Arbutoid Mycorrhizas Research Articles

Ericaceous Plants: A Review for the Bioprospecting of Ericoid Mycorrhizae from Ecuador

Ericaceae are a group of plants with biotechnological and commercial importance. These plants establish symbiotic associations with a wide group of mycorrhizal fungi. National and global studies have focused on two of them: arbuscular endomycorrhizae and ectomycorrhizae. The most recent type of mycorrhiza recorded is the cavendishioid ectendomycorrhizae. The cavendishioid is one of the least-studied and understood mycorrhizae, along with monotropoid and arbutoid mycorrhizae. Among the potentialities of these ectendomycorrhizae are the alleviation of environmental stress, the facilitation of establishment, survival, the ability to form dual mycorrhizae, and their importance as a fundamental biological element of plant ecological successions. However, the factors that influence or correlate with the diversity of some of these fungal guilds, as well as the functional significance of their associations, are still unknown. The present review focuses on ericoid mycorrhiza from Ecuador in order to promote their study and thus take advantage of the benefits that they provide to endemic species and those of commercial interest. This material contributes significantly to reducing research gaps and channeling applied projects in the biological sciences.

Moneses uniflora (L.) A. Gray on industrial dumps of the Middle Urals

The article presents the results of studying the coenopopulations of Moneses uniflora (L.) A. Gray (familyEricaceae Juss.) found during the monitoring of vegetation formation on the dumps of the mining and processing industryof the Sverdlovsk region (Middle Urals, southern taiga subzone). Studies have shown that the M. uniflora (family EricaceaeJuss.) coenopopulations in plant communities formed on industrial dumps and in natural phytocenosis are normal, fullfledged, young. The morphological parameters of generative individuals are significantly influenced by coenotic andedaphic factors. Arbutoid mycorrhiza was found in the thin roots of M. uniflora in all the studied habitats.

Synthesis and ultrastructural observation of arbutoid mycorrhizae of black truffles (Tuber melanosporum and T. aestivum)

Arbutus unedo (the strawberry tree) is a Mediterranean shrub which forms arbutoid mycorrhizae with a variety of Asco- and Basidiomycetes. After the discovery of the mycorrhizal symbiosis between A. unedo and Tuber borchii, in this study, arbutoid mycorrhizae were synthetized in greenhouse with Tuber aestivum and Tuber melanosporum. Six months after inoculation, both species colonized the roots of all inoculated A. unedo seedlings, but mature mycorrhizae were only observed after 12 months. Ultrastructure analysis of Tuber arbutoid mycorrhizae was described for the first time, showing, as observed in typical endosymbiosis, a rearrangement of host cells and the creation of an interface compartment with both truffle species. Immunolabelling experiments suggested that pectins are not present in the interface matrix surrounding the intracellular hyphae. Thus, the ability to establish symbiosis with A. unedo seems to be a common feature in the genus Tuber, opening up the possibility to use this plant for mycorrhization with valuable truffles. This could represent an important economic opportunity in Mediterranean areas by combining the production of truffles, edible fruits and valued honey.

Arbutoid mycorrhizas of the genus Cortinarius from Costa Rica.

Arbutoid mycorrhizas of Comarostaphylis arbutoides (Arbutoidea, Ericaceae) from neotropical montane forests are rarely described. To date, only mycorrhizal associations with the fungal species Leccinum monticola, Leotia lubrica and Sebacina sp. are known from literature. The genus Cortinarius is one of the most species-rich ectomycorrhizal taxa with over 2000 assumed species. In this study, two sites in the Cordillera de Talamanca of Costa Rica were sampled, where Com. arbutoides is endemic and grows together with Quercus costaricensis. Using a combined method of rDNA sequence analysis and morphotyping, 33 sampled mycorrhizal systems of Cortinarius were assigned to the subgenera Dermocybe, Phlegmacium and Telamonia. Specific plant primers were used to identify the host plant. Here, we present the phylogenetic data of all found Cortinarii and describe four of the arbutoid mycorrhizal systems morphologically and anatomically.

Mycorrhizal synthesis between Lactarius deliciosus and Arbutus unedo L.

