Different roles of the phytohormone gibberellin in the wide-spread arbuscular mycorrhiza and in orchid mycorrhiza.

Gibberellin (GA) is a classical plant hormone that regulates many physiological processes, such as plant growth, development, and environmental responses. GA inhibits arbuscular mycorrhizal (AM) symbiosis, the most ancient and widespread type of mycorrhizal symbiosis. Knowledge about mycorrhizal symbioses at the molecular level has been obtained mainly in model plants such as legumes and rice. In contrast, molecular mechanisms in non-model plants are still unclear. Recent studies have revealed the novel roles of GA in mycorrhizal symbioses: its positive effect in Paris-type AM symbiosis in Eustoma grandiflorum and its negative effect on both seed germination and mycorrhizal symbiosis in orchids. This review focuses on the recent data on GA function in AM and orchid mycorrhizal symbioses.

In Vitro Hyphal Branching Assay Using Rhizophagus irregularis.

Most terrestrial plants are associated with symbiotic Glomeromycotina fungi, commonly known as arbuscular mycorrhizal (AM) fungi. AM fungi increase plant biomass in phosphate-depleted conditions by allocating mineral nutrients to the host; therefore, host roots actively exude various specialized metabolites and orchestrate symbiotic partners. The hyphal branching activity induced by strigolactones (SLs), a category of plant hormones, was previously discovered using an in vitro assay system. For this bioassay, AM fungi of the Gigaspora genus (Gigasporaeae) are commonly used due to their linear hyphal elongation and because the simple branching pattern is convenient for microscopic observation. However, many researchers have also used Glomeraceae fungi, such as Rhizophagus species, as the symbiotic partner of host plants, although they often exhibit a complex hyphal branching pattern. Here, we describe a method to produce and quantify the hyphal branches of the popular model AM fungus Rhizophagus irregularis. In this system, R. irregularis spores are sandwiched between gels, and chemicals of interest are diffused from the surface of the gel to the germinating spores. This method enables the positive effect of a synthetic SL on R. irregularis hyphal branching to be reproduced. This method could thus be useful to quantify the physiological effects of synthesized chemicals or plant-derived specialized metabolites on R. irregularis. Key features • Development of an in vitro hyphal branching assay using germinating spores of Rhizophagus irregularis. • This in vitro assay system builds upon a method developed by Kameoka et al. [1] but modified to make it more applicable to hydrophilic compounds. • Optimized for R. irregularis to count the hyphal branches. • This bioassay requires at least 12 days to be done.

Colonization by orchid mycorrhizal fungi primes induced systemic resistance against necrotrophic pathogen.

Orchids and arbuscular mycorrhiza (AM) plants evolved independently and have different structures and fungal partners, but they both facilitate nutrient uptake. Orchid mycorrhiza (OM) supports orchid seed germination, but unlike AM, its role in disease resistance of mature plants is largely unknown. Here, we examined whether OM induces systemic disease resistance against a necrotrophic pathogen in a similar fashion to AM. We investigated the priming effect of mycorrhizal fungi inoculation on resistance of a terrestrial orchid, Bletilla striata, to soft rot caused by Dickeya fangzhongdai. We found that root colonization by a compatible OM fungus primed B. striata seedlings and induced systemic resistance against the infection. Transcriptome analysis showed that priming was mediated by the downregulation of jasmonate and ethylene pathways and that these pathways are upregulated once infection occurs. Comparison with the reported transcriptome of AM fungus-colonized rice leaves revealed similar mechanisms in B. striata and in rice. These findings highlight a novel aspect of commonality between OM and AM plants in terms of induced systemic resistance.

Chitin nanofibers promote rhizobial symbiotic nitrogen fixation in Lotus japonicus.

Chitin, an N-acetyl-D-glucosamine polymer, has multiple functions in living organisms, including the induction of disease resistance and growth promotion in plants. In addition, chitin oligosaccharides (COs) are used as the backbone of the signaling molecule Nod factor secreted by soil bacteria rhizobia to establish a mutual symbiosis with leguminous plants. Nod factor perception triggers host plant responses for rhizobial symbiosis. In this study, the effects of chitins on rhizobial symbiosis were examined in the leguminous plants Lotus japonicus and soybean. Chitin nanofiber (CNF), retained with polymeric structures, and COs elicited calcium spiking in L. japonicus roots expressing a nuclear-localized cameleon reporter. Shoot growth and symbiotic nitrogen fixation were significantly increased by CNF but not COs in L.japonicus and soybean. However, treatments with chitin and cellulose nanofiber, structurally similar polymers to CNF, did not affect shoot growth and nitrogen fixation in L.japonicus. Transcriptome analysis also supported the specific effects of CNF on rhizobial symbiosis in L.japonicus. Although chitins comprise the same monosaccharides and nanofibers share similar physical properties, only CNF can promote rhizobial nitrogen fixation in leguminous plants. Taking the advantages on physical properties, CNF could be a promising material for improving legume yield by enhancing rhizobial symbiosis.

