Acetylene Reduction Assay Research Articles

Ocean acidification rapidly reduces dinitrogen fixation associated with the hermatypic coral Seriatopora hystrix

Since productivity and growth of coral-associated dinoflagellate algae is nitrogen (N)-limited, dinitrogen (N2) fixation by coral-associated microbes is likely crucial for maintaining the coraldinoflagellate symbiosis. It is thus essential to understand the effects future climate change will have on N2 fixation by the coral holobiont. This laboratory study is the first to investi- gate short-term effects of ocean acidification on N2 fixation activity associated with the tropical, hermatypic coral Seriatopora hystrix using the acetylene reduction assay in combination with cal- cification measurements. Findings reveal that simulated ocean acidification (p CO2 1080 µatm) caused a rapid and significant decrease (53%) in N2 fixation rates associated with S. hystrix com- pared to the present day scenario (p CO2 486 µatm). In addition, N2 fixation associated with the coral holobiont showed a positive exponential relationship with its calcification rates. This sug- gests that even small declines in calcification rates of hermatypic corals under high CO2 conditions may result in decreased N2 fixation activity, since these 2 processes may compete for energy in the coral holobiont. Ultimately, an intensified N limitation in combination with a decline in skeletal growth may trigger a negative feedback loop on coral productivity exacerbating the negative long-term effects of ocean acidification.

Nitrogen fixation rates in algal turf communities of a degraded versus less degraded coral reef

Algal turf communities are ubiquitous on coral reefs in the Caribbean and are often dominated by N2-fixing cyanobacteria. However, it is largely unknown (1) how much N2 is actually fixed by turf communities and (2) which factors affect their N2 fixation rates. Therefore, we compared N2 fixation activity by turf communities at different depths and during day and night-time on a degraded versus a less degraded coral reef site on the island of Curacao. N2 fixation rates measured with the acetylene reduction assay were slightly higher in shallow (5–10-m depth) than in deep turf communities (30-m depth), and N2 fixation rates during the daytime significantly exceeded those during the night. N2 fixation rates by the turf communities did not differ between the degraded and less degraded reef. Both our study and a literature survey of earlier studies indicated that turf communities tend to have lower N2 fixation rates than cyanobacterial mats. However, at least in our study area, turf communities were more abundant than cyanobacterial mats. Our results therefore suggest that turf communities play an important role in the nitrogen cycle of coral reefs. N2 fixation by turfs may contribute to an undesirable positive feedback that promotes the proliferation of algal turf communities while accelerating coral reef degradation.

Hartmannibacter diazotrophicus gen. nov., sp. nov., a phosphate-solubilizing and nitrogen-fixing alphaproteobacterium isolated from the rhizosphere of a natural salt-meadow plant.

A phosphate-mobilizing, Gram-negative bacterium was isolated from rhizospheric soil of Plantago winteri from a natural salt meadow as part of an investigation of rhizospheric bacteria from salt-resistant plant species and evaluation of their plant-growth-promoting abilities. Cells were rods, motile, strictly aerobic, oxidase-positive and catalase-negative. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain E19(T) was distinct from other taxa within the class Alphaproteobacteria. Strain E19(T) showed less than 93.5 % 16S rRNA gene sequence similarity with members of the genera Rhizobium (≤93.5 %), Labrenzia (≤93.1 %), Stappia (≤93.1 %), Aureimonas (≤93.1 %) and Mesorhizobium (≤93.0 %) and was most closely related to Rhizobium rhizoryzae (93.5 % 16S rRNA gene sequence similarity to the type strain). The sole respiratory quinone was Q-10, and the polar lipids comprised phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an aminolipid and an unidentified phospholipid. Major fatty acids were C18 : 1ω7c (71.4 %), summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1; 8.3 %), C20 : 0 (7.9 %) and C16 : 0 (6.1 %). The DNA G+C content of strain E19(T) was 59.9±0.7 mol%. The capacity for nitrogen fixation was confirmed by the presence of the nifH gene and the acetylene reduction assay. On the basis of the results of our polyphasic taxonomic study, the new isolate represents a novel genus and species, for which the name Hartmannibacter diazotrophicus gen. nov., sp. nov. is proposed. The type strain of Hartmannibacter diazotrophicus is E19(T) ( = LMG 27460(T) = KACC 17263(T)).

Screening of Nitrogen Fixing Endophytic Bacteria in Oryza sativa L.

Nitrogen (N) is an essential element for the growth and yield of rice. Some endophytic bacteria can fix N2 from the air and convert to nitrogen compounds that can be utilized by plants. In this study, endophytic bacteria were isolated from one-month-old seedlings of five rice (Oryza sativa L.) varieties (Muey Nong 24, Muey Nong 25, Pathum Thani 1, Suphan Buri 1 and Chai Nat 1) growing without nitrogen fertilizer in the farmers’ field. One hundred and twenty-three isolates of endophytic bacteria were obtained from the roots, stems and leaves of these rice varieties. Nitrogenase activity of the bacteria in N-free culture medium was determined by acetylene reduction assay. Seven isolates of the bacteria with highest nitrogenase activity were identified by phylogenetic analysis of the 16S rRNA genes, and found to belong to Burkholderia cepacia (CS5), Citrobacter sp. (CR9), Citrobacter sp. (SS5), Citrobacter sp. (SS6), Bacillus amyloliquefaciens (25R14), B. amyloliquefaciens (SR1) and B. thuringiensis (25R2). Inoculation of Bu. cepacia (CS5) and Citrobacter sp. (CR9) to the seedlings of local rice variety (Muey Nong 24) significantly increased nitrogen concentration in the roots of rice.

Identification and characterization of endophytic bacteria from corn (Zea mays L.) roots with biotechnological potential in agriculture.

Six endophytic bacteria of corn roots were identified as Bacillus sp. and as Enterobacter sp, by sequencing of the 16S rRNA gene. Four of the strains, CNPSo 2476, CNPSo 2477, CNPSo 2478 and CNPSo 2480 were positive for the nitrogen fixation ability evaluated through the acetylene reduction assay and amplification of nifH gene. Two Bacillus strains (CNPSo 2477 and CNPSo 2478) showed outstanding skills for the production of IAA, siderophores and lytic enzymes, but were not good candidates as growth promoters, because they reduced seed germination. However, the same strains were antagonists against the pathogenic fungi Fusarium verticillioides, Colletotrichum graminicola, Bipolaris maydis and Cercospora zea-maydis. As an indication of favorable bacterial action, Enterobacter sp. CNPSo 2480 and Bacillus sp. CNPSo 2481 increased the root volume by 44% and 39%, respectively, and the seed germination by 47% and 56%, respectively. Therefore, these two strains are good candidates for future testing as biological inoculants for corn.

Symbiotic activity of pea (Pisum sativum) after application of Nod factors under field conditions.

Growth and symbiotic activity of legumes are mediated by Nod factors (LCO, lipo-chitooligosaccharides). To assess the effects of application of Nod factors on symbiotic activity and yield of pea, a two-year field experiment was conducted on a Haplic Luvisol developed from loess. Nod factors were isolated from Rhizobium leguminosarum bv. viciae strain GR09. Pea seeds were treated with the Nod factors (10−11 M) or water (control) before planting. Symbiotic activity was evaluated by measurements of nitrogenase activity (acetylene reduction assay), nodule number and mass, and top growth by shoot mass, leaf area, and seed and protein yield. Nod factors generally improved pea yield and nitrogenase activity in the relatively dry growing season 2012, but not in the wet growing season in 2013 due to different weather conditions.

Microbial ecology and biogeochemistry of continental Antarctic soils.

The Antarctica Dry Valleys are regarded as the coldest hyperarid desert system on Earth. While a wide variety of environmental stressors including very low minimum temperatures, frequent freeze-thaw cycles and low water availability impose severe limitations to life, suitable niches for abundant microbial colonization exist. Antarctic desert soils contain much higher levels of microbial diversity than previously thought. Edaphic niches, including cryptic and refuge habitats, microbial mats and permafrost soils all harbor microbial communities which drive key biogeochemical cycling processes. For example, lithobionts (hypoliths and endoliths) possess a genetic capacity for nitrogen and carbon cycling, polymer degradation, and other system processes. Nitrogen fixation rates of hypoliths, as assessed through acetylene reduction assays, suggest that these communities are a significant input source for nitrogen into these oligotrophic soils. Here we review aspects of microbial diversity in Antarctic soils with an emphasis on functionality and capacity. We assess current knowledge regarding adaptations to Antarctic soil environments and highlight the current threats to Antarctic desert soil communities.

Dynamics of soil diazotrophic community structure, diversity, and functioning during the cropping period of cotton (Gossypium hirsutum)

The soil sampled at different growth stages along the cropping period of cotton were analyzed using various molecular tools: restriction fragment length polymorphism (RFLP), terminal restriction length polymorphism (T-RFLP), and cloning-sequencing. The cluster analysis of the diazotrophic community structure of early sampled soil (0, 15, and 30 days) was found to be more closely related to each other than the later sampled one. Phylogenetic and diversity analysis of sequences obtained from the first (0 Day; C0) and last soil sample (180 day; C180) confirmed the data. The phylogenetic analysis revealed that C0 was having more unique sequences than C180 (presence of γ-Proteobacteria exclusively in C0). A relatively higher richness of diazotrophic community sequences was observed in C0 (S(ACE) : 30.76; S(Chao1) : 20.94) than C180 (S(ACE) : 18.00; S(Chao1) : 18.00) while the evenness component of Shannon diversity index increased from C0 (0.97) to C180 (1.15). The impact of routine agricultural activities was more evident based on diazotrophic activity (measured by acetylene reduction assay) than its structure and diversity. The nitrogenase activity of C0 (1264.85 ± 35.7 ηmol of ethylene production g(-1) dry soil h(-1) ) was statistically higher when compared to all other values (p < 0.05). There was no correlation found between diazotrophic community structure/diversity and N2 fixation rates. Thus, considerable functional redundancy of nifH was concluded to be existing at the experimental site.

Highlighting the threat from current and near-future ozone pollution to clover in pasture

Globally, the legume–rhizobia symbiosis, contained within specialised organs called root nodules, is thought to add at least 30 Tg N annually to agricultural land. The growth and functioning of a modern white clover (Trifolium repens cv. Crusader) and red clover (T. pratense cv. Merviot) cultivar were investigated in current and future ozone scenarios in solardomes. Both cultivars developed leaf injury and had significant reductions in root biomass and root nodule number in response to ozone, with Crusader also displaying a reduced size and mass of nodules. In-situ measurements of N-fixation in Crusader by acetylene reduction assay revealed reduced N-fixation rates in a future scenario with an increased background and moderate peaks of ozone. The implications for the sustainability of temperate pasture are discussed.

Pontibacter diazotrophicus sp. nov., a Novel Nitrogen-Fixing Bacterium of the Family Cytophagaceae

Few diazotrophs have been found to belong to the family Cytophagaceae so far. In the present study, a Gram-negative, rod-shaped bacterium that forms red colonies, was isolated from sands of the Takalamakan desert. It was designated H4XT. Phylogenetic and biochemical analysis indicated that the isolate is a new species of the genus Pontibacter. The 16S rRNA gene of H4XT displays 94.2–96.8% sequence similarities to those of other strains in Pontibacter. The major respiratory quinone is menaquinone-7 (MK-7). The DNA G+C content is 46.6 mol%. The major cellular fatty acids are iso-C15∶0, C16∶1ω5c, summed feature 3 (containing C16∶1ω6c and/or C16∶1ω7c) and summed feature 4 (comprising anteiso-C17∶1B and/or iso-C17∶1I). The major polar lipids are phosphatidylethanolamine (PE), one aminophospholipid (APL) and some unknown phospholipids (PLs). It is interesting to see that this bacterium can grow very well in a nitrogen-free medium. PCR amplification suggested that the bacterium possesses at least one type of nitrogenase gene. Acetylene reduction assay showed that H4XT actually possesses nitrogen-fixing activity. Therefore, it can be concluded that H4XT is a new diazotroph. We thus referred it to as Pontibacter diazotrophicus sp. nov. The type strain is H4XT ( = CCTCC AB 2013049T = NRRL B-59974T).

English

The association of Cymbopogon winterianus (Citronella), a member of the Poaceae family, with the N2-fixing bacteria, Azospirillum was examined to evaluate possible benefits for agriculture. The nodules on citronella were induced using plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 ppm in nitrogen free Hoagland&rsquo;s solution. Concentration of 2,4-D greater than 1.0 ppm caused stunning and death, while concentration of 2,4-D less than 1.0 ppm has no obvious effect on seedling growth. The presence of nitrogenase activity (acetylene reduction assay) and leghemoglobin (hemoprotein&nbsp;found in the&nbsp;nitrogen-fixing&nbsp;root nodules)&nbsp;was noticed in the plants treated with Azospirillum either alone or with nodulated plants, but higher activity was noticed in nodulated Azospirillum treated plants. These results confirmed the nitrogenase activity of Azospirillum brasilense in association with 2,4-D induced nodules of citronella. After transplanting to pots, it was noticed that nodulated and Azospirillum - treated plants showed higher chlorophyll content in leaf, enhanced nitrate reductase (NR) activity, leading to higher yield as compared to the control plants (non-nodulated). The nodulated plants treated with Azospirillum also had higher physiological activities as compared to plants treated only with Azospirillum. The results provide evidence that the plants belonging to the family Poaceae are potentially able to create a symbiosis with diazotropic bacteria, which colonize para-nodule tissue intracellularly, promoting a higher level of nitrogen (N2) fixation leading to better growth and plant development. &nbsp; Key words: Cymbopogon, nodulation, 2,4-D, Azospirillum brasilense, nitrogen fixation, oil yield.

Dissolved hydrogen and nitrogen fixation in the oligotrophic North Pacific Subtropical Gyre

The production of hydrogen (H2) is an inherent component of biological dinitrogen (N2) fixation, and there have been several studies quantifying H2 production relative to N2 fixation in cultures of diazotrophs. However, conducting the relevant measurements for a field population is more complex as shown by this study of N2 fixation, H2 consumption and dissolved H2 concentrations in the oligotrophic North Pacific Ocean. Measurements of H2 oxidation revealed microbial consumption of H2 was equivalent to 1-7% of ethylene produced during the acetylene reduction assay and 11-63% of (15)N2 assimilation on a molar scale. Varying abundances of Crocosphaera and Trichodesmium as revealed by nifH gene abundances broadly corresponded with diel changes observed in both N2 fixation and H2 oxidation. However, no corresponding changes were observed in the dissolved H2 concentrations which remained consistently supersaturated (147-560%) relative to atmospheric equilibrium. The results from this field study allow the efficiency of H2 cycling by natural populations of diazotrophs to be compared to cultured representatives. The findings indicate that dissolved H2 concentrations may depend not only on the community composition of diazotrophs but also upon relevant environmental parameters such as light intensity or the presence of other H2-metabolizing microorganisms.

