Cell wall synthesis in cotton roots after infection with Fusarium oxysporum

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.     

Observations on the Responses of Lentil Root Cells to Hyphae of Fusarium oxysporum

The infection of lentil roots by Fusarium oxysporum Schlecht and the responses of the host cells to invading hyphae were examined by light microscopy. Hyphae from inoculum placed on the zone of cell elongation entered the roots at the juncture of epidermal cells within 8 h after inoculation. Although swollen hyphal apices were observed on the epidermal cells, root penetration occurred without formation of these structures or appressoria. The sheath of material found on the surface of uninoculated roots was absent from inoculated roots penetrated by hyphae. Prior to penetration, the epidermal cells became irregular in shape and their cytoplasm appeared to be plasmolysed or granular. Hyphae were observed in the cortex 10—12 h after inoculation and non–penetrated cortical cells were distinctly lobate. Often these lobed cells had a broad, peripheral band of diffuse cytoplasm. When hyphae were first observed in the cortical cells, the walls were ruptured and only slightly stained or unstained by toluidine blue. The inability of such walls to bind the stain may have been the result of the removal of wall components by fungal enzymes. Although extensive proliferation of hyphae was evident throughout the cortex after 24 h of incubation, the endodermis and vascular cylinder were free of hyphae for at least 72 h. Hyphae from inoculum placed on the root hairs or the root apex failed to penetrate the roots during the first 24 h of incubation. The cytological results herein are discussed in relation to the infection of field plantings by this pathogen.     

Infection of Onion by the White Rot Pathogen, Sclerotium cepivorum

Infection of onion tissue by Sclerotium cepivorum occurred from germ tubes penetrating between adjacent epidermal cell walls or directly, via penetration pegs produced from slightly swollen hyphal tips or from beneath dome shaped infection cushions. After passing through the cuticle, the infection peg enlarged to form an infection hypha within the primary cell wall. Extensive degradation of the epidermal cell wall occurred, often at a distance of 2–3 cells from the advancing hyphae. As infection advanced, hyphae spread rapidly from the epidermis to the cortex growing between and within dead/dying host cells. Extensive host cell death resulted in localized collapse of the tissue around infection points. Complete colonization of the internal tissues of the root and stem base occurred within 5–7 days of inoculation.     

Studies on turfgrass snow mold caused byTyphula ishikariensis. II. Microscopical observation of infected bentgrass leaves

Light and transmission electron microscopy revealed thatTyphula ishikariensis penetrated into bentgrass leaves either through cuticles or stomata either by single hyphae or infection cushions formed on host surfaces. Time course study on infected leaves showed that penetration through stomatal subsidiary cells and their adjacent cells seemed to occur earlier than that through epidermal cells located farther from stomata. More than 30% of epidermal cells were infected by 10 days after inoculation. When hyphae penetrated through an intact cuticle of epidermal cells, they seemed to dissolve host cell walls enzymatically at penetration sites. Physical pressure also seemed to be involved in penetration.     

Mycoparsitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui

Summary Mycoparasitic behaviour ofFusarium oxysporum Schlechtendahl towardsAspergillus luchuensis Inui was studiedin vitro. Growth of the hyphae ofF. oxysporum inside the hyphae and conidiophores ofA. luchuensis and formation of resting bodies ofF. oxysporum in conidiophores and hyphae ofA. luchuensis were observed. Bursting of the host hypha attacked by the parasitic hyphae was also noticed.     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Penetration of Phytophthora cinnamomi into disease tolerant and susceptible eucalypts

The mechanisms of penetration of Phytophthora cinnamomi Rands into seedling eucalypt roots were studied by light and electron microscopy. Culture grown seedlings of root-rot tolerant Eucalyptus st johnii and root-rot susceptible Eucalyptus obliqua were inoculated with both zoospores and mycelium. Zoospores encysted on roots of both species and the germ tubes penetrated without the formation of appressoria. Swellings, previously described as appressoria, were formed when the germ tube was slow to enter the host by intracellular penetration. Vegetative hyphae penetrated both inter- and intracellularly into the zones of root elongation and differentiation, often through root hairs. Evidence of hydrolysis of the host cell-wall at the point of penetration was observed in electron micrographs. Several hours after the germ tube penetrated the epidermis, a thick plug of amorphous material formed in the germ tube slightly below the level of the outer walls of the epidermal cells, sealing off the hypha within the root. Behaviour of zoospores and germ tubes and the mechanism of penetration were similar on both hosts. Micrographs do not suggest any kind of a hypersensitive reaction by the host cells during the early stages of infection.     

