A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.     

Dynamics of mycorrhizae during development of riparian forests along an unregulated river

In this study, we explore two mycorrhizal groups during development of riparian soils along a freely-flowing river. We provide the first documentation of a shift in abundance between arbuscular mycorrhizae and ectomycorrhizae during floodplain succession. We used a chronosequence spanning 0–70 yr along a river in northwestern Montana, USA, to test the hypothesis that abundance of arbuscular mycorrhizal fungi (AMF) is greatest in early stages of soil development, and abundance of ectomycorrhizal fungi (ECMF) is greatest later in floodplain succession. We also measured the AMF-mediated process of formation of soil aggregates during site development. AMF colonization of the dominant tree (black cottonwood, Populus trichocarpa ) remained low (<5%), while AMF colonization of understory species was high (45–90%), across the chronosequence. Mycorrhizal inoculum potential (MIP) and hyphal length of AMF in soil peaked within the first 13 yr of succession and then declined. No single variable significantly correlated with AMF abundance, but AMF tended to decline as litter and soil organic matter increased. Density of ectomycorrhizal root tips in soil increased linearly throughout the chronosequence, and ectomycorrhizal colonization of cottonwood roots increased rapidly in early stages of succession. These patterns suggest that ECMF are not limited by dispersal, but rather influenced by abundance of host plants. Formation of water stable aggregates increased rapidly during the first third of the chronosequence, which was the period of greatest AMF abundance in the soil. The peak in AMF infectivity and hyphal length during early succession suggests that regular flooding and establishment of new sites promotes AMF abundance in this ecosystem. Regulation of rivers that eliminates creation of new sites may reduce contributions of AMF to riparian areas.     

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.     

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.     

Tolerance of ectomycorrhizal fungus mycelium to low temperature and freezing-thawing

Very little is known about the tolerance of ectomycorrhizal fungi to the freezing and repetitive freezing-thawing in northern coniferous forests. Isolates of Cortinarius multiformis , Russula densifolia , Suillus granulatus , and Lactarius deliciosus were exposed to a series of temperatures between +4 and -40 °C. The relative electrolyte leakage test indicated that the lethal temperature for 50% of samples was between -7.6 and -13.7 °C. Resume growth experiments showed that the 4 species of ectomycorrhizal fungi had a relatively high tolerance to the low temperatures, with L. deliciosus having the highest tolerance and C. multiformis the lowest. The repeated freezing-thawing delayed the growth of mycelium, which decreased with an increase in the number of freeze-thaw cycles.     

Molecular phylogenetic biodiversity assessment of arctic and boreal ectomycorrhizal Lactarius Pers. (Russulales; Basidiomycota) in Alaska, based on soil and sporocarp DNA

Despite the critical roles fungi play in the functioning of ecosystems, especially as symbionts of plants and recyclers of organic matter, their biodiversity is poorly known in high-latitude regions. In this paper, we discuss the molecular diversity of one of the most diverse and abundant groups of ectomycorrhizal fungi: the genus Lactarius Pers. We analysed internal transcribed spacer rDNA sequences from both curated sporocarp collections and soil polymerase chain reaction clone libraries sampled in the arctic tundra and boreal forests of Alaska. Our genetic diversity assessment, based on various phylogenetic methods and operational taxonomic unit (OTU) delimitations, suggests that the genus Lactarius is diverse in Alaska, with at least 43 putative phylogroups, and 24 and 38 distinct OTUs based on 95% and 97% internal transcribed spacer sequence similarity, respectively. Some OTUs were identified to known species, while others were novel, previously unsequenced groups. Non-asymptotic species accumulation curves, the disparity between observed and estimated richness, and the high number of singleton OTUs indicated that many Lactarius species remain to be found and identified in Alaska. Many Lactarius taxa show strong habitat preference to one of the three major vegetation types in the sampled regions (arctic tundra, black spruce forests, and mixed birch-aspen-white spruce forests), as supported by statistical tests of UniFrac distances and principal coordinates analyses (PCoA). Together, our data robustly demonstrate great diversity and nonrandom ecological partitioning in an important boreal ectomycorrhizal genus within a relatively small geographical region. The observed diversity of Lactarius was much higher in either type of boreal forest than in the arctic tundra, supporting the widely recognized pattern of decreasing species richness with increasing latitude.     

