Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Soil Organic Carbon is Increased in Mixed-Species Plantations of Eucalyptus and Nitrogen-Fixing Acacia

Soil organic carbon (soil C) sequestration in forests is often higher under nitrogen (N₂)-fixing than under non-N₂-fixing tree species. Here, we examined whether soil C could be increased using mixed-species plantations compared to monocultures, which are less productive aboveground than mixtures. In addition, we compared soil C sequestration under N₂-fixing trees with non-N₂-fixing trees that received N fertilizer. Monocultures of Eucalyptus globulus (E) and the N₂-fixing Acacia mearnsii (A) and mixtures of these species were planted in a replacement series: 100%E, 75%E + 25%A, 50%E + 50%A, 25%E + 75%A and 100%A. Soil samples were also collected from fertilized monoculture treatments (100%EFer) of E. globulus (250 kg N ha^?1). Total organic C, N and phosphorus were determined at age 8 years at two soil depths (0–10 cm and 10–30 cm) and three density fractions of soil organic matter (SOM) were quantified for 0–5 cm depth. Soil C was highest in the 50%E + 50%A mixed stand and was highly correlated with aboveground biomass, not to the percentage of A. mearnsii in mixtures. This was largely due to soil C at 10–30 cm because there were no treatment effects on soil C at 0–10 cm. All density fractions of SOM at 0–5 cm increased with the percentage of A. mearnsii. In E. globulus monocultures, N fertilization did not increase soil C when compared with unfertilized stands. These results indicate that the inclusion of N₂-fixing trees into eucalypt plantations may increase soil C stocks through increased productivity.     

Belowground competitive suppression of seedling growth by grass in an African savanna

Coexistence of N₂-fixing legumes and non-legume trees with grasses in African savannas results in intense competition between these life-forms. We hypothesised that belowground competition might induce different nutritional constraints in N₂- versus non-N₂-fixing species. A field (Hluhluwe-imFolozi nature reserve, South Africa) competition experiment with two N₂-fixing legume species (Acacia burkei and Acacia karroo) and two non-N₂-fixing species (Schotia brachypetala and Spirostachys africana) both with clipped grass and without grass was established. Plants were supplied with no fertilizer, or generous amounts of fertilizer (200?kg?N?ha^?1, 100?kg P₂O₅ ha^?1, 7.1?kg K₂O ha^?1) supplied as either 28?C10 (N?CK), P or a combination of these fertilizers (NPK). Regularly clipped grass suppressed growth (by more than 90?%) of both N₂- and non-N₂-fixing seedlings equally. Biomass accumulation of seedlings grown with grass and the grasses themselves responded positively to NK and/or NPK, but not P, although P-fertilization did have effects on foliar [N] and ??¹⁵N values of trees and grasses showing that plants accessed the fertilizer. Tree ??¹⁵N values and foliar [N] were also modified by NPK, demonstrating access to fertilizer. However, the ameliorative effects of NPK on grass competition-induced biomass suppression were only partial. This may be due to ??non-resource competition?? (i.e. root gaps) imposed by dense grass roots. The fact that nutrients were able to partially ameliorate the effects of grass competition, however, indicates that such ??non-resource competition?? may be partially overcome by even more generous supply of fertilizers.     

An assessment of soil enrichment by actinorhizal N2 fixation using δ15N values in a chronosequence of deglaciation at Glacier Bay,Alaska

