Lecture #13

Introduction to the Ascomycetes:

Ascomycota is the largest phylum of fungi

Ascomycota and Basidiomycota share a number of characters:

compartmentalized mycelium

dikaryotic stage in the life cycle

plectenchymatous structures associated with spore production, conidia

the two phyla have diverged from a common ancestor

Primary morphological character that distinguishes members of the Ascomycota are the ascus a sac-like cell containing the ascospores cleaved from within by free cell formation after karyogamy and meiosis. Eight ascospores typically are formed within the ascus, but this number may vary from one to over a thousand according to the species.

(IMAGE) Mycelial ascomycetes are characterized by a compartmentalized mycelium with distinctive walls, septa having simple pores, and the presence of Woronin bodies

saprobes - biotrophic
masters of symbioses: mutualists - commensals - parasitic
terrestrial - aquatic - marine

Pneumocystis carinii
Cryphonectria parasitica
Annisogramma

Many ascomycetes are closely associated with insects. Some, such as Ophiostoma, Ambrosiella, Raffaelea, and Symbiotaphrina, are found in insect mycangia and provide or detoxify the food of the insects.

Ascomycetes produce most of the known plant growth regulators; disease symptoms called leaf curls and witches' brooms, seeing the effects of these fungal products that induce the plants to differentiate in unusual ways. Gibberellin, the growth regulator involved in stem elongation, was first discovered in rice infected by Gibberella fujikuroi, the cause of foolish seedling disease.

Fungal secondary products may act as pheromones, some of which provide signals to fungus-associated insects or mammals. E.g., truffles

General Characteristics

Ascomycetes may have two distinct reproductive phases, one sexual involving the formation of the asci and ascospores mentioned earlier, and the other asexual, with spore production occurring at different times on the same mycelium.

(IMAGE - Asco life cycle)

Ascomycetes are delimited and classified by their sexual reproductive structures; vast numbers of ascomycetes known only by their asexual stages can be difficult, and the practice has been to place these taxa in the artificial group called Deuteromycota or Fungi Imperfecti.

Somatic Structures Somatic stages of ascomycetes may be single-celled, mycelial or dimorphic A large proportion of the cell walls of filamentous ascomycetes is chitin In the yeasts (Saccharomycetales) mannans and ß-1,3 glucans are the principal wall polysaccharides, and only limited amounts of chitin are present, often restricted to bud scars

In ascomycetes the hyphal wall appears two-layered, with a thick translucent inner layer and a dense, thin outer layer. Some basidiomycete walls are characterized by several interspersed dense and translucent layers, but this is not universal. Hyphae of ascomycetes are divided into compartments by septa that form from the hyphal periphery and advance toward the center, thus invaginating the plasma membrane. In most ascomycetes a small circular opening or pore is left near the center of the septum through which the plasma membrane and cytoplasm extend from one hyphal compartment to the next

In the filamentous ascomycete septum, the potential exists for cytoplasmic continuity between all parts of the mycelium; septal pores may be plugged or blocked by various types of membrane-bound structures

(IMAGE)

Woronin bodies are spherical, hexagonal, or rectangular membrane bound structures with a crystalline protein matrix that usually are associated with the septum; elevated levels of nitrogen, sulfur, and phosphorus in Woronin bodies when they were compared to the adjacent cell walls of the species they studied. Woronin bodies frequently plug the septal pores of hyphae and it is believed that they serve to separate aging or damaged hyphae from the rest of the mycelium, but details of their exact function remain unknown.

In addition to the Woronin bodies that may plug septal pores, a more complex structure occurs in some filamentous ascomycetes;septal pore organelles, often shaped like pulley wheels, that are distributed in parts of the mycelium so that structures involved in sexual reproduction routinely are isolated from other regions of the mycelium. They usually are found at the base of asci and sterile parts of the hymenium of the ascocarp.

