MicrobiologyBytes: Microbiology Notes: Mycology Updated: April 8, 2009 Search

Introduction to Mycology

Fungi have the several features that distinguish them from other organisms.

They have a filamentous branching system of cells, with apical growth, lateral branching and a heterotrophic nutrition. They are characterized by a life cycle that begins with germination from a spore or resting structure, followed by a period of growth as a substrate is exploited to produce biomass. Finally there is a period of sporulation, where propagules are formed that can be disseminated from the parent mycelium - Fig. 1. Schematic of a fungal life cycle:

They are vital to the biosphere for many reasons, not least for their decomposing activities on dead substrates that ensure the release of nutrients like carbon, minerals and nitrogen back into the biosphere.

MicrobiologyBytes: Fungi

Taxonomy

The origin of the fungi appears to be very ancient. They first appear in the fossil record coincidentally with the appearance of the land plants. This was in the Devonian period around 400 million years ago. The early microscopists found that fungi were simple to study, and they first observed division of cells by watching yeasts under the microscope.

They were one of the earliest groups to be classified, and the earliest scheme was drawn up in 1588. Early biological science was much obsessed with categorizing things and researchers constructed taxonomic systems based solely on structure. However, for the fungi, this created a polyphyletic group that contained microorganisms that had very different ancestors.

Taxonomic thinking today now prefers a monophyletic classification, where all groups within a phylum are descendants of one ancestor. Therefore current taxonomy relies on studying characters that are features or attributes of an individual organism that can be used to compare it with another organism. These features can be morphological, anatomical, ultra-structural, biochemical or based on sequences of nucleic acids.

Fungi are referred to as the True Fungi, or Eumycota, and are currently divided into four (plus one form class) major phyla on the basis of the morphology and sexual reproduction (see Table 1). New information based on presence and type of mitochondria, and the DNA sequencing of ribosomal RNA, place the members of the fungi into a complex phylogenetic tree.

Table 1. Features of the main groups of fungi.

Group:

Perforate septae +/-

Asexual sporulation:

Sexual sporulation:

‘Lower fungi’ :

Zygomycotina

-

non-motile sporangiospores

zygospore

Chytridiomycotina

-

motile zoospores

oospore

‘Higher fungi’ :

Ascomycotina

+

conidiospores

ascospore

Basidiomycotina

+

rare

basidiospore

Deuteromycotina

+

conidiospores

none

 

Two of the phyla are sometimes incorrectly described as Lower fungi, the Zygomycotina and the Chytridiomycotina. In the lower fungi vegetative mycelium is non-septate, and complete septa are only found in reproductive structures. Asexual reproduction is by the formation of sporangia, sexual reproduction by the formation of zygospores. We will return to these details later.

The other two phyla are sometimes described as the Higher fungi and have a more complex mycelium with elaborate, perforate septa. They are divided into the Ascomycotina and the Basidiomycotina. Members of the Ascomycotina produce asexual conidiospores and sexual ascospores in sac-shaped cells called asci. Fungi from the Basidiomycotina rarely produce asexual spores, and produce their sexual spores from club-shaped basidia in complex fruit bodies.

A particular problem for some of the fungi is that they do not possess sexual stages, and this lead to the construction of an artificial taxonomic group based on the asexual, mitosporic stage of the fungus. This group is a form group called the Deuteromycetes, or Imperfect fungi. However, using the current cladistic approaches mycologists are slowly establishing links between these species and other species with sexual stages.

It is important to know the taxonomic terms for the kingdom.

Kingdom:

Fungi

Division:

Eumycota

(sub-division)

e.g. Basidiomycotina

Class:

e.g. Basidiomycetes

Order:

e.g. Agaricales

Family:

e.g. Agaricaceae

Genus:

e.g. Agaricus
Species: e.g. Agaricus bisporus
- The mushroom!

 

Cell biology of the fungi

Their basic cellular unit is described as a hypha. This is usually a tubular cell which is surrounded by a rigid, chitin-containing cell wall. The hypha extends by tip growth, and multiplies by branching, creating a fine network called a mycelium. Hyphae contain nuclei, mitochondria, ribosomes, Golgi and membrane-bound vesicles within a plasma-membrane bound cytoplasm. The sub-cellular structures are supported and organized by micro-tubules and endoplasmic reticulum. If you have forgotten what these structures look like and what they do, please check them in a basic text book.

