动物的共生菌固氮

介绍了共生菌固氮涉及的动物和微生物类群、动物共生菌固氮的性质和机理。应用乙炔还原法和固氮酶基因检测等研究表明,所涉及的动物有7门13纲23目50科99属174种。动物肠道具有丰富的微生境,供不同生理需求的固氮菌生长发育,所蕴含的共生固氮菌类群也十分丰富,涵盖植物共生固氮菌、植物内生固氮菌、植物根际固氮菌、自生固氮菌等生态类型。一般认为动物共生固氮菌来源于环境,其性质属于联合共生固氮。动物共生固氮菌一般与其他共生生物形成复合体,以满足固氮过程中对电子和质子供体、能量供给、固氮酶活性保护以及氨阻遏解除等方面的需求。动物共生菌固氮产物氨的同化也需要多种共生物的协同作用,可能通过谷氨酰胺合成酶/谷氨酸合成酶等途径。总体上,食物氮、非蛋白氮和共生菌固氮相互协调,形成营养和解毒的代谢网络,共同维持动物体内氮素营养的动态平衡,并对未来研究提出展望。     

Heterotrophic n(2) fixation and distribution of newly fixed nitrogen in a rice-flooded soil system

Rice (Oryza sativa L.) plants growing in pots of flooded soil were exposed to a ¹⁵N₂-enriched atmosphere for 3 to 13 days in a gas-tight chamber. The floodwater and soil surface were shaded with a black cloth to reduce the activity of phototrophic N₂-fixing micro-organisms. The highest ¹⁵N enrichments were consistently observed in the roots, although the total quantity of ¹⁵N incorporated into the soil was much greater. The rate of ¹⁵N incorporation into roots was much higher at the heading than at the tillering stage of growth. Definite enrichments were also found in the basal node and in the lower outer leaf sheath fractions after 3 days of exposure at the heading stage. Thirteen days was the shortest time period in which definite ¹⁵N enrichment was observed in the leaves and panicle. When plants were exposed to ¹⁵N₂ for 13 days just before heading and then allowed to mature in a normal atmosphere, 11.3% of the total ¹⁵N in the system was found in the panicles, 2.3% in the roots, and 80.7% in the subsurface soil. These results provide direct evidence of heterotrophic N₂ fixation associated with rice roots and the flooded soil and demonstrate that part of the newly fixed N is available to the plant.     

Effect of Rice Plants on Nitrogenase Activity of Flooded Soils

In samples of flooded soil containing blue-green algae (cyanobacteria), the presence of rice plants did not influence the nitrogenase activity of the algae. Nitrogenase activity of heterotrophic bacteria was enhanced by the presence of rice plants, but this activity was not affected by changes in plant density. The rate of nitrogen fixation in the rhizosphere, however, varied significantly among the 16 rice varieties tested. A simple method was devised to test the nitrogen-fixing activity in the root zone of rice varieties, and data were obtained showing marked differences in the activities of the 16 varieties. In tests of two varieties with dissimilar rates of nitrogen fixation in their rhizospheres, the variety which had the greater root weight and lesser shoot weight and which supported greater methane formation had the greater nitrogenase activity.     

