Sarah C. Watkinson

The Plant Cell Wall–Decomposing Machinery Underlies the Functional Diversity of Forest Fungi

Comparative genomic analysis of “dry rot” fungus shows both convergent evolution and divergence among fungal decomposers. Brown rot decay removes cellulose and hemicellulose from wood—residual lignin contributing up to 30% of forest soil carbon—and is derived from an ancestral white rot saprotrophy in which both lignin and cellulose are decomposed. Comparative and functional genomics of the “dry rot” fungus Serpula lacrymans, derived from forest ancestors, demonstrated that the evolution of both ectomycorrhizal biotrophy and brown rot saprotrophy were accompanied by reductions and losses in specific protein families, suggesting adaptation to an intercellular interaction with plant tissue. Transcriptome and proteome analysis also identified differences in wood decomposition in S. lacrymans relative to the brown rot Postia placenta. Furthermore, fungal nutritional mode diversification suggests that the boreal forest biome originated via genetic coevolution of above- and below-ground biota.

Yeast forms dominate fungal diversity in the deep oceans

Fungi are the principal degraders of biomass in most terrestrial ecosystems. In contrast to surface environments, deep-sea environmental gene libraries have suggested that fungi are rare and non-diverse in high-pressure marine environments. Here, we report the diversity of fungi from 11 deep-sea samples from around the world representing depths from 1500 to 4000 m (146–388 atm) and two shallower water column samples (250 and 500 m). We sequenced 239 clones from 10 fungal-specific 18S rRNA gene libraries constructed from these samples, from which we detected only 18 fungal 18S-types in deep-sea samples. Our phylogenetic analyses show that a total of only 32 fungal 18S-types have so far been recovered from deep-sea habitats, and our results suggest that fungi, in general, are relatively rare in the deep-sea habitats we sampled. The fungal diversity detected suggests that deep-sea environments host an evolutionarily diverse array of fungi dominated by groups of distantly related yeasts, although four putative filamentous fungal 18S-types were detected. The majority of our new sequences branch close to known fungi found in surface environments. This pattern contradicts the proposal that deep-sea and hydrothermal vent habitats represent ancient ecosystems, and demonstrates a history of frequent dispersal between terrestrial and deep-sea habitats.

Fungi in Biogeochemical Cycles: The role of wood decay fungi in the carbon and nitrogen dynamics of the forest floor

decomposed amino acid that tracks the mycelial free amino acid pool. Its movement can be imaged by counting photon emissions from a scintillant screen in contact with the mycelial system. This method allows real-time imaging at high temporal and spatial resolution, for periods of weeks and areas up to 1 m 2 , in microcosms that mimic the mineral/organic soil interface of the forest floor. The results reveal a hitherto unsuspected dynamism and responsiveness in amino acid flows through mycelial networks of cord-forming, wood-decomposing basidiomycetes. We interpret these in the light of current understanding of the pivotal role of fungi in boreal and temperate forest floor nutrient cycling, and attempt to formulate key questions to investigate the effects of mycelial nitrogen translocation on forest floor decomposition and nitrogen absorption.

The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi.

ABSTRACT Mycelial fungi have a growth form which is unique among multicellular organisms. The data presented here suggest that they have developed a unique solution to internal solute translocation involving a complex, extended vacuole. In all filamentous fungi examined, this extended vacuole forms an interconnected network, dynamically linked by tubules, which has been hypothesized to act as an internal distribution system. We have tested this hypothesis directly by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker. Predictive simulation models were then used to determine the transport characteristics over extended length scales. This modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimeters to centimeters. Flux through the pathway is regulated by the dynamic tubular connections involving homotypic fusion and fission. There is also a strongly predicted interaction among vacuolar organization, predicted diffusion transport distances, and the architecture of the branching colony margin.

Fungi in the environment.

1. Imaging complex nutrient dynamics in mycelial networks D. P. Bebber, M. Tlalka, J. Hynes, P. R. Darrah, A. Ashford, S. C. Watkinson, L. Boddy and M. D. Fricker 2. Natural history of the fungal hypha: how Woronin bodies support a multicellular lifestyle Gregory Jedd 3. Environmental sensing and the filamentous fungal lifestyle Nick D. Read 4. Mineral transformations and biogeochemical cycles: a gomycological perspective Geoffrey M. Gadd, Euan P. Burford, Marina Fomina and Karrie Melville 5. Mycelial responses in heterogeneous environments: parallels with macroorganisms Lynne Boddy and T. Hefin Jones 6. Natural abundance of 15N and 13C in saprotrophic fungi: what can they tell us? Andy F. S. Taylor and Petra M. A. Fransson 7. Berkeley Award Lecture: mathematical modelling of the form and function of fungal mycelia Fordyce A. Davidson 8. Mycorrhizas and the terrestrial carbon cycle: roles in global carbon sequestration and plant community composition Jonathan R. Leake 9. Water relations in lichens Rosmarie Honegger 10. Development of the arbuscular mycorrhizal symbiosis: insights from genomics Jinyuan Liu, Melina Lopez-Meyer, Ignacio Maldonado-Mendoza and Maria J. Harrison 11. Functional genomics of plant infection by the rice blast fungus Magnaporthe grisea Joanna M. Jenkinson, Richard Wilson, Zachary Cartwright, Darren M. Soanes, Michael J. Kershaw, Amy E. Davies and Nicholas J. Talbot 12. Exploring the interaction between nematode-trapping fungi and nematodes using DNA microarrays Anders Tunlid 13. Role of (1-3) glucan in Aspergillus fumigatus and other human fungal pathogens Anne Beauvais, David S. Perlin and Jean Paul Latge 14. Plagues upon houses and cars: the unnatural history of Meruliporia incrassata, Serpula lacrymans, and Sphaerobolus stellatus Nicholas P. Money 15. Fungal species: thoughts on their recognition, maintenance and selection John W. Taylor, Elizabeth Turner, Anne Pringle, Jeremy Dettman, and Hanna Johannesson 16. Multilocus Sequence Typing (MLST) and Multilocus Microsatellite Typing (MLMT) in fungi Matthew C. Fisher 17. Fungi in the hidden environment: the gut of beetles Meredith Blackwell, Sung-Oui Suh and James B. Nardi 18. A saltmarsh decomposition system and its ascomycetous laccase genes Steven Y. Newell, Justine I. Lyons and Mary Ann Moran.

Imaging complex nutrient dynamics in mycelial networks

Transport networks are vital components of multi‐cellular organisms, distributing nutrients and removing waste products. Animal cardiovascular and respiratory systems, and plant vasculature, are branching trees whose architecture is thought to determine universal scaling laws in these organisms. In contrast, the transport systems of many multi‐cellular fungi do not fit into this conceptual framework, as they have evolved to explore a patchy environment in search of new resources, rather than ramify through a three‐dimensional organism. These fungi grow as a foraging mycelium, formed by the branching and fusion of threadlike hyphae, that gives rise to a complex network. To function efficiently, the mycelial network must both transport nutrients between spatially separated source and sink regions and also maintain its integrity in the face of continuous attack by mycophagous insects or random damage. Here we review the development of novel imaging approaches and software tools that we have used to characterise nutrient transport and network formation in foraging mycelia over a range of spatial scales. On a millimetre scale, we have used a combination of time‐lapse confocal imaging and fluorescence recovery after photobleaching to quantify the rate of diffusive transport through the unique vacuole system in individual hyphae. These data then form the basis of a simulation model to predict the impact of such diffusion‐based movement on a scale of several millimetres. On a centimetre scale, we have used novel photon‐counting scintillation imaging techniques to visualize radiolabel movement in small microcosms. This approach has revealed novel N‐transport phenomena, including rapid, preferential N‐resource allocation to C‐rich sinks, induction of simultaneous bi‐directional transport, abrupt switching between different pre‐existing transport routes, and a strong pulsatile component to transport in some species. Analysis of the pulsatile transport component using Fourier techniques shows that as the colony forms, it self‐organizes into well demarcated domains that are identifiable by differences in the phase relationship of the pulses. On the centimetre to metre scale, we have begun to use techniques borrowed from graph theory to characterize the development and dynamics of the network, and used these abstracted network models to predict the transport characteristics, resilience, and cost of the network.

