Nitrogen Fixation Associated with Rinsed Roots and Rhizomes of the Eelgrass Zostera marina

Nitrogen fixation was associated with the rinsed roots and rhizomes of the seagrass, Zostera marina L. Nitrogenase activity (acetylene reduction) was greater on rhizomes compared to roots, and on older roots and rhizomes relative to younger tissue. Compared to aerobic assays, anaerobic or microaerobic conditions enhanced the rate of acetylene reduction by rhizomes with attached roots, with the highest activity (100 nanomoles per gram dry weight per hour) occurring at pO₂ = 0.01 atmosphere. Addition of glucose, sucrose, or succinate also increased the rate of acetylene reduction under anaerobic conditions, with glucose providing the most stimulation. In one experiment, comparison of acetylene reduction assays with ¹⁵N₂ incorporation yielded a ratio of about 2.6:1. Seagrass communities are thought to be limited by the availability of nitrogen and, therefore, nitrogenase activity directly associated with their roots and rhizomes suggests the possibility of a N₂-fixing flora which may subsidize their nutritional demand for nitrogen.     

Nitrogen Fixation Associated with the New Zealand Mangrove (Avicennia marina (Forsk.) Vierh. var. resinifera (Forst. f.) Bakh.)

Nitrogenase activity in mangrove forests at two locations in the North Island, New Zealand, was measured by acetylene reduction and ¹⁵N₂ uptake. Nitrogenase activity (C₂H₂ reduction) in surface sediments 0 to 10 mm deep was highly correlated (r = 0.91, n = 17) with the dry weight of decomposing particulate organic matter in the sediment and was independent of light. The activity was not correlated with the dry weight of roots in the top 10 mm of sediment (r = −0.01, n = 13). Seasonal and sample variation in acetylene reduction rates ranged from 0.4 to 50.0 μmol of C₂H₄ m⁻² h⁻¹ under air, and acetylene reduction was depressed in anaerobic atmospheres. Nitrogen fixation rates of decomposing leaves from the surface measured by ¹⁵N₂ uptake ranged from 5.1 to 7.8 nmol of N₂ g (dry weight)⁻¹ h⁻¹, and the mean molar ratio of acetylene reduced to nitrogen fixed was 4.5:1. Anaerobic conditions depressed the nitrogenase activity in decomposing leaves, which was independent of light. Nitrogenase activity was also found to be associated with pneumatophores. This activity was light dependent and was probably attributable to one or more species of Calothrix present as an epiphyte. Rates of activity were generally between 100 and 500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ in summer, but values up to 1,500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ were obtained.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

Physiology of Root-Associated Nitrogenase Activity in Oryza sativa

An intact method for measuring immediately linear rates of acetylene reduction was used to investigate the relationship between temperature, pH, O₂ concentration, and light intensity with the rate of root-associated nitrogenase activity in rice (Oryza sativa L.). Nitrogenase activity varied over a temperature range of 10 to 50°C and optimal rates of acetylene reduction were recorded at 35°C. Nitrogenase activity was also influenced by the pH of the liquid surrounding the roots prior to assay. Maximal rates of acetylene reduction were recorded over a pH range from 5.8 to 7.5. Nitrogenase activity was significantly reduced by concentrations of O₂ 0.5% (v/v) or more when the intact plant assay method was used, and no optimum was detected. However, when the plant tops were removed and the cut ends sealed from the atmosphere for 4 hours, acetylene reduction rates were maximal at 0.25% O₂ (v/v). When plants were moved from sunlight (1,400 microeinsteins per square meter per second) to shade (9.6) root-associated nitrogenase activity at 35° C significantly decreased 15 min later to one-fourth the rate and recovered upon return to sunlight. When the light intensity reaching the leaf canopy was progressively reduced from 1,050 to 54 microeinsteins per square meter per second the rate of root-associated nitrogenase activity decreased from 550 ± 135 to 192 ± 55 nanomoles ethylene per gram dry root per hour. The study suggests that the rate of root-associated nitrogenase activity in rice at constant temperature may well be mediated by variations in the concentration of O₂ resulting from changes in the rate of photosynthesis as well as variations in the rate of transport of photosynthate.     

