Contribution Of Biological Nitrogen Fixation Research Articles

Biological nitrogen fixation in rice paddy soils is driven by multiple edaphic factors and available phosphorus is the greatest contributor

Biological nitrogen fixation (BNF) is important for sustainable rice cultivation. Various edaphic factors have been individually evaluated for their effects on BNF in paddy soils. However, no single factor could fully explain the different soil outcomes. Paddy BNF is more likely to be simultaneously influenced to various degrees by combinations of several factors; however, the relative importance of the interaction of multiple edaphic factors on the regulation of BNF in rice soils is still unclear. Twenty-seven paddy soil samples with different soil properties were collected from major cropping areas in southwest and northeast China. Rice was transplanted into pots of these soils and grown in a 15N2 enriched airtight chamber. Estimation of BNF was based on measurements of 15N enrichment in the different soils and rice plants at the end of a 77-d incubation period. BNF amounts ranged from 0.66 to 12.3 kg ha−1. BNF had a significant positive relationship with available phosphorus (AP) and significant quadratic relationships with available molybdenum (AMo) and total soil nitrogen (TN). AP explained 42% of the observed variation in BNF, TN explained 17%, and AMo explained 13%. The specific interaction between the soil cation exchange capacity (CEC) and available soil N (ASN, as determined by rice N uptake) accounted for 28% of the variation. BNF was reduced when AP was < 14 mg kg−1, AMo < 0.09 mg kg−1, or when TN was > 3.2 g kg−1. These results provide valuable benchmarks that could be used to guide farmers in managing their soils to improve the potential contribution of paddy BNF to soil fertility.

Biochar amendment reduces biological nitrogen fixation and nitrogen use efficiency in cadmium-contaminated paddy fields

Cadmium (Cd) contamination poses a considerable threat to human health through grain enrichment and limits biological nitrogen fixation (BNF) in paddy fields. Biochar has shown great potential for agricultural soil remediation because it inactivates Cd, but uncertainties remain as to how biochar amendments affect BNF and grain N use efficiency in paddies. To elucidate these issues, we investigated the effects of biochar amendment on the structure and function of diazotrophic bacterial communities in different rice growth stages in Cd-contaminated paddy fields, and evaluated the contribution of BNF to grain N use efficiency under biochar amendment. The results showed that biochar amendment significantly increased the abundance of diazotrophic bacteria in the tillering and jointing stages. Furthermore, the community structure of soil diazotrophic bacteria markedly changed with biochar amendment, with a significant reduction in the abundances of Euryarchaeota, Desulfobacterales (Proteobacteria), and Sphingomonadales (Bacteroidetes) in the tillering stage. Changes in the soil carbon/nitrogen (C/N) ratio was the main factor driving diazotrophic microbial community characteristics caused by the release of available C from biochar at the tillering stage, rather than the Cd. Moreover, biochar amendment increased the efficiency of BNF (especially for autotrophic N2 fixation) in the vegetative phase of rice growth. Notably, biochar amendment significantly decreased BNF efficiency during the filling stage and reduced grain N use efficiency. The limited available nutrients in biochar and the toxicity of polycyclic aromatics and phenols in biochar-derived dissolved organic matter were responsible for the varied impacts of biochar on BNF in different rice growth stages. For the first time, we report that biochar amendment in paddy soils reduces Cd toxicity but also inhibits BNF and thereby decreases N use efficiency. Therefore, before applying biochar to inactivate Cd in paddy fields, there should be a trade-off between agricultural production and ecological safety to achieve sustainable agriculture.

Mechanisms for Generating Low Potential Electrons across the Metabolic Diversity of Nitrogen-Fixing Bacteria.