Plant growth and ectomycorrhiza formation by transplants on deglaciated land near Exit Glacier, Alaska

 Ectomycorrhiza (EM) formation on plant roots in successional communities may vary with plant species, plant age, and age of vegetation after disturbance. To evaluate differences in mycorrhizal fungus communities on roots of same-aged seedlings across a deglaciated chronosequence, indoor-grown bioassay plants of four dominant species Epilobium latifolium L. (dwarf fireweed), Populus balsamifera ssp. trichocarpa (Torr.&Gray) Hult. (black cottonwood), Alnus sinuata (Regel) Rydb. (Sitka alder), and Picea sitchensis (Bong.) Carr. (Sitka spruce) were transplanted into five successional stages (Barren, Isolated Plant, Patchy, Alder, and Cottonwood) following deglaciation near Exit Glacier, Alaska. The species were selected for their successional status and mycorrhiza formation potential [EM or arbuscular mycorrhiza (AM) or both]. Seedlings were transplanted in June 1992, and half were harvested at the ends of the two subsequent growing seasons. The EM communities on P. balsamifera differed across the chronosequence while those of the other species did not. Morphotype B dominated the EM on P. balsamifera after the Barren stage, and the greatest EM colonization was in the Isolated Plant and Patchy stages. No AM were found. The EM observed on even-aged seedlings in this study were a subset of the EM found on naturally occurring plants (seedlings to mature trees) in a prior study, and some were common to multiple plant species. Most plant growth responses were not significant across stages or were inconsistent among plant species. Accepted: 25 December 1998     

Mycorrhiza and root-associated fungi in Spitsbergen

Summary Roots of 76 plant species collected in West Spitsbergen (Svalbard), in the middle-northern Arctic zone, were examined for mycorrhiza and root-associated fungi. Dryas octopetala, Pedicularis dasyantha and Salix polaris were ectomycorrhizal and Cassiope tetragona and Empetrum hermaphroditum ericoid mycorrhizal. Pedicularis dasyantha was only slightly infected. Structures resembling vesicular-arbuscular mycorrhizal (VAM) fungi were not found in the roots, and soil samples screened for VAM fungi contained only one spore. Root endophytic fungi commonly occurred in Spits-bergen, but only Olpidium brassicae, Pleospora herbarum, Papulaspora, Microdochium bolleyi and Rhizoctonia solani were identified with reasonable certainty. A sterile endophytic dark-septate fungus (DSF) was in 39.5% of the flowering-plant species examined, especially in the Brassicaceae, Caryophyllaceae, Saxifragaceae and Poaceae. DSF were categorized into four slightly overlapping groups. Sterile endophytic hyaline septate fungi were rare. In the literature it is suggested that at least some of the DSF species or the hyaline septate fungi are functionally mutualistic rather than saprophytic or pathogenic. The literature on ectomycorrhizal fungi and plants in Spitsbergen is reviewed, including about 50 species, mainly of the genera Cortinarius, Hebeloma, Inocybe, Laccaria, Lactarius and Russula. These are symbiotic with the above-mentioned ectomycorrhizal plants. Four further ectomycorrhizal plants (Betula nana, Salix spp.) are very rare in the area.     

Contrasting primary successional trajectories of fungi and bacteria in retreating glacier soils

Early community assembly of soil microbial communities is essential for pedogenesis and development of organic legacies. We examined fungal and bacterial successions along a well‐established temperate glacier forefront chronosequence representing ~70 years of deglaciation to determine community assembly. As microbial communities may be heavily structured by establishing vegetation, we included nonvegetated soils as well as soils from underneath four plant species with differing mycorrhizal ecologies (Abies lasiocarpa, ectomycorrhizal; Luetkea pectinata, arbuscular mycorrhizal; Phyllodoce empetriformis, ericoid mycorrhizal; Saxifraga ferruginea, nonmycorrhizal). Our main objectives were to contrast fungal and bacterial successional dynamics and community assembly as well as to decouple the effects of plant establishment and time since deglaciation on microbial trajectories using high‐throughput sequencing. Our data indicate that distance from glacier terminus has large effects on biomass accumulation, community membership, and distribution for both fungi and bacteria. Surprisingly, presence of plants rather than their identity was more important in structuring bacterial communities along the chronosequence and played only a very minor role in structuring the fungal communities. Further, our analyses suggest that bacterial communities may converge during assembly supporting determinism, whereas fungal communities show no such patterns. Although fungal communities provided little evidence of convergence in community structure, many taxa were nonrandomly distributed across the glacier foreland; similar taxon‐level responses were observed in bacterial communities. Overall, our data highlight differing drivers for fungal and bacterial trajectories during early primary succession in recently deglaciated soils.     

