Cyanobacterial–Bacterial Complexes in Plant Syncyanoses

The morphology and ultrastructure of associative microsymbiont complexes (AMC) isolated from the ferns Azolla pinnata and Azolla sp. and the apogeotropic roots of the cycad Cycas revoluta were studied. The composition of the AMC obtained includes the cyanobionts (symbiotic cyanobacteria) and satellite bacteria (SB). It was found that two types of cyanobacteria that substantially differ in their morphological organization are likely present as cyanobionts in the coralloids of C. revoluta. The isolated cyanobiont strains exhibited the morphological traits and regularities of development typical of the genus Nostoc; they were characterized by the ability of their cells to divide in mutually perpendicular planes. When isolating AMC from different morphological zones of C. revoluta apogeotropic roots, SB growth was revealed only around the pieces corresponding to the coralloid apical zone. No AMC components were revealed around the segments of the basal growth zone. Pure cyanobiont cultures were obtained from the AMC of C. revoluta coralloids. The AMC isolated from the ferns A. pinnata and Azolla sp. are characterized by obligate mutual dependence of the partners (the cyanobiont and SB).     

Characteristics of infection of plants and their cultured tissue with cyanobacterial-bacterial symbiotic associations]

The infection of tobacco, nightshade, rice plants, and their tissue cultures with the cyanobacteria-bacteria symbiotic associations (CBSA) isolated from natural syncyanoses (the ferns Azolla pinnata and Azolla sp. and the cycad Encephalartos ferox) was studied. The inoculation of the intact plants or their cuttings with CBSA led to the colonization of the plant roots, stems, and leaves by cyanobacteria and their bacterial symbionts (referred to as satellite bacteria, SB). The sites of the long-term contact of plant organs with cyanobacteria were characterized by the formation of copious slime. On the roots of infected plants, one could observe the callus growth of cortical parenchyma cells and the formation of pseudonodules, in which SB cells gradually accumulated. In mixed cultures of plant callus tissues and the CBSA isolated from the ferns A. pinnata and Azolla sp., the callus tissue specifically influenced the growth of the CBSA components, causing (depending on the plant species and strain) either their balanced growth, or their cyclic growth, or the predominant growth of one of the CBSA components (either cyanobacteria or satellite bacteria). This phenomenon is proposed to be used for the dissociation of stable multicomponent natural symbiotic complexes and the selection of their particular components.     

Genetic diversity among and within cultured cyanobionts of diverse species of <Emphasis Type="Italic">Azolla</Emphasis>

The cyanobionts isolated from 10 Azolla accessions belonging to 6 species (Azolla mexicana, A. microphylla, A. rubra, A. caroliniana, A. filiculoides, A. pinnata) were cultured under laboratory conditions and analyzed on the basis of whole cell protein profiles and molecular marker dataset generated using repeat sequence primers (STRR(mod) and HipTG). The biochemical and molecular marker profiles of the cyanobionts were compared with those of the free-living cyanobacteria and symbiotic Nostoc strains from Anthoceros sp., Cycas sp. and Gunnera monoika. Cluster analysis revealed the genetic diversity among the selected strains, and identified 3 distinct clusters. Group 1 included cyanobionts from all the 10 accessions of Azolla, group 2 comprised all the symbiotic Nostoc strains, while group 3 included the free-living cyanobacteria belonging to the genera Nostoc and Anabaena. The interrelationships among the Azolla cyanobionts were further revealed by principal component analysis. Cyanobionts from A. caroliniana-A. microphylla grouped together while cyanobionts associated with A. mexicana-A. filiculoides along with A. pinnata formed another group. A. rubra cyanobionts had intermediate relationship with both the subgroups. This is the first study analyzing the diversity existing among the cultured cyanobionts of diverse Azolla species through the use of biochemical and molecular profiles and also the genetic distinctness of these free-living cyanobionts as compared to cyanobacterial strains of the genera Anabaena and Nostoc.     

