泛析提高共生固氮效率

泛析提高共生固氮效率徐秀华（辽宁省农业管理干部学院,沈阳１１０１６１）当前人们对共生固氮效率与豆科植物种类、根瘤菌品系、固氮环境的关系已具有深刻的认识。值得探讨的是如何深入地发掘共生固氮潜力,提高共生固氮效率,为创造真正的＂绿色食品＂开拓门路。一共生...     

与某些非豆科植物共生的弗兰克氏菌(Frankia sp.)的泡囊及其固氮作用

非豆科植物的共生固氮作用大多数是由弗兰克氏菌的侵染形成根瘤引起。其中泡囊是弗氏菌在根瘤内的固氮场所(Akkermans等1977,Tjepkema等1981)。我们采用光学及电子显微镜对泡囊进行了观察,并对根瘤不同部位的泡囊数量及其固氮活性与其它生理活性的关系进行了研究。     

新疆干旱地区根瘤菌资源研究I．根瘤菌种类及其共生固氮作用

从新疆不同生态条件下生长的31属109种豆科植物根瘤分离获得373株根瘤菌,其中88株是从未报道过结瘤情况的39种豆科植物根瘤分离获得。它们与豆科寄主植物共生结瘤,95％以上为有效根瘤。不同种的根瘤固氮活性差异较大,黄芪属根瘤固氮活性较高,最高者为大豆根瘤固氮活性的42倍。 20属37种豆科植物根瘤中97％有吸氢活性。根瘤固氮活性、吸氢活性均与寄主植物生长发育期有相关性。     

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。     

弗兰克氏菌在构建新的共生固氮物种研究中的优越性

弗兰克氏菌在构建新的共生固氮物种研究中的优越性解放军石家庄医学高等专科学院０５００８１曲东明范浩南四川师范大学细胞研究室６１００６８韩善华弗兰克氏菌（Ｆｒａｎｋｉａ）能与多种木本被子植物建立共生固氮关系,它形成的根瘤具有较强的固氮能力,有些超过大豆根...     

糖蜜草(Melinis minutiflora Beauv.)内生固氮菌分离鉴定

糖蜜草（Melinis minutiflora Beauv．）是热带地区的一种优良牧草。采用选择性培养基在厌氧和好氧两种培养条件下,从糖蜜草根、茎中都可分离得到具有较强固氮酶活性的菌株。通过SDS-PAGE全细胞蛋白电泳技术快速聚类分析表明,来源于糖蜜草中的菌株为同一类群。16S rDNA序列分析和总DNA的G＋c％含量进一步确定糖蜜草中所分离的菌株属于固氮螺菌属（Azospirillum）,与产脂固氮螺菌（Azospirillum lipoferum）亲缘关系较近。BIOLOG板测定结果显示,糖蜜草菌株TMCY243对多种碳源具有很强的适应性,与产脂固氮螺菌（A．lipoferum）的模式菌株DSM 1691存在着较大的差异。以上结果表明,糖蜜草内生固氮菌为固氮螺菌属的一个新类群。     

甘蔗器官固氮酶活性及其对接种固氮菌的响应

以巴西固氮甘蔗品种B1、B8和广西主栽甘蔗品种ROC22和ROC16为材料,通过桶栽砂培方法,研究不同品种甘蔗及接种固氮菌R16SH后不同生育期和不同器官(根、茎、叶片、叶鞘)的固氮酶活性.结果显示,4个品种甘蔗及接菌后各器官的固氮酶活性均在伸长初期或伸长盛期达到高峰,且以根和茎较高,叶、叶鞘次之;B1、B8根的接菌效果最佳,其固氮酶活性分别比对照增加13.22%和12.42%,而B1和ROC16茎的固氮酶活性分别比对照提高12.19%和12.02%,叶片提高了8%~9%,叶鞘的固氮酶活性变化最小;4个品种甘蔗固氮酶活性表现为B8>B1>ROC22>ROC16,且品种B8与ROC22和ROC16存在显著或极显著差异;相对于品种ROC22和ROC16,固氮菌R16SH对B1与B8的株高以及B1的茎径有更好的生长诱导效应,且在伸长期表现最明显.研究表明,甘蔗的固氮酶活性在品种、器官及生育期间均存在明显差异,接种固氮菌能不同程度提高各品种器官的固氮酶活性,且各品种根、茎以及B1和B8的叶片固氮酶活性显著或极显著差异增加;巴西固氮甘蔗品种表现出比广西主栽甘蔗品种更强的固氮酶活性,且对接种固氮菌反应更敏感.     

