Intracellular Hyphae Research Articles

Initial Infection of Falcataria moluccana Leaves and Acacia mangium Phyllodes by Uromycladium tepperianum Fungi in a Laboratory Trial

Sengon is a fast growing species that is cultivated widely in Indonesia. Lately, sengon is severely attacked by fungus Uromycladium tepperianum that causing gall rust disease. It is also known to attack various types of acacia. This study aims to determine the fungal infection process U. Tepperianum on sengon leaves and the possibility of infection on Acacia mangium in the laboratory trial. Leave samples and fungal pathogen teliospores obtained from Cangkringan, Sleman, Yogyakarta. Several approach procedures conducted to achieve these objectives were: (1) identification of diseased trees, (2) collecting samples of diseased leaves, branches, twigs, and stems, and (3) artificial inoculation and investigating the infection process of U. tepperianum teliospores in the laboratory. The results showed that the process of infection in sengon started by teliospores germination and germ tube formation. Successive germ tube forming penetration pegs. In the plant tissue, the penetration peg formed hypha and further developed into intracellular and intercellular hyphae. The artificial inoculation on A. mangium leaf surface showed few spores can germinate. However, none of them managed to penetrate.

The vacuoles containing multivesicular bodies: a new observation in interaction between Ustilago esculenta and Zizania latifolia

Ustilago esculenta infects Zizania latifolia plants and induces swelling on their upper culms. As an edible aquatic vegetable, these swellings are called jiaobai (white bamboo). The process of fungal development in Huijiao (grey bamboo) plants from seedlings to upper culm swelling was investigated using knife cut tissues, light and electron microscopy. All Huijiao plants were relatively taller than all jiaobai plants and in knife-cut sections sori development differed after culm swelling in Huijiao plants. Light microscope examination show that hyphae were both intracellular and intercellular, forming hyphal aggregations, and were more abundant in nodes than in internodes. Rapid increase of hyphal aggregations occurred 15 days before culm swelling, causing the death of abundant host cells, and hyphal aggregations differentiated into sori initiates. Ultrastructural observation showed that intracellular hyphae at the interface of fungus-host interaction were encased in a sheath with outer electron-opaque and inner electron-dense layer. Scale-like material and membrane-bounded vesicles were often present in an outer electron-opaque matrix of the sheath. Vacuoles, lipid globules (LGs) and multivesicular bodies (MVBs) were occasionally seen in these fungal cells. In summer, abundant electron-dense MVBs, LGs and fewer vacuoles containing MVBs were present in a fungal cell. As the temperature decreased, some MVBs became electron-opaque, some internal vesicles were also electron-opaque, and subsequently, the MVBs disappeared, leaving irregular remnant materials in vacuoles. During the rapid increase in hyphal aggregations, the irregular remnants of material within the vacuoles disappeared and LGs were rare. At the beginning of sori development, LGs once again became abundant in the cells of hyphal aggregation. The process of sorus and teliospore development was observed systematically and relationships between each developmental stage examined from knife cut tissues, and cytological and ultrastructural sections. Sorus development occurred with the commencement of upper culm swelling and differed in the same tissues. Teliospore development was also uneven in a sorus. The inner region of a sorus developed earlier than the outer part.

Simultaneous transcriptome analysis of Sorghum and Bipolaris sorghicola by using RNA-seq in combination with de novo transcriptome assembly.

The recent development of RNA sequencing (RNA-seq) technology has enabled us to analyze the transcriptomes of plants and their pathogens simultaneously. However, RNA-seq often relies on aligning reads to a reference genome and is thus unsuitable for analyzing most plant pathogens, as their genomes have not been fully sequenced. Here, we analyzed the transcriptomes of Sorghum bicolor (L.) Moench and its pathogen Bipolaris sorghicola simultaneously by using RNA-seq in combination with de novo transcriptome assembly. We sequenced the mixed transcriptome of the disease-resistant sorghum cultivar SIL-05 and B. sorghicola in infected leaves in the early stages of infection (12 and 24 h post-inoculation) by using Illumina mRNA-Seq technology. Sorghum gene expression was quantified by aligning reads to the sorghum reference genome. For B. sorghicola, reads that could not be aligned to the sorghum reference genome were subjected to de novo transcriptome assembly. We identified genes of B. sorghicola for growth of this fungus in sorghum, as well as genes in sorghum for the defense response. The genes of B. sorghicola included those encoding Woronin body major protein, LysM domain-containing intracellular hyphae protein, transcriptional factors CpcA and HacA, and plant cell-wall degrading enzymes. The sorghum genes included those encoding two receptors of the simple eLRR domain protein family, transcription factors that are putative orthologs of OsWRKY45 and OsWRKY28 in rice, and a class III peroxidase that is a homolog involved in disease resistance in the Poaceae. These defense-related genes were particularly strongly induced among paralogs annotated in the sorghum genome. Thus, in the absence of genome sequences for the pathogen, simultaneous transcriptome analysis of plant and pathogen by using de novo assembly was useful for identifying putative key genes in the plant–pathogen interaction.