Autoactivation of mycorrhizal symbiosis signaling through gibberellin deactivation in orchid seed germination.

Orchids parasitically depend on external nutrients from mycorrhizal fungi for seed germination. Previous findings suggest that orchids utilize a genetic system of mutualistic arbuscular mycorrhizal (AM) symbiosis, in which the plant hormone gibberellin (GA) negatively affects fungal colonization and development, to establish parasitic symbiosis. Although GA generally promotes seed germination in photosynthetic plants, previous studies have reported low sensitivity of GA in seed germination of mycoheterotrophic orchids where mycorrhizal symbiosis occurs concurrently. To elucidate the connecting mechanisms of orchid seed germination and mycorrhizal symbiosis at the molecular level, we investigated the effect of GA on a hyacinth orchid (Bletilla striata) seed germination and mycorrhizal symbiosis using asymbiotic and symbiotic germination methods. Additionally, we compared the transcriptome profiles between asymbiotically and symbiotically germinated seeds. Exogenous GA negatively affected seed germination and fungal colonization, and endogenous bioactive GA was actively converted to the inactive form during seed germination. Transcriptome analysis showed that B. striata shared many of the induced genes between asymbiotically and symbiotically germinated seeds, including GA metabolism- and signaling-related genes and AM-specific marker homologs. Our study suggests that orchids have evolved in a manner that they do not use bioactive GA as a positive regulator of seed germination and instead autoactivate the mycorrhizal symbiosis pathway through GA inactivation to accept the fungal partner immediately during seed germination.

Monoterpene glucosides in Eustoma grandiflorum roots promote hyphal branching in arbuscular mycorrhizal fungi.

Host plant-derived strigolactones trigger hyphal branching in arbuscular mycorrhizal (AM) fungi, initiating a symbiotic interaction between land plants and AM fungi. However, our previous studies revealed that gibberellin-treated lisianthus (Eustoma grandiflorum, Gentianaceae) activates rhizospheric hyphal branching in AM fungi using unidentified molecules other than strigolactones. In this study, we analyzed independent transcriptomic data of E. grandiflorum and found that the biosynthesis of gentiopicroside (GPS) and swertiamarin (SWM), characteristic monoterpene glucosides in Gentianaceae, was upregulated in gibberellin-treated E. grandiflorum roots. Moreover, these metabolites considerably promoted hyphal branching in the Glomeraceae AM fungi Rhizophagus irregularis and Rhizophagus clarus. GPS treatment also enhanced R. irregularis colonization of the monocotyledonous crop chive (Allium schoenoprasum). Interestingly, these metabolites did not provoke the germination of the root parasitic plant common broomrape (Orobanche minor). Altogether, our study unveiled the role of GPS and SWM in activating the symbiotic relationship between AM fungi and E. grandiflorum.

Production of copper nanoparticle-immobilized chitin nanofibers and their role in plant disease control.

Chitin is used in agriculture to improve crop production; however, its use is limited due to difficulties in its handling. A chitin nanofiber (CNF) overcomes this issue and, due to its elicitor activity, has great potential for crop protection. To expand CNF utilization, a copper nanoparticles-based antimicrobic CNF (CuNPs/CNF) was prepared using a chemical reduction method. The formation of CuNPs was confirmed via scanning electron microscopy. Thermogravimetric analysis revealed that the amount of CuNPs on the CNF was dose-dependent on the precursor salt, copper acetate. CuNPs endowed the CNF with strong antimicrobial activity against Alternaria brassicicola and Pectobacterium carotovorum. Moreover, the CuNPs/CNF reduced pathogen infection in cabbage. The antimicrobial activity and disease prevention of the CuNPs/CNF was increased compared to the corresponding CNF or commercial agrochemical Bordeaux treatment. These results indicate that CuNPs conferred antimicrobial activity on the CNF and increased the efficacy of plant disease protection.

