Microbiological strategies for enhancing biological nitrogen fixation in nonlegumes

In an agro-ecosystem, industrially produced nitrogenous fertilizers are the principal sources of nitrogen for plant growth; unfortunately these also serve as the leading sources of pollution. Hence, it becomes imperative to find pollution-free methods of providing nitrogen to crop plants. A diverse group of free-living, plant associative and symbiotic prokaryotes are able to perform biological nitrogen fixation (BNF). BNF is a two component process involving the nitrogen fixing diazotrophs and the host plant. Symbiotic nitrogen fixation is most efficient as it can fix nitrogen inside the nodule formed on the roots of the plant; delivering nitrogen directly to the host. However, most of the important crop plants are nonleguminous and are unable to form symbiotic associations. In this context, the plant associative and endophytic diazotrophs assume importance. BNF in nonlegumes can be encouraged either through the transfer of BNF traits from legumes or by elevating the nitrogen fixing capacity of the associative and endophytic diazotrophs. In this review we discuss mainly the microbiological strategies which may be used in nonleguminous crops for enhancement of BNF.     

Trends in biological nitrogen fixation research and application

In the world each year 17.2×10⁷ tons of N are biologically fixed. Biological nitrogen fixation (BNF) contributes to plant production in arable lands and in natural ecosystems. Research to improve BNF is progressing through the breeding of efficient N-fixing organisms and host plants, selection of the best combinations of host plant and microsymbiont, and by the improvement of inoculation techniques and field management. Biotechnology is useful for the creation of promising N₂-fixing organisms. However, to increase plant production through enhanced BNF the constraints in establishing effective N₂-fixing systems in the field should be understood and eliminated.     

Biological nitrogen fixation in mixed legume/grass pastures

Biological nitrogen fixation (BNF) in mixed legume/grass pastures is reviewed along with the importance of transfer of fixed nitrogen (N) to associated grasses. Estimates of BNF depend on the method of measurement and some of the advantages and limitations of the main methods are outlined. The amounts of N fixed from atmospheric N₂ in legume/grass pastures throughout the world is summarised and range from 13 to 682 kg N ha^-1 yr^-1. the corresponding range for grazed pastures, which have been assessed for white clover pastures only, is 55 to 296 kg N ha^-1 yr^-1.Biological nitrogen fixation by legumes in mixed pastures is influenced by three primary factors; legume persistence and production, soil N status, and competition with the associated grass(es). These factors and the interactions between them are discussed. Legume persistence, production and BNF is also influenced by many factors and this review centres on the important effects of soil moisture status, soil acidity, nutrition, and pests and disease.Soil N status interacts directly with BNF in the short and long term. In the short-term, increases in soil inorganic N occurs during dry conditions and where N fertiliser is used, and these will reduce BNF. In the long-term, BNF leads to accumulation of soil N, grass dominance, and reduced BNF. However, cyclical patterns of legume and grass dominance can occur due, at least in part, to temporal changes in plant-available N levels in soil. Thus, there is a dynamic relationship between legumes and grasses whereby uptake of soil N by grass reduces the inhibitory effect of soil N on BNF and competition by grasses reduces legume production and BNF. Factors affecting the competition between legumes and grasses are considered including grass species, grazing animals, and grazing or cutting management.Some fixed N is transferred from legumes to associated grasses. The amount of N transferred below-ground, predominantly through decomposition of legume roots and nodules, has been estimated at 3 to 102 kg N ha^-1 yr^-1 or 2 to 26% of BNF. In grazed pasture, N is also transferred above-ground via return in animal excreta and this can be of a similar magnitude to below-ground transfer.Increased BNF in mixed legume/grass pastures is being obtained through selection or breeding of legumes for increased productivity and/or to minimise effects of nutrient limitations, low soil moisture, soil acidity, and pests and disease. Ultimately, this will reduce the need to modify the pasture environment and increase the role of legumes in low-input, sustainable agriculture.     

