Prospects and potentials for systems of biological nitrogen fixation in sustainable rice production

The N requirement of rice crops is well known. To overcome acute N deficiency in rice soils, this element is usually supplied to the rice crop as the commercially available fertilizer urea. But unfortunately a substantial amount of the urea-N is lost through different mechanisms causing environmental pollution problems. Utilization of biological N fixation (BNF) technology can decrease the use of urea-N, reducing the environmental problems to a considerable extent. Different BNF systems have different potentials to provide a N supplement, and it is necessary to design appropriate strategies in order to use BNF systems for efficient N supply to a rice crop. Research has been conducted around the world to evaluate the potential of different BNF systems to supply N to rice crops. This paper reviews salient findings of these works to assess all the current information available. This review indicates that the aquatic biota Cyanobacteria and Azolla can supplement the N requirements of plants, replacing 30–50% of the required urea-N. BNF by some diazotrophic bacteria like Azotobacter, Clostridium, Azospirillum, Herbaspirillum and Burkholderia can substitute for urea-N, while Rhizobium can promote the growth physiology or improve the root morphology of the rice plant. Green manure crops can also fix substantial amounts of atmospheric N. Among the green manure crops, Sesbania rostrata has the highest atmospheric N₂-fixing potential, and it has the potential to completely substitute for urea-N in rice cultivation.     

稻田养萍模式下不同施氮量对稻田氨挥发及红萍生物固氮作用的影响

红萍对水体铵态氮浓度较为敏感，稻田放养红萍模式下，红萍的生物固氮作用及其抑制氨挥发的作用对不同施氮量的响应未知。红萍为水生蕨藻共生体，具有很强的生物固氮能力。红萍可作为优质绿肥放养于稻田，以替代部分化学氮肥，起到节能减排的效应。为明确稻田养萍模式下不同施氮量对红萍生物固氮作用和田间氨挥发的影响，采用盆栽试验设置了0、75、150、225、300kg/hm²共5个施氮(以纯N量计)水平，监测了稻田放养红萍和水稻单种各处理的氨挥发量、生物固氮速率和水稻产量。结果表明：(1)同一施氮水平下，稻田放养红萍可显著降低氨挥发日通量峰值及氨挥发总量。在施氮量为225 kg/hm²时，稻田放养红萍对氨挥发总量的抑制作用最大，与水稻单种相比，抑制幅度可达83.2%。(2)红萍的生物固氮速率及固氮总量与施氮量呈线性负相关关系，随施氮量的增加，固氮速率和固氮量逐渐降低，施氮量300 kg/hm²并放养红萍处理得到的固氮速率及总量同不施氮肥不养萍处理之间无显著差异。(3)与不养萍处理相比，放养红萍组各处理的水稻产量都明显增加，其中施氮量为225...     

Mineral nitrogen impairs the biological nitrogen fixation in soybean of determinate and indeterminate growth types

Soybean (Glycine max Merrill) crop production in Brazil relies mainly on biological nitrogen fixation (BNF) for nitrogen (N) supply. Recent adoption of indeterminate growth-type genotypes has raised doubts on the need for supplemental mineral N that might negatively affect the BNF. We assessed the effects of mineral N on BNF attributes of soybean genotypes grown in central and southern Brazil. Genotypes were inoculated with Bradyrhizobium sp. and/or received mineral N in three sets of experiments. In the first set, two genotypes received increasing rates of mineral N in nutrient solution, which consistently reduced the BNF. In the second set, mineral N applied at sowing and/or topdressing reduced nodulation and ureides-N in determinate and indeterminate growth-type genotypes. In the third set, mineral N applied at R5.3 stage, foliar or as topdressing, did not increase grain yield in four field experiments. Mineral N impaired BNF irrespective of the growth type and had no effect on grain yield.     

利用15N2直接标记法研究水稻种植对稻田固氮量和固氮活性的影响

稻田固氮对土壤维持肥力有着重要的作用,但水稻种植与固氮菌及其活性之间的关系尚不清楚。本试验利用~(15)N_2直接标记法测定了下位砂姜土发育的简育水耕人为土在种水稻和不种水稻条件下的生物固氮量,及其在土壤不同层次(0～1、1～5、5～15 cm)和水稻中的分配,并通过实时荧光定量PCR技术测定了土壤中固氮菌nifH DNA及RNA基因数量。结果表明:种水稻处理显著提高了土壤各层固氮量,尤其提高了1～5 cm和5～15 cm土层土壤固氮量对总固氮量的贡献;种水稻处理的总固氮量是不种水稻处理的10.3倍;水稻植株中生物固定的氮占总固氮量的31.48%;在0～1 cm土层,种水稻处理显著提高了nifH RNA基因数量,而对nifH DNA基因数量的增加不显著。可见,水稻种植没有增加固氮菌的数量,稻田固氮量的增加是因为水稻种植极大地促进了固氮菌nifH基因的表达,提高了固氮菌的固氮活性。     

