The (oxalato)aluminate complex as an antimicrobial substance protecting the “shiro” of Tricholoma matsutake from soil micro-organisms

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, “matsutake” in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called “shiro” in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.     

Does ectomycorrhizal fungal community structure vary along a Japanese black pine (Pinus thunbergii) to black locust (Robinia pseudoacacia) gradient?

In this study we examined the role of the nitrogen-fixing tree, Robinia pseudoacacia (black locust), in ectomycorrhizal (ECM) formation and ECM community of Pinus thunbergii (Japanese black pine) seedlings. Two 200 m(2) experimental plots were established at the border between a Japanese black pine- and a black locust-dominated area in a coastal forest. The ECM fungal community of pine seedlings was examined by PCR-RFLP and sequence analysis. We analyzed the relationship between ECM formation, ECM community, growth, and nutrient status of pine seedlings and environmental conditions using the Mantel test and structural equation model. Percentages of ECM root tips, the number of ECM fungal species and ECM diversity on pine seedlings decreased in the black locust-dominated area. Cenococcum geophilum and Russula spp. were dominant in the Japanese black pine-dominated area, whereas Tomentella spp. were dominant in the black locust-dominated area. Nitrogen (N) concentration in soils or pine seedlings strongly influenced the percentage of ECM root tips, the number of ECM fungal species and ECM fungal similarity. These results imply the long-term eutrophication caused by N-fixing trees can change ECM formation and ECM community structure.     

A molecular survey of ectomycorrhizal hyphae in a California Quercus–Pinus woodland

Ectomycorrhizal (ECM) hyphal communities have not been well characterized. Furthermore, there have been few studies where the ECM hyphal community is compared to fungi detected as sporocarps or ECM-colonized root tips. We investigated fungi present as hyphae in a well-studied California Quercus–Pinus woodland. Hyphal species present were compared to those found as sporocarps and ECM root tips at the same site. Hyphae were extracted from root-restrictive nylon mesh in-growth bags buried in the soil near mature Quercus douglasii, Quercus wislizeni, and Pinus sabiniana. Taxa were identified using PCR, cloning, and DNA sequencing of internal transcribed spacer and 28s rDNA. Among the 33 species detected, rhizomorph-forming ECM fungi dominated the hyphal community, especially species of Thelephoraceae and Boletales. Most fungi in soils near Quercus spp. and P. sabiniana were ECM basidiomycetes, but we detected two ECM ascomycetes and three non-mycorrhizal fungi. Many ECM species present as hyphae were also previously detected at this site as sporocarps (18%) or on ECM root tips (58%). However, the hyphal community was mostly dominated by different taxa than either the sporocarp or ECM root communities.     

Genetic host-tree effects on the ectomycorrhizal community and root characteristics of Norway spruce

A greenhouse experiment was used to study the effects of host genotype on short root formation and ectomycorrhizal (ECM) fungal community structure in Norway spruce (Picea abies (L.) Karst.). Rooted cuttings representing 55 clones were inoculated with a mix of vegetative hyphae of five ECM fungal species (Laccaria sp., Amphinema byssoides, Piloderma sp., Cadophora finlandia, Paxillus involutus). After one growing season, the ECM fungal community structure was determined by amplifying the fungal internal transcribed spacer (ITS) of ribosomal DNA directly from ECM root tips. Restriction profiles of obtained amplicons were then compared to those of the inoculated strains. Spruce clones differed in their ECM fungal community composition; we found a statistically significant clone-specific effect on ECM fungal diversity and dominating fungal species. Nevertheless, the broad sense heritabilities of the levels of Laccaria sp., Piloderma sp. and A. byssoides colonisations as well as the ECM fungal community structure were low (H ²?=?0.04?0.11), owing to the high within-clone variation. As nitrogen concentration of needles correlated negatively with ECM fungal richness, our results imply that in the experimental conditions nutrient acquisition of young trees may benefit from colonisation with only one or two ECM fungal species. The heritability of short root density was moderate (H ²?=?0.41) and highest among all the measured shoot and root growth characteristics of Norway spruce cuttings. We suggest that the genetic component determining root growth and short root formation is significant for the performance of young trees in natural environments as these traits drive the formation of the below-ground symbiotic interactions.     

