Microbial Complexes Occurring on the Apogeotropic Roots and in the Rhizosphere of Cycad Plants

The microbial complexes of soil, the rhizosphere, and the rhizoplane of the apogeotropic (coralloid) roots of cycad plants were comparatively studied. The aseptically prepared homogenates of the surface-sterilized coralloid roots did not contain bacterial microsymbiont, indicating that in the root tissues the symbiosis is a two-component one (plant–cyanobacteria). At the same time, associated bacteria belonging to different taxonomic groups were detected in increasing amounts in the cycad rhizoplane, rhizosphere, and the surrounding soil. The bacterial communities found in the cycad rhizoplane and the surrounding soil were dominated by bacteria from the genus Bacillus. The saprotrophic bacteria and fungi colonizing the cycad rhizosphere and rhizoplane were dominated by microorganisms capable of degrading the plant cell walls. The local degradation of the cell wall was actually observed on the micrographs of the thin sections of cycad roots in the form of channels through which symbiotic cyanobacterial filaments can penetrate into the cortical parenchyma.     

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.     

Highly diverse endophytes in roots of <Emphasis Type="Italic">Cycas bifida</Emphasis> (Cycadaceae), an ancient but endangered gymnosperm

As an ancient seed plant, cycads are one of the few gymnosperms that develop a root symbiosis with cyanobacteria, which has allowed cycads to cope with harsh geologic and climatic conditions during the evolutionary process. However, the endophytic microbes in cycad roots remain poorly identified. In this study, using next-generation sequencing techniques, we investigated the microbial diversity and composition of both the coralloid and regular roots of Cycas bifida (Dyer) K.D. Hill. Highly diverse endophytic communities were observed in both the coralloid and regular roots. Of the associated bacteria, the top five families were the Nostocaceae, Sinobacteraceae, Bradyrhizobiaceae, Bacillaceae, and Hyphomicrobiaceae. The Nectriaceae, Trichocomaceae, and Incertae sedis were the predominant fungal families in all root samples. A significant difference in the endophytic bacterial community was detected between coralloid roots and regular roots, but no difference was observed between the fungal communities in the two root types. Cyanobacteria were more dominant in coralloid roots than in regular roots. The divergence of cycad root structures and the modified physiological processes may have contributed to the abundance of cyanobionts in coralloid roots. Consequently, the colonization of cyanobacteria inhibits the assemblage of other endophytes. Our results contribute to an understanding of the species diversity and composition of the cycad-endophyte microbiome and provide an abbreviated list of potential ecological roles of the core microbes present.     

Microbial complexes from apogeotropic roots and from rhizosphere of cycad plants

The microbial complexes of soil, the rhizosphere, and the rhizoplane of the apogeotropic (coralloid) roots of cycad plants were comparatively studied. The aseptically prepared homogenates of the surface-sterilized coralloid roots did not contain bacterial microsymbiont, indicating that it was absent in the root tissues. At the same time, associated bacteria belonging to different taxonomic groups were detected in increasing amounts in the cycad rhizoplane, rhizosphere, and the surrounding soil. The bacterial communities found in the cycad rhizoplane and the surrounding soil were dominated by bacteria from the genus Bacillus. The saprotrophic bacteria and fungi colonizing the cycad rhizosphere and rhizoplane were dominated by microorganisms capable of degrading the plant cell walls. The local degradation of the cell wall was actually observed on the micrographs of the thin sections of cycad roots in the form of channels, through which symbiotic cyanobacterial filaments can penetrate into the cortical parenchyma.     

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).     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

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).     

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.     

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C₂H₂-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2–3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.Abbreviation GS glutamine synthetase     

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.     

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.     

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.     

健康与凋萎病杨梅树体及根围菌群的差异

凋萎病是制约杨梅产业发展的严重病害。为了有效防控凋萎病,本研究分析了杨梅健康和感染凋萎病树体各部位及根表土和根围土中细菌和真菌群落的丰富度与多样性的差异。结果表明: 与健康树相比,病树根围土、根表土、根、枝干、枝皮和叶片的细菌和真菌丰富度均发生了显著变化,其中,根表土细菌和枝皮内真菌的丰富度和多样性均显著降低,而枝皮内细菌和根表土的真菌丰富度和多样性均显著升高。病树各部位及根表、根围土细菌和真菌的优势菌相对丰度在门、纲和属水平上发生了明显的变化,在病树枝干、根和根表土中的假单胞菌属及根表土、根围土中的镰刀菌属的相对丰度明显降低,病树根表土及根围土中青霉菌属的相对丰度明显增加。与凋萎病菌同属的拟盘多毛孢菌在病树根内显著减少,而在其他位置均大量增殖,其相对丰度与多数相对丰度较高的真菌呈正相关。本研究结果将为开发杨梅凋萎病的生态改良、培育健康树体和生物防治技术提供有效的理论依据。     

