Nitrogen-fixing Bacterial Strains Research Articles

OsCIPK2 mediated rice root microorganisms and metabolites to improve plant nitrogen uptake

Crop roots are colonized by large numbers of microorganisms, collectively known as the root-microbiome, which modulate plant growth, development and contribute to elemental nutrient uptake. In conditions of nitrogen limitation, the over-expressed Calcineurin B-like interacting protein kinase 2 (OsCIPK2) gene with root-specific promoter (RC) has been shown to enhance growth and nitrogen uptake in rice. Analysis of root-associated bacteria through high-throughput sequencing revealed that OsCIPK2 has a significant impact on the diversity of the root microbial community under low nitrogen stress. The quantification of nifH gene expression demonstrated a significant enhancement in nitrogen-fixing capabilities in the roots of RC transgenetic rice. Synthetic microbial communities (SynCom) consisting of six nitrogen-fixing bacterial strains were observed to be enriched in the roots of RC, leading to a substantial improvement in rice growth and nitrogen uptake in nitrogen-deficient soils. Forty and twenty-three metabolites exhibiting differential abundance were identified in the roots and rhizosphere soils of RC transgenic rice compared to wild-type (WT) rice. These findings suggest that OSCIPK2 plays a role in restructuring the microbial community in the roots through the regulation of metabolite synthesis and secretion. Further experiments involving the exogenous addition of citric acid revealed that an optimal concentration of this compound facilitated the growth of nitrogen-fixing bacteria and substantially augmented their population in the soil, highlighting the importance of citric acid in promoting nitrogen fixation under conditions of low nitrogen availability. These findings suggest that OsCIPK2 plays a role in enhancing nitrogen uptake by rice plants from the soil by influencing the assembly of root microbial communities, thereby offering valuable insights for enhancing nitrogen utilization in rice cultivation.

Regulation of an endophytic nitrogen-fixing bacteria GXS16 promoting drought tolerance in sugarcane

BackgroundDrought limits crop growth and is an important issue in commercial sugarcane (Saccharum officinarum) production. Drought tolerance in sugarcane induced by endophytic nitrogen-fixing bacteria is a complex biological process that ranges from altered gene expression and cellular metabolism to changes in growth and productivity.ResultsIn this study, changes in physiological features and transcriptome related to drought tolerance in sugarcane conferred by the Burkholderia endophytic nitrogen-fixing bacterial strain GXS16 were investigated. Sugarcane samples inoculated with GXS16 exhibited significantly higher leaf relative water content than those without GXS16 inoculation during the drought stages. Sugarcane treated with GXS16 had lower levels of H2O2 and higher levels of abscisic acid than sugarcane not treated with GXS16 in the non-watering groups. Transcriptomic analysis of sugarcane roots identified multiple differentially expressed genes between adjacent stages under different treatments. Moreover, both trend and weighted correlation network analyses revealed that carotenoid biosynthesis, terpenoid backbone biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction strongly contributed to the drought-tolerant phenotype of sugarcane induced by GXS16 treatment. Accordingly, a gene regulatory network including four differentially regulated genes from carotenoid biosynthesis (crtB, crtZ, ZEP and CYP707A) and three genes from terpenoid backbone biosynthesis (dxs, dxr, and PCME) was constructed.ConclusionsThis study provides insights into the molecular mechanisms underlying the application of GXS16 treatment to enhance drought tolerance in sugarcane, which will lay the foundation for crop development and improve productivity.

Valorization of chick feather wastes by Geobacillus thermodenitrificans PS41 to enhance the growth of Vigna unguiculata plant and Cyprinus carpio fish.