Ectomycorrhizal community structure varies among Norway spruce (Picea abies) clones

In northern boreal forests, the diversity of ectomycorrhizal (ECM) species is much greater than that of their host trees. This field study investigated the role of individual trees in shaping the ECM community. We compared ECM communities of eight Norway spruce (Picea abies) clones planted in a clear-cut area in 1994 with a randomized block design. In 2003, the ECM fungi were identified from randomly sampled root tips using denaturing gradient gel electrophoresis (DGGE) and rDNA internal transcribed spacer (ITS) sequence similarity. ECM diversity varied among clone groups, showing twofold growth differences. Moreover, according to detrended correspondence analysis (DCA), ECM community structure varied not only among but also within slow-growing or fast-growing clones. Results suggest that ECM diversity and community structure are related to the growth rate or size of the host. A direct or indirect influence of host genotype was also observed, and we therefore suggest that individual trees are partly responsible for the high diversity and patchy distribution of ECM communities in boreal forests.     

Genet Distribution of Ectomycorrhizal Fungus Suillus grevillei Populations in Two Larix kaempferi Stands over Two Years

Suillus grevillei in two Larix kaempferi stands was determined over two years by inter-simple sequence repeat (ISSR) polymorphism analysis using primers, (GTG)₅, (GCC)₅ and (GACA)₄. Thirty-five genets were identified from 67 sporocarps at the older stand (stand A in which the distribution of S. grevillei genet in 1997 was analyzed previously) in 1998, and 14 genets from 52 sporocarps at the younger stand (stand B) in 1997 and 1998. The characteristics of S. grevillei genets in stand A in 1998 were similar to those in 1997. A single genet was represented by 1.8 and 3.7 sporocarps on average in stands A and B, respectively. In stand A, 42 out of 61 genets, i.e., about 70% were represented by individual sporocarps compared to five out of 14 genets, i.e., about 35% in stand B. The largest and the average genet sizes was 6.8 m and less than 1 m in stand A, and 11 m and 2.3 m in stand B, respectively. A t-test showed the genet size in stand A to be significantly smaller than that in stand B. The above results indicate that the smaller genets of the S. grevillei population in stand A might be due to environmental conditions not genetic traits specific to this species. Observations over two years showed that although some genets formed sporocarps in both 1997 and 1998, many formed sporocarps only in one of the two years. Emerging positions of sporocarps in 1997 and 1998, which belonged to the same genet, were similar but not identical, about 2 m apart, suggesting mobility in the subterranean parts of ECM fungal genets. Received 10 April 2000/ Accepted in revised form 31 August 2000     

Ectomycorrhizal fungi of the Seychelles: diversity patterns and host shifts from the native Vateriopsis seychellarum (Dipterocarpaceae) and Intsia bijuga (Caesalpiniaceae) to the introduced Eucalyptus robusta (Myrtaceae), but not Pinus caribea (Pinaceae)

Ectomycorrhizal (ECM) fungi form highly diverse communities in temperate forests, but little is known about their community ecology in tropical ecosystems. Using anatomotyping and rDNA sequencing, ECM fungi were identified on root tips of the introduced Eucalyptus robusta and Pinus caribea as well as the endemic Vateriopsis seychellarum and indigenous Intsia bijuga in the Seychelles. Sequencing revealed 30 species of ECM fungi on root tips of V. seychellarum and I. bijuga, with three species overlapping. Eucalyptus robusta shared five of these taxa, whereas P. caribea hosted three unique species of ECM fungi that were likely cointroduced with containerized seedlings. The thelephoroid (including the anamorphic genus Riessiella), euagaric, boletoid and hymenochaetoid clades of basidiomycetes dominated the ECM fungal community of native trees. Two species of Annulatascaceae (Sordariales, Ascomycota) were identified and described as ECM symbionts of V. seychellarum. The low diversity of native ECM fungi is attributed to deforestation and long-term isolation of the Seychelles. Native ECM fungi associate with exotic eucalypts, whereas cointroduced ECM fungi persist in pine plantations for decades.     