矿质养分输入对森林生物固氮的影响

生物固氮是森林生态系统重要的氮素来源,并且在全球氮循环中占有重要的地位。近代以来,因人类活动加剧而导致氮沉降的增加以及其它矿质养分元素(如磷、钼、铁等)输入的改变已成为影响森林生态系统生物固氮的重要因素之一,并引起了学术界的普遍关注。综述了国内外关于森林生物固氮对矿质养分输入的响应及机理。主要内容包括:(1)森林生物固氮的概念及主要的测定方法;(2)矿质养分输入对森林生物固氮的影响。整体上讲,氮素输入抑制了森林生物固氮,磷和其他营养元素输入则表现为促进作用。氮和磷、磷和微量元素同时添加均提高了森林的固氮量;(3)矿质养分改变森林生物固氮的机理。包括生物作用机制(如改变地表层固氮菌的数量或群落丰度、改变结瘤植物的根瘤生物量和附生植物的丰度或盖度)和环境作用机制(如引起土壤酸化、改变碳源物质的含量);(4)探讨了矿质养分输入对森林生物固氮影响研究中所存在的问题,并对未来该领域的研究提出建议。     

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N₂-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions. In this paper we review some recent progress in this field, particularly at our institute in Rio de Janeiro, concerning specificity of selected Azospirillum strains in the infection of cereal roots and the promotion of responses in the host plants. The possible mechanisms of plant response are discussed including the possibility that plant growth substances or bacterial nitrate reductase are involved. The application of¹⁵N and N balance techniques to the quantification of plant associated BNF are considered and the possible strategies that may be adopted to further the understanding of true N₂-fixing plant/diazotroph associations. The recent discovery of many more plant-associated N₂-fixing bacteria suggests that further research in this area may eventually lead to the development of such associations with applications for agricultural productivity.     

Biological nitrogen fixation for sustainable agriculture: A perspective

The economic and environmental costs of the heavy use of chemical N fertilizers in agriculture are a global concern. Sustainability considerations mandate that alternatives to N fertilizers must be urgently sought. Biological nitrogen fixation (BNF), a microbiological process which converts atmospheric nitrogen into a plant-usable form, offers this alternative. Nitrogen-fixing systems offer an economically attractive and ecologically sound means of reducing external inputs and improving internal resources. Symbiotic systems such as that of legumes and Rhizobium can be a major source of N in most cropping systems and that of Azolla and Anabaena can be of particular value to flooded rice crop. Nitrogen fixation by associative and free-living microorganisms can also be important. However, scientific and socio-cultural constraints limit the utilization of BNF systems in agriculture. While several environmental factors that affect BNF have been studied, uncertainties still remain on how organisms respond to a given situation. In the case of legumes, ecological models that predict the likelihood and the magnitude of response to rhizobial inoculation are now becoming available. Molecular biology has made it possible to introduce choice attributes into nitrogen-fixing organisms but limited knowledge on how they interact with the environment makes it difficult to tailor organisms to order. The difficulty in detecting introduced organisms in the field is still a major obstacle to assessing the success or failure of inoculation. Production-level problems and socio-cultural factors also limit the integration of BNF systems into actual farming situations. Maximum benefit can be realized only through analysis and resolution of major constraints to BNF performance in the field and adoption and use of the technology by farmers.     

Does legume nitrogen fixation underpin host quality for the hemiparasitic plant Rhinanthus minor?

The high quality of leguminous hosts for the parasitic plantRhinanthus minor (in terms of growth and fecundity), comparedwith forbs (non-leguminous dicots) has long been assumed tobe a function of the legume's ability to fix atmospheric nitrogen(N) from the air and the potential for direct transfer of compatibleamino compounds to the parasite. Using associations betweenRhinanthus minor and Vicia faba (Fabaceae) that receive N eitherexclusively via symbiotic associations with rhizobia supplyingorganic N fixed from N₂ or exclusively through the supply ofinorganic nitrate to the substrate, the underlying reasons forthe quality of legumes as hosts for this parasite are unravelled.It is shown that sole dependence of the host, V. faba, on Nfixation results in lower growth of the attached parasite thanwhen the host is grown in a substrate supplied exclusively withinorganic N. In contrast, the host plants themselves achieveda similar biomass irrespective of their N source. The physiologicalbasis for this is investigated in terms of N and abscisic acid(ABA) partitioning, haustorial penetration, and xylem sap aminoacid profiles. It is concluded that legume N fixation does notunderpin the quality of legumes as hosts for Rhinanthus butrather the well-developed haustorium formed by the parasite,coupled with the lack of defensive response of the host tissuesto the invading haustorium and the presence of sufficient nitrogenouscompounds in the xylem sap accessible to the parasite haustoria,would appear to be the primary factors influencing host qualityof the legumes. Key words: ABA, haustorium, legume, nitrogen fixation, nodules, parasitic plant Received 14 November 2007; Revised 7 January 2008 Accepted 8 January 2008     

