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.     

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.     

Symbiotic algal nitrogenase activity and heterocyst frequency in sevenAzolla species after phosphorus fertilization

Seven species ofAzolla (A. caroliniana, A. microphylla, A. nilotica, A. filiculoides, A. mexicana, A. rubra, A. pinnata the last from both Malaysia and India) grown in pots of flooded soil were subjected to three different treatments with respect to P: none, single application, split application. The experiments were carried out under greenhouse conditions. Heterocyst frequency inAnabaena azollae and acetylene reducing activity (ARA) were studied in successiveAzolla leaves. Both variables increased from the first leaf (shoot apex) to the last one (before branch) in all species in the presence or absence of P. However, heterocyst frequency, ARA andAzolla biomass were all less in the treatment lacking P. Heterocyst frequency inA. azollae, ARA and biomass ofAzolla were higher when P was applied in split doses than in the other treatments.Azolla plants exhibited more ARA than the isolated leaves.     

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.     

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     

Variation revealed by RAPD‐PCR profiles of microsymbiont Anabaena azollae strains from four N2 fixing Azolla cultures

Nitrogen fixing Anabaena azollae strains isolated from four different Azolla cultures were characterized based on their total protein profile and RAPD profile to study the existing variation among them. As expected, the isolates showed almost similar protein banding patterns, but exhibited differences in 40–70 KDa protein subunits. Polymerase chain reaction of the DNA of the isolates, using four different primers, amplified specific sequences of DNA and showed clear polymorphism among the isolates. The RAPD profile generated the fingerprinting pattern characteristic of each strain based on the sequence of the primers used. Common band sharing observed between the strains A. azollae‐RS‐KK‐SK‐AM and A. azollae‐RS‐KK‐SK‐RP probably represents maternal inheritance of DNA to the progeny. The polymorphic bands were generated specifically for the isolates A. azollae‐RS‐KK‐SK‐RP and A. azollae‐RS‐KK‐SK‐AM with primers numbered 2 and 4, respectively, which could be developed as possible markers for these isolates.     

Ultrastructural ontogeny of leaf cavity trichomes inAzolla implies a functional role in metabolite exchange

Summary Anabaena azollae is associated with two types of multicellular epidermal trichomes inAzolla leaf cavities, the simple and branched hairs. The observation of transfer cell ultrastructure in some hair cells led to speculation that the cavity hairs might participate in metabolite exchange between the symbionts. The developmental ontogeny of cavity trichomes is described here, using transmission electron microscopy, with a goal of improving our understanding of possible functions of these structures in the symbiosis. The observations have established that all cells of simple and branched hairs develop the structural characteristics of transfer cells, but not simultaneously. Rather, there is an acropetal succession of transfer cell ultrastructure beginning in terminal cells, moving to body cells where present, and ending in stalk cells. The transfer cell stage is followed immediately by senescence in all hair cells. The timing of transfer cell differentiation, considered together with information from other studies, suggests that branched hairs may be involved in exchange of fixed nitrogen between the symbionts, while simple hairs may participate in exchange of fixed carbon fromAzolla toAnabaena. Contribution no. 869 from the Battelle-C. F. Kettering Research Laboratory.     

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.     

Spheroplast induction inAnabaena variabilis Kütz andA. azollae stras

Summary Spheroplasts were obtained by lysozyme treatment of 48 hour (4– 8cells) akinete germlings of the cultured cyanobacteriaAnabaena variabilis andA. azollae originally isolated from the leaf cavity of the fernAzolla pinnata. The osmotic stabilizer was 0.5 M sucrose. At least 50% of the cells in a short filament became spheroplasts after 1–4 hours in lysozyme (1 mg/ml) in incubation medium at 34 °C, with greater than 75% viability after 2 hours. The spheroplasts were osmotically fragile and showed intense chlorophyll autofluorescence in UV light. In phase microscopy, treated cells appeared larger, became spherical and lost some of their optical refraction. Transmission electron microscopy confirmed the loss of the peptidoglycan layer and the partial remains of the outer membrane after lysozyme exposure. We previously obtained protoplasts ofAzolla fern leaf cells so that we now can study the recognition sites in both members of theAzolla/Anabaena nitrogen fixing symbiosis during cell wall degradation and regeneration.     

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.     

The occurrence of coryneform bacteria in the leaf cavity of Azolla

Coryneform bacteria were found associated with the nitrogen fixing blue-green alga, Anabaena azollae in the leaf cavity of Azolla caroliniana. Plate counts indicated ca. 7,400±1,900 bacterial cells per mature leaf cavity or approximately 1 bacterial cell for every algal cell. No other type of bacterium was found in these cavities.     

Antigenic similarity among Anabaena azollae separated from different species of Azolla

Direct fluorescent antibody (FA) reaction results of 5 FAs against symbiotic Anabaena azollae indicated that all the A. azollae freshly separated from 32 specimens of Azolla collected worldwide (belonging to 6 different species) shared identical and highly specific antigens. None of these FAs exhibited cross-reaction with any of the free-living blue-green algae tested. FA absorption results confirmed these results and also indicate the existence of cross-reactive antigens between Azolla leaves and the surfaces of A. azollae. Antibodies made against free-living A. azollae did not cross-react with any of the symbiotic A. azollae indicating either: (i) these isolates are not true isolates, or (ii) their antigenic properties were altered during isolation and culturing. Such possibilities and their implications are discussed.     

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.     

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.     

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.     

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 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.     

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.     

Fructose supports glycogen accumulation,heterocysts differentiation,N2 fixation and growth of the isolated cyanobiont Anabaena azollae

Cells of the cyanobiont Anabaena azollae isolated from the water fern Azolla filiculoides were found to take up and utilize fructose in the light for mixotrophic growth. Fructose was favored by the cyanobiont as a substrate over sucrose and glucose. Cell growth in the presence of 8 mM fructose led to glycogen accumulation in the cells which approached 20% of the cell dry weight within 2 to 3 days, followed by reduction of glycogen content during the fourth day. Glucose-6-phosphate dehydrogenase activity was increased 5–6-fold in the fructose grown cells from the third day of growth onwards. The frequency of heterocysts in fructose-grown cells increased from 6 to 18%, and acetylene reduction by nitrogenase was increased 3-fold in the presence of fructose as compared with control cells, with maximum values observed between the third and fifth day of mixotrophic growth. Fructose-supported growth yielded a 2–4-fold increase in cell dry weight over controls.It is suggested that fructose-supported development and growth of the cyanobiont in batch cultures may resemble its mixotrophic growth and development in situ in the leaf cavity of the host fern Azolla.Abbreviation G6PDH glucose-6-phosphate dehydrogenase     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons