Occurrence of arbuscular mycorrhizal fungi in bromeliad species from the tropical Atlantic forest biome in Brazil

The mycorrhizal status of epiphytic, rupicolous, and terrestrial bromeliad species from the Brazilian Atlantic Rain Forest has been examined. Roots of 13 species of bromeliads were analyzed for the presence of mycorrhizal structures such as arbuscules, hyphae, and vesicles as well as other fungal structures. Rhizosphere soil was sampled to identify arbuscular mycorrhizal fungal (AMF) species associated only with terrestrial bromeliad species. Most specimens collected were epiphytic bromeliads in the genera Aechmea, Bilbergia, Nidularium, Tillandsia, and Vriesea. Differentiating structures of AMF were found in only three species of bromeliads. The pattern of mycorrhizal colonization was mainly internal, and external mycelium and arbuscules were observed only in the terrestrial Nidularium procerum. Root endophytes with dark brown septate mycelium, thin external hyphae, and Rhizoctonia-like sclerotia were also detected in some root segments. A total of ten spore morphotypes were recovered from the rhizosphere of N. procerum, with Acaulospora mellea, A. foveata, and Glomus sp. being the most common species recovered. Our study demonstrated that most of the epiphytic species are not associated with AMF. We attribute this mainly to the exposed bare root conditions found in epiphytic bromeliads.     

Communities of arbuscular mycorrhizal fungi in grassland: Seasonal variability and effects of environment and host plants

The seasonal dynamics of a community of endomycorrhizal fungal morphotypes in the roots of three grassland species (Achillea millefolium, Poa angustifolia, Plantago lanceolata) was evaluated, together with the effects of experimental treatment (mowing and phosphorus application) and the host plant properties. Strong seasonal variability was found in the fungal community, where clear seasonal patterns can be distinguished for several fungal morphotypes. The sampling date explained 20 to 30% of the total compositional variability for all three host species. ThePlantago roots host the highest number of arbuscular mycorrhizal (AM) fungal populations. There are two co-dominant fungal morphotypes inAchillea roots (assigned to the generaScutellospora andGlomus) and only one strongly dominant morphotype (assigned toGlomus), in thePoa roots. All three host species have a comparable pattern of richness of AM morphotypes with a single peak in the summer, possibly aligned with the flowering time of the host.     

Ectomycorrhizal morphotypes of naturally grownAbies firma seedlings

Ectomycorrhizas of naturally grown Momi fir (Abies firma) seedlings were characterized based on morphological features of fungal partners. A total of 128 seedlings were collected over three years (1995–1997) from a 10×30 m plot where occurrences of ectomycorrhizal fungal fruitbodies were monitored for the same period. Thirty-seven morphologically distinct ectomycorrhizal types were distinguished based mainly on the color of ectomycorrhizas and the characteristics of fungal mantles. Type 37 was thought to beCenococcum geophilum because of the jet-black mycorrhizas and the characteristic structure of mantle surfaces. For half of the classified morphotypes, fungal partners were inferred to be the generaLactarius, Russula, andTuber, and unidentified Basidiomycetes, based on earlier references.     

Defoliation effects on the community structure of arbuscular mycorrhizal fungi based on 18S rDNA sequences

The effects of defoliation on arbuscular mycorrhizal (AM) associations in the field were investigated in terms of the community structure of AM fungi colonizing roots of grassland plants; the carbohydrate balance of the host plants was also determined. We focused on two plant species dominating Japanese native grasslands: the grazing-intolerant species Miscanthus sinensis and the grazing-tolerant species Zoysia japonica. Community structures of AM fungi were determined from 18S rRNA gene sequences. The dominant fungal group in both plant species was the Glomus clade, which was classified into several phylogenetic groups based on genetic distances and topology. In Miscanthus roots, the Glomus-Ab, Glomus-Ac, and Glomus-Ad groups were detected almost equally. In Zoysia roots, the Glomus-Ab group was dominant. Defoliation effects on the community structure of AM fungi differed between the plant species. In Miscanthus roots, the percentage of root length colonized (%RLC) by the Glomus-Ac and Glomus-Ad groups was significantly reduced by defoliation treatment. On the other hand, AM fungal group composition in Zoysia roots was unaffected by defoliation except on the last sampling date. Decreased %RLC by Glomus-Ac and Glomus-Ad coincided with decreased non-structural carbohydrate (NSC) levels in host plants; also, significant positive correlations were found between the %RLC and some NSC levels. On the other hand, the %RLC by Glomus-Ab in both plant species was unaffected by the NSC level. These results suggest that AM fungal groups have different carbohydrate requirements from host plants.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00572-003-0286-x     