The availability of fixed nitrogen is a limiting factor in the net primary production of all ecosystems. Diazotrophs overcome this limit through the conversion of atmospheric dinitrogen to ammonia. Diazotrophs are phylogenetically diverse bacteria and archaea that exhibit a wide range of lifestyles and metabolisms, including obligate anaerobes and aerobes that generate energy through heterotrophic or autotrophic metabolisms. Despite the diversity of metabolisms, all diazotrophs use the same enzyme, nitrogenase, to reduce N2. Nitrogenase is an O2-sensitive enzyme that requires a high amount of energy in the form of ATP and low potential electrons carried by ferredoxin (Fd) or flavodoxin (Fld). This review summarizes how the diverse metabolisms of diazotrophs utilize different enzymes to generate low potential reducing equivalents for nitrogenase catalysis. These enzymes include substrate-level Fd oxidoreductases, hydrogenases, photosystem I or other light-driven reaction centers, electron bifurcating Fix complexes, proton motive force-driven Rnf complexes, and Fd:NAD(P)H oxidoreductases. Each of these enzymes is critical for generating low potential electrons while simultaneously integrating the native metabolism to balance nitrogenase's overall energy needs. Understanding the diversity of electron transport systems to nitrogenase in various diazotrophs will be essential to guide future engineering strategies aimed at expanding the contributions of biological nitrogen fixation in agriculture.

An agronomic study of legacy effects from annual legume pastures in acid soils

AbstractThe use of acid soil‐tolerant annual legume pasture species and liming to raise soil pH can have legacy effects that improve subsequent wheat crop growth in acid soils. We established a two‐year experiment in a semi‐arid environment with and without historical lime application to investigate how acid soils (±lime) influence the legacy effects of annual legume pastures. We measured legume and cereal growth and yield, soil‐borne disease incidence, rhizosheath organic acid type and amount, legume biological nitrogen fixation (BNF) and soil moisture in Year 1, and soil microbial biomass carbon (MBC) and wheat yield and quality in Year 2. Minor differences in soil moisture and soil‐borne diseases occurred between cereals and legumes, and the poor BNF contribution of all pasture legumes improved in lime treatments. Pasture legumes produced more organic acids than cereals, which increased in limed treatments. Soil MBC in Year 2 was greater following legumes than cereals. Year 2 wheat grain yield increased following pasture legumes, more so in limed treatments. The contribution of organic acid exudation by annual legume pastures to microbial community legacy effects warrants further scrutiny in semi‐arid environments, particularly when acid soils limit typical rotational benefits.

Microbiome of Nodules and Roots of Soybean and Common Bean: Searching for Differences Associated with Contrasting Performances in Symbiotic Nitrogen Fixation.

Biological nitrogen fixation (BNF) is a key process for the N input in agriculture, with outstanding economic and environmental benefits from the replacement of chemical fertilizers. However, not all symbioses are equally effective in fixing N2, and a major example relies on the high contribution associated with the soybean (Glycine max), contrasting with the low rates reported with the common bean (Phaseolus vulgaris) crop worldwide. Understanding these differences represents a major challenge that can help to design strategies to increase the contribution of BNF, and next-generation sequencing (NGS) analyses of the nodule and root microbiomes may bring new insights to explain differential symbiotic performances. In this study, three treatments evaluated in non-sterile soil conditions were investigated in both legumes: (i) non-inoculated control; (ii) inoculated with host-compatible rhizobia; and (iii) co-inoculated with host-compatible rhizobia and Azospirillum brasilense. In the more efficient and specific symbiosis with soybean, Bradyrhizobium presented a high abundance in nodules, with further increases with inoculation. Contrarily, the abundance of the main Rhizobium symbiont was lower in common bean nodules and did not increase with inoculation, which may explain the often-reported lack of response of this legume to inoculation with elite strains. Co-inoculation with Azospirillum decreased the abundance of the host-compatible rhizobia in nodules, probably because of competitiveness among the species at the rhizosphere, but increased in root microbiomes. The results showed that several other bacteria compose the nodule microbiomes of both legumes, including nitrogen-fixing, growth-promoters, and biocontrol agents, whose contribution to plant growth deserves further investigation. Several genera of bacteria were detected in root microbiomes, and this microbial community might contribute to plant growth through a variety of microbial processes. However, massive inoculation with elite strains should be better investigated, as it may affect the root microbiome, verified by both relative abundance and diversity indices, that might impact the contribution of microbial processes to plant growth.

