Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. IV. Host-endophyte interactions during arbuscular deterioration 1,2.

Scanning electron stereoscopy and transmission electron microscopy were used to correlate morphological alterations and cytological phenomena associated with deterioration of arbuscules in yellow poplar mycorrhizae. Arbuscular degradation was initiated at the tips of the finest branches and progressed basipetally. Cytoplasm in arbuscular hyphae progressively deteriorated and was followed by collapse of the fungal walls. Degraded portions of the arbuscules aggregated into clumps comprised of host wall material and the distorted fungal walls. Host nuclei, abundant mitochondria, and proplastids were closely associated with arbuscular branches undergoing cytoplasmic deterioration and with clumped portions of the arbuscule which contained degraded hyphal branches. Most of the arbuscules observed had deteriorated to the clumped stage. Some cortical cells contained several clumped arbuscules and nearly mature, intact arbuscules which indicated that reinfection occurs even as degradative phenomena are in progress. It is suggested that substantial quantities of mineral nutrients may be made available to the host via degradation of fungal cytoplasm in the arbuscular hyphae preceding aggregation of degraded hyphae into discrete clumps.     

Metabolic activity of Glomus intraradices in Arum- and Paris-type arbuscular mycorrhizal colonization

Colonization of two plant species by Glomus intraradices was studied to investigate the two morphological types (Arum and Paris), their symbiotic interfaces and metabolic activities. Root pieces and sections were stained to observe the colonization and metabolic activity of all mycorrhizal structures. There were no growth responses observed in the plants caused by mycorrhizal symbiosis. The two morphological types had a similar percentage of root colonized, but the Arum-type had higher metabolic activity. Most of the mycorrhizal structures (88%) showed succinate dehydrogenase activity; about half showed acid phosphatase activity; and a small percentage showed alkaline phosphatase activity. Phosphatase activity was highest in arbuscules and low in intercellular hyphae in the Arum-type colonization. In the Paris-type, hyphal coils and arbusculate coils showed a similar intermediate percentage of phosphatase activity. We conclude that acid phosphatase is more important than alkaline phosphatase in both colonization types. We discuss the possibility that, whereas arbuscules in Arum-type are the main site for phosphorus release to the host plant, both the hyphal and arbusculate coils may be involved in the Paris-type.     

Growth and sporulation of the arbuscular mycorrhizal fungus Glomus caledonium in dual culture with transformed carrot roots

Glomus caledonium was established in a dual culture with Ri T-DNA-transformed carrot roots. A modification of the minimal M medium buffered at pH 6.50 with 10 mM MES and solidified with 0.4% unpurified gellan gum allowed spore germination and formation of the symbiosis, together with the development of an extensive extramatrical mycelium and sporulation. Spore production increased with culture generation and most spores were viable. These spores colonized carrot roots and completed the fungal life cycle. In many cultures, sporulation was accompanied by the formation of arbuscule-like structures on short and thickened lateral branches of main hyphae. Root colonization was of the Paris-type with hyphae spreading intracellularly. Most colonized root cells contained coils of thickened hyphae, sometimes surrounded by fine hyphae, but no typical arbuscules were observed. Accepted: 26 January 2000     

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.     

Co-occurrence of three fungal root symbionts in Gaultheria poeppiggi DC in central Argentina

The roots of Gaultheria poeppiggi (Ericaceae) were examined for fungal symbiont colonization. Typical structures of ericoid mycorrhizas (hyphae and intracellular coil hyphae complexes), dark septate fungal endophytes (hyphae and sclerotia), and arbuscular mycorrhizas (hyphae, coils, vesicles and arbuscules) were found in the roots of all the individuals examined. The evolutionarily derived position of Gaultheria within the Ericales may suggest that G. poeppiggi recently acquired the ability to form arbuscular mycorrhizas rather than having retained it from ancestral lines.     

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. III Host-endophyte interactions during arbuscular development.

Scanning- and transmission-electron microscopy were used to examine developing and mature functional arbuscules in mycorrhizal roots of yellow poplar. Arbuscules developed from intracellular hyphae which branched repeatedly upon penetration into the host cells. Intermediate and late stages of developemnt were characterized by the production of numerous, short, bifurcate hyphae throughout the arbuscule. Mature arbuscules exhibited a coralloid morphology which resulted in a considerable increase in the surface area of the endophyte exposed within the host cells. Distinctive ultrastructural features of arbuscular hyphae included osmiophilic walls, nuclei, abundant cytoplasm, glycogen, and numerous small vacuoles. All arbuscular components were enclosed by host wall material and cytoplasm during development and at maturity. In infected cells, host nuclei were enlarged and the cytoplasm associated with the arbuscular branches typically contained abundant mitochondria, endoplasmic reticulum, and proplastids. Ultrastructural observations suggested that nutrient transfer may be predominantly directed toward the fungal endophyte during arbuscular development and while mature arbuscules remain functional.     

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.     

Studies on the Character istic of the Structures in the Cortical Cells of Gastrodia clara after Infection of Armillaria mellea

Aided by the techniques of thin and ultrathin sectioning and electron microscopy, the characteristic of structures in the cortical cells of Gastrodia elata was further investigated after infection of Armillaria mellea. It was found that the “hyphal coils”, observed with light microscope, in the cortical cells of G. elata were saccate structures deriv- ed from the cytoplasm of cortical cells and enclosed hyphae. And the cell walls of hyphae were digested in these sacs. Then, these hyphae without cell wall were cut into protoplast fragments in inner-most cortical cells. The results indicated that the cortical cells of G. elata possess digestive function.     

Gene transfer from a parasitic flowering plant to a fern

The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts.     

The H+-ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis

A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H⁺-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.     

Ultrastructural studies of Phellinus sulphurascens infection of Douglas-fir roots and immunolocalization of host pathogenesis-related proteins

Interactions between roots of Douglas-fir (DF; Pseudotsuga menziesii) seedlings and the laminated root rot fungus Phellinus sulphurascens were investigated using scanning and transmission electron microscopy and immunogold labelling techniques. Scanning electron micrographs revealed that P. sulphurascens hyphae colonize root surfaces and initiate the penetration of root epidermal tissues by developing appressoria within 2 d postinoculation (dpi). During early colonization, intra- and intercellular fungal hyphae were detected. They efficiently disintegrate cellular components of the host including cell walls and membranes. P. sulphurascens hyphae penetrate host cell walls by forming narrow hyphal tips and a variety of haustoria-like structures which may play important roles in pathogenic interactions. Ovomucoid–WGA (wheat germ agglutinin) conjugated gold particles (10 nm) confirmed the occurrence and location of P. sulphurascens hyphae, while four specific host pathogenesis-related (PR) protein antibodies conjugated with protein A–gold complex (20 nm) showed the localization and abundance of these PR proteins in infected root tissues. A thaumatin-like protein and an endochitinase-like protein were both strongly evident and localized in host cell membranes. A DF-PR10 protein was localized in the cell walls and cytoplasm of host cells while an antimicrobial peptide occurred in host cell walls. A close association of some PR proteins with P. sulphurascens hyphae suggests their potential antifungal activities in DF roots.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

A dorsiventral mycorrhizal root in the achlorophyllous<Emphasis Type="Italic"> Sciaphila polygyna</Emphasis> (Triuridaceae)

