单细胞稳定同位素标记技术在固氮微生物中的应用研究

生物固氮是指固氮微生物将大气中氮气还原为生物可利用氨的过程,是环境中新氮的主要来源,调控初级生产力并影响氮储库的收支平衡。由于环境中大部分固氮微生物不可纯培养,不依赖培养且具有高空间分辨率水平的单细胞技术,成为研究固氮微生物的有力手段。~(15)N_2稳定同位素标记技术,以微生物对~(15)N的同化量或速率为依据,是表征微生物固氮活性的最直接手段。本文对~(15)N_2稳定同位素标记结合两种单细胞技术,即纳米二次离子质谱(Nano SIMS)和单细胞拉曼光谱,用于固氮微生物研究的最新进展进行了综述,内容包括揭示环境中高活性固氮微生物、空间分布、与其他生物的共生关系、细胞生理状态等,并进一步对近期发展的基于单细胞拉曼光谱的固氮微生物研究进行了展望。     

蓝藻固氮对人工湿地颗粒物有机氮的贡献

固氮蓝藻能为水生态系统输入新氮,生物固氮对水体氮的贡献观点不一。人工湿地中固氮蓝藻输入的新氮对除氮效率的影响仍不明晰。本研究通过连续监测,比较了北京野生动物救护中心水禽栖息的人工湖和净化其水质的人工湿地中的浮游植物组成、颗粒物有机氮(PON)和水体固氮速率(Rn),分析固氮蓝藻鱼腥藻属(Anabaena)和非固氮蓝藻微囊藻属(Microcystis)藻细胞密度的季节变化,及其与水体PON和Rn相关性。通过IsoSource软件分析15N自然丰度,计算了各个来源(大气N_2、鸟粪、沉积物和水体硝酸盐)对人工湖和人工湿地中PON的贡献率。结果显示,人工湖中鱼腥藻和微囊藻交替爆发,Rn与PON和鱼腥藻细胞密度均呈正相关(P0.05),说明鱼腥藻输入的新氮可能被微囊藻利用转化为PON。大气N_2对生长季人工湖和人工湿地PON贡献率分别为0.5%~82.0%和50.0%~86.0%,9月水体固氮速率达到最高时大气N_2对PON贡献率可达80%以上。本研究表明,人工湖中的鱼腥藻固氮能为微囊藻提供可利用氮,可能是人工湖夏季微囊藻水华的原因之一。由于浮游植物在表流人工湿地较难沉淀和吸附,浮游植物增多使人工湿地去除PON的能力降低,因此鱼腥藻固氮可能间接降低人工湿地除氮能力。     

水位和施氮对若尔盖高寒湿地土壤非共生固氮的影响

高寒湿地中土壤微生物固氮是氮元素进入生态系统的主要途径之一,环境因子变化对土壤固氮功能的影响仍不明晰。在四川若尔盖高寒湿地搭建了由27个生态模拟箱组成的中宇宙实验系统,通过控制水位和模拟氮沉降,研究水位变化和施氮对土壤非共生固氮的影响。实验设计了3水位水平×3施氮水平共9个处理,测定了生态模拟箱中表层土壤的非共生固氮速率,土壤碳、氮含量,以及地上植物生物量和植物氮含量,比较不同水位和施氮处理下非共生固氮速率的变化规律并分析其与土壤和植物碳、氮含量的关系。研究发现：生态模拟箱中土壤非共生固氮速率范围是0.003-7.35 μg N g^-1 d^-1,从不淹水到淹水的处理土壤非共生固氮速率提高约2倍。施氮处理中固氮速率随土壤含水量升高而增强的敏感性高于施氮对照处理,且施氮处理下的生态模拟箱中土壤有机碳含量显著升高,据此推测施氮可能使淹水的生态模拟箱中的浮游植物提高生产力而释放可利用有机碳,从而间接促进土壤非共生固氮。本研究获得以下结论：（1）若尔盖高寒湿地中土壤水位是限制固氮速率的重要因子;（2）施氮背景下土壤含水量对非共生固氮的促进效应更明显。     