Arbutoid mycorrhizae were synthesized in vitro between Arbutus unedo L. and two isolates of Lactarius deliciosus. The fungal isolates were obtained from sporocarps collected under Pinus sylvestris and in a mixed forest stand of Quercus suber and Pinus pinea. Synthesis tubes filled with a mixture of sterilized peat, vermiculite, and perlite imbibed with nutrient solution were used. Two inoculation methods using solid and liquid media were tested. Shoots from an adult selected clone of A. unedo were used after in vitro rooting by auxin dipping. After 3 months of shoots transfer to the substrate, the root systems were examined for arbutoid mycorrhizae formation and later on ex vitro conditions, 9 months after acclimatization. The inoculum treatment with liquid medium improved the mycorrhizal development for both isolates, in vitro. Sterilized substrate for plant acclimatization increased the mycorrhizal development. The arbutoid mycorrhizae were observed in vitro as well as 9 months after acclimatization. Standard arbutoid mycorrhiza features were observed: pale yellow mantle, typical cruciform appearance, Hartig net (HN), and intracellular hyphal complexes, both confined to the epidermis. L. deliciosus mycorrhizae synthetized in vitro persisted 9 months after plant acclimatization. Morphological observations were confirmed by molecular techniques.

Leotia cf. lubrica forms arbutoid mycorrhiza with Comarostaphylis arbutoides (Ericaceae).

Arbutoid mycorrhizal plants are commonly found as understory vegetation in forests worldwide where ectomycorrhiza-forming trees occur. Comarostaphylis arbutoides (Ericaceae) is a tropical woody plant and common in tropical Central America. This plant forms arbutoid mycorrhiza, whereas only associations with Leccinum monticola as well as Sebacina sp. are described so far. We collected arbutoid mycorrhizas of C. arbutoides from the Cerro de la Muerte (Cordillera de Talamanca), Costa Rica, where this plant species grows together with Quercus costaricensis. We provide here the first evidence of mycorrhizal status for the Ascomycete Leotia cf. lubrica (Helotiales) that was so far under discussion as saprophyte or mycorrhizal. This fungus formed arbutoid mycorrhiza with C. arbutoides. The morphotype was described morphologically and anatomically. Leotia cf. lubrica was identified using molecular methods, such as sequencing the internal-transcribed spacer (ITS) and the large subunit (LSU) ribosomal DNA regions, as well as phylogenetic analyses. Specific plant primers were used to confirm C. arbutoides as the host plant of the leotioid mycorrhiza.

Characterization of Tuber borchii and Arbutus unedo mycorrhizas

For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies.

Sebacina sp. is a mycorrhizal partner of Comarostaphylis arbutoides (Ericaceae)

Sebacina (Sebacinales) forms ectomycorrhiza, arbutoid, ericoid, jungermannioid, cavendishioid and orchid mycorrhiza with diverse plant species. The woody plant Comarostaphylis arbutoides (Ericaceae) forms arbutoid mycorrhiza with Leccinum monticola. However, further morphotypes have hitherto not been described. C. arbutoides grows in tropical Central America at an elevation of 2,500–3,430 m a.s.l., where it is found as understory vegetation in forests or forms extensive thickets. It shares ectomycorrhizal fungi with Quercus species, thereby being a refuge for these fungi after forest clearance of the oaks. We collected arbutoid mycorrhizas of C. arbutoides from the Cerro de la Muerte (Cordillera de Talamanca) in Costa Rica, where it grows together with Quercus costaricensis. Sebacina sp. was identified after sequencing the internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions, and their phylogenetic analyses. The morphotype Sebacina sp.-C. arbutoides was described morphologically and anatomically.

Mycorrhizal synthesis between Pisolithus arhizus and adult clones of Arbutus unedo in vitro and in nursery

