Contrasting below-ground views of an ectomycorrhizal fungal community

Ectomycorrhizal fungal communities have been characterized in a number of ways. Here we compare colonized root-tip and mycelia views of an ectomycorrhizal fungal community. Ectomycorrhizal fungi, both as mycelia and colonized root tips, were identified in soil samples taken from a pine plantation. We determined that for some ectomycorrhizal fungal species multiple root tips from a single soil sample were not independent. Therefore in the comparison of root-tip and mycelia views, we considered species to be present or absent from each soil sample irrespective of the number of root tips colonized by the species. We observed 39 ectomycorrhizal fungal species in total, but 12 were observed exclusively as mycelia and 11 exclusively colonizing root tips. The relative frequencies of 10 species occurring as both mycelia and root tips were not independent of the method of observation. Our results suggest that ectomycorrhizal fungal species differ in their spatial distributions on root tips, and that root-tip and mycelia views of the community are different.     

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.     

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.     

Determining the outcome of field-based competition between two Rhizopogon species using real-time PCR

Interest in the ecology of ectomycorrhizal (ECM) fungi has increased considerably, but little is known about interspecific interactions among ECM species. We examined competitive interactions between Rhizopogon occidentalis and R. salebrosus at Point Reyes National Seashore, California, USA. At three field sites, species abundances were compared in single- and two-species treatments on Pinus muricata seedlings inoculated with spores. Competition for root tips was assessed using real-time polymerase chain reaction (PCR) of internal transcribed spacer rDNA. In general, we found strong competitive exclusion of R. salebrosus by R. occidentalis, with >or= 75% of the seedlings in the two-species treatment colonized exclusively by R. occidentalis after 5 and 10 months. However, on the seedlings that were co-colonized, we observed no significant difference in the abundances of R. salebrosus and R. occidentalis, suggesting that once R. salebrosus was established, it was no longer competitively inferior. There were no significant differences in survival, growth, or percentage leaf nitrogen of seedlings colonized with either Rhizopogon species, but both growth and percentage leaf nitrogen were significantly higher for ECM than non-ECM seedlings. We also observed strong positive correlations between actual ECM root tip weight and that inferred from real-time PCR for both species, indicating that this method provided an accurate assessment of root tip occupation and hence ECM competitive dynamics. In conjunction with a previous experiment, our results indicate that competition between these two Rhizopogon species occurs similarly in both field and laboratory settings and that when colonizing from spore, timing largely determines the outcome of initial competitive interactions.     

Morphological and molecular analyses of ectomycorrhizal diversity in <Emphasis Type="Italic">Pinus densiflora</Emphasis> seedlings

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification) and molecular analysis (internal transcribed spacer region amplification and sequencing), we assessed diversity and composition of the ectomycorrhizal fungal community colonizing P. densiflora seedlings (from 1 to 5 years ) in a naturally regenerating area of a mature forest, in Dongning County of Hei Longjiang Province, China. A total of 22 ectomycorrhizal taxa were determined on the roots of 100 seedlings. The most frequent species was Cenococcum geophilum, which colonized 31.2% of the root tips and occurred in 93.8% of the all samples. It was followed by Clavulina cinerea, Thelephora sp.2, and Russula albidula. They colonized in 22.7%, 17.5%, 13.4% of the root tips and occurred in 63.4%, 42.6%, 29.3% of the samples respectively. These results suggested that ectomycorrhizal diversity increased in P. densiflora seedlings with increasing age.     

Ectomycorrhizal fungus communities of Quercus liaotungensis Koidz of different ages in a northern China temperate forest

Ectomycorrhizal (ECM) fungal communities of Quercus liaotungensis of different ages (seedlings, young trees and mature trees) in the growing seasons (June and September) between 2007 and 2009 were studied in a temperate forest of northern China. A total of 66 ECM fungal taxa were identified based on ECM morphotyping, PCR-RFLP, and DNA sequence data. Of these fungal taxa, 51 were Basidiomycetes (77.3%) and 15 were Ascomycetes (22.7%). Cenococcum geophilum was the dominant species. Thelephoraceae (16 taxa), Sebacinaceae (12 taxa) and Russulaceae (seven taxa) were the most species-rich and abundant ECM fungi, accounting for 19.5%, 17.6% and 8.3% of the total ECM root tips, respectively. Results of multiple response permutation procedure (MRPP) analysis indicated that there were marginally significant effects of tree ages (A?=?0.01801, P?=?0.054) and growing seasons (A?=?0.01908, P?=?0.064) on the ECM fungal species composition of Q. liaotungensis in a temperate forest.     

Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior alaska and correlation of different fungi with host growth responses

The role of common mycorrhizal networks (CMNs) in postfire boreal forest successional trajectories is unknown. We investigated this issue by sampling a 50-m by 40-m area of naturally regenerating black spruce (Picea mariana), trembling aspen (Populus tremuloides), and paper birch (Betula papyrifera) seedlings at various distances from alder (Alnus viridis subsp. crispa), a nitrogen-fixing shrub, 5 years after wildfire in an Alaskan interior boreal forest. Shoot biomasses and stem diameters of 4-year-old seedlings were recorded, and the fungal community associated with ectomycorrhizal (ECM) root tips from each seedling was profiled using molecular techniques. We found distinct assemblages of fungi associated with alder compared with those associated with the other tree species, making the formation of CMNs between them unlikely. However, among the spruce, aspen, and birch seedlings, there were many shared fungi (including members of the Pezoloma ericae [Hymenoscyphus ericae] species aggregate, Thelephora terrestris, and Russula spp.), raising the possibility that these regenerating seedlings may form interspecies CMNs. Distance between samples did not influence how similar ECM root tip-associated fungal communities were, and of the fungal groups identified, only one of them was more likely to be shared between seedlings that were closer together, suggesting that the majority of fungi surveyed did not have a clumped distribution across the small scale of this study. The presence of some fungal ribotypes was associated with larger or smaller seedlings, suggesting that these fungi may play a role in the promotion or inhibition of seedling growth. The fungal ribotypes associated with larger seedlings were different between spruce, aspen, and birch, suggesting differential impacts of some host-fungus combinations. One may speculate that wildfire-induced shifts in a given soil fungal community could result in variation in the growth response of different plant species after fire and a shift in regenerating vegetation.     

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.     

Assessment of ectomycorrhizal fungal communities in the natural habitats of Tuber magnatum (Ascomycota, Pezizales)

The ectomycorrhizal (ECM) fungal communities of four natural Tuber magnatum truffle grounds, located in different Italian regions (Abruzzo, Emilia-Romagna, Molise, and Tuscany), were studied. The main objective of this study was to characterize and compare the ECM fungal communities in the different regions and in productive (where T. magnatum ascomata were found) and nonproductive points. More than 8,000 (8,100) colonized root tips were counted in 73 soil cores, and 129 operational taxonomic units were identified using morphological and molecular methods. Although the composition of the ECM fungal communities studied varied, we were able to highlight some common characteristics. The most plentiful ECM fungal taxa belong to the Thelephoraceae and Sebacinaceae families followed by Inocybaceae and Russulaceae. Although several ectomycorrhizas belonging to Tuber genus were identified, no T. magnatum ectomycorrhizas were found. The putative ecological significance of some species is discussed.     

Structure and species composition of ectomycorrhizal fungal communities colonizing seedlings and adult trees of Pinus montezumae in Mexican neotropical forests

Mexico is a center of diversity for pines, but few studies have examined the ectomycorrhizal (ECM) fungal communities associated with pines in this country. We investigated the ECM communities associated with Pinus montezumae seedlings and mature trees in neotropical forests of central Mexico and compared their structure and species composition. Root tips were sampled on both planted seedlings and naturally occurring adult trees. A total of 42 ECM operational taxonomic units (OTUs) was found on P.?montezumae. Diversity and similarity indices showed that community structure was similar for both plant growth stages, but phylogenetic diversity and Chao-estimated richness were higher for seedlings. Species composition differed between communities. The dominant OTUs belonged to the families Atheliaceae, Cortinariaceae, and Sebacinaceae, although different taxa appeared to colonize seedlings and adults. Only 12 OTUs were shared between seedlings and adults, which suggests that ECM fungi which colonize seedlings are still not fully incorporated into mycelial networks and that ECM taxa colonizing young individuals of P.?montezumae are likely to come from fungal propagules. Intra-generic diversity could be an insurance mechanism to maintain forest productivity under stressed conditions. This is the first report describing the abundance of Atheliaceae in tree roots in neotropical ecosystems.     

Establishment of ectomycorrhizal fungal community on isolated Nothofagus cunninghamii seedlings regenerating on dead wood in Australian wet temperate forests: does fruit-body type matter?

Decaying wood provides an important habitat for animals and forms a seed bed for many shade-intolerant, small-seeded plants, particularly Nothofagus. Using morphotyping and rDNA sequence analysis, we compared the ectomycorrhizal fungal community of isolated N. cunninghamii seedlings regenerating in decayed wood against that of mature tree roots in the forest floor soil. The /cortinarius, /russula-lactarius, and /laccaria were the most species-rich and abundant lineages in forest floor soil in Australian sites at Yarra, Victoria and Warra, Tasmania. On root tips of seedlings in dead wood, a subset of the forest floor taxa were prevalent among them species of /laccaria, /tomentella-thelephora, and /descolea, but other forest floor dominants were rare. Statistical analyses suggested that the fungal community differs between forest floor soil and dead wood at the level of both species and phylogenetic lineage. The fungal species colonizing isolated seedlings on decayed wood in austral forests were taxonomically dissimilar to the species dominating in similar habitats in Europe. We conclude that formation of a resupinate fruit body type on the underside of decayed wood is not necessarily related to preferential root colonization in decayed wood. Rather, biogeographic factors as well as differential dispersal and competitive abilities of fungal taxa are likely to play a key role in structuring the ectomycorrhizal fungal community on isolated seedlings in decaying wood.     

Quercus rubra-associated ectomycorrhizal fungal communities of disturbed urban sites and mature forests

The presence and quality of the belowground mycorrhizal fungal community could greatly influence plant community structure and host species response. This study tests whether mycorrhizal fungal communities in areas highly impacted by anthropogenic disturbance and urbanization are less species rich or exhibit lower host root colonization rates when compared to those of less disturbed systems. Using a soil bioassay, we sampled the ectomycorrhizal fungal (EMF) communities associating with Quercus rubra (northern red oak) seedlings in soil collected from seven sites: two mature forest reference sites and five urban sites of varying levels of disturbance. Morphological and polymerase chain reaction–restriction fragment length polymorphism analyses of fungi colonizing root tips revealed that colonization rates and fungal species richness were significantly lower on root systems of seedlings grown in disturbed site soils. Analysis of similarity showed that EMF community composition was not significantly different among several urban site soils but did differ significantly between mature forest sites and all but one urban site. We identified a suite of fungal species that occurred across several urban sites. Lack of a diverse community of belowground mutualists could be a constraint on urban plant community development, especially of late-successional woodlands. Analysis of urban EMF communities can add to our understanding of urban plant community structure and should be addressed during ecological assessment before pragmatic decisions to restore habitats are framed.     

Post-fire seedlings of Nothofagus alpina in Southern Chile show strong dominance of a single ectomycorrhizal fungus and a vertical shift in root architecture

We investigated belowground responses of Nothofagus alpina seedlings to post-fire conditions during natural regeneration after a wildfire in Chile, focusing on mycorrhizal community and root architecture. The complete root systems of 2-year-old N. alpina seedlings were extracted from a post-fire site with natural regeneration and compared to roots of seedlings from undisturbed forest nearby. Mycorrhizal morphotype richness was determined in each seedling. Morphometric parameters of tertiary root structure and dry biomass of whole root systems were determined in 5 cm vertical intervals and in four lateral root classes. With 43.5% of colonized vital mycorrhizal root tips, the Basidiomycete Descolea antarctica was the most abundant fungal symbiont on post-fire seedlings. Tertiary root morphology of these seedlings was distinct from control plants and characterized by a deep-reaching tap root with rather evenly distributed lateral branches whereas seedlings from the undisturbed site had shallower root systems with most lateral roots concentrated in the upper soil layers. Post-fire seedlings had more mycorrhizal rootlets and mycorrhiza-bearing third order lateral roots than control plants which was expressed in a 34% higher total root number but only a 10% higher total root biomass, although both values were not statistically significant. A major part of these fine roots in seedlings from burnt forest was found in deeper soil horizons, compared to the seedlings from undisturbed forest. According to our results, post-fire conditions clearly favour Descolea antarctica as an early ectomycorrhizal colonizer of Nothofagus seedlings at the studied site. As no significant changes in soil chemistry could be observed at the burnt site, the deep-reaching tertiary root architecture of these seedlings may be interpreted as a response to other abiotic factors like reduced moisture in surface soil.     

Changes in ectomycorrhizal community structure on two containerized oak hosts across an experimental hydrologic gradient

Shifts in ectomycorrhizal (ECM) community structure were examined across an experimental hydrologic gradient on containerized seedlings of two oak species, Quercus montana and Quercus palustris, inoculated from a homogenate of roots from mature oak trees. At the end of one growing season, seedlings were harvested, roots were sorted by morphotype, and proportional colonization of each type was determined. DNA was subsequently extracted from individual root tips for polymerase chain reaction, restriction fragment length polymorphism, and rDNA sequencing of the ITS1/5.8S/ITS2 region to determine identities of fungal morphotypes. Twelve distinct molecular types were identified. Analysis of similarity showed that ECM fungal assemblages shifted significantly in composition across the soil moisture gradient. Taxa within the genus Tuber and the family Thelephoraceae were largely responsible for the changes in fungal assemblages. There were also significant differences in ECM community assemblages between the two oak host species. These results demonstrate that the structure of ECM fungal communities depends on both the abiotic and biotic environments and can shift with changes in soil moisture as well as host plant, even within the same genus.     

Distribution of lead in mycorrhizal and non-mycorrhizal Norway spruce seedlings

Non-mycorrhizal spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus (Scop.) Fr. or two strains of Paxillits involutus (Batsch) Fr. were grown in an axenic silica sand culture system with frequently renewed nutrient solution. After successful mycorrhizal colonization, the seedlings were exposed to 1 μM PbCI₂ for 19 weeks. The degree of infection in all of the mycorrhizal treatments approached 100% during the experiment and was not affected by exposure to Pb. However, the number of root tips per root dry weight and the shoot: root ratio, both in the non-mycorrhizal and the mycorrhizal seedlings, had decreased after the 19 week treatment with PbCl₂ Using X-ray microanalysis, the distribution and concentration of Pb in the tissues of mycorrhizal and non-mycorrhizal root tips were compared. In the mycorrhizae of seedlings exposed to Pb no significant accumulation of Pb in the hyphal mantle or in fungal cell walls of the Hartig net were detected. Lead accumulated primarily in the cortex cell walls both of non-mycorrhizal and mycorrhizal root tips. No significant difference of Pb concentrations in root cortex cell walls of non-mycorrhizal and mycorrhizal seedlings was found; except for seedlings colonized with Paxillus involutus strain 537. However, at the endodermis no effect of mycorrhizal fungal colonization on the Pb tissue concentration was detected. The presence of the fungal sheath did not prevent Pb from reaching the root cortex. The endodermis acted as a barrier to Pb radial transport in both mycorrhizal and non-mycorrhizal seedling roots.     

Spatial patterns of gene expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycorrhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil microcosms

Functional compartmentation of the extramatrical mycelium of ectomycorrhizal (ECM) fungi is considered important for the operation of ECM associations, although the molecular basis is poorly characterized. Global gene expression profiles of mycelium colonizing an ammonium sulphate ((NH4)2SO4) nutrient patch, rhizomorphs and ECM root tips of the Betula pendula-Paxillus involutus association were compared by cDNA microarray analysis. The expression profiles of rhizomorphs and nutrient patch mycelium were similar to each other but distinctly different from that of mycorrhizal tips. Statistical analyses revealed 337 of 1075 fungal genes differentially regulated among these three tissues. Clusters of genes exhibiting distinct expression patterns within specific tissues were identified. Genes implicated in the glutamine synthetase/glutamate synthase (GS/GOGAT) and urea cycles, and the provision of carbon skeletons for ammonium assimilation via beta-oxidation and the glyoxylate cycle, were highly expressed in rhizomorph and nutrient patch mycelium. Genes implicated in vesicular transport, cytoskeleton organization and morphogenesis and protein degradation were also differentially expressed. Differential expression of genes among the extramatrical mycelium and mycorrhizal tips indicates functional specialization of tissues forming ECM associations.     

Influence of inoculation with a Trichoderma bio‐inoculant on ectomycorrhizal colonisation of Pinus radiata seedlings

Trichoderma species, through mechanisms such as mycoparasitism, antibiosis, induced resistance and competition, are able to suppress the growth and activity of soil microbes and therefore have the potential to influence ectomycorrhizal (ECM) fungal colonisation. ECM colonisation potential of Pinus radiata seedlings inoculated with the commercially available Trichoderma species bio‐inoculant, ArborGuard?, was investigated in a commercial containerised nursery setting and in a separate glasshouse experiment. Application of ArborGuard? to P. radiata seedlings in both the containerised nursery and glasshouse experiment had no detrimental effect on the ability of the naturally occurring ECM fungi to colonise the seedlings. Thelephora terrestris was the only ECM species colonising the P. radiata root tips in the containerised system while Tomentella ellisii was the dominant species found colonising the P. radiata root tips in the glasshouse experiment. No seedling growth promotion was observed with the addition of ArborGuard?. The results show that the Trichoderma bio‐inoculant ArborGuard? does not affect ECM colonisation of P. radiata seedlings by Th. terrestris and T. ellisii in a containerised nursery system.     

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.     

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.