Endophytic Nitrogen Fixation Research Articles

Potential effect of novel endophytic nitrogen fixing diverse species of Rahnella on growth promotion of wheat (Triticum aestivum L.)

Potential effect of novel endophytic nitrogen fixing diverse species of Rahnella on growth promotion of wheat (Triticum aestivum L.)

Evidence of endophytic nitrogen fixation as a potential mechanism supporting colonization of non-nodulating pioneer plants on a glacial foreland

Evidence of endophytic nitrogen fixation as a potential mechanism supporting colonization of non-nodulating pioneer plants on a glacial foreland

Genotypic Variation of Endophytic Nitrogen-Fixing Activity and Bacterial Flora in Rice Stem Based on Sugar Content.

Enhancement of the nitrogen-fixing ability of endophytic bacteria in rice is expected to result in improved nitrogen use under low-nitrogen conditions. Endophytic nitrogen-fixing bacteria require a large amount of energy to fix atmospheric nitrogen. However, it is unknown which carbon source and bacteria would affect nitrogen-fixing activity in rice. Therefore, this study examined genotypic variations in the nitrogen-fixing ability of rice plant stem as affected by non-structural carbohydrates and endophytic bacterial flora in field-grown rice. In the field experiments, six varieties and 10 genotypes of rice were grown in 2017 and 2018 to compare the acetylene reduction activity (nitrogen-fixing activity) and non-structural carbohydrates (glucose, sucrose, and starch) concentration in their stems at the heading stage. For the bacterial flora analysis, two genes were amplified using a primer set of 16S rRNA and nitrogenase (NifH) gene-specific primers. Next, acetylene reduction activity was correlated with sugar concentration among genotypes in both years, suggesting that the levels of soluble sugars influenced stem nitrogen-fixing activity. Bacterial flora analysis also suggested the presence of common and genotype-specific bacterial flora in both 16S rRNA and nifH genes. Similarly, bacteria classified as rhizobia, such as Bradyrhizobium sp. (Alphaproteobacteria) and Paraburkholderia sp. (Betaproteobacteria), were highly abundant in all rice genotypes, suggesting that these bacteria make major contributions to the nitrogen fixation process in rice stems. Gammaproteobacteria were more abundant in CG14 as well, which showed the highest acetylene reduction activity and sugar concentration among genotypes and is also proposed to contribute to the higher amount of nitrogen-fixing activity.

Current Progress in Nitrogen Fixing Plants and Microbiome Research.

In agroecosystems, nitrogen is one of the major nutrients limiting plant growth. To meet the increased nitrogen demand in agriculture, synthetic fertilizers have been used extensively in the latter part of the twentieth century, which have led to environmental challenges such as nitrate pollution. Biological nitrogen fixation (BNF) in plants is an essential mechanism for sustainable agricultural production and healthy ecosystem functioning. BNF by legumes and associative, endosymbiotic, and endophytic nitrogen fixation in non-legumes play major roles in reducing the use of synthetic nitrogen fertilizer in agriculture, increased plant nutrient content, and soil health reclamation. This review discusses the process of nitrogen-fixation in plants, nodule formation, the genes involved in plant-rhizobia interaction, and nitrogen-fixing legume and non-legume plants. This review also elaborates on current research efforts involved in transferring nitrogen-fixing mechanisms from legumes to non-legumes, especially to economically important crops such as rice, maize, and wheat at the molecular level and relevant other techniques involving the manipulation of soil microbiome for plant benefits in the non-legume root environment.

Endophytic nitrogen fixation - a possible 'hidden' source of nitrogen for lodgepole pine trees growing at unreclaimed gravel mining sites.

