Fungal Coils Research Articles

Plant and fungal gene expression coupled with stable isotope labeling provide novel information on sulfur uptake and metabolism in orchid mycorrhizal protocorms.

Orchid mycorrhiza (OM) represents an unusual symbiosis between plants and fungi because in all orchid species carbon is provided to the host plant by the mycorrhizal fungus at least during the early stages of orchid development, named a protocorm. In addition to carbon, orchid mycorrhizal fungi provide the host plant with essential nutrients such as phosphorus and nitrogen. In mycorrhizal protocorms, nutrients transfer occurs in plant cells colonized by the intracellular fungal coils, or pelotons. Whereas the transfer of these vital nutrients to the orchid protocorm in the OM symbiosis has been already investigated, there is currently no information on the transfer of sulfur (S). Here, we used ultra-high spatial resolution secondary ion mass spectrometry (SIMS) as well as targeted gene expression studies and laser microdissection to decipher S metabolism and transfer in the model system formed by the Mediterranean orchid Serapias vomeracea and the mycorrhizal fungus Tulasnella calospora. We revealed that the fungal partner is actively involved in S supply to the host plant, and expression of plant and fungal genes involved in S uptake and metabolism, both in the symbiotic and asymbiotic partners, suggest that S transfer most likely occurs as reduced organic forms. Thus, this study provides originalinformation about the regulation of S metabolism in OM protocorms, adding a piece of the puzzle on the nutritional framework in OM symbiosis.

Metabolomic adjustments in the orchid mycorrhizal fungus Tulasnella calospora during symbiosis with Serapias vomeracea.

All orchids rely on mycorrhizal fungi for organic carbon, at least during early development. In fact, orchid seed germination leads to the formation of a protocorm, a heterotrophic postembryonic structure colonized by intracellular fungal coils, thought to be the site of nutrient transfer. The molecular mechanisms underlying mycorrhizal interactions and metabolic changes induced by this symbiosis in both partners remain mostly unknown. We studied plant-fungus interactions in the mycorrhizal association between the Mediterranean orchid Serapias vomeracea and the basidiomycete Tulasnella calospora using nontargeted metabolomics. Plant and fungal metabolomes obtained from symbiotic structures were compared with those obtained under asymbiotic conditions. Symbiosis induced substantial metabolomic alterations in both partners. In particular, structural and signaling lipid compounds increased markedly in the external fungal mycelium growing near the symbiotic protocorms, whereas chito-oligosaccharides were identified uniquely in symbiotic protocorms. This work represents the first description of metabolic changes occurring in orchid mycorrhiza. These results - combined with previous transcriptomic data - provide novel insights on the mechanisms underlying the orchid mycorrhizal association and open intriguing questions on the role of fungal lipids in this symbiosis.

Cell-specific expression of plant nutrient transporter genes in orchid mycorrhizae

Orchid mycorrhizal protocorms and roots are heterogeneous structures composed of different plant cell-types, where cells colonized by intracellular fungal coils (the pelotons) are close to non-colonized plant cells. Moreover, the fungal coils undergo rapid turnover inside the colonized cells, so that plant cells containing coils at different developmental stages can be observed in the same tissue section. Here, we have investigated by laser microdissection (LMD) the localization of specific plant gene transcripts in different cell-type populations collected from mycorrhizal protocorms and roots of the Mediterranean orchid Serapias vomeracea colonized by Tulasnella calospora. RNAs extracted from the different cell-type populations have been used to study plant gene expression, focusing on genes potentially involved in N uptake and transport and previously identified as up-regulated in symbiotic protocorms. Results clearly showed that some plant N transporters are differentially expressed in cells containing fungal coils at different developmental stages, as well as in non-colonized cells, and allowed the identification of new functional markers associated to coil-containing cells.

Studies on the mycorrhiza of Geodorum densiflorum (Lam.) Schltr. from Western Ghats of Karnataka, India.