Relation between nitrogenase synthesis and activity in a marine unicellular diazotrophic strain, Gloeothece sp. 68DGA (Cyanophyte), grown under different light/dark regimens

The relationship between the abundance of nitrogenase and its activity was studied in the marine unicellular cyanobacterium Gloeothece sp. 68DGA cultured under different light/dark regimens. The Fe‐ and MoFe‐protein of nitrogenase and nitrogen (N₂)‐fixing (acetylene reduction) activity were detected only during the dark phase when the cells were grown under a 12 h light/12 h dark cycle (12L/12D). Nitrogenase activity appeared about 4 h after entering the dark phase. Maximum nitrogenase activity occurred at around the middle of the dark phase, and the activity rapidly decreased to zero before the start of the light phase. The rapid decrease of nitrogenase activity and the Fe‐protein of nitrogenase near the end of the dark phase in 12L/12D were partly recovered by the addition of l ‐methionine‐sulfoximine, an inhibitor of glutamine synthetase. Diurnal oscillation of the abundance of nitrogenase was maintained in the first subjective dark phase (i.e. the period corresponding to the dark phase) after the cells were transferred from 12L/12D to continuous illumination. However, enzyme activity was detected only when photosynthetic oxygen (O₂) evolution was completely suppressed by reducing the light intensity or by the addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Nitrogenase always appeared in the cells about 16 h after starting the light phase, even when the 12L/12D cycle was modified by the addition or subtraction of a single 6 h period of light or dark. These results suggest the following: (i) N₂‐fixation by Gloeothece sp. 68DGA is primarily regulated by an endogenous circadian oscillator at the level of nitrogenase synthesis. (ii) The endogenous circadian rhythm resets on a shift of the timing of the light phase. (iii) Nitrogenase activity is not always reflected in the presence of nitrogenase. (iv) The activity of nitrogenase is negatively regulated by fixed nitrogen and the concentration of ambient O₂.     

Biochemistry and physiology of nitrogen fixation with particular emphasis on nitrogen-fixing phototrophs

Summary This paper presents an overview of aspects of N₂-fixation in phototrophic N₂-fixers. Nitrogenase is little different in phototrophs from other organisms. Evidence suggests that fixed carbon dissimilation rather than direct photoreduction from oxidised inorganic compounds or exogenous photosynthetic electron donors is the major route of reductant supply to nitrogenase in phototrophs; inRhodospirillum rubrum pyruvate is a possible electron donor to nitrogenase; in cyanobacteria the oxidative pentose phosphate pathway is important, although some recent evidence implicates glycolysis and the tricarboxylic acid cycle in reductant supply in heterocystous cyanobacteria. In photosynthetic organisms light modulation of various enzymes occurs-some Calvin cycle enzymes are light activated, some oxidative pentose phosphate pathway and glycolytic enzymes are deactivated and some tricarboxylic acid cycle enzymes are activated. Reduced levels of thioredoxin in heterocysts may contribute to the sustained functioning of the oxidative pentose phosphate pathway in heterocysts in the light and dark. In photosynthetic bacteria such asRhodospirillum rubrum an activating enzyme which removes a modifying group from inactive Fe protein can activate nitrogenase. O₂ and NH ₄ ⁺ both inhibit N₂-fixation and there is some evidence in cyanobacteria that O₂ stability of whole cell nitrogenase can be achieved by prolonged incubation of cultures at high O₂.     

Effect of temperature on h(2) evolution and acetylene reduction in pea nodules and in isolated bacteroids

Nitrogenase (EC 1.7.99.2) activity in pea (Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H₂ evolution in air and H₂ evolution in Ar:O₂. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H₂ evolution in Ar:O₂ was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H₂ evolution in Ar:O₂ with increased temperature, indicating that either acetylene reduction or H₂ evolution in Ar:O₂ are inadequate measures of nitrogenase activity at higher temperature.     