Not all fungi are multicellular, some are unicellular and are termed yeasts. These grow by binary fission or budding, creating new individuals from the parent cell. Simplified diagram of a vegetative yeast cell and a budding cell:

Fungal metabolism

Fungi prefer moist habitats and they are largely mesophyllic, preferring temperatures between 15°C and 35°C. The carbon needs of fungi for energy metabolism and biosynthesis has to be met heterotrophically by one of three lifestyles:-

a. parasitism of plants or animals (causing disease)

b. saprophytism, growing on dead animal, plant or microbial biomass

c. symbiosis, growing together with algae, plants or insects

Soluble carbohydrates must enter hyphae by diffusion, and this is followed by active uptake across the fungal membrane. For the saprophytic fungi most carbon in the environment is present as a complex polymer like cellulose, chitin or lignin. These materials must be broken down enzymically before they can be utilized. Therefore fungi release degradative enzymes into their environments including cellulases, chitinases, proteases and multi-component lignin degrading enzymes, depending on the type of substrate the fungus is growing on. Regulation of these enzymes is by substrate-induction and end-product inhibition. Most fungal carbohydrate metabolism is via glycolysis and the tricarboxylic acid cycle, although both fermentative pathways and anaerobic pathways have been found in specialized fungi.

Fungi cannot fix gaseous nitrogen. They can however utilize nitrate, ammonia and some amino acids by direct uptake across the hyphal membrane. More complex nitrogen sources, such as proteins and peptides, can be utilized. once extracellular proteases have degraded them into amino acids. Fungi require both macro and micronutrients, but these are usually available to excess in their environments, and some fungi may have other requirements, for example thiamin and biotin, sterols, riboflavin, nicotinic acid and folic acid.

Fungi are capable of the formation and secretion of many types of secondary metabolites. These compounds can be produced by many different metabolic pathways.

Cell wall structure and growth: the structure of the fungal cell wall

The living part of a fungus consists of the apical cell of the mycelium and a few cells that are immediately behind it. Cells that are beyond about 5 cross walls, that is septae, and usually moribund unless they have special survival functions. The cells are very variable in size, but usually fall between 3-10 µm wide and 50 µm long. However, the apical cell of the mycelium is usually 300 to 400 µm long.

The septae that separate the cells are usually perforate, and have varying structures to protect the cell from loosing its contents if there was a disruption of a neighbouring cell. In the Ascomycetes the septae are protected by a Woronin body, an osmophilic oily structure which can plug the septum if required, or in the Basidiomycetes there is a complex wall structure to the septum and also a complex of endoplasmic reticulum associated with the septal pore. This is called a dolipore septum. The septae of fungi:

The lower fungi do not have perforate septae, they have complete ones to isolate reproductive or vacuolated regions of mycelium from the rest of their mycelium.

Food material is absorbed from all over this living region, but mostly from around the tip. This creates a concentration gradient around the mycelium, especially from around the tip, and this creates the drive for the mycelium to grow forwards from the tip up the concentration gradient for nutrients and towards fresh substrates. Zones of depletion around a mycelium:

From light and electron microscopy it is apparent that fungal cell walls are made up of a complex of fibrillar materials with an amorphous matrix. Using enzymatic and chemical digestions it has been established that the fibrils in the true fungi are chitin, chains of N-acetyl-glucosamine. This is an acetylated amino sugar derived from glucose. This polymer is also found in insect exoskeletons.

The structure of the b 1-4 linkages allows for microfibrils to form, with many parallel molecules aligning. These microfibrils can be between 10 and 25 nm in diameter. They are embedded in an amorphous matrix, and the end effect is much like a fibreglass mat. The exact composition of the wall varies between the major taxonomic groups. In the Zygomycetes the outer wall layer contains b 1-3 and b 1-6 linked amorphous glucans with a chitin microfibrillar inner layer. In the chytrids the outer layer is similar, but the fibrillar component is cellulose. In the higher fungi the outer layer of the Ascomycete cell wall is again b 1-3 and b 1-6 linked amorphous glucans, but the inner layer contains proteins above the chitin microfibrils. Basidiomycetes differ in that their outer layer is composed of a 1-3 linked amorphous glucans above the layer of b 1-3 and b 1-6 linked amorphous glucans, proteins and cellulose microfibrils. These differences are important, especially in the development of atopy and allergy.