Determination of nitrogen fixation effectiveness in selected <Emphasis Type="Italic">Medicago truncatula</Emphasis> isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on ¹⁵N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This ¹⁵N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.     

Sustainable agriculture in the semi-arid tropics through biological nitrogen fixation in grain legumes

Sustainable agriculture relies greatly on renewable resources like biologically fixed nitrogen. Biological nitrogen fixation plays an important role in maintaining soil fertility. However, as BNF is dependent upon physical, environmental, nutritional and biological factors, mere inclusion of any N₂-fixing plant system does not guarantee increased contributions to the soil N pool. In the SAT where plant stover is also removed to feed animals, most legumes might be expected to deplete soil N. Yet beneficial legume effects in terms of increased yields in succeeding cereal crops have been reported. Such benefits are partly due to N contribution from legumes through BNF and soil N saving effect. In addition, other non-N rotational benefits, for example, improved nutrient availability, improved soil structure, reduced pests and diseases, hormonal effects are also responsible. In this paper we have reviewed the research on the contribution of grain legumes in cropping systems and the factors affecting BNF. Based on the information available, we have suggested ways for exploiting BNF for developing sustainable agriculture in the semi-arid tropics (SAT). A holistic approach involving host-plant, bacteria, environment and proper management practices including need based inoculation for enhancing BNF in the cropping systems in the SAT is suggested.     

Fertilizers and biological nitrogen fixation as sources of plant nutrients: Perspectives for future agriculture

Biological nitrogen fixation (BNF) has an assured place in agriculture, mainly as a source of nitrogen for legumes. Legumes are currently grown mostly as a source of vegetable oil and as food for humans and animals, but not as nitrogen source.Other crops with BNF capability may be eventually be developed eventually. Such crops will also need mineral fertilizers to maintain a good status of soil nutrients, but their possible effects to the environment is also a concern. Fertilizers, however, will remain a necessary and sustainable input to agriculture to feed the present and increasing human population. It is not a case of whether BNF is better or worse than mineral fertilizers because both plays an important role in agriculture.     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

The reallocation of carbon in P deficient lupins affects biological nitrogen fixation

It is not known how phosphate (P) deficiency affects the allocation of carbon (C) to biological nitrogen fixation (BNF) in legumes. The alteration of the respiratory and photosynthetic C costs of BNF was investigated under P deficiency. Although BNF can impose considerable sink stimulation on host respiratory and photosynthetic C, it is not known how the change in the C and energy allocation during P deficiency may affect BNF. Nodulated Lupinus luteus plants were grown in sand culture, using a modified Long Ashton nutrient solution containing no nitrogen (N) for ca. four weeks, after which one set was exposed to a P-deficient nutrient medium, while the other set continued growing on a P-sufficient nutrient medium. Phosphorus stress was measured at 20 days after onset of P-starvation. During P stress the decline in nodular P levels was associated with lower BNF and nodule growth. There was also a shift in the balance of photosynthetic and respiratory C toward a loss of C during P stress. Below-ground respiration declined under limiting P conditions. However, during this decline there was also a shift in the proportion of respiratory energy from maintenance toward growth respiration. Under P stress, there was an increased allocation of C toward root growth, thereby decreasing the amount of C available for maintenance respiration. It is therefore possible that the decline in BNF under P deficiency may be due to this change in resource allocation away from respiration associated with direct nutrient uptake, but rather toward a long term nutrient acquisition strategy of increased root growth.     

