根瘤菌菌剂的研究与开发现状

根瘤菌与豆科植物共生成为豆科植物固氮的重要方式,它可以为豆科植物提供所需氮量的1/2～1/3。因此,土壤中有效根瘤菌的数量是决定豆科植物产量的重要因素,而根瘤菌菌剂的使用可以有效地提高土壤中根瘤菌数量。本文从根瘤菌菌剂制备中高效菌种的选育及匹配、高密度菌剂的制备、菌剂保存方法等方面进行综述。比较了自然选育、杂交选育和诱变选育等各类选育方法及琼脂试管配对法和水培配对法的优缺点;总结了菌剂制备的一般过程和方法;论述了菌剂保藏过程中冷冻干燥法和各种保护剂的使用对菌剂保藏效果的影响。本文阐述了根瘤菌菌剂的制备工艺和发展方向,为根瘤菌剂的研制提供重要参考。     

Global inputs of biological nitrogen fixation in agricultural systems

Biological dinitrogen (N₂) fixation is a natural process of significant importance in world agriculture. The demand for accurate determinations of global inputs of biologically-fixed nitrogen (N) is strong and will continue to be fuelled by the need to understand and effectively manage the global N cycle. In this paper we review and update long-standing and more recent estimates of biological N₂ fixation for the different agricultural systems, including the extensive, uncultivated tropical savannas used for grazing. Our methodology was to combine data on the areas and yields of legumes and cereals from the Food and Agriculture Organization (FAO) database on world agricultural production (FAOSTAT) with published and unpublished data on N₂ fixation. As the FAO lists grain legumes only, and not forage, fodder and green manure legumes, other literature was accessed to obtain approximate estimates in these cases. Below-ground plant N was factored into the estimations. The most important N₂-fixing agents in agricultural systems are the symbiotic associations between crop and forage/fodder legumes and rhizobia. Annual inputs of fixed N are calculated to be 2.95 Tg for the pulses and 18.5 Tg for the oilseed legumes. Soybean (Glycine max) is the dominant crop legume, representing 50% of the global crop legume area and 68% of global production. We calculate soybean to fix 16.4 Tg N annually, representing 77% of the N fixed by the crop legumes. Annual N₂ fixation by soybean in the U.S., Brazil and Argentina is calculated at 5.7, 4.6 and 3.4 Tg, respectively. Accurately estimating global N₂ fixation for the symbioses of the forage and fodder legumes is challenging because statistics on the areas and productivity of these legumes are almost impossible to obtain. The uncertainty increases as we move to the other agricultural-production systems—rice (Oryza sativa), sugar cane (Saccharum spp.), cereal and oilseed (non-legume) crop lands and extensive, grazed savannas. Nonetheless, the estimates of annual N₂ fixation inputs are 12–25 Tg (pasture and fodder legumes), 5 Tg (rice), 0.5 Tg (sugar cane), <4 Tg (non-legume crop lands) and <14 Tg (extensive savannas). Aggregating these individual estimates provides an overall estimate of 50–70 Tg N fixed biologically in agricultural systems. The uncertainty of this range would be reduced with the publication of more accurate statistics on areas and productivity of forage and fodder legumes and the publication of many more estimates of N₂ fixation, particularly in the cereal, oilseed and non-legume crop lands and extensive tropical savannas used for grazing.     

Symbiotic nitrogen fixation in legumes: Perspectives for saline agriculture

Saline agriculture provides a solution for at least two environmental and social problems. It allows us to return to agricultural production areas that have been lost as a consequence of salinization and it can save valuable fresh water by using brackish or salt water to irrigate arable lands. Sea water contains (micro) nutrients that can provide the additional benefit of a reduced need of fertilization in saline agriculture. However, nitrogen is only present in very low quantities in seawater. A salt tolerant nitrogen-fixing legume used as a vegetable crop, fodder or green manure could increase the availability of soil nitrogen as well as the sustainability of saline agriculture while minimizing the application of inorganic fertilizer. Besides the use of salt-tolerant legumes as green manure, such species could also be useful in salinized areas as fodder and/or human food.In this review, we assess the feasibility of the use of legumes in saline agriculture. Most legumes are sensitive to salinity, as is the process of nitrogen fixation by microorganisms in the nodules of the legumes. First, we identify different steps in nodulation and their respective sensitivity to salinity. We will then look at the sensitivity of the process of nitrogen fixation in various crop and non-crop legumes, differing in their tolerance to salinity. We will also look into the differential response of nitrogen fixation and biomass production to salinity. Finally, a list of salt tolerant legumes is presented (derived from the HALOPH database). We then evaluate the applicability and perspective of salt tolerant legumes in saline agriculture considering the diversity in growth forms, ecotypes and economic uses.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Enhancing crop legume N2 fixation through selection and breeding