Penetration of Hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans Without the Formation of Appressoria

In a study using scanning electron microscopy (SEM), the mode of hyperparasitism of Coniothyrium minitans on its host Sclerotinia sclerotiorum was investigated. The SEM micrographs confirm previous reports, from light microscopic studies, that hyphal tips of C. minitans invade the host hypha by direct penetration, without developing appressoria, and that indentation of the host cell wall at the point of penetration is often evident. There is no functional distinction between amain branch and a side branch hypha of the hyperparasite and tips of either type of hyphae are capable of invading host hyphae by direct penetration.     

Scanning electron microscopy of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of the Alnus crispa var. mollis Fern. were studied by scanning electron microscopy (SEM). The critical point drying of glutaraldehyde-osmium fixed nodular tissue permitted an excellent morphological preservation of the three-dimensional structures of the host and endophyte cells. The nodule endophyte was observed as two forms: the hypha which can be branched, and the vesicle which developed at the parental hypha tip. The actinomycetal endophyte penetrated through the host cortical cell wall and became enveloped by a membrane. This enclosing membrane is suggested to be the invaginated host plasmalemma. Perforations of the cell wall of the host infected cell were observed. These perforations are suggested to be the result of an enzymatic degradation process, probably regulated by the penetrating endophyte hyphae. In addition to the polymorphic endophyte, endogenous bacterial contaminants were observed in the nodular tissue. The present SEM study confirms previous light microscopy and transmission electron microscopy studies of the same species of root nodule symbiosis.     

Development of Stem Nodules in a Tropical Forage Legume, Aeschynomene afraspera

Rhizobial infection occurred on the stem of Aeschynomene afrasperaat the site of emergence of adventitious root primordia. Rhizobiainvaded cortical cells at the base of the root primordium. Thefirst infected cell enlarged and collapsed after rhizobia hadmultiplied in large numbers. At this time, a meristematic zonewas initiated some distance beneath the first infected cell.Rhizobial penetration into the deeper cortex was by progressivecollapse of infected cells towards the meristematic zone; rhizobiaentering the cortical cells by invagination of the host cellwall. At the entry point, rhizobia were embedded in digitatecell wall material. These infection structures were restricted,always originating from the cell wall of an adjacent infectedcell. Soon after infection, the cell collapsed progressivelyforming infection strand-like structures which developed upto the meristematic zone. When infection had reached the meristematiczone, invaded host cells ceased to collapse but divided repeatedlyto form the nodule. Key words: Aeschynomene afraspera, stem nodulation     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a -1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.Abbreviations AGL Aplysia gonad lectin - PGPR plant growth-promoting rhizobacteria The authors wish to thank Sylvain Noël for excellent technical assistance. This study was supported by grants from the Fonds Québécois pour la formation de chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC) and the Ministère de l'Industrie, du Commerce, de la Science et de la Technologie (SYNERGIE).     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

The fine structure of Culicinomyces clavisporus invading mosquito larvae

Electron microscope observations were made of the Australian and U.S. strains of Culicinomyces clavisporus infecting mosquito larvae. The wall of the conidium is composed of an inner (primary) layer, an outer (secondary) layer, and an exterior coating of a mucopolysaccharide substance believed responsible for conidial adhesion to the host cuticle prior to germination and penetration. In some instances the wall of the conidium is ruptured during germination and new wall layers and mucoid coating form around the germ tube whereas in other specimens the conidial wall layers extend around the germ tube without fracturing. The most common invasion site is through the larval foregut following ingestion of conidia. The apex of the germ tube presses tightly against the surface of the foregut cuticle and the mucilaginous coating is stripped away. There is evidence to suggest that the host epicuticle, which disappears across the zone of contact with the germ tube, is utilized for nutrition of the invading fungus. A collar of cuticle forms around the germ tube apex and a narrow penetrant hyphae extends into the procuticle. It is believed that cuticular penetration is primarily enzymatic assisted by mechanical pressure. The penetrant hypha swells into an oval cell in the hypodermal region and vegetative hypha then invade the hemocoel. The cells of the hypodermis develop signs of degeneration presumably due to the secretion of toxic substances from the invading hyphae. Host reactions, involving melanization of the host tissues, are sometimes evident among the invading penetrant hyphae in the cuticle or in the hypodermal cells in contact with the fungus. Melanized capsules form around some of the hyphae within the hemocoel. These latter reactions do not directly involve host blood cells and are examples of “humoral encapsulation” similar to that described by other authors during invasion of pathogenic organisms into mosquito larvae and chironomid larvae.     