Rheinheimera hassiensis sp. nov. and Rheinheimera muenzenbergensis sp. nov., two species from the rhizosphere of Hordeum secalinum

Two motile, Gram-staining-negative, aerobic, rod-shaped bacteria designated strains E48(T) and E49(T) were isolated from the rhizosphere of Hordeum secalinum from a natural salt meadow near Münzenberg, Germany. 16S rRNA gene sequence similarity analysis revealed that strains E48(T) and E49(T) shared similarities of 97.6 % with Rheinheimera pacifica KMM 1406(T) and 98.5 % with Rheinheimera nanhaiensis E407-8(T), respectively. Major fatty acids of strain E48(T) were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C17 : 1ω8c, and of strain E49(T) were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The DNA G+C contents were 50.5 mol% (E48(T)) and 50.0 mol% (E49(T)). Strains E48(T) and E49(T) grew at 4-37 °C (optimum 28 °C) and with 0-6 % NaCl (optimum 0-3 %) and 0-5 % NaCl (optimum 0-3 %), respectively. The potential for nitrogen fixation by strains E48(T) and E49(T) was evaluated by molecular techniques and the acetylene reduction assay. The DNA-DNA hybridization, physiological and molecular data demonstrated that strains E48(T) and E49(T) represent two novel species of the genus Rheinheimera, and therefore the names Rheinheimera hassiensis sp. nov. (type strain E48(T) = LMG 27268(T) = KACC 17070(T)) and Rheinheimera muenzenbergensis sp. nov. (type strain E49(T) = LMG 27269(T) = KACC 17071(T)) are proposed.

Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system

In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus Nostoc associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus Peltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N2 fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data.

Nitrogen fixation in the mucus of Red Sea corals

Scleractinian corals are essential constituents of tropical reef ecological diversity. They live in close association with diazotrophs [dinitrogen (N2)-fixing microbes], which can fix high rates of N2. Whether corals benefit from this extrinsic nitrogen source is still under debate. Until now, N2 fixation rates have been indirectly estimated using the acetylene reduction assay, which does not permit assessment of the amount of nitrogen incorporated into the different compartments of the coral holobiont. In the present study, the (15)N2 technique was applied for the first time on three Red Sea coral species. Significant (15)N enrichment was measured in particles released by corals to the surrounding seawater. N2 fixation rates were species specific and as high as 1.6-2 ng N day(-1) l(-1). However, no significant enrichment was measured in the symbiotic dinoflagellates or the coral host tissues, suggesting that corals do not benefit from diazotrophic N2 fixation.

Tolerância da bactéria diazotrófica Gluconacetobacter diazotrophicus a herbicidas utilizados na cultura da cana-de-açúcar

The objective of this work was to identify herbicides used in the sugarcane crop that affects neither the growth of nor the process of biological nitrogen fixation (BNF) by the diazotrophic bacterium Gluconacetobacter diazotrophicus. Eighteen herbicides (paraquat, ametryne, amicarbazone, diuron, metribuzin, [hexazinone + diuron], [hexazinone + clomazone], clomazone, isoxaflutole, sulfentrazone, oxyfluorfen, imazapic, imazapyr, [trifloxysulfuron sodium + ametryne], s-metolachlor, glyphosate, MSMA e 2.4-D) were tested in their respective commercial doses. For this, we determined the duration of lag phase, generation time and maximum cell density of G. diazotrophicus. We also evaluated the impact of herbicides on nitrogenase activity of G. diazotrophicus measured by the technique of acetylene reduction assay. Diuron, metribuzin, 2.4-D and MSMA caused a small reduction in maximum growth of G. diazotrophicus in vitro. Amicarbazone, [hexazinone + clomazone] and clomazone showed some bacteriostatic effect on G. diazotrophicus. Treatments containing the herbicide paraquat and glyphosate resulted inhibitory effect on BFN by G. diazotrophicus.

