Comparative anatomy of ectomycorrhizas synthesized on Douglas fir by Rhizopogon spp. and the hypogeous relative Truncocolumella citrina

The morphology and anatomy of ectomycorrhizas of Rhizopogon parksii , Rhizopogon vinicolor and Rhizopogon subcaerulescens , and a hypogeous relative, Truncocolumella citrina , synthesized on Douglas fir in glasshouse conditions using spore slurries as inoculum, are described and compared. Mycorrhizas formed with R. parksii and R. vinicolor did not exhibit their characteristic subtuberculate morphology in these tests, but rather had a pinnate form. All species had diagnostic features of ectomycorrhizas: a well-developed Hartig net and a fungal mantle. In addition, several species exhibited crystal inclusions in the outer mantle, usually at the interface between the mantle and soil. Truncocolumella citrina had crystal-like inclusions within the mantle but external to fungal hyphae, a feature rarely described in ectomycorrhizas.     

Regional specialization of Sarcodes sanguinea (Ericaceae) on a single fungal symbiont from the Rhizopogon ellenae (Rhizopogonaceae) species complex

We have sampled the mycorrhizal roots of 76 snow plants (Sarcodes sanguinea, Monotropoideae, Ericaceae) in two areas of the Sierra Nevada of California that are ～180 km apart. To identify the fungal symbionts associated with these plants, we first analyzed restriction fragment length polymorphisms (RFLPs) of the internal transcribed spacer region (ITS) of the fungal nuclear ribosomal repeat. Fungal ITS-RFLPs were successfully produced from 57 of the 76 plants sampled, and all symbionts shared the same DNA fragment pattern. The morphology of S. sanguinea mycorrhizae was consistent with that expected from a Rhizopogon species in section Amylopogon. To confirm and refine this identification, a total of six fungal ITS sequences were determined from S. sanguinea mycorrhizae. These sequences were analyzed together with eight existing and eight newly determined ITS sequences from Rhizopogon section Amylopogon. The newly determined sequences include an ITS sequence from the fungal symbiont of pine drops (Pterospora andromedea, Monotropoideae, Ericaceae), a plant that was previously reported to be exclusively associated with the Rhizopogon subcaerulescens group. When these sequences were analyzed together, the Sarcodes symbionts grouped tightly with several collections of R. ellenae including the holotype, one collection of R. idahoensis, and one collection of R. semireticulatus. A different lineage comprised collections of R. subgelatinosus, R. subcaerulescens, another collection of R. semireticulatus, and the Pterospora symbiont. We conclude that S. sanguinea associates exclusively with a single species in the R. ellenae species complex throughout our sampling range. These results indicate a much higher level of specificity in S. sanguinea than was previously reported and confirm the emerging pattern that nonphotosynthetic, monotropoid plants generally associate very specifically with a narrow range of ectomycorrhizal fungi.     

Effects of cadmium on growth and glucose utilisation of ectomycorrhizal fungi in vitro

Effects of Cd on growth and glucose utilisation of Paxillus involutus, Rhizopogon subcaerulescens and Suillus bovinus were investigated in vitro in liquid culture. S. bovinus was the species most sensitive to Cd in terms of dry matter production and P. involutus was less sensitive than R. subcaerulescens. Greater production of hyphae of P. involutus than the other fungi appeared to confer some degree of Cd resistance, possibly by binding Cd onto cell walls. Growth of the three fungi was increased by glucose addition. While Cd significantly reduced dry matter production of the fungi, there were no significant differences in glucose consumption caused by Cd treatment. This suggests that the use of glucose might have been diverted to detoxification and/or repair mechanisms. Further studies on respiration rates and energy metabolites of these fungi under Cd exposure are needed in order to clarify the results of the present study.     

Ectomycorrhizas of Cortinarius helodes and Gyrodon monticola with Alnus acuminata from Argentina

Field ectomycorrhizas of Cortinarius helodes Moser, Matheny & Daniele (sp. nov) and Gyrodon monticola Sing. on Alnus acuminata Kunth (Andean alder, aliso del cerro) are described based on morphological and anatomical features. Ectomycorrhizal roots were sampled beneath fruitbodies of C. helodes and G. monticola from two homogeneous A. acuminata forest sites located in Tucumán and Catamarca Provinces in Argentina. C. helodes ectomycorrhizas showed a thick white to beige mantle exuding a milky juice when injured, were bluish toward the apex, and had hyphal strands in the mantle. G. monticola ectomycorrhizas showed some conspicuous features like highly differentiated rhizomorphs, inflated brown cells on the mantle surface, and hyaline and brown emanating hyphae with dolipores. Restriction fragment length polymorphism analysis of the nuclear rDNA internal transcribed spacer provided a distinctive profile for each of the collections of fruitbodies and the mycorrhizal morphotypes.     

Mycorrhizal association of isolates from sporocarps and ectomycorrhizas withPinus densiflora seedlings

Thirty-two isolates from sporocarps of 27 species of macromycetes, 43 isolates from ectomycorrhizas ofPinus densiflora (Japanese red pine) and 1 isolate from an ectomycorrhiza ofQuercus myrsinaefolia were tested for the ability to form mycorrhizas withP. densiflora seedlings in glass tubes. Ten isolates from sporocarps ofHebeloma sp.,Laccaria bicolor, Lactarius chrysorrheus, Suillus granulatus, Scleroderma areolatum, Russula mariae andR. nigricans had formed ectomycorrhizas by 8 months after transplantation. Twenty isolates taken from mycorrhizas including ofCenococcum geophilum, R. mariae andR. nigricans formed ectomycorrhizas. The synthesized mycorrhizas were classified based on morphological characteristics such as hyphal arrangement of their fungal sheath, and appearance of cystidia and emanating hyphae. Twenty-one mycorrhizal types were recognized.Contribution No. 122, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Morphological classification of ectomycorrhizas ofPinus densiflora

Morphological classification of ectomycorrhizas ofPinus densiflora was conducted. Fifty soil samples containing pine ectomycorrhizas, and 40 pine seedlings were collected randomly in two separate reforested stands ofP. densiflora (45 yr old) from May 1992 to October 1994. Fifty-six types of ectomycorrhizas could be classified based upon microscopically observable morphological characteristics. Fifty percent of the types showed cystidia or other specific characteristics such as laticiferous hyphae in the fungal sheaths, verrucose emanating hyphae and a positive hyphal reaction to UV irradiation. Four mycorrhizal types were confirmed to be formed by the fungiRussula delica, R. mariae, R. nigricans, andCenococcum geophilum, respectively. Although the other 52 types were unidentified mycobionts at species level, it was inferred that they were formed by the fungiHebeloma, Lactarius, Russula andTuber. There was a slight difference in the observed mycorrhizal types between the tree ages. Contribution No. 127, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Tracking mycorrhizas and extraradical mycelium of the edible fungus <Emphasis Type="Italic">Lactarius deliciosus</Emphasis> under field competition with <Emphasis Type="Italic">Rhizopogon</Emphasis> spp

The objective of this study is to evaluate the field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus in competition with two ubiquitous soil fungi. Couples of plants inoculated with either L. deliciosus, Rhizopogon roseolus, or R. luteolus were transplanted, 10 cm apart, in two different sites at the following combinations: L. deliciosus-R. roseolus, L. deliciosus-R. luteolus, L. deliciosus-control (non-inoculated), control-R. roseolus, control-R. luteolus, and control-control. Eight months after transplantation, root colonization and extraradical soil mycelium for each fungal species were quantified. For mycelium quantification, soil cores equidistant to the two plants in each couple were taken, and total deoxyribonucleic acid (DNA) was extracted. Real-time polymerase chain reaction analysis was performed using specific primers and TaqMan Minor groove binding (MGB) probes designed in the ribosomal DNA internal transcribed spacer region of each fungal species. Field site significantly influenced persistence of both mycorrhizas and extraradical mycelium of L. deliciosus. Extraradical mycelium quantity was positively correlated with the final percentage of ectomycorrhizas for the three fungal species. Different competitive pressure between the two Rhizopogon species on L. deliciosus persistence was observed, with R. luteolus having no effect on L. deliciosus survival. Negative correlation between the final percentage of mycorrhizas of L. deliciosus and R. roseolus was observed. However, no relationship was determined between extraradical mycelia of both fungal species. The results obtained suggest that competition between L. deliciosus and R. roseolus takes place in the root system, for ectomycorrhiza formation in available roots, rather than in the extraradical phase.     

Comparative studies of ectomycorrhiza formation in Alnus glutinosa and Pinus resinosa with Paxillus involutus

 Mycorrhiza ontogeny and details of Hartig net and mantle structure were compared in ectomycorrhizas synthesized in growth pouches between the broad host range fungus Paxillus involutus and the tree species European black alder (Alnus glutinosa) and red pine (Pinus resinosa). In Alnus glutinosa, a paraepidermal Hartig net was restricted to the proximal (basal) portion of first-order laterals; the hypodermal layer appeared to be a barrier to fungal penetration. Phi-thickenings were present in some cortical cells but these were not related to lack of fungal ingress into the cortex. The mantle was often present close to the root apex but in many roots it was loosely organized and patchy. In several instances, the mantle formed around the root apex was only temporary; renewed root growth occurred without the formation of a mantle. In Pinus resinosa, the Hartig net developed between cortical cell layers of monopodial and dichotomously branched first–order laterals. Fungal hyphae in the Hartig net exhibited a complex labyrinthine mode of growth. The mantle had a pseudoparenchymatous structure and covered the root, including apices of dichotomously branched roots. The Paxillus–Pinus resinosa interaction had all the characteristics of a compatible ectomycorrhizal association. The Paxillus–Alnus glutinosa interaction, however, showed only aspects of superficial ectomycorrhizas, including the presence of a minimal (sometimes absent) and mostly proximal Hartig net and variable mantle development. Sclerotia were produced in the extraradical mycelium of Paxillus involutus when associated with either Alnus glutinosa or Pinus resinosa. Accepted: 22 October 1998     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

Russulaceae and Thelephoraceae form ectomycorrhizas with members of the Nyctaginaceae (Caryophyllales) in the tropical mountain rain forest of southern Ecuador

* Three members of the Nyctaginaceae, two Neea species and one Guapira species, occurred scattered within a very species-rich neotropical mountain rain forest. The three species were found to form ectomycorrhizas of very distinctive characters, while all other tree species examined formed arbuscular mycorrhizas. * The ectomycorrhizas were structurally typified according to light and transmission electron microscope investigations. The internal transcribed spacer (ITS) rDNA and part of the nuclear large subunit (LSU, 28S) rDNA of the mycorrhiza forming fungi were amplified and sequenced. Phylogenetic analyses were carried out. * Neea species 1 was found to form typical ectomycorrhizas with five different fungal species, Russula puiggarii, Lactarius sp., two Tomentella or Thelephora species, and one ascomycete. Neea species 2 and the Guapira species were associated with only one fungus each, a Tomentella/Thelephora species clustering closely together in an ITS-neighbour-joining tree. The long and fine rootlets of the Guapira species showed proximally a hyphal mantle and a Hartig net, but distally intracellular fungal colonization of the epidermis and root hair development. The ectomycorrhizal segments of the long roots of Neea species 2 displayed a hyphal mantle and a Hartig net around alive root-hair-like outgrowths of the epidermal cells. * The distribution and the evolution of ectomycorrhizas in the predominantly neotropic Nyctaginaceae are discussed.     

Aseptic ectomycorrhizal synthesis betweenAbies firma andCenococcum geophilum in artificial culture

A simple in vitro system is described for the synthesis ofAbies firma-Cenococcum geophilum ectomycorrhizas. SterilizedA. firma seedlings on both MMN and FH media were inoculated with hyphal discs from actively growing margins ofC. geophilum colonies. Typical ectomycorrhizas formed on seedlings on FH medium after 3 mo of incubation. By light microscopy, the synthesized mycorrhizas were seen to possess a thin mantle from which emanated extraradicle hyphae and highly branched, rarely septate intracortical Hartig net mycelium, characteristic ectomycorrhizal features. This is the first report of aseptic ectomycorrhization ofA. firma seedlings byC. geophilum. This model system will facilitate detailed studies on ectomycorrhizal development ofAbies species.     

Ectomycorrhizas of Cercocarpus ledifolius (Rosaceae)

? Premise of the study: Woody species in the Rosaceae form ectomycorrhizal associations, but the fungal symbionts are unknown. The species of fungi determine whether host plants are isolated from other ectomycorrhizal species in the plant community or linked with other trees through mycorrhizal networks. In this study we identified the fungi that form ectomycorrhizas with Cercocarpus ledifolius (curl-leaf mountain mahogany). ? Methods: Soil samples were collected under canopies of C. ledifolius. Ectomycorrhizas were described by morphology and by DNA sequences of the ITS region. Host species were confirmed by rbcL sequences. ? Key results: Sixteen species of fungi were identified from ectomycorrhizas of Cercocarpus ledifolius. The ectomycorrhizal community was distinguished by the presence of a Geopora species situated in the G. arenicola clade and by the absence of Rhizopogon, suilloids, and Sebacinales. Of the species on C. ledifolius, two also occurred on trees of Quercus garryana var. breweri and four on Arctostaphylos sp. ? Conclusions: The presence of fungal species in common with other ectomycorrhizal hosts shows that C. ledifolius, Q. garryana var. breweri, and Arctostaphylos species could be linked by a mycorrhizal network, allowing them to exchange nutrients or to share inoculum for seedling roots and new fine roots. Single-host fungi limited to C. ledifolius may improve resource acquisition and reduce competition with other ectomycorrhizal hosts. The finding of a Geopora species as a frequent mycobiont of C. ledifolius suggests that this fungus might be appropriate for inoculating seedlings for habitat restoration.     

Anatomical studies on ectomycorrhizas

Ectomycorrhizas of 5-month-old Pinus patula plants grown in autoclaved shola soil (forest soil) inoculated with basidiospores of Rhizopogon luteolus and Scleroderma citrinum are described. The main emphasis is on the organisation of the mantle tissue as seen in plan view and the features of associated hyphae and strands. The different layers in the mantle produced by the different fungi are described and illustrated.     

The influence of the ectomycorrhizal fungus Rhizopogon subareolatus on growth and nutrient element localisation in two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in response to manganese stress

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (-50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.     

Rhizopogon spore bank communities within and among California pine forests

In this study we examine the distribution of Rhizopogon species in spore banks from five California pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

The use of laser scanning confocal microscopy to characterize mycorrhizas of Pinus strobus L. and to localize associated bacteria

 Laser scanning confocal microscopy (LSCM), light microscopy (LM) and epifluorescence microscopy (FM) were used to observe the extramatrical hyphae, mantle patterns and associated bacteria on mycorrhizal tips of Pinus strobus L. seedlings grown in pot cultures. Laccaria sp. and Tuber sp. formed ectomycorrhizas with Pinus strobus, while Phialophora finlandia Wang & Wilcox and E-strain (sensu Danielson 1982) formed ectendomycorrhizas. Distinct mantle patterns and cystidia were observed with greater resolution using LSCM, and intracellular hyphae were visualized in three dimensions. Trypan blue penetrated fresh whole mounts to 20 μm and was an excellent stain for visualizing fungal hyphae and bacteria with LSCM. Fluorescein isothiocyanate and acridine orange were used in conjunction with LSCM and FM to localize bacteria on ectomycorrhizal tips. With LSCM, bacteria were visible in the surface mucigel, and optical sectioning through the root tip showed that bacteria were also present within the mantle. LSCM is a non-intrusive and fast method for visualizing mycorrhizal structures and their associated bacteria on fresh, whole root tips. Accepted: 17 July 1996     

Host islands within the California Northern Channel Islands create fine-scale genetic structure in two sympatric species of the symbiotic ectomycorrhizal fungus Rhizopogon

We have examined fine-scale genetic structure of the symbiotic ectomycorrhizal fungi Rhizopogon occidentalis and R. vulgaris on two of the California Channel Islands using five and six microsatellite loci, respectively. Both Rhizopogon species are sympatric on Santa Cruz and Santa Rosa Islands and are ectomycorrhizal with bishop pine (Pinus muricata) on both islands or Santa Rosa Island Torrey pine (P. torreyana ssp. insularis) on Santa Rosa. The combination of disjunct pine host distributions and geographic barriers within and among the islands have created highly structured Rhizopogon populations over very short distances (8.5 km on Santa Cruz Island; F(ST) = 0.258, F(ST) = 0.056, R. occidentalis and R. vulgaris, respectively). Both species show similar patterns of genetic differentiation as a result of limited dispersal between host populations as revealed by a significant isolation by distance relationship (r = 0.69, P < 0.04; r = 0.93, P < 0.001, R. occidentalis and R. vulgaris, respectively) and Bayesian clustering analyses, and is most likely a function of the small foraging range of the few mammals that disperse Rhizopogon on these islands and the enormous spore bank characteristic of Rhizopogon species.