THE HOST-PARASITE INTERFACE OF ALBUGO CANDIDA ON RAPHANUS SATIVUS

The fine structure of the intercellular hyphae of the obligate parasite Albugo candida infecting radish does not differ markedly from that described previously for cells of Peronospora manshurica. The stalked, capitate haustoria do not contain nuclei and are packed with mitochondria and lomasomes. The fungal plasma membrane and cell wall are continuous from the intercellular hypha throughout the haustorium except that there is no evidence of fungal cell wall around a portion of the haustorial stalk proximal to the haustorial head. Within the vacuolate host mesophyll cell, the haustorium is always surrounded by host plasma membrane and with at least a thin layer of host cytoplasm. The host cell wall invaginates at the point of haustorial penetration to form a short sheath around the region of penetration, but normally there is no host cell wall around the balance of the haustorium. About 1% of the haustoria observed were necrotic, and these were invariably walled-off completely from host cytoplasm by host cell wall. An amorphous, moderately electron-dense encapsulation lies between the haustorium proper and the host plasma membrane and extends into the penetration region between the sheath and the fungal cell wall. Invaded host cells contain more ribosomal-rich ground cytoplasm than uninfected cells. Glandular-like systems of tubules and connecting vesicles are often numerous in host cytoplasm in the vicinity of haustorial heads. These tubules open into the encapsulation, their limiting unit membranes being continuous with the host plasma membrane. We suggest that these represent a secretory mechanism of the host specifically induced by the parasite.     

A mycorrhizal fungus changes microtubule orientation in tobacco root cells

Summary Cortical cells of mycorrhizal roots undergo drastic morphological changes, such as vacuole fragmentation, nucleus migration, and deposition of cell wall components at the plant-fungus interface. We hypothesized that the cytoskeleton is involved in these mechanisms leading to cell reorganization. We subjected longitudinal, meristem to basal zone, sections of uninfectedNicotiana tabacum roots to immunofluorescence methods to identify the microtubular (MT) structures associated with root cells. Similar sections were obtained from tobacco roots grown in the presence ofGigaspora margarita, an arbuscular mycorrhizal fungus which penetrates the root via the epidermal cells, but mostly develops in the inner cortical cells. While the usual MT structures were found in uninfected roots (e.g., MTs involved in mitosis in the meristem and cortical hoops in differentiated parenchyma cells), an increase in complexity of MT structures was observed in infected tissues. At least three new systems were identified: (i) MTs running along large intracellular hyphae, (ii) MTs linking hyphae, (iii) MTs binding the hyphae to the host nucleus. The experiments show that mycorrhizal infection causes reorganization of root MTs, suggesting their involvement in the drastic morphological changes shown by the cortical cells.     

Identification of the endophypte ofDryas andRubus (Rosaceae)

Summary Root nodules ofDryas drummondii are of the coralloid type (Alnus type). The endophyte is present in the middle cortical cells of the root-nodule tissue. Transmission electron micrographs revealed an actinorhizal endophyte with septate hyphae and non-septate spherical or ovoid vesicles. Vesicles always possess at the base a septum; septa formation in the endophyte is always associated with the presence of mesosomes. Branching of the endophyte is not necessarily correlated with septum formation. Hyphal structures are more prominent in the apical part of the root nodule and vesicles are more numerous in a broad zone below this. In the middle and towards the base of the root nodule the endophytic structures appear in a stage of disintegration. Vesicles appear in a broad region near the periphery of the host cell and regularly show no strict orientation towards the host-cell wall. In the center of the host cells only hyphae occur. In the intercellular spaces between the host cells theFrankia endophyte produces spore-like structures although the outline of the sporangia is often faint.The coralloid root ofRubus ellipticus shows characteristically a basal rootlet initiated below the dichotomous branching of the nodular lobes, but extending beyond the root nodule. The endophyte is only present in the outer cortex of the root nodule in a 1–2 cell wide layer. This endophytic layer is bounded, internally as well as externally, with a 4–5 cell wide layer of tannin-filled host cells. The implications of this situation are discussed. Tannin-filled cells occur regularly inRubus species and their arrangement has been used for taxonomic purposes within the genus. TheRubus endophyte is aFrankia species with septate hyphae and distinctly septate spherical vesicles. The ultrastructure of the vesicles of theRubus endophyte is very similar to that of theAlnus endophyte.     

Self-penetration by the mycoparasiteDimargaris Cristalligena

Summary Ultrastructural evidence is presented for self-penetration by hyphae of the mycoparasiteDimargaris cristalligena. Plasmalemmal invagination occurs in a manner similar to that observed during penetration of a conventional host, and is accompanied by the synthesis of a penetration jacket. During self-penetration, however, there is little collar formation and the invading hypha fails to differentiate into a haustorial body.