agrobacterium, Bacillus, Erwinia, Pseudomonas, Rahnella, Vibrio, and Xanthomonas—only Bacillus megatherium reduced acetylene in pure culture. Our findings suggest that nitrogen fixation in the phyllosphere of the sampled epiphytes occurs under suitable conditions and that most of the bacteria involved are primarily soil and water inhabitants. The results also suggest a relationship between the composition of the nitrogen-fixing microbial communities grown on the leaf and the different development of the leaf area in Tillandsia due to the aerial components (wings) of the trichomes.     

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.     

Novel endophytic nitrogen-fixing clostridia from the grass Miscanthus sinensis as revealed by terminal restriction fragment length polymorphism analysis

Anaerobic nitrogen-fixing consortia consisting of N2-fixing clostridia and diverse nondiazotrophic bacteria were previously isolated from various gramineous plants (K. Minamisawa, K. Nishioka, T. Miyaki, B. Ye, T. Miyamoto, M. You, A. Saito, M. Saito, W. Barraquio, N. Teaumroong, T. Sein, and T. Tadashi, Appl. Environ. Microbiol. 70:3096-3102, 2004). For this work, clostridial populations and their phylogenetic structures in a stand of the grass Miscanthus sinensis in Japan were assessed by a 16S rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable-number (MPN) counts. PCR primers and restriction enzymes were optimized for analyses of the plant clostridia. Clostridia were detected in strongly surface-sterilized leaves, stems, and roots of the plants at approximately 10(4) to 10(5) cells/g of fresh weight; they made up a large proportion of N2-fixing bacterial populations, as determined by MPN counts associated with an acetylene reduction assay. Phylogenetic grouping by MPN-TRFLP analysis revealed that the clostridial populations belonged to group II of cluster XIVa and groups IV and V of cluster I; this result was supported by a culture-independent TRFLP analysis using direct DNA extraction from plants. When phylogenetic populations from M. sinensis and the soil around the plants were compared, group II clostridia were found to exist exclusively in M. sinensis.     

Isolation and 16S rRNA sequence analysis of the beneficial bacteria from the rhizosphere of rice

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 10(5)-10(6) counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using 15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.     

NITROGEN FIXATION IN THE BAYBERRY (MYRICA PENSYLVANICA) AND ITS ROLE IN COASTAL SUCCESSION

The nodules on roots of Myrica pensylvanica (bayberry) contain a bacterial endophyte. By using the acetylene reduction technique these plant endophyte associations were shown to be capable of fixing nitrogen. As nodulation was plentiful and fixation vigorous, it is proposed that the success of M. pensylvanica as an early successional plant of dunes and impoverished coastal soils is due in part to the nitrogen-fixing capacity of its nodular association.     

A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane

AIMS: To isolate and identify endophytic nitrogen-fixing bacteria in sugarcane growing in Cuba without chemical fertilizers. METHODS AND RESULTS: Two N2-fixing isolates, 9C and T2, were obtained from surface-sterilized stems and roots, respectively, of sugarcane variety ML3-18. Both isolates showed acetylene reduction and H2 production in nitrogen-free media. Nitrogenase activity measured by H2 production was about 15 times higher for isolate 9C than for T2 or for Gluconoacetobacter diazotrophicus (PAL-5 standard strain, ATCC 49037). The nifH gene segment was amplified from both isolates using specific primers. Classification of both T2 and 9C was made on the basis of morphological, biochemical, PCR tests and 16S rDNA sequence analysis. CONCLUSIONS: Isolate 9C was identified as a Pantoea species from its 16S rDNA, but showed considerable differences in physiological properties from previously reported species of this genus. For example, 9C can be cultured over a wide range of temperature, pH and salt concentration, and showed high H2 production (up to 67.7 nmol H2 h(-1) 10(10) cell(-1)). Isolate T2 was a strain of Gluconacetobacter diazotrophicus. SIGNIFICANCE AND IMPACT OF THE STUDY: A new N2-fixing endophyte, i.e. Pantoea, able to produce H2 and to grow in a wide range of conditions, was isolated from sugarcane stem tissue and characterized. The strain with these attributes may well be valuable for agriculture.     

Acetylene reduction by Rhodospirillaceae from the Aswan High Dam Lake

Potential nitrogen-fixing freshwater photosynthetic bacteria of 32 isolates from the Aswan High Dam Lake were investigated using the acetylene reduction technique to ascertain the magnitude of their contribution of fixed nitrogen to the ecosystem. The superior nitrogen-fixing Rhodospirillaceae wereRhodocyclus gelatinosus andRhodomicrobium vannielii followed byRhodobacter capsulatus, Rhodopseudomonas viridis andRhodopseudomonas palustris. High and moderate nitrogenase activities (534 to 1528 nmol ethylene produced/4 ml/h) were found in 56% of the isolates, while the remainder showed low activities (76 to 477 nmol ethylene produced/4 ml/h).     

Ecology of heterotrophic dinitrogen fixation in the rhizosphere of mangrove plant community at the Ganges river estuary in India

Summary Heterotrophic dinitrogen fixation in root associations of successional stages of the tropical mangrove plant community at the Ganges river estuary in India was investigated by excised-root acetylene reduction assay, and enumeration and identification of diazotrophic bacteria from sediment, root and tidal water samples. High to very high rates of nitrogenase activity (64–130 nmol C₂H₄/g dry root/h) were associated with washed excised roots of seven common early-successional mangrove species at the inundated swamps. Declining, late-successional mangroves at the occasionally inundated ridges had considerably lower values and the “declined” mangroves and other non-littoral species at embankment protected highlands had very low to insignificant values of root nitrogenase activity. Total and inorganic nitrogen contents of the mangrove sediments were low and were positively related to the stages of physiographic succession. Plant-associated sediments of particularly the old formation swamps had very high C/N ratios. Nine isolates of nitrogen-fixing bacteria belonging to all known O₂ response groups were distinguished from a large population of diazotrophs associated with roots of mangroves and other associate plant species of the community. The isolates differed with respect to their N₂-fixation efficiency and halotolerance in pure culture. There was no specificity of any of the bacterial isolates to any of the plant species of the community but a higher number of efficient isolates were seen to be associated with mangroves at the swampy succession. Sediment-free tidal water also contained a large population of microaerophilic and anaerobic N₂-fixing bacteria.     

动物的共生菌固氮

介绍了共生菌固氮涉及的动物和微生物类群、动物共生菌固氮的性质和机理。应用乙炔还原法和固氮酶基因检测等研究表明,所涉及的动物有7门13纲23目50科99属174种。动物肠道具有丰富的微生境,供不同生理需求的固氮菌生长发育,所蕴含的共生固氮菌类群也十分丰富,涵盖植物共生固氮菌、植物内生固氮菌、植物根际固氮菌、自生固氮菌等生态类型。一般认为动物共生固氮菌来源于环境,其性质属于联合共生固氮。动物共生固氮菌一般与其他共生生物形成复合体,以满足固氮过程中对电子和质子供体、能量供给、固氮酶活性保护以及氨阻遏解除等方面的需求。动物共生菌固氮产物氨的同化也需要多种共生物的协同作用,可能通过谷氨酰胺合成酶/谷氨酸合成酶等途径。总体上,食物氮、非蛋白氮和共生菌固氮相互协调,形成营养和解毒的代谢网络,共同维持动物体内氮素营养的动态平衡,并对未来研究提出展望。     

Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.