Fine-scale diversity and distribution of ectomycorrhizal fungal mycelium in a Scots pine forest.

Ectomycorrhizal (ECM) mycelium is a key component of the ectomycorrhizal symbiosis, yet we know little regarding the fine-scale diversity and distribution of mycelium in ECM fungal communities. We collected four 20 × 20 × 2-cm(3) (800-cm(3)) slices of Scots pine (Pinus sylvestris) forest soil and divided each into 100 2 × 2 × 2-cm(3) (8-cm(3)) cubes. The presence of mycelium of ECM fungi was determined using an internal transcribed spacer (ITS) database terminal restriction fragment length polymorphism (T-RFLP) approach. As expected, many more ECM fungi were detected as mycelium than as ectomycorrhizas in a cube or slice. More surprisingly, up to one-quarter of the 43 species previously detected as ectomycorrhizas over an area of 400 m(2) could be detected in a single 8-cm(3) cube, and up to three-quarters in a single 800-cm(3) slice. ECM mycelium frequency decreased markedly with depth and there were distinct 'hotspots' of mycelium in the moss/F1 layer. Our data demonstrate a high diversity of ECM mycelium in a small (8-cm(3) ) volume of substrate, and indicate that the spatial scale at which ECM species are distributed as mycelium may be very different from the spatial scale at which they are distributed as tips.

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 ß-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.

Effect of arsenic contaminated irrigation water on Lens culinaris L. and toxicity assessment using lux marked biosensor.

Contamination of irrigation water represents a major constraint to Bangladesh agriculture, resulting in elevated levels in the terrestrial systems. Lux bacterial biosensor technology has previously been used to measure the toxicity of metals in various environmental matrices. While arbuscular mycorrhizal fungi have their most significant effect on phosphorus uptake, but showed alleviated metal toxicity to the host plant. The study examined the effects of arsenic and inoculation with an arbuscular mycorrhizal fungus, Glomus mosseae, on lentil (Lens culinaris L. cv. Titore). Plants were grown with and without arbuscular mycorrhizal inoculum for 9 weeks in a sand and terra-green mixture (50:50, V/V) and watered with five levels of arsenic (0, 1, 2, 5, 10 mg As/L arsenate). The results showed that arsenic addition above 1 mg/L significantly reduced percentage of mycorrhizal root infection. On further analysis a close relationship was established with the vegetative and reproductive properties of lentil (L. culinaris) plants compared to the percentage bioluminescence of the soil leachate. However, arbuscular mycorrhizal fungal inoculation reduced arsenic concentration in roots and shoots. Higher concentrations of arsenic (5, 10 mg As/L arsenate) reduced the mycorrhizal efficiency to increase phosphorus content and nitrogen fixation. Therefore, this study showed that increased concentration of arsenic in irrigation water had direct implications to the lentil (L. culinaris) plants overall performance. Moreover the use of bioassay demonstrated that mycorrhiza and clay particle reduced arsenic bioavailability in soil.

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.

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.

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.

Small genetic differences between ericoid mycorrhizal fungi affect nitrogen uptake by Vaccinium.

Ericoid mycorrhizal fungi have been shown to differ in their pattern of nitrogen (N) use in pure culture. Here, we investigate whether this functional variation is maintained in symbiosis using three ascomycetes from a clade not previously shown to include ericoid mycorrhizal taxa. Vaccinium macrocarpon and Vaccinium vitis-idaea were inoculated with three fungal strains known to form coils in Vaccinium roots, which differed in their patterns of N use in liquid culture. (15)N was used to trace the uptake of -N, -N and glutamine-N into shoots. (15)N transfer differed among the three fungal strains, including two that had identical internal transcribed spacer (ITS) sequences, and was quantitatively related to fungal growth in liquid culture at low carbon availability. These results demonstrate that functional differences among closely related ericoid mycorrhizal fungi are maintained in symbiosis with their hosts, and suggest that N transfer to plant shoots in ericoid mycorrhizas is under fungal control.

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.

The electronic medical office: optimizing solutions.