Chitin-induced systemic disease resistance in rice requires both OsCERK1 and OsCEBiP and is mediated via perturbation of cell-wall biogenesis in leaves.

Chitin is a well-known elicitor of disease resistance and its recognition by plants is crucial to perceive fungal infections. Chitin can induce both a local immune response and a systemic disease resistance when provided as a supplement in soils. Unlike local immune responses, it is poorly explored how chitin-induced systemic disease resistance is developed. In this study, we report the systemic induction of disease resistance against the fungal pathogen Bipolaris oryzae by chitin supplementation of soils in rice. The transcriptome analysis uncovered genes related to cell-wall biogenesis, cytokinin signaling, regulation of phosphorylation, and defence priming in the development of chitin-induced systemic response. Alterations of cell-wall composition were observed in leaves of rice plants grown in chitin-supplemented soils, and the disease resistance against B. oryzae was increased in rice leaves treated with a cellulose biosynthesis inhibitor. The disruption of genes for lysin motif (LysM)-containing chitin receptors, OsCERK1 (Chitin elicitor receptor kinase 1) and OsCEBiP (Chitin elicitor-binding protein), compromised chitin-induced systemic disease resistance against B. oryzae and differential expression of chitin-induced genes found in wild-type rice plants. These findings suggest that chitin-induced systemic disease resistance in rice is caused by a perturbation of cell-wall biogenesis in leaves through long-distance signalling after local recognition of chitins by OsCERK1 and OsCEBiP.

Orchid mycorrhizal fungi and ascomycetous fungi in epiphytic Vanda falcata roots occupy different niches during growth and development.

Epiphytic orchids are commonly found in exposed environments, which plausibly lead to different root fungal community structures from terrestrial orchids. Until recently, few studies have been conducted to show the fungal community structure during the growth of a photosynthetic and epiphytic orchid in its natural growing site. In this study, the Vanda falcata (commonly known as Neofinetia falcata), one of Japan's ornamental orchids, was used to characterize the fungal community structure at different developmental stages. Amplicon sequencing analysis showed that all development stages contain a similar fungal community: Ascomycota dominate half of the community while one-third of the community belongs to Basidiomycota. Rhizoctonia-like fungi, a polyphyletic basidiomycetous fungal group forming mycorrhizas in many orchids, exist even in a smaller portion (around one-quarter) compared to other Basidiomycota members. While ascomycetous fungi exhibit pathogenicity, two Ceratobasidium strains isolated from young and adult plants could initiate seed germination in vitro. It was also found that the colonization of mycorrhizal fungi was concentrated in a part of the root where it directly attaches to the phorophyte bark, while ascomycetous fungi were distributed in the velamen but never colonized cortical cells. Additionally, the root parts attached to the bark have denser exodermal passage cells, and these cells were only colonized by mycorrhizal fungi that further penetrated into the cortical area. Therefore, we confirmed a process that physical regulation of fungal entry to partition the ascomycetes and mycorrhizal fungi results in the balanced mycorrhizal symbiosis in this orchid.

Disease resistance and growth promotion activities of chitin/cellulose nanofiber from spent mushroom substrate to plant.

Some studies have reported the method for treating the spent mushroom substrate (SMS). However, the effective use as a functional raw material based on properties of SMS remains a formidable challenge. In this study, we investigated the usefulness of SMS in agriculture to develop a new method for treating and utilizing it. First, we attempted to isolate chitin/cellulose nanofiber complex (CCNFC) from SMS using chemical pretreatment and mechanical fibrillation. The characterization results like SEM, FT-IR, and XRD showed that we successfully isolated the CCNFC from SMS. Second, we explored the biological activities of the CCNFC for its potential application as a functional agricultural nanomaterial. CCNFC water dispersion with low concentration (0.1 and 1 mg/mL) exhibited significant plant disease resistance and plant growth promotion activities. Our results suggested that SMS may provide a useful source of functional agricultural nanomaterial, which may contribute to treating and applying it in agriculture.

Conserved and Diverse Transcriptional Reprogramming Triggered by the Establishment of Symbioses in Tomato Roots Forming Arum-Type and Paris-Type Arbuscular Mycorrhizae.