水稻田中固氮蓝藻固氮活力的昼夜变化

蓝藻的固氮和光合作用密切联系,为了解固氮蓝藻在田间固氮活力的变化,我们对田间的蓝藻在不同生境下的固氮活力和昼夜变化进行现场(in situ)测定发现: 1.在不同生境中生长的各种固氮蓝藻的固氮活力的昼夜变化节律不尽相同,一般为一天出现一个活力高峰,在薄膜复盖培养池中高峰出现在中午(13∶00),而露田则在9∶00—12∶00之间;个别还出现两峰现象。2.不同种类的固氮蓝藻在田间的固氮活力有明显差别,混合藻种的酶活力最高,类颤鱼腥藻次之。3.不同生长期的类颤鱼腥藻的酶活力有明显不同。生长旺盛的活力高,衰老的活力低。4.稻田中不同小生境对蓝藻固氧酶活力有显著的影响,在水底生长的类颤鱼腥藻移至水面,其活力明显降低。根据蓝藻在田间的固氮活力的昼夜交化以及生境改变和生长状态对活力的影响,依估算生长良好的蓝藻在水田的固氮量最高可达285克氮/天/1000斤藓藻,最低51克氮,平均163克氮。假如在田间生长连续10天,即可固定1630克氮,相当于16.3斤的硫酸铵。但和测定的较高固氮活力(=258克氮/天相当于25.8斤硫铵)相比,固氮活力的潜力是相当大的。如能提高固氮蓝藻在田间的产量, 则其固氮量可大大增加。     

稳定碳同位素技术在土壤-植物系统碳循环中的应用

碳作为重要的生命元素,在土壤-植物系统物质循环中发挥重要作用.作为一种天然的示踪物,稳定碳同位素（13C）较放射性同位素具有安全、无污染、易控制的优点,在土壤-植物生态系统碳循环研究中得到广泛应用.通过检测土壤-植物体系中稳定碳同位素的自然丰度或采用稳定碳同位素标记有机材料,能够较真实地了解植物的光合特性、光合产物在土壤-植物体系中的运转及其在土壤中的分解、转化等过程.本文概述了稳定碳同位素技术在植物光合作用及光合产物运转、古气候重建、土壤有机质周转以及植物-根际微生物相互作用等方面的研究进展,并针对当前研究中存在的问题提出了今后的研究展望.     

稳定碳同位素技术在土壤-植物系统碳循环中的应用

碳作为重要的生命元素,在土壤 植物系统物质循环中发挥重要作用.作为一种天然的示踪物,稳定碳同位素（¹³C）较放射性同位素具有安全、无污染、易控制的优点,在土壤 植物生态系统碳循环研究中得到广泛应用.通过检测土壤 植物体系中稳定碳同位素的自然丰度或采用稳定碳同位素标记有机材料,能够较真实地了解植物的光合特性、光合产物在土壤 植物体系中的运转及其在土壤中的分解、转化等过程.本文概述了稳定碳同位素技术在植物光合作用及光合产物运转、古气候重建、土壤有机质周转以及植物 根际微生物相互作用等方面的研究进展,并针对当前研究中存在的问题提出了今后的研究展望.     

柱孢鱼腥藻固氮酶防氧的呼吸保护

柱孢鱼腥藻生长在缺氮情况下,发现其固氮活性增加的同时也减少了对氧的敏感性。缺氮生长细胞的乙炔还原活性给氧抑制一半时的氧分压(pO2)是0．5atm．,而有氮生长细胞的半抑制浓度为0．35atm．。这表明蓝藻有可能通过增加呼吸耗氧而提高了它的固氮酶活性。呼吸作用与固氮酶活性之间存在着密切的关系。无论在有氮、缺氮还是光诱导固氮酶形成的情况下,其固氮活性均随着呼吸速率的变化而变化。本研究结果,支持了柱孢鱼腥藻固氮酶的主要防氧手段是呼吸保护的观点。     

柱孢鱼腥藻固氮酶防氧的呼吸保护

柱孢鱼腥藻生长在缺氮情况下,发现其固氮活性增加的同时也减少了对氧的敏感性。缺氮生长细胞的乙炔还原活性给氧抑制一半时的氧分压(pO_2)是0.5atm.,而有氮生长细胞的半抑制浓度为0.35atm.。这表明蓝藻有可能通过增加呼吸耗氧而提高了它的固氮酶活性。呼吸作用与固氮酶活性之间存在着密切的关系。无论在有氮、缺氮还是光诱导固氮酶形成的情况下,其固氮活性均随着呼吸速率的变化而变化。本研究结果,支持了柱孢鱼腥藻固氮酶的主要防氧手段是呼吸保护的观点。     