Lodgepole pine (Pinus contorta var. latifolia) trees have been thriving on unreclaimed gravel mining sites in British Columbia, Canada, with tissue nitrogen-content and growth-rate unaffected by extremely low soil nitrogen-levels. This indicates that pine trees could be accessing a hidden nitrogen source to fulfill their nitrogen requirements - possibly via endophytic nitrogen-fixation. Endophytic bacteria originally isolated from native pine trees growing at gravel sites were selected (n = 14) for in vitro nitrogen-fixation assays and a year long greenhouse study to test the overall hypothesis that naturally occurring endophytic nitrogen-fixing bacteria sustain pine tree growth under nitrogen-limited conditions. Each of the 14 bacteria colonized the internal tissues of pine trees in the greenhouse study and fixed significant amounts of nitrogen from atmosphere (23%-53%) after one year as estimated through 15N isotope dilution assay. Bacterial inoculation also significantly enhanced the length (31%-64%) and biomass (100%-311%) of pine seedlings as compared to the non-inoculated control treatment. In addition, presence of the nifH gene was confirmed in all 14 bacteria. Our results support the possibility that pine trees associate with nitrogen-fixing bacteria, capable of endophytic colonization, to survive at unreclaimed gravel mining pits and this association could potentially be utilized for effective reclamation of highly disturbed sites in a sustainable manner.

Whole genome shotgun sequence of Bacillus paralicheniformis strain KMS 80, a rhizobacterial endophyte isolated from rice (Oryza sativa L.).

Bacillus paralicheniformis strain KMS 80 (MTCC No. 12704) is an isolate from the root tissues of rice (Oryza sativa L.) that displays biological nitrogen fixation and plant growth promoting abilities. Here, we report the complete genome sequence of this strain, which contains 4,566,040 bp, 4424 protein-coding genes, 8692 promoter sequences, 67 tRNAs, 20 rRNA genes with six copies of 5S rRNAs along with a single copy of 16S-23S rRNA and genome average GC-content of 45.50%. Twenty one genes involved in nitrogen metabolism pathway and two main transcriptional factor genes, glnR and tnrA responsible for regulation of nitrogen fixation in Bacillus sp. were predicted from the whole genome of strain KMS 80. Analysis of the ~ 4.57 Mb genome sequence will give support to understand the genetic determinants of host range, endophytic colonization behaviour as well as to enhance endophytic nitrogen fixation and other plant beneficial role of B. paralicheniformis in rice.

Draft Genome Sequence of Pseudomonas stutzeri Strain KMS 55, an Endophytic Diazotroph Isolated from Rice Roots.

ABSTRACTPseudomonas stutzeri strain KMS 55 (MTCC 12703) is an isolate from the root tissues of rice (Oryza sativa L.) that displays a high biological nitrogen fixation ability. Here, we report the complete genome sequence of this strain, which contains 4,637,820 bp, 4,289 protein-coding genes, 5,006 promoter sequences, 62 tRNAs, a single copy of 5S-16S-23S rRNA, and a genome average GC content of 51.18%. Analysis of the ~4.64-Mb genome sequence will give support to increased understanding of the genetic determinants of host range, endophytic colonization behavior, endophytic nitrogen fixation, and other plant-beneficial roles of Pseudomonas stutzeri.

Effect of Consortium of Endophytic Nitrogen Fixing Bacteria on Plant Nutrient Concentration of Seasonal (Suru) Sugarcane under Drip Irrigation.

Effect of Consortium of Endophytic Nitrogen Fixing Bacteria on Plant Nutrient Concentration of Seasonal (Suru) Sugarcane under Drip Irrigation.

Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer.

Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N2 -fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P.flexilis-AAB association is consistent across years, we re-sampled P.flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N2 , we incubated twigs with (13) N2 -enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using (13) N. We used the acetylene reduction assay to test for nitrogenase activity within P.flexilis twigs four times from June to September. We found evidence for N2 fixation in P.flexilis foliage. N2 diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8-13.6μgNm(-2) d(-1) to P.flexilis stands. AAB dominated the P.flexilis needle endophyte community. We propose that foliar endophytes represent a low-cost, evolutionarily stableN2 -fixing strategy for long-lived conifers. This novel source of biological N2 fixation has fundamental implications for understanding forest N budgets.

Application of ionomics to plant and soil in fields under long-term fertilizer trials.