Sugarcane Genotypes with Contrasting Biological Nitrogen Fixation Efficiencies Differentially Modulate Nitrogen Metabolism, Auxin Signaling, and Microorganism Perception Pathways.

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes.

Elite Bradyrhizobium strains boost biological nitrogen fixation and peanut yield in tropical drylands.

Peanut (Arachis hypogaea L.) is an important crop for the family-based systems in the tropics, mainly in Brazil. In the Brazilian drylands, peanuts are cropped in low technological systems, and cheap and efficient technologies are needed to improve crop yield and sustainability. Despite this importance, few data are available on selecting efficient peanut rhizobia in experiments under different edaphoclimatic conditions. This work evaluated the agronomic efficiency and the biological nitrogen fixation (BNF) by two elite Bradyrhizobium strains under four different fields in the Brazilian semiarid region. We compared a new efficient strain Bradyrhizobium sp. ESA 123 with the reference strain B. elkanii SEMIA 6144, currently used in peanut rhizobial inoculants in Brazil. Besides the inoculated treatments, two uninoculated controls were assessed (with and without 80kgha-1 of N-urea). The BNF was estimated by the δ15N approach in three out of four field assays. BNF contribution was improved by inoculation of both Bradyrhizobium strains, ranging from 42 to 51% in Petrolina and 43 to 60% in Nossa Senhora da Glória. Peanuts' yields benefited from the inoculation of both strains and N fertilization in all four assays. Nevertheless, the results showed the efficiency of both strains under different edaphoclimatic conditions, indicating the native strain ESA 123 as a potential bacterium for recommendation as inoculants for peanuts in Brazil, mainly in drylands.

Soybean relay‐cropped with winter camelina reduces biological nitrogen fixation

AbstractThe contribution of biological nitrogen fixation (BNF) in soybean [Glycine max (L.) Merr.] to plant nutrition and its N credit for the next crop is significant. The amount of N fixed via BNF in monocropped soybean is well documented, but it is unknown when soybean is relayed into winter camelina [Camelina sativa (L.) Crantz]. Therefore, BNF in soybean relayed into fertilized (WCFerti) and unfertilized (WCUnfert) winter camelina grown for seed, and soybean seeded after winter camelina terminated as a cover crop (WCCC) were compared with a monocrop (control) soybean. The results showed that N‐fixing bacteria successfully infected soybean roots and were potentially active in N fixation for all treatments. Ureide‐N abundance was in the order of control &gt; WCFerti = WCUnfert &gt; WCCC. The control had 38 and 44% more ureide‐N (indicating greater BNF) than WCFerti and WCUnfert, respectively. Relayed soybean had the same grain protein and oil concentrations as the control. However, the control had greater soybean grain, protein, and oil yields than relayed treatments due to grain yield differences. Fertilized winter camelina resulted in greater total (soybean + camelina) oil yield (859 kg ha–1) than the control (587 kg ha–1). The lower ureide‐N abundance and grain yields for relayed soybean than the control was due to differences in belowground and aboveground biomass. Further research is needed to develop earlier‐maturing winter camelina genotypes, better row spacing, and more shade‐tolerant soybean cultivars to minimize competition between the two species, enhance BNF, and increase relayed‐soybean yield.

Bradyrhizobium strains from Brazilian tropical soils promote increases in nodulation, growth and nitrogen fixation in mung bean

Mung bean [Vigna radiata (L.) Wilczek] is a legume of Asian origin cultivated in several countries, including Brazil, where its commercial cultivation has expanded in recent years. It has a promiscuous N2-fixing association with rhizobia, but nodulation in Brazilian soils has been highly variable. To achieve satisfactory yields with low production costs, studies are being carried out to select rhizobia from soils in Brazil that result in efficient symbiotic associations with mung bean. Previous studies indicate that only Bradyrhizobium strains were able to nodulate mung bean in Brazilian soils. The objective of this study was to evaluate the effect of 31 Bradyrhizobium strains and increasing levels of N fertilizer on nodulation, plant growth and N2 fixation. Bradyrhizobium strains resulted in increases of up to 71, 79, 43, 66, 40 and 55%, respectively, in nodule number and nodule, root and shoot biomasses, accumulated N and biological nitrogen fixation (BNF) contribution compared to the uninoculated control, however, there was no increase in grain yield. Compared to the control with N fertilizer, inoculated plants had lower biomass and accumulated N, despite the high BNF contribution. In general, strains of Bradyrhizobium japonicum superclade, especially those close to B. yuanmingense B071, showed greater efficiency compared to those of the superclade of B. elkanii. The application of N fertilizer increased root and shoot biomasses, however, significantly reduced nodulation. Bradyrhizobium strains isolated from Brazilian tropical soils showed potential for mung bean inoculation, however, the BNF contribution was not sufficient to meet the plant's N demand.

Bacterial Indole-3-Acetic Acid Influences Soil Nitrogen Acquisition in Barley and Chickpea.

Farming of barley and chickpea is nitrogen (N) fertilizer dependent. Using strategies that increase the nitrogen use efficiency (NUE) and its components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE) would reduce the N fertilizer application in the soil and its adverse environmental effects. We evaluated the effects of three different strains of diazotroph Klebsiella (K.p. SSN1, K.q. SGM81, and K.o. M5a1) to understand the role of biological nitrogen fixation (BNF) and bacterial indole-3-acetic acid (IAA) on NUE of the plants. A field study revealed that K.p. SSN1 results in profound increment of root surface area by eightfold and threefold compared to uninoculated (control) in barley and chickpea, respectively. We measured significant increase in the plant tissue nitrogen, chlorophyll content, protein content, nitrate reductase activity, and nitrate concentration in the inoculated plants (p ≤ 0.05). Treated barley and chickpea exhibited higher NUE and the components compared to the control plants (K.p. SSN1 ≥ K.q. SGM81> K.o. M5a1). Specifically, K.q. SGM81 treatment in barley increased NUpE by 72%, while in chickpea, K.p. SSN1 increased it by 187%. The substantial improvement in the NUpE and NUE by the auxin producers K.p. SSN1 and K.q. SGM81 compared with non-auxin producer K.o. M5a1 was accompanied by an augmented root architecture suggesting larger contribution of IAA over marginal contribution of BNF in nitrogen acquisition from the soil.

Agronomic evaluation of Herbaspirillum seropedicae strain ZAE94 as an inoculant to improve maize yield in Brazil

Diazotrophic bacteria applied as a seed inoculant can improve the grain yield of several crops including maize. The current study aimed to test the agronomic efficiency and contribution of biological nitrogen fixation (BNF) of the endophytic diazotroph Herbaspirillum seropedicae strain ZAE94 to maize under field conditions. Eighteen field assays were conducted in four different locations during consecutive years on two hybrids and two varieties of maize in a random block design with four replicates using a peat-based inoculant. The inoculant containing the ZAE94 strain was applied without nitrogen (N) fertilization or with 40 kg N ha–1 and was compared to the application of 40 and 80 kg N ha–1 without inoculation. Crop productivity and N accumulation in the grain were evaluated in addition to 15 N natural abundance (°15 N) to evaluate BNF in the treatments without N fertilization. Fertilization at 40 kg N ha–1 plus bacterial inoculation produced crop yields similar to the treatment with 80 kg N ha–1 and increased grain N content, especially in the off-season with 40 kg N ha–1. The inoculation treatments showed lower °15N values than the non-inoculated treatments, which was most evident in the off-season. The BNF contributed about 30% of N accumulated in plants inoculated with ZAE94. On average, 64% of the N fertilized plots showed an increase of the parameters evaluated in the inoculated treatments, compared with the control. Inoculation also increased root length, root volume, and leaf area, and these parameters were positively correlated with plant weight using a hydroponic assay. This study revealed that the application of H. seropedicae inoculant increased the amount of N in plants owing to BNF, and there is a better chance of yield response to inoculation under low N fertilizer application in the off-season.