The extent of transfer of fixed N between N₂-fixing and non-N₂-fixing plant species is largely unknown in successional studies. In order to redress this deficiency at a locale intensively studied ecologically, leaf tissue samples were collected from actinorhizal N₂-fixing (Alnus, Shepherdia, and Dryas) and two non-N₂-fixing (Salix) woody species within research plots located along a chronosequence of deglaciated fjord in Glacier Bay National Park, Alaska. The tissue samples were analyzed for ¹⁵N content, and the resulting data analyzed for trends in plant tissue N. Among the non-N₂-fixing Salix species, ¹⁵N values increased from the most recently deglaciated sites to converge with the temporally more-stable values for the symbiotic N₂-fixing species on sites at about 40 years after deglaciation. The lower ¹⁵N values of sequestered N in plant tissues suggested that N derived from N₂-fixing plants accounts for the major portion of N in associated plants up to 40 years after deglaciation. The ¹⁵N isotopic data also suggested that Shepherdia canadensis depends least on soil N, D. drummondii the most, and A. viridis ssp. sinuata somewhere between those two species. The presence of a sere dominated by dense thickets of A. viridis ssp. sinuata at the convergence of ¹⁵N values for the N₂-fixing and non-N₂-fixing species indicated that this species is most responsible for accumulation of fixed N in soil at Glacier Bay. This paper is dedicated to the memory of Steven J. Kohls who died prior to publication of this research.     

Arbuscular mycorrhizal fungi affect seedling recruitment: a potential mechanism by which N deposition favors the dominance of grasses over forbs

Background and aims

Nitrogen (N) deposition usually alters plant community structure and reduces plant biodiversity in grasslands. Seedling recruitment is essential for maintaining species richness and determines plant community composition. Arbuscular mycorrhizal fungi (AMF) are widespread symbiotic fungi and could facilitate seedling establishment. Here we conducted an experiment to address whether the influence of AMF on seedling recruitment depends on N addition and plant species.

Methods

Leymus chinensis were cultivated for 5 months in the microcosms that were inoculated with or without AMF at five N addition rates. Seeds of three main species (two C₃ grasses and one non-N₂-fixing forb) of the Eurasian steppe were sown to the 5-month-old microcosms. Seedling establishment was estimated by shoot biomass, N and P contents 7 weeks after seedling germination.

Results

AMF promoted seedlings recruitment of two C₃ grasses at addition rates above 0.5 g N m^?2. In contrast, seedling recruitment of the non-N₂-fixing forb was increased by AMF at addition rates below 0.5 g N m^?2 but was decreased above 2.5 g N m^?2.

Conclusions

These results partly explain why N addition favored the dominance of grasses over forbs in perennial grassland communities. Our study indicates that AMF have the potential to influence plant community composition by mediating revegetation in the face of N deposition.     

Elevated CO2 alleviates the effects of low P on the growth of N2-fixing Acacia auriculiformis and Acacia mangium

Nodulated seedlings of Acacia auriculiformis Cunn. ex Benth and Acacia mangium Willd were grown with different phosphorus (P) regimes for 90 days, and half of them were exposed to elevated CO₂ (800 μl l⁻¹) during the last 30 days. Under ambient CO₂, plant growth and the amount of N fixed symbiotically in N₂-fixing seedlings decreased with the decrease of supplied P; this relationship did not occur under elevated CO₂. The increase in plant biomass by elevated CO₂ at low P was accompanied by the increase in internal P use efficiency, the amount of N fixed symbiotically and N use efficiency. Elevated CO₂ recovered the low P-induced reduction in leaf dry matter per unit area or unit fresh weight, but it had no effect on the low P-induced increase in partitioning dry matter to roots. These results suggest that elevated CO₂ alleviates the low P effect mainly by increasing the use efficiency of internal P for plant growth and symbiotic N₂ fixation, and the source-sink relationship is possibly an important driving force for this effect of elevated CO₂ in A. auriculiformis and A. mangium.     