The success of BNF in soybean in Brazil

Approximately forty years after commercial cropping of soybean in Brazil began, the total area under this crop has reached over 13 M ha with a mean productivity of 2400 kg ha^–1. Soybean varieties introduced from the USA and varieties rescued from early introductions in Brazilian territory were part of the Brazilian soybean-breeding programme which spread the crop from high to low latitudes. Disease-resistance, pest-resistance, tolerance to low fertility soils, as well as production of plants with pods sufficiently high above the ground for efficient mechanical harvesting, were all aims of the programme. Although BNF was not explicitly considered as a trait for selection in the breeding/selection programme, maximisation of biological nitrogen fixation (BNF) was favoured by conducting selection and breeding trials on soils low in N, in which the seeds were inoculated with efficient Bradyrhizobium inoculants but without N fertiliser application. Several efficient imported Bradyrhizobium strains were found to be unable to compete with native soil micro-flora and other previously-introduced Bradyrhizobium strains. Surprisingly, after being in the soil for many years one or two of these strains had become more competitive while maintaining their high BNF capacity. Today, these strains are included amongst the recommended Brazilian inoculants and have promoted significant improvements in grain yields. The breeding of soybeans in conditions that made grain yield highly dependent on BNF, and the continuous attention paid to the selection of Bradyrhizobium strains appropriate for the newly released varieties, have been the main contributors to today's high yields and their great benefit to the Brazilian economy. There seems to be no reason why this ongoing research programme should not serve as an appropriate model to improve BNF inputs to grain legumes in other countries of the world.     

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

Summary Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N₂ into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.     

Biological nitrogen fixation in mixed legume-cereal cropping systems

Cereal/legume intercropping increases dry matter production and grain yield more than their monocultures. When fertilizer N is limited, biological nitrogen fixation (BNF) is the major source of N in legume-cereal mixed cropping systems. The soil N use patterns of component crops depend on the N source and legume species. Nitrogen transfer from legume to cereal increases the cropping system's yield and efficiency of N use. The use of nitrate-tolerant legumes, whose BNF is thought to be little affected by application of combined N, may increase the quantity of N available for the cereal component. The distance between the cereal and legume root systems is important because N is transferred through the intermingling of root systems. Consequently, the most effective planting distance varies with type of legume and cereal. Mutual shading by component crops, especially the taller cereals, reduces BNF and yield of the associated legume. Light interception by the legume can be improved by selecting a suitable plant type and architecture. Planting pattern and population at which maximum yield is achieved also vary among component species and environments. Crops can be mixed in different proportions from additive to replacement or substitution mixtures. At an ideal population ratio a semi-additive mixture may produce higher gross returns.     

Managing native and legume-fixed nitrogen in lowland rice-based cropping systems

Lowlands comprise 87% of the 145 M ha of world rice area. Lowland rice-based cropping systems are characterized by soil flooding during most of the rice growing season. Rainfall distribution, availability of irrigation water and prevailing temperatures determine when rice or other crops are grown. Nitrogen is the most required nutrient in lowland rice-based cropping systems. Reducing fertilizer N use in these cropping systems, while maintaining or enhancing crop output, is desirable from both environmental and economic perspectives. This may be possible by producing N on the land through legume biological nitrogen fixation (BNF), minimizing soil N losses, and by improved recycling of N through plant residues. At the end of a flooded rice crop, organic- and NH₄-N dominate in the soil, with negligible amounts of NO₃. Subsequent drying of the soil favors aerobic N transformations. Organic N mineralizes to NH₄, which is rapidly nitrified into NO₃. As a result, NO₃ accumulates in soil during the aerobic phase. Recent evidence indicates that large amounts of accumulated soil NO₃ may be lost from rice lowlands upon the flooding of aerobic soil for rice production. Plant uptake during the aerobic phase can conserve soil NO₃ from potential loss. Legumes grown during the aerobic phase additionally capture atmospheric N through BNF. The length of the nonflooded season, water availability, soil properties, and prevailing temperatures determine when and where legumes are, or can be, grown. The amount of N derived by legumes through BNF depends on the interaction of microbial, plant, and environmental determinants. Suitable legumes for lowland rice soils are those that can deplete soil NO₃ while deriving large amounts of N through BNF. Reducing soil N supply to the legume by suitable soil and crop management can increase BNF. Much of the N in legume biomass might be removed from the land in an economic crop produce. As biomass is removed, the likelihood of obtaining a positive soil N balance diminishes. Nonetheless, use of legumes rather than non-legumes is likely to contribute higher quantities of N to a subsequent rice crop. A whole-system approach to N management will be necessary to capture and effectively use soil and atmospheric sources of N in the lowland rice ecosystem.IRRI-NifTAL-IFDC joint contribution.     

