Microbiological strategies for enhancing biological nitrogen fixation in nonlegumes

In an agro-ecosystem, industrially produced nitrogenous fertilizers are the principal sources of nitrogen for plant growth; unfortunately these also serve as the leading sources of pollution. Hence, it becomes imperative to find pollution-free methods of providing nitrogen to crop plants. A diverse group of free-living, plant associative and symbiotic prokaryotes are able to perform biological nitrogen fixation (BNF). BNF is a two component process involving the nitrogen fixing diazotrophs and the host plant. Symbiotic nitrogen fixation is most efficient as it can fix nitrogen inside the nodule formed on the roots of the plant; delivering nitrogen directly to the host. However, most of the important crop plants are nonleguminous and are unable to form symbiotic associations. In this context, the plant associative and endophytic diazotrophs assume importance. BNF in nonlegumes can be encouraged either through the transfer of BNF traits from legumes or by elevating the nitrogen fixing capacity of the associative and endophytic diazotrophs. In this review we discuss mainly the microbiological strategies which may be used in nonleguminous crops for enhancement of BNF.     

作物根际联合固氮芽孢杆菌的分离鉴定及生态分布

本文报导一种富集和分离根际联合固氮芽孢杆菌的方法,该方法以果胶为唯一碳源的培养基进行富集,中性红为指示剂的培养基进行分离,根据固氮芽孢杆菌能产气以及在分离培养基平板上菌落为红色两个重要的鉴别性特征即可检出,准确性高,简便快速;应用此法从１５个地点６种作物共８８个根系样品中分离出５１株固氮芽孢杆菌,经鉴定均为多粘芽孢杆菌（Ｂａｃｉｌｕｓｐｏｌｙｍｙｘａ）;还初步讨论了多粘芽孢杆菌在几种作物根际的分布情况。     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

Insights into the history of the legume‐betaproteobacterial symbiosis

The interaction between legumes and rhizobia has been well studied in the context of a mutualistic, nitrogen‐fixing symbiosis. The fitness of legumes, including important agricultural crops, is enhanced by the plants’ ability to develop symbiotic associations with certain soil bacteria that fix atmospheric nitrogen into a utilizable form, namely, ammonia, via a chemical reaction that only bacteria and archaea can perform. Of the bacteria, members of the alpha subclass of the protebacteria are the best‐known nitrogen‐fixing symbionts of legumes. Recently, members of the beta subclass of the proteobacteria that induce nitrogen‐fixing nodules on legume roots in a species‐specific manner have been identified. In this issue, Bontemps et al. reveal that not only are these newly identified rhizobia novel in shifting the paradigm of our understanding of legume symbiosis, but also, based on symbiotic gene phylogenies, have a history that is both ancient and stable. Expanding our understanding of novel plant growth promoting rhizobia will be a valuable resource for incorporating alternative strategies of nitrogen fixation for enhancing plant growth.     

Isolation of genomic DNA from medicinal plants without liquid nitrogen

Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the lambda260/lambda280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.     

Global database of plants with root‐symbiotic nitrogen fixation: NodDB

Plants associated with symbiotic N‐fixing bacteria play important roles in early successional, riparian and semi‐dry ecosystems. These so‐called N‐fixing plants are widely used for reclamation of disturbed vegetation and improvement of soil fertility in agroforestry. Yet, available information about plants that are capable of establishing nodulation is fragmented and somewhat outdated. This article introduces the NodDB database of N‐fixing plants based on morphological and phylogenetic evidence (available at https://doi.org/10.15156/bio/587469 ) and discusses plant groups with conflicting reports and interpretation, such as certain legume clades and the Zygophyllaceae family. During angiosperm evolution, N‐fixing plants became common in the fabid rather than in the ‘nitrogen‐fixing’ clade. The global GBIF plant species distribution data indicated that N‐fixing plants tend to be relatively more diverse in savanna and semi‐desert biomes. The compiled and re‐interpreted information about N‐fixing plants enables accurate analyses of biogeography and community ecology of biological N fixation.     