Arbuscular mycorrhizal (AM) fungi allocate mineral nutrients to their host plants, and the hosts supply carbohydrates and lipids to the fungal symbionts in return. The morphotypes of intraradical hyphae are primarily determined on the plant side into Arum- and Paris-type AMs. As an exception, Solanum lycopersicum (tomato) forms both types of AMs depending on the fungal species. Previously, we have shown the existence of diverse regulatory mechanisms in Arum- and Paris-type AM symbioses in response to gibberellin (GA) among different host species. However, due to the design of the study, it remained possible that the use of different plant species influenced the results. Here, we used tomato plants to compare the transcriptional responses during Arum- and Paris-type AM symbioses in a single plant species. The tomato plants inoculated with Rhizophagus irregularis or Gigaspora margarita exhibited Arum- and Paris-type AMs, respectively, and demonstrated similar colonization rates and shoot biomass. Comparative transcriptomics showed shared expression patterns of AM-related genes in tomato roots upon each fungal infection. On the contrary, the defense response and GA biosynthetic process was transcriptionally upregulated during Paris-type AM symbiosis. Thus, both shared and different transcriptional reprogramming function in establishing Arum- and Paris-type AM symbioses in tomato plants.

Simultaneous mutations in SMN1 and SUMM2 fully suppress the dwarf and autoimmune phenotypes of Arabidopsis mpk4 mutant.

Disruption of the Arabidopsis mitogen-activated protein kinase pathway, MEKK1-MKK1/MKK2-MPK4 (hereafter designated as MEKK1 pathway), leads to the activation of distinct NLRs (nucleotide-binding and leucine-rich repeat receptors), TNL (TIR-type NLR) SMN1, and CNL (CC-type NLR) SUMM2, resulting in dwarf and autoimmune phenotypes. Unlike mekk1 and mkk1mkk2 mutants, the dwarf and autoimmune phenotypes of mpk4 are only partially suppressed by the summ2 mutation, suggesting a significant contribution of SMN1 to the mpk4 phenotypes. However, full suppression of mpk4 by the smn1summ2 double mutation remains to be elucidated. To address this key question, we generated a mpk4smn1summ2 triple mutant and analyzed the dwarf and constitutive cell death phenotypes. The mpk4smn1summ2 triple mutant showed restoration of plant size with no detectable cell death, indicating full suppression of the dwarf and autoimmune phenotypes. These results suggest that SMN1 and SUMM2 constitute a robust surveillance system for the MEKK1 pathway against pathogen infection.

Conservation and Diversity in Gibberellin-Mediated Transcriptional Responses Among Host Plants Forming Distinct Arbuscular Mycorrhizal Morphotypes.

Morphotypes of arbuscular mycorrhizal (AM) symbiosis, Arum, Paris, and Intermediate types, are mainly determined by host plant lineages. It was reported that the phytohormone gibberellin (GA) inhibits the establishment of Arum-type AM symbiosis in legume plants. In contrast, we previously reported that GA promotes the establishment of Paris-type AM symbiosis in Eustoma grandiflorum, while suppressing Arum-type AM symbiosis in a legume model plant, Lotus japonicus. This raises a hitherto unexplored possibility that GA-mediated transcriptional reprogramming during AM symbiosis is different among plant lineages as the AM morphotypes are distinct. Here, our comparative transcriptomics revealed that several symbiosis-related genes were commonly upregulated upon AM fungal colonization in L. japonicus (Arum-type), Daucus carota (Intermediate-type), and E. grandiflorum (Paris-type). Despite of the similarities, the fungal colonization levels and the expression of symbiosis-related genes were suppressed in L. japonicus and D. carota but were promoted in E. grandiflorum in the presence of GA. Moreover, exogenous GA inhibited the expression of genes involved in biosynthetic process of the pre-symbiotic signal component, strigolactone, which resulted in the reduction of its endogenous accumulation in L. japonicus and E. grandiflorum. Additionally, differential regulation of genes involved in sugar metabolism suggested that disaccharides metabolized in AM roots would be different between L. japonicus and D. carota/E. grandiflorum. Therefore, this study uncovered the conserved transcriptional responses during mycorrhization regardless of the distinct AM morphotype. Meanwhile, we also found diverse responses to GA among phylogenetically distant AM host plants.

In Vitro Symbiotic Germination: A Revitalized Heuristic Approach for Orchid Species Conservation.