Survival and colonization of inoculated bacteria in kallar grass rhizosphere and quantification of N2-fixation

Two experiments have been conducted, one in semi-solid Hoagland nutrient medium and the other in shallow pots containing saline soil. N₂-fixing bacteria belonging toAzospirillum, Azotobacter, Klebsiella andEnterobacter were inoculated separately on kallar grass grown in semi-solid nutrient medium. It was shown that inoculation affects root proliferation and also results in¹⁵N isotopic dilution. The % Ndfa ranged from 47–70 whereas no significant effect on the total nitrogen uptake was observed. The bacterial colonization of the root surface and the presence of enteric bacteria inside the root hair cells is reported. In a soil pot experiment, non-N₂-fixingPolypogon monspeliensis was used as a reference plant (control). A treatment receiving a high rate of nitrogen was also used as a non-N₂-fixing control.¹⁵N-labelled ammonium sulphate at 20 kg N ha^–1 and 90 kg N ha^–1 was used. The % Ndfa in the aerial parts of kallar grass was 12–15 whenP. monspeliensis was used as reference plant whereas 37–39% Ndfa was estimated when the treatment receiving high nitrogen fertilizer was used as a non-N₂-fixing control. These investigations revealed some problems of methodology which are discussed.     

Contribution of 15N-labelled leaf litter to N turnover, nitrous oxide emissions and N sequestration in a beech forest during eleven years

Aims

Decomposition of leaf litterfall plays a major role for nitrogen (N) dynamics in soils. However, little is known as to which extent beech leaf litter contributes to N turnover and nitrous oxide (N₂O) emissions within one decade after litterfall.

Methods

In 1997, we exchanged recently fallen leaf litter by ¹⁵N-labelled litter in a beech stand (Fagus sylvatica) at the Solling, Germany. Measurements were conducted 2–3 and 10–11 years after litter exchange.

Results

Two years after litter exchange, 92 % of added ¹⁵N was recovered in the surface 10 cm of the soil. The labelled N was primarily found in the upper part of the F layer of the moder type humus. Eleven years after litter exchange, 73 % of the added ¹⁵N was lost and the remaining 27 % was mainly recovered in the lower part of the F layer indicating N sequestration. The remaining leaf litter N was subject to measurable N mineralisation (2–3 % of litter N) and N₂O production (0.02 %). Between 0.3 % (eleventh year) and 0.6 % (second year) of total annual N₂O emissions were attributed to beech leaf litter of a single year.

Conclusions

Most of the annual N₂O emissions (1.33–1.54 kg N ha^?1 yr^?1) were probably derived from older soil N pools.     

Contrasting growth response of an N2-fixing and non-fixing forb to elevated CO2: dependence on soil N supply

With the ability to symbiotically fix atmospheric N₂, legumes may lack the N-limitations thought to constrain plant response to elevated concentrations of atmospheric CO₂. The growth and photosynthetic responses of two perennial grassland species were compared to test the hypotheses that (1) the CO₂ response of wild species is limited at low N availability, (2) legumes respond to a greater extent than non-fixing forbs to elevated CO₂, and (3) elevated CO₂ stimulates symbiotic N₂ fixation, resulting in an increased amount of N derived from the atmosphere. This study investigated the effects of atmospheric CO₂ concentration (365 and 700 mol mol^–1) and N addition on whole plant growth and C and N acquisition in an N₂-fixing legume (Lupinus perennis) and a non-fixing forb (Achillea millefolium) in controlled-chamber environments. To evaluate the effects of a wide range of N availability on the CO₂ response, we incorporated six levels of soil N addition starting with native field soil inherently low in N (field soil + 0, 4, 8, 12, 16, or 20 g N m^–2 yr^–1). Whole plant growth, leaf net photosynthetic rates (A), and the proportion of N derived from N₂ fixation were determined in plants grown from seed over one growing season. Both species increased growth with CO₂enrichment, but this response was mediated by N supply only for the non-fixer, Achillea. Its response depended on mineral N supply as growth enhancements under elevated CO₂ increased from 0% in low N soil to +25% at the higher levels of N addition. In contrast, Lupinus plants had 80% greater biomass under elevated CO₂ regardless of N treatment. Although partial photosynthetic acclimation to CO₂ enrichment occurred, both species maintained comparably higher A in elevated compared to ambient CO₂ (+38%). N addition facilitated increased A in Achillea, however, in neither species did additional N availability affect the acclimation response of A to CO₂. Elevated CO₂ increased plant total N yield by 57% in Lupinus but had no effect on Achillea. The increased N in Lupinus came from symbiotic N₂ fixation, which resulted in a 47% greater proportion of N derived from fixation relative to other sources of N. These results suggest that compared to non-fixing forbs, N₂-fixers exhibit positive photosynthetic and growth responses to increased atmospheric CO₂ that are independent of soil N supply. The enhanced amount of N derived from N₂ fixation under elevated CO₂ presumably helps meet the increased N demand in N₂-fixing species. This response may lead to modified roles of N₂-fixers and N₂-fixer/non-fixer species interactions in grassland communities, especially those that are inherently N-poor, under projected rising atmospheric CO₂.     

