Thomas R. Horton

The molecular revolution in ectomycorrhizal ecology: peeking into the black‐box

Molecular tools have now been applied for the past 5 years to dissect ectomycorrhizal (EM) community structure, and they have propelled a resurgence in interest in the field. Results from these studies have revealed that: (i) EM communities are impressively diverse and are patchily distributed at a fine scale below ground; (ii) there is a poor correspondence between fungi that appear dominant as sporocarps vs. those that appear dominant on roots; (iii) members of Russulaceae, Thelephoraceae, and/or non‐thelephoroid resupinates are among the most abundant EM taxa in ecosystems sampled to date; (iv) dissimilar plants are associated with many of the same EM species when their roots intermingle — this occurs on a small enough spatial scale that fungal individuals are likely to be shared by dissimilar plants; and (v) mycoheterotrophic plants have highly specific fungal associations. Although, these results have been impressive, they have been tempered by sampling difficulties and limited by the taxonomic resolution of restriction fragment length polymorphism methods. Minor modifications of the sampling schemes, and more use of direct sequencing, has the potential to solve these problems. Use of additional methods, such as in situ hybridization to ribosomal RNA or hybridization coupled to microarrays, are necessary to open up the analysis of the mycelial component of community structure.

Lack of belowground mutualisms hinders Pinaceae invasions.

Why particular invasions succeed and others fail is not well understood. The role of soil biota has been proposed as important. However, the role of mutualists has received much less attention than that of pathogens. Here we report that lack of adequate ectomycorrhizal fungi hinders invasion by exotic Pinaceae on Isla Victoria, Argentina, by reducing both the probability of establishment and growth of invading individuals. More than one hundred exotic tree species were introduced to this island ca. 80 years ago, but invasive trees are found in high densities only in areas adjacent to plantations. With a series of greenhouse and field experiments we found lower mycorrhizal colonization levels and few fungal species far from original plantings, and key fungal mutualists are confined to areas near plantations, probably owing to dispersal limitations. Low inoculum levels far from the plantations are retarding the invasion. Our experiments indicate that positive interactions belowground can play a key but underappreciated role in invasion dynamics.

Detection of forest stand-level spatial structure in ectomycorrhizal fungal communities

Ectomycorrhizal fungal (EMF) communities are highly diverse at the stand level. To begin to understand what might lead to such diversity, and to improve sampling designs, we investigated the spatial structure of these communities. We used EMF community data from a number of studies carried out in seven mature and one recently fire-initiated forest stand. We applied various measures of spatial pattern to characterize distributions at EMF community and species levels: Mantel tests, Mantel correlograms, variance/mean and standardized variograms. Mantel tests indicated that in four of eight sites community similarity decreased with distance, whereas Mantel correlograms also found spatial autocorrelation in those four plus two additional sites. In all but one of these sites elevated similarity was evident only at relatively small spatial scales (< 2.6 m), whereas one exhibited a larger scale pattern ( approximately 25 m). Evenness of biomass distribution among cores varied widely among taxa. Standardized variograms indicated that most of the dominant taxa showed patchiness at a scale of less than 3 m, with a range from 0 to < or =17 m. These results have implications for both sampling scale and intensity to achieve maximum efficiency of community sampling. In the systems we examined, cores should be at least 3 m apart to achieve the greatest sampling efficiency for stand-level community analysis. In some cases even this spacing may result in reduced sampling efficiency arising from patterns of spatial autocorrelation. Interpretation of the causes and significance of these patterns requires information on the genetic identity of individuals in the communities.

Early effects of prescribed fire on the structure of the ectomycorrhizal fungus community in a Sierra Nevada ponderosa pine forest

The eects of a prescribed fire on the presence of ectomycorrhizal fungi on the root tips of ponderosa pine were investigated one year after a prescribed ground fire. Ectomycorrhizas were sampled within 1 m# plots before and one year after the fire, and in nearby control plots that were not burned. The cores were divided into litter}organic, upper mineral, and lower mineral layers. The total ectomycorrhizal biomass in the control plots did not dier between year one and year two samples for any core layer, while in the fire plots the destruction of the litter}organic layer resulted in an eight-fold reduction in total ectomycorrhizal biomass. Mycorrhizal biomass in the two mineral layers was not significantly reduced by the fire. We used molecular tools to identify fungi directly from the ectomycorrhizas. In unburned plots members of the Russulaceae and Thelephoraceae were among the most frequent and abundant ectomycorrhizal types; species of most other taxa were rare. In the control plots these two families were among the dominant species in both years, but patchiness on a fine spatio-temporal scale caused some major changes in ranking of individual species between sample years. Rhizopogon subcaerulescens was the most pronounced example; its biomass in the control plot samples was seven times greater in year two than in year one because of an exceptionally large cluster of mycorrhizas encountered in a single core. The eect of fire on individual species was dicult to assess because of this patchiness and because all species were low in abundance after the fire. The most abundant pre-fire species were reduced to undetectable post-fire levels, while several less abundant species, including R. subcaerulescens, Cenococcum geophilum, and several unknown types were not substantially reduced by the fire. We speculate that the more abundant species, Martellia sp., thelephoroid 1, and Tomentella sublilacina, were dierentially aected because their dominance was most prominent at the litter and organic layers. In any case, a short-term eect of this fire appears to be increased species evenness.