Chicken feather meal has had a significant biofertilizer approach in recent years. The current study aims to assess feather biodegradation to promote plant and fish growth. The Geobacillus thermodenitrificans PS41 strain was more efficient in feather degradation. Feather residues were separated after degradation and evaluated under a scanning electron microscope (SEM) to detect bacterial colonization on feather degradation. It was observed that the rachi and barbules were entirely degraded. The complete degradation by PS41 suggests a relatively more efficient feather degradation strain. According to Fourier-transform infrared spectroscopy (FT-IR) studies, PS41 biodegraded feathers contain the functional groups of aromatic, amine, and nitro compounds. The present study suggested that biologically degraded feather meal improved plant growth. The feather meal combined with nitrogen-fixing bacterial strain showed the highest efficiency. The biologically degraded feather meal and Rhizobium combination induced physical and chemical changes in the soil. It is directly involved in soil amelioration, plant growth substance, and soil fertility, enhancing a healthy crop environment. The feather meal 4 and 5% was used as a feed diet of common carp (Cyprinus carpio) to increase growth performances and feed utilization parameters. In hematological and histological studies of formulated diets, significantly no toxic effects occurred in fish blood, gut, or fimbriae.

Siderophore Synthesis Ability of the Nitrogen-Fixing Bacterium (NFB) GXGL-4A is Regulated at the Transcriptional Level by a Transcriptional Factor (trX) and an Aminomethyltransferase-Encoding Gene (amt).

Kosakonia radicincitans GXGL-4A, a gram-negative nitrogen-fixing (NF) bacterial strain is coated with a thick capsulatus on the surface of cell wall, which becomes a physical barrier for exogenous DNA to enter the cell, so the operation of genetic transformation is difficult. In this study, an optimized Tn5 transposon mutagenesis system was established by using a high osmotic HO-1 medium combined with the electroporation transformation. Eventually, a mutant library containing a total of 1633 Tn5 insertional mutants were established. Of these mutants, the mutants M81 and M107 were found to have an enhanced capability to synthesize siderophore through the CAS agar plate assay and the spectrophotometric determination. The bacterial cells of two mutants were applied in cucumber growth-promoting experiment. Cucumber seedlings treated with M81 and M107 cells had a significant increase in biomass including seedling height, seedling fresh weight, root fresh weight, and root length. The whole genome sequencing of the mutants M81 and M107 showed that the integration sites of Tn5 transposon element were located in MmyB-like helix-turn-helix transcription regulator (locus tag: A3780_19720, trX) and aminomethyltransferase-encoding genes (locus tag: A3780_01680, amt) in the genome of GXGL-4A, respectively. The ability of siderophore synthesis of the target mutants was improved by Tn5 insertion mutagenesis, and the mutants obtained showed a good plant growth-promoting effect when applied to the cucumber seedlings. The results suggest that the identified functional genes regulates the biosynthesis of siderophore in azotobacter GXGL-4A, and the specific mechanism needs to be further investigated.

Genetic and symbiotic characterization of rhizobia nodulating legumes in a mining area in southeast Brazil

Identification and selection of nitrogen-fixing bacterial strains for inoculation into native leguminous tree species can assist in the recovery of degraded areas. Additionally, native strains from these areas are genetic resources adapted to these conditions and are thus suitable for selection. The aim of this study was to symbiotically and genetically characterize 18 bacterial strains from the Rhizobium and Bradyrhizobium genera isolated from Machaerium nyctitans, Platypodium elegans, and Ormosia arborea grown in a nursery in an iron mining area. Three experiments were conducted under axenic conditions in a greenhouse. The nodulation capacity of the strains was evaluated by the number (NN) and dry matter (NDM) of nodules. Symbiotic efficiency was evaluated based on the following parameters: SPAD index (SPAD), shoot dry matter (SDM), root dry matter (RDM), and total dry matter (TDM) of the plants, relative efficiency (RE), shoot nitrogen content (SNC), and total nitrogen content in the plant (TNC). The atpD and gyrB housekeeping genes and the nifH gene were sequenced for phylogenetic analysis, and the nodC and nodD symbiotic genes of the strains were amplified. Out of the 18 strains, 16 were authenticated by nodulation capacity in the species of origin. The SPAD variable allowed for the detection of differences between treatments before the SDM. Additionally, the SPAD index showed correlation with TNC, and the strain Bradyrhizobium sp., UFLA01-839, which may represent a new species, was outstanding in Machaerium nyctitans. The nifH, nodD, and nodC genes were detected in UFLA01-839.