Prospects of using leguminous green manuring crop Sesbania rostrata for supplementing fertilizer nitrogen in rice production and control of environmental pollution

Abstract

Modern rice cultivation relies on chemical nitrogen (N) fertilizer that ultimately degrades soil health and creates environmental pollution. The increasing concerns about environmental pollution, restoring soil health, and conservation of energy, public attention is now towards green manures as supplement for N fertilizer. Sesbania rostrata is a popular leguminous green manuring crop that fixes atmospheric N₂, improves soil health, and rice productivity through incorporation of its biomass in to soil. About 40–60?days plants provide 5–6 tons dry biomass that can supplement 50–100% N requirement. In world, it is adapted as pre-rice or post-rice green manuring crop. Long-term use of S. rostrata improves soil health and increased rice yield by 9–11% over chemical fertilizer. Use of S. rostrata in large scale for rice production can reduce environmental pollution problems as it can cut down chemical N requirement and thus reduction dispersal of Nr to the environment.     

Interaction among free-living N-fixing bacteria isolated from Drosera villosa var. villosa and AM fungi (Glomus clarum) in rice (Oryza sativa)

Rice is usually grown in N-deficient soils, demanding that the element be supplied to the field by commercially available N fertilizers. Unfortunately, a substantial amount of the urea-N or NO₃-N applied as fertilizers is lost through different mechanisms, causing environmental pollution problems. Utilization of biological N₂ fixation (BNF) technology can decrease the application of N fertilizers, reducing environmental risks. This study evaluated the effects of four free-living N-fixing bacterial species, isolated from oligotrophic soil conditions, as single inoculants or combined with arbuscular mycorrhizal fungi (Glomus clarum), on the development of rice plants grown as flooded or upland rice, in the greenhouse. Upland rice roots were inoculated with Methylobacterium sp., Burkholderia sp. and Sphingomonas sp., whereas the species Burkholderia sp., Pseudomonas sp. and Sphingomonas sp., were inoculated on flooded rice. Inoculants consisted of individual bacterial species or their mixtures, with or without G. clarum. Controls included non-bacteria/non-AM fungi, and AM fungi alone. Experiments were carried out in five replicates. The presence of G. clarum decreased or did not significantly affect plant growth under the different culture conditions. The presence of AM fungi stimulated the N-fixing bacterial population of upland rice. Bacterial species had different effects, under both culture conditions, and some genera of N-fixing bacteria increased root and shoot growth at different plant growth stages. The level of mycorrhiza colonization had no influence on plant growth     

大豆生物固氮模型研究进展

定量描述大豆生物固氮量对于施肥管理以及农业非点源污染控制具有十分重要的意义。生物固氮模型作为一种重要工具,在定量预测大豆生物固氮速率方面具有不可替代的作用。国内研究者对不同环境因子对固氮的影响开展了一些工作,但多未从定量角度出发。国外的生物固氮模型按构建方式大致可以分为经验模型和机理模型两类。其中机理模型研究较多,也是目前生物固氮模型的主要发展方向。本文简述了这两类模型的研究现状,并着重介绍了5种目前国外使用较为广泛的田间尺度下的大豆生物固氮机理模型,比较了不同模型中采用的环境因子响应函数的差异,并对进一步的研究作了展望,以期为国内大豆生物固氮定量研究工作的开展提供借鉴。     

Developed microbial biofilms can restore deteriorated conventional agricultural soils

Nitrogen fixing bacteria play a key role in the growth and persistence of effective microbial communities in the soil by supplying N through biological nitrogen fixation (BNF). In the long run, chemical inputs, particularly N fertilisers are known to adversely affect N₂ fixers and hence maintenance of soil fertility and crop productivity. This study examined the effect of developed microbial biofilms with N₂ fixers on restoration of soils deteriorated by conventional agricultural practices in tea cultivation. Just reducing recommended chemical fertiliser use by 50% significantly increased soil microbial biomass and BNF, and decreased soil NO₃⁻ and pest infestation. The lower chemical fertiliser addition coupled with the biofilm-based biofertilisers known as biofilmed biofertilisers (BFBFs) further increased BNF significantly. The combined application significantly increased soil organic C by ca. 20%, and reduced leaf transpiration by ca. 40%. It also supported plant growth, rhizoremediation and soil moisture conservation in comparison to the 100% chemical fertilisation. Those improved performances were observed to be proportional to the increased density of soil bacteria, and have several agronomic and environmental implications. It is apparent from this study that replenishing the depleted soil microbial communities by applying such biofertilisers is likely to be beneficial in agroecosystems with chemical N fertiliser use, if they are to be sustained for crop production.     

