Adhesion of fimbriated nitrogen-fixing enteric bacteria to roots of grasses and cereals

Summary The role of fimbriae in enterobacterial adhesion to roots of grasses and cereals is discussed. All nitrogen-fixing enteric bacteria isolated in Finland had fimbriae. AllEnterobacter isolates had mannose-binding type-1 fimbriae, whereas most of theKlebsiella isolates had both type-1 and type-3 fimbriae. The strains were isolated from a total of ten different grass species, and no specific association was found between grass species and bacterial fimbriation, biogroup or serogroup. Purified, radiolabeled fimbriae bound to roots ofPoa pratensis in vitro, and bacterial adhesion was inhibited by Fab fragments specific for fimbriae.Klebsiella strains carrying type-3 fimbriae adhered to roots of various grasses and cereals more efficiently than type-1- or nonfimbriated strains, and it was concluded that type-3 fimbriae are the major adhesions ofKlebsiella. Immunofluorescence studies revealed that the bacteria preferentially adhered to root hairs, and to a lesser extent, to the zone of elongation and the root cap mucilage. No strict host specificity in enterobacterial adhesion was observed.     

Nutrition of sorghum plants fertilized with nitrogen or inoculated withAzospirillum brasilense

Summary Sorghum plants were inoculated withAzospirillum brasilense or received an N-amended nutrient solution. Azospirillum inoculation increased plant dry weight and nitrogen assimilation by 25%. Most plant growth responses to Azospirillum were comparable to application of 2.0 mM N. Increased scavenging of nutrients, altered root permeability or nitrogen fixation are possible explanations for these effects.This work was supported by the United States Department of Agriculture, Agricultural Research Service (CRIS No. 5102-20170-001) in collaboration with the University of California, Berkeley. Requests for reqrints to G. J. Bethlenfalvay.     

化感水稻PI312777苗期根系分泌物中化学成分分析

在接近自然栽培的田间土壤条件下,以无种植水稻秧苗的田间土壤为对照,采用循环法分别收集化感水稻品种PI312777(PI)苗期(3～4叶)根系分泌物和对照土壤溶液,经乙醚萃取,GC-MS分析和仪器谱图库(NIST98 & WILEY)检索.结果表明,在水稻PI根系分泌物的乙醚萃取物中检测到3个化合物,其中萜类9个(峰面积10.97%)、酚醌类8个(5.87%)、酯类个(10.8%)、醛酮类3个(1.44%)、杂环类4个(8.04%)、醇类2个(1.23%)、醚类2个(0.57%)和其他物质2个(1.20%),萜类之间、酚醌类之间的化合物结构具有明显的相似性.在对照土壤溶液的乙醚萃取物中检测到39个化合物,其中有7个化合物与水稻PI根系分泌物中化合物相同.此外,还讨论了水稻根系分泌物与对照土壤溶液之间物质的差异和各类物质的化感作用机理.     

联合固氮菌叶面接种剂的优化及其在玉米叶际的定殖

【背景】联合固氮菌由于不具有宿主专一性,在土壤、叶际中广泛存在,对生态系统氮素供应有着重要贡献,它还可以通过分泌生长激素等间接作用促进植物生长,可作为重要的农业生产菌剂。土壤接种剂由于受土著微生物的竞争和土壤抑菌物质等的影响,接种效果不稳定,难以推广使用。相比于土壤环境,叶际生境相对简单且表面积巨大,进行叶际接种是固氮菌剂推广应用的一个新思路。【目的】优化联合固氮菌菌株W12接种添加剂,制备液体接种剂并研究其在玉米叶际的定殖效果。【方法】对菌株W12进行菌落PCR测序,构建系统发育树并确定分类地位。分别在培养液中添加不同浓度梯度的羧甲基纤维素(Carboxymethyl cellulose,CMC)和甘油(Glycerol,Gly),测量菌株W12生长曲线和固氮酶活性,优化添加剂浓度并制备液体接种剂,对接种剂的有效保存时间进行检测。将接种剂喷洒到玉米叶际,测量其对玉米产量和植株含氮量的影响,并通过低氮培养基进行回收计数。【结果】固氮菌菌株W12的16S r RNA基因序列与变栖克雷伯氏菌(Klebsiella variicola)的相似性高达99%,在培养液中添加CMC和甘油对菌株W12的生长无明显促进和抑制效果,但均提高了固氮酶活性。添加甘油制备的接种剂在盆栽和大田玉米叶面喷施后,在玉米生长末期叶际回收到的W12类似菌分别为4.3×105 CFU/g叶片和1.7×105 CFU/g叶片,显著高于未接种的处理;而且大田玉米籽粒、茎部和叶片的含氮量高于不接种的对照处理。经过90 d贮藏后,4°C保存的接种剂剩余活菌数均高于1.0×108 CFU/m L。【结论】羧甲基纤维素和甘油的添加不仅有利于固氮菌液体接种剂在叶片的附着,并能显著提高联合固氮菌菌株W12的固氮酶活性,低温冷藏可保证液体接种剂的有效活菌数;液体接种剂在玉米叶际喷施后,菌株W12能够成功定殖,并显著提高玉米植株和籽粒含氮量。研究结果为固氮菌叶面接种剂的制备和应用,以及实现农业氮肥减施保产的目标提供了借鉴意义。     

