单细胞水平解析土壤固氮鱼腥藻的碳氮固存过程研究进展

固氮鱼腥藻作为光能自养型微生物,具备良好的碳氮固存能力。施加固氮鱼腥藻能够提升土壤肥力并减少化肥施用量。然而,解析固氮鱼腥藻在土壤中的碳氮固存机制以及不同菌株固存效率差异仍有待深入研究。因此,从单细胞水平对土壤固氮鱼腥藻菌株进行筛选和碳氮固存过程的研究至关重要。针对固氮鱼腥藻在单细胞水平上发生的复杂而动态的元素变化过程,本研究综述了土壤固氮鱼腥藻碳氮固存过程,并探讨了利用纳米二次离子质谱法与稳定同位素标记结合(nano-secondary ion mass spectrometry-stable isotopic probing, NanoSIMS-SIP)技术和拉曼光谱成像与稳定同位素标记结合(Raman spectroscopy imaging-stable isotopic probing, Raman-SIP)技术解析单细胞水平上的碳氮元素的时空分布的原理、进展与难点。重点关注了单细胞稳定同位素技术定量可视化固氮鱼腥藻碳氮固存的最新技术发展与应用。同时,对该类可视化技术的未来研究进行了展望。本研究对于理解固氮鱼腥藻在土壤中的碳氮固存机制和固氮效率差异具有重要的科学意义,为农业生产中减少化肥使用、提高土壤肥力提供理论依据。     

多环芳烃(PAHs)微生物降解的原位表征方法

多环芳烃(polycyclic aromatic hydrocarbons,PAHs)是一类在环境中广泛存在的持久性有机污染物,微生物降解是去除环境中多环芳烃污染的主要途径。传统的有关PAHs微生物降解的研究主要依靠分离培养技术,难以准确认识PAHs微生物降解的原位过程及机制。近年来发展起来的原位表征方法可以在基因及单细胞水平研究PAHs在复杂环境中的微生物降解过程,能够原位表征具有PAHs降解功能的微生物及其功能基因和代谢活性,是阐明PAHs原位降解过程及分子机制的强有力的手段。该文综述了宏基因组技术(meta-genomics)、稳定同位素探针技术(stable isotope probe,SIP)、荧光原位杂交技术(fluorescence in situ hybridization,FISH)、拉曼光谱技术(Raman spectra)以及二次离子质谱技术(secondary ion mass spectrometry,SIMS)等原位表征技术在PAHs微生物降解研究领域的应用及其存在的问题和发展趋势等。PAHs微生物降解过程及机制的原位表征将为缓解与修复PAHs污染提供科学基础。     

Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Nitrogen fixation in Pseudomonas stutzeri

A recently developed oxygen gradient system and a complex medium were used to isolate a microaerobically N₂-fixing heterotrophic bacterium from the rhizosphere of a high fixing Sorghum nutans cultivar. The isolate was identified as nif(+) phenotype of Pseudomonas stutzeri on the basis of cultural, physiological and biochemical characteristics, including DNA/DNA hybridization. N₂ fixation was demonstrated by assimilation of ¹⁵N₂ into cellular protein; the physiology of nitrogen fixation was studied. The isolate contains one 30 MD plasmid and can be cured with associated loss of N₂ fixation capability.Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Dinitrogen fixation in white clover grown in pure stand and mixture with ryegrass estimated by the immobilized 15N isotope dilution method

Dinitrogen fixation in white clover (Trifolium repens L.) grown in pure stand and mixture with perennial ryegrass (Lolium perenne L.) was determined in the field using ¹⁵N isotope dilution and harvest of the shoots. The apparent transfer of clover N to perennial ryegrass was simultaneously assessed. The soil was labelled either by immobilizing ¹⁵N in organic matter prior to establishment of the sward or by using the conventional labelling procedure in which ¹⁵N fertilizer is added after sward establishment. Immobilization of ¹⁵N in the soil organic matter has not previously been used in studies of N₂ fixation in grass/clover pastures. However, this approach was a successful means of labelling, since the ¹⁵N enrichment only declined at a very slow rate during the experiment. After the second production year only 10–16% of the applied ¹⁵N was recovered in the harvested herbage. The two labelling methods gave, nonetheless, a similar estimate of the percentage of clover N derived from N₂ fixation. In pure stand clover, 75–94% of the N was derived from N₂ fixation and in the mixture 85–97%. The dry matter yield of the clover in mixture as percentage of total dry matter yield was relatively high and increased from 59% in the first to 65% in the second production year. The average daily N₂ fixation rate in the mixture-grown clover varied from less than 0.5 kg N ha⁻¹ day⁻¹ in autumn to more than 2.6 kg N ha⁻¹ day⁻¹ in June. For clover in pure stand the average N₂ fixation rate was greater and varied between 0.5 and 3.3 kg N ha⁻¹ day⁻¹, but with the same seasonal pattern as for clover in mixture. The amount of N fixed in the mixture was 23, 187 and 177 kg N ha⁻¹ in the seeding, first and second production year, respectively, whereas pure stand clover fixed 28, 262 and 211 kg N ha⁻¹ in the three years. The apparent transfer of clover N to grass was negligible in the seeding year, but clover N deposited in the rhizosphere or released by turnover of stolons, roots and nodules, contributed 19 and 28 kg N ha⁻¹ to the grass in the first and second production year, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of mycorrhizal associations on foliar δ15N values of legume and non-legume shrubs and trees in the fynbos of South Africa: Implications for estimating N2 fixation using the 15N natural abundance method

In this study, we examined the use of the ¹⁵N natural abundance method to quantify the percentage N derived from fixation of atmospheric N₂ in honeybush (Cyclopia spp.) shrubs and trees in the fynbos, South Africa. Non-fixing shrubs and trees of similar phenology to the Cyclopia species were chosen as reference plants. These reference plants were selected to cover a range of mycorrhizal associations (ericoid mycorrhizal, arbuscular mycorrhizal and non-mycorrhizal). Isotopic analysis revealed a wide range of foliar ¹⁵N values for the reference plants, including many very negative values. The marked differences in ¹⁵N values were defined by the mycorrhizal status of the reference plant species, with the ericoid and arbuscular mycorrhizal plants showing lower foliar ¹⁵N values relative to their non-mycorrhizal counterparts. In contrast, the ¹⁵N values of the N₂-fixing Cyclopia species were uniformly clustered around zero, from –0.11 to –1.43. These findings are consistent with the observation that mycorrhizal fungi discriminate against the heavier ¹⁵N isotope during transfer of N from the fungus to the host plant, leaving the latter depleted in ¹⁵N (i.e. with a more negative ¹⁵N value). However, a major assumption of the ¹⁵N natural abundance method for estimating N₂ fixation is that both legume and reference plant should have the same level of fractionation associated with N uptake. But, because mycorrhizal associations may strongly affect the level of fractionation during N uptake and transfer, the test legume should belong to the same mycorrhizal group as the chosen reference plant species. As shown in this study, if the mycorrhizal status of the legume and the reference plant differs, or cannot be assessed, then the ¹⁵N natural abundance technique cannot be used to quantitatively estimate N₂ fixation.     

15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.     

15N2 incorporation and acetylene reduction by Azospirillum isolated from rice roots and soils

Summary Nitrogen fixation by strains of Azospirillum isolated from several rice soils and rice cultivars was investigated by¹⁵N₂ incorporation and C₂H₂ reduction. C₂H₂ reducing ability markedly varied among the strains obtained from soils differing widely in their physico-chemical properties. Large variations in¹⁵N₂ incorporation by Azospirillum isolated from the roots of several rice cultivars were also noticed. The present study reveals that rice cultivars harbour Azospirillum with differential N₂-fixing ability and that plant genotype is of importance for optimal associations.     

The effect of a single application of cow urine on annual N2 fixation under varying simulated grazing intensity, as measured by four 15N isotope techniques

The effects of dairy cow urine and defoliation severity on biological nitrogen fixation and pasture production of a mixed ryegrass-white clover sward were investigated over 12 months using mowing for defoliation. A single application of urine (equivalent to 746 kg N ha^–1), was applied in late spring to plots immediately after light and moderately-severe defoliation (35 mm and 85 mm cutting heights, respectively) treatments were imposed. Estimates of percentage clover N derived from N₂ fixation (%Ndfa) were compared by labelling the soil with ¹⁵N either by applying a low rate of ¹⁵N-labelled ammonium sulphate, immobilising ¹⁵N in soil organic matter, adding ¹⁵N to applied urine, or by utilising the small differences in natural abundance of ¹⁵N in soil. Urine application increased annual grass production by 85%, but had little effect on annual clover production. However, urine caused a marked decline in %Ndfa (using an average of all ¹⁵N methods) from 84% to a low of 22% by 108 days, with recovery to control levels taking almost a year. As a result, total N fixed (in above ground clover herbage) was reduced from 232 to 145 kg N ha^–1 yr^–1. Moderately–severe defoliation had no immediate effect on N₂ fixation, but after 108 days the %Ndfa was consistently higher than light defoliation during summer and autumn, and increased by up to 18%, coinciding with an increase in growth of weeds and summer-grass species. Annual N₂ fixation was 218 kg N ha^–1 yr^–1 under moderately-severe defoliation compared to 160 kg N ha^–1 yr^–1 under light defoliation. Estimates of %Ndfa were generally similar when ¹⁵N-labelled or immobilised ¹⁵N were used to label soil regardless of urine and defoliation severity. The natural abundance technique gave highly variable estimates of %Ndfa (–56 to 24%) during the first 23 days after urine application but, thereafter, estimates of %Ndfa were similar to those using ¹⁵N-labelling methods. In contrast, in urine treated plots the use of ¹⁵N-labelled urine gave estimates of %Ndfa that were 20–30% below values calculated using conventional ¹⁵N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in ¹⁵N distribution with depth. This study shows that urine has a prolonged effect on reducing N₂ fixation in pasture. In addition, defoliation severity is a potential pasture management tool for strategically enhancing N₂ fixation.