Simulated nitrogen deposition affects wood decomposition by cord-forming fungi

Anthropogenic nitrogen (N) deposition affects many natural processes, including forest litter decomposition. Saprotrophic fungi are the only organisms capable of completely decomposing lignocellulosic (woody) litter in temperate ecosystems, and therefore the responses of fungi to N deposition are critical in understanding the effects of global change on the forest carbon cycle. Plant litter decomposition under elevated N has been intensively studied, with varying results. The complexity of forest floor biota and variability in litter quality have obscured N-elevation effects on decomposers. Field experiments often utilize standardized substrates and N-levels, but few studies have controlled the decay organisms. Decomposition of beech (Fagus sylvatica) blocks inoculated with two cord-forming basidiomycete fungi, Hypholoma fasciculare and Phanerochaete velutina, was compared experimentally under realistic levels of simulated N deposition at Wytham Wood, Oxfordshire, UK. Mass loss was greater with P. velutina than with H. fasciculare, and with N treatment than in the control. Decomposition was accompanied by growth of the fungal mycelium and increasing N concentration in the remaining wood. We attribute the N effect on wood decay to the response of cord-forming wood decay fungi to N availability. Previous studies demonstrated the capacity of these fungi to scavenge and import N to decaying wood via a translocating network of mycelium. This study shows that small increases in N availability can increase wood decomposition by these organisms. Dead wood is an important carbon store and habitat. The responses of wood decomposers to anthropogenic N deposition should be considered in models of forest carbon dynamics.

Nuclear microscopy of biological specimens

Recent developments in technology have enabled the scanning proton microprobe to scan at submicron spatial resolution on a routine basis. The use of the powerful combination of techniques PIXE (proton induced X-ray emission), nuclear (or Rutherford) backscattering (RBS), and secondary electron detection operating at this resolution will open up new areas in many scientific disciplines. This paper describes some of the work carried out in the biological sciences over the last year, using the Oxford SPM facility. Collaborations with biological scientists have drawn attention to the wealth of information that can be derived when these techniques are applied to micro-organisms, cells and plant tissue. Briefly described here are investigations into the uptake of heavy metals by the alga Pandorina morum, the structure of the diatom Stephanopyxis turris, the presence of various types of crystal structures within the cells of Spirogyra, the heavy metal uptake of a mycorrhizal fungus present in the bracken (Pteridium aquilinum) root, the role of sphagnum moss in the absorption of inorganic elements, the measurement of heavy metals in environmentally-adapted cells of the yeast Saccharomyces cerevisiae, and the elemental distribution in the growing tip of a spore from the plant Equisetum arvense, with special emphasis placed on the visual interpretation of the elemental and secondary-electron maps provided by the nuclear microscopical techniques.

New approaches to investigating the function of mycelial networks

Fungi play a key role in ecosystem nutrient cycles by scavenging, concentrating, translocating and redistributing nitrogen. To quantify and predict fungal nitrogen redistribution, and assess the importance of the integrity of fungal networks in soil for ecosystem function, we need better understanding of the structures and processes involved. Until recently nitrogen translocation has been experimentally intractable owing to the lack of a suitable radioisotope tracer for nitrogen, and the impossibility of observing nitrogen translocation in real time under realistic conditions. We have developed an imaging method for recording the magnitude and direction of amino acid flow through the whole mycelial network as it captures, assimilates and channels its carbon and nitrogen resources, while growing in realistically heterogeneous soil microcosms. Computer analysis and modeling, based on these digitized video records, can reveal patterns in transport that suggest experimentally testable hypotheses. Experimental approaches that we are developing include genomics and stable isotope NMR to investigate where in the system nitrogen compounds are being acquired and stored, and where they are mobilized for transport or broken down. The results are elucidating the interplay between environment, metabolism, and the development and function of transport networks as mycelium forages in soil. The highly adapted and selected foraging networks of fungi may illuminate fundamental principles applicable to other supply networks.

Quantifying dynamic resource allocation illuminates foraging strategy in Phanerochaete velutina

Saprotrophic woodland fungi forage for mineral nutrients and woody resources by extension of a mycelial network across the forest floor. Different species explore at different rates and establish networks with qualitatively differing architecture. However, detailed understanding of fungal foraging behaviour has been hampered by the absence of tools to quantify resource allocation and growth accurately and non-invasively. To solve this problem, we have used photon-counting scintillation imaging (PCSI) to map and quantify nutrient allocation and localised growth simultaneously in heterogeneous resource environments. We show that colonies spontaneously shift to an asymmetric growth pattern, even in the absence of added resources, often with a distinct transition between the two growth phases. However, the extent of polarisation was much more pronounced and focussed in the presence of an additional cellulose resource. In this case, there was highly localised growth, often at the expense of growth elsewhere in the colony, and marked accumulation of (14)C-AIB in the sector of the colony with the added resource. The magnitude of the response was greatest when resource was added around the time of the endogenous developmental transition. The focussed response required a metabolisable resource, as only limited changes were seen with glass fibre discs used to mimic the osmotic and thigmotropic stimuli upon resource addition. Overall the behaviour is consistent with an adaptive foraging strategy, both to exploit new resources and also to redirect subsequent foraging effort to this region, presumably with an expectation that the probability of finding additional resources is increased.