Effect of phosphorus management in rice–mungbean rotations on sandy soils of Cambodia

Nitrogen (N) and phosphorus (P) deficiencies are key constraints in rainfed lowland rice (Oryza sativa L.) production systems of Cambodia. Only small amounts of mineral N and P or of organic amendment are annually applied to a single crop of rainfed lowland rice by smallholder farmers. The integration of leguminous crops in the pre‐rice cropping niche can contribute to diversify the production, supply of C and N, and contribute to soil fertility improvement for the subsequent crop of rice. However, the performance of leguminous crops is restricted even more than that of rice by low available soil P. An alternative strategy involves the application of mineral P that is destined to the rice crop already to the legume. This P supply is likely to stimulate legume growth and biological N₂ fixation, thus enhancing C and N inputs and recycling N and P upon legume residue incorporation. Rotation experiments were conducted in farmers' fields in 2013–2014 to assess the effects of P management on biomass accumulation and N₂ fixation (δ¹⁵N) by mungbean (Vigna radiata L.) and possible carry‐over effects on rice in two contrasting representative soils (highly infertile and moderately fertile sandy Fluvisol). In the traditional system (no legume), unamended lowland rice (no N, + 10 kg P ha^?1) yielded 2.8 and 4.0 t ha^?1, which increased to 3.5 and 4.7 t ha^?1 with the application of 25 kg ha^?1 of urea‐N in the infertile and the moderately fertile soil, respectively. The integration of mungbean as a green manure contributed up to 9 kg of biologically fixed N (17% Nfda), increasing rice yields only moderately to 3.5–4.6 t ha^?1. However, applying P to mungbean stimulated legume growth and enhanced the BNF contribution up to 21 kg N ha^?1 (36% Nfda). Rice yields resulting from legume residue incorporation (“green manure use”–all residues returned and “grain legume use”–only stover returned) increased to 4.2 and 4.9 t ha^?1 in the infertile and moderately fertile soil, respectively. The “forage legume use” (all above‐ground residues removed) provided no yield effect. In general, legume residue incorporation was more beneficial in the infertile than in the moderately fertile soil. We conclude that the inclusion of mungbean into the prevailing low‐input rainfed production systems of Cambodia can increase rice yield, provided that small amounts of P are applied to the legume. Differences in the attributes of the two major soil types in the region require a site‐specific targeting of the suggested legume and P management strategies, with largest benefits likely to accrue on infertile soils.     

禾豆间作氮素高效利用机理及农艺调控途径研究进展

为保障粮食安全,农业生产中化肥使用极为普遍,但过量使用,往往引起地下水污染、温室效应加剧、生物多样性降低等多种环境和生态问题。禾豆间作系统由于不同作物生物学特性和氮素利用存在差异,进行合理调控可充分发挥生物固氮优点,从而减少化肥投入,提高生产效益,是一种稳产、高产、高效可持续的种植体系。该系统中"氮转移"、"氮阻遏"消减和氮素时空分异是目前研究的热点,也是促进豆科作物固氮、减少化肥投入的有效途径,可实现禾、豆两种作物对氮素的高效利用。特别是该系统中作物品种、施氮制度、空间布局以及种植密度等农艺措施是对种间关系进行调控的必要手段,合理优化可有效促进禾/豆间作竞争与互补协同作用,增强氮素协调利用,从而挖掘两种作物对氮素高效利用的生物学潜力。为此,本文基于前人研究成果和农业可持续发展观点,重点综述了国内外有关禾豆间作氮素高效利用主要机理及相关农艺调控途径的研究现状,旨在为构建简易、高产、高效、氮肥节约型禾豆间作模式提供有力的科学依据和理论支撑。     

共生固氮在农牧业上的作用及影响因素研究进展

共生固N是生物固N的主体部分,具有固N效率高、应用范围广等特点。叙述了主要豆科作物年固N量及固N量占豆科作物本身所吸收N的比例,阐述了豆科作物在与非豆科作物间套轮作中固定N素的转移及对非豆科作物的影响,并介绍了影响豆科作物-根瘤菌共生体共生固N效率的主要因素。开展豆科作物-根瘤菌共生体系方面的研究对农业可持续发展具有重要意义。     