Ectomycorrhizal fungal succession in mixed temperate forests

Ectomycorrhizal (ECM) fungal communities of Douglas-fir (Pseudotsuga menziesii) and paper birch (Betula papyrifera) were studied along a chronosequence of forest development after stand-replacing disturbance. Previous studies of ECM succession did not use molecular techniques for fungal identification or lacked replication, and none examined different host species. Four age classes of mixed forests were sampled: 5-, 26-, 65-, and 100-yr-old, including wildfire-origin stands from all four classes and stands of clearcut origin from the youngest two classes. Morphotyping and DNA sequences were used to identify fungi on ECM root tips. ECM fungal diversities were lower in 5-yr-old than in older stands on Douglas-fir, but were similar among age classes on paper birch. Host-specific fungi dominated in 5-yr-old stands, but host generalists were dominant in the oldest two age classes. ECM fungal community compositions were similar in 65- and 100-yr-old stands but differed among all other pairs of age classes. Within the age range studied, site-level ECM fungal diversity reached a plateau by the 26-yr-old age class, while community composition stabilized by the 65-yr-old class. Simple categories such as 'early stage', 'multi stage', and 'late stage' were insufficient to describe fungal species' successional patterns. Rather, ECM fungal succession may be best described in the context of stand development.     

Strong host preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed by DNA barcoding and taxon-specific primers

Ectomycorrhizal (ECM) symbiosis is a widespread plant nutrition strategy in Australia, especially in semiarid regions. This study aims to determine the diversity, community structure and host preference of ECM fungi in a Tasmanian wet sclerophyll forest. Ectomycorrhizal fungi were identified based on anatomotyping and rDNA internal transcribed spacer (ITS)-large subunit (LSU) sequence analysis using taxon-specific primers. Host tree roots were identified based on root morphology and length differences of the chloroplast trnL region. A total of 123 species of ECM fungi were recovered from root tips of Eucalyptus regnans (Myrtaceae), Pomaderris apetala (Rhamnaceae) and Nothofagus cunninghamii (Nothofagaceae). The frequency of two thirds of the most common ECM fungi from several lineages was significantly influenced by host species. The lineages of Cortinarius, Tomentella-Thelephora, Russula-Lactarius, Clavulina, Descolea and Laccaria prevailed in the total community and their species richness and relative abundance did not differ by host species. This study demonstrates that strongly host-preferring, though not directly specific, ECM fungi may dominate the below-ground community. Apart from the richness of Descolea, Tulasnella and Helotiales and the lack of Suillus-Rhizopogon and Amphinema-Tylospora, the ECM fungal diversity and phylogenetic community structure is similar to that in the Holarctic realm.     

EcM fungal community structure, but not diversity, altered in a Pb-contaminated shooting range in a boreal coniferous forest site in Southern Finland

Boreal forests contain diverse fungal communities that form essential ectomycorrhizal symbioses with trees. To determine the effects of lead (Pb) contamination on ectomycorrhizal fungal communities associated with the dominant pine (Pinus sylvestris L.), we surveyed sporocarps for 3 years, analyzed morphotyped ectomycorrhizal root tips by direct sequencing, and 454-sequenced fungal communities that grew into in-growth bags during a 2-year incubation at a shooting range where sectors vary in the Pb load. We recorded a total of 32 ectomycorrhizal fungi that formed conspicuous sporocarps, 27 ectomycorrhizal fungal phylotypes from 294 root tips, and 116 ectomycorrhizal fungal operation taxonomic unit (OTUs) from a total of 8194 internal transcribed spacer-2 454 sequences. Our ordination analyses by nonparametric multidimensional scaling (NMS) indicated that the Pb enrichment induced a shift in the ectomycorrhizal community composition. This was visible as indicative trends in the sporocarp and root tip data sets, but was explicitly clear in the communities observed in the in-growth bags. The compositional shift in the ectomycorrhizal community was mainly attributable to an increase in the frequencies of OTUs assigned to genus Thelephora and to a decrease in the OTUs assigned to Pseudotomentella, Suillus, and Tylospora in Pb-contaminated areas when compared with the control. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem function remain undetermined.     