Cytology of infection of apple leaves by Diplocarpon mali

Diplocarpon mali, the causal agent of Marssonina leaf blotch of apple, causes severe defoliation during the growing season. Little information is available on the mode of infection and the infection process. In this study, the infection strategies of D. mali in apple leaves were investigated using fluorescence and electron microscopy. Conidia attached to leaf surface apparently by mucilage and germinated on both sides of leaves 6 h post-inoculation (hpi). The pathogen penetrated the cuticle by infection pegs formed either in germ tubes or appressoria in 6 hpi, and then formed haustoria in host epidermal and mesophyll cells accompanied by extension of subcuticular and intercellular hyphae. Five days post-inoculation (dpi), the intracellular hyphae were observed. At the same time, the subcuticular hyphal strands (SHS) were produced as a means for fast expansion and reproduction. About 7 dpi, acervuli formed on inoculated leaves. This was the first observation that D. mali formed haustoria and SHS as infection strategies. Our results suggest that D. mali may behave like a hemibiotroph, which can use both biotrophic and necrotrophic strategies to establish infections on apple leaves.

Arabidopsis thaliana model system reveals a continuum of responses to root endophyte colonization

We surveyed the non-mycorrhizal model plant Arabidopsis thaliana microscopically for its ability to form dark septate endophyte (DSE) symbioses in field, greenhouse, and laboratory studies. The laboratory studies were also used to estimate host growth responses to 34 Periconia macrospinosa and four Microdochium sp. isolates. Consistent with broad host range observed in previous experiments, field-, greenhouse-, and laboratory-grown A. thaliana were colonized by melanized inter- and intracellular hyphae and microsclerotia or chlamydospores indicative of DSE symbiosis. Host responses to colonization were variable and depended on the host ecotype. On average, two A. thaliana accessions (Col-0 and Cvi-0) responded negatively, whereas one (Kin-1) was unresponsive, a conclusion consistent with our previous analyses with forbs native to the field site where the fungi originate. Despite the average negative responses, examples of positive responses were also observed, a conclusion also congruent with earlier studies. Our results suggest that A. thaliana has potential as a model for more detailed dissection of the DSE symbiosis. Furthermore, our data suggest that host responses are controlled by variability in the host and endophyte genotypes.

New insights into the survival strategy of the invasive soilborne pathogen Phytophthora cinnamomi in different natural ecosystems in Western Australia

SummaryDespite its importance as one of the most notorious, globally distributed, multihost plant pathogens, knowledge on the survival strategy of Phytophthora cinnamomi in seasonally dry climates is limited. Soil and fine roots were collected from the rhizosphere of severely declining or recently dead specimens of 13 woody species at 11 dieback sites and two dieback spots and from healthy specimens of five woody species at four dieback‐free sites in native forests, woodlands and heathlands of the south‐west of Western Australia (WA). Phytophthora cinnamomi was recovered from 80.4, 78.1 and 100% of tested soil, fine root and soil–debris slurry samples at the 11 dieback sites, in some cases even after 18‐month storage under air‐dry conditions, but not from the small dieback spots and the healthy sites. Direct isolations from soil–debris slurry showed that P. cinnamomi colonies exclusively originated from fine roots and root fragments not from free propagules in the soil. Microscopic investigation of P. cinnamomi‐infected fine and small woody roots and root fragments demonstrated in 68.8, 81.3 and 93.8% of samples from nine woody species the presence of thick‐walled oospores, stromata‐like hyphal aggregations and intracellular hyphae encased by lignitubers, respectively, while thin‐walled putative chlamydospores were found in only 21.2% of samples from five woody species. These findings were confirmed by microscopic examination of fine roots from artificially inoculated young trees of 10 woody species. It is suggested that (i) the main function of chlamydospores is the survival in moderately dry conditions between consecutive rain events and (ii) selfed oospores, hyphal aggregations, and encased hyphae and vesicles in infected root tissue of both host and non‐host species are the major long‐term survival propagules of P. cinnamomi during the extremely dry summer conditions in WA.

