Estimation of Biological Nitrogen Fixation Capacity by Sugarcane Using 15N

[ Objective] The study aimed to reveal the biological nitrogen fixation capacity by sugarcane from Brazil under the ecological conditions of Guangxi, and to provide reference for study on the biological nitrogen fixation capacity by sugarcane and related generalization and application. [ Method] The ^15N isotopic fertilizer was solely applied on plants of three sugarcane cultivars planted in greenhouse with no other fertilizer forms applied, meanwhile virus-free stem seedling was regarded as control, to measure their biological nitrogen fixation capacity using ^15N isotope. [ Result ] The nitrogen fixation rate of B8 from Brazil reached 26.91%, while Guitang 11 and RIC16 presented no or poor nitrogen fixation capacity. [ Conclusion] The sugarcane eultivar B8 from Brazil showed some nitrogen fixation capacity under the ecological conditions of Guangxi.

“Biological Nitrogen Fixation” Book Summary

Biological nitrogen fixation is a very valuable alternative to nitrogen fertilizer. This process will be discussed in the “Biological Nitrogen Fixation” book. A wide array of free-living and associative nitrogen fixing organisms (diazotrophs) will be covered. The most extensively studied and applied example of biological nitrogen fixation is the symbiotic interaction between nitrogen fixing “rhizobia” and legume plants. While legumes are important as major food and feed crops, cereals such as wheat, maize and rice are the primary food crops, but do not have this symbiotic nitrogen fixing interaction with rhizobia. It has thus been a “holy grail” to transfer the ability to fix nitrogen to the cereals and this topic will be also addressed in these books.

Biological nitrogen fixation in cereal crops:Progress,strategies,and perspectives

Nitrogen is abundant in the atmosphere but is generally the most limiting nutrient for plants.The inability of many crop plants,such as cereals,to directly utilize freely available atmospheric nitrogen gas means that their growth and production often rely heavily on the application of chemical fertilizers,which leads to greenhouse gas emissions and the eutrophication of water.By contrast,legumes gain access to nitrogen through symbiotic association with rhizobia.These bacteria convert nitrogen gas into biologically available ammonia in nodules through a process termed symbiotic biological nitrogen fixation,which plays a decisive role in ecosystem functioning.Engineering cereal crops that can fix nitrogen like legumes or associate with nitrogen-fixing microbiomes could help to avoid the problems caused by the overuse of synthetic nitrogen fertilizer.With the development of synthetic biology,various efforts have been undertaken with the aim of creating so-called‘‘N-self-fertilizing’’crops capable of performing autonomous nitrogen fixation to avoid the need for chemical fertilizers.In this review,we briefly summarize the history and current status of engineering N-self-fertilizing crops.We also propose several potential biotechnological approaches for incorporating biological nitrogen fixation capacity into non-legume plants.

What do we know about biological nitrogen fixation in insects? Evidence and implications for the insect and the ecosystem

Many insects feed on a low‐nitrogen diet,and the origin of their nitrogen supply is poorly understood.It has been hypothesized that some insects rely on nitrogen‐fixing bacteria(diazotrophs)to supplement their diets.Nitrogen fixation by diazotrophs has been extensively studied and convincingly demonstrated in termites,while evidence for the occurrence and role of nitrogen fixation in the diet of other insects is less conclusive.Here,we summarize the methods to detect nitrogen fixation in insects and review the available evidence for its occurrence(focusing on insects other than termites).We distinguish between three aspects of nitrogen fixation investigations:(i)detecting the presence of potential diazotrophs;(ii)detecting the activity of the nitrogen‐fixing enzyme;and(iii)detecting the assimilation of fixed nitrogen into the insect tissues.We show that although evidence from investigations of the first aspect reveals ample opportunities for interactions with potential diazotrophs in a variety of insects,demonstrations of actual biological nitrogen fixation and the assimilation of fixed nitrogen are restricted to very few insect groups,including wood‐feeding beetles,fruit flies,leafcutter ants,and a wood wasp.We then discuss potential implications for the insect's fitness and for the ecosystem as a whole.We suggest that combining these multiple approaches is crucial for the study of nitrogen fixation in insects,and argue that further demonstrations are desperately needed in order to determine the relative importance of diazotrophs for insect diet and fitness,as well as to evaluate their overall impact on the ecosystem.

Plant growth and metabolism of exotic and native Crotalaria species for mine land rehabilitation in the Amazon

Despite its enormous benefits,mining is respon-sible for intense changes to vegetation and soil properties.Thus,after extraction,it is necessary to rehabilitate the mined areas,creating better conditions for the establishment of plant species which is challenging.This study evaluated mineral and organic fertilization on the growth,and carbon and nitrogen(N)metabolism of two Crotalaria species[Cro-talaria spectabilis(exotic species)and Crotalaria maypu-rensis(native species from Carajás Mineral Province(CMP)]established on a waste pile from an iron mine in CMP.A control(without fertilizer application)and six fertilization mixtures were tested(i=NPK;ii=NPK+micronutrients;iii=NPK+micronutrients+organic compost;iv=PK;v=PK+micronutrients;vi=PK+micronutrients+organic compost).Fertilization contributed to increased growth of both species,and treatments with NPK and micronutrients had the best results(up to 257%cf.controls),while organic fertilization did not show differences.Exotic Crotalaria had a greater number of nodules,higher nodule dry mass,chlorophyll a and b contents and showed free ammonium as the predominant N form,reflecting greater increments in biomass compared to native species.Although having lower growth,the use of this native species in the rehabilitation of mining areas should be considered,mainly because it has good development and meets current government legislation as an opportunity to restore local biodiversity.

Relay-intercropping soybean with maize maintains soil fertility and increases nitrogen recovery efficiency by reducing nitrogen input

Optimized nitrogen(N)management can increase N-use efficiency in intercropping systems.Legume-nonlegume intercropping systems can reduce N input by exploiting biological N fixation by legumes.Measurement of N utilization can help in dissecting the mechanisms underlying N uptake and utilization in legume-nonlegume intercropping systems.An experiment was performed with three planting patterns:monoculture maize(MM),monoculture soybean(SS),and maize-soybean relay intercropping(IMS),and three N application levels:zero N(NN),reduced N(RN),and conventional N(CN)to investigate crop N uptake and utilization characteristics.N recovery efficiency and 15N recovery rate of crops were higher under RN than under CN,and those under RN were higher under intercropping than under the corresponding monocultures.Compared with MM,IMS showed a lower soil N-dependent rate(SNDR)in 2012.However,the SNDR of MM rapidly declined from 86.8%in 2012 to 49.4%in 2014,whereas that of IMS declined slowly from 75.4%in 2012 to 69.4%in 2014.The interspecific N competition rate(NCRms)was higher under RN than under CN,and increased yearly.Soybean nodule dry weight and nitrogenase activities were respectively 34.2%and 12.5%higher under intercropping than in monoculture at the beginning seed stage.The amount(Ndfa)and ratio(%Ndfa)of soybean N2 fixation were significantly greater under IS than under SS.In conclusion,N fertilizer was more efficiently used under RN than under CN;in particular,the relay intercropping system promoted N fertilizer utilization in comparison with the corresponding monocultures.An intercropping system helps to maintain soil fertility because interspecific N competition promotes biological N fixation by soybean by reducing N input.Thus,a maize-soybean relay intercropping system with reduced N application is sustainable and environmentally friendly.

Transfer characteristics of nitrogen fixed by leguminous green manure crops when intercropped with maize in northwestern China