Ionomics is the study of elemental accumulation in living organisms using high-throughput elemental profiling. In the present study, we examined the ionomic responses to nutrient deficiency in maize grown in the field in long-term fertilizer trials. Furthermore, the available elements in the field soils were analyzed to investigate their changes under long-term fertilizer treatment and the ionomic relationships between plant and soil. Maize was cultivated in a field with the following five long-term fertilizer treatments: complete fertilization, fertilization without nitrogen, without phosphorus, without potassium, and no fertilization. Concentrations of 22 elements in leaves at an early flowering stage and in soils after harvest were determined. The fertilizer treatments changed the availabilities of many elements in soils. For example, available cesium was decreased by 39 % and increased by 126 % by fertilizations without nitrogen and potassium, respectively. Effects of treatments on the ionome in leaves were evaluated using the translocation ratio (the concentration in leaves relative to the available concentration in soils) for each element. Nitrogen deficiency specifically increased the uptake ability of molybdenum, which might induce the enhancement of nitrogen assimilation and/or endophytic nitrogen fixation in plant. Potassium deficiency drastically enhanced the uptake ability of various cationic elements. These elements might act as alternatives to K in osmoregulation and counterion of organic/inorganic anions. Two major groups of elements were detected by multivariate analyses of plant ionome. Elements in the same group may be linked more or less in uptake and/or translocation systems. No significant correlation between plant and soil was found in concentrations of many elements, even though various soil extraction methods were applied, implying that the interactions between the target and other elements in soil must be considered when analyzing mineral dynamics between plant and soil.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1562-x) contains supplementary material, which is available to authorized users.

Biodiversity of Endorhizospheric Plant Growth Promoting Bacteria

Endorhizospheric bacteria also known as endophytic bacteria that resides within living plant tissues without substantially harming plants have found a large number of applications in today’s agriculture such as nutrient cycling, tolerance to biotic and abiotic stress as well as promotion of plant growth. In present study an attempt has been made to test biodiversity of endophytic plant growth promoting bacteria from different plant species. As nitrogen fixation is one of the prime mechanism of plant growth promoting activity exerted by microorganisms, endophytic nitrogen fixing bacteria were isolated from from surface sterilized plant parts viz. root, stem and leaves of species Cynodon dactylon (Durva), Pothos scandens (Money plant), Ipomea batata (Sweet potato), Saccharum officinarum (Sugarcane) cv. CO.LK-8001 and CO.-84135, Musa paradica (Banana) and Zea mays (maize) cv. GM-6 by using nitrogen free media selective for growth of nitrogen fixing bacteria. In all total 10 isolates were obtained form different plant species and parts and showed variation in their biochemical and physiological characteristics. All the isolates were found to fix nitrogen and nitrogen fixation rates of all the isolates ranged from 4.0 to 36.3 mg N fixed / gm of sugar consumed. Moreover, they also showed solubilizing tri calcium phosphate in Pikovskaya’s broth.

Genome Sequence of Enterobacter sp. Strain SP1, an Endophytic Nitrogen-Fixing Bacterium Isolated from Sugarcane

Enterobacter sp. strain SP1 is an endophytic nitrogen-fixing bacterium isolated from a sugarcane stem and can promote plant growth. The draft genome sequence of strain SP1 presented here will promote comparative genomic studies to determine the genetic background of interactions between endophytic enterobacteria and plants.

Isolation and identification of diazotrophic bacteria from internal tissues ofPinus contortaandThuja plicata

Lodgepole pine ( Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar ( Thuja plicata Donn ex D. Don) are capable of growing in soils that are severely nitrogen-limited. A rarely evaluated but possible nitrogen source for gymnosperms growing at nutrient poor sites is nitrogen fixation by endophytic bacteria. To investigate this possibility, we looked for nitrogen-fixing bacteria in surface-sterilized needle, stem, and root tissues of naturally regenerating lodgepole pine and western red cedar seedlings (2–4 years old) and trees (>20 years old) growing near Williams Lake, Chilliwack Lake, and Boston Bar, British Columbia, Canada. Ninety-nine bacterial isolates were obtained by plating surface-sterilized plant tissue extracts on tryptic soy agar (TSA) and combined carbon medium agar (CCMA), a nitrogen-free medium. Bacterial isolates were identified using gas chromatographic fatty acid methyl ester (GC-FAME) and 16S rRNA gene analyses and were evaluated for nitrogenase activity using an acetylene reduction assay. Representatives of the genera Bacillus and Paenibacillus dominated the culturable endophytic bacterial community in tissues of both tree species, with three Paenibacillus isolates and one Dyadobacter fermentans strain demonstrating consistently high acetylene-reduction activities. Our results raise the possibility that endophytic nitrogen fixation provides a supplementary nitrogen source for naturally regenerated lodgepole pine and western red cedar.

