Microscopy analyses
Stereomicroscopy was used to observe the anatomy of Spiranthes spiralis underground tubers. These appeared to be externally colonized by fungal hyphae of very diverse sizes. Both thick and thin hyphae seemed to enter into S. spiralis roots by appressoria-like structures. Internal root colonization was investigated by light, fluorescence and confocal microscopy. Light microscopy of thin sections confirmed intracellular colonization by fungal hyphae. Convoluted structures, apparently consistent with clumped thin hyphae, were usually visible in the centre of the root cells close to nuclei ( Fig. 1A) and in most cases pelotons appeared to be partially digested. Fluorescence (Fig. 1B, D) and confocal (Fig. 1C) microscopy were used to visualize better fungal colonization. Roots were cross-sectioned and stained with acridine orange. Blue light excitation of acridine orange-incubated sections revealed that the stained filling material was consistent with fungal hyphae structures. Growth of the fungus appears restricted to cortical cells of plant roots. Fungal hyphae were visible penetrating the cell walls of root cells and forming characteristic coils within these (Fig. 1D). Details of mycelial structures coiling inside cells, and hyphae passing through plant cell walls were seen using confocal laser microscopy (Fig. 1C). Electron microscopy allowed us to distinguish the presence of basidiomycete type septa with the typical dolipore structure with entire parenthesomes and cell wall bulges at the edge of the pore (Fig. 1F, G). Hyphae inside cells of explants from surface-sterilized roots gave rise to fungal cultures (Fig. 1E) formed by basidiomycete hyphae, as seen in Fig. 1F.
การวิเคราะห์ไมโครสโคStereomicroscopy was used to observe the anatomy of Spiranthes spiralis underground tubers. These appeared to be externally colonized by fungal hyphae of very diverse sizes. Both thick and thin hyphae seemed to enter into S. spiralis roots by appressoria-like structures. Internal root colonization was investigated by light, fluorescence and confocal microscopy. Light microscopy of thin sections confirmed intracellular colonization by fungal hyphae. Convoluted structures, apparently consistent with clumped thin hyphae, were usually visible in the centre of the root cells close to nuclei ( Fig. 1A) and in most cases pelotons appeared to be partially digested. Fluorescence (Fig. 1B, D) and confocal (Fig. 1C) microscopy were used to visualize better fungal colonization. Roots were cross-sectioned and stained with acridine orange. Blue light excitation of acridine orange-incubated sections revealed that the stained filling material was consistent with fungal hyphae structures. Growth of the fungus appears restricted to cortical cells of plant roots. Fungal hyphae were visible penetrating the cell walls of root cells and forming characteristic coils within these (Fig. 1D). Details of mycelial structures coiling inside cells, and hyphae passing through plant cell walls were seen using confocal laser microscopy (Fig. 1C). Electron microscopy allowed us to distinguish the presence of basidiomycete type septa with the typical dolipore structure with entire parenthesomes and cell wall bulges at the edge of the pore (Fig. 1F, G). Hyphae inside cells of explants from surface-sterilized roots gave rise to fungal cultures (Fig. 1E) formed by basidiomycete hyphae, as seen in Fig. 1F.
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