植物内生固氮菌在绿色农业生产建设中的应用

化学氮肥在解决人类温饱问题中起了极为重要的作用,但近年发现过量施用化肥有较大的负面影响.植物内生固氮菌是近年发现的一类非豆科植物中的高效固氮微生物,这一新型固氮系统为新型绿色农业生产建设提供了一种新的生物固氮途径.本研究综述了化学氮肥在农业生产建设中的危害、生物固氮作用、植物内生同氮菌及其固氮机制、植物内生固氮菌在绿色农业生产建设中的应用,并对应用植物内生固氮菌的可能性和意义,以及需要注意的问题作了一些探讨.     

生物固氮及在可持续农业中的应用

氮是限制农业生产的重要营养元素.生物固氮指某些原核生物能利用体内的固氮酶将空气中的氮气还原为氨,为植物生长提供氮素.自然界中存在多种具有固氮能力的微生物,依据其固氮方式分为自生固氮、共生固氮和联合固氮三种类型.联合固氮茵通过趋化定殖在植物根表,并生长、固氮.     

禾谷类作物与微生物联合共生固氮作用的研究

生物固氮作用对农业生产的重要意义是众所周知的。近十年来,在生物固氮研究的各个方面都有许多重要的进展。细菌与植物联合固氮作用的发现,为非豆科植物扩大利用生物固氮的前景开辟了一条新的途径。联台固氮体系是介于自生固氮体系与共生固氮体系的中间类型。固氮细菌与相应联合的植物之间具有较密切的相互影响,但是它们又不像根瘤菌与豆科植物那样形成特异的共生构造一根瘤。这种固氮     

15N同位素稀释法评估甘蔗的生物固氮量

为评估甘蔗生物固氮量,采用15N同位素稀释法,以木薯为参比植物,进行温室桶栽试验.结果表明:甘蔗全生育期植株固氮11.3514％ Ndfa,固氮量每桶0.9269 g.甘蔗根、茎、叶的固氮百分率和固氮量大小依序为叶＞茎＞根.叶的固氮百分率(13.2668％ Ndfa)略高于植株,但两者差异不显著.甘蔗植株全氮量中来自空气氮(生物固氮)、肥料氮和土壤氮的比例分别为11.3514％、7.6857％、80.9629％.甘蔗的氮肥利用率为58.7583％.甘蔗根、茎、叶各部位均有固氮现象,生产上可以用叶代替植株来评估甘蔗的生物固氮量.     

细菌-植物联合固氮研究进展

生物固氮过程对农业生产的重要意义是众所周知的。工业固氮一年约提供4千万吨氮肥,而生物固氮则每年贡献约1亿吨。从生态学观点看,自然界存在三种固氮体系:自生固氮(如自生固氮菌),共生固氮(如根瘤菌和豆科植物共生)和联合固氮(如雀稗和雀稗固氮菌的联合)。本文只介绍联合固氮体系的研究进展。联合固氮体系是自生固氮和共生固氮体系的中间类型。固氮细菌与相应联合的植物之间具有较密切的     

禾本科植物联合固氮研究及其应用现状展望

综述了近年来从禾本科植物体内和根际发现的内生固氮菌和根际固氮菌的种类、特征及对宿主的促生机理,以及固氮菌接种剂在农业生产中的应用现状和存在的问题,指出影响联合固氮菌接种效果的主要因素有土著微生物的竞争;植物基因型差异和环境条件的变化,如结合态氮(氨、亚硝酸盐、硝酸盐等)对固氮酶的合成阻遏和较高的氧分压对联合固氮菌的固氮效率影响．提出了发掘和利用禾本科植物的生物固氮潜力的努力方向：从自然界分离筛选获得广谱高效固氮菌株;应用基因工程构建耐铵、泌铵型联合固氮菌;诱导禾本科植物形成固氮根瘤;充分发挥植物内生固氮菌的优势．     

内蒙古典型草原的生物量与生产力

氮素是植物生长和作物高产的限制因素。氮素来源主要是生物固氮和化学固氮。生物固氮是地球表面氮素的主要来源。在生物固氮中,目前最有效的还是豆科根瘤菌固氮。但近年来,禾本科植物根系与固氮菌的联合固氮作用引起了人们的广泛重视。联合固氮是自生固氮和共生固氮体系的中间类型,固氮细菌与相应联合植物之间具有较密切的相互影响,但又不象形成根瘤那样具有共生结构。这种联合固氮作用在自然界中广泛存在,各种作物,热带和亚热带牧草的根际和根表均有联合固氮菌存在,它们能提供土壤氮素。经测定水稻根际每个生长季非藻类的生物固氮量达25—30     