As one of the largest families of flowering plants, Orchidaceae is well-known for its high diversity and complex life cycles. Interestingly, such exquisite plants originate from minute seeds, going through challenges to germinate and establish in nature. Alternatively, orchid utilization as an economically important plant gradually decreases its natural population, therefore, driving the need for conservation. As with any conservation attempts, broad knowledge is required, including the species' interaction with other organisms. All orchids establish mycorrhizal symbiosis with certain lineages of fungi to germinate naturally. Since the whole in situ study is considerably complex, in vitro symbiotic germination study is a promising alternative. It serves as a tool for extensive studies at morphophysiological and molecular levels. In addition, it provides insights before reintroduction into its natural habitat. Here we reviewed how mycorrhiza contributes to orchid lifecycles, methods to conduct in vitro study, and how it can be utilized for conservation needs.

Nanofibrillation Is an Effective Method to Produce Chitin Derivatives for Induction of Plant Responses in Soybean.

Chitin, an N-acetylglucosamine polymer, is well-known to have unique biological functions, such as growth promotion and disease resistance induction in plants. Chitin has been expectedly used for improving crop yield using its functions; however, chitin derivatives, such as chitin oligosaccharide (CO) and chitosan, are widely used instead since chitin is difficult to handle because of its insolubility. Chitin nanofiber (CNF), produced from chitin through nanofibrillation, retains its polymeric structure and can be dispersed uniformly even in water. Here, the effects of CO and CNF on plant responses were directly compared in soybeans (Glycine max) to define the most effective method to produce chitin derivatives for plant response induction. The growth promotion of aerial parts was observed only in CNF-treated plants. The transcriptome analysis showed that the number of differentially expressed genes (DEGs) in CNF-treated soybeans was higher than in CO-treated soybeans. Notably, the expression patterns of DEGs were mostly similar but were strongly induced by CNF treatment as compared with the CO group. These results reveal that CNF can induce stronger plant response to chitin than CO in soybeans, suggesting nanofibrillation, rather than oligomerization, as a more effective method to produce chitin derivatives for plant response induction.

The effects of gibberellin on the expression of symbiosis-related genes in Paris-type arbuscular mycorrhizal symbiosis in Eustoma grandiflorum.

Arbuscular mycorrhiza (AM) is a symbiotic interaction in terrestrial plants that is colonized by fungi in the Glomeromycotina. The morphological types of AM, including the Arum-type and Paris-type, are distinct, depending on the host plant species. A part of the regulatory pathways in Arum-type AM symbiosis has been revealed because most model plants form the Arum-type AM with a model AM fungus, Rhizophagus irregularis. Moreover, gibberellin (GA) is known to severely inhibit AM fungal colonization in Arum-type AM symbiosis. Recently, we showed that exogenous GA treatment significantly promoted AM fungal colonization in Paris-type AM symbiosis in Eustoma grandiflorum. In this study, we focused on the transcriptional changes in AM symbiosis-related genes in GA-treated E. grandiflorum. The expression levels of all examined E. grandiflorum genes were maintained or increased by GA treatment compared with those of the control treatment. Our new results suggest that signaling pathway(s) required for establishing AM symbiosis in E. grandiflorum may be distinct from the well-characterized pathway for that in model plants.

Arabidopsis SMN2/HEN2, Encoding DEAD-Box RNA Helicase, Governs Proper Expression of the Resistance Gene SMN1/RPS6 and Is Involved in Dwarf, Autoimmune Phenotypes of mekk1 and mpk4 Mutants.

In Arabidopsis thaliana, a mitogen-activated protein kinase pathway, MEKK1-MKK1/MKK2-MPK4, is important for basal resistance and disruption of this pathway results in dwarf, autoimmune phenotypes. To elucidate the complex mechanisms activated by the disruption of this pathway, we have previously developed a mutant screening system based on a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Here, we report that the second group of mutants, smn2, had defects in the SMN2 gene, encoding a DEAD-box RNA helicase. SMN2 is identical to HEN2, whose function is vital for the nuclear RNA exosome because it provides non-ribosomal RNA specificity for RNA turnover, RNA quality control and RNA processing. Aberrant SMN1/RPS6 transcripts were detected in smn2 and hen2 mutants. Disease resistance against Pseudomonas syringae pv. tomato DC3000 (hopA1), which is conferred by SMN1/RPS6, was decreased in smn2 mutants, suggesting a functional connection between SMN1/RPS6 and SMN2/HEN2. We produced double mutants mekk1smn2 and mpk4smn2 to determine whether the smn2 mutations suppress the dwarf, autoimmune phenotypes of the mekk1 and mpk4 mutants, as the smn1 mutations do. As expected, the mekk1 and mpk4 phenotypes were suppressed by the smn2 mutations. These results suggested that SMN2 is involved in the proper function of SMN1/RPS6. The Gene Ontology enrichment analysis using RNA-seq data showed that defense genes were downregulated in smn2, suggesting a positive contribution of SMN2 to the genome-wide expression of defense genes. In conclusion, this study provides novel insight into plant immunity via SMN2/HEN2, an essential component of the nuclear RNA exosome.