不同CO2浓度和N肥水平对粳稻茎鞘非结构性碳水化合物含量与积累的影响

为了解CO2浓度升高和N肥水平对水稻茎鞘内非结构性碳水化合物(NSC)含量和积累量的影响,利用开顶式气室(OTC),以常规粳稻"南粳9108"为试验材料,设置3个CO2浓度水平:对照T0(背景大气)、T0+120μmol·mol-1(T1)和T0+200μmol·mol-1(T2)。在OTC内采用盆栽方式,设置3个氮(N)肥水平:10 g N·m^-2(N1)、20 g N·m^-2(N2)和30g N·m^-2(N3)。分别于水稻抽穗期、灌浆期(抽穗后20 d)和成熟期对地上部分各器官生物量、茎鞘NSC含量以及顶部四张叶片的N含量进行分析。结果表明:CO2浓度升高对抽穗期叶N含量总体无显著影响,但显著降低灌浆期N2和N3水平的叶N含量;CO2浓度升高对抽穗期茎鞘NSC含量和积累量无显著影响,抽穗期置换到高CO2浓度环境使灌浆期茎鞘NSC积累显著增加,置换到低CO2浓度环境使NSC积累显著减少。同一CO2浓度条件下,NSC含量和积累量均为N1>N2>N3,且N1处理均显著高于N3处理,CO2浓度升高和N水平的交互作用对灌浆期茎鞘NSC含量影响显著。水稻产量在不同CO2浓度水平间无显著差异,但随施氮水平的提高而增加。抽穗期与灌浆期水稻茎鞘NSC含量和积累量与茎鞘干重呈极显著正相关,与叶N含量呈极显著负相关;叶N衰减越慢,灌浆期水稻茎鞘NSC残留比(RNSC)越低;结实率和产量与RNSC呈显著负相关,RNSC越大,茎鞘NSC转移的越少,结实率和产量越低。     

谷氨酰胺合成酶活性与水稻杂种优势预测

从杂种优势预测生理指标需具备的３个基本条件入手,对杂交水稻及其亲本生长发育过程中的谷氨酰胺合成酶（ＧＳ,ＥＣ６、３、１、２）活性进行研究,结果表明：水稻ＧＳ活性在整个生育期中的变化呈现一定的规律性,并且杂种酶活性是由其双亲遗传性所决定的;同时,ＧＳ活性与多个生理性状及产量性状具有一定的相关性;在孕穗期和抽穗期与产量性状的关系尤为密切,因此,在生殖生长期ＧＳ活性在一定程度上可以反映产量性状的优势水平,可望作为水稻杂种优势预测的一个辅助指标。     

鸭稻共作方式对水稻生长的影响

对鸭稻共作生产方式下水稻几个生育期的株高、叶面积指数、生物量、根系活力、根系形态特征指标等进行了对比研究。结果表明,鸭稻共作生态模式中由于鸭子的活动,对水稻生长发育具有一定影响。与常规水稻栽培方式相比,鸭稻共作方式下水稻株高、叶面积指数、生物量有减小的趋势,而且在有效分蘖期和幼穗分化期存在着显著或极显著差异。鸭子活动对水稻根系生长状况(根/冠比、根系活力)也具有一定作用,与常规水稻栽培方式相比,鸭稻共作方式下水稻根冠比和根系活力有增加趋势,根冠比在幼穗分化期达到显著差异,根系活力在有效分蘖期存在显著差异;鸭稻共作方式下水稻根系总长变化无明显规律,根系表面积与常规栽培方式下相比无显著差异。     

不同生育期水分胁迫对水稻根叶渗透调节物质变化的影响

 干旱是限制水稻(Oryza sativa)作物产量的主要生态因子之一,渗透调节是作物适应干旱逆境的生理机制之一。在人为控制水分的盆栽条件下, 对水稻生长的分蘖期、幼穗分化期、抽穗期、结实期分别进行水分胁迫,研究水稻根系及叶片渗透调节物质的变化规律。结果表明, 不同生育期 干旱胁迫后叶片水势均显著下降,根系和叶片的有机渗透调节物质如可溶性糖、游离氨基酸、脯氨酸和无机渗透调节物质包括K⁺、Mg²⁺等含量 均大幅度上升,而且幼穗分化期和抽穗期这两个对水分胁迫最敏感的时期上升幅度最大,其中又以有机渗透调节物质变化最显著。不同生育期渗 透调节大小的顺序为：抽穗期>幼穗分化期>结实期>分蘖期,反映了不同生育时期渗透调节能力的差异。同时幼穗分化期和抽穗期水分胁迫结束 后再复水后根系和叶片的有机渗透调节物质含量仍长期明显高于对照,而无机离子则变化规律比较复杂,有的升高有的则降低。叶片的渗透调 节能力大于根系,无论是叶片或根系都是K⁺对渗透调节的贡献最大;其次是Ca²⁺, 6 种渗透调节物质含量大小排列顺序为K⁺ > Ca²⁺ >可溶性糖 > Mg²⁺ > 游离氨基酸 > 脯氨酸。     