禾本科植物联合固氮研究及其应用现状展望

综述了近年来从禾本科植物体内和根际发现的内生固氮菌和根际固氮菌的种类、特征及对宿主的促生机理,以及固氮菌接种剂在农业生产中的应用现状和存在的问题,指出影响联合固氮菌接种效果的主要因素有土著微生物的竞争;植物基因型差异和环境条件的变化,如结合态氮(氨、亚硝酸盐、硝酸盐等)对固氮酶的合成阻遏和较高的氧分压对联合固氮菌的固氮效率影响．提出了发掘和利用禾本科植物的生物固氮潜力的努力方向：从自然界分离筛选获得广谱高效固氮菌株;应用基因工程构建耐铵、泌铵型联合固氮菌;诱导禾本科植物形成固氮根瘤;充分发挥植物内生固氮菌的优势．     

Use of celluloselytic nitrogen-fixing bacteria in the enrichment of roughage with protein

A new strain of acid-tolerant facultative anaerobic cellulose-degrading bacteria Bacillus cytaseus 21 (Mc Bethe ef Scales, 1912), which are capable to fixing atmospheric nitrogen, was isolated. This strain is intended for solid-phase fermentation and enrichment with protein of cellulose-containing waste of plant cultivation.     

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.     

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.     

Use of Cellulose-Degrading Nitrogen-Fixing Bacteria in the Enrichment of Roughage with Protein

A new strain of acid-tolerant facultative anaerobic cellulose-degrading bacteria Bacillus cytaseus 21 (Mc Bethe ef Scales, 1912), which are capable to fixing atmospheric nitrogen, was isolated. This strain is intended for solid-phase fermentation and enrichment with protein of cellulose-containing waste of plant cultivation.     

固氮植物绿豆对核桃幼苗生长、叶片气孔气体交换及水力特征的作用

采用砂培方法, 在温室内将一年生核桃(Juglans regia)嫁接苗木和绿豆(Vigna radiata)进行间作, 研究绿豆对核桃苗木生长、水分平衡和光合特性的影响。该研究设有5种处理, 即: 对照(核桃单作, 正常供应氮素); 核桃单作, 不添加氮素; 核桃绿豆间作, 不添加氮素; 核桃绿豆间作, 正常供应氮素; 绿豆单作, 不添加氮素。结果显示: 种植绿豆可以增加土壤氮含量和核桃茎内氮含量, 但对核桃叶和根系中的氮含量影响不明显。种植绿豆显著增加不施氮核桃的高生长和直径生长, 但降低了正常供氮核桃的生长。无论种植绿豆与否, 不供氮处理降低了核桃的总叶面积, 提高了根冠比。核桃叶片气孔气体交换对各处理的响应和生长有相同的趋势。缺氮显著降低了核桃叶柄在中午的导水率、提高了导水损失率; 种植绿豆显著提高不供氮核桃的导水率而且明显降低了其导水损失率。然而, 种植绿豆使正常供氮的核桃降低了导水率, 加剧了导水损失率。同时, 绿豆受到间作的竞争压力, 产量和生物量有所下降。由研究结果可知, 在贫瘠的土壤上, 固氮植物绿豆改善了间作的核桃的氮营养, 有益于核桃木质部发育、水分平衡以及光合代谢。但是在氮充足的土壤中, 种植绿豆反而降低了核桃的水分供应, 影响其气体交换和生长。     

Soil characteristics of Rocky Mountain National Park grasslands invaded by Melilotus officinalis and M. alba

Aim Invasion of nitrogen‐fixing non‐native plant species may alter soil resources and impact native plant communities. Altered soils may be the driving mechanism that provides a suitable environment to facilitate future invasions and decrease native biodiversity. We hypothesized that Melilotus invasion would increase nitrogen availability and produce soil microclimate and biochemical changes, which could in turn alter plant species composition in a montane grassland community. Location Our research addressed the effects of white and yellow sweet clover (Melilotus officinalis and M. alba) invasion on soil characteristics and nitrogen processes in the montane grasslands in Rocky Mountain National Park. Methods We sampled soil in replicate sites of Melilotus‐invaded and control (non‐invaded) patches within disturbed areas in montane grassland habitats. Soil composites were analysed for available nitrogen, net nitrogen mineralization, moisture, carbon/nitrogen (C : N ratio), texture, organic matter and pH. Data were recorded at three sample dates during the growing seasons of 1998 and 1999. Results Contrary to our expectations, we observed lower nitrogen availability and mineralization in invaded patches, and differences in soil moisture content and soil C : N. Soil C : N ratios were higher in invaded plots, in spite of the fact that Melilotus had the lowest C : N ratios of other plant tissue analysed in this study. Main conclusions These findings provide land managers of natural areas with a better perspective on the possibilities of nitrogen‐fixing species impact on soil nutrient levels.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Bacteriophage in relation to nitrogen fixation by red clover

Summary Cultures of Rhizobium trifolii resistant to the action of a given bacteriophage, may vary appreciably in their nitrogen fixing ability in association with the proper host plant. Likewise, cultures of Rh. trifolii, sensitive to bacteriophage may or may not benefit the host plant as judged by nitrogen fixation.Since sensitive and resistant cultures may be comparable in their nitrogen fixing capacity, it appears that the behavior of a culture of Rh. trifolii toward the lytic action of a specific bacteriophage in vitro cannot be correlated with its nitrogen fixing ability in association with the host plant.If a bacteriophage is added to a sensitive strain of Rh. trifolii used for inoculation of red clover, the cultures recovered from the nudules are often only of the resistant type. When this occurs the nitrogen fixed through association of plant and bacteria is decreased in the case of a good strain, but is unaffected if the strain is of the poor type. The addition of phage to a resistant strain of Rh. trifolii used for inoculation of red clover plants does not change the resistant type of culture recovered from the nodules, nor is the fixation of nitrogen by plant and bacteria affected.     

The effects of insects,nutrients, and plant invasion on community structure and function above‐ and belowground

Soil nutrient availability, invasive plants, and insect presence can directly alter ecosystem structure and function, but less is known about how these factors may interact. In this 6‐year study in an old‐field ecosystem, we manipulated insect abundance (reduced and control), the propagule pressure of an invasive nitrogen‐fixing plant (propagules added and control), and soil nutrient availability (nitrogen added, nitrogen reduced and control) in a fully crossed, completely randomized plot design. We found that nutrient amendment and, occasionally, insect abundance interacted with the propagule pressure of an invasive plant to alter above‐ and belowground structure and function at our site. Not surprisingly, nutrient amendment had a direct effect on aboveground biomass and soil nutrient mineralization. The introduction of invasive nitrogen‐fixing plant propagules interacted with nutrient amendment and insect presence to alter soil bacterial abundance and the activity of the microbial community. While the larger‐scale, longer‐term bulk measurements such as biomass production and nutrient mineralization responded to the direct effects of our treatments, the shorter‐term and dynamic microbial communities tended to respond to interactions among our treatments. Our results indicate that soil nutrients, invasive plants, and insect herbivores determine both above‐ and belowground responses, but whether such effects are independent versus interdependent varies with scale.     