Ustilago maydis: Dissecting the Molecular Interface between Pathogen and Plant

Fungal diseases of plants represent one of the most eminent threats to agriculture. Given the food needs of a growing world population and that more and more crops are devoted to fuel production, the necessity to develop crops with better resistance to disease is increasing. To accomplish this, the mechanisms that plant pathogenic fungi use to colonize plants need to be elucidated. As of now, there are only few examples/models in which this can be done on a functional, genome-wide level, taking into account both the pathogen and its host plant [1]. The fungus Ustilago maydis (U. maydis) is one of these examples. It is a member of the smut fungi: a large group of parasites infecting mostly grasses, including several important crop plants such as maize (Figure 1B), wheat, barley, and sugar cane. Smut fungi are biotrophs, i.e., parasites that need the living host plant to complete their sexual life cycle [2], [3]. They do not establish prominent feeding structures like the related, haustoria-forming rust fungi. During penetration, the host plasma membrane invaginates and completely encases the intracellular hyphae (Figure 1A), establishing an extended interaction zone [4] mediating the exchange of molecules between fungus and host. In contrast to most smut fungi that cause a systemic infection, remaining symptomless until the plant flowers, U. maydis can infect all above-ground parts of the maize plant but fails to spread systemically. U. maydis induces local tumors in which spores develop (Figure 1B) – a unique feature that allows detection of symptoms in corn seedlings less than a week after syringe infection with high levels of inoculum. This, together with the toolbox developed for reverse genetics, cell biology, and functional studies, has contributed to its status as a model for biotrophic basidiomycete fungi [5]. Here the current level of our understanding of the elaborate molecular crosstalk between U. maydis and its host plant will be discussed. Figure 1 Disease symptoms and schematic presentation of effector cocktail use in different maize organs and tissues infected by U. maydis.

Plant wounding and Ophiostoma mitovirus 3a (OMV3a) influence infection of creeping bentgrass by Sclerotinia homoeocarpa

Colonization and lesion development by virulent, asymptomatic and hypovirulent isolates of Sclerotinia homoeocarpa on nonwounded (NW) and wounded (W) leaves of creeping bentgrass were characterized. Hypovirulent and asymptomatic isolates contain the fungal virus, Ophiostoma mitovirus 3a, and virulent isolates are virus-free. On NW leaves, all isolates infected leaves with appressoria along cell walls and through stomata by 48 hours post-inoculation (hpi). Inter- and intracellular hyphae formed on heavily colonized, NW leaves at 96 hpi. Wound-inoculated grass had a colonization front characterized by inter- and intracellular hyphal colonization within nonsymptomatic tissues at 8 hpi by direct infection of the wound site. The colonization front grew ahead of symptom development for 50 hpi on both NW and W leaves. In contrast to virulent and asymptomatic isolates, the hypovirulent isolate seldom colonized more than 30% of leaf tissue of the NW or W leaves. Callose accumulation under fungal appressoria, oxalate oxidase activity, and hydrogen peroxide accumulation at lesion borders were also demonstrated. The results indicate that S. homoeocarpa is capable of colonizing creeping bentgrass without causing visible symptoms. In addition, the fungus causes disease more rapidly on W than NW grass, and the etiology of virulent, asymptomatic and hypovirulent isolates was similar. The results of this study contribute to our understanding of the basic biology of S. homoeocarpa and may influence integrated management practices.

Correction: Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Correction: Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Arbuscular mycorrhiza formation in cordate gametophytes of two ferns, Angiopteris lygodiifolia and Osmunda japonica

Mycorrhizal symbiosis is common among land plants including pteridophytes (monilophytes and lycophytes). In pteridophytes with diplohaplontic life cycle, mycorrhizal formations were mostly reported for sporophytes, but very few for gametophytes. To clarify the mycorrhizal association of photosynthetic gametophytes, field-collected gametophytes of Angiopteris lygodiifolia (Marattiaceae, n=52) and Osmunda japonica (Osmundaceae, n=45) were examined using microscopic and molecular techniques. Collected gametophytes were mostly cut into two pieces. One piece was used for light and scanning microscopic observations, and the other for molecular identification of plant species (chloroplast rbcL sequences) and mycorrhizal fungi (small subunit rDNA sequences). Microscopic observations showed that 96% (50/52) of Angiopteris and 95% (41/43) of Osmunda gametophytes contained intracellular hyphae with arbuscules and/or vesicles and fungal colonization was limited to the inner tissue of the thick midribs (cushion). Fungal DNA analyses showed that 92% (48/52) of Angiopteris and 92% (35/38) of Osmunda have sequences of arbuscular mycorrhizal fungi, which were highly divergent but all belonged to Glomus group A. These results suggest that A. lygodiifolia and O. japonica gametophytes consistently form arbuscular mycorrhizae. Mycorrhizal formation in wild fern gametophytes, based on large-scale sampling with molecular identification of host plant species, was demonstrated for the first time.

