The importance of individuals: intraspecific diversity of mycorrhizal plants and fungi in ecosystems.

A key component of biodiversity is the number and abundance of individuals (i.e. genotypes), and yet such intraspecific diversity is rarely considered when investigating the effects of biodiversity of mycorrhizal plants and fungi on ecosystem processes. Within a species, individuals vary considerably in important reproductive and functional attributes, including carbon fixation, mycelial growth and nutrient utilization, but this is driven by both genetic and environmental (including climatic) factors. The interactions between individual plants and mycorrhizal fungi can have important consequences for the maintenance of biodiversity and regulation of resource transfers in ecosystems. There is also emerging evidence that assemblages of genotypes may affect ecosystem processes to a similar extent as assemblages of species. The application of whole-genome sequencing and population genomics to mycorrhizal plants and fungi will be crucial to determine the extent to which individual variation in key functional attributes is genetically based. We argue the need to unravel the importance of the diversity (especially assemblages of different evenness and richness) of individuals of both mycorrhizal plants and fungi, and the need to take a 'community genetics' approach to better understand the functional significance of the biodiversity of mycorrhizal symbioses.

Genetic population structure of the ectomycorrhizal fungus Pisolithus microcarpus suggests high gene flow in south-eastern Australia.

Pisolithus are ectomycorrhizal fungi that associate with roots of numerous plant species in natural and plantation forests worldwide. Despite the fact that Pisolithus spp. are present in plantation forests in many countries, knowledge of the genetic population structure of Pisolithus spp. remains limited. In this study, we have tested the hypothesis that a propensity for long-distance spore dispersal in Pisolithus microcarpus, along with the widespread distribution of potential eucalypt and acacia plant hosts in south-eastern Australia facilitates gene flow that limits population differentiation. Five polymorphic simple sequence repeat markers were used to investigate the population structure of P. microcarpus. Isolates were grouped according to geographical origin and isolate genotypes were analysed among the geographical populations. Pairwise F (ST) estimates indicated limited genetic differentiation among the geographical populations. Analysis of molecular variance revealed that most of the genetic variation present was within geographical populations, with only 1.3% of the genetic variation among P. microcarpus geographical populations. This was particularly pronounced for four geographical populations within a ca 7,000 km(2) area New South Wales, which were each separated by < 100 km and appeared to be genetically homogeneous. The lack of population structure is suggested to be due to a high degree of gene flow, via basidiospores, between the New South Wales geographical populations.

Ectomycorrhizal fungi: exploring the mycelial frontier.

Ectomycorrhizal (ECM) fungi form mutualistic symbioses with many tree species and are regarded as key organisms in nutrient and carbon cycles in forest ecosystems. Our appreciation of their roles in these processes is hampered by a lack of understanding of their soil-borne mycelial systems. These mycelia represent the vegetative thalli of ECM fungi that link carbon-yielding tree roots with soil nutrients, yet we remain largely ignorant of their distribution, dynamics and activities in forest soils. In this review we consider information derived from investigations of fruiting bodies, ECM root tips and laboratory-based microcosm studies, and conclude that these provide only limited insights into soil-borne ECM mycelial communities. Recent advances in understanding soil-borne mycelia of ECM fungi have arisen from the combined use of molecular technologies and novel field experimentation. These approaches have the potential to provide unprecedented insights into the functioning of ECM mycelia at the ecosystem level, particularly in the context of land-use changes and global climate change.

Bacteria associated with ectomycorrhizas of slash pine (Pinus elliottii) in south-eastern Queensland, Australia.