鱼腥藻(Anabaena 7120)DNA的转化作用

用化学方法从固氮蓝藻鱼腥藻(Anabaena 7120)细胞中有效地提取了DNA,以此为供体DNA,用它的氧敏感固氮突变种鱼腥藻一1(Anabaena-l)为受体进行转化实验。在大量的转化实验中,仅有两次获得转化后的突变种在空气中、在无氮培养基上能生长,其转化频率为10~(-6)—10~(-5)。转化子在有氧条件下的乙炔还原活力相当于野生种。它表明突变种的除氧系统通过转化而得到恢复。推测鱼腥藻7120突变种可能具有吸收和整合外源DNA的能力。对丝状蓝藻转化困难的原因进行了探讨,结果表明受体藻胞外DNA酶活力水平高于其他单细胞蓝藻,这可能是影响有效地转化作用的重要原因之一。     

菌渣化肥配施对稻田土壤微生物量碳氮和可溶性碳氮的影响

菌渣作为一种养分丰富的有机物料还田,可减少化肥施用,同时保持土壤肥力;而土壤微生物量碳、氮和可溶性碳、氮是土壤活性碳氮库的重要组成部分,其含量和比例变化对土壤肥力均具有重要作用。因此,探讨不同比例菌渣化肥配施对土壤微生物量碳、氮及可溶性碳、氮的影响,评价菌渣在优化土壤肥力方面的生态作用具有重要意义。本研究在水稻田间定位试验条件下,设置3个化肥水平(C) 0%、50%、100%,菌渣相对用量(F) 0%、50%、100%,共9个处理,分析了各处理土壤微生物量碳(MBC)、氮(MBN)和可溶性碳(DOC)、氮(DON)的变化特征,及其占土壤有机碳(SOC)和全氮(TN)的比例与相关关系。结果表明:菌渣化肥配施后,微生物量碳和可溶性碳、氮均在C100F50最高,微生物量氮在C50F100最高,与不施肥处理相比,分别显著增加了49.40%、43.65%、83.52%、207.19%;MBC/SOC和DOC/SOC均随着菌渣化肥配施量的增加而减少,MBN/TN和DON/TN均在C100F50最高。相关分析表明,MBC、DOC与SOC,MBN与TN均呈极显著正相关,DON和TN呈显著正相关。总体来讲,菌渣化肥配施能够显著提高土壤微生物量碳、氮和可溶性碳、氮含量,但不是随着用量的增加一直呈增加趋势,高量菌渣或者化肥下会有降低趋势;菌渣化肥配施降低了土壤微生物量和可溶性碳氮比,因此适宜的菌渣化肥配施是提高土壤有机碳周转速度、微生物活性及其氮素供应能力和有效性的最佳选择。     

Intrapopulation variation in carbon and nitrogen stable isotope ratios in the earthworm Aporrectodea longa

The natural abundance variations in carbon and nitrogen stable isotope ratios in a population of the earthworm Aporrectodea longa, a species known to feed on both soil and plant litter, is reported in this paper. Worms were collected from a small land area of an old white clover field and body tissue and mucus were analyzed separately. The range of isotopic values was small, but patterns of variation were not random. Tissue carbon and nitrogen isotope ratios were significantly higher in adult than in juvenile A. longa and tissue nitrogen isotope ratios tended to increase with increasing biomass of individuals. Further, carbon and nitrogen isotope ratios were positively correlated in both tissue and mucus. Possible causes of the observed patterns, including physiological effects, body composition and assimilation of C and N from different plant, soil and microbial sources are discussed. It is concluded that the causes of natural variability in isotopic composition must be understood and validated experimentally before natural abundance stable isotope methods can be used for the analysis of trophic relations among detritivorous soil invertebrates.     