To ascertain the possibility of cultivating maize using biological nitrogen fixation(BNF)by leguminous green manure crops in maize/leguminous green manure intercropping systems,BNF and nitrogen(N)transfer were studied in Xining and Wuwei,two typical northwestern Chinese cities.The experimental treatments included monocultured maize,monocultured green manures(hairy vetch and common vetch),and their intercropping systems.The proportions of N derived from the atmosphere(%N_(dfa))in intercropping systems were not significantly different from that in monocultured green manure systems at either experimental site,except for that in hairy vetch(HV)in Xining.The amount of N derived from the atmosphere(N_(dfa))of common vetch(CV)significantly decreased from 1.16 and 1.10 g/pot in monoculture to 0.77 and 0.55 g/pot when intercropped with maize,in Xining and Wuwei,respectively,and the N_(dfa) of HV when intercropped significantly decreased from 1.02 to 0.48 g/pot in Xining.In the intercropping systems in Xining and Wuwei,the amounts of N transferred(N_(transfer))from CV to maize were 21.54 and 26.81 mg/pot,accounting for 32.9 and 5.9%respectively of the N accumulation in maize,and the values of N_(transfer) from HV to maize were 39.61 and 46.22 mg/pot,accounting for 37.0 and 23.3%,respectively,of the N accumulation in maize.Path analysis showed that soil nutrient and green manure biomass were mainly related to N_(dfa),and thatδ^(15) N had a primary relationship with N_(transfer).We found that 5.9-37.0%of N accumulation in maize was transferred from green manures,and that the N transfer ability to maize of HV was higher than that of CV.In conclusion,intercropping with leguminous green manures provided a feasible way for maize to effectively utilize biologicallyfixed N.

The role of green manure nitrogen use by corn and sugarcane crops in Brazil

Three assays were developed from April 3, 1995 to October 10, 2005. The work with corn was conducted in a greenhouse, using velvet bean (Mucuna aterrima) and sunn hemp (Crotalaria juncea) as green manure with 15N labeling of either shoots or roots, in two soils with contrasting textural classes. The mineralization of N from legume plants incorporated into the two soils was investigated too. This work included two green manures: velvet bean and sunn hemp, and the common bean (Phaseolus vulgaris) residues. Nitrogen from the velvet bean accounted for a greater proportion of the soil inorganic N;shoots were responsible for most of N accumulated. Common bean residues caused immobilization of inorganic N. The leguminous species added were intensively and promptly mineralized, preserving the soil native nitrogen. One hundred days after emergence of the corn, velvet bean provided higher accumulation of nitrogen in the soil, higher absorption by corn plants and accumulation in the aerial part. The green manure decomposition was more intense in the medium textured soil. In this soil, highest nitrogen losses were also observed. The sugarcane (Saccharum spp.) was cultivated for five years in the field and was harvested three times;15N recovery was evaluated in the first two harvests. The combination of inorganic fertilizer and green manure resulted in higher sugarcane yields than either N source applied separately;however, in the second cutting the yields were higher where sunn hemp was used than in plots with ammonium sulfate. The recovery of N by the first two consecutive harvests accounted for 19% to 21% of the N applied as sunn hemp and 46% to 49% of the N applied as ammonium sulfate. Very little inorganic N was present in the 0-40 cm soil layer with both N sources.

Co-Inoculation of Soybean with Bradyrhizobium and Azospirillum Promotes Early Nodulation

Soybean inoculation with elite strains of Bradyrhizobium to improve nodulation, N2 fixation, and grain yield is well established worldwide. However, when grown in soils where N is deficient, soybean undergoes an initial phase of N starvation that may last up to 20 days after seedling germination due to the lack of synchronism between the phase when seed N reserves are exhausted and the moment when plants begin to benefit from the nitrogen fixed by the bacteria. Practices that promote early nodulation may play a key role in reducing the N starvation period. Azospirillum is a plant growth promoting rhizobacteria (PGPR) that can stimulate root hair formation and root growth, creating more sites for early root infection and nodule formation by N2-fixing Bradyrhizobium spp. In this study, the effects of co-inoculating soybeans with Bradyrhizobium spp. and Azospirillum brasilense on nodulation precocity and N2 fixation were evaluated under greenhouse and field conditions. Nodule number and dry weight, as well as plant and root dry weight and N accumulated in shoots at 15, 18, 21, 24 and 30 days after emergence (DAE) were evaluated in response to inoculation with Bradyrhizobium spp. alone or when co-inoculated with Azospirillum sp. In the greenhouse, co-inoculated plants nodulated precociously as indicated by a significant increase (p Azospirillum helps plants to overcome environmental stresses.