Asymbiotic biological nitrogen fixation in a temperate grassland as affected by management practices

Estimates of asymbiotic biological N fixation (BNF) in temperate grasslands are few with large variations. In the past six decades, European grasslands have been subjected to intensive management practices and presently it is not known how asymbiotic BNF is influenced by these practices. Our objective was to assess the impact of fertilizer application and mowing frequency on asymbiotic BNF in a Central European grassland. In 2008, we established a three-factorial experiment with two fertilizer treatments (no fertilizer application and combined nitrogen (N), phosphorus (P) and potassium (K) fertilization at 180–30–100 kg ha−1 yr−1), two mowing frequencies (cut once and thrice per year) and three sward compositions through the application of herbicides (control, monocot- and dicot-enhanced swards). Three years after the initial sward manipulation, there was no more difference in functional group composition. Between June 2011 and May 2012, we measured in-situ asymbiotic BNF using the acetylene reduction assay, calibrated with 15N2-fixation method. Across treatments, asymbiotic BNF rates in the 0–5-cm soil depth ranged from 1.7 (±0.2 SE) kg ha−1 yr−1 for fertilized plots cut once a year to 5.7 (±2.3 SE) kg ha−1 yr−1 for unfertilized plots cut thrice a year. Fertilization decreased asymbiotic BNF, suggesting that the potential positive effect of increased soil P levels might have been overruled by the negative effect of increased soil mineral N levels. Intensive mowing stimulated asymbiotic BNF, which was probably due to an increase in rhizodeposition. Our calibration of the acetylene reduction assay with the 15N2-fixation method resulted in a conversion factor of 0.61, which largely deviates from the theoretical conversion factor of 3. Furthermore, laboratory incubations under increased soil moisture and temperature conditions overestimated BNF rates compared to in-situ measurements. Thus, laboratory measurements with altered soil moisture, temperature or disturbed soil may lead to strong biases in estimates of asymbiotic BNF. Our results suggest that input of N through BNF may be considerable in temperate grasslands. We conclude that BNF studies should be conducted in-situ and that the acetylene reduction assay should be calibrated against 15N2-fixation calibration for reliable estimates.

Isolation of heterotrophic diazotrophic bacteria from estuarine surface waters

The wide distribution of diverse nitrogenase (nifH) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N2 -fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N2 -fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N2 -fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9 × 10(4) and 4.7 × 10(4) nifH copies l(-1) respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.

Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies.

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.

Cellvibrio diazotrophicus sp. nov., a nitrogen-fixing bacteria isolated from the rhizosphere of salt meadow plants and emended description of the genus Cellvibrio

Two Gram-reaction-negative, aerobic, nitrogen-fixing, rod-shaped bacteria, designated strains E20 and E50(T), were isolated from the rhizosphere of salt meadow plants Plantago winteri and Hordeum secalinum, respectively, near Münzenberg, Germany. Based on the 16S rRNA gene sequence analysis both strains E20 and E50(T) are affiliated with the genus Cellvibrio, sharing the highest similarity with Cellvibrio gandavensis LMG 18551(T) (96.4%) and (97.1%), respectively. Strains E20 and E50(T) were oxidase and catalase-positive, grew at a temperature range between 16 and 37 °C and in the presence of 0-5% NaCl (w/v). The DNA G+C contents were 52.1 mol% (E20) and 51.6 mol% (E50(T)). Major fatty acids of strains E20 and E50(T) were summed feature 3 (C16 : 1ω7c and/or iso-C(15 : 0) 2-OH), C(16 : 0), C(18 : 1)ω7c, C(12 : 0), C(18 : 0) and C(12 : 0) 3-OH. The DNA-DNA relatedness of the strains to Cellvibrio gandavensis LMG 18551(T) was 39% for strain E20 and 58% for strain E50(T). The nitrogen fixation capability of strains E20 and E50(T) was confirmed by the acetylene reduction assay. On the basis of our polyphasic taxonomic study, strains E20 and E50(T) represent a novel species of the genus Cellvibrio, for which the name Cellvibrio diazotrophicus is proposed. The type strain of Cellvibrio diazotrophicus is E50(T) ( = LMG 27267(T) = KACC 17069(T)). An emended description of the genus Cellvibrio is proposed based on the capability of fixing nitrogen and growth in presence of up to 5% NaCl (w/v).