一种高效研究大豆根瘤共生固氮的营养液栽培体系

为建立一种既可高效结瘤固氮, 又具有一定产量的大豆(Glycine max)营养液栽培系统, 设计并进行了2个试验。首先在不同供氮条件下, 研究了接种根瘤菌对大豆的结瘤状况、固氮能力、生物量及产量的影响。结果表明, 供氮过高或过低, 均影响大豆生长、产量形成及根瘤固氮; 并且植物生长所需的最适供氮水平远高于生物固氮所需的最适供氮水平。此外, 大豆生物固氮活性最高的时期在生殖期第一期(R1期)之前。由此推断, 大豆R1期前, 供应较低的氮, 有利于根瘤形成及固氮; 而从R1期起, 应提高供氮水平, 以促进植物生长及产量的形成。在此基础上开展第2个试验, 对供氮条件进行了优化处理(即R1期前低氮供应、R1期开始中氮供应)。结果表明, 与持续供应高氮相比, 优化供氮处理不仅可获得较多固氮酶活性较高的大根瘤, 还能保持较好的生长、获得更高的百粒重及维持80%左右的产量。研究结果不仅可为高效研究大豆根瘤共生固氮提供营养液配方, 还可为大豆高产高效栽培提供试验依据。     

几种生态因素对西北干旱地区豆科植物结瘤固氮的影响

通过对西北干旱地区栽培和野生豆科植物不同环境条件固氮状况的调查表明,栽培豆科植物一般能自然结瘤,野生豆科植物种的结瘤率极低。根瘤颜色栽培植物多为粉红色,而野生植物多为白色、黄色或棕色。通过对水分、光照强度和温度等不同条件下根瘤ARA测定,表明根瘤固氮活性与生态条件关系密切,而土壤水分是限制根瘤固氮活性表达的主要因素。     

新疆干旱区豆科植物结瘤的固氮特性

调查了新疆干旱区72种豆科植物的结瘤固氮活性,其中33种尚未见报道。这些植物所结根瘤在外形上多数不规则,以皮层厚和白色、棕色者居多,与非干旱区的根瘤形态显著不同。根瘤固氮活力相差较大,但比一般豆科植物根瘤活性高,最高者可达当地大豆根瘤的42倍。根瘤活性与宿主的抗逆境能力有关。此外,从11种豆科植物根瘤观察到10种具有吸氢活性。对干旱区豆科共生固氮生理生态的特性进行了讨论。     

Biological nitrogen fixation in trees in agro-ecosystems

The integration of trees, especially nitrogen fixing trees (NFTs), into agroforestry and silvo-pastoral systems can make a major contribution to sustainable agriculture by restoring and maintaining soil fertility, and in combating erosion and desertification as well as providing fuelwood. The particular advantage of NFTs is their biological nitrogen fixation (BNF), their ability to establish in nitrogen-deficient soils and the benefits of the nitrogen fixed (and extra organic matter) to succeeding or associated crops.The importance of NFTs leads to the question of how we can maximise or optimize their effects and how we can manage BNF and the transfer of nitrogen to associated or succeeding plantings. To be able to achieve these goals, suitable methods of measuring BNF in trees are necessary. The total nitrogen difference (TND) method is simple, but is better suited for low than high soil N conditions. The acetylene reduction assay (ARA), although sensitive and simple, has many technical limitations especially for NFTs, and the estimates of BNF have generally been very low, compared to other methods. For NFTs, the ¹⁵N techniques are still under development, but have already given some promising results (e.g., has been used to measure large genetic variability in BNF within different NFTs).Various factors affect BNF in trees. They include the age of trees, the microbial component, soil moisture, temperature, salinity, pH, soil N level and plant nutrient deficiencies. Some of the factors, e.g. temperature, affect the symbiosis more than plant growth, and differences in the effects of these factors on BNF in different NFT genotypes have been reported. These factors and research needs for improving BNF in trees are discussed.