Sugarcane Genetic Controls Involved in the Association with Beneficial Endophytic Nitrogen Fixing Bacteria

Sugarcane is an economically important culture in Brazil, being the world most important source of sugar and ethanol production. Brazilian sugarcane culture is able to obtain large and significant contributions of nitrogen from plant-associated Biological Nitrogen Fixation (BNF), reducing the use of nitrogen fertilizers and leading to an increase in energy balance of the culture. Different populations of endophytic N2-fixing bacteria associated with sugarcane were identified. Besides BNF, these bacteria exhibit growth promoting traits by mechanisms involving nutrient solubilization, plant hormone production and pathogens antagonistic activity. BNF contribution efficiency to sugarcane is controlled by plant and bacteria genotypes as well as environmental conditions. Here, there will be reviewed the advances made in studies on molecular, cellular and physiological mechanisms by which sugarcane controls establishment and efficiency of endophytic association. Transcriptome profile analysis indentified several differentially expressed sugarcane genes during early stages of the association. Regulatory networks involved in various plant processes such as plant-microorganism recognition, defense, plant hormone signaling, plant growth and nitrogen metabolism were reported to be responsive to endophytic diazotrophic bacteria colonization. The data indicate that sugarcane actively participates in the association with these bacteria. Comprehension of how various sugarcane regulatory mechanisms are coordinated and connected to genotype and environmental signals, in order to control the establishment of a beneficial and endophytic type of association is still a big challenge. Nevertheless, the knowledge being accumulated may guide studies to improve plant association with endophytic diazotrophic bacteria and, possibly, to extend it to other crops.

Two genome sequences of the same bacterial strain, Gluconacetobacter diazotrophicus PAl 5, suggest a new standard in genome sequence submission

Gluconacetobacter diazotrophicus PAl 5 is of agricultural significance due to its ability to provide fixed nitrogen to plants. Consequently, its genome sequence has been eagerly anticipated to enhance understanding of endophytic nitrogen fixation. Two groups have sequenced the PAl 5 genome from the same source (ATCC 49037), though the resulting sequences contain a surprisingly high number of differences. Therefore, an optical map of PAl 5 was constructed in order to determine which genome assembly more closely resembles the chromosomal DNA by aligning each sequence against a physical map of the genome. While one sequence aligned very well, over 98% of the second sequence contained numerous rearrangements. The many differences observed between these two genome sequences could be owing to either assembly errors or rapid evolutionary divergence. The extent of the differences derived from sequence assembly errors could be assessed if the raw sequencing reads were provided by both genome centers at the time of genome sequence submission. Hence, a new genome sequence standard is proposed whereby the investigator supplies the raw reads along with the closed sequence so that the community can make more accurate judgments on whether differences observed in a single stain may be of biological origin or are simply caused by differences in genome assembly procedures.

Social bacteria and asocial eukaryotes

The end of 2007 brought us draft genome sequences of two eukaryotic microorganisms, Babesia bovis and Malassezia globosa, as well as complete genomes of the ammonia-oxidizing archaeal chemoautotroph Nitrosopumilus maritimus, and several environmental bacteria (Table 1). In terms of genome size, this list covers both sides of the spectrum: the 245 kb genome of an obligate insect symbiont Sulcia muelleri is the second smallest microbial genome sequenced so far, whereas the 13 033 kb chromosome of the social myxobacterium Sorangium cellulosum breaks the record as the largest bacterial genome – and the largest DNA molecule in the prokaryotic world.