First catalogue of the rust fungi of French Guiana, northern South America

An annotated list of the rust fungi (Uredinales or Pucciniales) of French Guiana is presented. It enumerates 68 species of which 57 are new reports for the department and 3, Aecidium plukenetiae, Puccinia kourouensis and P. parianicola, are new to science. Dicheirinia guianensis and Hapalophragmium angylocalycis are excluded from the French Guianan mycobiota. New host plants are reported for Batistopsora crucis-filii, B. pistila, Cerotelium ficicola, C. sabiceae, Crossopsora piperis, Desmella aneimiae, Endophyllum guttatum, Kweilingia divina, Puccinia lateritia, Uredo anthurii and Uromyces anguriae. Previously undescribed characters are presented for Achrotelium lucumae, Chaconia ingae, Cerotelium sabiceae, Prospodium amapaensis, Sphenospora smilacina and Uromyces wulffiae-stenoglossae. Chaconia ingae showed haustorial complexes comprising both intracellular hyphae and D-haustoria. In Cerotelium sabiceae, the haustorial mother cells retained the nuclei while D-haustoria were enucleate. The occurrence of these haustorial types in tropical rust fungi is discussed. Internal basidium formation is described for the first time in Sphenospora: teliospores of S. smilacina produced external or internal basidia. The species richness and composition of the French Guianan rust mycobiota are discussed in a neotropical context.

Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum.

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

Fungal associations in gametophytes and young sporophytic roots of the fern Nephrolepis exaltata

Fungal associations in gametophytes and young sporophytic roots of the fernNephrolepis exaltataInformation is limited on the presence of endophytic fungal associations in green gametophytes and young sporophytes of extant ferns. Nothing is known about their presence in Polypodiales, the largest order among extant ferns. We screened chlorophyllous gametophytes and young sporophytes ofNephrolepis exaltata(L.) Schott., (Lomariopsidaceae, Polypodiales) growing naturally on soil, brick and coir for the presence of fungal endophytes. Gametophytes and young sporophytes growing on different substrates were invariably colonized by septate endophytic fungi. Hyaline or brown, regularly septate, inter- or intracellular hyphae with moniliform cells or microsclerotia characterized septate endophytic fungi. However, only the roots of young sporophytes growing on soil and bricks harboured arbuscular mycorrhizal (AM) fungi. The AM morphology conformed to the intermediate type with intracellular hyphal coils, arbusculate coils and intercellular hyphae. No AM fungal spores could be retrieved from the soil on which gametophytes and young sporophytes were growing. The observations in this study support the idea that the septate fungal endophytes could confer an ecological advantage on colonized individuals, especially on nutrient deficient substrates.

Fungal endophytes in Astromyelon-type (Sphenophyta, Equisetales, Calamitaceae) roots from the Upper Pennsylvanian of France

A distinctive fungal endophyte, Cashhickia acuminata nov. gen. et sp., is described from permineralized calamite roots from the Upper Pennsylvanian Grand-Croix cherts of France. Heavily infected roots contain numerous intracellular hyphae in the outer cortex that arise from a meshwork-like mycelium extending between cortical cells. All intracellular hyphae are oriented toward the root center; none occur on the inner periclinal host cell walls. Other roots of the same type show localized infection by this fungus in which isolated cortical cells contain or give rise to intracellular fungal growth. Within the cortical cells are host responses in the form of callosities that indicate the roots were alive at the time of infection. Other endophytes are present in the same host tissue but are less frequent. The discovery of this association provides the first detailed account on the morphology of a Carboniferous fungal root endophyte, as well as the spatial distribution within the host, and infection pathways within the cortical tissues.

Multiple Exocytotic Markers Accumulate at the Sites of Perifungal Membrane Biogenesis in Arbuscular Mycorrhizas

Arbuscular mycorrhizas (AMs) are symbiotic interactions established within the roots of most plants by soil fungi belonging to the Glomeromycota. The extensive accommodation of the fungus in the root tissues largely takes place intracellularly, within a specialized interface compartment surrounded by the so-called perifungal membrane, an extension of the host plasmalemma. By combining live confocal imaging of green fluorescent protein (GFP)-tagged proteins and transmission electron microscopy (TEM), we have investigated the mechanisms leading to the biogenesis of this membrane. Our results show that pre-penetration responses and symbiotic interface construction are associated with extensive membrane dynamics. They involve the main components of the exocytotic machinery, with a major participation of the Golgi apparatus, as revealed by both TEM and in vivo GFP imaging. The labeling of known exocytosis markers, such as v-SNARE proteins of the VAMP72 family and the EXO84b subunit of the exocyst complex, allowed live imaging of the cell components involved in perifungal membrane construction, clarifying how this takes place ahead of the growing intracellular hypha. Lastly, our novel data are used to illustrate a model of membrane dynamics within the pre-penetration apparatus during AM fungal penetration.