Hyphal compartments often are uninucleate, but mycelia consisting of multinucleate cells are well known. The perforations in the hyphal septa permit nuclei to migrate from one compartment of a hypha to another; the ability of nuclei to migrate throughout the mycelium is important in the phenomenom of heterokaryosis Ascomycete mycelium may be organized into fungal tissues (plectenchyma); If such a tissue is loosely woven and the mycelial strands are more or less evident, it is known as prosenchyma. If, however, the hyphae have lost their individuality and the cells are more or less isodiametric, closely resembling the parenchyma of plants, it is known as pseudoparenchyma.

Sexual Reproduction - unifactorial

Two compatible nuclei are brought together in the same cell by one of several methods:

1. Two morphologically similar gametangia touch at their tips or coil around each other and fuse. The fusion cell develops into the ascus. No dikaryotic phase is developed in most species because karyogamy takes place immediately after plasmogamy. In the yeasts, which are mostly unicellular, the somatic cells themselves act as gametangia with the zygote becoming transformed directly into the ascus

2. Some species produce morphologically differentiated uninucleate or multinucleate gametangia. These are called antheridia and ascogonia. Asci develop from outgrowths of the ascogonium, which is designated the female gametangium. The male nucleus passes from the antheridium into the ascogonium at the point of fusion between the two gametangia. The ascogonium bears a specialized hypha, the trichogyne that receives the male nucleus. This process does not involve formation of a fusion cell, and a dikaryotic stage may persist for a while before karyogamy occurs.

3. In some species a process called spermatization involves a single detached male cell that becomes attached to the female receptive organ--whether a trichogyne or somatic hypha--and empties its nucleus into the receptive cell. The male nucleus or nuclei migrate to the ascogonium through the septal pores. The male cells are dispersed by insects, wind, or water to the receptive female organs. The functional male gamete may be a spermatium, microconidium, or conidium. Spermatia are minute, spherical or elongated, uninucleate, male sex cells incapable of germination by a germ tube. It generally is assumed that they are evolutionary reduced from conidia. Spermatia may be formed on the mycelium or in specialized structures called spermogonia. Microconidia have been described as minute conidia that behave as spermatia, but which also are capable of germinating and giving rise to mycelium. Conidia also may function as spermatia by attaching themselves to the receptive organs and emptying nuclei into them.

4. Plasmogamy - sometimes referred to as somatogamy - involves the fusion of unspecialized somatic hyphae of two compatible mycelia with the nuclei migrating to the ascogonia through the septal perforations. This is a method of plasmogamy that may not be prevalent in ascomycetes, but is usual in many basidiomycetes.

In the ascomycetes, with yeasts being the major exception, the two nuclei may remain in close association and undergo successive divisions that result in a number of dikaryotic cells.

Nuclear fusion eventually takes place in the young ascus. Meiosis in the diploid zygote nucleus occurs almost immediately after fusion, and results in the production of four haploid nuclei. These four nuclei then divide mitotically, resulting in the formation of eight nuclei that will become incorporated into the eight ascospores during ascosporogenesis.

Incompatibility Systems

Homogenic incompatibility- is a process that promotes outcrossing in sexual fusions and is controlled by mating type genes.

Heterogenic incompatibility- the process that governs fusion of like somatic or vegetative hyphae; referred to as somatic or vegetative incompatibility.

Sexual reproduction in heterothallic ascomycetes requires the participation of genetically different strains. The mating system is controlled by a single genetic locus (MAT) which specifies one of two alternative mating types, and is termed unifactorial or bipolar.

The first mating type locus cloned was from Saccharomyces cerevisae. Haploid cells are designated either MAT A or MAT a. Each mating type signals its presence to a cell of the opposite mating type by producing specific peptide pheromones. The a cells secrete a factor that is recognized by a receptor protein in A cells, and conversely A cells produce A factor that binds to a receptor in a type cells. The binding of both pheromones to the receptors results in a transient arrest of the cell cycle and induces the production of cell surface molecules that facilitate conjugation and fusion of the two cells

In the ascomycetes that have been studied, the genes at the MAT loci are completely dissimilar and cannot be regarded as homologous alleles. The term "idiomorph" has been coined to describe such alternative forms of a locus that lack significant sequence homology, but occupy the same site in the genome

Other points about ascomycete mating loci.