甘蔗固氮内生菌——重氮营养醋杆菌的研究进展

在巴西,许多地区甘蔗的栽培已有几十甚至几百年的历史,尽管氮素供应明显不足,但甘蔗的产量和土壤中的氮素储备并没有随时间的推移而下降。认为甘蔗可能得益于生物联合固氮。通过氮平衡和１５Ｎ技术已证明生物的联合固氮作用对牧草和甘蔗等农作物的生长有重要的农学意义。认为甘蔗所吸收的氮素中有８０％可能来自生物固氮作用,只有少数来自施放到土壤中的化合态氮［１］。迄今已从甘蔗根际中分离到１１个属的固氮细菌,但是这些存在于甘蔗根际固氮菌的数量都不足以解释发生于甘蔗中的高效固氮作用．对甘蔗与固氮菌的联合共生固氮作用的深…     

广西甘蔗根际高效联合固氮菌的筛选及鉴定

对广西主要甘蔗产区的根际联合固氮细菌进行了收集和评价,拟筛选获得对甘蔗具有潜在促生性能的联合固氮菌,为甘蔗生产节肥减耗提供依据。结合nifH基因扩增和固氮酶活性分析方法筛选获得36个固氮细菌菌株;进一步对所获得固氮菌株的固氮能力、溶磷性、分泌植物生长素IAA的特性等促进植物生长潜能进行评价,获得了5个同时具有较强固氮能力、降解无机磷和分泌植物生长激素IAA的功能菌株;通过Biolog鉴定系统和16S rRNA序列分析对5个具有较好应用潜力的固氮菌进行分类鉴定。结果表明这5个菌株分别属于Klebsiella sp.、Bacillus megaterium、Pseudomonas sp.、Pantoea sp.和Burkholderia sp.。本研究结果表明广西甘蔗根际联合固氮菌具有较大的开发利用潜力。     

生物固氮研究中的几个热点问题

氮素化肥在农业生产中一直发挥重要作用,为了发展持续生态农业,全世界的研究者都在进行着长期不懈的努力,不断优化和拓展生物固氮系统。介绍固氮研究中的4个热点问题：⑴联合固氮;⑵根际微生物量氮及微生物活度;⑶通过豆科植物凝集素基因转化扩大根瘤菌宿主范围;⑷结瘤固与“类根瘤”固氮。     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.     

Nitrogen fixation in rice systems: state of knowledge and future prospects

Rice is the most important cereal crop. In the next three decades, the world will need to produce about 60% more rice than today's global production to feed the extra billion people. Nitrogen is the major nutrient limiting rice production. Development of fertilizer-responsive varieties in the Green Revolution, coupled with the realization by farmers of the importance of nitrogen, has led to high rates of N fertilizer use on rice. Increased future demand for rice will entail increased application of fertilizer N. Awareness is growing, however, that such an increase in agricultural production needs to be achieved without endangering the environment. To achieve food security through sustainable agriculture, the requirement for fixed nitrogen must increasingly met by biological nitrogen fixation (BNF) rather than by using nitrogen fixed industrially. It is thus imperative to improve existing BNF systems and develop N₂-fixing non-leguminous crops such as rice. Here we review the potentials and constraints of conventional BNF systems in rice agriculture, as well as the prospects of achieving in planta nitrogen fixation in rice.     

一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

[背景] 我国甘蔗生产中氮肥过量施用严重,导致生产成本居高不下,充分发挥甘蔗与内生固氮菌的联合固氮作用,减少氮肥施用量,对促进我国甘蔗产业可持续发展具有重要意义。[目的] 筛选优势甘蔗内生固氮菌,对其基本特性、联合固氮效率及促生长功能进行评价。[方法] 从甘蔗根系分离到一株内生固氮菌GXS16,利用乙炔还原法测定固氮酶活性,通过PCR扩增nifH基因确定菌株为固氮菌;通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类;通过接种盆栽甘蔗检测菌株的促生长作用,采用¹⁵N同位素稀释法检测菌株相对固氮效率。[结果] 菌株GXS16固氮酶活性为2.42μmol-C₂H₄/（h·mL）,根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果,菌株GXS16属于伯克氏菌属（Burkholderia）;菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶（1-Aminocyclopropane-1-Carboxylate Deaminase,ACC）活性及合成生长素吲哚乙酸（Indoleacetic Acid,IAA）、降解无机磷的功能;接种GXS16处理甘蔗植株的株高比对照增长15%以上,干重增长20%以上,¹⁵N同位素测定显示甘蔗根、茎、叶从空气中获得氮的百分比分别为7.69%、15.64%和8.72%,效率显著优于模式菌株G.diazotrophicus PAL5。[结论] Burkholderia sp.GXS16是一株高效甘蔗内生固氮菌,具有良好应用前景。     

Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production?