Pine microsatellite markers allow roots and ectomycorrhizas to be linked to individual trees

Linking roots and ectomycorrhizas (EcM) to individual host trees in the field is required to test whether individual trees support different ectomycorrhizal communities. Here we describe a method that identifies the source of EcM roots by PCR of polymorphic pine nuclear microsatellite loci using fluorescently labelled primers and high-throughput fragment analysis. ITS-PCR can also be performed on the same EcM DNA extract for fungal identification. The method was tested on five neighbouring Scots pine (Pinus sylvestris var scotica) trees in native woodland. Successful host tree identification from DNA extracts of EcM root tips was achieved for 93% of all root fragments recovered from soil cores. It was estimated that each individual mature pine sampled was colonised by between 15 and 19 EcM fungi. The most abundant fungal species were found on all five trees, and within the constraints of the sampling scheme, no differences between trees in EcM fungal community structure or composition were detected.     

Spatial Distribution of the Subterranean Mycelia and Ectomycorrhizae of Suillus grevillei Genets

Suillus grevillei , and found that one, SG-5, is species-specific to S. grevillei and polymorphic. Using the SG-5 marker, we analyzed for the first time the horizontal and vertical distribution of the subterranean parts of several S. grevillei genets. The spatial distribution of S. grevillei genets in the soil demonstrated that the development of S. grevillei sporocarps is correlated with that of extra-radical mycelia and ectomycorrhizae of the same genet, which are distributed in a narrow area. However, sporocarps are not always centered over the subterranean parts and the amount of subterranean mycelia and mycorrhizae is not always correlated to the number of sporocarps formed on them. No S. grevillei mycelia and mycorrhizae were detected beneath the positions where S. grevillei sporocarps emerged in the previous year. The observation indicates the rapid alteration of the subterranean parts of S. grevillei genets, and suggests that S. grevillei genets change location rather than merely extend their habitat year after year. Received 13 October 2000/ Accepted in revised form 15 March 2001     

Diversity and composition of ectomycorrhizal community on seedling roots: the role of host preference and soil origin

As the main source of inocula, ectomycorrhizal (ECM) fungal propagules are critical for root colonization and seedling survival in deforested areas. It is essential to know factors that may affect the diversity and composition of ECM fungal community on roots of seedlings planted in deforest areas during reforestation. We quantitatively evaluated the effect of host plant and soil origin on ECM fungal propagule community structure established on roots of Castanopsis fargesii, Lithocarpus harlandii, Pinus armandii, and Pinus massoniana growing in soils from local natural forests and from sites deforested by clear-cut logging in the 1950s and 1960s. ECM root tips were sampled in April, July, and October of 2006, and ECM fungal communities were determined using ECM root morphotyping, internal transcribed spacer (ITS)-RFLP, and ITS sequencing. A total of 36 ECM fungal species were observed in our study, and species richness varied with host species and soil origin. Decreased colonization rates were found in all host species except for L. harlandii, and reduced species richness was found in all host species except for P. armandii in soil from the deforested site, which implied the great changes in ECM fungal community composition. Our results showed that 33.3% variance in ECM fungal community composition could be explained by host plant species and 4.6% by soil origin. Results of indicator species analysis demonstrated that 14 out of 19 common ECM fungal species showed significant preference to host plant species, suggesting that the host preference of ECM fungi was one of the most important mechanisms in structuring ECM fungal community. Accordingly, the host plant species should be taken into account in the reforestation of deforested areas due to the strong and commonly existed host preference of ECM fungi.     