自生固氮菌的生态分布及其对农药抗性的研究

将不同地点的土样均匀地撒在无氮培养基平板上,分离并鉴定自生固氮菌。同时采用抑菌圈法和分光光度计测透光率的方法,检测6种常用的田间农药对自生固氮菌的生长影响;还运用茚三酮显色反应检测农药对自生固氮菌固氮效果的影响。结果表明,不同农药及其不同浓度对自生固氮菌生长及固氮量的影响有所不同。为添加固氮菌以提高农作物产量,监测固氮菌含量来评估作物产量,以及田间正确使用农药提供了理论依据。     

植物内生固氮菌及其固氮机理研究进展

氮素是植物生长必不可少的元素,植物内生固氮菌不仅能够在植物体内产生氮素以供植物利用,而且在自然界氮素循环过程中发挥积极作用,对农业可持续发展具有重要意义。近年来,植物内生固氮菌逐渐成为研究热点。由植物内生固氮菌的发现、作物共生、侵入途径、固氮机理、促生作用机制等方面系统地综述了植物内生固氮菌的研究进展,探讨了植物内生固氮菌新的研究思路以及一些尚未解决的问题,以期为植物内生固氮菌及生物固氮研究提供参考。     

Enterobacteria-mediated nitrogen fixation in natural populations of the fruit fly Ceratitis capitata

Nitrogen, although abundant in the atmosphere, is paradoxically a limited resource for multicellular organisms. In the Animalia, biological nitrogen fixation has solely been demonstrated in termites. We found that all individuals of field-collected Mediterranean fruit flies (Ceratitis capitata) harbour large diazotrophic enterobacterial populations that express dinitrogen reductase in the gut. Moreover, nitrogen fixation was demonstrated in isolated guts and in live flies and may significantly contribute to the fly's nitrogen intake. The presence of similar bacterial consortia in additional insect orders suggests that nitrogen fixation occurs in vast pools of terrestrial insects. On such a large scale, this phenomenon may have a considerable impact on the nitrogen cycle.     

What do we know about biological nitrogen fixation in insects? Evidence and implications for the insect and the ecosystem

Many insects feed on a low‐nitrogen diet, and the origin of their nitrogen supply is poorly understood. It has been hypothesized that some insects rely on nitrogen‐fixing bacteria (diazotrophs) to supplement their diets. Nitrogen fixation by diazotrophs has been extensively studied and convincingly demonstrated in termites, while evidence for the occurrence and role of nitrogen fixation in the diet of other insects is less conclusive. Here, we summarize the methods to detect nitrogen fixation in insects and review the available evidence for its occurrence (focusing on insects other than termites). We distinguish between three aspects of nitrogen fixation investigations: (i) detecting the presence of potential diazotrophs; (ii) detecting the activity of the nitrogen‐fixing enzyme; and (iii) detecting the assimilation of fixed nitrogen into the insect tissues. We show that although evidence from investigations of the first aspect reveals ample opportunities for interactions with potential diazotrophs in a variety of insects, demonstrations of actual biological nitrogen fixation and the assimilation of fixed nitrogen are restricted to very few insect groups, including wood‐feeding beetles, fruit flies, leafcutter ants, and a wood wasp. We then discuss potential implications for the insect's fitness and for the ecosystem as a whole. We suggest that combining these multiple approaches is crucial for the study of nitrogen fixation in insects, and argue that further demonstrations are desperately needed in order to determine the relative importance of diazotrophs for insect diet and fitness, as well as to evaluate their overall impact on the ecosystem.     

Changes in Asymbiotic, Heterotrophic Nitrogen Fixation on Leaf Litter of Metrosideros polymorpha with Long-Term Ecosystem Development in Hawaii

We measured nitrogenase activity (acetylene reduction) of asymbiotic, heterotrophic, nitrogen-fixing bacteria on leaf litter from the tree Metrosideros polymorpha collected from six sites on the Hawaiian archipelago. At all sites M. polymorpha was the dominant tree, and its litter was the most abundant on the forest floor. The sites spanned a soil chronosequence of 300 to 4.1 million y. We estimated potential nitrogen fixation associated with this leaf litter to be highest at the youngest site (1.25 kg ha^-1 y^-1), declining to between 0.05 and 0.22 kg ha^-1 y^-1 at the oldest four sites on the chronosequence. To investigate how the availability of weathered elements influences N fixation rates at different stages of soil development, we sampled M. polymorpha leaf litter from complete, factorial fertilization experiments located at the 300-y, 20,000-y and 4.1 million–y sites. At the youngest and oldest sites, nitrogenase activity on leaf litter increased significantly in the plots fertilized with phosphorus and “total” (all nutrients except N and P); no significant increases in nitrogenase activity were measured in leaf litter from treatments at the middle-aged site. The results suggest that the highest rates of N fixation are sustained during the “building” or early phase of ecosystem development when N is accumulating and inputs of geologically cycled (lithophilic) nutrients from weathering are substantial. Received 4 February 1999; accepted 29 March 2000.     