Carbon assimilation, 𝛅13C and water relations of Elaeagnus angustifolia grown at two groundwater depths in the Minqin desert,China

Abstract

We investigated the physiological responses of Elaeagnus angustifolia to variation in groundwater depth. Elaeagnus angustifolia seedlings were grown in the Minqin desert in lysimeters supplied with underground water at the soil depth of 1.40 m and 3.40 m. Results showed that constant access to groundwater allowed plants supplied with water at the lower soil layer to meet their water requirement and, consequently, they were not affected by water stress. There were no differences in A _max (the net CO₂ assimilation rate under conditions of photosynthetically photon flux density and CO₂ saturation), J _max (maximum rate of electron transport) and stomatal conductance between the two underground water treatments. However, plants with deeper groundwater had a significantly higher V _cmax (i.e. a higher carboxylation efficiency of Rubisco) and mesophyll conductance resulting in increased photosynthesis measured at the CO₂ growth condition (A) and, consequently higher intrinsic water-use efficiency (WUE). However, respiration was also increased in plants grown with deeper groundwater. This may have offset the increased A and led to a similar long-term WUE, as expressed by carbon isotope discrimination (δ¹³C), between the two ground water treatments. In the present study, we also found a high foliage nitrogen concentration in the E. angustifolia plants (3.75% on average), that may be very significant ecologically in improving soil properties. The physiological traits of E. angustifolia found in this study confirm that the use of perennial phreatophytic, nitrogen fixing species has significant potential to positively impact soil fertility and carbon sequestration under environmental conditions found in the Minqin desert.     

Long-term integrated soil fertility management in South-western Nigeria: Crop performance and impact on the soil fertility status

Crop response, tree biomass production and changes in soil fertility characteristics were monitored in a long-term (1986–2002) alley-cropping trial in Ibadan, Nigeria. The systems included two alley cropping systems with Leucaena leucocephala and Senna siamea on the one hand and a control (no-trees) system on the other hand, all cropped annually with a maize–cowpea rotation. All systems had a plus and minus fertilizer treatment. Over the years, the annual biomass return through tree prunings declined steadily, but more drastically for Leucaena than for Senna. In 2002, the nitrogen contribution from Leucaena residues stabilized at about 200 kg N/ha/year, while the corresponding value for Senna was about 160 kg N/ha/year. On average, the four Leucaena prunings were more equal in biomass as well as in amounts of N, P and cations, while the first Sennapruning was always contributing up to 60% of the annual biomass or nutrient return. Maize crop yields declined steadily in all treatments, but the least so in the Senna + fertilizer treatment where in 2002 still 2.2 tonnes/ha of maize were obtained. Nitrogen fertilizer use efficiency was usually higher in the Senna treatment compared to the control or the Leucaena treatment. Added benefits due to the combined use of fertilizer N and organic matter additions were observed only for the Sennatreatment and only in the last 6 years. At all other times, they remained absent or were even negative in the Leucaenatreatments for the first 3 years. Most chemical soil fertility parameters decreased in all the treatments, but less so in the alley cropping systems. The presence of trees had a positive effect on remaining carbon stocks, while they were reduced compared to the 1986 data. Trees had a positive effect on the maintenance of exchangeable cations in the top soil. Exchangeable Ca, Mg and K – and hence ECEC – were only slightly reduced after 16 years of cropping in the tree-based systems, and even increased in the Senna treatments. In the control treatments, values for all these parameters reduced to 50% or less of the original values after 16 years. All the above points to the Senna-based alley system with fertilizers as the more resilient one. This is reflected in all soil fertility parameters, in added benefits due to the combined use of fertilizer nitrogen and organic residue application and in a more stable maize yield over the years, averaging 2.8 tonnes/ha with maximal deviations from the average not exceeding 21%.     

辣椒镰孢根腐病防病促生细菌的筛选及其效应

【目的】筛选辣椒(Capsicum annuum L.)根腐病防病促生细菌并明确其防病促生效应。【方法】采集健康辣椒根围土壤样品,以辣椒根腐病病原真菌茄镰孢(Fusarium solani)和尖镰孢(Fusarium oxysporum)为指示菌,采用平板对峙法筛选生防细菌,采用选择性培养基筛选溶无机磷、溶有机磷、固氮菌和解钾菌等促生菌,钼锑抗比色法测定溶磷量,凯氏定氮法测定固氮量,火焰原子吸收光谱法测定解钾量。对特性良好组合的菌株进行16S rDNA序列分析鉴定并制作菌剂,最后采用盆栽法测定菌剂防病促生效果。【结果】共筛选得到323株特性良好的功能菌株,拮抗菌78株,溶有机磷菌87株,溶无机磷菌107株,固氮菌128株,解钾菌123株,部分菌株同时具有多个功能特性。互作组合得到6个特性良好的菌株组合,包括8株功能菌株,鉴定发现XP271和XP181为枯草芽胞杆菌(Bacillus subtilis),XP125为特基拉芽胞杆菌(Bacillus tequilensis),XP236为耐盐芽胞杆菌(Bacillus halotolerans),XP79为巨大芽胞杆菌(Bacillus ...     