Multiple-host fungi are the most frequent and abundant ectomycorrhizal types in a mixed stand of Douglas fir (Pseudotsuga menziesii) and bishop pine (Pinus muricata)

The ectomycorrhizal fungal associations of Douglas fir ( Pseudotsuga menziesii D. Don) and bishop pine ( Pinus muricata D. Don) were investigated in a mixed forest stand. We identified fungi directly from field-collected ectomycorrhizal (ECM) root tips using PCR-based methods. Sixteen species of fungi were found, of which twelve associated with both hosts. Rhizopogon parksii Smith was specific to Douglas fir. Three other species colonized only one of the hosts, but were too infrequent to draw conclusions about specificity. Seventy-four percent of the biomass of ECM root tips sampled in the stand were colonized by members of the Thelephoraceae and Russulaceae. All 12 species of fungi that associated with both tree species did so within a 10×40 cm soil volume, suggesting that individual fungal genotypes linked the putatively competing tree hosts.     

Long-term effect of apatite on ectomycorrhizal growth and community structure

Ectomycorrhizal (ECM) fungi are efficient at taking up phosphorus (P) from mineral sources, such as apatite, which are not easily available to the host trees. Since ECM fungal species differ in P uptake rates, it can be expected that the composition of the ECM fungal community will change upon exposure to apatite, provided that the P transfer is rewarded by more carbon being transferred to the fungal symbiont. Control and apatite-amended mesh bags were buried in pairs in the humus layer of a P-poor Norway spruce forest. The ECM fungal community that colonized these bags was analyzed by DNA extraction, PCR amplification of the internal transcribed spacer (ITS) region, cloning, and random sequencing. Fungal biomass was estimated by ergosterol analysis. No change in the ECM fungal community structure was seen after 5?years of apatite exposure, although the fungal biomass increased threefold upon apatite amendment. Our results indicate that host trees enhance carbon allocation to ECM fungi colonizing P sources in P-poor forests but the lack of change in the composition of the ECM fungal community suggests that P transfer rates were similar among the species. Alternatively, higher P transfer among certain species was not rewarded with higher carbon transfer from the host.     

Fungal sporocarps house diverse and host-specific communities of fungicolous fungi

Sporocarps (fruit bodies) are the sexual reproductive stage in the life cycle of many fungi. They are highly nutritious and consequently vulnerable to grazing by birds and small mammals, and invertebrates, and can be infected by microbial and fungal parasites and pathogens. The complexity of communities thriving inside sporocarps is largely unknown. In this study, we revealed the diversity, taxonomic composition and host preference of fungicolous fungi (i.e., fungi that feed on other fungi) in sporocarps. We carried out DNA metabarcoding of the ITS2 region from 176 sporocarps of 11 wood-decay fungal host species, all collected within a forest in northeast Finland. We assessed the influence of sporocarp traits, such as lifespan, morphology and size, on the fungicolous fungal community. The level of colonisation by fungicolous fungi, measured as the proportion of non-host ITS2 reads, varied between 2.8–39.8% across the 11 host species and was largely dominated by Ascomycota. Host species was the major determinant of the community composition and diversity of fungicolous fungi, suggesting that host adaptation is important for many fungicolous fungi. Furthermore, the alpha diversity was consistently higher in short-lived and resupinate sporocarps compared to long-lived and pileate ones, perhaps due to a more hostile environment for fungal growth in the latter too. The fungicolous fungi represented numerous lineages in the fungal tree of life, among which a significant portion was poorly represented with reference sequences in databases.Subject terms: Microbial ecology, Molecular evolution     

Similar taxonomic richness but different communities of ectomycorrhizas in native forests and non-native plantation forests

This investigation sought to examine if there was a difference between the ectomycorrhizal (ECM) communities in plots of native oak and introduced Scots pine and Sitka spruce forest. The ECM communities in four plots of each forest type were described, from five soil cores collected in each plot, by morphotyping, internal transcribed spacer (ITS)-restriction fragment length polymorphism matching of mycorrhizas and sporocarps and ITS sequencing. Fifty-one distinct taxa were distinguished; 25 were identified to species level, 11 to genus and 15 remained unidentified. Seventy-one ECM species were recorded as sporocarps from the forest plots; most (43 species) were found in the Sitka spruce plots. The below-ground ECM communities of the different forest types did not differ significantly with respect to species richness of taxa on roots, but differed in species composition. Multivariate analysis produced a clear separation of the communities of the different forest types using below-ground data, but the above-ground sporocarp data did not separate the forest types. Moreover, results of a Mantel test found no relationship between the above- and below-ground similarity matrices. The oak plots had the most distinctive ECM community, with Laccaria amethystina and Elaphomyces granulatus being frequent. The Sitka spruce plots showed the lowest intra-forest type similarity and were often dominated by "nursery type" ectomycorrhizas. There was only 10% similarity between the above- and below-ground ECM species in these plots, different colonisation methods of ectomycorrhizal taxa and insufficient below-ground sampling being possible reasons for this disparity. Our results indicate that plantations of non-native Sitka spruce can support similar levels of ECM diversity as native forests.     