Bacterial communities associated with ectomycorrhizal and uncolonized roots of Pinus elliottii (slash pine) collected from a plantation in south-east Queensland, Australia, were investigated, using cultivation-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene PCR products obtained using a cultivation-independent approach revealed that bacterial communities associated with ectomycorrhizal root tips differed significantly from those associated with roots uncolonized by ectomycorrhizal fungi. DGGE analysis of cultivable bacterial communities revealed no significant difference between ectomycorrhizal and uncolonized roots. Neither analytical approach revealed significant differences between the bacterial communities associated with ectomycorrhizal roots colonized by a Suillus sp. or an Atheliaceae taxon. Cloned bacterial 16S rRNA genes revealed sequence types closely related with that of Burkholderia phenazinium, common in both ectomycorrhizal-colonized and -uncolonized roots, while sequence types most similar to the potentially phyopathogenic bacteria Burkholderia andropogonis and Pantoea ananatis were only detected in ectomycorrhizal roots. These results highlight the possibility of global movement of microorganisms, including putative pathogens, as a result of the introduction of exotic pine plantations.

Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest.

Fungi were isolated from the roots of 17 plant species from the families Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae-Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae and Stylidiaceae at a sclerophyll forest site in New South Wales, Australia. Internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) and sequence comparisons indicated that the isolated fungi had affinities to a range of ascomycetes, basidiomycetes and zygomycetes. Four RFLP types had closest affinities to previously identified Helotiales ericoid mycorrhizal (ERM) or Oidiodendron spp. Isolates representing six RFLP types, which were variously isolated from all 17 plant species, formed ERM coils in hair root epidermal cells of Woollsia pungens (Ericaceae) under gnotobiotic conditions. Three of these isolates formed intercellular hyphae, intracellular hyphae and/or microsclerotia, which are typical of dark septate endophyte infection, in roots of Stylidium productum (Stylidiaceae), indicating an ability to form different types of association with roots of different hosts. Overall the data indicate that a broad range of plant taxa may act as repositories for ERM fungi in sclerophyll forest soil.

Biology of mycorrhizal associations of epacrids (Ericaceae).

Epacrids, a group of southern hemisphere plants formerly considered members of the separate family Epacridaceae, are in fact most closely allied to the Vaccinioid tribe (Ericaceae). Epacrids and other extant ericoid mycorrhiza-forming plants appear to have a monophyletic origin. In common with many Ericaceae they form ericoid mycorrhizas. ITS sequence data indicate that the fungi forming ericoid mycorrhizas with epacrids and other extant Ericaceae are broadly similar, belonging to a poorly defined group of ascomycetes with phylogenetic affinities to Helotiales. The basic development and structure of ericoid mycorrhizal infections in epacrids is similar to other Ericaceae. However, data are limited on the structure and physiology of both hair roots and ericoid mycorrhizas for all Ericaceae. Relatively little is known about the functional significance of ericoid mycorrhizas in epacrids in southern hemisphere habitats that are often poor in organic matter accumulation. However the abilities of fungal endophytes of epacrids to utilize organic N and P substrates equal those of endophytes from northern hemisphere heathland plant hosts. Investigations using 15 N/13 C-labelled organic N substrates suggest that mycorrhizal endophytes are important, at least, to the N nutrition of their epacrid hosts in some habitats. Contents Summary 305 I. Epacrid plant hosts 306 II. Evolution of ericoid mycorrhizas in epacrids 306 III. Epacrid hair roots and their mycorrhizal associations 307 IV. Seasonality and incidence of mycorrhizal infection 310 V. Structure and development of mycorrhizal associations 311 VI. Nature of the mycorrhizal fungal endophytes 315 VII. Community and population biology of mycorrhizal endophytes 318 VIII. Functional aspects of mycorrhizas in epacrids 319 IX. Conclusions 322 Acknowledgements 322 References 322.

Ectomycorrhizal fungal communities in young forest stands regenerating after clearcut logging.

