Genetic diversity and phylogeny analysis of Anabaena azollae based on RFLPs detected in Azolla-Anabaena azollae DNA complexes using nif gene probes

The cyanobacterium Anabaena has both symbiotic and free-living forms. The genetic diversity of Anabaena strains symbiotically associated with the aquatic fern Azolla and the evolutionary relationships among these symbionts were evaluated by means of RFLP (restriction fragment length polymorphism) experiments. Three DNA fragments corresponding to nif genes were cloned from the free-living cyanobacterium Anabaena PCC 7120 and used as probes. A mixture of Azolla, Anabaena and bacterial DNA was extracted from Azolla fronds and digested with two restriction enzymes. Single-copy RFLP signals were detected with two of the probes in all Azolla Anabaena examined. Multiple-copy RFLP signals were obtained from the third probe which corresponded to a part of the nif N gene. A total of 46 probe/enzyme combinations were scored as present or absent and used to calculate pairwise Nei's genetic distances among symbiotic Anaebaena strains. Phylogenetic trees summarizing phenetic and cladistic relationships among strains were generated according to three different evolutionary scenarios: parsimony, UPGMA and neighbour joining. All trees revealed identical phylogenetic relationships. Principal component analysis was also used to evaluate genetic similarities and revealed three groups: group one contains the cyanobacteria associated with plants from the Azolla section, group two contains those associated with plants from the pinnata species and group three contains those associated with plants from the nilotica species. The same groups had already been identified earlier in a random amplified polymorphic DNA (RAPD) analysis of Azolla-Anbaena DNA complexes, suggesting that the present Azolla taxonomy should be revised. We now suggest a taxonomy of Anabaena azollae that is parallel to such a revised Azolla taxonomy. An Azolla chloroplast DNA sequence derived from Oryza sativa was also used as an RFLP probe on Azolla DNA to confirm the presence of plant DNA in the total genomic DNA extracted from ferns with or without the symbiont. Our results also suggest that total DNA extracted from the Azolla-Anabaena complexes includes both plant and symbiont DNA and can be used equally well for RFLP analysis of host plant or symbiotic cyanobacteria.     

A comparative study of nitrogen fixation by the Anabaena-Azolla symbiosis and free-living populations of Anabaena spp. in Lake Ngahawa,New Zealand

Summary The symbiotic fern Azolla filiculoides var. rubra, which contains a blue-green nitrogen fixing alga Anabaena azollae, fixed 164 Kg N·ha^-1·ann^-1 in the littoral zone of a small eutrophic lake. Associated planktonic Anabaena spp. blooms, dominated by Anabaena spiroides, fixed 29.5Kg N·ha^-1·ann^-1. Nitrogen fixation in both organisms was not obviously related to ambient dissolved inorganic nitrogen levels. By comparing ¹⁵N–N₂ and acetylene reduction techniques, we determined a ratio of 3 moles C₂H₂ reduced to 1 mole of N₂ fixed. Combining this with results from one diurnal investigation, it was estimated that 24% of the total daily fixation by Azolla occurred at night. Highest nitrogen fixation rates in Azolla occurred when plant density was lowest. Nitrogen fixation by planktonic Anabaena spp. generally paralleled changes in biomass. Frond breakage due to wind caused a decrease in Azolla nitrogen fixation and growth which was followed by a bloom of planktonic Anabaena spp. A second Anabaena spp. bloom was instrumental in the summer decline of Azolla. Maximum growth and nitrogen fixation of both organisms did not occur simultaneously. If physical disruption to the Azolla mat does not occur, it is likely that growth of the population would continue throughout the year.This work was completed at the Department of Scientific and Industrial Research, Freshwater Section, PO Box 415, Taupo, New Zealand, with partial assistance of N.S.F. Grant BMS-74-20745 to C.R. Goldman     

MSX-resistant mutants of Anabaena 7120 with derepressed heterocyst development and nitrogen fixation

To investigate the role of ammonium-assimilating enzyme in heterocyst differentiation, pattern formation and nitrogen fixation, MSX-resistant and GS-impaired mutants of Anabaena 7120 were isolated using transposon (Tn5-1063) mutagenesis. Mutant Gs1 and Gs2 (impaired in GS activity) exhibited a similar rate of nitrogenase activity compared to that of the wild type under dinitrogen aerobic conditions in the presence and absence of MSX. Filaments of Gs1 and Gs2 produced heterocysts with an evenly spaced pattern in N₂-grown conditions, while addition of MSX altered the interheterocyst spacing pattern in wild type as well as in mutant strains. The wild type showed complete repression of heterocyst development and nitrogen fixation in the presence of NO₃ ^– or NH₄ ⁺, whereas the mutants Gs1 and Gs2 formed heterocysts and fixed nitrogen in the presence of NO₃ ^– and NH₄ ⁺. Addition of MSX caused complete inhibition of glutamine synthetase activity in wild type but Gs1 and Gs2 remained unaffected. These results suggest that glutamine but not ammonium is directly involved in regulation of heterocyst differentiation, interheterocyst spacing pattern and nitrogen fixation in Anabaena.     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

Differences in growth rate,nitrogen fixation and numbers of cyanobionts and heterocysts among three Azolla pinnata var. pinnata strains

The differences in nitrogen fixation, growth rate and numbers of cyanobionts and heterocysts among three Azolla pinnata var. pinnata strains were examined. The relative growth rate (RGR) and nitrogen fixation of PP7002 and PP7003 were significantly low compared with those of PP7005. The application of ammonium ions at 0.2 mM or more increased the growth rate of PP7002 and PP7003, but not PP7005. The numbers of cyanobionts and heterocysts in the mature region of PP7002 and PP7003 were statistically lower than those of PP7005. The low nitrogen-fixing activity of PP7002 and PP7003 as compared with PP7005 might be related to the restricted number of heterocysts. In PP7002 and PP7003, nitrogen fixation might be insufficient for full growth.     

How does glutamine synthetase activity determine plant tolerance to ammonium?

The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants’ capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.     

Influence of urea on biological N2 fixation and N transfer from Azolla intercropped with rice

The N₂ fixed by Azolla before and after urea application during the rice cycle, the mineralisation of Azolla-N as well as its availability to rice was studied in two greenhouse experiments conducted in 1996 and 1997 and in June 1998 in Goettingen (Germany). Dry matter production of the various rice parts of experiment 1 showed a clear positive synergism between treatment with Azolla and urea with a resulting apparent N recovery by rice increasing from 40% (without Azolla) to 57% in the presence of Azolla. Part of this increase may be due to N fixed biologically by Azolla and transferred to the rice. The second experiment shed some light on the role of BNF. Using an iterative method of estimation, the daily rate of N fixation was estimated at 0.6 – 0.7 kg N ha^–1. The rate was not so much affected by the age of the Azolla crop. At this rate, the BNF would amount to up to 100 kg N ha^–1 over a 130-day season. Assuming that BNF may be inhibited for a period of 5 – 10 days following urea application due to high levels of N in the floodwater, this might reduce the BNF by between 6 and 14 kg N ha over the season. Using the mean-pool-abundance concept, it was estimated that around 75 – 80% of the Azolla-N mineralized during the growth period was actually absorbed by the rice plants. Of the N taken up by rice around 28% was derived from the biologically fixed Azolla N, the remainder was urea N cycled through the Azolla. Azolla also seems to help sustain the soil N supply by returning N to the soil in quantities roughly equal to those extracted from the soil by the rice plant.     

Pigment distribution and nitrogen fixation inAnabaena azollae

Summary The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity.In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N₂-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla).Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O₂ evolution and the absence of photo-fixation of CO₂ in the cyanobionts.Pigment composition and N₂-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility.It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.     

Evaluation of the availability ofAzolla-N and urea-N to rice using15N

Summary The symbiotic association of the water fernAzolla with the blue-green algaAnabaena azollae can fix 30–60 kg N ha^–1 per rice cropping season. The value of this fixed N for rice production, however, is only realized once the N is released from theAzolla biomass and taken up by the rice plants. The availability of N applied asAzolla or as urea was measured in field experiments by two¹⁵N methods. In the first,Azolla caroliniana (Willd.) was labelled with¹⁵N in nutrient solution and incorporated into the soil at a rate of 144 kg N ha^–1. The recovery ofAzolla-N in the above ground parts of rice [Oryza sativa (L) cv. Nucleoryza] was found to be 32% vs. 26% for urea applied at a rate of 100 kg N/ha; there was no significant difference in recovery. In the second, 100 kg N/ha of¹⁵N-urea was applied separately or in combination with either 250 or 330 kg N ha^–1 of unlabelledAzolla. At the higher rate, the recovery ofAzolla-N was significantly greater than that of urea. There was a significant interaction when both N sources were applied together, which resulted in a greater recovery of N from each source in comparison to that source applied separately. Increasing the combined urea andAzolla application rate from 350 kg N ha^–1 to 430 kg N ha^–1 increased the N yield but had no effect on the dry matter yield of rice plants. The additional N taken up at the higher level of N application accumulated to a greater extent in the straw compared to the panicles. Since no assumptions need to be made about the contribution of soil N in the method using¹⁵N-labelledAzolla, this method is preferable to the¹⁵N dilution technique for assessing the availability ofAzolla-N to rice. Pot trials usingAzolla stored at –20°C or following oven-drying showed that both treatments decreased the recovery of N by one third in comparison to freshAzolla.     

DNA amplification fingerprinting of the Azolla-Anabaena symbiosis

The Azolla-Anabaena symbiosis has been used for centuries as a nitrogen biofertilizer in rice paddies. Genetic improvement of the symbiosis has been limited by the difficulty in identifying Azolla-Anabaena accessions and Anabaena azollae strains. The recently developed technique of DNA amplification fingerprinting (DAF) was applied to this problem. DAF uses single, short, oligonucleotide primers of arbitrary sequence to direct amplification of a characteristic set of DNA products by a thermostable DNA polymerase in a thermocycling reaction. The products are separated in polyacrylamide gels and detected by silver staining. DAF could easily distinguish and positively identify accessions of Azolla-Anabaena with DNA extracted from the intact symbioses. The contribution of prokaryotic Anabaena sequences to the fingerprint of the intact symbioses, however, ranged from 0 to 77%, depending on the primer sequence. Therefore, DNA extracted from the intact symbioses would not be suitable for Azolla taxonomy studies. The fingerprints of Anabaena strains isolated by sucrose gradient centrifugation from different species of Azolla could be easily distinguished, and DAF patterns were used to confirm the maternal pattern of transmission of Anabaena in a sexual hybrid. Template DNA extracted from roots was used to produce fingerprints for Azolla without interference from the microsymbiont. Comparison of the patterns from the parents and a hybrid gave strong evidence confirming sexual hybridization.     

Content of Oxalate in Actinidia Deliciosa Plants Grown in Nutrient Solutions with Different Nitrogen Forms

Kiwifruit plants (Actinidia deliciosa cv. Hayward) were grown in Hoagland nutrient solution with calcium nitrate, potassium nitrate, ammonium nitrate or ammonium chloride as the nitrogen source. Plants grown in the solution with nitrate nitrogen displayed a higher oxalate content, greater shoot length and leaf area, and higher content of ascorbic acid and NO₃ ^– ions in the leaves. Plants grown in the solution with ammonium nitrate, and particularly with ammonium chloride, showed low oxalate content, low content of ascorbic acid and NO₃ ^–, high content of Cl^– and Na⁺, low shoot length and leaf area. Oxalate formation appeared to be connected with the assimulation of nitrate, more precisely with nitrate reduction, while ammonium nitrogen assimilation did not induce the synthesis of oxalic acid.     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Sucrose synthase and RuBisCo expression is similarly regulated by the nitrogen source in the nitrogen-fixing cyanobacterium Anabaena sp.

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.     

Nitrogen fixation by Spirillum sp. from rice roots

Nitrogen fixation by Spirillum sp. obtained from the roots of rice plants grown at different levels of combined nitrogen was studied. The roots of rice plants exposed to low levels of combined nitrogen (20–40 kg N/ha) harboured Spirillum sp. possessing higher nitrogen-fixing efficiency as compared to the cultures from plants receiving 60–100 kg N/ha. More-over, the nitrogen-fixing efficiency of these Spirillum spp. varied with age of the plant, irrespective of the dosage of combined nitrogen.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Diagnosis of nitrogen deficiency and toxicity of Eucalyptus globulus seedlings by foliar analysis