Improving nitrogen uptake efficiency by chitin nanofiber promotes growth in tomato.

Chitin, an N-acetyl-D-glucosamine polymer, has been known to enhance plant growth. However, this polysaccharide has not been used extensively in experimental work or agriculture practices because its hydrophobic nature makes it difficult to handle. Chitin nanofiber (CNF), which disperses well in water, can feasibly be used to evaluate the effect of chitin on the promotion of plant growth. In this study, we analysed the contents of inorganic elements and global gene expression to obtain an overview of the growth-promoting action of chitins in plants. Significant increases in the biomass of aerial parts and concentration of chlorophyll following treatment with CNF or short-chain chitin oligomers were observed in tomatoes that were hydroponically cultivated under ultralow nutrient concentrations. The results of the quantification of inorganic elements demonstrated that concentrations of nitrogen and carbon significantly increased in whole tomato plant under chitin treatment. Transcriptome analysis of CNF-treated tomatoes by RNA sequencing showed that the expression levels of genes related to nitrogen acquisition and assimilation, nutrient allocation and photosynthesis were altered. These results indicate that the growth-promoting action of chitin treatment is caused by an improvement in nitrogen uptake efficiency and that CNF could be a useful material for nutrient management in tomato production.

Gibberellin Promotes Fungal Entry and Colonization during Paris-Type Arbuscular Mycorrhizal Symbiosis in Eustoma grandiflorum.

Arbuscular mycorrhizas (AMs) are divided into two types according to morphology: Arum- and Paris-type AMs. Gibberellins (GAs) mainly inhibit the establishment of Arum-type AM symbiosis in most model plants, whereas the effects of GAs on Paris-type AM symbiosis are unclear. To provide insight into the mechanism underlying this type of symbiosis, the roles of GAs were investigated in Eustoma grandiflorum when used as the host plant for Paris-type AM establishment. Eustoma grandiflorum seedlings were inoculated with the model AM fungus, Rhizophagus irregularis, and the effects of GA and the GA biosynthesis inhibitor uniconazole-P on the symbiosis were quantitatively evaluated. Exogenous GA significantly increased hyphopodium formation at the epidermis, thus leading to the promotion of fungal colonization and arbuscule formation in the root cortex. By contrast, the suppression of GA biosynthesis and signaling attenuated fungal entry to E. grandiflorum roots. Moreover, the exudates from GA-treated roots strongly induced the hyphal branching of R. irregularis. Our results show that GA has an contrasting effect on Paris-type AM symbiosis in E. grandiflorum compared with Arum-type AM symbiosis. This finding could be explained by the differential regulation of the early colonization stage, where fungal hyphae make contact with and penetrate the epidermis.

Bletilla striata (Orchidaceae) Seed Coat Restricts the Invasion of Fungal Hyphae at the Initial Stage of Fungal Colonization.

Orchids produce minute seeds that contain limited or no endosperm, and they must form an association with symbiotic fungi to obtain nutrients during germination and subsequent seedling growth under natural conditions. Orchids need to select an appropriate fungus among diverse soil fungi at the germination stage. However, there is limited understanding of the process by which orchids recruit fungal associates and initiate the symbiotic interaction. This study aimed to better understand this process by focusing on the seed coat, the first point of fungal attachment. Bletilla striata seeds, some with the seed coat removed, were prepared and sown with symbiotic fungi or with pathogenic fungi. The seed coat-stripped seeds inoculated with the symbiotic fungi showed a lower germination rate than the intact seeds, and proliferated fungal hyphae were observed inside and around the stripped seeds. Inoculation with the pathogenic fungi increased the infection rate in the seed coat-stripped seeds. The pathogenic fungal hyphae were arrested at the suspensor side of the intact seeds, whereas the seed coat-stripped seeds were subjected to severe infestation. These results suggest that the seed coat restricts the invasion of fungal hyphae and protects the embryo against the attack of non-symbiotic fungi.