Ectomycorrhizal, vesicular-arbuscular and dark septate fungal colonization of bishop pine (Pinus muricata) seedlings in the first 5 months of growth after wildfire

Abstract We followed the colonization frequency of ectomycorrhizal (EM), vesicular-arbuscular mycorrhizal (VAM), and dark septate (DS) fungi in 1- to 5-month-old bishop pine seedlings reestablishing after a wildfire. Seedlings were collected on a monthly basis at either a VAM-dominated chaparral scrub site or an EM-dominated forest site, both of which were burned. In both vegetation types, fully developed EM were observed from the third month after germination. EM fungi observed on the seedlings from the scrub site were limited to Rhizopogon subcaerulescens, R. ochraceorubens and Suillus pungens. Seedlings from the forest were colonized by a greater variety of EM fungi including Amanita spp., Russula brevipes and a member of the Cantharellaceae. VAM structures (vesicles, arbuscules or hyphal coils) were observed in the seedling root systems beginning 1 month after germination at the scrub site and 3 months after germination at the forest site. Seedlings from the scrub site consistently had more frequent VAM fungal colonization than those from the forest site through the fifth month after germination. DS fungi were observed in most seedlings from both the scrub and forest sites beginning in the first month post-germination. We propose that these fungi survived as a resident inoculum in the soils and did not disperse into the sites after the fire.

Ectomycorrhizal fungi introduced with exotic pine plantations induce soil carbon depletion

Abstract Exotic pine plantations are promoted for their presumed capacity to provide a net sink of atmospheric C. Millions of hectares worldwide will be subjected to conversion into plantations during the next decades. However, pine introductions are known to result in a marked depletion of soil C, a phenomenon which has remained unexplained. We studied plantations in paramo grasslands of Ecuador, where the effect of the exotic introduction of radiata pines ( Pinus radiata ) and their accompanying ectomycorrhizal fungi can be studied in isolation from other ecosystem disturbances. We suggest that ectomycorrhizal fungi can extract C previously accumulated by paramo grasslands based on (a) a drastic simplification of the ectomycorrhizal community shown by direct DNA identification, (b) a loss of up to 30% soil C within 2 levels.

Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale

In most ectomycorrhizal (EM) community studies involving molecular identification methods there is a poor correspondence between fungi that appear dominant as sporocarps and those that appear dominant on EM roots and the species richness belowground is higher than that above ground. As a consequence, many fungi from root tip samples remain unidentified. In most studies, genetic data from multiple samples of an EM morphotype collected from various sampling locations are compared to genetic data from one to a few sporocarps of each species for identification purposes. The mismatch between above- and belowground species richness may be influenced by these different sampling efforts. To address this, intra-specific variation in the ITS region first investigated in Kårén et al. (1997) is revisited here, but at a spatial scale in which variation is expected to be low. Sporocarps were collected across a 7 km region of the Oregon Dunes National Recreation Area in western North America. ITS–RFLP data are presented for 3-18 sporocarps from each of 44 EM species in 18 genera. A total of 311 sporocarps were analyzed. Fifty-three ITS–RFLP types were observed. Of the 44 species, 38 (86% of total) yielded a single, species specific, RFLP type. No 2 species had the same RFLP type. Polymorphic ITS–RFLP types were observed in six species (14%). The following three species had two ITS-RFLP types with one type dominating: Inocybe lacera, Laccaria proxima, and Rhizopogon subcaerulescens. The following three species had three RFLP types with no type dominating: Laccaria laccata, Lactarius deliciosus, and Tricholoma flavovirens. A phylogenetic analysis of ITS sequences in Tricholoma revealed that two of the RFLP types in T. flavovirenswere apparently the result of intra-specific variation, while the third RFLP type was likely a cryptic species. All the other RFLP types observed in Tricholoma represented unique phylogenetic species. These results suggest that ITS–RFLP data from single samples (sporocarp or EM) are robust for characterizing most of the species at this scale. However, restriction endonucleases detect a limited amount of existing nucleotide variation and thus have limited value to detect cryptic species. Therefore, additional analyses of sequence data should be added to the RFLP matching technique to identify unknown RFLP types. These data also suggest that polymorphic RFLP types within species do not adequately explain the mismatch between above- and belowground views of EM species richness.