不同供氮水平对水稻/花生间作系统中氮素行为的影响

水稻旱作／花生间作栽培是一种新兴的节水农业技术。用”N稀释标记法在盆栽条件下研究了间作系统在15kghm^-2、75kghm^-2和150kghm^-23个氮素供应水平条件下花生生物固氮以及水稻旱作／花生间作系统中氮素的转移，同时用^15N的富积标记法研究了花生根系腐解对间作系统氮素转移的贡献。结果表明，在15和75kghm^-22个氮素水平下，间作水稻比单作水稻的干物质量分别增加了23．5％和12．2％，在P=0．05的水平有显著差异。间作水稻和单作水稻的氮素吸收量分别为135、143mg株 ^-1和117、131mg株^-1，分别比单作增加14．8％和8．8％。不同栽培方式对花生的干物质积累和氮素吸收影响很小。在3个氮素水平下间作花生和单作花生的固氮量分别为76．1％、53．3％、50．7％和72．8％、56．5％、35．4％，在低氮水平下的生物固氮显著高于高氮条件，间作对花生的生物固氮有一定促进作用。间作系统中的氮素转移率和转移量在3个氮素水平分别为12．2％、9．2％、6．2％和16．3、13．0、10．4mg株^-1，氮素的转移率和转移的数量显著地随氮素水平的增加而减少。用^15N花生叶片标记直接证明了氮素从花生体内向水稻的转移，随刈割时间氮素转移量显著下降，表明花生根系腐解对间作系统的氮素转移有积极作用。     

稻草和尿素配施时水稻对肥料氮和土壤氮的吸收利用

利用＾１５Ｎ同位素示踪技术,研究了稻草和尿素配施时水稻对肥料和土壤氮的吸收。结果表明,稻草单施导致土壤速效氮的生物固定,氮素供应不足是影响水稻分蘖成穗的限制因子,稻草配合尿素施用,明显改善肥料氮和土壤氮的供应,既有利于当季水稻增产,也有利于培养土壤肥力,还有利于后茬物作产量的提高 。     

Increased phosphorus availability from sewage sludge ashes to maize in a crop rotation with clover

A recycling of Phosphorus (P) from the human food chain is mandatory to secure the future P supply for food production. However, many available recycled P fertilizers from sewage sludge do not have an adequate P bioavailability and, thus, are not suitable for their application in soils with pH >5.5–6.0, unless being combined with efficient mobilization measures. The aim of the study was to test the P mobilization ability of red clover (Trifolium pratense L.) from two thermally recycled P fertilizers for a subsequently grown maize. Two sewage sludge ashes (SSA) were investigated in a pot experiment at soil pH 7.5 with red clover differing in its nitrogen (N) supply (added N fertilizer or biological N₂ fixation (BNF)), followed by maize (Zea maize L.). Shoot dry matter of maize was almost doubled when N supply of previous grown clover was covered by BNF, instead of receiving added N fertilizer. Similarly, shoot P removal of maize following clover with BNF was significantly increased. It is suggested that the P mobilization is related to the BNF, and a proton release of N₂ fixing clover roots led to the measured decrease in soil pH and thereby increased P availability of the tested fertilizers.     

Paddy System with a Hybrid Rice Enhances Cyanobacteria Nostoc and Increases N2 Fixation

Biological nitrogen(N) fixation(BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight,field-based ~(15)N_2-labelling growth chamber experiments were used to assess the BNF capacity of different rice varieties. In addition,both the 16 S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based ~(15)N_2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha~(-1) in inbred japonica(W23) and hybrid indica(IIY) rice cultivars planted in the rice-soil systems, respectively, and only 1%–2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil(0–1 cm) under IIY than under W23. Sequencing of the 16 S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23.Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar(IIY) promoted BNF to a greater extent than the inbred rice cultivar(W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.     

太湖地区稻田绿肥固氮量及绿肥还田对水稻产量和稻田土壤氮素特征的影响

通过田间试验,利用15N自然丰度法,研究了太湖地区水稻土冬季绿肥的固氮量,以及绿肥还田后配施氮肥对水稻产量、稻田土壤供氮能力及土壤氮素淋失特征的影响。试验结果表明,紫云英和蚕豆当季分别能固定氮约32.8和68.8 kg km-2进入稻田生态系统以培肥土壤和供下季水稻利用。蚕豆秸秆还田后基本能满足水稻生长所需的氮,紫云英和蚕豆还田施氮120 kg km-2时,既可保证水稻较高产量,又节约当季化学氮肥45%～55%。紫云英和蚕豆还田不施氮肥处理,整个生长期耕层土壤溶液NH+4-N、NO-3-N和TN浓度均低于配施氮肥的处理;蚕豆还田处理土壤溶液TN浓度高于紫云英还田处理。随氮肥用量增加,NH+4-N、NO-3-N和TN浓度有增加趋势,不同施氮量间差异不显著。绿肥-水稻轮作,紫云英和蚕豆还田土壤氮素淋溶显著降低。配施氮肥增加了土壤氮的淋失量,尤其施氮300 kg km-2处理,土壤淋溶液NH+4-N、TN浓度显著高于施氮0～240 kg km-2的处理。     