Vertical distributions of stable isotopic compositions and bacteriochlorophyll homologues in suspended particulate matter in saline meromictic Lake Abashiri

We determined chloropigment composition as well as stable carbon and nitrogen isotopic compositions of dissolved and particulate species in saline meromictic Lake Abashiri. We observed a sharp peak of bacteriochlorophyll e in a narrow redox boundary zone and the upper monimolimnion, indicating a dense population of brown-colored strains of green sulfur bacteria around the chemocline. Nitrogen isotopic records of particulate nitrogen and dissolved ammonium suggested that the green sulfur bacteria in the redox boundary zone assimilated either ammonium or dinitrogen through the nitrogen fixation pathway. In the anoxic monimolimnion, several lines of evidence suggest that a major portion of particulate organic matter originated from the overlying mixolimnion and redox boundary zone.     

三种蓝莓根际细菌的分离及其对蓝莓苗生长发育的影响

【目的】从3种蓝莓根际土壤中分离细菌,探究蓝莓根际土壤细菌多样性,并筛选具有产酸、促生长、抑菌性能的菌株,为蓝莓专用微生物肥料的研究提供优质菌株资源和理论基础。【方法】选用5种培养基分离3种蓝莓根际土壤细菌,并进行16S rRNA基因测序和系统发育分析。筛选产酸、产吲哚-3-乙酸（indole-3-acetic acid,IAA）和铁载体、固氮、溶磷和抑制灰葡萄孢生长的菌株,挑选最适菌株制备菌剂进行蓝莓苗盆栽实验验证促生能力,并检测菌剂对蓝莓元素吸收和根际土壤肥力的影响。【结果】从3种蓝莓根际土壤分离得到124株细菌,挑选70株代表性菌株进行16S rRNA基因测序,分布于3个门21个属,其中芽孢杆菌属（Bacillus）、假单胞菌属（Pseudomonas）、链霉菌属（Streptomyces）和红球菌属（Rhodococcus）为优势分离菌群。代表性菌株中,21.4%的菌株能产酸,21.4%的菌株产吲哚-3-乙酸,47.1%的菌株具有固氮潜力,65.7%的菌株具有解磷能力,14.3%的菌株能产铁载体。少量菌株同时具有产酸、产IAA、固氮、解磷和抑菌等能力。选取具有产酸和多种促生特征的菌株绿针假单胞菌CSM-70和双鱼假单胞菌CSM-129进行盆栽蓝莓苗处理,发现2株菌均能显著促进蓝莓苗的生长发育并调控根际土壤pH,其中菌株CSM-70处理还显著促进了蓝莓叶片氮、磷元素的吸收,提升了土壤速效钾、碱解氮的含量。【结论】蓝莓根际细菌多样性高且蕴藏着丰富的促生长菌株,绿针假单胞菌CSM-70和双鱼假单胞菌CSM-129能够促进蓝莓苗生长、调控根际土壤pH和肥力,并促进植株养分吸收,具有蓝莓专用微生物菌剂研制与应用的潜力。     

薇甘菊根际可培养固氮菌和氨化细菌的分离鉴定与促生作用

【目的】固氮菌和氨化细菌是氮循环产生生物有效氮的关键起始环节,直接影响了外来入侵植物的生长速度和扩散进程。然而,关于典型入侵植物薇甘菊根际可培养固氮菌和氨化细菌的研究尚未见报道,这在很大程度上制约了我们对薇甘菊根际高效的氮素转化机制的深刻理解。【方法】采用传统平板涂布培养法对野外采集的薇甘菊根际土壤中的可培养固氮菌和氨化细菌进行了分离鉴定,并进行了接种验证实验。【结果】结果表明,入侵植物薇甘菊根际土壤中的固氮菌和氨化细菌的菌群密度显著高于两个本地伴生植物(火炭母和鸡屎藤),其固氮效率及有机氮矿化效率也优于2个本地种;系统发育分析表明：薇甘菊根际的固氮菌菌株归类于5个属,分别为伯克霍尔德氏菌属(Burkholderia)、肠杆菌属(Enterobacter)、植物杆菌属(Phytobacter)、新肠杆菌属(Kosakonia)和根瘤菌属(Rhizobium);氨化细菌归类于7个属,分别为沙雷氏菌属(Serratia)、不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、博德特氏菌属(Bordetella)、寡养单胞菌属(Stenotrophomonas)、苍...     

南亚热带格木、马尾松幼龄人工纯林及其混交林生态系统碳氮储量

研究比较了南亚热带6年生格木(Erythrophleum fordii)、马尾松(Pinus massoniana)幼龄人工纯林及马尾松与格木混交林生态系统碳氮储量及其分配特征。结果表明,生态系统总碳储量依次为马尾松-格木混交林(137.75 t/hm2)格木纯林(134.07 t/hm2)马尾松纯林(131.10 t/hm2),总氮储量则为格木纯林(10.19 t/hm2)马尾松-格木混交林(8.68 t/hm2)马尾松纯林(7.01 t/hm2)。3种人工林生态系统碳氮库空间分布基本一致,绝大部分储存于0—100 cm土壤层,平均占生态系统总储量的81.49%和96.91%,其次为乔木层(分别占17.52%和2.69%),林下植被和凋落物层所占比例最小。林地土壤碳主要集中于表土层,其中0—30 cm土层平均碳储量为52.52 t/hm2,占土壤总碳储量(0—100 cm)的47.99%,土壤氮的分布则无明显规律。相比于纯林,与固氮树种混交的营林方式表现出更大的碳储存能力。3种幼龄人工林生态系统较低的地上与地下部分碳氮分配比,表明其仍具有较强的碳氮固持潜力。     

不同施肥措施下亚热带稻田土壤碳、氮演变特征及其耦合关系

在湖南省稻田生态系统长期定位监测点研究了不同施肥措施下稻田土壤碳、氮演变及其耦合特征.结果表明：1986—2003年,无肥处理（对照,CK）稻田土壤有机碳和全氮含量略呈下降趋势;化肥（NPK）处理有机碳和全氮含量基本保持稳定,而有机 无机肥配施处理有机碳和全氮含量均呈增加趋势.与对照相比,化肥处理的土壤有机碳和全氮含量分别提高13%和18%,低量有机肥（LOM）处理分别提高54%和45%,高量有机肥（HOM）处理分别提高89%和67%.统计分析表明,土壤有机碳和全氮含量呈显著正相关（P<0.01）.稻田土壤C/N为8.5～12.9,多数分布在10左右.研究表明,有机-无机肥配合施用能在一定程度上促进稻田土壤碳、氮的固定与积累;稻田土壤碳、氮具有较好的耦合关系.     

The use of carbon isotope ratios to evaluate legume contribution to soil enhancement in tropical pastures

Soil carbon distribution with depth, stable carbon isotope ratios in soil organic matter and their changes as a consequence of the presence of legume were studied in three 12-year-old tropical pastures (grass alone —Brachiaria decumbens (C₄), legume alone —Pueraria phaseoloides (C₃) and grass + legume) on an Oxisol in Colombia. The objective of this study was to determine the changes that occurred in the¹³C isotope composition of soil from a grass + legume pasture that was established by cultivation of a native savanna dominated by C₄ vegetation. The¹³C natural abundance technique was used to estimate the amount of soil organic carbon originating from the legume. Up to 29% of the organic carbon in soil of the grass + legume pasture was estimated to be derived from legume residues in the top 0–2-cm soil depth, which decreased to 7% at 8–10 cm depth. Improvements in soil fertility resulting from the soil organic carbon originated from legume residues were measured as increased potential rates of nitrogen mineralization and increased yields of rice in a subsequent crop after the grass + legume pasture compared with the grass-only pasture. We conclude that the¹³C natural abundance technique may help to predict the improvements in soil quality in terms of fertility resulting from the presence of a forage legume (C₃) in a predominantly C₄ grass pasture.