Legume N₂ fixation is variable, but nonetheless is a valuable process in world agriculture. There is great potential to increase the contribution by the crop legumes to the world's supply of soil.N. This will be achieved by (i) increasing the area of legumes sown by farmers; (ii) improved management of the crops in order that the major determinants of productivity, e.g. land area, water availability, are converted to harvested product with maximum efficiency; and (iii) genetic modification of the commonly-grown species to ensure high dependence of the legume crop on N₂ fixation at all levels of productivity. Currently-used methods for measuring N₂ fixation and for assessing heritability and repeatability of N₂ fixation in breeding and selection programs are reviewed. Results from research programs to define genetic variation in N₂ fixation and to enhance N₂ fixation through selection and breeding are presented with particular emphasis on common bean (Phaseolus vulgaris) and soybean (Glycine max).     

Fine mapping of a major quantitative trait locus that regulates pod shattering in soybean

Legumes represent the second most important family of crop plants, accounting for ~27 % of the world’s crop production. While some legumes are grown as forages or vegetables, most crop legumes are grown for harvesting their nutritious seeds. The legume seeds are contained in the pod, which is composed of a single seed-bearing carpel that, when matures, splits open along two seams, a process called pod dehiscence or pod shattering. Pod shattering before or during harvest causes yield losses of grain legumes. Moreover, the dominant shattering trait of the wild progenitors is a limiting factor for efficient introgression of value-added traits into elite breeding lines. Knowledge of the genetic mechanisms underlying pod shattering will facilitate breeding of shattering-resistant varieties, expedite introgression of agronomically favorable traits from wild species to elite breeding lines, and enrich our understanding of the evolution of seed dispersal and crop domestication in diverse crop species. Here we report fine mapping of a major quantitative trait locus (designated as qPDH1) that regulates pod shattering in soybean (Glycine max). A combination of linkage and association mapping allowed us to delimit the qPDH1 locus within a 47-kb region on chromosome 16. The data reported here will facilitate positional cloning of the underlying gene and the development of breeder-friendly genetic markers for marker-assisted selection in soybean.     

On the efficiency of legume supernodulating mutants

The supernodulating mutants of legumes lack the internal regulation of the number of symbiotic root nodules that harbour N₂-fixing nodule bacteria. On one hand, these mutants represent an efficient tool for dramatic increase in the degree of rhizobial symbiosis development. The trait of released nodulation is often associated with the desirable resistance of nodule initiation and functioning to the inhibition by ambient nitrate. On the other hand, the more intense and stable atmospheric nitrogen fixation of supernodulated plants is devalued by plant growth depression that results from the disproportion between the photosynthetic capacity of the shoot and the catabolic demands of symbiotic nodules. The deleterious effects of excessive nodulation can be neutralised or alleviated by a breeding strategy aimed at creating an ideotype of N₂-fixing legume. The growth depression can be diminished by the reduction in the nodule number typical for supernodulators, that is, 6–10-fold of the wild type, to the level found permissive for the particular crop. This shift should be accompanied with breeding aimed at the increased photosynthetic capacity of the shoot. Forage varieties of legumes represent a reserve of high photosynthetic and shoot growth capacity, thanks to a long-term breeding history for green biomass accumulation. Moreover, the deleterious effects of supernodulation are less perceived after introgression into the background of forage varieties in view of different criteria in their evaluation, such as nitrogen accumulation and biomass production per crop area unit. The growth of supernodulators can be further corrected by breeding for auxiliary traits such as long-vine shoot architecture, a longer vegetation period and late flowering. The same strategy is applicable to the compensation for inherent pleiotropic changes in plant development, which are often associated with primarily symbiotic mutations. Supporting evidence for the efficiency of the described approach has already been reported.     