Molecular Biology in the Improvement of Biological Nitrogen Fixation by Rhizobia and Extending the Scope to Cereals.

The contribution of biological nitrogen fixation to the total N requirement of food and feed crops diminished in importance with the advent of synthetic N fertilizers, which fueled the “green revolution”. Despite being environmentally unfriendly, the synthetic versions gained prominence primarily due to their low cost, and the fact that most important staple crops never evolved symbiotic associations with bacteria. In the recent past, advances in our knowledge of symbiosis and nitrogen fixation and the development and application of recombinant DNA technology have created opportunities that could help increase the share of symbiotically-driven nitrogen in global consumption. With the availability of molecular biology tools, rapid improvements in symbiotic characteristics of rhizobial strains became possible. Further, the technology allowed probing the possibility of establishing a symbiotic dialogue between rhizobia and cereals. Because the evolutionary process did not forge a symbiotic relationship with the latter, the potential of molecular manipulations has been tested to incorporate a functional mechanism of nitrogen reduction independent of microbes. In this review, we discuss various strategies applied to improve rhizobial strains for higher nitrogen fixation efficiency, more competitiveness and enhanced fitness under unfavorable environments. The challenges and progress made towards nitrogen self-sufficiency of cereals are also reviewed. An approach to integrate the genetically modified elite rhizobia strains in crop production systems is highlighted.

Chronic Atmospheric Reactive Nitrogen Deposition Suppresses Biological Nitrogen Fixation in Peatlands.

Biological nitrogen fixation (BNF) represents the natural pathway by which mosses meet their demands for bioavailable/reactive nitrogen (Nr) in peatlands. However, following intensification of nitrogen fertilizer and fossil fuel use, atmospheric Nr deposition has increased exposing peatlands to Nr loading often above the ecological threshold. As BNF is energy intensive, therefore, it is unclear whether BNF shuts down when Nr availability is no longer a rarity. We studied the response of BNF under a gradient of Nr deposition extending over decades in three peatlands in the U.K., and at a background deposition peatland in Sweden. Experimental nitrogen fertilization plots in the Swedish site were also evaluated for BNF activity. In situ BNF activity of peatlands receiving Nr deposition of 6, 17, and 27 kg N ha-1 yr-1 was not shut down but rather suppressed by 54, 69, and 74%, respectively, compared to the rates under background Nr deposition of ∼2 kg N ha-1 yr-1. These findings were corroborated by similar BNF suppression at the fertilization plots in Sweden. Therefore, contribution of BNF in peatlands exposed to chronic Nr deposition needs accounting when modeling peatland's nitrogen pools, given that nitrogen availability exerts a key control on the carbon capture of peatlands, globally.

Differences in contribution of biological nitrogen fixation to yield performance of common bean cultivars as assessed by the 15N natural abundance technique

Identification of variability in biological N2 fixation (BNF) contribution among common bean (Phaseolus vulgaris L.) cultivars under field conditions requires a reliable methodology. This study aimed to assess different common bean cultivars for plant growth and grain yield and to quantify the BNF contribution to the crop using the 15N natural abundance technique. Two field experiments were conducted in an Oxisol in Brazil over two consecutive years, with eight common bean cultivars inoculated with rhizobium or fertilized with mineral N. Plants were analysed at mid-pod filling stage (two weeks after full-flowering) and at grain maturity. BNF was estimated by the 15N natural abundance technique. Average grain yields were 1614 or 2942 kg ha−1 in Experiment I, and 3284 or 3919 kg ha−1 in Experiment II, with rhizobium inoculation or mineral N, respectively. The average contributions of N derived from atmosphere were 14 and 26%, and amounts of N2-fixed were 7 and 22 kg N ha−1, in bean plants at mid-pod filling, respectively, in Experiment I and Experiment II. The contributions of BNF increase when common bean crop reached its optimum yield potential even though soil N was the most important source for the plants. There is good relationship between δ15N values of grains and shoots, provided cultivars are of similar growth habit. The 15N natural abundance technique allowed identifying cultivars with relatively high BNF capability for commercial crop and breeding purposes.

