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.     

Features of cyanobacterial-bacterial complexes of microsymbionts of 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).     

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).     

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.     

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.     

Association of non-heterocystous cyanobacteria with crop plants

Cyanobacteria have the ability to form associations with organisms from all domains of life, notably with plants, which they provide with fixed nitrogen, among other substances. This study was aimed at developing artificial associations between non-heterocystous cyanobacteria and selected crop plants. We isolated several non-heterocystous cyanobacteria from various rice fields. The cultures were tested for their capacity to produce the plant hormone indole-3-acetic acid (IAA), and the possible role of IAA in the association of cyanobacteria with seedling roots was evaluated. Axenic cultures were co-inoculated with 10-day-old plant seedlings of Triticum aestivum, Vigna radiata and Pisum sativum and incubated for 1 week. Cyanobacterial association with the roots of these seedlings was quantified by measuring chlorophyll-a. Cyanobacterial association with the roots was observed by light microscopy as well as by confocal laser scanning microscopy (CLSM). Based on sequence analysis of the 16S rRNA gene, the isolates were identified as Synechocystis sp., Chroococcidiopsis sp., Leptolyngbya sp., and Phormidium sp. CLSM observations revealed the intimate association of cyanobacteria with the seedling roots as well as invasion of the roots and root cells. Strains producing IAA were more efficient in the colonization of the roots than those that lacked this ability. IAA-producing cyanobacteria possess a tryptophan-dependent pathway, and these cyanobacteria showed IAA synthesis activity in the presence of roots in media lacking tryptophan. Based on the results of this study, we conclude that non-heterocystous cyanobacteria also have the potential for use in agriculture to improve the growth and yield of crop plants that do not naturally form associations with cyanobacteria.     

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana

Glutamine synthetase (GS) is the primary NH₄ ⁺ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.     

Azolla--a model organism for plant genomic studies

The aquatic ferns of the genus Azolla are nitrogen-fixing plants that have great potentials in agricultural production and environmental conservation. Azolla in many aspects is qualified to serve as a model organism for genomic studies because of its importance in agriculture, its unique position in plant evolution, its symbiotic relationship with the N2-fixing cyanobacterium, Anabaena azollae, and its moderate-sized genome. The goals of this genome project are not only to understand the biology of the Azolla genome to promote its applications in biological research and agriculture practice but also to gain critical insights about evolution of plant genomes. Together with the strategic and technical improvement as well as cost reduction of DNA sequencing, the deciphering of their genetic code is imminent.     

Models for plant tissue development with cell division orientation regulated by preprophase bands of microtubules

Abstract. Interesting new ideas have recently been put forward concerning the regulation of cell division orientation in plants by preprophase bands of microtubules and by their sites of attachment on cell edges. These ideas can be shown to be related to cellular models of development with edge label control. It is the purpose of this paper to elaborate on this relationship and to point out applications of these models to morphogenesis in plant tissues with regular division sequences, particularly in the apical regions of roots of the fern Azolla pinnata.     

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 .     

Scanning Electron Microscopic Observation of Symbiotic Relationship of Azolla-Anabaena AzoUae During the Vegetative Growth

The occurrence and development of the hair ceils on the shoot tips and in the leaf cavities of A. filiculoides, A. microphylla, A. pinnata and their algae-free cultures were examined by means of scanning electron microscopy with microdissect technique. The patterns of Anabacna moving into the leave cavities from the shoot tips were investigated on three species of Azolla during their vegetative growth. The results showed that the patterns of symbiotic Anabaena infecting the leaf cavities are similarity among three species of Azolla and may be divided to the four phases which are summarized as follows: 1. occurrence of primary branched hair and adhesion of Anabaena; 2. development of primary branched hair and spreding of Anabaena; 3. building of hair bridge and entrance of Anabaena into the cavities; 4. formation of secondary simple hair and transference of Anabaena within the cavity. These observations resulted in a hypothesis that hair induces and leads its partner. It is suggested that the hair cell is likely to be a structure of Azolla for attracting and recognizing its symbiont in addition to transport substance between fern and algae.     

Effect of bacterial polysaccharides on the growth ofGaeumannomyces graminis var.tritici and wheat roots

Agrobacterium sp. and related species which in the soil and in the rhizosphere of wheat accompany the fungus Gaemannomyces graminis var. tritici and cause take-all of the wheat roots produced polysaccharides in pure cultures (glucans, mannoglucans and galactomannoglucans). These polysaccharides were utilized better by the mycelium of G. graminis than glucose and polysaccharides of plant origin that occurred on the surface of wheat roots (the so-called mucigel). At lower concentrations these bacterial polysaccharides stimulated growth of wheat roots, higher concentrations (more than 0.1%) were inhibitory. Bacteria inoculated on the surface of wheat first inhibited and then stimulated the development of the plants and their growth. Changes in the growth rate of wheat, the rhizosphere of which was colonized by bacteria simultaneously with the fungus G. graminis and also some changes in the course of the disease of wheat roots caused by the fungus can be explained by the inhibitory or stimulatory effect of polysaccharides of accompanying bacteria.     