Ectomycorrhizal community structure in a xeric Quercus woodland based on rDNA sequence analysis of sporocarps and pooled roots

Quercus woodlands are key components of California's wild landscapes, yet little is known about ectomycorrhizal (EM) fungi in these ecosystems. We examined the EM community associated with Quercus douglasii using sporocarp surveys and by pooling EM roots and subjecting them to DNA extraction, polymerase chain reaction (PCR), cloning, restriction fragment length polymorphism (RFLP) screening and DNA sequencing. Ectomycorrhizal root symbionts were sampled four times in 2003-04. During this time, the below-ground community structure was relatively stable; we found no evidence of taxa adapted to winter or spring conditions and only one species varied widely in occurrence between years. The EM community from sporocarps and roots was diverse (161 species), rich in Ascomycota (46 species), and dominated by fungi with cryptic sporocarps. This included a large number of resupinate and hypogeous taxa, many of which were detected both above- and below-ground.     

Population genetic structure of an ectomycorrhizal fungus Amanita manginiana in a subtropical forest over two years

The population genetic structure of the late-stage fungus Amanita manginiana in a natural forest in Dujiangyan, southwest China was examined over two years using inter-simple sequence repeat (ISSR) markers. Seven ISSR primers were used and 170 bands were obtained in this population: 134/160 and 135/153 bands were polymorphic for sporocarps of 2001 and 2002, respectively. Each sporocarp represented a single genet in 2001 and 2002, and no identical genets were found between the two years. The results of genetic similarity comparison, using unweighted pair group method with arithmetic means, and analysis of molecular variance, indicated that although genetic variances were mainly within individuals of the same year the genetic variance between years was statistically significant (P<0.001). Relationships between genetic similarity and spatial distance of pairwise sporocarps were also found to be different in the two years. The differences in genetic structure and genetic similarity between individuals of the two years implied that the sporocarps were not likely to be derived from continuous generations, i.e., the sporocarps collected in 2002 were not developed from sexual spores dispersed by sporocarps of 2001. We suggest that the life-cycle traits of ectomycorrhizal (ECM) fungi should be considered in genetic studies on ECM fungal populations.     

Interactions between extraradical ectomycorrhizal mycelia, microbes associated with the mycelia and growth rate of Norway spruce (Picea abies) clones

Despite their ecological relevance, field studies of the extraradical mycelia of ectomycorrhizal (ECM) fungi are rare. Here we examined in situ interactions between ECM mycelia and host vigour. Ectomycorrhizal mycelia were harvested with in-growth mesh bags buried under Norway spruce (Picea abies) clones planted in 1994 in a randomized block design. Mycelial biomass was determined and fungal species were identified by denaturing gradient gel electrophoresis (DGGE) and sequencing of the internal transcribed spacer 1 (ITS1) region. Microbial community structure in the mycelium was investigated by phospholipid fatty acid (PLFA) profiling. Compared to slow-growing spruce clones, fast-growing clones tended to support denser mycelia where the relative proportions of Atheliaceae fungi and PLFAs indicative of Gram-positive bacteria were higher. Ascomycetes and PLFAs representative of Gram-negative bacteria were more common with slow-growing clones. In general, the ECM mycelial community was similar to the ECM root-tip community. Growth rate of the hosts, the ECM mycelial community and the microbes associated with the mycelium were related, suggesting multitrophic interactions between trees, fungi and bacteria.