Resource Optimization and Symbiotic Nitrogen Fixation

In temperate forests, symbiotic nitrogen (N) fixation is restricted to the early phases of succession despite the persistence of N limitation on production late in succession. This paradox has yet to be explained adequately. We hypothesized that the restriction of N fixation to early stages of succession results from the optimization of resource allocation in the vegetation. Because of this optimization, N fixation should be restricted to periods when fixation is less costly than N uptake. Our analysis differs from others in the way we calculate the cost of N uptake; we assess the cost of N uptake as the amount of carbon (C) that could be assimilated if the resources necessary to acquire one gram of N from the soil were allocated instead to photosynthesis. We then simulate N fixation as an asymptotic function of the difference in cost between N uptake and N fixation and proportional to the abundance of host tissues for the N-fixing symbionts. The factors that contribute to conditions that favor N fixation are (a) elevated-carbon dioxide (CO₂) concentrations, (b) an open canopy, (c) low available N in the soil, and (d) a soil volume already well exploited by roots. Our results indicate that changes in the relative cost of uptake vs fixation can explain most of the pattern in fixation through both primary and secondary succession, but that competitive interactions with nonfixing species play a role in the final exclusion of fixation in later stages of succession. Received 26 September 2000; accepted 31 January 2001.     

The Sensitivity of Moss-Associated Nitrogen Fixation towards Repeated Nitrogen Input

Nitrogen (N₂) fixation is a major source of available N in ecosystems that receive low amounts of atmospheric N deposition. In boreal forest and subarctic tundra, the feather moss Hylocomium splendens is colonized by N₂ fixing cyanobacteria that could contribute fundamentally to increase the N pool in these ecosystems. However, N₂ fixation in mosses is inhibited by N input. Although this has been shown previously, the ability of N₂ fixation to grow less sensitive towards repeated, increased N inputs remains unknown. Here, we tested if N₂ fixation in H. splendens can recover from increased N input depending on the N load (0, 5, 20, 80, 320 kg N ha^-1 yr^-1) after a period of N deprivation, and if sensitivity towards increased N input can decrease after repeated N additions. Nitrogen fixation in the moss was inhibited by the highest N addition, but was promoted by adding 5 kg N ha^-1 yr^-1, and increased in all treatments during a short period of N deprivation. The sensitivity of N₂ fixation towards repeated N additions seem to decrease in the 20 and 80 kg N additions, but increased in the highest N addition (320 kg N ha^-1 yr^-1). Recovery of N in leachate samples increased with increasing N loads, suggesting low retention capabilities of mosses if N input is above 5 kg N ha^-1 yr^-1. Our results demonstrate that the sensitivity towards repeated N additions is likely to decrease if N input does not exceed a certain threshold.     

Biological Nitrogen Fixation Prevents the Response of a Eutrophic Lake to Reduced Loading of Nitrogen: Evidence from a 46-Year Whole-Lake Experiment

A whole-ecosystem experiment in Lake 227 (L227) at the Experimental Lakes Area, ongoing since 1969, examined the roles of carbon (C), nitrogen (N), and phosphorus (P) in controlling eutrophication. During 2011, we conducted a series of sub-experiments and more intensive monitoring to improve estimates of N fixation and its ability to meet algal growth demands in the decades following the cessation of artificial N loading, while maintaining long-term high artificial P loading. Stoichiometric nutrient ratios indicated both moderate N and P limitation of the phytoplankton during spring, preceding a shift in phytoplankton community structure toward dominance by N fixing cyanobacteria. During bloom development, and for the remainder of the stratified period, stoichiometric nutrient ratios indicated moderate to strong P limitation. N fixation rates, corrected using ¹⁵N₂ methods, increased 2× after 1990, when N loading ceased. Ambient dissolved inorganic nitrogen prior to the bloom represented less than 3% of N demands of the phytoplankton. N fixation accounted for between 69–86% of total N loading to the epilimnion during the period of rapid bloom development, and 72–86% of total N loading during the May–October period. Phytoplankton biomass did not decline in L227 during the 40 years since artificial N loading was reduced, or the nearly 25 years since artificial N loads ceased entirely (1990–2013), and remained approximately 20× higher than four nearby reference lakes. These results suggest that despite constraints on biological N fixation, it retains a large capacity to offset potential N loading reductions in freshwaters.     