BACKGROUND MATCHING AND COLOR-CHANGE PLASTICITY IN COLONIZING FRESHWATER SCULPIN POPULATIONS FOLLOWING RAPID DEGLACIATION

Anthropogenic-induced change is forcing organisms to shift their distributions and colonize novel habitats at an increasing rate, which leads to complex interactions among evolutionary processes. Coastrange sculpin ( Cottus aleuticus ) have colonized recently deglaciated streams of Glacier Bay in Alaska within the last 220 years. We examined divergence among populations in background matching coloration and tested the hypothesis that observed variation is due to morphological color plasticity. To examine how color-change plasticity has interacted with other evolutionary processes, we also determined the influence of colonization on neutral genetic diversity. We observed clinal variation in substrate-matching fish color along the chronological continuum of streams. Microsatellites provided little evidence of genetic subdivision among sculpin populations. Fish color was significantly correlated to substrate color, but was not correlated to neutral population genetic structure. Furthermore, a laboratory experiment revealed that morphological color plasticity could explain much, but not all, of the observed fish color divergence. Our study demonstrates that sculpin in Glacier Bay have colonized and adapted to recently deglaciated habitat and suggests that color change plasticity has aided in this process. This research, therefore, highlights the important role phenotypic plasticity may play in the adaptation of species to rapid climate change.     

Biogeochemical Stoichiometry Reveals P and N Limitation Across the Post-glacial Landscape of Denali National Park,Alaska

Global warming has accelerated glacial retreat in high-elevation and high-latitude ecosystems, exposing new terrain that can undergo predictable patterns of ecosystem succession, especially in coastal areas with relatively mild climates. However, little work has been done in harsher high-elevation and inland areas where the rate of plant and microbial succession may be greatly slowed by dryness and low temperatures. The present study is the first to address microbial succession at a major glacial foreland (the Middle Fork Toklat Glacier) in the interior of Alaska. We used a spatially nested sampling regime to reveal the landscape patterns in microbial activity and biogeochemical pools during the pre-plant stage of primary succession along this high-elevation and high-latitude chronosequence. Recently deglaciated soils (0–10 years) were colonized by a diverse microbial community that included many chemoautotrophs that likely subsist on high levels of un-weathered minerals (for example, pyrite) found at this site. Rates of N-fixation and extracellular enzyme activities were very low in the youngest soils sampled, but increased during the first 20 years of succession coinciding with a decrease in TOC and C:N levels. In older soils (20–54 years), TOC and TON increased and IN became undetectable perhaps indicating N limitation. Indicators of microbial activity stopped increasing 20 years post de-glaciation and remained at levels well below those seen at lower elevation and lower latitude sites, perhaps indicating severe nutrient limitations. Stoichiometric analyses also indicated phosphorus and nitrogen limitation across the entire chronosequence, with no indication of carbon limitation of microbial activity. These results indicate that nutrient limitation, rather than the constraints of a severe climate, may be the dominant factor slowing the rate of succession at high-latitude and high-altitude glacial forelands.     

Detoxification of ferulic acid by ectomycorrhizal fungi

The ectomycorrhizal fungi Laccaria amethystina and Lactarius deterrimus grown in liquid culture were used to study the fate of added ferulic acid. Laccaria amethystina degraded ferulic acid to the major metabolite vanillic acid. The intermediate vanillin was not detected. Lactarius deterrimus showed a completely different detoxification pattern. Two dimers and one trimer of ferulic acid could be identified as polymerization products of this fungus. A bioassay of the possible biological activities of ferulic acid and vanillic acid on these fungi revealed that vanillic acid was less toxic than ferulic acid for Laccaria amethystina but that both phenolic acids were toxic for Lactarius deterrimus. The results are discussed with respect to ectomycorrhizal fungal growth in the organic layer of forest soils and between living root cells of ectomycorrhizas.     

Ericaceous plant–fungus network in a harsh alpine–subalpine environment

In terrestrial ecosystems, plant species and diverse root‐associated fungi form complex networks of host–symbiont associations. Recent studies have revealed that structures of those below‐ground plant–fungus networks differ between arbuscular mycorrhizal and ectomycorrhizal symbioses. Nonetheless, we still remain ignorant of how ericaceous plant species, which dominate arctic and alpine tundra, constitute networks with their root‐associated fungi. Based on a high‐throughput DNA sequencing data set, we characterized the statistical properties of a network involving 16 ericaceous plant species and more than 500 fungal taxa in the alpine–subalpine region of Mt. Tateyama, central Japan. While all the 16 ericaceous species were associated mainly with fungi in the order Helotiales, they varied remarkably in association with fungi in other orders such as Sebacinales, Atheliales, Agaricales, Russulales and Thelephorales. The ericaceous plant–fungus network was characterized by high symbiont/host preferences. Moreover, the network had a characteristic structure called ‘anti‐nestedness’, which has been previously reported in ectomycorrhizal plant–fungus networks. The results lead to the hypothesis that ericaceous plants in harsh environments can host unexpectedly diverse root‐associated fungal taxa, constituting networks whose structures are similar to those of previously reported ectomycorrhizal networks but not to those of arbuscular mycorrhizal ones.     