Mucilage acts to adhere cyanobacteria and cultured plant cells to biological and inert surfaces

Abstract The surfaces of cells of several species of cyanobacteria have been studied using low-temperature scanning electron microscopy (SEM), and have been shown to be covered in a layer of hydrated mucilage. This mucilage is observed in specimens of Anabaena azollae adhering to plant cells in their natural symbiotic niche (the cavity of the fronds of Azolla species) and in samples of the various species of cyanobacteria immobilised on polyurethane and polyvinyl support matrices. The mucilage appears to maintain the close contact observed between the cyanobacteria and these surfaces. Comparable films observed surrounding plant cells immobilised on similar polymeric surfaces are considered to be performing a similar function.     

Response of growth and antioxidant enzymes in Azolla plants (Azolla pinnata and Azolla filiculoides) exposed to UV-B

Effect of ultravilolet-B (0.4 Wm(-2)) irradiation on growth, flavonoid content, lipid peroxidation, proline accumulation and activities of superoxide dismutase and peroxidase was comparatively analysed in Azolla pinnata and Azolla filiculoides. Growth measured as increment in dry weight reduced considerably due to all UV-B treatments. However, the reduction was found to be severe in A. filiculoides as compared to A. pinnata. The level of UV-absorbing compound flavonoids increased significantly in A. pinnata plants whereas only a slight increase in the flavonoid content was observed in A. filiculoides. UV-B exposure led to enhanced production of malondialdehyde (MDA) and electrolyte leakage in A. filiculoides than A. pinnata. Proline accumulation also showed a similar trend. Marked differences in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD) was noticed in both the plants exposed to UV-B. Our comparative studies indicate A. pinnata to be better tolerant to UV-B as compared with A. filiculoides which appears to be sensitive.     

无藻萍的RAPD分析及其在三膘组满江红种间关系研究中的应用

从满江红Azolla Lam.萍-藻共生体中提取DNA进行的RAPD系统分析通常忽视了满江红样品的异质性。本研究通过获得无藻的满江红,比较有藻萍、无藻萍和离体藻之间的RAPD指纹图谱。发现从有藻萍中提取DNA的扩增反应来源于萍藻双方DNA的共同影响。依引物和植物样本的不同,共生双方对扩增产物的贡献结果不同,说明了用无藻萍进行RAPD检测的重要性。对满江红三膘组5个种的11个无藻萍样本进行了RAPD分析,由9个引物产生的127个DNA多态片段用于计算样本间的Jaccard相似系数和UPGMA树状聚类图。结果     

Programming good relations--development of the arbuscular mycorrhizal symbiosis

The majority of plants live in symbiotic associations with fungi or bacteria that improve their nutrition. Critical steps in a symbiosis are mutual recognition and subsequently the establishment of an intimate association, which involves the penetration of plant tissues and, in many cases, the invasion of individual host cells by the microbial symbiont. Recent advances revealed that in the arbuscular mycorrhizal symbiosis with soil fungi of the order Glomeromycota, plant-derived signals attract fungal hyphae and stimulate their growth. Upon physical attachment of the fungal symbiont to the root surface, an active plant developmental program prepares the epidermal cells for penetration by the fungus. Thus, plants actively help symbiotic fungi to colonize their roots rather than just tolerating them.     

Single-plant,Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.     

Effects of atmospheric SO2 on Azolla and Anabaena symbiosis

The water fern Azolla pinnata R. Br. was fumigated for 1 week with either 25, 50 or 100 nl 1⁻¹ SO₂. The symbiosis of Azolla with Anabaena azollae (spp.) was severely damaged by atmospheric SO₂ even at concentrations as low as 25 nl 1⁻¹, with significant reductions in growth, reduction of C₂H₂, NH₃ assimilation, protein synthesis, and heterocyst development. These disturbances appear to be mainly responsible for the extreme sensitivity of this fern to atmospheric SO₂. Changes in violaxanthin/antheraxanthin and epoxy-lutein/lutein ratios also indicate that free radical products are induced by atmospheric SO₂. These results suggest that the Azolla-Anabaena symbiotic system is a very responsive and reliable lower plant model to study the detailed effects of total sulphur deposition upon the balances between various important plant metabolic processes.     

Growth behaviour of Azolla pinnata at various salinity levels and induction of high salt tolerance

Azolla pinnata is an extremely NaCl-sensitive plant and cannot tolerate an external NaCl concentration beyond 30 mM. Preincubation of plants in 20 mM NaCl for 18 days, followed by stepwise transfer (10 mM NaCl per day) made them able to grow at an otherwise lethal NaCl concentration of 60 mM at rates comparable to the growth of unadapted plants in 20 mM NaCl. Plants, not preincubated in 20 mM NaCl or preincubated for a duration shorter than 18 days were unable to survive and did not grow in 60 mM external NaCl. Na+, K+ and Ca2+ concentrations in the control, NaCl-stressed and adapted plants differed significantly indicating that adaptation involved the development of a capability in the plants to regulate ion concentration.