15N同位素稀释法研究固氮菌接种对甘蔗生物固氮的影响

利用15N同位素稀释法,研究接种固氮菌klebsiella L03对甘蔗品种B8和ROC22的生物固氮的影响。结果表明： B8的固氮百分率最高为3128%Ndfa,从苗期开始就显著高于ROC22,分蘖期和伸长初期的固氮量和固氮百分率与ROC22相比差异达到极显著水平。B8和ROC22的根、茎、叶都可发生固氮,以伸长初期（60d）叶片中的固氮能力最强。接种L03能显著提高B8和ROC22各器官的含氮量,其中对ROC22叶片和B8茎的含氮量提高作用最明显。不同甘蔗品种、相同品种的不同器官甚至相同器官的不同生长时期固氮作用有很大差异。     

Evidence for enhanced N availability during switchgrass establishment and seeding year production following inoculation with rhizosphere endophytes

Improvement in sustainable production of switchgrass (SG, Panicum virgatum L), as a purpose-grown biomass feedstock crop, could be realized through investigation of plant–microbe interactions associated with plant growth promoting rhizobacteria (PGPR), capable of biological nitrogen fixation (BNF). The objective of this study is to increase establishment year production of SG biofuels by inoculation with a mixed PGPR inoculum. We isolated pure strains of N₂-fixing, and other PGPR, from SG rhizomes. The bacteria were identified as Paenibacillus polymyxa, an N₂-fixing bacterium, and other PGPR capable of solubilizing phosphate and/or producing auxins. Field trials utilizing these strains in a mixed PGPR inoculum showed that inoculated plants contained more N in tillers during anthesis but not at senescence, suggesting that more N could be cycled to belowground roots and rhizomes for winter storage. The amount of N removal in biomass and recovery of fertilizer N were also greater for inoculated than uninoculated plants. PGPR inoculation also resulted in positive N balances, suggesting improved access to N from non-fertilizer N sources, possibly through BNF and improved soil N uptake. Overall, inoculation of SG with PGPR enhanced N acquisition and could be an effective strategy to increase the establishment year production of this crop.     

Isotopic evidence of significant assimilation of atmospheric-derived nitrogen fixed by Azospirillum brasilense co-inoculated with phosphate-solubilising Pantoea dispersa in pepper seedling

The contribution of plant growth-promoting bacteria (PGPB) of the genus Azospirillum to the plant N budget through biological nitrogen fixation (BNF) is still controversial. The aim of this study was to determine the contribution of BNF by Azospirillum brasilense on pepper grown at different N levels, attained using the ¹⁵N natural abundance method. To this end, pepper plants were grown in a growth chamber and treated with A. brasilense combined with Pantoea dispersa and then irrigated at four different N levels (0, 1, 3 and 7 mM NO₃⁻). The assimilation of fixed N was clear from the lower δ¹⁵N values observed in bacteria-treated plants compared with those of non-bacteria treated plants. The percentage of BNF-derived N decreased with decreasing NO₃⁻ levels in the growth medium. BNF contribution to the total nitrogen content of plants was found to be as high as 46%. The results suggest that the bacteria have a potential to supply a considerable amount of N to pepper seedlings, as well as to stimulate plant growth and N uptake when Azospirillum–Pantoea treatment is combined with low NO₃⁻ levels.     

氮饥饿水稻利用不同形态氮素的差异及其生理机制

通过水培试验，研究了氮饥饿7d后，恢复供应不同形态氮源对水稻氮吸收和积累及氮同化中关键酶活性和光合色素的影响。结果表明，缺氮促进根系生长，增加根冠比。恢复供氮4d显著增加地上部生物量。铵硝混合营养促进了水稻对氮的吸收和转运，叶片和根系中全氮及叶片中铵态氮的含量以硝酸铵处理最高。与单一铵或硝营养相比，铵硝混合营养增强了根系的谷氨酰胺合成酶和叶片中硝酸还原酶的活性，提高了水稻同化和利用氮的能力。另外，与纯硝营养相比，供应铵态氮显著增加了叶片中总叶绿素，尤其是叶绿素a的含量。因此，改善水肥管理、平衡对水稻供氮的铵硝配比将提高水稻氮素的吸收和利用效率。