Mating type switching - occurs when a cell of the opposite mating type is not present and is due to "silent copies" of the second mating gene that are present at other loci in the genome of a single cell. Some ascomycetes are homothallic.

Vegetative or somatic incompatibility in ascomycetes prevents the fusion of genetically different mycelia (heterogenetic incompatibility) and is usually under multigenic control by a series of bi- or multiallelic genes at vegetative incompatibility (het or vic) loci. When two mycelia in different vegetative compatibility (vc) groups meet in a substrate, they may interact with varying degrees of antagonism, as would be expected of a phenotype under multigenic control. In some cases barrage reactions may be recognized by a clear zone between the two mycelial fronts due to the lysis of the interacting cells. Sometimes incomplete interactions occur at the margins and unstable dikaryons may exist for a short time. In some cases pigments or enhanced conidium production indicates the meeting of the two mycelia

How do heterothallic ascomycetes in different VC groups undergo sexual fusions. In the species that have been studied, separate individuals are maintained until the time of sexual reproduction when morphologically and physiologically differentiated sex organs, the ascogonia and antheridia, develop and apparently are not usually under the control of the vegetative incompatibility system.

Life Cycle Pattern

No "typical" life cycle to describe

Pyronema omphaloides as an example that illustrates the pattern of both sexual and asexual reproduction in a single life cycle

1. Ascospore germinates by one or more germ tubes to produce a mycelium. As germ tubes issue from a spore, the spore nucleus divides and the nuclear progeny are distributed within the growing hyphae. Soon after hyphal growth is initiated, septa are laid down to delimit uninucleate compartments, and hyphal branching takes place.

2. Growing mycelium may begin to produce conidia almost immediately. Conidia may be important in providing a continuous and abundant source of inoculum for the localized dispersal of the fungus for a period of months; many crops of conidia may be borne in a single season. The conidia are produced in enormous numbers, and are responsible for the propagation and dissemination of the individual for much of its life. Under favorable conditions a conidium germinates by a germ tube, and produces a mycelium that is in all ways similar to that produced by the germination of the ascospores. In this way, a large number of asexual generations are produced during the growing season.

3. At some time when appropiate conditions exist, the mycelium ceases conidium production and begins to undergo a complex series of physiological changes that will allow sexual reproduction to be initiated. The ascogonia are differentiated from the somatic hyphae. Compatible nuclei now are brought to the ascogonium by contact of an antheridium and an ascogonium and the passage of the antheridial nuclei into the trichogyne and eventually into the ascogonial base.

4. After plasmogamy the ascogonium produces a number of papillae just opposite groups of nuclei located in the periphery of the ascogonium. The ascogonial walls appear to be thinner at these points. As these papillae enlarge, nuclei from the ascogonium begin to pass into them one by one. Eventually the papillae elongate into ascogenous hyphae in which a leading pair of nuclei can be detected, followed often by a second pair. The nuclei in the ascogenous hyphae and those still in the ascogonium soon undergo simultaneous mitosis. Primary septa are formed between daughter nuclei in such a way that the tip cell of the ascogenous hypha is uninucleate and is followed by a series of binucleate cells that contain pairs of non-sister nuclei. It is probable that one nucleus in each binucleate cell of the ascogenous hyphae is ascogonial in origin and the other antheridial. This is the dikaryotic phase of the life cycle.

5. In a large number of ascomycetes, including P. omphaloides, one of the binucleate cells of the ascogenous hypha elongates and bends over to form a hook or crozier. The two nuclei in this hooked cell divide in such a way that their spindles are oriented more or less vertically and parallel to one another, so that two of the daughter nuclei-one from each spindle and, therefore, of different origin-are close to one another at the bend of the hook, while one of the other two nuclei is located at the tip and one near the basal septum of the hook. Two septa are laid down, separating the hook into three cells. The tip and basal cells are uninucleate, one containing an antheridial nucleus and the other, an ascogonial nucleus; the crook cell is binucleate. This binucleate crook cell is the young ascus.

