Mycology is a curious discipline; a peculiar division has arisen in what the word means. Meaning seems to depend on who you are. Around the world, biochemists, molecular biologists and even clinical scientists study fungi in great detail, but don't call themselves mycologists. They call themselves biochemists, molecular biologists or clinicians! Yet they study only fragments of the organism. If the molecular biology (or biochemistry, or pathology) is to be properly understood and exploited then at some stage those fragments have to be assembled into an overall, whole organism, view. At which point we are in danger of needing one of those ‘field mycologists’ that are so undervalued that: ‘In many parts of the world mycologists are an endangered species’ (Minter, 2001) and consideration needs to be given to ‘promoting the conservation of taxonomists themselves’ (Courtecuisse, 2001).

In this textbook we have attempted to provide the overall view of each topic from the start; we like to think of this as the real fungal biology. Now, in this chapter, it is time to be divisive. Throughout this book we have attempted to provide molecular details and molecular interpretations of as many as possible of the situations we have described. Knowing what we know by this stage about the overall biology of fungi we can afford to examine some of the fragments in much more detail and ask how they can be manipulated to our advantage.

Events at the hyphal tip are crucial to the extension of the hypha; so it is vital that we describe the molecular processes taking place in the hyphal tip as far as we can, and this is the main purpose of Chapter 5.

In this chapter we will give you a complete outline of eukaryotic cell biology with emphasis on how fungal cells work and how the cell biology contributes to mycelial growth. Because they are eukaryotes that are easy to cultivate in the laboratory, several fungi have been adopted as model organisms for experimentation and we will show how yeasts, in particular, have been used in this way since the nineteenth century. We discuss the essentials of cell structure in some detail, emphasising the molecular biology of the nucleus, nucleolus, nuclear import and export, and mRNA translation and protein sorting. We also briefly cover nuclear genetics and mitotic and meiotic nuclear division. The plasma membrane and signalling pathways, and endomembrane systems, cytoskeletal systems and molecular motors form major topics because directed and rapid transport of materials needed for hyphal tip extension is a crucial and characteristic feature of highly polarised filamentous growth. Other features of cell biology that are specific to fungi include the fungal cell wall, the cell biology of the hyphal apex, the nature of hyphal fusions and mycelial interconnections, the meaning of cytokinesis in fungi, and septation and the yeast–mycelial dimorphism.

In this chapter on ecosystem mycology we cover fungi as saprotrophs, and the mutualisms between plants and fungi, concentrating on fungi as recyclers that can make the earth move. Fungi also cause food contamination and deterioration through their formation of toxins, although some of these, like statins and strobilurins, are exploited commercially for our own practical purposes.

The ability of fungi to degrade wood makes them responsible for the decay of structural timber in dwellings, but on the other hand enables them to be used to remediate toxic and recalcitrant wastes. A downside, though, is that wood decay fungi release chlorohydrocarbons, potent greenhouse gases, to the atmosphere and thereby potentially contribute to global warming.

Interactions with plants dominate the rest of the chapter. We describe all types of mycorrhiza: arbuscular (AM) endomycorrhizas, ericoid endomycorrhizas, arbutoid endomycorrhizas, monotropoid endomycorrhizas, orchidaceous endomycorrhizas, ectomycorrhizas and ectendomycorrhizas. The effects of mycorrhizas and their commercial applications, and the impact of environmental and climate changes are also discussed. Finally, we introduce lichens, endophytes and epiphytes.

Evidently, fungi contribute to a broad and vibrant network of interactions with all members of the plant, animal and bacterial kingdoms (Prosser, 2002). Because of their unique attributes, fungi in particular play vital roles in most ecosystems.

The evolutionary origins and phylogenetics of fungi are the topic of this chapter, and we present it against the background of global evolution in the hope of improving appreciation of the timescale involved.

Reading this chapter could be the longest task you'll have in your life as a student, because we plan to deal with all the time that has ever existed. We are doing this to provide some context to the enormous lengths of time that we have to think about when discussing the origins of one of the major eukaryotic kingdoms. Using the most recent molecular phylogenetic analyses, we describe fungi as an ancient and successful lineage, arguably the first terrestrial eukaryotes. To do this we have to talk in billions (109) of years, and we find it difficult to envisage a billion years. It's even less easy, given the amazing changes witnessed during a pitifully short human lifetime, to imagine the sorts of changes that can occur in a billion years.

It's slightly easier to think in terms of fractions and percentages and to take in the lifetime of the universe by equating a billion years to a little over 7% of the total age of the Universe, and extending that to realise that the lifetime of our Sun is about one-third of the age of the universe.

Well, we did say only slightly easier!