Selecting matched root architecture in tree pairs to be used for assessing N2 fixation based on soil-15N-labelling

It is commonly assumed that soil-¹⁵N-labelling provides reliable estimates of N₂ fixation in trees by matching N₂-fixing and non-N₂-fixing tree pairs. As root system is a key parameter in determining suitability of the tree pairs, we compared root architecture of Acacia cyanophylla Lindl. and Casuarina glauca Sieber ex. Spreng. (two N₂-fixing trees) with Eucalyptus camaldulensis Dehn. and Ceratonia siliqua L. (two non-N₂-fixing trees) at 4-year-old in Mediterranean-semiarid zone. The rhizobium strain used appeared more motile than Frankia strain. A. cyanophylla and E. camaldulensis had extensive rooting area and volume of fine roots, and both species tended to develop marked horizontal rooting, compared to C. glauca and C. siliqua. Characteristics of fine- and horizontal-root components can be used in selecting matched root systems of N₂-fixing and reference-paired trees. Root architecture of C. glauca was more similar to C. siliqua, than to E. camaldulensis, and that of A. cyanophylla was more similar to E. camaldulensis than to C. siliqua. Accordingly, E. camaldulensis is an appropriate reference to estimate actual N₂ fixation by A. cyanophylla, and C. siliqua is an appropriate reference for C. glauca, when using soil-¹⁵N-labelling method in the prevailing site environment.     

Dominance of legume trees alters nutrient relations in mixed species forest restoration plantings within seven years

Failures in reforestation are often attributed to nutrient limitation for tree growth. We compared tree performance and nitrogen and phosphorus relations in adjacent mixed-species plantings of contrasting composition, established for forest restoration on Ultisol soil, originally covered by tropical semi-deciduous Atlantic Forest in Southeast Brazil. Nutrient relations of four tree species occurring in both planting mixtures were compared between a legume-dominated, species-poor direct seeding mixture of early-successional species (“legume mixture”), and a species-diverse, legume-poor mixture of all successional groups (“diverse mixture”). After 7 years, the legume mixture had 6-fold higher abundance of N₂-fixing trees, 177% higher total tree basal area, 22% lower litter C/N, six-fold higher in situ soil resin-nitrate, and 40% lower in situ soil resin-P, compared to the diverse mixture. In the legume mixture, non-N₂-fixing legume Schizolobium parahyba (Fabaceae-Caesalpinioideae) had significantly lower proportional N resorption, and both naturally regenerating non-legume trees had significantly higher leaf N concentrations, and higher proportional P resorption, than in the diverse mixture. This demonstrate forms of plastic adjustment in all three non-N₂-fixing species to diverged nutrient relations between mixtures. By contrast, leaf nutrient relations in N₂-fixing Enterolobium contortisiliquum (Fabaceae-Mimosoideae) did not respond to planting mixtures. Rapid N accumulation in the legume mixture caused excess soil nitrification over nitrate immobilization and tighter P recycling compared with the diverse mixture. The legume mixture succeeded in accelerating tree growth and canopy closure, but may imply periods of N losses and possibly P limitation. Incorporation of species with efficient nitrate uptake and P mobilization from resistant soil pools offers potential to optimize these tradeoffs.     

Neotropical Legume Tree Inga edulis Forms N2-fixing Symbiosis with Fast-growing Bradyrhizobium Strains

Inga edulis Mart. is a tropical legume tree used for shade in coffee and cacao plantations and as a hedgerow in alley-cropping practices. Little information can be found concerning N₂ fixation in this species. This study was conducted to characterize the rhizobia of I. edulis and determine if it is capable of fixing substantial amounts of N₂. Four strains of fast-growing, Gram-negative rhizobia-type bacteria were isolated from I. edulis nodules. The strains were identified by sequencing of partial 16S–23S rDNA internal spacer region. Nitrogenase activity was determined using acetylene reduction assay (ARA). Dinitrogen fixation was measured under controlled conditions by the ¹⁵N isotope dilution technique using two non-N₂-fixing reference species, Vochysia guatemalensis Donn. Sm, and Gmelina arborea Roxb. ex. Sm. Seedlings were grown in three growth media: native soil and naturally N-depleted sand amended to a low and high N level. The four strains of symbiotic bacteria were closely related to Bradyrhizobium japonicum and to Bradyrhizobium liaoningense. Nodules demonstrated nitrogenase activity as measured by ARA. Vochysia guatemalensis was a better non-N₂-fixing reference than G. arborea. When V. guatemalensis was used as the non-N₂-fixing reference, the estimate of the percentage of N fixed from atmosphere out of total N in I. edulis seedlings was ca. 40 in the two sand media treatments and 10 in the native soil.     

N and P colimitation of N2-fixing and N-supplied fynbos legumes from the Cape Floristic Region

Background and aims

Legume species in the fynbos vegetation of the Cape Floristic Region, that fix N₂ in soils with low P, may have evolved for enhanced acquisition and efficient use of P. It was hypothesized that N₂-fixing and combined-N supplied (N-supplied) A. linearis, P. calyptrata and C. genistoides are adapted to low P and would be relatively unresponsive to increased P of 100 μM.

Methods

18 legume species were evaluated for their nodulation response to low P availability. The N X P interaction was then examined in A. linearis, P. calyptrata and C. genistoides reliant on either N₂-fixation or 300 μM N (NH₄NO₃), and receiving 0.1, 1.0, 10 and 100 μM P (NaH₂PO₄).

Results

In the species selection experiment, A. linearis, P. calyptrata and C. genistoides, with the greatest nodule fresh weight (FW) and nodule FW to root FW ratio, were the most prolific nodulating species. In the N X P experiment, with low P supply, the biomass of N₂-fixing P. calyptrata and C. genistoides was consistently greater than that of N-supplied plants. In contrast, with high P supply of 100 μM P, all N-supplied plants accumulated more biomass than the corresponding N₂-fixing plants. High P-use efficiency, poor down-regulation of P uptake and P storage was evident in A. linearis and P. calyptrata.

Conclusion

The growth response to P and the significant N X P interactions indicate that N₂-fixing and N-supplied plants were not adapted to low P, but rather colimited by both N and P.     

Spatial variation of the stable nitrogen isotope ratio of woody plants along a topoedaphic gradient in a subtropical savanna

Variation in the stable N isotope ratio (δ¹⁵N) of plants and soils often reflects the influence of environment on the N cycle. We measured leaf δ¹⁵N and N concentration ([N]) on all individuals of Prosopis glandulosa (deciduous tree legume), Condalia hookeri (evergreen shrub), and Zanthoxylum fagara (evergreen shrub) present within a belt transect 308 m long × 12 m wide in a subtropical savanna ecosystem in southern Texas, USA in April and August 2005. Soil texture, gravimetric water content (GWC), total N and δ¹⁵N were also measured along the transect. At the landscape scale, leaf δ¹⁵N was negatively related to elevation for all the three species along this topoedaphic sequence. Changes in soil δ¹⁵N, total N, and GWC appeared to contribute to this spatial pattern of leaf δ¹⁵N. In lower portions of the landscape, greater soil N availability and GWC are associated with relatively high rates of both N mineralization and nitrification. Both soil δ¹⁵N and leaf [N] were positively correlated with leaf δ¹⁵N of non-N₂ fixing plants. Leaf δ¹⁵N of P. glandulosa, an N₂-fixing legume, did not correlate with leaf [N]; the δ¹⁵N of P. glandulosa’s leaves were closer to atmospheric N₂ and significantly lower than those of C. hookeri and Z. fagara. Additionally, at smaller spatial scales, a proximity index (which reflected the density and distance of surrounding P. glandulosa trees) was negatively correlated with leaf δ¹⁵N of C. hookeri and Z. fagara, indicating the N₂-fixing P. glandulosa may be important to the N nutrition of nearby non-N₂-fixing species. Our results indicate plant ¹⁵N natural abundance can reflect the extent of N retention and help us better understand N dynamics and plant-soil interactions at ecosystem and landscape scales.     

Effects of nitrogen deposition and management practices on leaf litterfall and N and P return in a Moso bamboo forest

The effects of nitrogen (N) deposition and management practices on leaf litterfall and N and P return in Moso bamboo forest are not yet known. In this study, we investigated the effects of four levels of simulated N deposition, including low-N (N30, 30 kg ha^?1 year^?1), medium-N (N60, 60 kg ha^?1 year^?1), and high-N (N90, 90 kg ha^?1 year^?1), and a control with no N (N-free addition). The experiment was performed in a Moso bamboo forest under conventional management (CM) and intensive management (IM). The results showed that leaf litterfall and N and P return occurred mainly from March to June and accounted for 78.2–82.2, 78.5–82.1, and 85.6–94.6% of annual leaf litterfall, N return, and P return, respectively. Unlike CM, IM significantly increased leaf litterfall and N and P return. The positive effects were further amplified by low- and medium-N deposition, but not high-N deposition. The combination of low- and medium-N deposition and IM significantly increased N and P return, but not litterfall. Our results indicated that the interaction of anthropogenic management practices and N deposition need to be considered when estimating the effects of N deposition on the biogeochemical cycle of a forest ecosystem.     

长期氮沉降下杉木人工林凋落物与土壤的C、N、P化学计量特征

为研究长期氮沉降条件下林木凋落物与土壤养分之间的关系,该文以亚热带杉木(Cunninghamia lanceolata)人工林为研究对象,分析了模拟氮沉降处理第12年时杉木林凋落物不同组分(叶、枝、果)与不同土层土壤(0～20 cm、20～40 cm、40～60 cm)的C、N、P含量及其化学计量比。氮沉降处理分4个水平,分别为N0(0 kg N·hm^-2·a^-1)、N1(60 kg N·hm^-2·a^-1)、N2(120 kg N·hm^-2·a^-1)、N3(240 kg N·hm^-2·a^-1),每处理重复3次。结果表明:(1)凋落物各组分的C、N、P含量及其化学计量比均高于土壤; 凋落物和土壤化学计量比均表现为C/P>C/N>N/P; 凋落物不同组分的C、N含量表现为叶>果>枝,而P含量表现为叶>枝>果。(2)12 a氮沉降增加了凋落物叶、枝和果的N含量,增幅分别为4.24%、15.97%、6.47%; 同时增加了凋落物枝N/P,降低了凋落物枝C含量、C/N和C/P; 中-高氮沉降(N2、N3)增加了土壤N含量,低氮沉降(N1)增加了土壤C/P、N/P。(3)相关性分析表明凋落物N与土壤N显著正相关,土壤C/P与凋落物C/P、N/P显著负相关,土壤P与凋落物N/P显著负相关。综上结果说明凋落物N是土壤N的重要N素来源之一,而土壤N可能是决定长期氮沉降后凋落物N/P的主要因素。     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.     