Diazotroph Endophytic Bacteria in Cultivated and Wild Rice in Thailand

The population size of endophytic nitrogen fixing or diazotrophic bacteria was determined for three varieties of cultivated rice and four populations of wild rice. The dynamics of the bacterial populations from both types of rice was estimated at different stages of plant growth. The number of diazotrophic bacteria in roots, stems and leaves of all varieties of cultivated rice increased with ageing of the plants to a maximum at the heading stage (60 days after transplanting). However, nitrogenase activity could not be detected in bacterial isolates from cultivated rice. The highest bacterial population (5.25 × 10 6 per gram fresh material) was found in the roots of Oryza rufipogon, and this population showed the highest nitrogenase activity. In wild rice genotypes the populations of endophytic diazotrophs were stable, with the higher population in O. rufipogon as compared to O. rufipogon (18883) and O. nivara (18852). Cultures of diazotrophic bacteria were isolated and characterized as species of the genera Azospirillum, Herbaspirillum, Beijerinckia and Pseudomonas. All isolates were Gram negative and motile, and produced both pectinase and cellulase. Optimum growth and nitrogen fixation activity for Azospirillum sp. was recorded at 30-35 °C and pH 6.0-7.0, for Beijerinckia sp. at 25-30 °C and pH 6.0-7.5, and for Herbarspirillum sp. at 30-35 °C and pH 6.0-7.5.

Evaluation of the Nitrogen-fixing Ability of Endophytic Clostridia based on Acetylene Reduction and Reverse Transcription-PCR Targeting the nifH Transcript and Ribosomal RNA

To examine whether plant-derived clostridia and their consortium fix nitrogen in plants, an inoculation system was developed using the grass Miscanthus sinensis under aseptic conditions. Among 13 clostridial strains previously isolated from M. sinensis, Clostridium sp. strain Kas107-1 was selected as the best colonizer in the plant with the non-diazotrophic bacterium Entrobacter sp. strain B901-2. Nitrogen-fixing (acetylene-reducing) activity was not observed in the plants inoculated with Kas107-1 without a carbon source. On the other hand, nitrogenfixing activity was detected when carbon sources were supplied to the roots. To confirm the endophytic nitrogenfixing activity, we cloned nifH genes and monitored their expression in strain Kas107-1. Although this bacterium possessed at least two copies of nifH (nifH1 and nifH2), the nifH1 transcript was exclusively detected in free-living cells and endophytic cells in the plants by reverse transcription (RT)-PCR analysis. RT-PCR analysis of ribosomal RNA suggested the endophytic colonization of the plants by Kas107-1. These results indicate that Clostridium sp. strain Kas107-1 can potentially fix nitrogen in plants. A RT-PCR analysis targeting both functional gene transcripts and the ribosomal RNA molecule is useful for researching endophyte ecology, because their function and colonization in plants can be examined simultaneously with a single preparation of RNA.

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N2-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N2-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and 15N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N2-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N2 fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N2-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.

Endophytic colonization of plant roots by nitrogen-fixing bacteria

Nitrogen-fixing bacteria are able to enter into roots from the rhizosphere, particularly at the base of emerging lateral roots, between epidermal cells and through root hairs. In the rhizosphere growing root hairs play an important role in symbiotic recognition in legume crops. Nodulated legumes in endosymbiosis with rhizobia are amongst the most prominent nitrogen-fixing systems in agriculture. The inoculation of non-legumes, especially cereals, with various non-rhizobial diazotrophic bacteria has been undertaken with the expectation that they would establish themselves intercellularly within the root system, fixing nitrogen endophytically and providing combined nitrogen for enhanced crop production. However, in most instances bacteria colonize only the surface of the roots and remain vulnerable to competition from other rhizosphere micro-organisms, even when the nitrogen-fixing bacteria are endophytic, benefits to the plant may result from better uptake of soil nutrients rather than from endophytic nitrogen fixation. Azorhizobium caulinodans is known to enter the root system of cereals, other non-legume crops and Arabidopsis, by intercellular invasion between epidermal cells and to internally colonize the plant intercellularly, including the xylem. This raises the possibility that xylem colonization might provide a non-nodular niche for endosymbiotic nitrogen fixation in rice, wheat, maize, sorghum and other non-legume crops. A particularly interesting, naturally occurring, non-nodular xylem colonising endophytic diazotrophic interaction with evidence for endophytic nitrogen fixation is that of Gluconacetobacter diazotrophicus in sugarcane. Could this beneficial endophytic colonization of sugarcane by G. diazotrophicus be extended to other members of the Gramineae, including the major cereals, and to other major non-legume crops of the World?