Efficiency of Inoculation with Rhizobium in Peanuts(Arachis hypogaea L.)Grown in Brazilian Cerrado Soil

In Brazil, the peanut culture has been expanding mainly in the state of São Paulo, responsible for 80% of the national production. It is an edible culture produces oil of excellent quality, contains proteins rich in amino acids that make up to 36% of weight of the grain. Furthermore, it is associated with nitrogen-fixing bacteria. However, the practice of inoculation is not very common in peanuts, being this species considered susceptible to nodularity with a large number of rhizobia. However, known’s that inoculation with selected strains can increase the efficiency of symbiosis and peanut yield. Thus, the present study was proposed to evaluate the development of peanut inoculated with rhizobia and grown in Cerrado’s Oxisol. The experiment was conducted in a greenhouse, in a completely randomized design, five treatments, one cultivar (IAC 505) and four replicates. The treatments were composed of three strains of rhizobia and two controls, one absolute and the other with nitrogen fertilizer (50 mg⋅N⋅dm3). Each experimental plot has been consisted an 8 dm3 pot with three plants. The inoculation was done, applying 3 mL of bacterial broth (108 mL−1 cells) in the root system of each plant. The SPAD index, number and dry weight of nodules, dry mass of shoots and roots, number and weight of grains were analyzed. The response of Peanuts inoculated was significantly, as all inoculated plants showed similar development to nitrogen-fertilized, emphasizing the potential for supplying nitrogen in peanut development.

Trends in carbon sink along the Belt and Road in the future under high emission scenario

Over the past three decades,the drawdown of atmospheric CO_(2) in vegetation and soil has fueled net ecosystem production(NEP).Here,a global land-surface model(CABLE)is used to estimate the trend in NEP and its response to atmospheric CO_(2),climate change,biological nitrogen(N)fixation,and N deposition under future conditions from 2031 to 2100 in the Belt and Road region.The trend of NEP simulated by CABLE decreases from 0.015 Pg carbon(C)yr^(-2) under present conditions(1936–2005)to−0.023 Pg C yr^(-2) under future conditions.In contrast,the trend in NEP of the CMIP6 ensemble changes from 0.014 Pg C yr^(-2) under present conditions to−0.009 Pg C yr^(-2) under future conditions.This suggests that the trend in the C sink for the Belt and Road region will likely decline in the future.The significant difference in the NEP trend between present and future conditions is mainly caused by the difference in the impact of climate change on NEP.Considering the responses of soil respiration(RH)or net primary production(NPP)to surface air temperature,the trend in surface air temperature changes from 0.01℃ yr^(-1) under present conditions to 0.05℃ yr^(-1) under future conditions.CABLE simulates a greater response of RH to surface temperature than that of NPP under future conditions,which causes a decreasing trend in NEP.In addition,the greater decreasing trend in NEP under future conditions indicates that the C-climate-N interaction at the regional scale should be considered.It is important to estimate the direction and magnitude of C sinks under the C neutrality target.

Biotechnological Potential of Endophytic Bacteria to Improve the Micropropagated Seedling of Variety RB92579 Sugarcane(Saccharum officinarum L.)

Endophytic bacteria may influence agricultural production in several ways, including promoting plant growth. Two experiments were conducted in order to evaluate the combination of endophytic bacteria from the Brazilian Northeast region aims at the commercial introduction of the inoculation of these bacteria in micropropagated sugarcane plants using a temporary immersion bioreactor. One experiment was done in tubes with sterile commercial substrate, and the other was done in pots with soil;both were installed in a greenhouse. A mixed inoculation was performed in six inoculated endophytic diazotrophic bacteria in micropropagated sugarcane plants, variety RB92579. In the experiment with soil, the mixed inoculation significantly increased the shoot dry matter of plants without the addition of nitrogen fertilizer. However, the accumulation of total-N in the tissues showed no significant differences between treatments with and without nitrogen fertilization. The evaluation of micropropagated seedlings showed no increases in the parameters tested. The results showed that the response of inoculation in temporary immersion bioreactor micropropagation is possible, and that the application of homologous strains may have contributed to a better response by the interaction of endophytic bacteria with sugarcane RB92579. Further studies should be conducted to improve the methodology, which indicates a great potential to optimize this process on a commercial scale.

Isolation and Molecular Characterization of Elite Indigenous Rhizobia Nodulating Phaseolus bean (Phaseolus vulgaris L.)