Infection of Plants and Plant Tissue Cultures with Cyanobacteria–Bacteria Complexes

The infection of tobacco, nightshade, rice plants, and their tissue cultures with the cyanobacteria–bacteria associative microsymbiont complexes (AMC) isolated from natural syncyanoses (the ferns Azolla pinnataand Azollasp. and the cycad Encephalartos ferox) was studied. The inoculation of the intact plants or their cuttings with AMC led to the colonization of the plant roots, stems, and leaves by cyanobacteria and their bacterial symbionts (referred to as satellite bacteria, SB). The sites of the long-term contact of plant organs with cyanobacteria were characterized by the formation of copious slime. On the roots of infected plants, one could observe the callus growth of cortical parenchyma cells and the formation of pseudonodules, in which SB cells gradually accumulated. In mixed cultures of plant callus tissues and the AMC isolated from the fernsA. pinnataand Azollasp., the callus tissue specifically influenced the growth of the AMC components, causing (depending on the plant species and strain) either their balanced growth, or their cyclic growth, or the predominant growth of one of the AMC components (either cyanobacteria or satellite bacteria). This phenomenon is proposed to be used for the dissociation of stable multicomponent natural symbiotic complexes and the selection of their particular components.     

Nitrogenase in free-living and symbiotic cyanobacteria: Immunoelectron microscopic localization

Abstract Nitrogenase (Fe-protein) was localized in the free-living cyanobacterium Anabaena cylindrica and in the cyanobionts of Cycas revoluta and Peltigera aphthosa , using colloidal gold as an immunocytochemical marker. The Fe-protein was found to be evenly distributed throughout the heterocyst cytoplasma in A. cylindrica and in both the cyanobionts, including multiple heterocysts of the C. revoluta cyanobiont. No label was observed in the vegetative cells of free-living A. cylindrica or of the cyanobionts, although the cyanobionts apparently live under microaerobic conditions.     

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths >?10?cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.     

Growth and biochemical characterization of associations between cyanobionts and wheat seedlings in co-culturing experiments

N₂-fixing cyanobacteria are unique in their capacity to form symbiotic associations with a wide range of eukaryotic hosts belonging to different plant groups. The present study was undertaken to analyze the interactions of the cyanobiont PI 01 (from Azolla pinnata) and Nostoc PCC 9229 (from Gunnera monoika) with wheat seedlings, in co-culturing experiments. Each of the cyanobionts enhanced significantly the volume of root and shoot biomass in the experimental cultures. The transverse sections of roots in the co-cultured seedlings revealed the presence of aseriate packets of cyanobionts below the root epidermis. The investigated cyanobionts excreted amino acids (His, Met, Val) and sugars into the medium, while indoleacetic acid was detected when the cyanobionts were grown in a tryptophan containing medium. During the co-culturing, sugars and proline were detected in the extracellular filtrates. It can be hypothesized that these sugars and amino acids may serve as signal substances in the development of functional associations between the relevant cyanobionts and the wheat seedlings.     

Formation of Phenolic Compounds in Apogeotropic Roots of Cycad Plants

Formation and location of phenolic compounds in apogeotropic roots (coralloid roots) were studied in six cycad species, which belong to the genera Cycas, Encephalartos, and Ceratozamia. Total contents of soluble phenolic compounds in coralloid roots in all species studied varied insignificantly, with except for Ceratozamia mexicana that accumulated three times higher amounts of phenolic compounds. Phenolic compounds were accumulated in cell walls of cortical parenchyma of coralloid roots, in intercellular spaces, and in specialized storage cells, found in all zones of apogeotropic roots. The greatest number of phenol-storing cells was situated in the cortical parenchyma of the central part of coralloid roots, adjacent to a zone where active symbiotic cyanobacteria were localized, and in the coralloid root basal region lacking viable forms of cyanobionts. It was suggested that phenolic compounds affect the formation of symbiosis between cyanobacteria and apogeotropic roots of cycad plants, as well as their metabolism.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria

Lectins were extracted from whole fern grindings ofAzolla pinnata (AP) andAzolla filiculoides (AF) by precipitation with ammonium sulfate to 20% of saturation. At high pH both lectins dissociate into inactive subunits (5000 mol wt) which reassociate into active aggregates (>500,000 mol wt) following concentration by ammonium sulfate precipitation or freezing and thawing. Although amino sugars inhibited hemagglutinating activity of both AP and AF lectins,d-fructose was inhibitory only to the AP lectin hemagglutinating activity, andd-galactose was slightly inhibitory to the AP lectin but not to the AF lectin. Both lectins exhibited specificity for freshly extracted cyanobionts from homologous fern species: AP lectin agglutinated cyanobiont filaments from AP, but not from AF; AF lectin agglutinated cyanobiont filaments from AF, but not AP. Neither lectin reacted with cultured cyanobionts from either fern species. Hemagglutinating titers were likewise reduced by adsorption of these lectins to homologous cyanobiont cells. This report provides strong suggestive evidence for specificity in this N-fixing symbiosis between aquatic fern and cyanobacterium.     

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.     

Symbiosis in cycads: the origin and development of coralloid roots in Macrozamia communis (Cycadaceae)

Coralloid roots of Macrozamia have more apparent developmental stages than those of many other cycad genera, providing an ideal study vehicle for obtaining a better understanding of the growth and development of symbiotic cycad coralloid roots. In M. communis L. Johnson, the process begins with initiation of young apogeotropic, papillose roots called “precoralloids” and involves phases of maturation, cyanobacterial invasion, coralloid formation, senescence, and regeneration. Active precoralloid apices continue to produce papillose tissue, but during precoralloid maturation, the prominent papillose sheath is gradually replaced by a thin, dermal layer with scattered lenticels. Cyanobacterial invasion has been observed at different stages of precoralloid maturation and stimulates further, irreversible development of precoralloids into coralloids. Newly invaded precoralloids in the process of transition may be readily identified by their distinctive apical lenticels. Coralloid development involves transformation of the original, apogeotropic precoralloid tissue as well as production of new coralloid tissue by apical meristems. Although continuous, these two coralloid regions may be recognized by their external morphology. New coralloid growth involves cessation of papillose sheath production, change in gravitropic response, proliferation of lenticels, and early differentiation of a conspicuous cyanobacterial zone. Three mechanisms enabling continuity of the coralloid root system are: 1) production of new precoralloids and coralloids from bases of existing roots of the same kind; 2) initiation of atypical roots from within the internal tissues of degenerating coralloids; and 3) development of internal secondary periderm during decortication of aging coralloid tissue.     

Atypical cell forms overproducing extracellular substances in population of cycad cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural changes indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was proteinous. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by channels in the cytoplasmic membrane to form, respectively, a slimy extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

Heterotrophic metabolism and regulation of uptake hydrogenase activity in symbiotic cyanobacteria

The H₂ uptake activity of three cyanobionts isolated fromCycas revoluta, C. circinalis andazolla filiculoides was shown to be related primarily to the growth rate and independent of the main mode of carbon nutrition. Significant H₂ uptake was found in the coralloid roots ofCycas revoluta andZamia furfuracea (3 and 22 times higher than the respective C₂H₂ reduction activities). The results attained allow us to conclude that in cyanobacteria, in contrast to most nitrogen-fixing heterotrophs, uptake hydrogenase activity is not repressed by carbon substrates and that cyanobacteria in association seem to be endowed with sufficient H₂ uptake capacity to recover all of the H₂ released during the process of N₂-fixation.     

Distribution of sym-homosperimidine in eubacteria, cyanobacteria, algae and ferns

Abstract Polyamines were analyzed in 12 of N₂-fixing aerobic eubacteria and other eubacteria, cyanobacteria, algae and ferns. sym -Homospermidine (homospermidine) was found to be widely distributed as a major polyamine in various N₂-fixing eubacteria which belong to Azospirillum, Agromonas, Beijerinckia, Bradyrhizobium, Rhizobium and Xathnbacter . 3 species of Azotobater contained spermidine but not homospermidine, though they are N₂-fixing eubactera. Homospermidine is also distributed in some eubacteria, i.e., the photosynthetic Rhodopseudomanas rutila and the sulfur-oxidizing Thiobacillus denitrificans , a cyanobacterium, Synechococcus sp., and in the cyanobacterium-symbiotic ferns, Azolla imbircatta and Azolla japonica .     

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae)

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400?bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.     

Atypical Cell Forms Overproducing Extracellular Substances in Populations of Cycad Cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural properties indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was protein-like. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by ruptures in the cytoplasmic membrane to form, respectively, a mucilagious extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

Cyanobiont diversity within and among cycads of one field site.

Limited diversity was found among cyanobionts from a cultivated population of cycads at a field site in Florida. All isolates were classified as Nostoc but were different from the one Nostoc species found in the soil. These cyanobacteria were root endophytes of several plants of Zamia integrifolia and one of Dioon. The isolates were similar morphologically and in their reactions to four fluorescein isothiocyanate conjugated lectins. Electrophoretic protein profiles and zymograms distinguished one cyanobiont and the soil Nostoc. A tenacious Anabaena epiphyte was also discovered inhabiting the surfaces of root nodules.     

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen‐fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well‐supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.