The Effect of Organic and Bio-Fertilization on some Physical and Chemical Properties of Calendula officinalis L. Plant

The effect of organic fertilization (using three rates of poultry manure) and biological fertilization (using a nitrogen-fixing bacterial strain Azospirillum brasilense, mycorrhizal (VAM) fungi and another bacterial strain dissolving phosphate(Bacillus megatherium) on Calendula officinalis L. plant during two consecutive seasons were studied. The results showed that all organic and biological fertilization treatments improved different vegetative growth characteristics and enhanced flowering factors. The various chemical components (Chlorophyll A&B) and carotenoids, as well as an increase in carbohydrates, minerals percentage and oleanolic acid. Hence, organic and biological fertilizers can be used as an alternative to chemical fertilization, which reduces production costs and pollution the agricultural environment and leads to improvement of the natural and chemical properties.

Biotechnological potential of cellulolytic nitrogen-fixing Klebsiella sp. C-3 isolated from paddy soil

In the present study, a new thermo-adaptive cellulolytic nitrogen-fixing bacterial strain designated as Klebsiella sp. C-3, which could degrade cellulose and hemicellulose in rice straw (RS) without an added nitrogen source, was isolated from paddy soil. The highest activities of enzymes (FPase, 0.92 IU/mL, and Xylanase, 17.1 IU/mL) produced by Klebsiella sp. C-3 were recorded at 37 °C. In RS hydrolysate, the % of the total nitrogen content increased from 0.65% to 3.7% after 7-day fermentation with Klebsiella sp. C-3. The main reducing sugar products of RS hydrolysate were glucose, xylose, and cellobiose. Unexpectedly, Klebsiella sp. C-3 had much lower water loss (30%) than the control group (61.85%) and other bacterial isolates at 45 °C after a 7-day incubation period. Hence, Klebsiella sp. C-3 could provide a feasible pathway to manage lignocellulosic biomass in soil and improve water retention in thermophilic biological processes and arid soils.

Bradyrhizobium campsiandrae sp. nov., a nitrogen-fixing bacterial strain isolated from a native leguminous tree from the Amazon adapted to flooded conditions.

The nitrogen-fixing bacterial strain UFLA 01-1174T was isolated from nodules of Campsiandra laurilifolia Benth. originating from the Amazon region, Brazil. Its taxonomic position was defined using a polyphasic approach. Analysis of the 16S rRNA gene placed the strain in the Bradyrhizobium genus, the closest species being B. guangdongense CCBAU 51649T and B. guangzhouense CCBAU 51670T, both with 99.8% similarity. Multilocus sequence analysis (MLSA) of recA, gyrB, glnII, rpoB, atpD, and dnaK indicated that UFLA 01-1174Tis a new species, most closely related to B. stylosanthis BR 446T (94.4%) and B. manausense BR 3351T (93.7%). Average nucleotide identity (ANI) differentiated UFLA 01-1174T from the closest species with values lower than 90%. The G + C content in the DNA of UFLA 01-1174Tis 63.6mol%. Based on this data, we conclude that the strain represents a new species. The name proposed is Bradyrhizobium campsiandrae, with UFLA 01-1174T (= INPA 394BT = LMG 10099T) as type strain.

Efficient Nitrogen-Fixing Bacteria Isolated from Soybean Nodules in the Semi-arid Region of Northeast Brazil are Classified as Bradyrhizobium brasilense (Symbiovar Sojae).