95% of basidiospores fall within 1 m of the cap: a field- and modeling-based study

Plant establishment patterns suggest that ectomycorrhizal fungal (EMF) inoculant is not found ubiquitously. The role of animal vectors dispersing viable EMF spores is well documented. Here we investigate the role of wind in basidiospore dispersal for six EMF species, Inocybe lacera, Laccaria laccata, Lactarius rufus, Suillus brevipes, Suillus tomentosus and Thelephora americana. Basidiospores adhered to microscope slides placed on three 60 cm transects radiating from sporocarps. Morphological characteristics of species as well as average basidiospore volume were recorded. Number of basidiospores was quantified at specific distances to produce actual dispersal gradients. We found a negative exponential decay model using characteristics for each species fit the field data well. The 95% modeled downwind dispersal distance of basidiospores was calculated for each species. The 95% modeled downwind dispersal distance increased with increasing cap height and decreasing basidiospore volume for the species sampled, with 95% of basidiospores predicted to fall within 58 cm of the cap. Differences in anatomical characteristics of EMF species influence how far basidiospores are dispersed by wind. We discuss the role of wind dispersal leading to patterns of EMF establishment during primary succession.

Pezizalean mycorrhizas and sporocarps in ponderosa pine (Pinus ponderosa) after prescribed fires in eastern Oregon, USA

Post-fire Pezizales fruit commonly in many forest types after fire. The objectives of this study were to determine which Pezizales appeared as sporocarps after a prescribed fire in the Blue Mountains of eastern Oregon, and whether species of Pezizales formed mycorrhizas on ponderosa pine, whether or not they were detected from sporocarps. Forty-two sporocarp collections in five genera (Anthracobia, Morchella, Peziza, Scutellinia, Tricharina) of post-fire Pezizales produced ten restriction fragment length polymorphism (RFLP) types. We found no root tips colonized by species of post-fire Pezizales fruiting at our site. However, 15% (6/39) of the RFLP types obtained from mycorrhizal roots within 32 soil cores were ascomycetes. Phylogenetic analyses of the 18S nuclear ribosomal DNA gene indicated that four of the six RFLP types clustered with two genera of the Pezizales, Wilcoxina and Geopora. Subsequent analyses indicated that two of these mycobionts were probably Wilcoxina rehmii, one Geopora cooperi, and one Geopora sp. The identities of two types were not successfully determined with PCR-based methods. Results contribute knowledge about the above- and below-ground ascomycete community in a ponderosa pine forest after a low intensity fire.

Douglas-fir ectomycorrhizae in 40- and 400-year-old stands: mycobiont availability to late successional western hemlock

We investigated ectomycorrhizal (EM) fungi in forest stands containing both early successional Douglas-fir and late successional western hemlock at two points in the typical stand development by identifying EM fungi from roots of Douglas-fir and western hemlock in mixed stands. In an early seral stage forest, EM roots of western hemlock seedlings and intermingling 40-year-old Douglas-fir were sampled. In a late seral stage forest, EM roots of trees of both species were sampled in a 400-year-old stand. We use molecular approaches to identify the symbionts from field samples in this descriptive study. In the early seral stage study, >95% of the western hemlock root tips by biomass were colonized by fungi also colonizing Douglas-fir roots. This result supports the prediction that western hemlock can associate with fungi in Douglas-fir EM networks. In the same study, fungi specific to Douglas-fir colonized 14% of its EM root tips. In the late seral stage study, 14% of the western hemlock root tips were colonized by fungi also observed in association with Douglas-fir, a result strongly influenced by sampling issues and likely represents a conservative estimate of multiple host fungi in this old growth setting. Fungi specific to Douglas-fir colonized 25% of its root tip biomass in the old growth study, in tight coralloid clusters within five of the 24 soil samples. The trends revealed in this study corroborate earlier studies suggesting a predominance of multiple host fungi in mixed communities of EM plants. The role of host-specific fungi in these stands remains unclear.