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。     

Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules

Summary Nitrogen-fixing peanut root nodules are characterized by their unique structural organization, distinct from other legume nodules. The focus of this study has been in and around the hostsymbiont interface, where the bacterioid and the host cell surface (peribacteroid membrane envelope) interact during symbiosis. The infected nodule cells have revealed the presence of lipid bodies (oleosomes) in intimate association with the peribacteroid membrane, which encloses the large spherical bacteroids with a relatively narrow peribacteroid space. Electron dense structures, referred to as dense bodies have been found attached to the bacteroid outer membranes at the host-symbiont interface. The dense bodies are osmiophilic, amorphous and 3,3-diaminobenzidine positive. The isolated intact bacteroids with dense bodies attached to their cell wall showed significant catalase activity. Many microbodies showing DAB-positive reaction have been found in the host cytoplasm, associated closely with the peribacteroid membrane. These ultrastructural and cytochemical characteristics of peanut root nodules suggest that lipids are utilized during symbiosis and the dense bodies and microbodies may be involved in the catabolic process.Abbreviation DAB 3,3-diaminobenzidine     

Ammonium effects on function and structure of nitrogen-fixing root nodules of Alnus incana (L.) Moench

Cloned plants of Alnus incana (L.) Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammonium on the function and structure of the root nodules were studied by adding 20 mM NH₄Cl (20 mM KCl=control) for four days. Nitrogenase activity decreased to ca. 50% after one day and to less than 10% after two days in ammonium treated plants, but was unaffected in control plants. The results were similar at photon flux densities of 200 and 50 mol m^-2 s^-1. At the higher light level the effect was concentration dependent between 2 and 20 mM NH₄Cl. The recovery was slow, and more than 11 d were needed for plants treated with 20 mM ammonium to reach initial activity. The distribution of ¹⁴C to the root nodules after assimilation of ¹⁴CO₂ by the plants was not changed by the ammonium treatment. Microscopical studies of root nodules showed high frequencies of endophyte vesicles being visually damaged in nodules from ammonium-treated plants, but not in nodules from control plants. When nitrogenase activity was restored, visually damaged vesicles were again few, whereas young developing vesicles were numerous. The slow recovery, the ¹⁴C-translocation pattern, and the structural changes of the endophyte indicate a more complex mechanism of ammonium influence than simply a short-term reduction in supply of carbon compounds to the nodules.     

Endophytic colonization of plant roots by nitrogen-fixing bacteria

Plant and Soil - Nitrogen-fixing bacteria are able to enter into roots from the rhizosphere, particularly at the base of emerging lateral roots, between epidermal cells and through root hairs. In...     

不同供氮水平下水曲柳（Fraxinus mandushurica Rupr.）幼苗根系呼吸季节动态

根呼吸是林木根系获得吸收养分和水分所需能量的重要生理活动.为了探讨林木根系呼吸速率的季节变化及其影响因素,采用离体根系法(Li-6400-06叶室连接到Li-6400便携式CO2/H2O分析系统)研究了水曲柳(Fraxinus mandushurica Rupr.)苗木各径级根呼吸速率在不同供氮水平下季节变化规律.结果表明:水曲柳苗木根呼吸速率表现出明显的季节动态,且与气温的季节变化规律相同,其中比根呼吸速率在0.5732 μmolCO2 · g-1 · s-1(直径≤2 mm,10月份)～7.1861 μmolCO2 · g-1 · s-1(直径≤2 mm,7月份)之间变化,表面积呼吸速率也是7月份最高,达到0.6848 μmolCO2 · cm-2 · s-1(直径>5 mm),10月份最低,仅为0.0132 μmolCO2 · cm-2 · s-1(直径≤2 mm);比根呼吸速率随根直径变大而降低,表面积呼吸速率变化规律则完全相反.供氮水平对水曲柳苗木根呼吸速率的影响随气温升高明显增强,其在6～8月份各径级根系中均达到显著水平(0.00072mm)Q10值范围为2.07～2.96,Q10值随根系径级增大而降低的现象表明水曲柳苗木细根对温度变化反应更为敏感;细根Q10值在供氮水平间差异显著(P=0.0392<0.05),粗根则不明显,表明土壤供氮水平主要影响细根的Q10值变化.     

根土空间对高粱根系生理特性及产量的影响

采用尼龙袋（允许水分和养分自由通过）装土栽培作物的方法,研究了根系生长空间对高粱根系生理特性及产量的影响.结果表明,限制根系生长空间影响了高粱的生长,不同程度地降低了高粱的株高、叶面积、花后旗叶SOD及POD活性、总根长、根系吸收面积、根系与地上部干重、养分吸收量及最终产量,但增加了根系活力及活性吸收面积占总吸收面积的百分数.施肥有利于改善高粱根系在空间胁迫下的生长,增加根系吸收面积和活力,促进根系对养分的吸收,在一定程度上延缓根系生长空间不足所造成的不良影响.     

Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges

The potential of nitrogen-fixing (NF) bacteria to form a symbiotic relationship with leguminous plants and fix atmospheric nitrogen has been exploited in the field to meet the nitrogen requirement of the latter. This phenomenon provides an alternative to the use of the nitrogenous fertiliser whose excessive and imbalanced use over the decades has contributed to green house emission (N(2)O) and underground water leaching. Recently, it was observed that non-leguminous plants like rice, sugarcane, wheat and maize form an extended niche for various species of NF bacteria. These bacteria thrive within the plant, successfully colonizing roots, stems and leaves. During the association, the invading bacteria benefit the acquired host with a marked increase in plant growth, vigor and yield. With increasing population, the demand of non-leguminous plant products is growing. In this regard, the richness of NF flora within non-leguminous plants and extent of their interaction with the host definitely shows a ray of hope in developing an ecofriendly alternative to the nitrogenous fertilisers. In this review, we have discussed the association of NF bacteria with various non-leguminous plants emphasizing on their potential to promote host plant growth and yield. In addition, plant growth-promoting traits observed in these NF bacteria and their mode of interaction with the host plant have been described briefly.     

Most probable numbers of Azospirillum associated with the roots of inoculated pearl millet

Summary Inoculation of pearl millet (Pennisetum americanum (L.) Leeke) with Azospirillum significantly increased the numbers of this organism in the rhizosphere, rhizoplane, washed and crushed roots and surface sterilized and crushed roots. The maximum number of organisms plant^–1 were localized in the rhizosphere. The numbers of Azospirillum on the roots of inoculated plants grown under sterilized conditions were much higher than in the field grown plants. In both cases populations outside the roots were higher than in the surface sterilized roots. The highest numbers per unit root weight were recorded between 60–75 days of growth. N₂-ase activity throughout the growth cycle was very low and was not related to the populations of Azospirillum on the roots. Root exudates and extracts of pearl millet showed a stimulatory effect on the growth of Azospirillum suggesting their possible involvement in the colonization of this organism on the roots of inoculated plants.     

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.     

Utilization of simple phenolics for dinitrogen fixation by soil diazotrophic bacteria

Summary A microaerobic diazotrophic bacterium tentatively identified as aPseudomonas species was isolated from a forest soil. Its nitrogenase (C₂H₂ reduction) activity in liquid medium was significantly supported by phenolic compounds when compared with glucose-, mannitol- or malate-supported activity. The utilization of phenolics was dependent on substrate induction and the appropriate oxygen concentration. At a pO₂ of 0.05 protocatechuate was a better carbon source for N₂ fixation than glucose. In the case ofLignobacter protocatechuate was a better carbon source for N₂ fixation than glucose at pO₂ 0.2 but not at pO₂ 0.05. It is suggested that certain monomeric phenols can support nitrogenase activities in many carbon-limited soil environments.Contribution No. 1484 from the Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Canada.     

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia , the nitrogen-fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O₂ transport.     

Effects of increased atmospheric CO2, temperature,and soil N availability on root exudation of dissolved organic carbon by a N-fixing tree (Robinia pseudoacacia L.)

Root exudation has been hypothesized as one possible mechanism that may lead to increased inputs of organic C into the soil under elevated atmospheric CO₂, which could lead to greater long-term soil C storage. In this study, we analyzed exudation of dissolved organic C from the roots of seedlings of the N-fixing tree Robinia pseudoacacia L. in a full factorial design with 2 CO₂ (35.0 and 70.0 Pa) × 2 temperature (26° and 30 °C during the day) × 2 N fertilizer (0 and 10.0 mM N concentration) levels. We also analyzed the decomposition rates of root exudate to estimate gross rates of exudation. Elevated CO₂ did not affect root exudation of organic C. A 4 °C increase in temperature and N fertilization did, however, significantly increase organic C exudation rates. Approximately 60% of the exudate decomposed relatively rapidly, with a turnover rate of less than one day, while the remaining 40% decomposed more slowly. These results suggest that warmer climates, as predicted for the next century, may accelerate root exudation of organic C, which will probably stimulate rapid C cycling and may make a minor contribution to intermediate to more long-term soil C storage. However, as these losses to root exudation did not exceed 1.2% of the net C fixed by Robinia pseudoacacia, root exudation of organic C appears to have little potential to contribute to long-term soil C sequestration. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 intracellularly 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.     

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.     

Memoir of a 1949 railway journey with photosynthetic bacteria

A serendipic observation at the Hopkins Marine Station of Stanford University in 1948 led to the discovery that anoxygenic photosynthetic bacteria can fix molecular nitrogen. To confirm the discovery, an unusual collaborative event was arranged between laboratories at Washington University (St. Louis) and the University of Wisconsin (Madison).