A fundamental shift has taken place in agricultural research and world food production. In the past, the principal driving force was to increase the yield potential of food crops and to maximize productivity. Today, the drive for productivity is increasingly combined with a desire for sustainability. For farming systems to remain productive, and to be sustainable in the long-term, it will be necessary to replenish the reserves of nutrients which are removed or lost from the soil. In the case of nitrogen (N), inputs into agricultural systems may be in the form of N-fertilizer, or be derived from atmospheric N₂ via biological N₂ fixation (BNF).Although BNF has long been a component of many farming systems throughout the world, its importance as a primary source of N for agriculture has diminished in recent decades as increasing amounts of fertilizer-N are used for the production of food and cash crops. However, international emphasis on environmentally sustainable development with the use of renewable resources is likely to focus attention on the potential role of BNF in supplying N for agriculture. This paper documents inputs of N via symbiotic N₂ fixation measured in experimental plots and in farmers' fields in tropical and temperate regions. It considers contributions of fixed N from legumes (crop, pasture, green manures and trees), Casuarina, and Azolla, and compares the relative utilization of N derived from these sources with fertilizer N.     

Insufficient nitrogen supply from symbiotic fixation reduces seasonal crop growth and nitrogen mobilization to seed in highly productive soybean crops

Nitrogen (N) supply can limit the yields of soybean [Glycine max (L.) Merr.] in highly productive environments. To explore the physiological mechanisms underlying this limitation, seasonal changes in N dynamics, aboveground dry matter (ADM) accumulation, leaf area index (LAI) and fraction of absorbed radiation (fAPAR) were compared in crops relying only on biological N₂ fixation and available soil N (zero-N treatment) versus crops receiving N fertilizer (full-N treatment). Experiments were conducted in seven high-yield environments without water limitation, where crops received optimal management. In the zero-N treatment, biological N₂ fixation was not sufficient to meet the N demand of the growing crop from early in the season up to beginning of seed filling. As a result, crop LAI, growth, N accumulation, radiation-use efficiency and fAPAR were consistently higher in the full-N than in the zero-N treatment, leading to improved seed set and yield. Similarly, plants in the full-N treatment had heavier seeds with higher N concentration because of greater N mobilization from vegetative organs to seeds. Future yield gains in high-yield soybean production systems will require an increase in biological N₂ fixation, greater supply of N from soil or fertilizer, or alleviation of the trade-off between these two sources of N in order to meet the plant demand.     

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.     

Comparison of isotope techniques and non-nodulating isolines to study the effect of ammonium fertilization on dinitrogen fixation in soybean,Glycine max

Summary Two experiments were carried out with two nodulating and non-nodulating soybean isolines, with three different levels of N as (¹⁵NH₄)₂SO₄ at the equivalent of 0, 25 and 50 kg N/ha. In the first experiment three seeds were sown in each pot and the plants harvested at 35, 55 and 75 days. In the second experiment only one seed was sown per pot and harvested at 75 days.Isotope dilution technique and in certain cases natural isotope variation (¹⁵N) was used to determine directly the origin of nitrogen in the plant, whether from soil, fertilizer or biological N₂-fixation. The use of nodulating and non-nodulating isolines enabled comparison with the classical method of estimating N₂-fixation by difference from total plant N. Results at the 75 day harvest were similar for either method, but at the earlier harvests, particularly at 35 days, the total-N method was inadequate. The isotope method appeared more sensitive while the total-N method suffered from greater variability with correspondingly high standard errors and significant differences.It was found that by the 35 and 55 day harvests hardly any N₂-fixation had taken place, plant nitrogen being almost entirely derived from soil or fertilizer N. Plants in competition used up soil fertilizer N more rapidly, thus stimulating symbiotic nitrogen fixation. When only one plant was grown in each pot it had a greater proportion of N derived from soil or fertilizer, and less N derived from fixation. In general the¹⁵N data showed that only about 25% of the applied fertilizer N was absorbed by the plant.The nodulating isoline absorbed more N than the non-nodulating plants. This suggests a possible synergistic effect of N₂-fixation on N derived from other sources, giving an increase in total-N content of nudulated plants. The N derived from N₂-fixation was scarcely detectable in the roots but appeared to be translocated almost entirely to shoots and pods.With 25 kg N/ha the greater proportion of the nitrogen in the pods was derived from N₂-fixation. Even with 50 kg N/ha the nitrogen in the pods derived from fixation remained high, that being derived from fertilizer being less than 15%. About 80% of the nitrogen in the nodules was due to fixation.In the present experiment the application of 25 kg N/ha appeared sufficient to give maximum N absorption by both isolines. At this level symbiotic fixation by Rhizobium remained high in nodulating plants, while the proportion of total N due to fixation was reduced with 50 kg N/ha.UNDP/IAEA Project BRA 78/006.     