Arbuscular mycorrhizal status of spring ephemerals in the desert ecosystem of Junggar Basin, China

A survey was made of the arbuscular mycorrhizal (AM) status of 73 spring ephemeral plant species that grow in the desert ecosystem of Junggar Basin, northwest China. The proportion of AM colonization ranged from 7 to 73% with a mean value of 30%. A total of 65 plant species studied were AM with coils/arbuscules or vesicles and the remaining eight species were possibly AM with no coils/arbuscules or vesicles but with fungal mycelia in the root cortex. AM fungal spores were isolated from rhizosphere samples of all 73 plant species and identified. The mean spore density was 22 per 20 ml of air-dried soil, ranging from 0 to 120. Colonization and spore density of perennials were slightly higher than of annuals and varied among different plant families. A total of 603 AM fungal spore (or sporocarp) specimens were isolated belonging to six genera, Acaulospora, Archaeopora, Entrophospora, Glomus, Paraglomus, and Scutellospora; Glomus was the dominant genus. We conclude that spring ephemerals may be highly dependent on AM associations for survival in the very infertile and arid soils of this desert ecosystem.Electronic supplementary material Supplementary material is available for this article at     

Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic,medicinal orchid (<Emphasis Type="Italic">Dendrobium nobile</Emphasis>): estimation and characterization

Although the terrestrial and temperate orchids–fungal biology have been largely explored, knowledge of tropical epiphytic orchids–fungus relationships, especially on the ecological roles imparted by non-mycorrhizal fungal endophytes, is less known. Exploitation of the endophytic fungal mycobiota residing in epiphytic orchid plants may be of great importance to further elucidate the fungal ecology in this special habitat as well as developing new approaches for orchid conversations. The composition of fungal endophytes associated with leaves, stems and roots of an epiphytic orchid (Dendrobium nobile), a famous Chinese traditional medicinal plant, was investigated. Microscopic imaging, culture-dependant method and molecular phylogeny were used to estimate their entity and diversity. Totally, there were 172 isolates, at least 14 fungal genera and 33 different morphospecies recovered from 288 samples. Ascomycetes, coelomycetes and hyphomycetes were three major fungal groups. There were higher overall colonization and isolation rates of endophytic fungi from leaves than from other tissues. Guignardia mangiferae was the dominant fungal species within leaves; while the endophytic Xylariaceae were frequently observed in all plant tissues; Colletotrichum, Phomopsis and Fusarium were also frequently observed. Phylogenetic analysis based on ITS gene revealed the high diversity of Xylariacea fungi and relatively diverse of non-Xylariacea fungi. Some potentially promising beneficial fungi such as Clonostachys rosea and Trichoderma chlorosporum were found in roots. This is the first report concerning above-ground and below-ground endophytic fungi community of an epiphytic medicinal orchid, suggesting the ubiquitous distribution of non-mycorrhizal fungal endophytes in orchid plants together with heterogeneity and tissue specificity of the endophyte assemblage. Possible physiological functions played by these fungal endophytes and their potential applications are also discussed briefly. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fern-associated arbuscular mycorrhizal fungi are represented by multiple Glomus spp.: do environmental factors influence partner identity?

Symbioses involving arbuscular mycorrhizal fungi (AMF) are among the most important ecological associations for many plant species. The diversity of AMF associated with ferns, however, remains poorly studied. Using recently designed Glomus-specific primers, we surveyed the AMF community associated with ferns from deciduous, broad-leaved second-growth forest habitats at the eastern edge of the piedmont region of central Virginia, USA. Results indicate that this molecular approach may be a useful tool for detecting AMF in ferns compared to traditional techniques based on morphology. Over 30 potential fungal ribotypes were identified from eight fern species using denaturing gradient gel electrophoresis. Fungal ribotypes were found to differ widely in terms of (1) the number of fern partners with which they interact and (2) their relative frequency within each fern. Sequence analysis of fungal isolates from three species of fern indicated that the primers were generally highly specific for Glomus species but some non-target DNA was also amplified. Cloned polymerase chain reaction (PCR) products from Polystichum acrostichoides and Osmunda regalis revealed several phylogenetically distinct Glomus species. A single Glomus species was identified in the cloned PCR products from Botrychium virginianum. These findings challenge the hypothesis that the extent or degree of fern–fungal symbiosis is somehow tied to root complexity. Environmental factors appear to influence the suite of AMF that form partnerships with ferns. Some species of fern from similar habitats associated with dissimilar fungal partners (e.g., P. acrostichoides and Athyrium filix-femina var. asplenioides), whereas others harbored uniform fungal communities (e.g., Asplenium platyneuron). The significance of these data in terms of ecological and evolutionary dynamics of the AMF–fern symbiosis is discussed. Brittany West, Jessica Brandt, and Kay Holstien contributed equally to this work.     

