I describe several studies that investigate effects of collembolan on morphology and nutrient partitioning in mycelia of cord-forming fungi. The collembola (Folsomia candida, Protaphorura armata and Proisotoma minuta) and fungi (Phanerochaete velutina, Hypholoma fasciculare, Phallus impudicus and Resinicium bicolor) used occur commonly in British woodlands. Each study employed two-species (one fungus: one collembola) microcosms containing compressed soil. The fungus was added in a colonized wood block and allowed to grow out across the soil before collembola were added. Fungal growth and morphology were characterized using image analysis techniques. Collembolan grazing occurred predominantly on the diffuse hyphae present at mycelial margins, except for R. bicolor where thick cords were regularly severed. Grazing reduced extension rate and hyphal coverage of mycelia; effects were more dramatic at high collembolan densities. The region of mycelium attacked was consistent across collembola species; the magnitude of grazing impacts varied. Fungal responses to grazing were species-specific. R. bicolor mycelia were often destroyed, P. velutina and H. fasciculare retained an intact cord system but their growth was modified, P. impudicus was little affected. Mycelial network architecture was altered. In addition to changes in fungal morphology, wood decay rate of H. fasciculare was decreased by grazing. Mycelial nutrient distribution was explored by pulse-labelling P. velutina mycelia with 15N. This showed that P. armata grazing reduced the uptake and transfer of 15N to outgrowth mycelium, and did not increase nitrogen release into the soil.

The effect of arrival of wood resources, precolonised by either Coholus versicolor, Phlebia radiata, Stereum hirsutum and Vuilleminia comedens, on mycelial systems of Phanerochaete velutina was studied in trays of non-sterile soil in the laboratory. Morphological responses and nutrient movement were quantified non-destructively using respectively image analysis and P monitoring with a scintillation probe, and subsequently destructively harvesting. The presence of a fungus occupying the new resource or state of decay of the resource arriving on an established system had major effects on deployment of biomass and on the uptake and allocation of phosphorus, the effects were specific to the species occupying the new resource. When new resources were added of constant size and decay state, massive redeployment of biomass away from regions with no new resource only occurred with three types of new resource: (1) uncolonised (2) colonised by V. comedens', and (3) to a lesser extent those colonised by S. hirsutum. P was taken up by P. velutina both in the vicinity of the inoculum and the new resource, and was translocated to the new resource from both sites of uptake, however the local supply contributed most. Bidirectional translocation also occurred. Initial P translocation to wood resources precolonised for three years by V. comedens and S. hirsutum was initially high, greater initial translocation of P occurred to younger precolonised resources of 8 weeks where a dramatic increase in P activity was apparent in systems containing the precoloniser V. comedens. Mycelial systems containing two resources, precolonised and/or uncolonised and at different states of decay had major effects on phosphorus translocation to these resources. Mycelial systems growing from different inoculum sizes effected the outcome of interactions between P. velutina and resource restricted fungi. Phosphorus translocation was also effected by inoculum to resource ratio within the system. 12 1 Lower P activity was observed in mycelial systems extending from 1cm inoculum when compared to systems extending from 4cm and 16 cm3 inoculum. An increase in inoculum to resource ratio increased the capability of cord systems of P. velutina to capture resources that were once occupied by other wood decay species. These responses are discussed in relation to mycelial foraging strategies, nutrient translocation and partitioning within mycelial cord systems.

Lignin degrading (white-rot) basidiomycete fungi are major agents of carbon cycling and play a key role in maintaining forest ecosystems. Trametes versicolor is a white- rot fungus of both industrial and ecological interest. The aims of this study were to investigate the effects of biotic and abiotic stress factors on growth, ligninolytic enzyme (laccase) production and gene expression in white-rot fungi, using T. versicolor as a model for most of the work. Mycelial interactions are important in defining community structure in wood-rotting fungi, and during these interactions volatile organic compounds (VOCs) are produced which can affect the growth of other fungi, at a distance. The effects of different combinations of interacting fungi on T. versicolor growth and ligninolytic enzyme production showed that effects were variable. Effects of VOCs from interaction on wood blocks were more significant than those on agar. Abiotic stress factors affect fungal metabolism and thereby regulate their biological activity. This study investigated the effect of abiotic stress factors on T. versicolor using different temperatures (low and high), osmotic pressure (KCl) and nutrients (no and low nitrogen). Growth rate, laccase production and expression of three genes (FRA19, Nox, and Lacc) were mainly reduced when the abiotic stress was imposed, although there was some variability in gene expression. Laccase is encoded by a gene family and differential expression of gene family members under stress treatments was investigated. Some evidence for an over-representation of γ group sequences following stress treatments was found. Future directions are discussed to further investigate the roles of biotic and abiotic stress in regulating the growth and underlying biological processes of white rot fungi.

Saprotrophic cord-forming basidiomycete fungi are major agents of wood decomposition in woodland and support the decomposer food-web. Limited resource availability and the abundance of mycelium in soil leads to competition between fungi. These fungal interactions are aggressive involving reallocation of mycelial biomass, pigment formation, changes in gene expression and enzyme synthesis. Collembola are abundant mycophagous invertebrates in woodlands and affect fungal morphology and growth. Experiments investigated the effects of collembola grazing on fungal interaction progression and the effects of these interactions on collembola behaviour and mortality. In British woodlands, the collembola Folsomia Candida and <italic>Protaphorura armata </italic> are common as are the cord-forming fungi <italic>Hypholoma fasciculare, Phallus impudicus, Phanerochaete velutina</italic> and Resinicium bicolor. Pairwise interactions between these fungi were investigated in agar and compressed soil microcosms. Multiple genetic isolates of two of the fungi studied were also used. Fungal morphology was affected by collembola grazing in soil- but less so in agar- microcosms. In particular, when interacting with <italic> H. fasciculare</italic>, grazing of <italic>P. velutina mycelia</italic> accelerated growth over the opposing mycelium but reduced extension over soil. This was associated with an increased ability to colonise the wood resource of <italic> H. fasciculare</italic>. Grazing did not reduce the transport efficiency of <italic> P. velutina</italic> but the estimated cost of biomass production rose more steeply with increasing area than in ungrazed systems. Despite changes in progression, interaction outcome was not generally substantially altered by grazing. Collembola exhibited strong preferences for certain mycelia during interactions but showed a change in preference in others. Collembola mortality on fungal interactions in agar microcosms also varied with the species interacting. There was limited evidence of attraction of collembola to the fungal interaction zone. Overall, the results suggest that collembola grazing may have important impacts on fungal species assemblage and their ability to extend in search of new resources.

(1) Fine structural and biochemical studies have been made on the infection of Vicia faba L. by Uromyces fabae (obligate parasite) and Botrytis fabae (facultative parasite). (2) Fine structural observations of host cells infected with U. fabae showed that they could be divided into two categories, (a) cells with increased capacity for synthesis (2-8 days after infection), which are characterised by an increase in the volume of the cytoplasm and by an increase in the size of cytoplasmic organelles (i.e. rough endoplasmic reticulum (ER), nucleus, mitochondria); (b) cells with decreased metabolic activities (12-20 days after infection) and characterised by senescence of the cytoplasm and by the reduction in the size of cytoplasmic organelles, especially chloroplasts. Numerous vesicles were found to be associated with the inner membrane of chloroplasts in such infected cells. (3) Electron microscopic observations showed that cells from leaves infected with B. fabae, can also be divided into two categories, (a) cells in the centre of the necrotic lesion which are characterised by blackening and the destruction of the tissues;(b) cells at the periphery of the necrotic lesion, which are characterised by an increase in the volume of the cytoplasm, an increase in the size of cytoplasmic organelles, by the presence of lomasomes, and by the presence of vesicular inclusions in the cytoplasm and electron dense bodies in the extracellular spaces.(4) The fine structure of U. fabae resembles that of other comparable obligate parasites, except for the presence of an electron dense ring in the middle part of the haustorial neck, and vesicular and tubular structures in the vacuoles of this fungus. The growth of B. fabae on the host tissues was found to be relatively limited as compared with its growth on artificial medium. Likewise, the cytoplasm and cytoplasmic organelles of B. fabae were found to be denser when the fungus was grown on artificial media. Lomasomes were found to be common in the cytoplasm of this fungus, and the relation of this structure to the hydrolysis of the host cell wall is discussed. (5) Several biochemical tests were made to assess the significance of some of the structural changes taking place in the host as a result of infection (i.e. amino acid composition, protein synthesis and photochemical activities of isolated chloroplasts). Special emphasis was placed on the effect of both infections on the latter process. The results can be summarised as follows:- (a) infection with U. fabae caused an increase in the rate of the Hill reaction, cyclic phosphorylation (2-10 days after infection), followed by a decrease in such activity (12-20 days after infection). The infection also caused an alteration to ATP:e(^-) ratio. .(b) infection with B. fabae caused a marked reduction in all photochemical activities tested, (i.e. Hill reaction, cyclic and uncyclic phosphorylation).(c) The observed effects were found to be independent of the chlorophyll concentration and the reduction in the photosynthetic area.