Biological Nitrogen Fixation in Sugar Cane: A Key to Energetically Viable Biofuel Production

The advantages of producing biofuels to replace fossil energy sources are derived from the fact that the energy accumulated in the biomass is captured directly from photosynthesis and is thus renewable, and that the cycle of carbon dioxide fixation by the crop, followed by burning of the fuel makes no overall contribution to atmospheric CO₂ or, consequently, to global warming. However, these advantages are negated if large quantities of fossil fuels need to be used to grow or process the biofuel crop. In this regard, the Brazilian bioethanol program, based on the fermentation/distillation of sugar cane juice, is particularly favorable, not only because the crop is principally hand harvested, but also because of the low nitrogen fertilizer use on sugar cane in Brazil. Recent ¹⁵N and N balance studies have shown that in some Brazilian cane varieties, high yields are possible without N fertilization because the plants are able to obtain large contributions of nitrogen from plant-associated biological N₂ fixation (BNF). The N₂-fixing acid-tolerant bacterium Acetobacter diazotrophicus was first found to occur within roots, stems, and leaves of sugar cane. Subsequently, two species of Herbaspirillum also have been found to occur within the interior of all sugar cane tissues. The discovery of these, and other N₂-fixing bacteria that survive poorly in soil but thrive within plant tissue (endophytic bacteria), may account for the high BNF contributions observed in sugar cane. Further study of this system should allow the gradual elimination of N fertilizer use on sugar cane, at least in Brazil, and opens up the possibility of the extension of this efficient N₂-fixing system to cereal and other crops with consequent immense potential benefits to tropical agriculture.     

Dominance of bacterial ammonium oxidizers and fungal denitrifiers in the complex nitrogen cycle pathways related to nitrous oxide emission

Organic compounds and mineral nitrogen (N) usually increase nitrous oxide (N₂O) emissions. Vinasse, a by‐product of bio‐ethanol production that is rich in carbon, nitrogen, and potassium, is recycled in sugarcane fields as a bio‐fertilizer. Vinasse can contribute significantly to N₂O emissions when applied with N in sugarcane plantations, a common practice. However, the biological processes involved in N₂O emissions under this management practice are unknown. This study investigated the roles of nitrification and denitrification in N₂O emissions from straw‐covered soils amended with different vinasses (CV: concentrated and V: nonconcentrated) before or at the same time as mineral fertilizers at different time points of the sugarcane cycle in two seasons. N₂O emissions were evaluated for 90 days, the period that occurs most of the N₂O emission from fertilizers; the microbial genes encoding enzymes involved in N₂O production (archaeal and bacterial amoA, fungal and bacterial nirK, and bacterial nirS and nosZ), total bacteria, and total fungi were quantified by real‐time PCR. The application of CV and V in conjunction with mineral N resulted in higher N₂O emissions than the application of N fertilizer alone. The strategy of vinasse application 30 days before mineral N reduced N₂O emissions by 65% for CV, but not for V. Independent of rainy or dry season, the microbial processes were nitrification by ammonia‐oxidizing bacteria (AOB) and archaea and denitrification by bacteria and fungi. The contributions of each process differed and depended on soil moisture, soil pH, and N sources. We concluded that amoA‐AOB was the most important gene related to N₂O emissions, which indicates that nitrification by AOB is the main microbial‐driven process linked to N₂O emissions in tropical soil. Interestingly, fungal nirK was also significantly correlated with N₂O emissions, suggesting that denitrification by fungi contributes to N₂O emission in soils receiving straw and vinasse application.