The Co-inoculation of Rhizobium and Bradyrhizobium Increases the Early Nodulation and Development of Common Beans

It has recently been shown that the co-inoculation of Rhizobium tropici and Bradyrhizobium spp. can benefit the nodulation, development, and biological nitrogen fixation of common beans (Phaseolus vulgaris L.). Here, we compared this type of co-inoculation with the co-inoculation of Azospirillum brasilense on two common bean cultivars and evaluated whether they can stimulate the early nodulation of this crop, aiming at anticipating the contribution of biological nitrogen fixation to plant nutrition. The co-inoculation with B. elkanii 29w increased the number of nodules in the V3 and V4 stages. Both co-inoculations stimulated a larger mass of nodules and larger shoot biomass during the V4 stage. The co-inoculation of Bradyrhizobium also stimulated root growth. The co-inoculation effects were consistent for the two tested cultivars, but some differences in response indicate a possible genotype effect. We can conclude that the co-inoculation of B. elkanii benefits the common bean during the early stages of its cycle. These effects are comparable with the effects of the co-inoculation with Azospirillum brasilense Sp 245.

Biological nitrogen fixation and urea-N recovery in 'Coastcross-1' pasture treated with Azospirillum brasilense

Abstract: The objective of this work was to quantify the inoculation effect of Azospirillum brasilense (Ab-V5 and Ab-V6 strains) on the forage yield, biological nitrogen fixation (BNF), and urea-15N recovery of the forage grass 'Coastcross-1'. The experiment was carried out in a 2 (with or without inoculation) × 2 (without N fertilizer and with 100 kg ha-1 N per year as urea) × 7 (cuts) factorial arrangement. The natural 15N abundance method was used to determine BNF; to determine urea-N recovery, 15N-labeled urea was applied in microplots. Forage yield was higher in grasses subjected to inoculation, with 7.4 Mg ha-1 dry matter per year, for the treatment without N fertilizer, and 8.0 Mg ha-1 dry matter per year for the treatment with 100 kg ha-1 N per year, respectively, which shows an additive effect of inoculation and N fertilization. The contribution of BNF was 23.0 and 53.8 kg ha-1 per year for the unfertilized treatment, both in uninoculated and inoculated plants, respectively. Urea-15N recovery was 13.7 and 16.5 kg ha-1 per year for uninoculated and inoculated treatments, respectively, corresponding to 13.7 and 16.5% of the urea-N applied. Inoculation with A. brasilense increases forage yield and the contribution of BNF to grass nutrition with N, as well as urea-N recovery by the forage grass.

Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

The Mo- and Fe-nitrogenases of the endophyte Kosakonia sp. UYSO10 are necessary for growth promotion of sugarcane

Sugarcane is a multipurpose crop primarily used to produce sugar, energy and bioethanol. It requires high amounts of N-fertilization for optimal growth, which increases production costs and environmental degradation. The contribution of biological nitrogen fixation to Uruguayan commercial sugarcane cultivars was demonstrated previously, and diazotrophic bacteria that were isolated from the stems were characterized and identified. From this collection, the isolate UYSO10 related to the Kosakonia genus (formerly Enterobacter) was described as a plant growth-promoting endophyte of sugarcane plants. To evaluate the effect of the inoculation of wild-type and nitrogenase-deficient strains of Kosakonia sp. UYSO10 on sugarcane growth promotion under non-sterile conditions. Kosakonia sp. UYSO10 was inoculated onto sugarcane setts for plant growth promotion greenhouse experiments. Single and double mutants resulting to the nitrogenase-encoding genes (nifH, anfH) were constructed, and the phenotypes were evaluated in vitro and in vivo. Kosakonia sp. UYSO10 is able to promote sugarcane growth under non-sterile conditions, that strain UYSO10 harbors two functional nitrogenases and the inactivation of both nitrogenase-encoding genes diminish its capacity of promoting growth on sugarcane. All together, the results obtained showed that the biological nitrogen fixation ability of Kosakonia sp. UYSO10 is required for sugarcane growth promotion.