Arbutoid mycorrhizae were synthesized between adult selected clones of Arbutus unedo L. and Pisolithus arhizus. Two micropropagated clones were tested: AL1, in vitro and C1 (acclimatized plants) in nursery and later in a field trial. In vitro, rooted shoots were transferred to test tubes containing the substrate previously inoculated with mycelium cultured on agar. In the nursery, two inoculation treatments were tested (vegetative inocula or dry sporocarps) and compared to control plants. In the field trial, plants from nursery inoculation treatments were compared and an additional control treatment using seedlings was implemented. Plant height was evaluated 4 months later in the nursery and 20 months later in the field trial. Roots were examined by morphological and histological studies: a) in vitro plantlets one month after inoculation and nine months after acclimatization; and b) 20 months after the field trial was established. Arbutoid mycorrhizae were observed in vitro one month after inoculation, indicating compatibility between A. unedo and P. arhizus. These showed the presence of a mantle, Hartig net, and intracellular hyphal complexes confined to the epidermal root cells. Arbutoid mycorrhizae were also observed nine months after acclimatization in inoculated and control plants. In order to confirm the identity of mycorrhizae, molecular techniques were used, in previously inoculated in vitro plants, 12 months after acclimatization. Thelephora and Hebeloma mycorrhizae, two types of highly competitive and widespread mycorrhizae on nurseries were identified. In the nursery, dry sporocarp treatment improved plant height after four months. In a field trial (20 months later), plants growth did not show significant differences. By this time, mycorrhized roots with Cenococcum geophilum and other types were identified. These results and their implications on A. unedo breeding program are discussed.

Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophicPyrola japonica(Ericaceae) growing under different light environments

Mixotrophy is a strategy whereby plants acquire carbon both through photosynthesis and heterotrophic exploitation of mycorrhizal fungi. In Euro-American Pyroleae species studied hitherto, heterotrophy levels vary according to species, sites of study, and possibly light conditions. We investigated mycorrhizal association and mixotrophy in the Asiatic forest species Pyrola japonica, and their plasticity under different light conditions. Pyrola japonica was sampled bimonthly in sunny and shaded conditions from a deciduous broadleaf forest. We microscopically assessed the rate of fungal colonization and sequenced the ITS to identify the mycorrhizal fungi. We measured (13)C and (15)N isotopic abundances in P. japonica as compared with neighboring autotrophic and mycoheterotrophic plants, to evaluate P. japonica's heterotrophy level. Pyrola japonica formed arbutoid mycorrhizas devoid of fungal mantles, with intracellular hyphal coils and a Hartig net. It tended to be more colonized by mycorrhizal fungi in spring and summer. Most associated fungi belonged to ectomycorrhizal taxa, and 84% of identified fungi were Russula spp. Rate of mycorrhizal colonization and Russula frequency tended to be higher in shaded conditions. Both δ(13)C and δ(15)N values of P. japonica were significantly higher in autotrophic plants, showing that about half of the carbon on average was received from mycorrhizal fungi. Both isotopic values negatively correlated with light availability, suggesting higher heterotrophy levels in shaded conditions. The mixotrophic P. japonica undergoes changes in mycorrhizal symbionts and carbon nutrition according to light availability. Our results suggest that during Pyroleae evolution, a tendency to increased heterotrophy emerged in the Pyrola/Orthilia clade.

The influence of mycorrhizal inoculation and paclobutrazol on water and nutritional status of Arbutus unedo L.

The purpose of this study was to analyze morphological and physiological aspects of Arbutus unedo L. plants treated with paclobutrazol (PAC), compounds characterized by their double activity as plant growth regulators and fungicides, and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch, which forms a special type of mycorrhizal colonization called arbutoid mycorrhiza. Native A. unedo L. seedlings were grown in a greenhouse and subjected to four treatments for 4 months: 0 or 60 mg of PAC and inoculated or not with P. tinctorius (Pers.) . The arbutoid mycorrhizal inoculation increased in plants treated with PAC. Paclobutrazol reduced shoot and root biomass, plant height, internode length, stem diameter, leaf area, total root length and number of tips. P. tinctorius increased plant height and had a beneficial effect on the root system (increasing root diameter and the number of tips). PAC treatment led to an increase in ion levels in the leaf tissue, while mycorrhizal inoculation induced lower K and higher P contents in the roots. Leaf water potentials (at predawn and at midday) increased with the combined treatment. The absence of water deficit conditions meant there was no osmotic adjustment. Higher photosynthesis ( P n) values were associated with higher stomatal conductance ( g s) values in the mycorrhizal plants, which influenced water uptake from the roots. However, g s decreased in the PAC-treated plants, reducing photosynthesis and, as a consequence, growth. The higher hydraulic conductivity ( L p) in the plants treated with PAC may have induced a better water energy status and good water transport. The combined treatment produced beneficial effects in the plants, improving their water and nutritional status.