Optimizing the care for patients in the orthopaedic clinical setting involves a wide range of issues. Surgical techniques, preoperative and postoperative care, long-term outcomes follow-up, continuing education, and patient communication are a few of the important areas that surgeons deal with on a regular basis. Successful management of this information has an impact on clinical outcomes, direct patient care, financial decisions, and management of the surgeon's time. The development of a comprehensive electronic medical office is a powerful and probably necessary tool to successfully manage such information and achieve the goals of an effective and safe orthopaedic practice.

Ectomycorrhizal fungi in culture respond differently to increased carbon availability.

Carbon (C) availability to ectomycorrhizal fungi is likely to increase at elevated atmospheric CO(2). To determine whether there are any broad patterns in species' responses that relate to their ecology, we compared growth, respiration, N uptake and C exudation of 17 fungal isolates in liquid culture. As a surrogate for increased C availability we used three different C:N ratios (10:1, 20:1 and 40:1), moving from conditions of C limitation to conditions of N limitation. Responses were species-specific, and suilloid fungi were the most responsive in terms of growth and respiration. In contrast, a group of eight isolates showed no growth increase above C:N 20:1. This inability to respond was not due to N limitation, although there were marked differences in N uptake between isolates. At higher C availability isolates generally became more efficient in converting C into biomass. Six isolates showed net release of exudates into the culture medium (up to 40% of the C in biomass and respiration). We conclude that the findings were in agreement with field observations, and suggest that pure culture observations can yield ecologically relevant information on how ectomycorrhizal fungi may respond under conditions of elevated CO(2).

Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris).

The diversity of cultivable endobacteria associated with four different ectomycorrhizal morphotypes (Suillus flavidus, Suillus variegatus, Russula paludosa and Russula sp.) of Scots pine (Pinus sylvestris) was analysed by restriction fragment length polymorphism profiling of PCR-amplified rDNA intergenic spacer regions and by sequence analyses of 16S rRNA genes. Ectomycorrhizal root tip surface-sterilization methods were developed and assessed for their efficiencies. Bacterial communities from surface-sterilized ectomycorrhizal root tips were different from those of ectomycorrhizal root tips without surface-sterilization for all the morphotypes studied. Endobacteria belonging to the genera Pseudomonas, Burkholderia and Bacillus were isolated from more than one ectomycorrhizal morphotype, whereas species of Rahnella, Janthinobacterium and Rhodococcus were only isolated from the single morphotypes of S. variegatus, R. paludosa and Russula sp., respectively. Some of the isolated endobacteria utilized fungal sugars more readily than typical plant sugars in carbon utilization assays.

Tibiotalocalcaneal Fusion for Severe Deformity and Bone Loss.

Tibiotalocalcaneal fusion is an effective salvage procedure for combined end-stage ankle and subtalar arthrosis and for the management of severe planar deformities of the ankle and hindfoot. Although the procedure results in a rigid ankle and hindfoot, it is often the only means of providing patients with a stable and painless foot and ankle for ambulation. Some patients who require the procedure have substantial bone loss that can be managed with a variety of autograft and allograft options. Options for tibiotalocalcaneal fixation include both internal and external devices, the selection of which depends on the underlying pathology, amount of bone loss, and type of bone graft selected. Relatively high complication rates associated with tibiotalocalcaneal fusion have been reported, with complications ranging from superficial infection to ultimate amputation; however, proper patient selection and careful graft and fixation planning can minimize the postoperative complications of the procedure.

"Smile" incision: an approach for open reduction and internal fixation of calcaneal fractures.