The growth of external AM fungal mycelium in sand dunes and in experimental systems

We estimated the biomass and growth of arbuscular mycorrhizal (AM) mycelium in sand dunes using signature fatty acids. Mesh bags and tubes, containing initially mycelium-free sand, were buried in the field near the roots of the dune grass Ammophila arenaria L. AM fungal mycelia were detected at a distance of about 8.5 cm from the roots after 68 days of growth by use of neutral lipid fatty acid (NLFA) 16:1ω5. The average rate of mycelium extension during September and October was estimated as 1.2 mm day⁻¹. The lipid and fatty acid compositions of AM fungal mycelia of isolates and from sand dunes were analysed and showed all to be of a similar composition. Phospholipid fatty acids (PLFAs) can be used as indicators of microbial biomass. The mycelium of G. intraradices growing in glass beads contained 8.3 nmol PLFAs per mg dry biomass, and about 15% of the PLFAs in G. intraradices, G. claroideum and AM fungal mycelium extracted from sand dunes, consisted of the signature PLFA 16:1ω5. We thus suggest a conversion factor of 1.2 nmol PLFA 16:1ω5 per mg dry biomass. Calculations using this conversion factor indicated up to 34 μg dry AM fungal biomass per g sand in the sand dunes, which was less than one tenth of that found in an experimental system with Glomus spp. growing with cucumber as plant associate in agricultural soil. The PLFA results from different systems indicated that the biomass of the AM fungi constitutes a considerable part of the total soil microbial biomass. Calculations based on ATP of AM fungi in an experimental growth system indicated that the biomass of the AM fungi constituted approximately 30% of the total microbial biomass. This revised version was published online in June 2006 with corrections to the Cover Date.     

Arbuscular and ectomycorrhizal colonization of two <Emphasis Type="Italic">Eucalyptus</Emphasis> species in semiarid Brazil

Our goal was to evaluate the mycorrhizal colonization, as well as the density of arbuscular mycorrhizal (AM) fungal spores, in Eucalyptus camaldulensis and E. grandis monocultures at 2 years in a semiarid part of Brazil. Soil and root samples were collected in 2 consecutive years. Eucalyptus camaldulensis showed varied AM colonization level according to season of sampling, and Glomus was dominant in spore numbers. Eucalyptus grandis showed dominant ectomycorrhizal (ECM) colonization and lower AM fungal spore density. Overall results suggest that E. camaldulensis has both AM and ECM dependencies, whereas E. grandis is solely ECM dependent in the monocultures.     

Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

The influence of crop rotation and soil fumigation on a mycorrhizal fungal community associated with soybean

Population densities of mycorrhizal fungal propagules in a western Kentucky field highly productive for soybean were measured by bioassay throughout a soybean production season. The primary experimental variables were crop rotation (soybeans in 1985, then 2 years in corn, milo, fescue, or soybean, then soybean in 1988 on all plots when populations of propagules were determined) and soil fumigation with 67% methyl bromide/33% chloropicrin. Of the 20 species in three genera found, Glomus predominated both in terms of number of species and population densities. Most species of Glomus occurred at higher population densities in rotated plots than in continuous soybean plots. In continuous soybean plots, species of Gigaspora made up a much higher proportion of the mycorrhizal fungal community than in rotated crops. Species richness and diversity were lower, and dominance and equitability higher, in nonfumigated continuous soybean plots than in rotated plots early in the season, but the differences were not present at the end of the season. Soil fumigation killed most propagules in the upper 15 cm of soil, but after production of a crop of soybeans, populations of total propagules and most Glomus spp. recovered to prefumigation densities. However, Gigaspora margarita and Gigaspora gigantea did not recover similarly. Fumigation reduced species richness and diversity and increased dominance, but the effects were ameliorated by the end of the season. Colonization of roots was low during vegetative growth but increased rapidly after the onset of soybean reproduction. There was no evidence for mutualism during the early half of the season, perhaps due to high soil P and low dependency of soybean. Fumigation increased soybean yields. A stable mycorrhizal fungal community appeared to become established with continuous soybean production, and both crop rotation and soil fumigation disrupted the community.     