The relationship between shoot growth and foliar nitrogen (N) in E. globulus seedlings was studied in the glasshouse to determine standard values for N deficiency and toxicity diagnosis. Seedlings were grown for 9 weeks in yellow sand, at 10 rates of N, applied as ammonium sulphate, calcium nitrate or ammonium nitrate. Shoot dry weight (DW) increased linearly with N rate for all forms of N in the deficiency range. Seedlings continued to respond to higher rates of ammonium and ammonium nitrate than to nitrate. Maximum shoot DW for nitrate fed plants and ammonium nitrate fed plants were 51% and 84% respectively of ammonium fed plants. Total N concentration in the youngest fully expanded leaf (YFEL) ranged from 1.0% to 3.3% in deficient and adequate plants. The critical N concentration for deficiency diagnosis (corresponding to 90% maximum yield) in the YFEL, determined from these growth response curves averaged over all N forms, was 2.6% N. For ammonium nitrate fed plants, total N concentration in the YFEL for the severely deficient, deficient, adequate, and toxic ranges were <1.4%, 1.4–2.5%, 2.6–3.5%, > 4.3%. High total N concentrations were associated with growth depression and toxicity symptoms, which differed with N form. For nitrate fed plants, a total N concentration above 3.3% in the YFEL was associated with severe growth depression, and leaf tip necrosis. The adequate concentration range for ammonium nitrate was similar to values found on a field trial with 7 month old E. globulus trees grown on an exforest site.     

The effects of immobilization on the biochemical,physiological and morphological features of Anabaena azollae

Anabaena azollae, a presumptive isolate from Azolla filiculoides, was immobilized in polyurethane foam, hydrophilic polyvinyl foam and alginate. When viewed by low-temperature scanning electron microscopy a thick mucilage layer covered the surface of both cells and matrix; this closely resembles the mode of attachment of the symbiont Anabaena in the Azolla leaf cavity. The heterocyst frequency of the immobilized A. azollae doubled relative to free-living cells and reached a level of 14–17%. Immobilization induced increases in both hydrogen production via nitrogenase or hydrogenase and in the rates and stabilization of acetylene reduction (N₂-fixation). Ammonia production by immobilized cells with L-methionine-D,L-sulfoximine (MSX) is greater than that of freeliving cells. Immobilized cells without MSX were, however, able to excrete ammonium at lower rates thus emulating the characteristic of the symbiotic cyanobacteria (A. azollae) in the leaf cavity of Azolla.Abbreviations Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-D,L-sulfoximine - SEM scanning electron microscopy - PU polyurethane - PV polyvinyl     

DIVERSITY AND HOST SPECIFICITY OF AZOLLA CYANOBIONTS1

A unique, hereditary symbiosis exists between the water fern Azolla and cyanobacteria that reside within a cavity in the dorsal leaf‐lobe of the plant. This association has been studied extensively, and questions have frequently been raised regarding the number and diversity of cyanobionts (cyanobacterial symbionts) among the different Azolla strains and species. In this work, denaturating gradient gel electrophoresis (DGGE) and a clone library based on the 16S rRNA gene were used to study the genetic diversity and host specificity of the cyanobionts in 35 Azolla strains covering a wide taxonomic and geographic range. DNA was extracted directly from the cyanobacterial packets, isolated after enzymatic digestion of the Azolla leaves. Our results indicated the existence of different cyanobiont strains among Azolla species, and diversity within a single Azolla species, independent of the geographic origin of the host. Furthermore, the cyanobiont exhibited host‐species specificity and showed most divergence between the two sections of genus Azolla, Azolla and Rhizosperma. These findings are in agreement with the recent redefinition of the taxon Azolla cristata within the section Azolla. With regard to the taxonomic status of the cyanobiont, the genus Anabaena of the Nostocaceae family was identified as the closest relative by this work.     

Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters

A set of 24 strains belonging to the genus Anabaena (Phylum Cyanobacteria), isolated from diverse geographic locations in India, were evaluated along with three International type strains of Anabaena (ATCC 29414, ATCC 29208 and ATCC 27899) for their morphological, physiological and biochemical diversity. The morphological dataset, consisting of 58 variants for 15 characters, and SDS-PAGE protein profiles comprising 17 polymorphic bands were utilized to differentiate the selected Anabaena strains and explore the patterns of diversity through cluster analysis. Physiological and biochemical characterization with respect to nitrogen fixation and accumulation of chlorophyll and phycobiliproteins led to the identification of some highly promising Anabaena strains for use as biofertilizers and source of pigments. The study highlighted the tremendous inter and intraspecific diversity within the Anabaena isolates and indicated the potential as well as constraints of the morphological and protein profiling datasets for unambiguous differentiation and analyses of diversity among the Anabaena strains.