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     

TheAzolla-Anabaena association: Historical perspective,symbiosis and energy metabolism

The heterosporous water-fern genusAzolla is one of the few symbioses with a cyanobacterium in the genusAnabaena. TheAzolla-Anabaena association includes six extant speciesof Azolla, which are widely distributed in relatively placid tropical and/or temperate freshwater environments. The earliest mention of the plant seems to be in an ancient Chinese dictionary that appeared about 2000 years ago.Azolla was used in about the 11th century in Vietnam. By 1980 renewed interest in this symbiotic association was shown by the demand for a less fossil energy-dependent agricultural technology. The importation of a variety ofA. filiculoides may have been a most significant breakthrough for the improvementof Azolla cultivation in China. The history of research may be divided into three periods and a new biotechnological stageof Azolla research has recently begun. Each mature dorsal leaf lobe has an ellipsoid cavity which containsAnabaena azollae throughout its development. HeterocystousA. azollae from sixAzolla species share identical and highly specific antigens.Azolla and its endophyte exhibit a coordinated pattern of differentiation and development. Epidermal hair cells of the host are probably interactive with the symbiont. The interior surface of a mature leaf cavity is lined with an envelope and covered by a mucilaginous layer.A. azollae shares the cavity with small populations of the bacteriaPseudomonas andAzotobacter. Endophyte-freeAzolla may rarely occur in nature and can be generated by aseptic techniques.Anabaena azollae can be isolated fromAzolla fronds by gentle pressure and by enzymatic digestion. The free living cultures derived from theAnabaena so obtained differ in some respects, however, from the freshly extracted symbiont, and might better be called the presumptive isolate. BothAzolla andAnabaena contain specific photosynthetic pigments. The optimum conditions for photosynthesis have been measured.Azolla is a C₃ plant and has high net photosynthesis. PSII activity in the symbiont is low. Nitrogenase is localized in the heterocysts of the symbiont and has some advantages compared with free-living cyanobacteria. SymbioticA. azollae has a high frequency of heterocysts. Unidirectional hydrogenase occurs in the symbiont and recycles electrons and ATP. Simultaneous measurements of N₂ fixation and photosynthesis show the dependence of nitrogenase on photosynthetically captured radiation for energy by an indirect dependence on CO₂ fixation. The host contains most of the total GS and GDH activities, and the symbiont excretes a substantial portion of its newly fixed nitrogen as ammonium. The two partners in the association exhibit a comparable developmental gradient and a mechanism of cooperative integration for their energy metabolism, thus improving the efficiency of solar energy conversion and presenting a unique model for biotechnology.     

A phytohaemagglutinin from the Azolla-anabaena symbiosis

Proteins recovered from cell-free extracts of the Azolla-Anabaena azollae symbiosis exhibited haemagglutination activity; galactose was the most effective carbohydrate tested in preventing haemagglutination. Extracts of cyanobacteria-free Azolla also caused haemagglutination but extracts of free-living or symbiotic Anabaena azollae did not. Symbiotic Azolla plants grown on NO₃^? showed lower haemagglutination activity than did those grown on N₂; activity increased on removal of NO₃^?. The lower activity of the NO₃^?-grown material may be due to NO₃su? exerting a direct effect on lectin activity/synthesis, or it may act indirectly by inhibiting the development of Anabaena which in turn affects the Azolla lectin. The purified lectin was shown to be composed of 6 sub-units, each of M.W. 21000.     

Taxonomic status ofAnabaena azollae: An overview

Despite the long-standing and widespread use of the symbiotic association between the aquatic fern Azolla and its cyanobacterial symbiontAnabaena azollae to augment nitrogen supplies in rice paddy soils, very little is known about taxonomic aspects of the symbiosis. The two partners normally remain associated throughout vegetative and reproductive development, limiting the opportunities for interchanges. We have used monoclonal antibodies and DNA/DNA hybridization techniques to show that the cyanobacterial partner is not uniform throughout the genus Azolla, and that substantial diversification has occurred. With these procedures it will be possible to characterize genotypes of the cyanobacterium and to monitor experiments aimed at synthesizing new combinations ofAzolla species andAnabaena azollae strains.     

The Azolla, Anabaena azollae Relationship: I. Initial Characterization of the Association

Cultures of Azolla caroliniana Willd. free of the symbiotic blue-green alga, Anabaena azollae, were obtained by treatment of Azolla fronds with a regimen of antibiotics. These symbiontfree plants can be maintained only on medium containing a combined nitrogen source.     

DNA Probes Show Genetic Variation in Cyanobacterial Symbionts of the Azolla Fern and a Closer Relationship to Free-Living Nostoc Strains than to Free-Living Anabaena Strains

Twenty-two isolates of Anabaena azollae derived from seven Azolla species from various geographic and ecological sources were characterized by DNA-DNA hybridization. Cloned DNA fragments derived from the genomic sequences of three different A. azollae isolates were used to detect restriction fragment length polymorphism among all symbiotic anabaenas. DNA clones were radiolabeled and hybridized against southern blot transfers of genomic DNAs of different isolates of A. azollae digested with restriction endonucleases. Eight DNA probes were selected to identify the Anabaena strains tested. Two were strain specific and hybridized only to A. azollae strains isolated from Azolla microphylla or Azolla caroliniana. One DNA probe was section specific (hybridized only to anabaenas isolated from Azolla ferns representing the section Euazolla), and five other probes gave finer discrimination among anabaenas representing various ecotypes of Azolla species. These cloned genomic DNA probes identified 11 different genotypes of A. azollae isolates. These included three endosymbiotic genotypes within Azolla filiculoides species and two genotypes within both A. caroliniana and Azolla pinnata endosymbionts. Although we were not able to discriminate among anabaenas extracted from different ecotypes of Azolla nilotica, Azolla mexicina, Azolla rubra and Azolla microphylla species, each of the endosymbionts was easily identified as a unique genotype. When total DNA isolated from free-living Anabaena sp. strain PCC7120 was screened, none of the genomic DNA probes gave detectable positive hybridization. Total DNA of Nostoc cycas PCC7422 hybridized with six of eight genomic DNA fragments. These data imply that the dominant symbiotic organism in association with Azolla spp. is more closely related to Nostoc spp. than to free-living Anabaena spp.     

A Cobalt Requirement for Symbiotic Growth of Azolla filiculoides in the Absence of Combined Nitrogen

Cobalt was shown to be essential for the symbiotic growth of Azolla filiculoides and Anabacna azollac in the absence of fixed nitrogen. Addition of 0.01 μg/liter cobalt resulted in large increases in yield, chlorophyll content and nitrogen fixation as compared to control cultures without cobalt. Cobalt was not required for the growth of Azolla when nitrate nitrogen was supplied. The number of Anabaena azollae cells in the fronds of Azolla appeared to be decreased by ommission of cobalt from the culture medium containing nitrate nitrogen. It is concluded that cobalt is essential for the symbiotic growth of Azolla in the absence of combined nitrogen and it is suggested that the cobalt requirement is associated with the growth of Anabaena azollae.     

Fingerprinting cyanobionts and hosts of theAzolla symbiosis by DNA amplification

Cyanobionts of six species of the aquatic fernAzolla were evaluated by specific and random DNA profiles amplified by the DNA polymerase chain reaction. Simultaneous examination of the prokaryoticAnabaena azollae and the host was achieved using primers for the chloroplast-encoded intron of the tRNA-Leucine (UAA) gene. These amplifiedtrnL intron sizes, restriction fragment length polymorphisms of the amplified 16s rRNA gene, and random amplified polymorphic DNAs demonstrated the capacity of this method for the rapid assessment of similarities amongAnabaena azollae and minorAnabaena isolates fromAzolla.     

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     

Surface lectin binding toAnabaena variabilis and to cultured and freshly isolatedAnabaena azollae

Five fluorescein isothiocyanate (FITC)-labeled lectins and Calcofluor white ST were tested for their binding abilities to vegetative cells and heterocysts of culturedAnabaena variabilis (AVA) and toA. azollae from four species ofAzolla; toAnabaena azollae freshly isolated fromAzolla pinnata (AP),A. caroliniana (AC),A. mexicana (AX), andA. filiculoides (AF); and to cultured akinetes ofAnabaena variabilis and four isolates ofA. azollae. Heterocysts of cultured cells of threeAnabaena isolates (APC, ACC, AXC) were most intensively surface-stained with soybean agglutinin fromGlycine max (SBA)-FITC; those of AX and AC were dimly stained with wheat germ agglutinin fromTriticum vulgaris (WGA); and only heterocysts of AX were dimly stained withDolichos biflorus agglutinin (DBA). Akinetes of cultured cells stained only with ConA. Vegetative cells and heterocysts of all four fresh isolates stained with Jack Beam aglutinin fromCanavalia ensiformis (ConA). None of the cell types were stained with either peanut agglutinin fromArachis hypogea (PNA) or Calcofluor.     

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.     

Growth,nitrogen fixation,and mass culture of isolatedAnabaena azollae

Summary An independent strain ofAnabaena azollae was evaluated for its potential as a biofertilizer in wetland rice fields. Sustained rapid growth (doubling time=10.5 h) and nitrogenase activity (32 nmol C₂H₄ h^–1 g^–1 chl) was recorded. Mass cultivation (up to 300 litres) for the first time with this species was also achieved.     

Cyanobacterial biofertilizers in rice agriculture

Floodwater and the surface of soil provide the sites for aerobic phototrophic nitrogen (N) fixation by free-living cyanobacteria and theAzolla-Anabaena symbiotic N₂-fixing complex. Free-living cyanobacteria, the majority of which are heterocystous and nitrogen fixing, contribute an average of 20–30 kg N ha^-1, whereas the value is up to 600 kg ha^-1 for theAzollaAnabaena system (the most beneficial cyanobacterial symbiosis from an agronomic point of view). Synthesis and excretion of organic/growth-promoting substances by the cyanobacteria are also on record. During the last two or three decades a large number of studies have been published on the various important fundamental and applied aspects of both kinds of cyanobacterial biofertilizers (the free-living cyanobacteria and the cyanobacteriumAnabaena azollae in symbiotic association with the water fernAzolla), which include strain identification, isolation, purification, and culture; laboratory analyses of their N₂-fixing activity and related physiology, biochemistry, and energetics; and identification of the structure and regulation of nitrogenfixing (nif) genes and nitrogenase enzyme. The symbiotic biology of theAzolla-Anabaena mutualistic N₂-fixing complex has been clarified. In free-living cyanobacterial strains, improvement through mutagenesis with respect to constitutive N₂ fixation and resistance to the noncongenial agronomic factors has been achieved. By preliminary meristem mutagenesis inAzolla, reduced phosphate dependence was achieved, as were temperature tolerance and significant sporulation/spore germination under controlled conditions. Mass-production biofertilizer technology of free-living and symbiotic (Azolla-Anabaena) cyanobacteria was studied, as were the interacting and agronomic effects of both kinds of cyanobacterial biofertilizer with rice, improving the economics of rice cultivation with the cyanobacterial biofertilizers. Recent results indicate a strong potential for cyanobacterial biofertilizer technology in rice-growing countries, which opens up a vast area of more concerted basic, applied, and extension work in the future to make these self-renewable natural nitrogen resources even more promising at the field level in order to help reduce the requirement for inorganic N to the bare minimum, if not to zero.     

Ammonium ion-excreting cyanobacterial mutant as a source of nitrogen for growth of rice: A feasibility study

Summary Growth of rice plants in a nitrogen-free medium was enhanced by inoculation with a nitrogenase-derepressed mutant (strain SA-1) of a cyanobacterium,Anabaena variabilis which excretes NH₄ ⁺ into the medium. Both total dry weight and nitrogen content of rice plants were substantially increased in the presence of the mutant strain but not with the wild type parent, strain SA-0.     

Sub-cellular distribution of nitrogen compounds inAzolla andAnabaena by ESI and EELS analysis

Summary The sub-cellular localization of some nitrogen compounds within the leaf cavities ofAzolla filiculoides Lam. was obtained by means of electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The analyses were performed on ultrathin unstained sections of differentAzolla leaf cavities which contain epidermal hairs,Anabaena azollae Strasb. and bacteria. Net nitrogen distributions were visualized by image analysis, and nitrogen peaks were evidenced in spectra recorded in the same areas. Different distributions of nitrogen compounds were observed within the leaf cavities along the stem, in particular inside the epidermal hairs ofAzolla and the vegetative cells and heterocysts ofA. azollae.     

Cellular differentiation and nitrogenase activity in the cyanobacteriumAnabaena

Nitrogenase activity at periods of differentiation of heterocysts and akinetes was assayed by the acetylene reduction technique. There was no nitrogenase activity in ammoniumgrown, non-heterocystousAnabaena sp.; the activity appeared only after a lag-phase of about 17 – 21 h after the ammonium-grown culture had been transferred to medium free of combined nitrogen. This activity started appearing as the proheterocysts were developing to mature heterocysts. Maximum nitrogenase activity was attained with exponential phase of culture and mature heterocysts. This activity gradually decreased with the differentiation of akinetes. Only insignificant nitrogenase activity was observed in old cultures in which most cells had matured into akinetes.     

The structural nif genes of four symbiotic Anabaena azollae show a highly conserved physical arrangement

Cloned DNA from Anabaena sp. PCC 7120 was used to determine the distribution of restriction sites around NifHDK genes of endosymbionts extracted from Azolla species. Although many of the restriction sites of the symbiotic Anabaena nifHDK genes differed from those of the free-living Anabaeba sp. PCC 7120, three apparently identical restriction sites were found in and around the nifD region. The arrangement of A. azollae and Anabaena sp. PCC 7120 nif H,D,K genes appears similar, with nifH and nifD linked and nifK some distance away from nifD. The A. azollae nifHDK genes appear strongly conserved among the Azolla species examined, regardless of the geographical origin of the ferns.     

Sediment and phytoplankton records of the cyanobacterial genus Anabaena in boreal Lake Pyhäjärvi

Past occurrence and quantities of Anabaena cyanobacteria in Lake Pyhäjärvi, SW Finland, were investigated using sediment and phytoplankton records. A short sediment core covering the past 20 years was examined for Anabaena resting spores (akinetes) in order to assess the utility of akinetes as a paleolimnological proxy. Sedimentary akinetes confirmed the past existence of Anabaena in water, but did not show a direct correlation with the amount of Anabaena spp. in water samples. The amount of planktonic Anabaena spp. correlated with total phosphorus and nitrogen concentrations in Lake Pyhäjärvi, but the number of akinetes was considered to have a relationship with low nutrient concentrations and dominant bloom-forming cyanobacteria. Akinetes are probably suitable for low-resolution and long time scale paleolimnological investigations, where they provide information of past cyanobacteria that cannot be directly attained otherwise.     

A CONTRIBUTION TO THE EARLY TERTIARY FOSSIL RECORD OF THE SALVINIACEAE

Megaspores and massulae of Azolla, Azollopsis, and Salvinia are recorded from the Paleocene and Eocene of Montana and the Dakotas. Three new species of Azolla are described and two species of Azolla are redescribed. The columella of the floating apparatus of the megaspore apparatuses of Azolla appears to be a phylogenetic localization of the perispore around the megaspore. A new section of Azolla (section Kremastospora) is created for species whose megaspore apparatuses have many floats and whose massulae have hooked (anchor-shaped) glochidia. Salvinia preauriculata is based only on leaf remains from the Eocene of North America. Megaspores and massulae which probably are conspecific with the leaf remains are described. The megaspores and massulae of the fossil species are much smaller than those of the living species, S. auriculata, with which S. preauriculata has been compared. The similar size of the megaspores and massulae of the fossil species suggests a less specialized condition than in living species, where these two structures differ greatly in size. Azollopsis tomentosa, previously known from the late Cretaceous, has been found in the Eocene.     

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.