苏铁Cycas revoluta珊瑚根内生真菌的多样性

苏铁类植物是目前发现的唯一一类具有珊瑚根结构的裸子植物,是探究植物-微生物互作的理想材料。本研究通过高通量测序技术对中国13个地区栽培苏铁Cycas revoluta珊瑚根中的内生真菌组成及多样性进行分析,结果表明苏铁内生真菌多样性较高,但各地区间内生真菌的组成及多样性差异不显著,显示苏铁和珊瑚根内生真菌的共生与栽培地无关。     

Secretion activity of white lupin’s cluster roots influences bacterial abundance,function and community structure

White lupin (Lupinus albus L. cv. Amiga) reacts to phosphate deficiency by producing cluster roots which exude large amounts of organic acids. The detailed knowledge of the excretion physiology of the different root parts makes it a good model plant to study plant-bacteria interaction. Since the effect of the organic acid exudation by cluster roots on the rhizosphere microflora is still poorly understood, we investigated the abundance, diversity and functions of bacteria associated with the cluster roots of white lupin, with special emphasis on the influence of root proximity (comparing root, rhizosphere soil and bulk soil fractions) and cluster root growth stages, which are characterized by different excretion activities. Plants were grown for five weeks in microcosms, in the presence of low phosphate concentrations, on acidic sand inoculated with a soil suspension from a lupin field. Plate counts showed that bacterial abundance decreased at the stage where the cluster root excretes high amounts of citrate and protons. In vitro tests on isolates showed that the frequencies of auxin producers were highest in juvenile and mature cluster roots and significantly decreased in senescent cluster roots. However, no significant difference in the frequency of auxin producers was found between cluster and non cluster roots. The diversity and structure of bacterial communities were investigated by DGGE of 16S rDNA and 16S rRNA. The diversity and community structure were mostly influenced by root proximity and, to a lesser extent, by cluster root stage. The richness of bacterial communities decreased with root proximity, whereas the proportion of active populations increased. The high citrate and proton excretion occurring at the mature stage of cluster roots had a strong impact on the structure and richness of the bacterial communities, both in the root and in the rhizosphere soil.     

Analogous wheat root rhizosphere microbial successions in field and greenhouse trials in the presence of biocontrol agents Paenibacillus peoriae SP9 and Streptomyces fulvissimus FU14

Two Pythium-infested soils were used to compare the wheat root and rhizosphere soil microbial communities from plants grown in the field or in greenhouse trials and their stability in the presence of biocontrol agents. Bacteria showed the highest diversity at early stages of wheat growth in both field and greenhouse trials, while fungal diversity increased later on, at 12 weeks of the crop cycle. The microbial communities were stable in roots and rhizosphere samples across both soil types used in this study. Such stability was also observed irrespective of the cultivation system (field or greenhouse) or addition of biocontrol coatings to wheat seeds to control Pythium disease (in this study soil infected with Pythium sp. clade F was tested). In greenhouse plant roots, Archaeorhizomyces, Debaryomyces, Delftia, and unclassified Pseudeurotiaceae were significantly reduced when compared to plant roots obtained from the field trials. Some operational taxonomic units (OTUs) represented genetic determinants clearly transmitted vertically by seed endophytes (specific OTUs were found in plant roots) and the plant microbiota was enriched over time by OTUs from the rhizosphere soil. This study provided key information regarding the microbial communities associated with wheat roots and rhizosphere soils at different stages of plant growth and the role that Paenibacillus and Streptomyces strains play as biocontrol agents in supporting plant growth in infested soils.     

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.     

Three-dimensional visualization and quantification of water content in the rhizosphere

? Despite the importance of rhizosphere properties for water flow from soil to roots, there is limited quantitative information on the distribution of water in the rhizosphere of plants. ? Here, we used neutron tomography to quantify and visualize the water content in the rhizosphere of the plant species chickpea (Cicer arietinum), white lupin (Lupinus albus), and maize (Zea mays) 12 d after planting. ? We clearly observed increasing soil water contents (θ) towards the root surface for all three plant species, as opposed to the usual assumption of decreasing water content. This was true for tap roots and lateral roots of both upper and lower parts of the root system. Furthermore, water gradients around the lower part of the roots were smaller and extended further into bulk soil compared with the upper part, where the gradients in water content were steeper. ? Incorporating the hydraulic conductivity and water retention parameters of the rhizosphere into our model, we could simulate the gradual changes of θ towards the root surface, in agreement with the observations. The modelling result suggests that roots in their rhizosphere may modify the hydraulic properties of soil in a way that improves uptake under dry conditions.     

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.