Yeast cell walls are rather different. They still contain the amorphous glucans, but there are instead of the linked fibrils of mannans, b 1-6 linked polymers which in turn are linked to proteins. This particular combination makes for a much more plastic wall and a spherical rather than tubular shape. It is the ability to move from mycelial to yeast form that allows many of the fungi to become pathogenic. This is called dimorphism, and I return to it later.

Growth

Growth occurs at the hyphal tip by the fusion of characteristic membrane-bound vesicles derived from the Golgi. These vesicles accumulate in the apical 10 µm of the hyphal tip. The organization of these vesicles varies in the different taxonomic groups. Hyphal tips from the three major taxa:

Cell wall synthesis:

As the hypha grows, the tip cell becomes larger and larger as it accumulates biomass. This gives rise to two phenomena. One we have already described, septation, and the other is branching. Branching occurs some way behind the first septum. Branching occurs by the formation of another apical cell growth point within a subapical cell. usually the branches are at a very precise angle to the parent hypha, characteristic of the karyotype of the cell.

Kinetics of growth

When fungi are filamentous their growth rate cannot be established by cell counting using a heamocytometer or by tubidometric measurements which can be used to measure bacterial and yeast growth . However, by measuring mass (M) changes with time (t) under excess nutrient conditions the specific growth rate (μ) for the culture can be calculated using the formula:

 dM = µM
 dt

Fungal growth in a given medium follows the growth phases of lag, acceleration, exponential, linear, retardation, stationary and decline phase. Phases of fungal growth:

Exponential growth occurs only for a brief period as a hyphae branches are initiated, and then the new hypha extends at a linear rate into uncolonised regions of substrate. Only hyphal tips contribute to extension growth. However, older hyphae can grow aerially or differentiate to produce sporing structures. Rates can be very rapid indeed. Some species may extend at 6mm/hour, but more often it is at around 0.1 to 2 mm/hour.

It is also possible to observe hyphal growth by microscopy, measuring tip growth and branching rates of mycelium. From this data the hyphal growth unit and the peripheral growth zone can be calculated. The hyphal growth unit (G), which is the average length of hypha which is required to support tip growth, is defined a ratio between the total length of mycelium and the total number of tips.

Total number of tips = hyphal growth unit
Total length of mycelium

In most fungi the hyphal growth unit includes the tip cell plus 2 to 3 sub-apical compartments. The ratio increases exponentially from germination, but stabilizes to give a constant figure for a particular strain under any given set of environmental conditions.

If you destroy the apical compartment of a fungal cell growth of that apex can no longer continue. It can however resume from the sub-apical compartment. The exact length of hyphae needed to maintain that apical growth can be found by making experimental incisions in a colony margin. This so called peripheral growth zone (PGZ)(w) is the region of mycelium behind the tip needed to support maximum growth of the hyphal tip. This zone permits radial extension at a rate equal to that of the specific growth rate of unicells in liquid culture. The mean rate of hyphal extension (E) is a function of the hyphal growth unit and the specific growth rate

E=Gm

The zone can be as much as 12 cells deep in some fungi, particularly in the lower fungi that have very few septae. Higher fungi with protected perforate septae can have very small PGZs .The radial extension rate (Kr) is a function of the peripheral growth zone and the specific growth rate (m ).

Kr = w m

Growth is a highly regulated process, and is affected by light, heat, changes in osmolarity, ionic strength of the media and by pressure. Growth is also accompanied by nuclear division and cross wall formation. The apical compartment of the hypha is often mutinucleate, sometimes there are over 50 nuclei. This is because there are repeated mitotic divisions of the nucleus in a parasynchronous wave, beginning at the tip and moving backwards towards the first septum. The mitotic division is very rapid, taking about 4 minutes, and the hypha continues to grow forwards whilst this is happening. Undifferentiated growth does not continue indefinitely. Fungal colonies show a differentiation from margin to the centre of the colony.

  1. The extension zone. Hyphae are advancing into fresh media. Branching patterns tend to be at a 90° angle and monopodial. The branches are arranged to give the most effective colonization and utilization of the substrate, and the growth of the hyphae is oriented to avoid the other hyphae. The mechanism by which this occurs is unknown, but probably relates to food gradients and toxin gradients.
  2. The productive zone. Here the major increase in biomass occurs. Aerial mycelium is formed, and the hyphae thicken.
  3. The fruiting zone. Biomass gain has ceased, and spores are formed. This is equivalent to the stationary phase of batch culture.
  4. Ageing zone. Vacuolate or empty hyphae are present, autolysis occurs. This is equivalent to the decline phase of batch culture.

 


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