Nitrogen fixation by marine cyanobacteria

Discrepancies between estimates of oceanic N(2) fixation and nitrogen (N) losses through denitrification have focused research on identifying N(2)-fixing cyanobacteria and quantifying cyanobacterial N(2) fixation. Previously unrecognized cultivated and uncultivated unicellular cyanobacteria have been discovered that are widely distributed, and some have very unusual properties. Uncultivated unicellular N(2)-fixing cyanobacteria (UCYN-A) lack major metabolic pathways including the tricarboxylic acid cycle and oxygen-evolving photosystem II. Genomes of the oceanic N(2)-fixing cyanobacteria are highly conserved at the DNA level, and genetic diversity is maintained by genome rearrangements. The major cyanobacterial groups have different physiological and ecological constraints that result in highly variable geographic distributions, with implications for the marine N-cycle budget.     

Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient

Background and aims

The selection of legume species and species mixtures influences agroecosystem nitrogen (N) and carbon cycling. We utilized a fertility gradient to investigate the effects of plant species interactions on biological N fixation of an annual and perennial legume in response to shifting soil resource availability.

Methods

Legume N fixation of annual field pea (Pisum sativum) and perennial red clover (Trifolium pratense) grown in monoculture and mixtures with oats (Avena sativa) or orchardgrass (Dactylis glomerata) was estimated using the ¹⁵N natural abundance method across 15 farm fields and we measured six soil N pools ranging from labile to more recalcitrant.

Results

Evidence of complementary and facilitative species interactions was stronger for the perennial red clover-orchardgrass mixture than for the annual field pea-oat mixture (N Land Equivalency Ratios were 1.6 and 1.2, respectively). We estimated that the transfer of fixed N from red clover to orchardgrass increased aboveground N fixation estimates by 15% from 33 to 38?kg?N ha^?1. Despite a more than 2-fold range in soil organic matter levels and more than 3-fold range in labile soil N pools across field sites, the N fertility gradient was not a strong predictor of N fixation. While grass N assimilation was positively correlated with soil N pools, we found only weak, inverse correlations between legume N fixation and soil N availability. In grass-legume mixtures, soil N availability indirectly influenced N fixation through plant competition.

Conclusions

These results suggest that increasing diversity of cropping systems, particularly through the incorporation of perennial mixtures into rotations, could improve overall agroecosystem N cycling efficiency.     

Anoxygenic Photosynthesis and Nitrogen Fixation by a Microbial Mat Community in a Bahamian Hypersaline Lagoon

Simultaneous measurements of photosynthesis (both oxygenic and anoxygenic) and N(inf2) fixation were conducted to discern the relationships between photosynthesis, N(inf2) fixation, and environmental factors potentially regulating these processes in microbial mats in a tropical hypersaline lagoon (Salt Pond, San Salvador Island, Bahamas). Major photoautotrophs included cyanobacteria, purple phototrophic bacteria, and diatoms. Chemosystematic photopigments were used as indicators of the relative abundance of mat phototrophs. Experimental manipulations consisted of light and dark incubations of intact mat samples exposed to the photosystem II inhibitor DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a dissolved organic carbon source (D-glucose), and normal seawater (37(permil)). Photosynthetic rates were measured by both O(inf2) and (sup14)C methods, and nitrogenase activity (NA) was estimated by the acetylene reduction assay. Moderate reductions in salinity (from 74 to 37(permil)) had no measurable effect on photosynthesis, O(inf2) consumption, or NA. CO(inf2) fixation in DCMU-amended samples was (symbl)25% of that in the control (nonamended) samples and demonstrated photosynthetic activity by anoxygenic phototrophs. NA in DCMU-amended samples, which was consistently higher (by a factor of 2 to 3) than the other (light and dark) treatments, was also attributed to purple phototrophic bacteria. The ecological implication is that N(inf2) fixation by anoxygenic phototrophs (purple phototrophic bacteria and possibly cyanobacteria) may be regulated by the activity of oxygenic phototrophs (cyanobacteria and diatoms). Consortial interactions that enhance the physiological plasticity of the mat community may be a key for optimizing production, N(inf2) fixation, and persistence in these extreme environments.     

Nitrogen economy of alpine plants on the north Tibetan Plateau: Nitrogen conservation by resorption rather than open sources through biological symbiotic fixation

Nitrogen (N) is one of the most important factors limiting plant productivity, and N fixation by legume species is an important source of N input into ecosystems. Meanwhile, N resorption from senescent plant tissues conserves nutrients taken up in the current season, which may alleviate ecosystem N limitation. N fixation was assessed by the ¹⁵N dilution technique in four types of alpine grasslands along the precipitation and soil nutrient gradients. The N resorption efficiency (NRE) was also measured in these alpine grasslands. The aboveground biomass in the alpine meadow was 4–6 times higher than in the alpine meadow steppe, alpine steppe, and alpine desert steppe. However, the proportion of legume species to community biomass in the alpine steppe and the alpine desert steppe was significantly higher than the proportion in the alpine meadow. N fixation by the legume plants in the alpine meadow was 0.236 g N/m², which was significantly higher than N fixation in other alpine grasslands (0.041 to 0.089 g N/m²). The NRE in the alpine meadows was lower than in the other three alpine grasslands. Both the aboveground biomass and N fixation of the legume plants showed decreasing trends with the decline of precipitation and soil N gradients from east to west, while the NRE of alpine plants showed increasing trends along the gradients, which indicates that alpine plants enhance the NRE to adapt to the increasing droughts and nutrient‐poor environments. The opposite trends of N fixation and NRE along the precipitation and soil nutrient gradients indicate that alpine plants adapt to precipitation and soil nutrient limitation by promoting NRE (conservative nutrient use by alpine plants) rather than biological N fixation (open sources by legume plants) on the north Tibetan Plateau.     

Nitrogen Limitation of Pond Ecosystems on the Plains of Eastern Colorado

Primary production in freshwater ecosystems is often limited by the availability of phosphorus (P), nitrogen (N), or a combination of both (NP co-limitation). While N fixation via heterocystous cyanobacteria can supply additional N, no comparable mechanism for P exists; hence P is commonly considered to be the predominant and ultimate limiting nutrient in freshwater ecosystems. However, N limitation can be maintained if P is supplied in stoichiometric excess of N (including N fixation). The main objective of this study was to examine patterns in nutrient limitation across a series of 21 vernal ponds in Eastern Colorado where high P fluxes are common. Across all ponds, water column dissolved inorganic N steadily decreased throughout the growth season due to biological demand while total dissolved P remained stable. The water column dissolved inorganic N to total dissolved P ratios suggested a transition from NP co-limitation to N limitation across the growth season. Periphyton and phytoplankton %C was strongly correlated with %N while %P was assimilated in excess of %N and %C in many ponds. Similarly, in nutrient addition bottle assays algae responded more strongly to N additions (11 out of 18 water bodies) than P additions (2 out of 18 water bodies) and responded most strongly when N and P were added in concert (12 out of 18 water bodies). Of the ponds that responded to nutrient addition, 92% exhibited some sort of N limitation while less than 8% were limited by P alone. Despite multiple lines of evidence for N limitation or NP co-limitation, N fixation rates were uniformly low across most ponds, most likely due to inhibition by water column nitrate. Within this set of 18 water bodies, N limitation or NP co-limitation is widespread due to the combination high anthropogenic P inputs and constrained N fixation rates.     

Controls Over Leaf Litter and Soil Nitrogen Fixation in Two Lowland Tropical Rain Forests

Global comparisons suggest that rates of N fixation in tropical rain forests may be among the highest on earth. However, data supporting this contention are rare, and the factors that regulate N fixation within the biome remain largely unknown. We conducted a full-factorial (N × P) fertilization experiment in two lowland tropical rain forests in Costa Rica to explore the effects of nutrient availability on rates of free-living N fixation in leaf litter and soil. P fertilization significantly increased N fixation rates in both leaf litter and soil, and the effect was dependent on sampling date. Fertilization with N did not affect rates of N fixation at any time. In addition, variation in N fixation rates measured in unfertilized plots at four sampling time points suggested seasonal variability in N fixation: leaf litter N fixation ranged from 0.36 kg/ha/yr in the dry season to 5.48 kg/ha/yr in the wet season. Soil N fixation showed similar patterns ranging from a dry season low of 0.26 kg/ha/yr to a wet season high of 2.71 kg/ha/yr. While the observed temporal variability suggests potential climatic control over free-living N fixation in these forests, data suggest that neither soil nor leaf litter moisture alone regulate N fixation rates. Instead, we hypothesize that a combination of ample C availability, low leaf litter N:P ratios, and high rainfall coincide during the latter portions of the rainy season and drive the highest free-living N fixation rates of the year.     

Biological Nitrogen Fixation in Pachyrhizus ahipa (Wedd.) Parodi

The patterns of plant growth and N₂ fixation capability in Pachyrhizusahipa (Wedd) Parodi inoculated with Bradyrhizobium ‘PachyrhizusSpec 1’ strains (Lipha Tech) were investigated in a zero-Nculture system under greenhouse conditions The P ahipa plantis day-neutral with respect to reproductive development Competitionoccurred between the two storage organs (legume and tuber) andprevented high tuber yield in P ahipa The symbiotic effectivenessof the association was high, as the profuse nodulation providedthe inoculated plants with adequate amounts of N Nodules werepresent throughout the cycle of P ahipa The change in rate ofN₂ fixation (RNF) and relative growth rate (RGR) was almostparallel during ontogenesis The developmental pattern of N₂fixation activity revealed that 65% of total N₂ fixation occurredafter N began to accumulate in the reproductive (pod wall plusseed) tissue During pod filling allocation of N compounds tothe seeds exceeded N₂ fixation, the pod walls being the primarysource of redistributed N, followed by the leaves. Pachyrhizus ahipa (Wedd) Parodi, ahipa, tuber crop, dinitrogen fixation, dry matter, N partitioning, reproductive growth     

Release of Dissolved Organic Nitrogen by Marine Diazotrophic Cyanobacteria, Trichodesmium spp

Trichodesmium sp. is a filamentous, colonial cyanobacterium which contributes substantially to the input of nitrogen in tropical and subtropical oceanic waters through nitrogen fixation (N(2) fixation). We applied a N tracer technique to assess the rate of release of dissolved organic nitrogen (DON) from this cyanobacterium and compared those rates with rates of N(2) fixation determined for the same assemblages at the same times of day. Rates of release of DON showed considerable variation within replicate experiments and were variable depending on time of day and duration of time course experiments. On average, rates of DON release were ca. 50% the rates of N(2) fixation. We also fractionated the DON released by using ultrafiltration and found that 60 to 80% of the total organic release was of the size class <10,000 Da. The release of these organic compounds by Trichodesmium spp. is likely a significant source of new nitrogen for the associated bacteria or the non-nitrogen-fixing filaments of the Trichodesmium colonies.     

Nitrogen cycling in the soil–plant system along a precipitation gradient in the Kalahari sands

Nitrogen (N) cycling was analyzed in the Kalahari region of southern Africa, where a strong precipitation gradient (from 978 to 230 mm mean annual precipitation) is the main variable affecting vegetation. The region is underlain by a homogeneous soil substrate, the Kalahari sands, and provides the opportunity to analyze climate effects on nutrient cycling. Soil and plant N pools, ¹⁵N natural abundance (δ¹⁵N), and soil NO emissions were measured to indicate patterns of N cycling along a precipitation gradient. The importance of biogenic N₂ fixation associated with vascular plants was estimated with foliar δ¹⁵N and the basal area of leguminous plants. Soil and plant N was more ¹⁵N enriched in arid than in humid areas, and the relation was steeper in samples collected during wet than during dry years. This indicates a strong effect of annual precipitation variability on N cycling. Soil organic carbon and C/N decreased with aridity, and soil N was influenced by plant functional types. Biogenic N₂ fixation associated with vascular plants was more important in humid areas. Nitrogen fixation associated with trees and shrubs was almost absent in arid areas, even though Mimosoideae species dominate. Soil NO emissions increased with temperature and moisture and were therefore estimated to be lower in drier areas. The isotopic pattern observed in the Kalahari (¹⁵N enrichment with aridity) agrees with the lower soil organic matter, soil C/N, and N₂ fixation found in arid areas. However, the estimated NO emissions would cause an opposite pattern in δ¹⁵N, suggesting that other processes, such as internal recycling and ammonia volatilization, may also affect isotopic signatures. This study indicates that spatial, and mainly temporal, variability of precipitation play a key role on N cycling and isotopic signatures in the soil–plant system.     