From there we go on to consider planet Earth as a habitat (your habitat) and the unique series of events that make this planet (the ‘Goldilocks planet’) so suitable for the sustained evolution of life.

Most fungal mycelia contain haploid nuclei. This is a characteristic of Kingdom Fungi; unlike the other major eukaryotic groups, most true fungi are haploid. Even in fungus-like organisms in the Oomycota (Kingdom Chromista) like Phytophthora infestans, the cause of potato blight, the nuclei are diploid. This difference in ploidy is an important contrast between ‘true’ and ‘non-true’ fungi. Of course, there are exceptions to every rule and some true fungi are diploid, like Candida albicans, a yeast which causes disease in humans; and rhizomorphs and fruit bodies of Armillaria mellea (a pathogen of trees that belongs to the Basidiomycota) (Peabody et al.,2000).

In this chapter compatibility and the individualistic mycelium will be our main concerns. Formation and breakdown of heterokaryons and the nature and maintenance of the dikaryon are major topics, as are the mechanisms that regulate these processes: vegetative compatibility and the incompatibility systems. We also discuss gene segregation during the mitotic division cycle, which culminates conceptually in what is known as the parasexual cycle. Finally, we consider the segregations of the cytoplasmic genetic entities, mitochondria, plasmids, viruses and prions.

Compatibility and the individualistic mycelium

Because of the difference in ploidy, the life cycles of true fungi and those of other major groups of eukaryotes differ significantly. For example, for most true fungi diploid nuclei are only produced transiently during sexual reproduction, whereas the haploid state is limited to the gametes in most animals and plants.

Introduction

Asexual sporulation is generally the most prolific reproductive mode for fungi. Asexual spores of higher fungi are called conidia, which are non-motile asexual propagules made from the side or tip of specialized sporogenous cells and do not form through progressive cleavage of the cytoplasm. The process of conidiation is complex and involves temporal and spatial regulation of gene expression, cell specialization and intercellular communication. However, the genetic mechanisms controlling fungal sporulation have only been addressed in detail in two wellstudied ascomycetes, Aspergillus nidulans and Neurospora crassa. In this chapter we will describe the genetic regulation of development in A. nidulans. It is presumed that variations on this theme will apply in many cases to understanding conidiogenesis in other fungi as well.

The A. nidulans asexual reproductive cycle can be divided into three conceptual stages: (1) a growth phase that is required for cells to acquire competence to respond to induction signals; (2) initiation of the developmental pathway; (3) the events leading to sporulation.

Colony formation

Vegetative growth in A. nidulans begins with the germination of a spore. Spore germination leads to the formation of tubular structures, termed hyphae, that grow in a polar fashion by apical extension to form a network of interconnected hyphae known as a mycelium. The mycelium forms a radially symmetrical colony that expands indefinitely at a constant rate of about 0.5 mm h-1 (at 37 °C).

The fungi are admirably equipped with an array of degradative and lytic weaponry to colonize and enter the animal body. The host, of course, has an equally impressive armoury not only of defensive, but also aggressive machinery to prevent such an invasion. Very few fungi are obligate animal pathogens, but many are opportunists, able to take advantage of any chink in the host's defences.

Fungal infections of humans

The dermatophytes

The dermatophytes are a group of fungi which are particularly well adapted for the infection of the superficial keratinized structures of the body, including the nails, hair shafts and the stratum corneum (the cornified layer) of the skin. The true dermatophytes all belong to one of three genera of fungi, and are all able to use keratin as a source of nutrients. They are distinguished from a number of other fungi which may colonize the same superficial sites, including Candida albicans, Malassezia furfur and Aspergillus spp., by the fact that the dermatophytes are not opportunists. They are well adapted to their pathological niche and able to infect the immunocompetent individual.

The three genera of dermatophytes are Trichophyton, Epidermophyton and Microsporum, which together contain over 40 species. These organisms are relatively similar, but can be distinguished by colony morphology, macroscopic appearance and some biochemical tests. Recently, discovery of the perfect, or sexual, stage of several species of dermatophyte has led to their reclassification into two new genera, Arthroderma, containing the perfect Trichophyton species, and Nannizzia, containing the perfect Microsporon species.

Introduction

Fungal species have been at the forefront of developments in microbiology in particular and biology in general ever since the middle of the sixteenth century. Fungi have vital roles in agriculture, medicine and biotechnology. This chapter provides an overview of the role of microbiology in history with emphasis on eukaryotes. After a short introduction into what microbiology encompasses, a historical survey of some milestones in the development of modern microbiology, especially mycology, is given. This also describes where other scientific fields were influenced by fungal research.

Microorganisms and microbiology

Microorganisms are the most widely distributed group of living beings. They are capable of occupying nearly every ecological niche due to the fact that individual microorganisms can grow under extreme conditions, such as temperatures ranging from – 15 °C to more than 100 °C, or in the absence of oxygen or light. Neither animals nor plants are so adaptable; this is also true as regards nutrition. Some microorganisms can even utilize exotic energy sources such as alkanes, inorganic salts, or substituted aromatic compounds.

Generally, the term ‘microorganism’ encompasses all living beings too small to be seen by the human eye without magnification, i.e. smaller than about 1 mm. They are either prokaryotic, e.g. bacteria, or eukaryotic, e.g. hyphal fungi, yeasts and protozoa. We focus here on fungi and yeasts. For historical reasons the term ‘fungus’ is used for filamentously growing microorganisms, whereas yeasts are single-celled eukaryotes. However, taxonomically both are fungi (see Chapter 2).

‘Fungi are often found in damp places; that is why they are shaped like umbrellas.’

This schoolboy howler, possibly aprocryphal, summarizes the parlous state of knowledge about fungi that all too often exists in university-level students, and it is with the aim of counteracting the perceived low status of fungal biology, in contrast to the biology of animals, plant and bacteria, that this book was conceived. It is our goal to demonstrate that fungal research is dynamic, active and exciting. Fungi have played major roles in developing central concepts in biology (see Chapter 1) and continue to be vital model organisms in many areas of research. They have vital roles in animal and plant disease, ecology and biotechnology.

The key to the resurgence of fungal biology has undoubtedly been the development of molecular biology tools for these organisms. A theme of this book is that molecular biology is almost always a vital tool in current research. Part of the reason for the difficulty in studying fungi is their diversity. This has made a succinct, useful definition of fungi impossible. The definition of fungi as the organisms studied by mycologists is an aphorism designed to confuse. Such confusion is now readily explained since, largely through the use of molecular tools (see Chapter 2) the relationship between the diverse fungal groups has become much clearer.

The rest of this book concentrates on the area of mycology where research is substantial and, largely using molecular biology, a detailed functional understanding is emerging.

Introduction

Fungal mycelia will continue to grow and invade new substrates for as long as satisfactory conditions prevail (Chapter 9), typically producing numerous asexual spores (Chapter 7) and other mitotically derived invasive, reproductive and/or resistant structures (strands, rhizomorphs, sclerotia, stromata, etc.). Except for the Mycelia Sterilia (Deuteromycotina, Chapter 2), under particular conditions a fungus enters a sexual pathway, resulting in genetic segregation and production of recombinant progeny.

Since most fungi seem to be haploid for most of their life cycles, the first step in this process is to bring together two haploids so that nuclei can coexist in the same cytoplasm, undergo karyogamy followed by the meiotic division, and then generate and distribute progeny spores. These processes are considered in this chapter (see also Carlile and Watkinson, 1994; Elliott, 1994; Moore, 1998).

Sex: what and why?

Most fungi produce abundant asexual spores which are extremely effective in dispersing the organism. We have to ask why so many fungi invest more resources in a more complex sexual reproduction. There are, indeed, many fungi which only reproduce asexually but the majority still have a sexual cycle. Sex must have selective advantage if sexual stages are not to be replaced by asexual ones entirely (Maynard Smith, 1978).

The crucial point which provides the contrast with asexual reproduction is fusion of nuclei derived from different individuals. If the individuals differ in genotype, the fusion nucleus will be heterozygous and the products of the meiotic division can have recombinant genotypes.

The hyphal mode of growth

The dominance of filamentous fungi within the ecosystem is attributed to their common mode of growth, extending as branched filaments (hyphae) which can rapidly spread across uncolonized substrates. The success of this growth habit for exploiting the natural environment can be judged on a number of factors: the extraordinary diversity of fungal species (estimated at three million, second only to the insects), their distribution in virtually every habitat on the planet and the parallel evolution of a similar growth habit by another important class of soil microorganisms, the prokaryotic streptomycetes. Clearly the ability of a microbe to rapidly colonize new substrates by concentrating growth at its apex, is well suited for life as a heterotroph in a heterogenous environment.

Spore dormancy and germination

Spores are products of both sexual and asexual reproduction and act as units of dispersal in fungi. The majority of spores germinate to produce one or more germ tubes and a new fungal mycelium when the spore settles on an appropriate substrate under favourable environmental conditions. When a spore is faced with unfavourable conditions such as lack of nutrients, low temperature, an unfavourable pH or the presence of an inhibitor (e.g. on a plant surface), the spore remains dormant. Spores under these conditions are exogenously dormant and will only germinate when the environmental conditions become favourable.

In less than two decades, beginning when Walker and Doolittle (1982) examined relationships among aquatic fungi based on RNA nucleotide sequence, comparative studies of nucleic acid variation have revolutionized evolutionary mycology. Although there is far more remaining to be done than has been done, the big picture of fungal evolution is coming into focus. In this chapter we combine a review of the main points found in the new phylogenetic trees of fungi with a look at the methods used to make the trees. The challenge is not just to learn the current trees, but to learn how they are made and evaluated. The concept of testing alternative evolutionary histories is as important as the new insights that have been gained on fungal evolution; specific trees will change as new data become available but the need to evaluate alternative trees will remain.

Phylogenetic patterns among fungi

To appreciate the phylogeny of fungi, it is necessary to consider them in relation to the rest of life on earth, which is believed to be a continuum beginning several billions of years (Gyr) ago and leading to the present. We will break the continuum into three levels, the ‘Big Picture’ of fungal evolution or how the organisms studied by mycologists relate to the rest of life, the four phyla that make the kingdom Fungi, and individual members of the four phyla.

Big picture of fungal evolution

The most basic question is, how are fungi related to other biota?

Introduction

The yeast Saccharomyces cerevisiae is arguably one of the most important fungal organisms used in biotechnological processes. Making bread and alcoholic beverages, yeast has served mankind for several thousands of years. Many enzymes and biological compounds useful in biochemical research have been produced from yeast cells. In the mid-1930s yeast was introduced as an experimental system for molecular biology (Roman, 1981) and has since received increasing attention. The elegance of yeast genetics and the ease of manipulation of yeast, and finally the technical breakthrough of yeast transformation to be used in reverse genetics, have substantially contributed to the enormous growth in yeast molecular biology (Broach et al., 1981; Strathern et al., 1981; Guthrie and Fink, 1991). This success is also due to the fact, which was not anticipated a couple of years ago, that the extent to which basic biological structures and processes have been conserved throughout eukaryotic life is remarkable.

Yeast: an experimental system for molecular biology

Yeast is a versatile eukaryotic model organism

It is now well established that yeast is an ideal system in which cell architecture and fundamental cellular mechanisms can be successfully investigated. Among all eukaryotic model organisms, S. cerevisiae combines several advantages. It is a unicellular organism which, unlike more complex eukaryotes, is amenable to mass production. It can be grown on defined media giving the investigator complete control over environmental parameters.

Introduction

It is evident that living organisms regulate their metabolism in order to balance the conflicting demands of providing building blocks for synthesis and growth whilst at the same time providing energy derived from catabolism. These metabolic pathways are regulated by well-characterized enzymatic methods such as feedback inhibition and, once appropriate levels of enzymes have been provided, can be efficiently regulated at the level of the control of metabolic flux by the myriad of interactions between the enzymes and metabolites. Many essential enzymes and proteins are produced constitutively, but others are regulated at the level of gene transcription and/or translation. It is likely that in the most primitive cells, metabolic pathways evolved before transcription regulatory mechanisms. Transcriptional control may have evolved, therefore, to act as a damping mechanism, smoothing out the effects of more rapid changes caused by the build-up and dissemination of pathway metabolites. Or such regulation may be required to target the production of enzymes to particular tissues or organelles, or to allow the organism to use preferred carbon or nitrogen sources. But one of the driving forces for the cell may have been energy conservation: ensuring that enzymes are only supplied when they are needed, and stopping unnecessary enzyme synthesis. In this possible latter role, the cell has had to develop metabolite recognition mechanisms and signal transduction pathways that are able to sense particular cellular metabolites and couple this to transcription control mechanisms.

Introduction

Scope

Biotechnology is defined here as the exploitation by man of biological systems for manufacture of biomass or derived products. Filamentous fungi have been used by man for centuries but the technology is now advancing at an unprecedented rate raising the potential for fungal applications to new heights. A successful product-based biotechnology relies upon the combination of several factors but, principally, being able to supply a desirable product at the right price. Although such economic realities pervade all biotechnology, they will concern us little in this chapter. Rather, we will concentrate on fungal products already available commercially and some which could become so. The aim of the chapter is to discuss the biological aspects of the formation of such products by fungi and to discuss the impact that modern molecular biology plays in improving their yields and creating novel products.

History

The long history of using filamentous fungi in the food industry indicates that many species are safe either for consumption or for the production of food components. Several species of edible mushrooms have long been cultivated but, in addition, others that do not form large fruiting bodies are also grown for human consumption. The other established technology using filamentous fungi is the production of fermented foods, e.g. soy sauce, where fungi are used as sources of enzymes that degrade complex substrates not otherwise available for use by bacteria and yeasts.