Nowadays application of biological nitrogen fixation (BNF) through rhizobia inoculums is highly promoted as a solution to solve the problem of poor soil fertility in areas where legumes are cultivated. This is due to the fact that, rhizobia enhance nitrogen fixation, induce disease resistance, reduce heavy metal in the soil, facilitate bioavailabity of iron in soil and is environmental friendly. To get rhizobia strains which are suitable for inoculants production, isolation and molecular characterization of elite rhizobia are highly needed. Molecular characterization acts as a spark plug for discovery of many microbes including Rhizobia. Multi Locus Sequence Analysis (MLSA), 16S rRNA gene sequence analysis, DNA-DNA hybridization and SDS-PAGE analysis of the whole-cell proteins are the molecular techniques mostly used in characterizing rhizobia. But before deciding to use or not to use rhizobia inoculants in certain areas, knowing the population size of indigenous rhizobia found in that area is very important, because this is a major factor which determines inoculums responses as well gives clues on which areas need or do not need inoculation. The Most Probable Number (MPN) method is mostly used in enumerating rhizobia population of the soil. Given that, in most of the developing countries, including Tanzania, Biological Nitrogen Fixation (BNF) technology is not fully flourished;more efforts in isolation, molecular characterization of elite rhizobia and estimation of indigenous rhizobia population in various areas are required.

Changes in soil free-living diazotrophic community and co-occurrence patterns along desert wetland degradation gradient in the Mu Us Desert,northern China

Climate change and human activity have led to the degradation of desert wetlands.Free-living diazotrophs are vital for soil nitrogen input.However,a comprehensive understanding of how soil free-living diazotrophic communities and their co-occurrence patterns respond to desert wetland degradation is lacking.Here,quantitative polymerase chain reaction(qPCR),amplicon sequencing targeting nitrogenase gene(nifH),and network analysis were used to investigate the abundance,diversity,community composition,and co-occurrence patterns of soil free-living diazotrophs along the wetland degradation gradient,i.e.,non-degraded(ND),lightly degraded(LD),moderately degraded(MD),and severely degraded(SD),in the southeastern Mu Us Desert,northern China.The abundance and Shannon,Simpson,Chao 1,and ACE indexes decreased(P<0.05)by 14.6%,20.7%,2.1%,46.5%,and 45.0%,respectively,in SD wetland,whereas no significant difference(P>0.05)was observed between ND and LD wetlands.The relative abundance of Proteobacteria generally decreased(by 53.5%–19.7%)across the different degradation levels,while the relative abundance of Cyanobacteria increased(by 6.2%–40.1%)from ND to MD levels.The abundance,diversity,and community composition of diazotrophs were most strongly related to soil organic carbon,followed by total nitrogen,moisture,and pH.The least number of network nodes and edges and the lowest density were observed for MD and SD wetlands,indicating that the complexity of free-living diazotrophic networks was reduced by continued degeneration.Overall,severe desert wetland degradation affected the abundance,diversity,and network complexity of soil free-living diazotrophs more negatively than light degradation.This degradation promoted the growth of autotrophic diazotrophs and inhibited the growth of heterotrophic diazotrophs.These changes were mostly related to the loss of soil organic carbon.

Synergistic Effects of Biochar and NPK Fertilizer on Soybean Yield in an Alkaline Soil

Biochar effects on legume growth and biological nitrogen fixation have been studied extensively, mostly in acidic soils with laboratory produced biochar. In the present study, a pot experiment in a full factorial experimental design was performed to examine soybean yield and nodulation of three genotypes grown with or without biochar and NPK fertilizers in an alkaline soil. We observed synergistic effects of biochar and NPK fertilizer applications on biomass and seed yields for all three soybean genotypes. Total biomass production and seed yield increased on average by 67% and 54%, respectively, with biochar and by 201% and 182% with NPK fertilizer application compared to the control. When applications of biochar and NPK fertilizer were combined, the increases were 391% and367%, respectively. However, the biomass production in the control was very low(692 kg ha-1) due to a high soil p H(8.80). The nodulation increased with biochar and NPK fertilizer applications, and was largest with the combined application. A correlation was found between leaf chlorophyll content(single photon avalanche diode value) and nodule number. We suggested that the synergistic increase in yield was due to a decrease in soil p H caused by biochar and NPK fertilizer applications thereby increasing P availability in this alkaline 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(δ^(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 δ^(15)N 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.