Field technique using the acetylene reduction method to assay nitrogenase activity and its association with the rice rhizosphere

Summary A plastic bag, used as a field assay chamber, assisted in the investigation of the acetylene reduction method as a technique to measure nitrogen-fixing activity in rice paddy fields. A study of the change in volume of the plastic bag and of the loss of acetylene and ethylene from the bag provided evidence that this plastic bag method was feasible in field assays. The field assay of rice at the grain ripening stage showed that the nitrogen-fixing activity increased linearly after a lag phase of 0 to 3 hours during a time-course experiment.Detachment of the aerial part of the rice plant from its root remaining in the field did not affect the nitrogen-fixing activity of the root.The markedly higher nitrogen-fixing activity in the planted areas of the field compared with the nonplanted areas between the plant rows indicated that the nitrogenase activity in the field is associated with the roots of the rice plant. re]19751204     

广东早稻高产的适宜气温指标

选取了广东典型生态区的曲江、高要、广州、汕头和湛江5个代表性试验点进行地理分期播种试验,各试验点均采用3个主栽品种、设置6个播期,观测早稻不同发育期、测定其产量及其产量构成因子。结合附近气象站同期观测资料,采用数理统计和变异系数等方法,分析平均气温对早稻产量及产量构成因子的影响,确定了广东早稻高产不同生育期的适宜气温指标。结果表明:全生育期平均气温为23～24℃,其中出苗-分蘖期为18～21℃、分蘖-孕穗期为21～25℃、孕穗-齐穗期为24～28℃、齐穗-成熟期为27～30℃。     

水稻纹枯病菌营养及寄主资源生态位

由于IPM概念的局限性,有害生物生态调控（EPM）理论和方法的提出发展了IPM,生态位原则是有害生物生态调控（EPM）的重要原则之一,生态位研究为EPM的具体实施提供了依据。应用可持续农业和EPM理论及生态位理论研究了水稻纹枯病的生态位,分析了水稻纹枯病菌氮肥营养生态位和寄主品种资源生态位,结果表明：以相对侵染效率作为指标,在水稻不同生育期,纹枯病的氮肥营养生态位宽度不同,其中以孕穗期的生态位宽度最小,为0.6979,拔节期、抽穗期、灌浆期和乳熟期的生态位宽度分别是0.9741,0.8884,0.7974和0.9815,表明水稻纹枯病在水稻不同生育阶段利用氮肥的效能不同。寄主品种资源生态位宽度在拔节期、孕穗期、抽穗期、乳熟期分别为0.9348,0.7677,0.8875和0.9962。以病情指数为指标,氮肥营养生态位宽度在拔节期、孕穗期、抽穗期、灌浆期和乳熟期分别为0.9379,0.9696,0.6775,0.6729和0.7691。其氮肥营养生态位宽度在拔节期与孕穗期最大,生态位宽度指数接近于1。寄主品种资源生态位宽度在各生育期均接近1,表明寄主品种资源生态位宽度在各生育期是相似的,即说明水长期稻纹枯病菌利用品种资源各状态的选择和利用效能是相似的。     

Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland and Dryland Rice

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice.     

Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates

AIMS: The aims of the study were to identify the effects of rice endophyte Pantoea agglomerans YS19 on host plant growth and allocations of photosynthates. METHODS AND RESULTS: Endophytic diazotrophic YS19 showed nitrogen-fixing activity in N-free medium, and produced four categories of phytohormones which were indole-3-acetic acid, abscisic acid, gibberellic acid and cytokinin in Luria-Bertani medium. Inoculation of YS19 improved the biomass of the 12-day-cultivated host rice seedlings by 63.4% on N-free medium or by 18.7% on N-supplemented medium. Spraying of YS19 cell culture onto the rice plants at the premilk stage enhanced the transportation of the photosynthetic assimilation product from the source (flag leaves) to the sink (stachys) significantly. The formation of the plant sink was obviously inhibited when YS19 cell culture was applied at the late milk stage. CONCLUSIONS: This research suggests that endophyte YS19 promotes host rice plant growth and affects allocations of host photosynthates. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggested that YS19 possesses the potential for increasing rice production in field application. Meanwhile, a suitable plant growth stage must be selected for the foliar spraying of YS19 cell culture.     

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

AIMS: To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. METHODS AND RESULTS: A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. CONCLUSIONS: This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.     

Relationships between nitrogen fixation and photosynthesis as the main components of the productivity in alfalfa

Alfalfa (Medicago sativa L.) plants were inoculated with Sinorhizobium meliloti Tn-5 mutants featuring various nitrogen-fixing effectiveness and then grown in sand culture to study relations between CO₂ exchange, plant productivity, and nitrogen fixation. At the flowering stage, the relationship between nitrogen fixation and photosynthesis of whole alfalfa plants was described with the logarithmic curve. At the same stage of plant development, a close relationship was observed between nitrogen fixation rate and plant weight; this relationship showed a trend toward saturation at high rates of nitrogen fixation. The increase in nitrogenase activity of root nodules was accompanied by stimulation of root respiration; the relation of respiration to nitrogen-fixing activity was manifested stronger than its relation to the total root weight. It is concluded that highly effective strains of root nodule bacteria can realize their potential only in combination with complementary plant genotypes featuring active photosynthesis that provides a balanced supply of assimilates for both the symbiotic apparatus and growth processes in the macrosymbiont.     

nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Use of 13C labeling to assess carbon partitioning in transgenic and nontransgenic (parental) rice and their rhizosphere soil microbial communities

Photosynthetic assimilation of CO₂ is a primary source of carbon in soil and root exudates and can influence the community dynamics of rhizosphere organisms. Thus, if carbon partitioning is affected in transgenic crops, rhizosphere microbial communities may also be affected. In this study, the temporal effects of gene transformation on carbon partitioning in rice and rhizosphere microbial communities were investigated under greenhouse conditions using the ¹³C pulse-chase labeling method and phospholipid fatty acid (PLFA) analysis. The ¹³C contents in leaves of transgenic (Bt) and nontransgenic (Ck) rice were significantly different at the seedling, booting and heading stages. There were no detectable differences in ¹³C distribution in rice roots and rhizosphere microorganisms at any point during rice development. Although a significantly lower amount of Gram-positive bacterial PLFAs and a higher amount of Gram-negative bacterial PLFAs were observed in Bt rice rhizosphere as compared with Ck at all plant development stages, there were no significant differences in the amount of individual ¹³C-PLFA between Bt and Ck rhizospheres at any growing stage. These findings indicate that the insertion of cry1Ab and marker genes into rice had no persistent or adverse effect on the photosynthate distribution in rice or the microbial community composition in its rhizosphere.     

Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation

Background and aims

Gluconacetobacter diazotrophicus is a nitrogen-fixing endophytic bacterium isolated from sugarcane, rice, elephant grass, sweet potato, coffee, and pineapple. These plants have high level of asparagine, which promotes microbial growth and inhibits nitrogenase activity. The regulation of intracellular concentrations of this amino acid is essential for growth and biological nitrogen fixation (BNF) in this diazotroph; however its asparagine metabolic pathway has not yet been clearly established.

Methods

The work reported here is the first to demonstrate the use of an alternative route for asparaginyl-tRNA (Asn-tRNA) and asparagine formation in an endophytic nitrogen-fixing bacterium by using in silico and in vitro analysis.

Results

The indirect route involves transamidation of incorrectly charged tRNA via GatCAB transamidase. Nitrogenase activity was completely inhibited by 20?mM Asn in LGI-P medium, which in contrast promotes protein synthesis and microbial growth.

Conclusions

The analysis carried out in this work shows that intracellular levels of asparagine regulate the expression of nitrogenase nifD gene (GDI0437), suggesting that the presence of an alternative route to produce asparagine might give the G. diazotrophicus a tighter control over cell growth and BNF, and may be of importance in the regulation of the endophytic plant-microbe interaction.     