The effects on the ectomycorrhizal fungal community of clearcut logging, which is used to harvest millions of hectares of ectomycorrhizal forest annually, has been studied for a number of years. Here, we review current knowledge of inoculum sources for ectomycorrhizal fungi in forests and then re-examine earlier studies of ectomycorrhizas on young trees in regenerating stands. We conclude that, taken separately from the effects of site preparation, the major impact of clearcut logging is to change the species composition of the ectomycorrhizal fungal community rather than to reduce the percentage of roots colonized. A thorough examination of site preparation treatments suggests that the changes in fungal species composition are driven by changes in the biology and chemistry of the soil environment after clearcutting as much as they are by loss or change in fungal inoculum. This is an important conclusion because it implies that these new ectomycorrhizal fungal communities are better adapted to the new conditions than the ones in the forest would have been. The shift in fungal species composition and diversity will have implications for seedling establishment and competition. The effects of individual fungi or diverse assemblages of fungi on seedling growth, and effects of changes in the ability of young trees to associate with a common mycelium are discussed. Contents Summary 399 I. Introduction 400 II. Population biology and inoculum potential of ectomycorrhizal fungi 401 III. Ectomycorrhiza development on seedlings regenerating after clearcut logging 402 IV. Which is the most important factor driving changes in the ECM fungal community after clearcut logging: inoculum loss or change in the below-ground environment? 406 V. Possible consequences for regenerating stands of species shifts in ectomycorrhizal fungi 414 VI. Conclusions 416.

Identification of laccase-like genes in ectomycorrhizal basidiomycetes and transcriptional regulation by nitrogen in Piloderma byssinum.

• Ectomycorrhizal (ECM) fungi were screened for laccase-like genes by polymerase chain reaction (PCR) using primers for white rot fungal laccase genes, and expression of the genes was examined by reverse transcriptase polymerase chain reaction (RT-PCR) for Piloderma byssinum in axenic culture under different nutrient conditions. • Laccase-like genes were present in Rhizopogon roseolus along with several Russulales and Atheliaceae taxa, and showed strong nucleotide sequence similarity to laccase genes in white rot fungi. Multiple laccase-like genes were only identified in Piloderma spp. • Laccase-like genes were expressed in Piloderma spp., with transcript levels some six times higher under high nitrogen conditions in P. byssinum than when nitrogen availability was lower. • The potential roles of laccases in nutrient mobilization and/or differentiation of multihyphal ECM fungal structures are discussed.

Toxic interactions of metal ions (Cd2+ , Pb2+ , Zn2+ and Sb3- ) on in vitro biomass production of ectomycorrhizal fungi.

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd2+ , Pb+2 , Zn2+ and Sb3- , measured as an inhibition of fungal biomass production, Isolates were grown in liquid media amended with the metals, Individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd2+ and Zn2+ was recorded, while Pb2+ and Sb3- individually had little effect. The presence of Pb2+ and Sb3- in the media did however, amcliorate Cd2+ and Zn2+ toxicity in some circumstances. Interactions between Cd+2 and Zn+2 were investigated further over a range of concentrations. Zn2+ was found to significantly ameliorate the toxicity of Cd2+ to three of the four isolates tested. The influence of Zn2+ varied between ECM species and with the concentrations of metals tested.

Identification of genes for lignin peroxidases and manganese peroxidases in ectomycorrhizal fungi.

• Genes for ligninolytic enzymes, normally associated with white-rot fungi, are shown to be widespread in a broad taxonomic range of ectomycorrhizal (ECM) fungi. • ECM fungi were screened for lignin peroxidase (LiP) and manganese peroxidase (MnP) genes by PCR using primers specific for known isozymes in the white-rot fungus Phanerochaete chrysosporium, with DNA sequencing used to confirm the identity of the amplified fragments. • Genes for LiPs were detected in ECM fungi representing the orders Agaricales, Aphyllophorales, Boletales, Cantharellales, Hymenochaetales, Sclerodermatales, Stereales and Thelephorales. MnP genes were detected in only Cortinarius rotundisporus and three ECM Stereales taxa. • The presence of genes for decomposer activities supports putative evolutionary relationships between ECM and saprotrophic fungi. Expression of the lignolytic genes may facilitate ECM fungal access to nutrients associated with dead plant material in soil and potentially a supplementary carbon supply. Strict functional boundaries between ECM and decomposer fungi may be less clear-cut than previously thought.

Interactive effects of preindustrial, current and future atmospheric CO2 concentrations and temperature on soil fungi associated with two Eucalyptus species.