Soybean (Glycine max L.) is an important legume that greatly benefits from inoculation with nitrogen-fixing bacteria. In a previous study, five efficient nitrogen-fixing bacterial strains, isolated from nodules of soybean inoculated with soil from semi-arid region, Northeast Brazil, were identified as a new group within the genus Bradyrhizobium. The taxonomic status of these strains was evaluated in this study. The phylogenetic analysis of the 16S rRNA gene showed the high similarity of the five strains to Bradyrhizobium brasilense UFLA03-321T (100%), B. pachyrhizi PAC48T (100%), B. ripae WR4T (100%), B. elkanii USDA 76T (99.91%), and B. macuxiense BR 10303T (99.91%). However, multilocus sequence analysis of the housekeeping genes atpD, dnaK, gyrB, recA, and rpoB, average nucleotide identity, and digital DNA-DNA hybridization analyses supported the classification of the group as B. brasilense. Some phenotypic characteristics allowed differentiating the five strains and the type strain of B. brasilense from the two neighboring species (B. pachyrhizi PAC48T and B. elkanii USDA 76T). The nodC and nifH genes' analyses showed that these strains belong to symbiovar sojae, together with B. elkanii (USDA 76T) and B. ferriligni (CCBAU 51502T). The present results support the classification of these five strains as Bradyrhizobium brasilense (symbiovar sojae).

Complete Genome Sequence of Kosakonia radicincitans GXGL-4A, a Nitrogen-Fixing Bacterium with Capability to Degrade TEX.

Kosakonia radicincitans GXGL-4A, a free-living nitrogen-fixing (NF) bacterial strain isolated from maize (Zea mays L.) roots was found to have ability to degrade aromatic hydrocarbons. In this study, we describe the main morphological characteristics of bacterium, aromatic hydrocarbon-degrading capability, and the complete genome of K. radicincitans GXGL-4A. The genome is consisted of only one 5,687,681bp linear chromosome with a G + C content of 53.96%. The strain has two genetically distinct nitrogenase systems, one based on molybdenum (Mo) similar to nitrogenase isolated from a wide range of nitrogen-fixing organisms, and the other contains iron (Fe). The differences in transcriptional level of several important nitrogen fixation (nif) genes between LB (nitrogen-rich, NR) and A15 nitrogen-free (nitrogen-limited, NL) culture conditions were detected using Real-time Quantitative Reverse Transcription PCR (qRT-PCR). The bacterial cells of GXGL-4A can grow well in LB liquid medium containing 1% toluene, ethylbenzene or xylene, suggesting a good resistance to the tested aromatic hydrocarbons. The results of GC-MS analysis showed that K. radicincitans GXGL-4A has a good capability to degrade toluene, ethylbenzene, and xylene (TEX). Completion of the genome sequencing will no doubt contribute to the deep exploration and comprehensive utilization of this NF bacterium in sustainable agriculture and bioremediation of aromatic pollutants.

Azospirillum palustre sp. nov., a methylotrophic nitrogen-fixing species isolated from raised bog.

Nitrogen-fixing bacterial strain, designated B2T, was isolated from methane-oxidation enrichment originating from a Sphagnum-dominated raised peatland in Tver region, Russia, and its phenotypic, chemotaxonomic and genomic characteristics were investigated. Cells of isolate were Gram-negative, aerobic, rod or spiral-shaped, with motility provided by a single polar flagellum in liquid media and peritrichous flagella on solid media. Strain was able to grow at 15-40 °C, pH 5.5-8.5 and tolerated NaCl to 2.0 % (w/v). Strain B2T gave positive amplification for dinitrogen reductase (nifH gene) and acetylene reduction activity was recorded up to 1250 nmol ethylene h-1 (mg protein)-1. Analysis of 16S rRNA showed that B2T represents a member of the genus Azospirillum and had the highest sequence similarity with A. humicireducens SgZ-5T (97.92 %). The predominant quinone system was ubiquinone Q-10 and the major fatty acids were C18 : 1ω7, C16 : 1ω7 and C16 : 0. The strain was facultative methylotrophic and used methanol and formate for the growth. Genome sequencing revealed a genome size of 8.0 Mbp and a G+C content of 67.8 mol%. The mxaFI genes encoding methanol dehydrogenase were absent, but a homologous xoxF gene was detected. The genes encoding enzymes involved in the biosynthesis of tetrahydromethanopterin (H4MPT) (formaldehyde oxidation) and NAD-linked formate dehydrogenase (fdsABG) were identified. Pairwise determined whole genome average nucleotide identity (gANI) values confirmed that strain B2T represents a novel species, for which we propose the name Azospirillum palustre sp. nov. with the type strain B2T (VKM B-3233T, КСТС 62613Т).

Bioreduction of toxicity influenced by bioactive molecules secreted under metal stress by Azotobacter chroococcum.

Heavy metal pollution destruct soil microbial compositions and functions, plant's performance and subsequently human health. Culturable microbes among many metal abatement strategies are considered inexpensive, viable and environmentally safe. In this study, nitrogen fixing bacterial strain CAZ3 recovered from chilli rhizosphere tolerated 100, 1000 and 1200 µg mL-1 of cadmium, chromium and nickel, respectively and was identified as Azotobacter chroococcum by 16S rDNA sequence analysis. Under metal stress, cellular morphology of A. chroococcum observed under SEM was found distorted and shrinkage of cells was noticed when grown with 50 µg mL-1 of Cd (cell size 1.7 µm) and 100 of µg mL-1 Ni (cell size 1.3 µm) compared to untreated control (cell size 1.8 µm). In the presence of 100 µg mL-1 of Cr, cells became elongated and measured 1.9 µm in size. Location of metals inside the cells was revealed by EDX. A dose dependent growth arrest and consequently the death of A. chroococcum cells was revealed under CLSM. A. chroococcum CAZ3 secreted 320, 353 and 133 µg EPS mL-1 when grown with 100 µg mL-1 each of Cd, Cr and Ni, respectively. The EDX revealed the presence of 0.4, 0.07 and 0.24% of Cd, Cr and Ni, respectively within EPS extracted from metal treated cells. Moreover, a dark brown pigment (melanin) secreted by A. chroococcum cells under metal pressure displayed tremendous metal chelating activity. The EDX spectra of melanin extracted from metal treated cells of A. chroococcum CAZ3 displayed 0.53, 0.22 and 0.12% accumulation of Cd, Cr and Ni, respectively. The FT-IR spectra of EPS and melanin demonstrated stretching vibrations and variations in surface functional groups of bacterial cells. The C-H stretching of CH3 in fatty acids and CH2 groups, stretching of N-H bond of proteins and O-H bond of hydroxyl groups caused the shifting of peaks in the EPS spectra. Similar stretching vibrations were recorded in metal treated melanin which involved CHO, alkyl, carboxylate and alkene groups resulting in significant peak shifts. Nuclear magnetic resonance (NMR) spectrum of EPS extracted from A. chroococcum CAZ3 revealed apparent peak signals at 4.717, 9.497, 9.369 and 9.242 ppm. However, 1H NMR peaks were poorly resolved due largely to the impurity/viscosity of the EPS. The entrapment of metals by EPS and melanin was confirmed by EDX. Also, the induction and excretion of variable amounts of metallothioneins (MTs) by A. chroococcum under metal pressure was interesting. Conclusively, the present findings establish- (i) cellular damage due to Cd, Cr and Ni and (ii) role of EPS, melanin and MTs in adsorption/complexation and concurrently the removal of heavy metals. Considering these, A. chroococcum can be promoted as a promising candidate for supplying N efficiently to plants and protecting plants from metal toxicity while growing under metal stressed environment.

Recognizing Patterns: Spatial Analysis of Observed Microbial Colonization on Root Surfaces

Root surfaces are major sites of interactions between plants and associated microorganisms. Here, plants and microbes communicate via signaling molecules, compete for nutrients, and release substrates that may have beneficial or harmful effects on each other. Whilst the body of knowledge on the abundance and diversity of microbial communities at root-soil interfaces is now substantial, information on their spatial distribution at the microscale is still scarce. In this study, a standardized method for recognizing and analyzing microbial cell distributions on root surfaces is presented. Fluorescence microscopy was combined with automated image analysis and spatial statistics to explore the distribution of bacterial colonization patterns on rhizoplanes of rice roots. To test and evaluate the presented approach, a gnotobiotic experiment was performed using a potential nitrogen-fixing bacterial strain in combination with roots of wetland rice. The automated analysis procedure resulted in reliable spatial data of bacterial cells colonizing the rhizoplane. Among all replicate roots, the analysis revealed an increasing density of bacterial cells from the root tip to the region of root cell maturation. Moreover, bacterial cells showed significant spatial clustering and tended to be located around plant root cell walls. The quantitative data suggest that the structure of the root surface plays a major role in bacterial colonization patterns. Possible adaptations of the presented approach for future studies are discussed along with potential pitfalls such as inaccurate imaging. Our results demonstrate that standardized recognition and statistical evaluation of microbial colonization on root surfaces holds the potential to increase our understanding of microbial associations with roots and of the underlying ecological interactions.

Genomic characterization of Nitrospirillum amazonense strain CBAmC, a nitrogen-fixing bacterium isolated from surface-sterilized sugarcane stems.

Nitrospirillum amazonense is a nitrogen-fixing bacterium that shows potential to promote plant growth when inoculated into sugarcane and rice plants. This microorganism has been the subject of biochemical and genetic characterization to elucidate important functions related to host plant interaction and growth promotion, including the determination of draft genome sequences of two strains, Y2 and CBAmC, the second of which is the aim of the present study. CBAmC has been isolated from sugarcane (Saccharum spp.), and is currently used in a sugarcane consortium inoculant with four other nitrogen-fixing bacterial strains. The present paper describes a significant improvement in the genome sequence and assembly for the N. amazonense strain CBAmC, and determination for the first time of a complete genome sequence for this bacterial species, using PacBio technology. The analysis of the genomic data obtained allowed the discovery of genes coding for metabolic pathways and cellular structures that may be determinant for the success of the bacterial establishment and colonization into the host sugarcane plant, besides conferring important characteristics to the inoculant. These include genes for the use of sucrose and N-glycans, biosynthesis of autoinducer molecules, siderophore production and acquisition, auxin and polyamine biosynthesis, flagellum, σ-fimbriae, a variety of secretion systems, and a complete denitrification system. Concerning genes for nitrogenase and auxiliary proteins, it was possible to corroborate literature data that in N. amazonense these probably had originated from horizontal gene transfer, from bacteria of the Rhizobiales order. The complete genomic sequence of the CBAmC strain of N. amazonense revealed that the bacterium harbors four replicons, including three chromosomes and one chromid, a profile that coincides with that of other two strains, according to literature data, suggesting that as a replicon pattern for the species. Finally, results of phylogenomic analyses in this work support the recent reclassification of the species, separating it from the Azospirillum genus. More importantly, results of the present work shall guide subsequent studies on strain CBAmC as well as the development of a sugarcane inoculant.

Saline soil microbiome: A rich source of halotolerant PGPR

Twenty halotolerant free-living, nitrogen-fixing bacterial strains were isolated from the Lonar Soda Lake and screened for growth at different NaCl concentrations. Out of 20, only Four isolates DL2, DL3, and DL6 showed optimum growth at 4% NaCl, and DL11 showed optimum growth at 6% NaCl concentration and tolerated 10% NaCl concentration. Biochemical and 16S rRNA sequencing identified DL2 as P. mendocina, DL3 as A. beijerinckii, DL6 as P. stutzeri, and DL11 as B. subtilis. Further, these four isolates were characterized for plant growth-promoting traits and all are indole acetic acid and ammonia producers and they also solubilize the phosphate under 4% NaCl concentration. The salt tolerance of these isolates was also increased from 10 to 14% by using a physical mutagen like UV and mutant strains were also screened for PGP traits. To determine the efficacy of these isolates as plant growth-promoting bacteria, Bioinoculum I (mixed culture of wild strains) and Bioinoculum II (mixed culture of mutant strains) were prepared and tested on Catharanthus roseus at 4% NaCl concentration. Plants treated with Bioinoculum II showed 12.61 and 82.35% increase in shoot and root length, respectively, and 71.42% increase in number of flowers as compared to control plants under saline conditions. These results indicate that halotolerant bacteria isolated from saline environments have the potential to enhance plant growth under saline stress through direct or indirect mechanisms and would be most appropriate as bioinoculants under such conditions.

SYMBIOTIC EFFICIENCY OF RHIZOBIA STRAINS WITH COWPEA IN SOUTHERN MARANHÃO

ABSTRACT The objective of this study was to evaluate the symbiotic efficiency of three novel nitrogen-fixing bacterial strains with cowpea cv. BRS Guariba as forage. Two experiments were carried out in the field in Balsas and São Raimundo das Mangabeiras, MA, in a randomized block design with seven treatments and four replications: three novel strains efficient in symbiotic nitrogen fixation with cowpea (UFLA 3 -153, UFLA 3-154 and UFLA 3-164), two strains already approved as inoculants by MAPA (UFLA 3-84/SEMIA 6461 and INPA 3-11B/SEMIA 6462), and two controls without inoculation, with and without mineral N. Symbiotic efficiency of strains was determined by the number of nodules, nodules dry matter, shoot dry matter, relative efficiency, crude protein content and shoot N content. Strains UFLA 3-154 and UFLA 3-164 were the most efficient in Balsas, whereas UFLA 3-153 and UFLA 3-164 were the most efficient in São Raimundo das Mangabeiras. Strains UFLA 3-153, UFLA 3-154 and UFLA 3-164 were more efficient in symbiotic nitrogen fixation with cowpea than the strains already approved as inoculants UFLA 3-84 and INPA 3-11B in the climatic and edaphic conditions of southern Maranhão. UFLA 3-164 is the most efficient strain in this region.

Azospirillum agricola sp. nov., a nitrogen-fixing species isolated from cultivated soil.

A polyphasic approach was used to characterize a novel nitrogen-fixing bacterial strain, designated CC-HIH038T, isolated from cultivated soil in Taiwan. Cells of strain CC-HIH038T were Gram-stain-negative, facultatively aerobic and spiral-shaped, with motility provided by a single polar flagellum. The 16S rRNA gene sequence analysis of strain CC-HIH038T showed highest sequence similarity to Azospirillum doebereinerae (98.0 %), Azospirillum thiophilum (97.5 %), Azospirillum rugosum (97.4 %) and Azospirillum zeae (97.2 %) and lower sequence similarity ( < 97.0 %) to all other species of the genus Azospirillum. According to DNA-DNA association, the relatedness values of strain CC-HIH038T with A. doebereinerae, A. thiophilum, A. rugosum and A. zeae were 51.8 %, 41.2 %, 56.5 % and 37.5 %, respectively. Strain CC-HIH038T was able to grow at 20-37 °C and pH 7.0-8.0. Strain CC-HIH038T gave positive amplification for dinitrogen reductase (nifH gene); the activity was recorded as 8.4 nmol ethylene h- 1. The predominant quinone system was ubiquinone Q-10 and the DNA G+C content was 68.8 mol%. The major fatty acids found in strain CC-HIH038T were C16 : 0, iso-C18 : 0, C16 : 0 3-OH, C14 : 0 3-OH/iso-C16 : 1 and C18 : 1ω7c/C18 : 1ω6c. Based on the distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence analysis, strain CC-HIH038T is considered to represent a novel species in the genus Azospirillum, for which the name Azospirillum agricola sp. nov. is proposed. The type strain is CC-HIH038T ( = BCRC 80909T = JCM 30827T).

Bradyrhizobium spp. Strains in Symbiosis with Pigeon Pea cv. Fava-Larga under Greenhouse and Field Conditions

ABSTRACT: Optimization of symbiosis between nitrogen-fixing bacteria and legumes has been extensively studied, seeking agricultural sustainability. To evaluate the symbiotic efficiency of nitrogen-fixing bacterial strains belonging to the Bradyrhizobium genus with pigeon pea (Cajanus cajan (L.) Millsp.) cv. Fava-Larga, experiments were conducted in Leonard jars (axenic conditions), pots with soil, and in the field. Ten strains were tested in Leonard jars, and three strains, in addition to BR 29, were selected according to their ability to promote the growth of [...]

Cultivable Nitrogen Fixing Bacteria from Extremely Alkaline-Saline Soils

Three soils with different Electrolytic Conductivity (EC) from the former Lake Texcoco (soil with low EC 1.2 dS·m-1, medium with EC 83.1 - 107.8 dS·m-1, and high with EC 137.3 - 152.5 dS•m-1) were used to isolate nitrogen-fixing bacterial strains through enrichment cultures in nitrogen-free media. The medium and high EC in the soil affected negatively the nitrogen-fixing activity, which was generally ten times lower compared to the activity in the soil with low EC. Twenty-one bacterial strains were isolated, identified and characterized for their nitrogen fixation capacity. The diazotrophic genetic potential of all isolates was confirmed by amplification and sequencing of partial nifH and nifD genes and diazotrophic activity quantified by the acetylene reduction assay. Azospirillum brasilense, and several species of Paenibacillus (P. fujiensis, P. durus, P. borealis, P. graminis, P. massiliensis and P. wynnii) were identified. Isolates belonging to the Paenibacillus genus were found in the three soils. Paenibacillus fujiensis and P. durus showed a high nitrogenase activity. The phylograms based on nifH and nifD gene sequences were consistent with 16S rRNA gene phylogeny.

Effects of a Formulated Bio-product Containing the Nitrogen Fixing Bacterial Strain Enterobacter oryzae 3LSO1 on Sugarcane Growth

Nitrogen (N) is an essential element for plant growth, widely applied as chemical N-fertilizer to improve yield of sugarcane. The elevated doses of fertilizers may have negative and unpredictable effects on the environment. Bio-fertilizers containing nitrogen fixing bacteria (NFB) are alternative and potential way to reduce application of chemical N-fertilizer. The NFB strain Enterobacter oryzae 3LSO1 isolated from rhizosphere soil of sugarcane can fix atmospheric dinitrogen (N2), produces indole-3-acetic acid as plant growth promoting substance. Therefore, this effective strain was developed as a vermiculite-based bio-product to enhance sugarcane growth. The bio-product was compared storing conditions at 10 and 30 °C for 30 or 60 days after formulation (DAF), and then its performance was evaluated. Result stated that the bio-product could be kept at 10 and 30 °C for up to 60 DAF without a decrease in the NFB population or a loss in its ability to fix N2. In a greenhouse experiment, commercial sugarcane variety Khon Kaen 3 applied with the E. oryzae 3LSO1 bio-product and planted with or without nitrogen fertilizer could increase the tiller number, plant height, fresh and dry weight, NFB population in soil and sugarcane, compared with the control. This E. oryzae 3LSO1 bio-product not only used as a bio-nitrogen fertilizer for sugarcane crops, but also reduced chemical N-fertilizer application for sustainable agriculture.