The star-like root system of the achlorophyllous Sciaphila polygyna (Triuridaceae) consists of roots up to 1.4 mm thick and 1 cm long seemingly radiating from a single origin. Internally, the roots show a bilateral symmetry when viewed in cross-section: the third root cell layer contains rather loose coils of the aseptate mycorrhizal fungus from the dorsal to the lateral sides, in contrast to the extremely dense coils of thin hyphae in its ventral part. Additionally, the hyphae develop vesicle-like swellings mainly in the central part of the dorsal side as well as the lateral parts of the third layer. The fourth root layer is anatomically heteromorphic, having exceptionally large cells, reaching up to 320×130 m in size (giant cells), in the lower lateral parts. The root-colonizing hyphae only degenerate in the fourth layer, most readily in the giant cells, where they may swell to 24 m in diameter, collapse and end as amorphous clumps. Hyphae in the third layer keep their definite structure. The structures are interpreted to be the result of a dynamic reaction of the root to the actual fungal penetration points in order to maximize the benefit from the subsequent colonization by compartmentation of the root tissue. The function of the third layer is to host the fungus and keep it alive within its cells, while mainly the giant cells serve for its digestion. Many indications suggest an arbuscular mycorrhiza for this association. Similarities and differences to other myco-heterotrophic species are discussed.     

Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas

During the establishment of vesicular-arbuscular mycorrhizas, fungal hyphae contact the root surface, form appressoria and initiate the internal colonization phase. Structural changes occur in the cell wall, the cytoplasm and the nucleus as the fungus progresses from a presymbiotic to a symbiotic phase. Nuclei in spores are in G₁ whereas in intraradical hyphae they are in G₁ and G₂. Changes in nuclear organization are evident in various stages in the colonization process. Dramatic changes in both symbionts occur as the nutrient exchange interface is established between arbuscules and root cortical cells. An interfacial matrix, consisting of molecules common to the primary wall of the cortical cell, separates the cortical cell plasma membrane from the fungal cell wall. Ectomycorrhizas are characterized structurally by the presence of a mantle of fungal hyphae enclosing the root and usually an Hartig net of intercellular hyphae characterized by labyrinthine branching. As hyphae contact the root surface, they may respond by increasing their diameter and switching from apical growth to precocious branching. The site of initial contact of hyphae may be either the root cap or the ‘mycorrhiza infection zone’. The mantle varies considerably in structure depending on both the plant and fungus genome. In some ectomycorrhizas, the mantle may be a barrier to apoplastic transport, and in most it may store polyphosphate, glycogen, lipids and perhaps protein.     

Schizophyllum commune Fr. as a destructive mycoparasite

Schizophyllum commune Fr. was shown, by light, scanning, and transmission electron microscopy, to be a destructive mycoparasite on several phytopathogenic and nematode-trapping fungi. The hyphae of S. commune coiled around host hyphae and fruiting structures and penetrated them by means of either unspecialized hyphae or by penetration pegs that developed from terminal appressoria. The host cell walls were usually chemically degraded after which the parasite grew through an electron-dense, papillate, reaction region and its underlying membrane(s) produce trophic hyphae inside the host 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.     

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.     

Positive association between mycorrhiza and foliar endophytes in Poa bonariensis, a native grass

The interaction between mycorrhiza and leaf endophytes (Neotyphodium sp.) was studied in three Poa bonariensis populations, a native grass, differing significantly in endophyte infection. The association between endophytes and mycorrhizal fungi colonisation was assessed by analysing plant roots collected from the field. We found that roots from endophyte-infected populations showed a significantly higher frequency of colonisation by mycorrhizal fungi and that soil parameters were not related to endophyte infection or mycorrhiza colonization. In addition, we did not observe significant differences in the number of AM propagules in soils of the three populations sites. We also report the simultaneous development of Paris-type and Arum-type mycorrhiza morphology within the same root systems of P. bonariensis. The co-occurrence of both colonisation types in one and the same root system found in the three populations, which differed in Neotyphodium infection, suggests that foliar endophytes do not determine AM morphology. The percentage of root length colonised by different types of fungal structures (coils, arbuscules, longitudinal hyphae and vesicles) showed significant and positive differences in arbuscular frequency associated with endophyte infection, whereas the much smaller amounts of vesicles and hyphal coils did not differ significantly.