Effects of a nitrogen fixing plant Vigna radiata on growth,leaf stomatal gas exchange and hydraulic characteristics of the intercropping Juglans regia seedlings

采用砂培方法, 在温室内将一年生核桃(Juglans regia)嫁接苗木和绿豆(Vigna radiata)进行间作, 研究绿豆对核桃苗木生长、水分平衡和光合特性的影响。该研究设有5种处理, 即: 对照(核桃单作, 正常供应氮素); 核桃单作, 不添加氮素; 核桃绿豆间作, 不添加氮素; 核桃绿豆间作, 正常供应氮素; 绿豆单作, 不添加氮素。结果显示: 种植绿豆可以增加土壤氮含量和核桃茎内氮含量, 但对核桃叶和根系中的氮含量影响不明显。种植绿豆显著增加不施氮核桃的高生长和直径生长, 但降低了正常供氮核桃的生长。无论种植绿豆与否, 不供氮处理降低了核桃的总叶面积, 提高了根冠比。核桃叶片气孔气体交换对各处理的响应和生长有相同的趋势。缺氮显著降低了核桃叶柄在中午的导水率、提高了导水损失率; 种植绿豆显著提高不供氮核桃的导水率而且明显降低了其导水损失率。然而, 种植绿豆使正常供氮的核桃降低了导水率, 加剧了导水损失率。同时, 绿豆受到间作的竞争压力, 产量和生物量有所下降。由研究结果可知, 在贫瘠的土壤上, 固氮植物绿豆改善了间作的核桃的氮营养, 有益于核桃木质部发育、水分平衡以及光合代谢。但是在氮充足的土壤中, 种植绿豆反而降低了核桃的水分供应, 影响其气体交换和生长。     

铜尾矿废弃地生物土壤结皮固氮微生物多样性

生物土壤结皮能够有效提高矿业废弃地有机质和氮的积累,促进植被的恢复.本研究基于固氮微生物的nifH基因多样性,以铜陵铜尾矿废弃地3种类型的生物土壤结皮(藻结皮、藓结皮和藻-藓混合结皮)为对象,利用变性梯度凝胶电泳(DGGE)技术研究生物土壤结皮中固氮微生物的多样性以及废弃地植物群落发育对其产生的影响.结果表明： 尾矿库裸地表面的藻结皮的固氮微生物多样性最高,其次为维管植物群落下的藻-藓混合结皮,苔藓结皮的固氮蓝藻多样性最低;随着维管植物群落高度和盖度的增加,固氮微生物多样性降低,铜尾矿废弃地的pH、水分、有机质、养分含量(氮和磷)以及有效态和总重金属浓度对固氮微生物多样性的影响均不显著.测序和系统发育分析表明,废弃地结皮中固氮微生物以蓝藻为主,主要为不具有异形胞的丝状蓝藻.
     

Soil Nitrogen Conditions Approach Preinvasion Levels following Restoration of Nitrogen-Fixing Black Locust (Robinia pseudoacacia) Stands in a Pine–Oak Ecosystem

Restoring native plant communities on sites formerly occupied by invasive nitrogen‐fixing species poses unique problems due to elevated soil nitrogen availability. Mitigation practices that reduce available nitrogen may ameliorate this problem. We evaluated the effects of tree removal followed by soil preparation or mulching on native plant growth and soil nitrogen transformations in a pine–oak system formerly occupied by exotic nitrogen‐fixing Black locust (Robinia pseudoacacia) trees. Greenhouse growth experiments with native grasses, Andropogon gerardii and Sorghastrum nutans, showed elevated relative growth rates in soils from Black locust compared with pine–oak stands. Field soil nutrient concentrations and rates of net nitrification and total net N‐mineralization were compared 2 and 4 years since Black locust removal and in control sites. Although soil nitrogen concentrations and total net N‐mineralization rates in the restored sites were reduced to levels that were similar to paired pine–oak stands after only 2 years, net nitrification rates remained 3–34 times higher in the restored sites. Other nutrient ion concentrations (Ca, Mg) and organic matter content were reduced, whereas phosphorus levels remained elevated in restored sites. Thus, 2–4 years following Black locust tree removal and soil horizon mixing achieved through site preparation, the concentrations of many soil nutrients returned to preinvasion levels. However, net nitrification rates remained elevated; cover cropping or carbon addition during restoration of sites invaded by nitrogen fixers could increase nitrogen immobilization and/or reduce nitrate availability, making sites more amenable to native plant establishment.