MATERIALS: A consecutive group of 73 patients (77 calcaneal fractures) treated with open reduction and internal fixation through a smile-shaped lateral approach to the hindfoot were reviewed retrospectively. Inclusion criteria were a closed displaced intra-articular fracture of the calcaneus, no compartment syndrome, and adequate followup. Followup ranged from 4 months to 4 years. RESULTS: One patient had a deep wound infection that required removal of hardware and intravenous antibiotics. No deep dehiscence or flap necrosis occurred. Superficial dehiscence occurred in three patients (4%) and superficial necrosis was seen in nine patients (12%); all resolved with continued casting to protect the wound. Six patients (8%) complained of numbness or pain in the sural nerve distribution. No symptomatic neuromas were seen. Reflex sympathetic dystrophy occurred in three patients (4%); all resolved with sympathetic blocks. Only one patient required an additional procedure for treatment of a wound-related problem or infection. CONCLUSION: The "smile" incision provides good fracture exposure for reduction without added morbidity.

Interaction between an isolate from the Hymenoscyphus ericae aggregate and roots of Pinus and Vaccinium.

• A fungal isolate was obtained from Piceirhiza bicolorata-like ectomycorrhizas on Pinus sylvestris in a 160-yr-old natural woodland. • The fungus was identified by sequencing the PCR-amplified rDNA ITS regions. The sequence was compared with similar known taxa and grouped with Cadophora finlandia in the Hymenoscyphus ericae aggregate. • The fungus formed P. bicolorata-like ectomycorrhizas in aseptic synthesis with P. sylvestris seedlings. When seedlings of Vaccinium myrtillus were exposed to mycelium arising from these ectomycorrhizas, or to mycelium in pure culture, the hyphae entered the cells of the hair roots and formed coils characteristic of ericoid mycorrhizas. The presence of the fungus stimulated Vaccinium root growth and altered root architecture. • This is the first full report of the ability of a fungus from the H. ericae aggregate simultaneously to form both ectomycorrhizas and what appear to be ericoid mycorrhizas.

Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere.

• Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs. • The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with 14 C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization. • Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms. • The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the 'Gadgil-effect', where retarded litter decomposition has been observed in the presence of EcM roots.

Intraspecific diversity regulates fungal productivity and respiration.

Individuals and not just species are key components of biodiversity, yet the relationship between intraspecific diversity and ecosystem functioning in microbial systems remains largely untested. This limits our ability to understand and predict the effects of altered genetic diversity in regulating key ecosystem processes and functions. Here, we use a model fungal system to test the hypothesis that intraspecific genotypic richness of Paxillus obscurosporus stimulates biomass and CO(2) efflux, but that this is dependent on nitrogen supply. Using controlled experimental microcosms, we show that populations containing several genotypes (maximum 8) of the fungus had greater productivity and produced significantly more CO(2) than those with fewer genotypes. Moreover, intraspecific diversity had a much stronger effect than a four-fold manipulation of the carbon:nitrogen ratio of the growth medium. The effects of intraspecific diversity were underpinned by strong roles of individuals, but overall intraspecific diversity increased the propensity of populations to over-yield, indicating that both complementarity and selection effects can operate within species. Our data demonstrate the importance of intraspecific diversity over a range of nitrogen concentrations, and the need to consider fine scale phylogenetic information of microbial communities in understanding their contribution to ecosystem processes.

Spatial distribution of sporocarps of stipitate hydnoid fungi and their belowground mycelium.

Interest in stipitate hydnoid fungi of the genera Bankera, Hydnellum, Phellodon and Sarcodon has increased due to the decline in numbers of sporocarps in Europe. Conservation of these fungi is hindered by a lack of understanding of their basic ecology. In particular, a better understanding of their belowground ecology is required. Real-time PCR in conjunction with spatially explicit sampling was used to quantify the relationship between sporocarps and mycelium of Hydnellum peckii and Phellodon tomentosus. Species-specific DNA of the target species was quantified in 100 soil samples collected on a 360 x 360 cm grid at five locations where sporocarps were present. All sporocarps within the grid and up to 2 m around the grid were mapped. Sporocarp production did not occur over the whole extent of the belowground mycelium of these two species, and mycelium extended up to 330 cm away from the immediate site of sporocarp production. Spatial analyses using Kernel-smoothing and Moran's I correlograms showed that, with a single exception, there was no quantitative relationship between sporocarp distribution and the belowground abundance of mycelium. These findings have important implications for the conservation of this rare group of fungi.

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.