Mycorrhizal association of Geodorum densiflorum (Lam.) Schltr. an endangered terrestrial orchid in the Western Ghats of Karnataka has been investigated. Anatomical studies of the fully grown orchid have revealed the presence of the fungal coils in the cells of the pseudobulb and in the cortical region of the root, indicating the continued association of the fungus with the plant. The degree of colonisation was extensive in the root . Pure culture of the fungus associated with the underground parts of the plant was obtained and identified as Rhizoctonia solani, a common mycorrhiza forming species with many orchids. The rhizosphere soil analysis of the nutrients was carried out which revealed the decreased level of phosphate when compared to nitrogen and potassium. Nutrient analysis supports the fact that mycorrhizal association occurs under the deficiency of soil nutrients like phosphate.

Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza‐forming Ceratobasidiaceae fungi

• We investigated the fungal symbionts and carbon nutrition of a Japanese forest photosynthetic orchid, Platanthera minor, whose ecology suggests a mixotrophic syndrome, that is, a mycorrhizal association with ectomycorrhiza (ECM)-forming fungi and partial exploitation of fungal carbon. • We performed molecular identification of symbionts by PCR amplifications of the fungal ribosomal DNA on hyphal coils extracted from P. minor roots. We tested for a (13)C and (15)N enrichment characteristic of mixotrophic plants. We also tested the ectomycorrhizal abilities of orchid symbionts using a new protocol of direct inoculation of hyphal coils onto roots of Pinus densiflora seedlings. • In phylogenetic analyses, most isolated fungi were close to ECM-forming Ceratobasidiaceae clades previously detected from a few fully heterotrophic orchids or environmental ectomycorrhiza surveys. The direct inoculation of fungal coils of these fungi resulted in ectomycorrhiza formation on P. densiflora seedlings. Stable isotope analyses indicated mixotrophic nutrition of P. minor, with fungal carbon contributing from 50% to 65%. • This is the first evidence of photosynthetic orchids associated with ectomycorrhizal Ceratobasidiaceae taxa, confirming the evolution of mixotrophy in the Orchideae orchid tribe, and of ectomycorrhizal abilities in the Ceratobasidiaceae. Our new ectomycorrhiza formation technique may enhance the study of unculturable orchid mycorrhizal fungi.

Grazing tolerance and mycorrhizal colonization: Effects of resource manipulation and plant size in biennial Gentianella campestris

As herbivory usually leads to loss of photosynthesizing biomass, its consequences for plants are often negative. However, in favorable conditions, effects of herbivory on plants may be neutral or even beneficial. According to the compensatory continuum hypothesis plants can tolerate herbivory best in resource-rich conditions. Besides herbivory, also primarily positive biotic interactions like mycorrhizal symbiosis, bear carbon costs. Tritrophic plant–fungus–herbivore interaction further complicates plant's cost-benefit balance, because herbivory of the host plant is expected to cause decline in mycorrhizal colonization under high availability of soil nutrients when benefits of symbiosis decline in relation to costs. To gain insight into above interactions we tested the effects of plant size and resource manipulation (simulated herbivory and fertilization) on both above-ground performance and on root fungal colonization of the biennial Gentianella campestris. Clipping caused allocation shift from height growth to branches in all groups except in large and fertilized plants. For large plants nutrient addition may have come too late, as the number of meristems was most likely determined already before the fertilization. Clipping decreased the amount of DSE (dark septate endophytic) fungi which generally are not considered to be mycorrhizal. The effect of clipping on total fungal colonization and colonization by arbuscular mycorrhizal (AM) fungal coils were found to depend on host size and resource level. Dissimilar mycorrhizal response to simulated herbivory in small vs. large plants could be due to more intensive light competition in case of small plants. Carbon limited small plants may not be able to maintain high mycorrhizal colonization, whereas large clipped plants allocate extra resources to roots and mycorrhizal fungi at the expense of above-ground parts. Our results suggest that herbivory may increase carbon limitation that leads re-growing shoots and fungal symbionts to function as competing sinks for the limited carbon reserves.

Heavy metal localisation in mycorrhizas ofEpipactis atrorubens (Hoffm.) Besser (Orchidaceae) from zinc mine tailings

The metal distribution within mycorrhizal and nonmycorrhizal roots of Epipactis atrorubens collected from zinc mine tailings and an area rich in heavy metal ores (both located in southern Poland) was investigated. The tailings, consisting of post-flotation material, were characterised by high levels of toxic elements such as Zn, Pb, and Cd, while soil outside the tailings was also strongly enriched in heavy metals. Atomic absorption spectrometry and proton-induced X-ray emission analysis revealed that heavy metals were mostly accumulated within orchid roots. Elemental maps from proton-induced X-ray emission showed that plant root epidermis and fungal coils which had developed within cortical cells of roots collected from the zinc mine tailings were the main places of Zn and Pb accumulation, associated with increased concentrations of Fe, Cd, Ti, Mn, Si, Ca, and S. The mean content of Pb and Zn in the coils was 4 to 5 times higher than in the root epidermis. In mycorrhizal roots from the tailings a statistically significant decrease in Pb and Zn content towards the inside of the root was observed. The mean content of Pb in coils from roots of plants growing outside the tailings was about 1% of the concentration in root coils from the tailings. Coils selected from orchid roots originating from a site outside the tailings contained comparatively high concentrations of Zn, Cd, and Cu, which was probably due to the high content of these elements in the soil. The results presented suggest a biofiltering effect against heavy metals by orchid mycorrhizal fungi.

Effects of Light Environment on Vesicular‐Arbuscular Mycorrhiza Development in Inga leiocalycina, a Tropical Wet Forest Tree1

ABSTRACTResults of a shadehouse experiment and a field study with seedlings and saplings of Inga leiocalycina Willd. (Legum‐inoseae), a canopy tree of American lowland tropical wet forests, indicated that plant light environment affects both (1) the degree of root colonization by vesicular‐arbuscular mycorrhizal (VAM) fungi and (2) the development of VAM structures in the root. Among shadehouse‐grown seedlings, VAM colonization increased with increasing light intensity, from a mean of 20 percent (21.5 cm colonized root) at a light level approximating forest understory environments to 47 percent (233.1 cm) at a light level found in centers of treefall gap environments. In contrast, VAM colonization levels were similar between gap and understory habitats among field‐collected sapling roots; however, the anatomy of the symbiosis differed between light environments. Roots of light gap saplings had significantly more intracellular fungal coils than understory sapling roots. At subsaturating levels for photosynthesis, light availability appeared to be a limiting resource for this symbiosis, restricting the extent of VAM fungal colonization and development of symbiotic structures. Despite this light‐mediated limitation, woody seedlings showed evidence of VAM fungi in 20 to 30 percent of their roots at light levels typical of the tropical wet forest ecosystem in general. I consider these results in the context of forest dynamics, and I suggest that, for woody seedlings, maintaining moderate levels of VAM in understory environments is of long‐term benefit because it enables them to respond rapidly to the increased nutrient demand imposed by their increased growth rate when canopy openings occur.ABSTRACTLos resultados de un experimento en cámara oscura y un estudio de campo con plántulas y arbolillos de Inga leiocalycina Willd. (Leguminoseae), un árbol del dosel de los bosques tropicales húmedos bajos de América, indicaron que el ambiente luminoso de la planta afecta tanto (1) el grado de colonización de la raíz por las micorrizas vesículo‐arbusculares (MVA) como (2) el desarrollo de sus estructuras en la raíz. Entre las plántulas de la cámara oscura, la colonizacion de MVA aumentó junto con la intensidad luminosa; de un promedio de 20 por ciento (21.5 cm de raíz colonizada), a un nivel de iluminación similar al del sotobosque, hasta 47 por ciento (233.1 cm), a un nivel similar al del centra de los claros de bosque. En contraste, los niveles de colonización de MVA fueron similares entre las raices de los arbolillos colectados en el campo, tanto en ambientes del sotobosque como en los claros de bosque, aunque la anatomía de la simbiosis fue diferente en los dos ambientes luminosos. Las raices de los arbolillos del claro del bosque tuvieron más ovillos micoticos que aquellas del sotobosque. A niveles subsaturados de fotosintesis, la disponibilidad de luz parece ser un recurso limitante para esta simbiosis, pues restringe la extensión de la colonización de MVA y el desarrollo de las estructuras simbióticas. A pesar de esta limitación, las plántulas leñosas muestran evidencia de MVA en 20–30 por ciento de sus raices a niveles de iluminación típicos de la mayoría de los bosques tropicales húmedos. Considerando estos resultados dentro del contexto de la dínamica forestal, puedo sugerir que mantener niveles moderados de MVA representa un beneficio a largo plazo para las plántulas leñosas del sotobosque, ya que les permite responder rápidamente a la demanda de nutrimentos que impone el incremento de su tasa de crecimiento cuando ocurren aperturas en el dosel.

Ultrastructure of the wall of the hyphal coils of the Microthyriaceae

Transmission electron microscopy of leaves of Canthium bearing mycelium of Echidnodella and Balladyna (Microthyriaceae) showed fungal coils in the epidermal cells that were connected to the external mycelium by slender penetration hyphae. The fungal coils were surrounded by a cellulose-containing layer that appeared to be an extension of the host epidermal cell wall.

Mycorrhizal differences between genuine roots and tuberous roots of adult plants ofSpiranthes sinensis var.amoena (Orchidaceae)

Genuine roots ofSpiranthes sinensis var.amoena were infected with the mycorrhizal fungusRhizoctonia repens immediately after root formation in autumn. Infection by the mycorrhizal fungus extended, reaching a maximum the following early summer. The amount of living mycorrhizal fungus in the genuine roots dramatically declined in the flowering season, and then the roots decomposed. Tuberous roots were formed in spring. Mycorrhizas were limited to local infections and did not spread along the roots. The infection level of living mycorrhizal fungus in the tuberous roots was less than in the genuine roots throughout the year. The amount of dead fungal coils in the tuberous roots increased as the tuberous roots aged. The mycorrhizal characteritics of tuberous roots ofS. sinensis var.amoena were totally different from those of genuine roots although the tuberous roots morphologically resembled the genuine roots.

Fungal coil formation ofRhizoctonia repens in seedlings ofGaleola septentrionalis (orchidaceae)

Protocorms or protocorms with roots of an achlorophyllous orchidGaleola septentrionalis were inoculated with isolates ofRhizoctonia repens, R. solani, andRhizoctonia spp. The seedlings were infected with eight of twelve isolates ofR. repens. Fungal coils were formed in the cells, which was suggestive of a symbiotic association. The other isolates caused soft rot or no infection to the protocorms or the protocorms with a root.

Ultrastructure of Mycorrhizas of Dracophyllum secundum R. Br. (Ericales:Epacridaceae)

Dracophyllum secundum R. Br. (Epacridaceae) often possessed ericoid mycorrhizas; fungal endophytes formed coils within cells of the epidermis of hair-roots. The plant plasma membrane extended around the hyphae. In some epidermal cells of hair-roots, both plant and fungal cells retained their structural integrity, both partners showing mitochondrial, vacuolar and lipid droplet profiles, and with much of the plant cytoplasm associated with the hyphal coils. In other epidermal cells of hair-roots, fungal coils were present but cytoplasmic features of both symbionts appeared to have broken down. Some epidermal cells showed no evidence of fungal infection. These three arrangements could occur in root-cells of the same age, and are interpreted as resulting from different stages in the development and degeneration of the infection by the mycorrhizal fungus. Two structural types of fungal endophyte here found in ericoid mycorrhizas in D. secundum: one with simple septa, Woronin bodies and two-layered walls (presumed to be an Ascomycete), and another with dolipore septa with imperforate parenthesomes (presumed to be a Basidiomycete). The possibilities that the mycorrhizas may be seasonal, and that mycorrhizal status varies from place to place, are discussed.