Molecular study of arbuscular mycorrhizal fungi colonizing the sporophyte of the eusporangiate rattlesnake fern (<Emphasis Type="Italic">Botrychium virginianum</Emphasis>, Ophioglossaceae)

The arbuscular mycorrhizal (AM) fungi colonizing the sporophytes of the eusporangiate rattlesnake fern (Botrychium virginianum, Ophioglossaceae) in its Hungarian population were investigated in the present study. Different regions of the nrRNA gene complex were analyzed using two different primer sets. These produced similar results for the detected AM fungi phylotypes. Several AM fungal lineages were associated with sporophytes of B. virginianum. Phylogenetic analyses of different partial small subunit datasets grouped one lineage into the Gigasporaceae, showing similarities with Scutellospora sequences. In addition to unidentified Scutellospora phylotypes, it is possible that S. gregaria also colonized the fern. Several AM fungal phylotypes colonizing the sporophytes grouped into Glomus group A. They did not form distinct clades but grouped with sequences of AM fungi with different geographic and host origins. One main lineage clustered into the widespread G. fasciculatum/G. intraradices group and one into the subgroup GlGrAc, while others had no affinity to the subgroups of Glomus group A. As AM fungal phylotypes associated with B. virginianum seem to belong to widespread AM fungal taxa and show no specificity to this fern, we suppose that the previously described special anatomy of AM of B. virginianum is determined by the plant.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

Variability of Nuclear SSU-rDNA Group Introns Within Septoria Species: Incongruence with Host Sequence Phylogenies

We report structural features and distribution patterns of 26 different group I introns located at three distinct nucleotide positions in nuclear small subunit ribosomal DNA (SSU-rDNA) of 10 Septoria and 4 other anamorphic species related to the teleomorphic genus Mycosphaerella. Secondary structure and sequence characteristics assigned the introns to the common IC1 and IE groups. Intron distribution patterns and phylogenetic relationships strongly suggested that some horizontal transfer events have occurred among the closely related fungal species sampled. To test this hypothesis, we used a comparative approach of intron- and rDNA-based phylogenies through MP- and ML-based topology tests. Our results showed two statistically well-supported major incongruences between the intron and the equivalent internal transcribed spacer (ITS) tree comparisons made. Such absence of a co-evolutive history between group I introns and host sequences is discussed relatively to the intron structures, the mechanisms of intron movement, and the biology of the Mycosphaerella pathogenic fungi. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Debashish Bhattacharya     

Morphological and Molecular Evidence of Arbuscular Mycorrhizal Fungal Associations in Costa Rican Epiphytic Bromeliads1

Arbuscular mycorrhizal fungi influence the growth, morphology, and fitness of a variety of plant species, but little is known of the arbuscular mycorrhizal (AM) fungal associations of plant species in forest canopies. Plant species' associations with AM fungi are most often elucidated by examining the roots for fungal structures; however, morphological data may provide a limited resolution on a plant's mycorrhizal status. We combined a traditional staining technique with a molecular marker (the 18S ribosomal gene) to determine whether or not a variety of epiphytic bromeliads form arbuscular mycorrhizal fungal associations. Using these methods we show that the epiphytic bromeliad Vriesea werkleana forms arbuscular mycorrhizal fungal associations with members of the genus Glomus. AM fungal sequences of this plant species formed three distinct clades nested within a larger Glomus clade; two of the clades did not group with any previously sequenced lineage of Glomus. Novel clades may represent novel species. Although Vriesea werkleana is associated with multiple AM fungal species, each individual plant is colonized by a single lineage. The combination of morphological and molecular methods provides a practical approach to the characterization of the mycorrhizal status of epiphytic bromeliads, and perhaps other tropical epiphytes.     

不同生境下川麦冬根围土壤丛枝菌根真菌多样性北大核心CSCD

采用高通量(Illumina Miseq)测序技术对栽培和野生2种生境下川麦冬根围的丛枝菌根(AM)真菌多样性和群落结构进行测定,并结合土壤理化因子进行相关性分析,以明确两种生境下川麦冬根围土壤AM真菌多样性和优势群落的分布特点,探讨AM真菌群落分布差异的驱动因子,为AM真菌应用于麦冬生产提供理论依据和技术支持。结果表明:(1)不同生境下川麦冬根围土壤中共鉴定出AM真菌3属10种,其中野生川麦冬根围土壤鉴定出的AM真菌3属7种,分别隶属于无梗囊霉属(Acaulospora)、多孢囊霉属(Diversispora)和球囊霉属(Glomus),而栽培环境下鉴定出AM真菌1属6种,隶属于球囊霉属。2个生境优势属均为球囊霉属。(2)不同生境下川麦冬根围AM真菌之间存在显著差异,野生生境下川麦冬根围土壤AM真菌多样性指数ACE和Shannon均显著高于人工栽培生境,而Simpson指数则相反。(3)相关性分析表明,AM真菌多样性指数及群落组成结构均与土壤理化因子存在相关性,其中全钾(TK)、全磷(TP)、全氮(TN)对AM真菌多样性指数和群落结构组成均存在显著影响。研究认为,不同生境下川麦冬根围AM真菌群落存在显著差异,球囊霉属为川麦冬互利共生的关键属,TK、TP、TN是不同生境川麦冬根围AM真菌群落差异的主要驱动因子。     

Detection of arbuscular mycorrhizal fungal spores in the air across different biomes and ecoregions

Aerial dispersal of fungal spores is common, but the role of wind and air movement in dispersal of spores of arbuscular mycorrhizal (AM) fungi is largely unknown. Several studies have examined the possibility of AM fungal spores being moved by wind vectors without observing spores taken from the air environment. For the first time this study observed the presence of AM fungal spores in the air. The frequency of AM fungal spores in the air was determined in six North American biomes composed of 18 ecoregions. Multiple samples were taken from both the air and the soil at each location. AM fungal spores were found in high abundance in the soil (hundreds of spores per gram of soil), however, they were rarely found in the air (most samples contained no AM fungal spores). Furthermore, only the Glomus morphotype was found in the air, whereas spores in the soil were taxomomically more diverse (Glomus, Acaulospora, Gigaspora, Scutellospora morphotypes were observed). The proportion of Glomus spores in the air relative to Glomus spores in the soil was highest in more arid systems, indicating that AM fungi may be more likely to be dispersed in the air in such systems. Nonetheless, the results indicate that the air is not likely a dominant mode of dispersal for AM fungi.     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Pityopus californicus</Emphasis>: structural characteristics of seed and seedling development in a myco-heterotrophic species

Pityopus californicus (Eastw.) H. F. Copel., a monotypic member of the Monotropoideae in the family Ericaceae, is a myco-heterotrophic species with distribution limited to the Pacific Northwest of the USA. Young embryos of P. californicus developed mycorrhizal associations in seed packets that had been buried for up to 681 days, suggesting that seeds of P. californicus may require the presence of a fungus to achieve germination. Samples of nongerminated seeds and early stages in embryo and root development were subsequently processed for light microscopy, histochemistry, and transmission electron microscopy (TEM). Nongerminated seeds possessed a thick testa, lacked a shoot and root meristem, and consisted of an embryo with large parenchymatous cells containing protein bodies and starch grains as storage reserves. In the earliest developmental stage (seed coat still attached), fungal hyphae were present on the testa surface and between the testa and embryo. This stage was followed by embryo elongation, the organization of a root apical meristem, and the development of a well-developed fungal mantle surrounding the elongated embryo. At least two morphotypes were identified based on structural characteristics of the mantle. One of these, with ascomycetous septa, had Cenococcum-like features. Late-stage embryo/early root development revealed a typical mantle and Hartig net, with fungal pegs penetrating the outer tangential walls of epidermal cells. Transfer cell-like deposits of wall material, similar to those described in Monotropa spp., enclosed fungal pegs. The development of a Hartig net and fungal pegs suggests that nutrient exchange interfaces are required for seedling development.