Soil microbial processes have a central role in global fluxes of the key biogenic greenhouse gases and are likely to respond rapidly to climate change. Whether climate change effects on microbial processes lead to a positive or negative feedback for terrestrial ecosystem resilience is unclear. In this study, we investigated the interactive effects of [CO(2)] and temperature on soil fungi associated with faster-growing Eucalyptus saligna and slower-growing Eucalyptus sideroxylon, and fungi that colonised hyphal in-growth bags. Plants were grown in native soil under controlled soil moisture conditions, while subjecting the above-ground compartment to defined atmospheric conditions differing in CO(2) concentrations (290, 400, 650 µL L(-1)) and temperature (26 and 30 °C). Terminal restriction fragment length polymorphism and sequencing methods were used to examine effects on the structure of the soil fungal communities. There was no significant effect of host plant or [CO(2)]/temperature treatment on fungal species richness (α diversity); however, there was a significant effect on soil fungal community composition (ß diversity) which was strongly influenced by eucalypt species. Interestingly, ß diversity of soil fungi associated with both eucalypt species was significantly influenced by the elevated [CO(2) ]/high temperature treatment, suggesting that the combination of future predicted levels of atmospheric [CO(2)] and projected increases in global temperature will significantly alter soil fungal community composition in eucalypt forest ecosystems, independent of eucalypt species composition. These changes may arise through direct effects of changes in [CO(2)] and temperature on soil fungi or through indirect effects, which is likely the case in this study given the plant-dependent nature of our observations. This study highlights the role of plant species in moderating below-ground responses to future predicted changes to [CO(2)] and temperature and the importance of considering integrated plant-soil system responses.

Identical genotypes of an ericoid mycorrhiza-forming fungus occur in roots of Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) in an Australian sclerophyll forest.

Assemblages of fungi associated with roots of cooccurring Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) seedlings at a sclerophyll forest site in New South Wales, Australia, were investigated by direct DNA extraction and analysis of rRNA gene internal transcribed spacer (ITS) products by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses. While ordination of the DGGE data suggested that the assemblages did not differ significantly between the two plant taxa, T-RFLP data provided marginal statistical support for the presence of different assemblages. Fungi isolated from roots of both plants were identified by ITS sequence comparisons largely as ascomycetes, several of which had close sequence identity to Helotiales ericoid mycorrhizal (ERM) fungi. One isolate morphotype from E. pulchella had close sequence similarity to ectomycorrhizal fungi in the Cenococcum geophilum complex, and neighbour-joining analysis grouped this strongly with other Australian C. geophilum-like sequences. Distribution of genotypes of an ERM Helotiales ascomycete in root systems of the two plant taxa was also investigated using inter-simple sequence repeat (ISSR)-PCR. Nineteen ISSR genotypes were identified, two of which were present in roots of both plant taxa. The results are discussed in the context of potential mycelial connections between Ericaceae and non-Ericaceae plants.

Long term repeated prescribed burning increases evenness in the basidiomycete laccase gene pool in forest soils.

Repeated prescribed burning alters the biologically labile fraction of nutrients and carbon of soil organic matter (SOM). Using a long-term (30 years) repeated burning experiment where burning has been carried out at a 2- or 4-year frequency, we analysed the effect of prescribed burning on gross potential C turnover rates and phenol oxidase activity in relation to shifts in SOM composition as observed using Fourier-transform infrared spectroscopy. In tandem, we assessed the genetic diversity of basidiomycete laccases. While the overall effect of burning was a decline in phenol oxidase activity, Shannon diversity and evenness of laccases was significantly higher in burned sites. Co-correspondence analysis of SOM composition and laccase operational taxonomic unit frequency data also suggested a strong correlation. While this correlation could indicate that the observed increase in laccase genetic diversity due to burning is due to increased resource diversity, a temporal replacement of the most abundant members of the assembly by an otherwise dormant pool of fungi cannot be excluded. As such, our results fit the intermediate disturbance hypothesis. Effects were stronger in plots burned in 2-year rotations, suggesting that the 4-year burn frequency may be a more sustainable practice to ensure the long-term stability of C cycling in such ecosystems.