Glacial loss and its effect on riparian vegetation of alpine streams

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Effects of repeated harvesting of forest residues on the ectomycorrhizal community in a Swedish spruce forest

The use of biofuels has been proposed as one possible substitute for fossil fuels, which contribute substantially to the increase in [CO₂] in the atmosphere. However, increased harvesting of forest residues for biofuel might affect the availability of base cations, P and N, as well as the development, community dynamics and function of ectomycorrhizas. This in turn might influence nutrient uptake and tree growth. In this study we investigated the effects of repeated forest residue harvesting on ectomycorrhizal species colonizing spruce roots in the humus layer of a 35-yr-old forest. Harvesting significantly decreased the thickness of the humus layer as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per unit humus volume. Changes in mycorrhizal community structure were studied by ITS typing with the use of PCR–RFLP analysis. In total, 19 different ITS types were found on two different sampling occasions (autumn and spring); 11 of these were common to both samplings. Nine of the ITS types were identified to at least the genus level by comparison with RFLP patterns of identified fruiting bodies or axenic cultures. Five species, Cortinarius sp. 2, Thelephora terrestris (Ehrenb.) Fr., Lactarius theiogalus (Bull.:Fr.) S. F. Gray s.st. Neuhoff, Tylospora fibrillosa Donk and Tö-96-12, occurred on over 5% of the total sampled root tips. Together these five types colonized 63% of the mycorrhizas screened. A similarity index assessment showed no shift in mycorrhizal community structure as a result of harvesting. Our findings suggest that the repeated removal of forest residues might have a strong effect on the quantity and development of ectomycorrhizal roots in the organic horizon, but little effect on the species composition of the community.     

Characterization of 'safe sites' for pioneers in primary succession on recently deglaciated terrain

1 We characterized safe sites for individuals of five early colonizers ( Abies lasiocarpa , Juncus drummondii , J. mertensianus , Saxifraga ferruginea , S. tolmiei ) that had survived at least one growing season on the recently deglaciated forefront of the Lyman Glacier in the North Cascade Mountains, Washington, USA.
2 Sites with concave surfaces, coarse surface substrate and in the vicinity of large rocks were more likely to be colonized by pioneering plant species.
3 We speculate that the distribution of plants is determined by the presence of sites that facilitate seed trapping and protect seeds and seedlings from desiccation.
4 The data identify the abiotic factors that determine initial recruitment and spatial distribution of plants. Such controls precede biotic interactions in this primary successional sere.     

中国南方地区松乳菇遗传多样性分析

松乳菇Lactarius deliciosus是一种对松树的生长具有重大意义的外生菌根真菌,具有很高的经济价值,因无法人工培育,过度的人工采摘造成产地生态的严重破坏。比较中国南方3个省9个不同地区的68个松乳菇的rDNA ITS区碱基序列的差异,以期研究各不同群体的ITS单倍体型多样性,不同地区群体之间的遗传关系,以及遗传分化与地理隔离的关系。遗传分析发现,得到的68个松乳菇样品的ITS序列可认定为7种单倍体型,这些单倍体型聚集为2个不同的系统进化群体,表现出两种不同的系统进化方向。7种单倍体型中,两个主     

The modified primary succession following sand mining: A validation of the use of chronosequence analysis

Changes in small mammal succession along a forested coastal dune system in the Myall Lakes-National Park, New South Wales, during 15 years of regeneration after sand mining, were determined by surveys in 1982 and 1987. The results clearly demonstrate a wave of succession moving along the mining path, and confirm that over the intervening 5 year period to 1987, small mammal succession has followed a path indicated by that of the 1982 chronosequence. The house mouse, Mus musculus quickly recolonizes disturbed areas, and peak abundance occurred on plots with a regeneration age of 3.7 years in 1982 and 3.4 years in 1987 (i.e. the 3.0–4.5 age class). Colonization by the New Holland mouse. Pseudomys novaehollandiae was first recorded on plots with 4.6 years regeneration in 1982 and 5.4 years in 1987. Peak Pseudomys numbers occurred on plots in the 7.5–9.0 age class in both 1982 and 1987. The common dunnart, Sminthopsis murina appears to be the third species to enter the succession. This modified primary succession closely resembles the mammalian secondary succession following fire where additional evidence suggests that the use of chronosequence analysis may also be appropriate.