不同生育阶段夜温升高对双季水稻产量的影响

利用两间玻璃温室内夜间不同的温度条件,研究了水稻不同生育阶段的夜温升高对双季早、晚稻产量的影响.结果表明：播种-幼穗分化（一次枝梗分化期）期间夜温升高,有利于双季水稻的分蘖,植株有效穗数显著增加,平均夜间最低温度每升高1 ℃,双季早、晚稻的产量分别提高10.02%～13.18%和6.52%～7.78%（P＜0.01）;幼穗分化-抽穗（10%稻穗抽出剑叶鞘）期间夜温升高,导致颖花退化,使每穗发育颖花数减少,平均夜间最低温度每升高1 ℃,双季早、晚稻的产量分别下降3.76%～6.67%和3.66%～6.94%（P＜0.01）;灌浆结实期（抽穗-成熟）夜温升高,双季早稻的结实率和产量显著下降,而双季晚稻的结实率和产量显著提高,平均夜间最低温度每升高1 ℃,双季早稻的产量下降2.07%～5.61%（P＜0.05）,双季晚稻的产量提高1.63%～2.28%（P＜0.05）.表明不同生育阶段的夜温升高对双季水稻产量的影响存在明显差异.     

氮肥处理对氮素高效吸收水稻根系性状及氮肥利用率的影响

2011—2012年在土培条件下,以氮素吸收效率差异较大的15个常规籼稻为供试材料,研究氮肥运筹对不同氮效率品种根系性状、成熟期吸氮量及氮肥利用率的影响,分析影响氮高效水稻氮素吸收的主要根系性状。结果表明:(1)各氮肥处理下,成熟期吸氮量均表现为氮高效品种氮中效品种氮低效品种。适量增施氮肥及基肥+促花肥处理有利于氮高效品种吸氮量的增加,氮素吸收受品种、氮肥处理的显著影响。(2)在施氮量处理下,氮高效品种单株不定根数、单株根干重、单株不定根总长大或较大,单株根活力在常氮(N2)、高氮(N3)处理下有一定的优势;在施氮时期处理下,氮高效品种单株不定根数、单株不定根总长、单株根干重、单株根系总吸收面积、单株根系活跃吸收面积、抽穗期冠根比多数处理有优势;增施氮肥有利于促进氮高效品种单株不定根总长和单株根活力的提高,适量施氮有利于单株不定根数、单株根干重增加,前期施氮可促进不定根的发生和伸长,后期施氮有利于不定根的充实和根系生理性状的提高。此外,增施氮肥可提高各类品种冠根比;(3)在常氮、高氮处理下,氮高效品种氮肥利用率大于氮中效、氮低效品种。(4)提高单株不定根数、单株不定根总长、单株根活力及抽穗期冠根比有利于各类品种吸氮量的提高,增加根干重对氮高效品种吸氮量的提高也有显著的促进作用。结合相关分析与通径分析结果,抽穗期冠根比及单株不定根数、单株根活力、单株不定根总长、单株根干重是影响氮高效品种吸氮能力的主要根系性状。     

Nitrogen fixation in lakes

Summary Determinations in the open waters of lakes using N¹⁵ as a tracer show that nitrogen fixation is generally associated with the presence of heterocystous blue-green algae and is light dependent. Although nitrogen-fixing blue-green algae tend to be abundant when the concentration of nitrate or ammonia in the water is low, fixation itself is not necessarily inhibited by the presence of these sources of combined nitrogen. The activity of nitrogen-fixing blue-green algae shows a direct relationship to concentration of dissolved organic nitrogen. As a result of the interaction of such factors, nitrogen fixation per unit area of lake surface per year tends to be greatest at an early stage of eutrophication. In relation to the total nitrogen budget of a lake the contribution of biological nitrogen fixation to the income is probably always small but at certain times and in particular water strata it may contribute a major part of the nitrogen assimilated by the phytoplankton.