Basidiomycete fungal communities in Australian sclerophyll forest soil are altered by repeated prescribed burning.

Soil basidiomycetes play key roles in forest nutrient and carbon cycling processes, yet the diversity and structure of below ground basidiomycete communities remain poorly understood. Prescribed burning is a commonly used forest management practice and there is evidence that single fire events can have an impact on soil fungal communities but little is known about the effects of repeated prescribed burning. We have used internal transcribed spacer (ITS) terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the impacts of repeated prescribed burning every two or four years over a period of 30 years on soil basidiomycete communities in an Australian wet sclerophyll forest. Detrended correspondence analysis of ITS T-RFLP profiles separated basidiomycete communities in unburned control plots from those in burned plots, with those burned every two years being the most different from controls. Burning had no effect on basidiomycete species richness, thus these differences appear to be due to changes in community structure. Basidiomycete communities in the unburned control plots were vertically stratified in the upper 20 cm of soil, but no evidence was found for stratification in the burned plots, suggesting that repeated prescribed burning results in more uniform basidiomycete communities. Overall, the results demonstrate that repeated prescribed burning alters soil basidiomycete communities, with the effect being greater with more frequent burning.

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 understory 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 understory. 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 understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.

Chitinolytic activities of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae).

Ericoid mycorrhizal endophytes and other root-associated fungi from Epacris pulchella (Ericaceae) in an eastern Australian sclerophyll forest, along with Hymenoscyphus ericae, were tested for their abilities to produce extracellular chitinolytic activities during growth in axenic culture. Two root-associated fungi produced activities that were active against only a monomeric 4-methylumbelliferyl (4-MU) glycoside of N-acetylglucosamine, suggesting exo-acting beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. All ericoid mycorrhizal fungi and two root-associated fungi produced activities against dimeric and trimeric 4-MU glycosides of N-acetylglucosamine, suggesting production of chitobiosidase and endo-acting chitinase (EC 3.2.1.14) respectively in addition to beta-N-acetylhexosaminidase. Specific activities for all ericoid mycorrhizal fungi, including H. ericae, were of the same order of magnitude, suggesting that their chitinolytic potential is broadly similar. Chitinase activities were only produced by an ericoid mycorrhizal fungus when chitin was included in the medium, however, no activity was produced if glucose was also present in the medium.

Influence of long-term repeated prescribed burning on mycelial communities of ectomycorrhizal fungi.

To demonstrate the efficacy of direct DNA extraction from hyphal ingrowth bags for community profiling of ectomycorrhizal (ECM) mycelia in soil, we applied the method to investigate the influence of long-term repeated prescribed burning on an ECM fungal community. DNA was extracted from hyphal ingrowth bags buried in forest plots that received different prescribed burning treatments for 30 yr, and denaturing gradient gel electrophoresis (DGGE) profiles of partial fungal rDNA internal transcribed spacer (ITS) regions were compared. Restriction fragment length polymorphism (RFLP) and sequence analyses were also used to compare clone assemblages between the treatments. The majority of sequences derived from the ingrowth bags were apparently those of ECM fungi. DGGE profiles for biennially burned plots were significantly different from those of quadrennially burned and unburned control plots. Analysis of clone assemblages indicated that this reflected altered ECM fungal community composition. The results indicate that hyphal ingrowth bags represent a useful method for investigation of ECM mycelial communities, and that frequent long-term prescribed burning can influence below-ground ECM fungal communities.

Basidiomycete mycelia in forest soils: dimensions, dynamics and roles in nutrient distribution.

Basidiomycete mycelia are ubiquitous in forest soils where they fulfil a range of key ecological functions. Population studies, based largely on basidiome collections, indicate that mycelia of many ectomycorrhizal and saprotrophic basidiomycetes can spread vegetatively for considerable distances through soil, but the extent to which these become physically or physiologically fragmented is unclear. This review considers aspects of the distribution, dynamics and translocatory activities of individual basidiomycete mycelia in forest soil, highlighting current gaps in our understanding and possible ways to address these.

Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots.

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.