Natural15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes

The¹⁵N abundance of plants usually closely reflects the¹⁵N abundance of their major immediate N source(s); plant-available soil N in the case of non-N₂-fixing plants and atmospheric N₂ in the case of N₂ fixing plants. The¹⁵N abundance values of these sources are usually sufficiently different from each other that a significant and systematic difference in the¹⁵N abundance between the two kinds of plants can be detected. This difference provides the basis for the natural¹⁵N abundance method of estimating the relative contribution of atmospheric N₂ to N₂-fixing plants growing in natural and agricultural settings. The natural¹⁵N abundance method has certain advantages over more conventional methods, particularly in natural ecosystems, since disturbance of the system is not required and the measurements may be made on samples dried in the field. This method has been tested mainly with legumes in agricultural settings. The tests have demonstrated the validity of this method of arriving at semi-quantitative estimates of biological N₂-fixation in these settings. More limited tests and applications have been made for legumes in natural ecosystems. An understanding of the limits and utility of this method in these systems is beginning to emerge. Examples of systematic measurements of differences in¹⁵N abundance between non-legume N₂-fixing systems and neighbouring non-fixing systems are more unusual. In principle, application of the method to estimate N₂-fixation by nodulated non-legumes, using the natural¹⁵N abundance method, is as feasible as estimating N₂-fixation by legumes. Most of the studies involving N₂-fixing non-legumes are with this type of system (e.g., Ceanothus, Chamabatia, Eleagnus, Alnus, Myrica, and so forth). Resuls of these studies are described. Applicability for associative N₂-fixation is an empirical question, the answer to which probably depends upon the degree to which fixed N goes predominantly to the plant rather than to the soil N pool. The natural¹⁵N abundance method is probably not well suited to assessing the contribution of N₂-fixation by free-living microorganisms in their natural habitat, particularly soil microorganisms.This work was supported in part by subcontracts under grants from the US National Science Foundation (DEB79-21971 and BSR821618)     

Influence of pruning management on P and N distribution and use efficiency by N2 fixing and non-N2 fixing trees used in alley cropping systems

Pruning of hedgerow trees is an important management practice for the successful establishment of an alley cropping system. Although pruning affects biomass production, only meager evidence of this management on distribution of nutrients among the different plant organs after tree regrowth is available. This study examined the effect of pruning on the distribution and use efficiency of N and P in a N₂ fixing leguminous tree species, Gliricidia sepium, and two non-N₂ fixing leguminous tree species, Senna siamea and S. spectabilis, grown in a field on an Alfisol (low in P) at Fashola (Guinea Savanna Zone), Southwestern Nigeria. Four P rates, 0, 20, 40 and 80 kg P ha^–1 as single superphosphate were used and management treatments included pruned versus unpruned plants. The ¹⁵N isotope dilution technique was used to measure N₂ fixation in G. sepium. Partitioning of total P among different plant organs was influenced by plant species and pruning management, but was not affected by P application rates. The distribution of total P in the various plant organs followed that of dry matter yield while N partitioning had a different pattern. Pruned plants distributed about 118% more total P to branches and had a higher physiological P use efficiency (PPUE) than unpruned plants. Leaves were the biggest sink for total N and N allocation in the other plant organs was influenced by plant species and pruning management, G. sepium had relatively more of its total N and P partitioned into roots (about double that of the non-N₂ fixing trees) but had a lower PPUE. Unpruned and pruned G. sepium derived 35 and 54% respectively of their total N from atmospheric N2, with about 54% of the fixed N2 being allocated to leaves and roots. Results showed that N and P pools turned over in the branches during plant regrowth after pruning but the causative factors associated with this phenomenon were not clear.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.