Sugarcane inoculated with endophytic diazotrophic bacteria: effects on yield, biological nitrogen fixation and industrial characteristics.

The growing need for the use of clean energy has led society to seek alternatives to fossil fuels in order to less pollute the environment. Sugarcane has been known to produce enough biomass and to have associative microorganisms, such as plant growth-promoting bacteria (PGPB), which have the potential to improve the yield of crops. The objective of this study is to evaluate the efficiency and the isotopic dilution of 15N of microbial inoculants containing endophytic diazotrophic bacteria, individually inoculated and in mixture, on productivity, technological quality and biological nitrogen fixation (BNF) of sugarcane variety RB92579. The experiment was conducted for three consecutive years, comprising different crop cycles, in a completely randomized block design with four replications. The evaluations were at 11 months after planting for plant-cane, and at 12 months after cutting for ratoon cane and second ratoon cane. The contribution of BNF by 15N natural abundance technique to inoculated treatments ranged from 18% to 57.31%. This study showed that no strain promoted the improvement on yield and biological nitrogen fixation during the three cycles of sugarcane (plant-cane, ratoon cane and second ratoon cane). However, industrial characteristics of sugar cane can be affectd by PGPB inoculation.

Rooting and growth of pre-germinated sugarcane seedlings inoculated with diazotrophic bacteria

Renovation of Brazilian sugarcane plantations using the pre-sprouted seedling technique (PSS) was undertaken to increase sanitation and productivity while reducing the cost of sugarcane planting. The objective of this study was to evaluate growth, nutrient acquisition, and contribution of biological nitrogen fixation (BNF) in two sugarcane cultivars RB867515 and IACSP95-5000, inoculated with five strains/species of diazotrophic bacteria, applied together and individually, for 60 days during growth. The cultivars were grown in a greenhouse in boxes filled with a sterile substrate followed by growth in tubes filled with a commercial substrate in the second phase; finally the plants were transferred to pots containing soil enriched with 15N and grown outdoors. The treatments used were control (no inoculation); mixed inoculation with the five strains, Gluconacetobacter diazotrophicus (PAL5T), Herbaspirillum rubrisubalbicans (HCC103), Herbaspirillum seropedicae (HRC54), Nitrospirillum amazonense (CBAmC) and Paraburkholderia tropica (PPe4T); and individual inoculation with each strain. Increases of up to 50% in the root dry mass was observed following mixed inoculation of the seedlings in the germination phase, with a significant increase in root initiation, volume, and area, especially of the fine roots. Inoculation of seedlings of both cultivars led to increased biomass in tubes of non-sterile substrate. In soil, a higher nitrogen accumulation was also observed in the cvar. IACSP95-5000 in all treatments except plants inoculated with N. amazonense. For the cvar. RB867515 shoots, the opposite was observed were the highest N accumulation was observed for N. amazonense, H. seropedicae followed by the mixture. In this experiment using soil labelled with 15N there was a response on N uptake caused by all bacteria used in one cvar but only by 2 of the 5 strains in other cvar. Using the 15N enrichment of the plants the results showed that the control and inoculated plants all derived 60–70% of N from the atmosphere, and inoculation had no effect on BNF contribution. For P and K levels, RB867515 showed no effect of inoculation, but the other cvar. did respond to inoculation.