Nitrogen fixation in soybean as influenced by cultivar and Rhizobium strain

Summary The¹⁵N-substratum labeling technique and other indirect methods were used to compare nitrogen (N₂) fixation in soybean varieties grown in the field in Greece and Romania. Significant variation in the amount (Ndfa) and proportion of N derived from fixation (% Ndfa) was found in different varieties. With 20 kg N/ha applied to soil, N₂ fixed ranged from 22 to 236 kg N/ha in Greece and from 17 to 132 kg N/ha in Romania. In general, varieties or treatments with higher dry matter yield supported greater fixation. Also, varieties with high Ndfa had high % Ndfa andvice versa. Breeding N₂-fixing legumes for high yields at low soil N levels therefore appears to be a reasonable strategy for enhancing N₂ fixation. Heavy applications of inorganic N fertilizer severely depressed N₂ fixation in two out of the three varieties used in Romania. One variety, F 74–412, however, derived slightly higher amounts of N₂ from fixation at 100 kg N/ha rate than when fertilized with 20 kg N/ha. In Greece, Chippewa, Williams and Amsoy-71 inoculated with a Nitragin inoculant fixed similar amounts of N₂ at both 20 and 100 kg N/ha fertilizer rates. However, when Chippewa and Williams were inoculated with amother, locally-isolated Rhizobium strain, N₂ fixation was substantially depressed at the higher N rate.     

The Role of Nitrogen Fixation in Cyanobacterial Bloom Toxicity in a Temperate,Eutrophic Lake

Toxic cyanobacterial blooms threaten freshwaters worldwide but have proven difficult to predict because the mechanisms of bloom formation and toxin production are unknown, especially on weekly time scales. Water quality management continues to focus on aggregated metrics, such as chlorophyll and total nutrients, which may not be sufficient to explain complex community changes and functions such as toxin production. For example, nitrogen (N) speciation and cycling play an important role, on daily time scales, in shaping cyanobacterial communities because declining N has been shown to select for N fixers. In addition, subsequent N pulses from N₂ fixation may stimulate and sustain toxic cyanobacterial growth. Herein, we describe how rapid early summer declines in N followed by bursts of N fixation have shaped cyanobacterial communities in a eutrophic lake (Lake Mendota, Wisconsin, USA), possibly driving toxic Microcystis blooms throughout the growing season. On weekly time scales in 2010 and 2011, we monitored the cyanobacterial community in a eutrophic lake using the phycocyanin intergenic spacer (PC-IGS) region to determine population dynamics. In parallel, we measured microcystin concentrations, N₂ fixation rates, and potential environmental drivers that contribute to structuring the community. In both years, cyanobacterial community change was strongly correlated with dissolved inorganic nitrogen (DIN) concentrations, and Aphanizomenon and Microcystis alternated dominance throughout the pre-toxic, toxic, and post-toxic phases of the lake. Microcystin concentrations increased a few days after the first significant N₂ fixation rates were observed. Then, following large early summer N₂ fixation events, Microcystis increased and became most abundant. Maximum microcystin concentrations coincided with Microcystis dominance. In both years, DIN concentrations dropped again in late summer, and N₂ fixation rates and Aphanizomenon abundance increased before the lake mixed in the fall. Estimated N inputs from N₂ fixation were large enough to supplement, or even support, the toxic Microcystis blooms.     