Exploring cycad foliage as an archive of the isotopic composition of atmospheric nitrogen

Molecular nitrogen (N₂) constitutes the majority of Earth's modern atmosphere, contributing ~0.79 bar of partial pressure (pN₂). However, fluctuations in pN₂ may have occurred on 10⁷–10⁹ year timescales in Earth's past, perhaps altering the isotopic composition of atmospheric nitrogen. Here, we explore an archive that may record the isotopic composition of atmospheric N₂ in deep time: the foliage of cycads. Cycads are ancient gymnosperms that host symbiotic N₂‐fixing cyanobacteria in modified root structures known as coralloid roots. All extant species of cycads are known to host symbionts, suggesting that this N₂‐fixing capacity is perhaps ancestral, reaching back to the early history of cycads in the late Paleozoic. Therefore, if the process of microbial N₂ fixation records the δ¹⁵N value of atmospheric N₂ in cycad foliage, the fossil record of cycads may provide an archive of atmospheric δ¹⁵N values. To explore this potential proxy, we conducted a survey of wild cycads growing in a range of modern environments to determine whether cycad foliage reliably records the isotopic composition of atmospheric N₂. We find that neither biological nor environmental factors significantly influence the δ¹⁵N values of cycad foliage, suggesting that they provide a reasonably robust record of the δ¹⁵N of atmospheric N₂. Application of this proxy to the record of carbonaceous cycad fossils may not only help to constrain changes in atmospheric nitrogen isotope ratios since the late Paleozoic, but also could shed light on the antiquity of the N₂‐fixing symbiosis between cycads and cyanobacteria.     

Acetylene Reduction by Soil Cores of Maize and Sorghum in Brazil

Nitrogenase activity was measured by the C₂H₂ reduction method in large soil cores (29 cm in diameter by 20 cm in depth) of maize (Zea mays) and sorghum (Sorghum vulgare). The activity was compared to that obtained by a method in which the roots were removed from the soil and assayed for nitrogenase activity after an overnight preincubation in 1% O₂. In a total of six experiments and 28 soil cores, the nitrogenase activity of the cores was an average of 14 times less than the activity of roots removed from the same cores and preincubated. Nitrogenase activity in the cores was very low and extrapolated to an average nitrogen fixation rate of 2.8 g of N/hectare per day. It was shown that inadequate gas exchange was not a reason for the lower activity in the soil cores, and the core method gave satisfactory results for nitrogenase activity of soybeans (Glycine max) and Paspalum notatum.     

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica

The mechanism of O₂ protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O₂ inhibition of nitrogenase activity correlated with the O₂ tension of the medium in which heterocyst formation was induced. O₂ resistance also correlated with the apparent K_m for acetylene, indicating that O₂ tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O₂ that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O₂ sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O₂ but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O₂ diffusion barrier and active respiration by the heterocyst.     

Bacterial Life and Dinitrogen Fixation at a Gypsum Rock

The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of Chroococcidiopsis spp. The organisms of the shards expressed nitrogenase activity (C₂H₂ reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of Chroococcidiopsis sp. strain PCC7203, C₂H₂ reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (nifH) indicated that Chroococcidiopsis possesses the alternative molybdenum nitrogenase 2, expressed in Anabaena variabilis only under reduced O₂ tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O₂ tension that allow N₂ fixation to proceed in the unicellular Chroococcidiopsis at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of nifH sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N₂ under microaerobic conditions only.     

Nitrogen Fixation Dynamics of Two Diazotrophic Communities in Mono Lake, California

Two types of diazotrophic microbial communities were found in the littoral zone of alkaline hypersaline Mono Lake, California. One consisted of anaerobic bacteria inhabiting the flocculent surface layers of sediments. Nitrogen fixation (acetylene reduction) by flocculent surface layers occurred under anaerobic conditions, was not stimulated by light or by additions of organic substrates, and was inhibited by O₂, nitrate, and ammonia. The second community consisted of a ball-shaped association of a filamentous chlorophyte (Ctenocladus circinnatus) with diazotrophic, nonheterocystous cyanobacteria, as well as anaerobic bacteria (Ctenocladus balls). Nitrogen fixation by Ctenocladus balls was usually, but not always, stimulated by light. Rates of anaerobic dark fixation equaled those in the light under air. Fixation in the light was stimulated by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and by propanil [N-(3,4-dichlorophenyl)propanamide]. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea-elicited nitrogenase activity was inhibited by ammonia (96%) and nitrate (65%). Fixation was greatest when Ctenocladus balls were incubated anaerobically in the light with sulfide. Dark anaerobic fixation was not stimulated by organic substrates in short-term (4-h) incubations, but was in long-term (67-h) ones. Areal estimates of benthic N₂ fixation were measured seasonally, using chambers. Highest rates (~29.3 μmol of C₂H₄ m⁻² h⁻¹) occurred under normal diel regimens of light and dark. These estimates indicate that benthic N₂ fixation has the potential to be a significant nitrogen source in Mono Lake.     