Asymbiotic nitrogen fixation in the litter and surface soil of a Pinus laricio Poiret forest (Sila,Calabria, Italy)

Abstract

Nitrogen fixation was measured in a Corsican pine (Pinus laricio Poiret) forest in Calabria (Southern Italy). Acetylene reduction activity (ARA) and CO₂ production levels were determined by incubation of litter and superficial (0–5 cm) soil layer samples in the field, at monthly intervals. ARA variations were not correlated to those of substrate moisture, air temperature and microbial respiration. In fact N₂ fixation presented phases of different intensity which irregularly followed each other. Both litter and soil showed similar rates of N₂ fixation. Based on a C₂H₂:N₂ ratio of 3:1 0.8 Kg N ha^–1 y^–1 in each layer are fixed in the Pinus laricio forest, thus contributing to the N status of the soil in this nutrient–poor forest.     

甘蔗联合固氮的研究进展及应用潜力

在农业生产中,化学氮肥的投入大幅度增加了粮食产量,然而过量或不合理的施肥措施对农业生态环境造成了严重破坏.因此,挖掘植物自身的生物学特性,寻求其他有效的氮素来源,对农业减肥增效至关重要.其中,植物与微生物之间的生物固氮作用,能为宿主提供大量的清洁氮源,在农业生产中发挥着不可替代的作用.本文以甘蔗为代表,综述了植物联合固...     

土壤pH调控固氮植物和非固氮植物间的氮转移

氮作为构成蛋白质的主要成分, 是植物生长的必要营养物质。陆地生态系统普遍存在土壤氮缺乏的现象, 混交种植模式中固氮植物可以将生物固定的氮转移给非固氮植物, 是满足非固氮植物氮需求的途径之一。明确固氮和非固氮植物间氮转移的影响因素有助于恢复退化生态系统, 构建稳定群落, 增加生态系统生产力。为了量化环境及生物等因素对氮转移的影响, 该研究采用文献调研法, 对118组氮转移比例(氮转移量占非固氮植物氮含量的比值, P_transfer)文献和实验数据(包括21种固氮植物和23种非固氮植物)进行了线性混合模型分析。结果表明土壤pH是影响P_transfer变化的最主要因素(解释量为44.04%), 其次为年平均温度(解释量为9.14%)以及固氮与非固氮植物生物量比值(解释量为2.95%), 而作为随机因素的固氮和非固氮植物物种差异的解释量为16.52%。此外, 碱性土壤中P_transferr显著高于酸性土壤。在酸性土壤中, 年平均温度(解释量为12.49%)和土壤总氮含量(解释量为11.72%)是影响P_transfer差异的主要因素, P_transfer随着年平均温度和土壤总氮含量的增加而显著增加。而在碱性土壤中, P_transfer差异主要受到固氮与非固氮植物生物量比值(解释量为13.29%)、年降水量(解释量为10.73%)和土壤总氮含量(解释量为9.33%)的调控。相对于酸性土壤, 碱性土壤能够显著增加固氮与非固氮植物生物量比值进而增加P_transfer。同时, 在碱性土壤中P_transfer与年降水量和土壤总氮含量呈显著正相关关系。这些结果对提高固氮和非固氮植物间的氮转移, 有效缓解土壤氮对非固氮植物生长的限制以及构建稳定群落具有重要意义。     

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。     

冬季低温及模拟升温对生物土壤结皮固氮活性的影响

以腾格里沙漠东南缘固沙植被区和相邻天然植被区发育的藻类和藓类结皮为研究对象,采用不同规格的OTCs研究了冬季低温及短期模拟升温对其固氮活性的影响。结果表明:不同规格的OTCs装置冬季全天气温升温幅度在1℃左右,不同深度土层升温幅度更加明显,约为3.2℃;冬季试验期,湿润条件下藻类和藓类结皮均具有固氮活性,平均固氮活性分别为1.2×104和0.4×104nmolC2H4·m-2·h-1,藻类结皮的固氮活性显著高于藓类结皮(P<0.01);试验期藻类和藓类结皮的固氮活性均与培养期气温显著正相关(P<0.001),与试验前3d降水量也呈显著正相关(P<0.001)。低温湿润冷冻环境下,结皮生物体胞内冰晶形成而导致的固氮酶体系受损可能是造成冬季结皮固氮活性降低的主要原因,冬季升温能促进结皮固氮活性的提高。本研究表明,在未来全球变暖和降水格局变化背景下,冬季升温能促进生物土壤结皮对区域生态系统的氮贡献。     

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems,and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intraceliularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers,we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus,with minimal or zero inputs.