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.

The infection process of Puccinia xanthii f. sp. ambrosiae-trifidae on Ambrosia trifida

Puccinia xanthii Schwein. f. sp. ambrosiae-trifidae S.W.T. Batra is an obligate parasitic rust fungus of Ambrosia trifida Linn. Field investigations in Liaoning Province, China, showed that it is an effective biocontrol agent of this alien invasive weed. Its infection of the plant was observed by light microscopy combined with Coomassie Brilliant Blue R-250 staining. We report the infection process, including teliospore germination and basidiospore formation on the host leaf surface, penetration of host tissue, and development of fungal hyphae within the host tissue. Fresh teliospores began to germinate from the germ pore within 1 h under suitable conditions and soon produced basidiospores or secondary basidiospores. Basidiospores falling on host leaves germinated from the end of the basidiospore opposite to the apiculus. Appressoria of germ tubes tended to orient along leaf epidermis cell ridges or at junctions near stomata rather than fixing randomly on the leaf surface. These germ tubes grew for short or longer distances before forming appressoria. The rust fungus directly penetrated the host epidermis by infectious pegs rather than through stomata. Within host tissues, the rust fungus formed intraepidermal vesicles, primary hyphae, intracellular hyphae, and M-haustoria. The intricate infectious structures formed by P. xanthii f. sp. ambrosiae-trifidae on or in host tissues suggest that the rust fungus is a suitable organism for researching the interaction between the pathogen and host plant.

A new endoconidial black meristematic genus,Atramixtia, associated with declining white spruce and phylogenetically allied to a lineage of dothidealean conifer pathogens

An endoconidial, black meristematic taxon Atramixtia arboricola gen. et. sp. nov. (Dothideales) from the black subicula found on twigs of declining white spruce, Picea glauca (Moench) Voss, in Alberta is described. It is morphologically distinguishable from other endoconidial taxa by the conidioma composed of clumps of endoconidial conidiogenous cells, scattered meristematically dividing cells, dematiaceous hyphae, abundant brown, granular matrix materials, and sometimes plant tissue. Endoconidia also occur in conidiogenous cellular clumps that are not organized into a conidioma but develop directly from stromatic cells on the bark. In culture, it forms similar endoconidial conidiomata and also a mycelial, blastic synanamorph that superficially resembles Hormonema . Atramixtia arboricola is a member of the Dothideales and shows phylogenetic affinities to a clade of conifer-stem and -needle pathogens, including Sydowia and Delphinella , although no teleomorph was found either on the natural substrate or in culture. It has not been determined whether A. arboricola is pathogenic to its host, but the occurrence of abundant intracellular hyphae in the host periderm suggests that the fungus is at least parasitic.

Asexual reproduction of Hygrophorus olivaceoalbus by intracellular microsclerotia in root cells of Picea abies – A winner of ozone stress?

Hygrophorus olivaceoalbus has long been known as an ectomycorrhizal fungus, formerly designated with the artificial binomen Piceirhiza gelatinosa. Recently, it has been found to be abundant and very frequent under double ambient ozone free air fumigation of mature Norway spruce trees. As it has already been reported that this fungus can form intercellular hyphae within the root meristem, a more detailed study was performed to clarify its type of root colonization. The present study not only revealed intercellular hyphae within the meristematic zone but also intracellular hyphae within root cortex cells which grow towards and into large tannin droplets—phenolic compounds usually deposited as defensive aids—to finally fill them completely. The hyphal assemblages become globular which at first sit as separated hyphal balls within cells still containing cytoplasm. Later on, they are apparently released from the root cells, presumably as microsclerotia for dispersal of the species. Old ectomycorrhizae (ECM) show an apical pore, and later a large orifice of a tube-like cylinder formed by the thick, persistent hyphal mantle. The root tissue is progressively degrading towards proximal parts. Disintegrating root cells apparently liberate the microsclerotia through the orifice. Further studies have to find out the mechanism by which the microsclerotia are liberated and whether they operate as asexual propagules and lastly how and by whom they are propagated. As these ECM are found under ozone stress, and with identical features at higher altitudes, stress impact on trees might be the causative agent for the high frequency and abundance of Hygrophorus olivaceoalbus ECM.

Mycena sp., a mycorrhizal fungus of the orchid Dendrobium officinale

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.