Oxygen-Poor Microzones as Potential Sites of Microbial N2 Fixation in Nitrogen-Depleted Aerobic Marine Waters

The nitrogen-deficient coastal waters of North Carolina contain suspended bacteria potentially able to fix N₂. Bioassays aimed at identifying environmental factors controlling the development and proliferation of N₂ fixation showed that dissolved organic carbon (as simple sugars and sugar alcohols) and particulate organic carbon (derived from Spartina alterniflora) additions elicited and enhanced N₂ fixation (nitrogenase activity) in these waters. Nitrogenase activity occurred in samples containing flocculent, mucilage-covered bacterial aggregates. Cyanobacterium-bacterium aggregates also revealed N₂ fixation. In all cases bacterial N₂ fixation occurred in association with surficial microenvironments or microzones. Since nitrogenase is oxygen labile, we hypothesized that the aggregates themselves protected their constituent microbes from O₂. Microelectrode O₂ profiles revealed that aggregates had lower internal O₂ tensions than surrounding waters. Tetrazolium salt (2,3,5-triphenyl-3-tetrazolium chloride) reduction revealed that patchy zones existed both within microbes and extracellularly in the mucilage surrounding microbes where free O₂ was excluded. Triphenyltetrazolium chloride reduction also strongly inhibited nitrogenase activity. These findings suggest that N₂ fixation is mediated by the availability of the appropriate types of reduced microzones. Organic carbon enrichment appears to serve as an energy and structural source for aggregate formation, both of which were required for eliciting N₂ fixation responses of these waters.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

LIGHT ACTION SPECTRA OF N2 FIXATION BY HETEROCYSTOUS CYANOBACTERIA FROM THE BALTIC SEA1

An on‐line, laser photo‐acoustic, trace gas detection system in combination with a stepper motor‐controlled monochromator was used to record semicontinuous light action spectra of nitrogenase activity in heterocystous cyanobacteria. Action spectra were made of cultures of Nodularia spumigena Mertens ex Bornet & Flahault, Aphanizomenon flos‐aquae Ralfs ex Bornet & Flahault, and Anabaena sp. and from field samples of a cyanobacterial bloom in the Baltic Sea. Nitrogenase activity was stimulated by monochromatic light coinciding the red and blue peaks of chl a, the phycobiliproteins phycocyanin (allophycocyanin) and phycoerythrin, and several carotenoids. Because nitrogenase is confined to the heterocyst, it was concluded that all photopigments must have been present in these cells, were involved in light harvesting and photosynthesis, and supplied the energy for N₂ fixation. The species investigated showed marked differences in their nitrogenase action spectra, which might be related to their specific niches and to their success in cyanobacterial blooms. Moreover, light action spectra of nitrogenase activity shifted during the day, probably as the result of changes in the phycobiliprotein content of the heterocyst relative to chl a. Action spectra of nitrogenase and changes in pigment composition are essential for the understanding of the competitive abilities of species and for the estimation of N₂ fixation by a bloom of heterocystous cyanobacteria.     

Nitrogen fixation by Nodularia spumigena Mertens (Cyanobacteriaceae). 2: Laboratory studies

The effects of changes in diurnal light patterns, salinity, and phosphorus on nitrogen fixation (as measured by acetylene reduction) by Nodularia spumigena Mertens were examined. As well, the effects of added inorganic nitrogen on growth, nitrogen fixation and heterocyt frequencies, and changes in nitrogen fixation and heterocyst frequencies during the growth cycle of Nodularia in cultures were determined.The diurnal pattern of nitrogenase activity in Nodularia was primarily light-induced, though dark activity did occur. Nitrogenase activity following a period of darkness exceeded the normal light rate (> 90 compared to 50 nmol · C₂H₂ reduced · ml^–1 · h^–1). Nitrogen fixation was reduced by high and very low salinities (5 to 10 was the optimum range), and added phosphorus stimulated nitrogenase in P-starved cells. Added nitrogen (ammonium or nitrate) had no effect on the growth of Nodularia, but in short term studies, ammonium completely inhibited nitrogenase activity. Heterocyst frequencies were greatest in the log phase of growth (to 40 per mm). During stationary phase, nitrogenase activity was negligable.     

Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp

Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO₂/C₂H₂ ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by ¹⁵N₂ incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms