Ian C. Anderson

Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community

ABSTRACT A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.

Spatial and temporal ecology of Scots pine ectomycorrhizas

Spatial analysis was used to explore the distribution of individual species in an ectomycorrhizal (ECM) fungal community to address: whether mycorrhizas of individual ECM fungal species were patchily distributed, and at what scale; and what the causes of this patchiness might be. Ectomycorrhizas were extracted from spatially explicit samples of the surface organic horizons of a pine plantation. The number of mycorrhizas of each ECM fungal species was recorded using morphotyping combined with internal transcribed spacer (ITS) sequencing. Semivariograms, kriging and cluster analyses were used to determine both the extent and scale of spatial autocorrelation in species abundances, potential interactions between species, and change over time. The mycorrhizas of some, but not all, ECM fungal species were patchily distributed and the size of patches differed between species. The relative abundance of individual ECM fungal species and the position of patches of ectomycorrhizas changed between years. Spatial and temporal analysis revealed a dynamic ECM fungal community with many interspecific interactions taking place, despite the homogeneity of the host community. The spatial pattern of mycorrhizas was influenced by the underlying distribution of fine roots, but local root density was in turn influenced by the presence of specific fungal species.

Use of molecular methods to estimate the size and distribution of mycelial individuals of the ectomycorrhizal basidiomycete Pisolithus tinctorius

A field study was conduced to determine the size and spatial distribution of mycelial individuals of Pisolithus tinctorius at a site in NSW, Australia. Following collection and mapping of carpophores and isolation of the fungi into axenic culture, genomic DNA was extracted and combined data from RAPD and microsatellite analyses used to identify and map mycelial individuals. Thirty-three genetically distinct individuals were recognized at the field site and, while one large individual (at least 30 m diam.) was identified, most individuals appeared relatively small ( P. tinctorius mycelia through soil.

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understorey vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co‐occurring C. vulgaris and Vaccinium myrtillus in the forest understorey. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understorey. These data demonstrate that forest understorey ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.

Changes in fungal community composition in response to vegetational succession during the natural regeneration of cutover peatlands

Despite the importance of peatlands as a major store of sequestered carbon and the role of fungi in releasing sequestered C, we know little about the community structure of fungi in peatlands. We investigated these across a gradient of naturally regenerating peatland vegetation using denaturing gradient gel electrophoresis (DGGE) and clone libraries of fragments of the fungal rRNA internal transcribed spacer (ITS) region. Significant changes in the fungal community structure of peat samples at different stages of regeneration were observed, which relate to the composition of the vegetation recolonizing these sites. Cloning and sequence analysis also demonstrated a potential shift in the relative abundance of the main fungal phyla. Some of the clones identified to genus level were highly related to fungi known to play a role in the degradation of plant litter or wood in similar ecosystems and/or form mycorrhizal associations. In addition, several fungal isolates highly related to peat clones were obtained, and their enzymic capacity to degrade structural plant tissues was assessed. Together, these results suggest that the fungal community composition of peat may be an important indicator of the status of regeneration and potential carbon sequestration of cutover peatlands.

Spatial analysis of ectomycorrhizal fungi reveals that root tip communities are structured by competitive interactions

Microbial ecology has made large advances over the last decade, mostly because of improvements in molecular analysis techniques that have enabled the detection and identification of progressively larger numbers of microbial species. However, determining the ecological patterns and processes taking place in communities of microbes remains a significant challenge. Are communities randomly assembled through dispersal and priority effects, or do species interact with each other leading to positive and negative associations? For mycorrhizal fungi, evidence is accumulating that stochastic and competitive interactions between species may both have a role in shaping community structure. Could the methodological approach, which is often incidence based, impact the outcomes detected? Here, we applied an incidence‐based Terminal Restriction Fragment Length Polymorphism (T‐RFLP) database approach to examine species diversity and ecological interactions within a community of ectomycorrhizal (ECM) fungi. Co‐occurrence analysis revealed that the ECM community colonizing root tips was strongly structured by competitive interactions, or ecological processes generating a similar spatial pattern, rather than neutral processes. Analysis of β‐diversity indicated that community structure was significantly more similar (spatially autocorrelated) at distances equal to or <3.41 m. The eight most frequently encountered species in the root tip community of ECM fungi displayed significant competitive interactions with at least one other species, showing that the incidence‐based approach was capable of detecting this sort of ecological information.

New insights into the mycorrhizal Rhizoscyphus ericae aggregate: spatial structure and co‐colonization of ectomycorrhizal and ericoid roots

• Fungi in the Rhizoscyphus ericae aggregate have been recovered from the roots of co-occurring ericaceous shrubs and ectomycorrhizal trees. However, to date, there is no evidence that the same individual genotypes colonize both hosts, and no information on the extent of the mycelial networks that might form. • Using spatially explicit core sampling, we isolated fungi from neighbouring Pinus sylvestris (ectomycorrhizal) and Vaccinium vitis-idaea (ericoid mycorrhizal) roots and applied intersimple sequence repeat (ISSR) typing to assess the occurrence and extent of shared genets. • Most isolates were identified as Meliniomyces variabilis, and isolates with identical ISSR profiles were obtained from neighbouring ericoid and ectomycorrhizal roots on a number of occasions. However, genet sizes were small (< 13  cm), and several genets were found in a single soil core. Genetic relatedness was independent of spatial separation at the scales investigated (< 43  m) and M. variabilis populations from sites 20  km apart were genetically indistinguishable. • We conclude that individual genets of M. variabilis can simultaneously colonize Scots pine and Vaccinium roots, but there is no evidence for the formation of large mycelial networks. Our data also suggest significant genotypic overlap between widely separated populations of this ubiquitous root-associated fungus.

Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas

The overstorey coniferous trees and understorey ericaceous dwarf shrubs of northern temperate and boreal forests have previously been considered to form mycorrhizas with taxonomically and functionally distinct groups of fungi. Here, we tested the hypothesis that Meliniomyces variabilis and Meliniomyces bicolor, isolated from Piceirhiza bicolorata ectomycorrhizas of pine, can function as ericoid mycorrhizal symbionts with Vaccinium vitis-idaea. We used split-compartment microcosms to measure the reciprocal exchange of (13)C and (15)N between V. vitis-idaea and three fungal isolates in the Hymenoscyphus ericae aggregate isolated from Scots pine ectomycorrhizas (M. variabilis and M. bicolor) or Vaccinium roots (M. variabilis). The extramatrical fungal mycelium of labelled mycorrhizal plants was significantly enriched in (13)C, and the leaves were significantly enriched in (15)N, compared with nonmycorrhizal and nonlabelled controls. * These findings show for the first time that fungi in the H. ericae aggregate, isolated from pine ectomycorrhizas, can transfer C and N and can thus form functional ericoid mycorrhizas in an understorey ericaceous shrub.

Intra- and interspecific variation in patterns of organic and inorganic nitrogen utilization by three Australian Pisolithus species

The ability of three Australian Pisolithus species, discriminated on the basis of ITS sequence data, to utilize a range of inorganic and organic nitrogen sources was assessed in liquid axenic culture. Both intra-, and putative interspecific, variation in nitrogen source utilization was observed. Most isolates demonstrated a preference for NH 4 + over NO 3 − , although some showed no significant preference for either inorganic source. All isolates utilized a range of amino acids. Species I isolates demonstrated a preference for acidic and/or neutral amino acids over basic acids, while species II and III isolates generally utilized amino acids poorly relative to species I. Although most isolates utilized BSA poorly, two species I isolates that had been maintained in axenic culture for > 10 y grew well on this substrate, suggesting possible changes in nitrogen utilization with extended storage in axenic culture.

Ecological understanding of root-infecting fungi using trait-based approaches

Classification schemes have been popular to tame the diversity of root-infecting fungi. However, the usefulness of these schemes is limited to descriptive purposes. We propose that a shift to a multidimensional trait-based approach to disentangle the saprotrophic-symbiotic continuum will provide a better framework to understand fungal evolutionary ecology. Trait information reflecting the separation of root-infecting fungi from free-living soil relatives will help to understand the evolutionary process of symbiosis, the role that species interactions play in maintaining their large diversity in soil and in planta, and their contributions at the ecosystem level. Methodological advances in several areas such as microscopy, plant immunology, and metatranscriptomics represent emerging opportunities to populate trait databases.