Fungal associates of Pyrola rotundifolia, a mixotrophic Ericaceae, from two Estonian boreal forests

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.

High diversity of ectomycorrhizal fungi associated with Arctostaphylos uva-ursi in subalpine and alpine zones: Potential inoculum for afforestation

Arctostaphylos uva-ursi plant associations have long been suggested to be a suitable location for afforestations, but systematic studies on the observed beneficial effect are still missing in Alpine areas. In this study we address the question which ectomycorrhizal (ECM) fungal communities exist in Austrian alpine and subalpine Arctostaphylos habitats, and if they persist in A. uva-ursi stands even hundred of years after disappearance of trees in these areas. The diversity of fungal communities associated with A. uva-ursi was assessed in four study sites (1800–2450 m a.s.l.) in the Central European Alps of Tyrol (Austria). Three approaches were applied: (i) collecting and identifying fungal fruit bodies; (ii) studying the arbutoid mycorrhiza morphotypes of A. uva-ursi; (iii) molecular identification of the most common arbutoid morphotypes. The detected diversity of ECM fungal species was very high (99 taxa), and the occurrence of e.g. Boletus, Cantharellus, Suillus, Tricholoma, Russula spp. in sites that have been treeless for centuries is especially remarkable. Fruit bodies of 82 fungal species were found in association with A. uva-ursi, 63 of them were ECM. Seventy arbutoid morphotypes were detected on 2072 mycorrhized root tips, and 39 taxa of basidiomycete mycobionts were identified with molecular methods. Twelve Cortinarius spp. clearly dominated the mycobiont species richness, followed by nine spp. of Thelephoraceae and four spp. of Boletaceae. Most mycobionts of A. uva-ursi were generalists, but fungal taxa (e.g. Lactarius deterrimus and Suillus spp.) known to be specifically associated with other host plants were also detected. We therefore consider A. uva-ursi as a “non-selective” host, providing mycorrhizal inoculum of highly specialized and effective mycorrhizal fungi for afforestation. This explains why A. uva-ursi plant associations represent suitable locations for forest regeneration and afforestation, especially in the harsh environmental conditions of subalpine and alpine sites.

Morphological and molecular evidence supporting an arbutoid mycorrhizal relationship in the Costa Ricanpáramo

This study examines evidence for a particular arbutoid mycorrhizal interaction in páramo, a high-altitude neotropical ecosystem important in hydrological regulation but poorly known in terms of its fungal communities. Comarostaphylis arbutoides Lindley (Ericaceae) often forms dense thickets in Central American páramo habitats. Based on phylogenetic classification, it has been suggested that C. arbutoides forms arbutoid mycorrhizae with diverse Basidiomycetes and Ascomycetes; however, this assumption has not previously been confirmed. Based on field data, we hypothesized an arbutoid mycorrhizal association between C. arbutoides and the recently described bolete Leccinum monticola Halling & G.M. Mueller; in this study, we applied a rigorous approach using anatomical and molecular data to examine evidence for such an association. We examined root samples collected beneath L. monticola basidiomes for mycorrhizal structures, and we also compared rDNA internal transcribed spacer (ITS) sequences between mycorrhizal root tips and leaf or basidiome material of the suspected symbionts. Root cross sections showed a thin hyphal sheath and intracellular hyphal coils typical of arbutoid mycorrhizae. DNA sequence comparisons confirmed the identity of C. arbutoides and L. monticola as the mycorrhizal symbionts. In addition, this paper provides additional evidence for the widespread presence of minisatellite-like inserts in the ITS1 spacer in Leccinum species (including a characterization of the insert in L. monticola) and reports the use of an angiosperm-specific ITS primer pair useful for amplifying plant DNA from mycorrhizal roots without co-amplifying fungal DNA.

Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi

The ectomycorrhizal basidiomycete species Lactarius deterrimus Gröger is considered to be a strictly host-specific mycobiont of Picea abies (L.) Karst. However, we identified arbutoid mycorrhiza formed by this fungus on the roots of Arctostaphylos uva-ursi (L.) Spreng. in a mixed stand at the alpine timberline; typical ectomycorrhiza of P. abies were found in close relation. A. uva-ursi is known as an extremely unspecific phytobiont. The mycorrhizae of both associations are described and compared morphologically. The mycorrhiza formed by L. deterrimus on both A. uva-ursi and P. abies show typical ectomycorrhizal features such as a hyphal mantle and a Hartig net. The main difference between the mycorrhizal symbioses with the different phytobionts is the occurrence of intracellular hyphae in the epidermal cells of A. uva-ursi. This emphasizes the importance of the plant partner for mycorrhizal anatomy. This is the first report of a previously considered host-specific ectomycorrhizal fungus in association with A. uva-ursi under natural conditions. The advantages of this loose specificity between the fungus and plant species is discussed.

Exploring structural definitions of mycorrhizas, with emphasis on nutrient-exchange interfaces

The roots or other subterranean organs of most plants develop symbioses, mycorrhizas, with fungal symbionts. Historically, mycorrhizas have been placed into seven categories based primarily on structural characteristics. A new category has been proposed for symbiotic associations of some leafy liverworts. An important feature of mycorrhizas is the interface involved in nutrient exchange between the symbionts. With the exception of ectomycorrhizas, in which fungal hyphae remain external to plant cell walls, all mycorrhizas are characterized by fungal hyphae breaching cell walls but remaining separated from the cell cytoplasm by a plant-derived membrane and an interfacial matrix that forms an apoplastic compartment. The chemical composition of the interfacial matrix varies in complexity. In arbuscular mycorrhizas (both Arum-type and Paris-type), molecules typical of plant primary cell walls (i.e., cellulose, pectins, β-1,3-glucans, hydroxyproline-rich glycoproteins) are present. In ericoid mycorrhizas, only rhamnogalacturonans occur in the interfacial matrix surrounding intracellular hyphal complexes. The matrix around intracellular hyphal complexes in orchid mycorrhizas lacks plant cell wall compounds until hyphae begin to senesce, then molecules similar to those found in primary cell walls are deposited. The interfacial matrix has not been studied in arbutoid mycorrhizas and ectendomycorrhizas. In ectomycorrhizas, the apoplastic interface consists of plant cell wall and fungal cell wall; alterations in these may enhance nutrient transfer. In all mycorrhizas, nutrients must pass into the symplast of both partners at some point, and therefore current research is exploring the nature of the opposing membranes, particularly in relation to phosphorus and sugar transporters.Key words: interface, apoplastic compartment, Hartig net, arbuscule, intracellular complex, nutrient exchange.

Copper resistance of the evergreen dwarf shrub Arctostaphylos uva-ursi: an experimental exposure

The copper (Cu) resistance of Arctostaphylos uva-ursi was tested in a pot experiment (lasting 8 weeks) using rooted cuttings originating from an area near the Harjavalta Cu–Ni smelter, SW Finland. The fine roots were moderately infected by arbutoid mycorrhizae. The plants were exposed to five Cu levels (1, 10, 22, 46 and 100 mg l −1) given repeatedly together with a nutrient solution. The critical Cu concentration in the nutrient solution inhibiting the growth of A. uva-ursi was below 10 mg l −1 Cu (EC 50 value for biomass production 3.3 mg l −1 Cu). This concentration was clearly lower than the value we have found earlier for other dwarf shrubs under similar experimental conditions. Most of the Cu given accumulated in the roots and old stems. The results suggest that A. uva-ursi cuttings were relatively sensitive to Cu despite the ability of the adult clones to grow in Cu-contaminated soil. The adult clones extend their roots into the less toxic deeper soil layers, which may facilitate the avoidance of heavy metals.

A comparison of ectomycorrhiza identification based on morphotyping and PCR-RFLP analysis