Nitrogen Fixation in Bryophytes, Lichens, and Decaying Wood along a Soil-age Gradient in Hawaiian Montane Rain Forest

We determined rates of acetylene reduction and estimated total nitrogen fixation associated with bryophytes, lichens, and decaying wood in Hawaiian montane rain forest sites with underlying substrate ranging in age from 300 to 4.1 million years. Potential N fixation ranged from ca 0.2 kg/ha annually in the 300‐year‐old site to ca 1 kg/ha annually in the 150,000‐year‐old site. Rates of acetylene reduction were surprisingly uniform along the soil‐age gradient, except for high rates in symbiotic/associative fixers at the 150,000‐year‐old site and in heterotrophic fixers at the 2100‐year‐old site. Low fixation at the youngest site, where plant production is known to be N‐limited, suggests that demand for N alone does not govern N fixation. Total N fixation was highest in sites with low N:P ratios in leaves and stem wood, perhaps because epiphytic bryophytes and lichens depend on canopy leachate for mineral nutrients and because heterotrophic fixation is partly controlled by nutrient supply in the decomposing substrate; however, differences in substrate cover, rather than in fixation rates, had the largest effect on the total N input from fixation at these sites.     

Nitrogen fixation by subclover associations fertilized with sulfur

Summary The effect of S fertilization on symbiotic N₂ fixation was measured with the¹⁵N technique and the N difference method in a lysimeter study using Josephine loam (Typic Haploxurults). Nitrogen fixation by subclover (Trifolium subterraneum L.) was strongly enhanced by added S. The association of soft chess (Bromus mollis L.) or filaree (Erodium botrys (Cav.) Bertol.) with subclover increased the percentage of N in subclover that was fixed, with the results that N₂ fixation was increased beyond that due to the mere increase in subclover biomass. Nitrogen fixation estimates by¹⁵N dilution and N difference methods were highly correlated (r²=0.97), and S fertilizer did not result in any significant differences in N₂-fixation estimation by the two methods. Both methods were useful in distinguishing between soil N uptake and N₂ fixation where S applications produced highly significant increases in both uptake and fixation. Application of sulfur fertilizers to much annual rangeland has the potential to increase pasture productivity through enhanced N₂ fixation. Contribution of the University of California Hopland Field Station and Department of Agronomy and Range Science, Univ. of California, Davis, CA 95616.     

Nitrogen Fixation by Rhodospirillum rubrum Grown in Nitrogen-limited Continuous Culture

Cell-free extracts of the photosynthetic bacterium Rhodospirillum rubrum were inconsistent in reducing N(2). An internally illuminated fermentor, designed for the continuous culture of this organism on N(2) under nitrogen-limited conditions, produced cells which yielded cell extracts with consistent activity for cell-free N(2) fixation. A nitrogen-limited continuous culture, supplied ammonia rather than N(2), gave cell-free extracts with even more active N(2) fixation. Extracts of cells grown in the fermentor with glutamate nitrogen as the limiting nutrient in continuous culture did not reduce N(2), but whole cells fixed (15)N-enriched N(2). The discovery that cells from ammonia and glutamate nitrogen-limited continuous cultures are capable of N(2) reduction suggests that R. rubrum cells produce the N(2)-reducing enzymes in response to conditions of nitrogen deficiency rather than in response to the presence of N(2). Examination of the effect of the pN(2) on N(2) reduction by cell-free preparations of R. rubrum indicated that the K(N(2)) is approximately 0.071 atm. Cell-free extracts from R. rubrum were tested for their ability to reduce substrates other than N(2).     

Nitrogen Stress and Apparent Photosynthesis in Symbiotically Grown Pisum sativum L

Pea plants (Pisum sativum L. cv. Alaska) were inoculated individually with one of 15 Rhizobium leguminosarum strains and grown under uniform environmental conditions in the absence of combined N. Differences in effectiveness of the Rhizobium strains produced plants with differing rates of whole plant apparent N₂ fixation and total N content at the same morphological stage of development. Plants were analyzed to determine interactions between N₂ fixation, N allocation, apparent photosynthesis, and growth. Total leaf N increased linearly with total N₂ fixation (R² = 0.994). The proportion of total N allocated to leaves, the per cent N content of individual leaves, and the photosynthetic efficiency of individual leaves showed a curvilinear response with increasing plant N content. Differences in allocation patterns of leaf N between plants with low and high N content resulted in differences in the relationship between total N content and plant dry weight. Results from this study show that N₂ fixation interacts with leaf photosynthetic efficiency and plant growth in a manner that is dependent upon the allocation of symbiotically fixed N.     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.