The first part of this investigation deals with the ultrastructural aspects of the dormant and germinating spores and the growing mycelia of the fungus Phycomyces blakesleeanus in submerged culture. Emphasis is focussed on the detection and behaviour of the protein ferritin in these stages particularly in the growing mycelia. The concomitant morphological and ultrastructural changes are described. Time course experiments on growing mycelia revealed that at certain stages of differentiation, detectable ferritin first disappeared in incipient germ hyphae. Its reappearance was found to depend upon the presence of added iron in the medium. A correlation between this reappearance and the availability of lipid droplets, in sections of mycelia, is described and discussed. Successive changes in the ultra- structure of mitochondria during growth and differentiation are also described. In the second part of this study, the electron microscopic observations of ferritin behaviour are backed with tangible biochemical evidences by isolating the protein at different times and conditions from the growing mycelia. The effect of the presence of high iron in the medium and other factors on ferritin synthesis are described. A sixfold de novo increase in apoferritin synthesis is attained by iron administration to growing mycelia. Factors affecting such a synthesis and radioactivity incorporation are described and discussed. By the use of polyacrylamide gel electrophoresis the subunit molecular weight of Phycomyces ferritin has been determined as 20,200 daltons.

Fungi are able to perceive and respond to changes in their environment through the activation of signal transduction pathways. Protein kinase C is a serine/threonine kinase found in all eukaryotes that is implicated in the regulation of signalling pathways. Here I report the identification of a protein kinase C-encoding gene, PKC1, which I have shown is cytoplasmically localised and expressed during conidial germination and appressorium formation in the rice blast fungus Magnaporthe oryzae. Targeted gene deletion of PKC1 was attempted unsuccessfully, therefore analysis of Pkc1 was carried out by RNAi-mediated gene silencing and selective kinase inhibition. A hairpin dsRNA–expressing construct was designed to target PKC1 and was introduced into Magnaporthe oryzae under the control of an inducible promoter. Silencing was variable but resulted in a loss of viability and when coupled with failure to obtain a gene replacement, suggests that Pkc1 is essential for viability. PKC1 gene silencing resulted in a severe hyphal growth defect and reduction in conidiogenesis. The phenotype was rescued by targeted gene deletion of the dicer- like gene, MDL2, the result is therefore consistent with gene silencing. A chemical genetics approach to selective kinase inhibition was also attempted. A previously characterised, non- essential kinase with a readily scorable phenoype, ATG1, was initially targeted to provide proof of concept in M. oryzae. Mutation of an amino acid residue in the ATP-binding site of the kinase resulted in increased susceptibility to chemically modified inhibitors. However, PKC1 does not tolerate the necessary modification to the ATP-binding site. Finally, analysis of the cell integrity pathway suggests PKC1 acts on this pathway but constitutive activation of the pathway only partially restored viability to the PKC1-gene silenced mutant and it is therefore likely to have other additional cellular targets.

The oomycete Pythium oligandrnm is used as a biocontrol agent because ofthe symbioses it forms with plants, fungi, and other oomycetes. It can parasitize phytopathogenic fungi and oomycetes providing protection to crop plants. P. oligandrnm grows as an endophyte within roots ofplants without causing disease symptoms or defense responses associated with pathogen attack. Growth of P. oligandrnm within plant tissues induces systemic resistance to pathogens and also stimulates plant growth. Little is known ofthe molecular processes that are involved in these proc7sses. Limited molecular tools were currently available to study this organism. The work set out to develop basic tools, and to acquire the fIrst signifIcant DNA sequencing data for this orgalllsm. P. oligandrnm expressed sequence tags derived from vegetative mycelia and a P. oligandrnm-P. infestans interaction were analyzed in an attempt to fmd sequences that may be involved in its biotic interactions. Many sequences with similarity to previously described effectors from fungi, oomycetes, and bacteria were revealed. ' A transformation protocol was adapted for use in P. oligandrnm, which was used to heterologously express green flourescent protein (gfp), producing florescent hyphae that could be used to follow the interaction with a plant and oomycete host. Several genes were identifIed from the EST libraries that were similar to nematode eggshell protein-encoding genes. Using the transformation protocol, it was possible to silence the expression ofthese genes by homology-dependent gene silencing. Oospores .from silenced strains displayed major ultrastructural abnormalities and were sensitive to degradative enzyme treatment. Using immunocytochemistry, these proteins were localized to the oogonial and oospore wall. It was therefore suggested that these proteins are integral components of the oospore/oogonial wall. From the EST libraries three sequences were identifIed that were similar to sea slug pheromones. Similar sequences were found to form a la:.ge gene family in the genomes ofPhytopthora infestans, P. sojae and P. ramorum. One of these genes in P. infestans, was shown to be up-regulated in the motile zoospore stage, leading to the hypothesis that this gene may be acting as a pheromone in zoospore autoaggregation. An initial characterization found no evidence to support this hypothesis.

The bacterial disease of potatoes known as 'black leg' is presently recognised simply in terms of pathogenic bacteria and a susceptible host. Closer investigation suggests that the relationship is not a simple one and the work described here provides a possible explanation.

No description available.