Two methods are currently being used to describe ECM fungal communities associated with root tips: molecular techniques and morphological classification. Previous studies have found that these two approaches give conflicting results, with several fungal genotypes being identified from different ectomycorrhizas within the same morphotype. This has led researchers to question the usefulness of the morphological approach. The objective of this study was to compare the two approaches on ectomycorrhizas collected from three plant species growing in two different environments. Specifically, mycorrhizas were classified using a detailed morphological approach and then were subjected to PCR-RFLP analysis of the ITS region of the rRNA gene repeat. Ectomycorrhizas of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were sampled from three widely dispersed sites with different soil types in the southern interior of British Columbia. Ectomycorrhizas of Douglas-fir and arbutoid mycorrhizas from Arctostaphylos uva-ursi were sampled from a fourth site in a different biogeoclimatic zone. For eight of eleven dominant morphotypes, one main RFLP banding pattern was observed. Ninety-three % of the mycorrhizas analyzed in these eight morphotypes would have been classified in the same way by either method. Five of the eight morphotypes were positively identified as Russula nigricans, Lactarius sp., Leccinum scabrum, Rhizopogon sect. Villosuli, and Thelephora terrestris by matching the RFLPs to those of fungal fruit bodies in our database or by sequencing the ITS region. The other morphotypes producing one dominant RFLP pattern were designated as Cenococcum, E-strain and Mycelium radicis atrovirens (MRA) based on their morphology. Morphotyping did not distinguish amongst major RFLP types for mycorrhizas classified as Amphinema-like, Piloderma-like and Rhizopogon-like A. We conclude that detailed morphological classification can be very useful as the primary method of ectomycorrhizal classification, when used in conjunction with molecular techniques. This approach will allow for an efficient use of research funds.

Comparison of ericoid and ectomycorrhizal colonization and ectomycorrhizal morphotypes in mixed conifer and pygmy forests on the northern California coast

On the northern California coast, mixed conifer forests occur on younger, relatively fertile terraces, whereas mesic and hydric pygmy forests occur on older, infertile, poorly drained terraces. We characterized mycorrhizal associations, ectomycorrhizal morphotypes, and colonization levels for nine plant species from three plant communities (mixed conifer, mesic pygmy, and hydric pygmy). Pinus contorta ssp. bolanderi (Parl.) Critchf. and Pinus muricata D. Don were ectomycorrhizal; all ericaceous plants formed ericoid mycorrhizas except Arctostaphylos nummularia A. Gray, which formed only arbutoid mycorrhizas. Arbuscular mycorrhizas were not observed, even on Cupressus goveniana ssp. pigmaea (Lemmon) J. Bartel. Ectomycorrhizal colonization was significantly lower in pygmy forests, while ericoid mycorrhizal colonization was significantly higher, as compared with the mixed conifer forest. Ectomycorrhizal and ericoid mycorrhizal colonization was greater on hummocks than in swales at hydric pygmy sites. Thirteen distinct ectomycorrhizal and arbutoid mycorrhizal morphotypes were observed: eight only on Pinus spp. and two only on A. nummularia. Two morphotypes were found only in mixed conifer, and eight were found only in pygmy communities. Distribution of morphotypes may reveal habitat and (or) host adaptation by ectomycorrhizal fungi.Key words: ectomycorrhizal morphotypes, ericoid mycorrhizas, pygmy forest, Ericaceae, mycorrhizal colonization, low-fertility soils.

Visualization of mycorrhizal fungal structures in resin embedded tissues with xanthene dyes using laser scanning confocal microscopy

The xanthene dyes sulforhordamine G, phloxine B, rose Bengal, and 4,5,6,7-tetrachloroflorescein were used as fluorochromes for laser scanning confocal microscopy of LR-White resin-embedded mycorrhizae. Sulforhodamine G was the most effective dye, giving an even staining of cell components throughout the material, with minimal background fluorescence of LR-White resin. Confocal microscopy of stained blocks of tissue on a slide, viewed without the use of a coverslip, revealed the three-dimensional nature of various mycorrhizal structures; these structures included arbuscules, vesicles, and coiled hyphae in arbuscular mycorrhizae; coiled hyphae in orchid mycorrhizae; mantle and Hartig net hyphae in ectomycorrhizae; and intracellular hyphae in arbutoid mycorrhizae. Sections mounted on slides viewed with confocal microscopy provided exceptional clarity of fungal form and cytoplasmic contents and showed the relationship to the plant cells, also with negligible background fluorescence. Mounting and staining blocks of resin-embedded material provided a fast and effective technique for the visualization of a variety of plant and fungal tissues. Stain penetration in whole-mounted samples was sufficient to reconstruct clear three-dimensional images using confocal microscopy.Key words: mycorrhizae, xanthene dyes, confocal microscopy, resin embedding.