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.     

Role of legumes in providing N for sustainable tropical pasture systems

Forage legumes have long been lauded for their ability to fix atmospheric nitrogen and contribute to the sustainability of agricultural production systems. However despite the benefits they bring in terms of increased herbage and animal production they are not widely used in temperate or tropical regions. In this review the amounts of biological nitrogen fixation (BNF) needed to sustain the soil-plant-animal system are discussed and related to the amounts fixed in tropical pastures. The data suggest that tropical forage legumes have the capacity to meet the requirements to balance the N cycle of grazed pastures. The actual amounts required will depend on the rate of pasture utilization and the efficiency of recycling via litter, excreta and internal remobilization. The efficiency of nitrogen fixation (% of legume N derived from fixation) is usually high in tropical pastures (>80%) and is unlikely to be affected by inorganic soil N in the absence of N fertilizer. Thus an estimate of the amoutns of N fixed could be obtained from simple estimates of legume biomass provided tissue levels of other nutrients such as phosphorus and potassium are adequate. Key factors for the achievement of sustainable grass/legume pastures include the selection of appropriate germplasm adapted to the particular environment and the judicious use of fertilizers such as phosphorus, potassium, calcium, magnesium and sulphur on acid infertile soils typical of the sub-humid and humid tropics. The main constraints to the widespread adoption of forage legumes include a lack of legume persistence, the presence of anti-quality factors such as tannins, variable Bradyrhizobium requirements and lack of acceptability by farmers. Strategies for the alleviation of these constrainst are discussed. Forage legumes can be used to recuperate degraded soils via their ability to improve the physical, chemical and biological properties of soils and these benefits could be of particular use for small-scale resource-poor farmers. The incorporation of forage legumes into agropastoral systems is discussed as an environmentally and economically attractive means to encourage the widespread adoption of legumes in the humid tropics.     

Nitrogen dynamics in grain crop and legume pasture systems under elevated atmospheric carbon dioxide concentration: A meta‐analysis

Understanding nitrogen (N) removal and replenishment is crucial to crop sustainability under rising atmospheric carbon dioxide concentration ([CO₂]). While a significant portion of N is removed in grains, the soil N taken from agroecosystems can be replenished by fertilizer application and N₂ fixation by legumes. The effects of elevated [CO₂] on N dynamics in grain crop and legume pasture systems were evaluated using meta‐analytic techniques (366 observations from 127 studies). The information analysed for non‐legume crops included grain N removal, residue C : N ratio, fertilizer N recovery and nitrous oxide (N₂O) emission. In addition to these parameters, nodule number and mass, nitrogenase activity, the percentage and amount of N fixed from the atmosphere were also assessed in legumes. Elevated [CO₂] increased grain N removal of C₃ non‐legumes (11%), legumes (36%) and C₄ crops (14%). The C : N ratio of residues from C₃ non‐legumes and legumes increased under elevated [CO₂] by 16% and 8%, respectively, but the increase for C₄ crops (9%) was not statistically significant. Under elevated [CO₂], there was a 38% increase in the amount of N fixed from the atmosphere by legumes, which was accompanied by greater whole plant nodule number (33%), nodule mass (39%), nitrogenase activity (37%) and %N derived from the atmosphere (10%; non‐significant). Elevated [CO₂] increased the plant uptake of fertilizer N by 17%, and N₂O emission by 27%. These results suggest that N demand and removal in grain cropping systems will increase under future CO₂‐enriched environments, and that current N management practices (fertilizer application and legume incorporation) will need to be revised.     

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

Mutation breeding of grain legumes

Genetic variation among existing cultivars and in germplasm collections is the outcome of selection during evolution and plant breeding. Mutagenesis offers the plant breeder a chance to tackle unconventional objectives, particularly those that were at a selection disadvantage in the past. Effective mutagens are available, but the bottleneck is the effective selection of rare desired variants from large mutagenized populations. Selection methods must be non-destructive. Grain legume mutation breeding has already led to improved cultivars with higher yield, better grain quality, or stronger resistance to pathogenens. Many mutations affecting nitrogen fixation related traits have also been reported. Some could be useful in breeding better cultivars, but the majority are being used to study the factors interacting in the complex process of symbiotic nitrogen fixation and to improve the strategy for producing cultivars with better fixation capacity.     

蚕豆种质资源、抗病育种和QTL定位及抗逆性研究进展

蚕豆是世界温带和亚热带地区一种重要的食用豆类作物,在中国的栽培历史超过2100年。中国是世界上蚕豆栽培面积最大、总产量最多的国家,蚕豆因其高效生物固氮、土壤改良和环境友好特性,已成为中国现代农业种植结构调整、西部经济欠发达地区和丘陵山区农民脱贫致富的重要经济作物。目前,多种DNA标记已广泛应用于大豆、菜豆、豌豆等豆类作物,并取得了一系列重要进展,但蚕豆分子遗传学的研究进展相对缓慢。本文对蚕豆的起源、分类、国内外蚕豆遗传多样性、遗传图谱构建,以及生长习性、抗病育种和QTL定位、抗逆性研究进行了综述,旨在为国内外蚕豆资源的深入研究和利用提供参考。     

Symbiotic performance of grain and wild herbaceous legumes in the Okavango Delta and Tswapong region of Botswana

The low inherent soil fertility, especially nitrogen (N) constrains arable agriculture in Botswana. Nitrogen is usually added to soil through inorganic fertilizer application. In this study, biological nitrogen fixation by legumes is explored as an alternative source of N. The objectives of this study were to measure levels of N₂ fixation by grain legumes such as cowpea, Bambara groundnut and groundnut in farmers’ fields as well as to estimated N₂ fixation by indigenous herbaceous legumes growing in the Okavango Delta. Four flowering plants per species were sampled from the panhandle part of the Okavango Delta and Tswapong area. Nitrogen fixation was measured using the ¹⁵N stable isotope natural abundance technique. The δ¹⁵N values of indigenous herbaceous legumes indicated that they fixed N₂ (?1.88 to +1.35 ‰) with the lowest value measured in Chamaecrista absus growing in Ngarange (Okavango Delta). The δ¹⁵N values of grain legumes growing on farmers’ fields ranging from ?1.2 ‰ to +3.3 ‰ indicated that they were fixing N₂. For grain legumes growing at most farms, %Ndfa were above 50% indicating that they largely depended on symbiotic fixation for their N nutrition. With optimal planting density, Bambara groundnuts on farmers’ fields could potentially fix over 90 kg N/ha in some parts of Tswapong area and about 60 kg N/ha in areas around the Okavango Delta. Results from this study have shown that herbaceous indigenous legumes and cultivated legumes play an important role in the cycling of N in the soil. It has also been shown that biological N₂ on farmer’s field could potentially supply the much needed N for the legumes and the subsequent cereal crops if plant densities are optimized with the potential to increase food security and mitigate climate change.     

Prospects and problems of simple linear models for estimating symbiotic N2 fixation by crop and pasture legumes

Symbiotic dinitrogen (N₂) fixation of crop and pasture legumes is a critical component of agricultural systems, but its measurement is expensive and labour intensive. Simple models which can provide approximations based on crop or pasture dry matter production would be useful for agrononomists and those interested in regional nitrogen (N) cycle fluxes. We investigate meta analysis of published data on legume shoot dry matter production, shoot %N and legume %N fixed (%Ndfa) and look for relationships among these, as a possible way of providing useful approximations of N₂ fixation. We restricted our analysis to Australian studies where we have ready access to the primary data and where cultivars, management and climate are more constrained compared to a universal dataset. Regression analysis between shoot dry matter and amounts of shoot N₂ fixed were strong for all crop and pasture legumes with significant differences in slope and intercept values being obtained between pastures and crops, and between chickpea (Cicer arietinium) and all other crop and pasture legumes. Annual pasture legumes showed the strongest linear relationship between N₂ fixation and shoot dry matter and had the greatest slope (20.2–24.3 kg N₂ fixed/t), compared to 18.7 kg N₂ fixed/t for the perennial pasture legume lucerne (alfalfa, Medicago sativa), and between 10.7 to 23.0 kg N₂/t for crop legumes, depending upon species. It was recognised that the use of such shoot-based relationships would underestimate the total amounts of N₂ fixed since the contributions of fixed N present in, or derived from, roots and nodules are not included. Furthermore there needs to be careful consideration of the validity of an intercept term, which might reflect suppression of N₂ fixation at low dry matter and high soil mineral N availability, or possibly the use of non-linear regression. For chickpea crops grown in north-eastern Australia, multiple regression indicated that N₂ fixation was much more closely correlated with %Ndfa than dry matter production. Evidence presented also indicated that %Ndfa of other crops and lucerne in this region may similarly be influenced by soil mineral N. The regression approach presented provides a statistical basis to approximate N₂ fixation in the first instance. This work highlights some of the dangers of fitting single regressions to aggregated datasets and using these to approximate symbiotic N₂ fixation. The analysis indicates that where pasture legumes are grown in mixtures with non-legumes, and driven to high dependence on N₂ fixation, simple linear regressions may be quite useful, provided that possible differences between species are investigated as the slopes of the regressions between these can be quite different. For crop legumes, where low dependence on N₂ fixation can occur at higher mineral N availability, there is a need to carefully consider the intercept term, obtain estimates of mineral N availability, and/or resort to non-linear models. The gross generalisations presented in scatter plots cannot be reliably applied any more specifically, even within the datasets from which they were generated, and in some cases even within legume species between regions. They cannot substitute for direct measurement where any certainty is required under a particular set of defined conditions.     

Developing a breeding strategy to exploit quantitative variation in symbiotic nitrogen fixation

Summary This paper examines evidence which quantifies the relative importance of legume and Rhizobium genotypes as determinants of phenotypic variation in symbiotic nitrogen fixation. It demonstrates potentially large and unpredictable effects of the Rhizobium genotype. The likely importance of such effects on crop yield is considered. The information is then used to assess ways in which legume breeding programmes may be altered to encompass the effects of genetic variation in Rhizobium.     

Unique and conserved features of floral evocation in legumes

Legumes, with their unique ability to fix atmospheric nitrogen, play a vital role in ensuring future food security and mitigating the effects of climate change because they use less fossil energy and produce less greenhouse gases compared with N-fertilized systems. Grain legumes are second only to cereal crops as a source of human and animal food, and they contribute approximately one third of the protein consumed by the human population. The productivity of seed crops, such as grain legumes, is dependent on flowering. Despite the genetic variation and importance of flowering in legume production, studies of the molecular pathways that control flowering in legumes are limited.Recent advances in genomics have revealed that legume flowering pathways are divergent from those of such model species as Arabidopsis thaliana. Here, we discuss the current understanding of flowering time regulation in legumes and highlight the unique and conserved features of floral evocation in legumes.     

Biological nitrogen fixation in resource-poor agriculture in South Africa

Farm lands of resource-poor communities in South Africa are depleted of nutrients due to continuous mono-cropping, limited use of fertilisers, and sometimes leaching caused by high rainfall. Despite the well-known advantages of biological nitrogen fixation (BNF) in cropping systems, less than 10% of the grain crops planted annually in these areas are legumes. Using a participatory research and development approach, resource-poor farmers were introduced to conservation agriculture (CA) practices, including BNF, that promoted zero (or reduced) tillage, increased retention of soil cover, as well as crop diversification. Because crop rotation and intercropping of legumes with cereals are known to contribute to soil fertility while enhancing food security, resource-poor farmers from various Provinces in South Africa were trained on the benefits of legume culture for eight years. As a result, these resource-poor farmers did not only get training in inoculation techniques, but were also supplied with inoculants for use on their farms. Data collected from Farmers Demonstration Trials at Belvedere, Dumbarton and Lusikisiki, showed that the grain and fodder yield of maize planted after legumes, and maize intercropped with legumes, were comparable to those of maize receiving high N fertilizer dose (i.e. 54 kg N at planting and 54 kg N as top-dressing). The same data further showed thatRhizobium inoculation, when combined with application of low levels of P and K, significantly increased crop yields within farmers’ trial plots. BNF therefore offers a great opportunity for resource-poor farmers in South Africa to increase their crop yields and thus improve the quality of their livelihoods through the adoption of affordable and sustainable biological technologies that enhance soil fertility.