6. Karyogamy takes place in the young ascus soon after the septa are formed in the hook, thus ending the dikaryotic phase. The ascus with its diploid, zygote nucleus begins to elongate, and the zygote nucleus undergoes meiosis. The four haploid products of meiosis each undergo a mitotic division so that a total of eight nuclei are formed. These will become incorporated in the ascospores that develop within the ascus by free cell formation.

In many species of filamentous ascomycetes each ascogenous hypha branches and rebranches in various ways, and produces a cluster of asci. This often is accomplished as follows: The crook cell elongates into a new hook instead of developing directly into an ascus, and the tip and basal hook cells fuse and form another hook by the side of the first. This process may be repeated several times, forming a cluster of hooks, the crook cells of which finally develop asci.

Ascosporogenesis.

Ascospores are formed within the ascus as a result of free cell formation or ascosporogenesis.

Two mechanisms involved:

1. Portions of cytoplasm, each typically containing a single nucleus, are delimited by an envelope consisting of two closely appressed unit membranes, and
2. The ascospore wall is deposited between these two membranes to separate them from one another as the spore matures.

In most of the filamentous ascomycetes examined these envelopes initially are part of a discontinuous cylinder that forms around the extreme periphery of the ascus very near the plasma membrane and surrounds all of the nuclei of the ascus. This cylinder, which consists of two closely spaced unit membranes is called the ascus vesicle or the enveloping membrane system (EMS).

The EMS invaginates and fragments into sheets that then cleave out the young ascospore; not all the cytoplasm within an ascus is incorporated into the ascospore initials. The portion that remains outside the spores is called epiplasm and possibly serves to nourish the developing spores and deposit the external ornamentation on the spores.

Once an ascospore initial has been delimited by the EMS, the inner membrane of the EMS becomes the spore plasma membrane while the outer membrane becomes what has been termed the "ascospore investing membrane". The investing membrane is displaced progressively from the plasma membrane of the spore as the spore wall develops between these two membranes. Although it appears that the inner portion of the ascospore wall is deposited by the spore itself, in some species at least part of the outer wall layer(s) as well as the spore ornamentation appear to be deposited by the epiplasm of the ascus.

Most common number of ascospores produced per ascus in most species is eight. However, some species routinely produce asci containing smaller numbers of spores including one, two, three, or four, while larger numbers of spores, up to possibly as many as a thousand, are produced in a few species. Ascospores come in a variety of sizes and shapes ranging from long, thin, and thread-like to globose and even hat-shaped in appearance. Ascospores may be one-celled or septate.

Croziers and clamp connections:

Crozier and clamp connections are homologous?
Ascogonial coils
Ascus mother cell - basidium
Endospores vs. exospores

(IMAGE - See p81)

Asci.

Asci may be spherical to elongated with cylindrical, ovoid, or globose forms

Asci may be stalked or sessile; they may arise at various levels within the ascocarp or from a single level.

A definite layer of asci, whether naked or enclosed in an ascocarp is called a hymenium.

Developmental studies have shown that sometimes asci may develop in a hymenium, but become rearranged at maturity so that they appear to be scattered.

Three basic types of asci can be defined from light microscopy studies: prototunicate, unitunicate, and bitunicate.

The prototunicate asci have a thin, delicate wall and release their spores by deliquescing.

The wall in both a unitunicate and a bitunicate ascus is said to consist of two layers: exotunica and endotunica. In the so-called unitunicate ascus these layers adhere closely throughout the life of the ascus, and the spores are released through a terminal pore, slit, or hinged cap (operculum).

In the bitunicate ascus the endotunica expands up to twice or more its original length, separating from the ruptured exotunica at the time of spore release. Spores are released through a pore in the endotunica. Because of this behavior, the bitunicate ascus has been called the Jack-in-the-box ascus or the fissitunicate ascus by lichen specialists.

Ascocarp

In general there are five ways that ascomycetes can be separated according to the way they bear their asci:

1. Those that bear naked asci without any fruiting body
2. Those that produce their asci inside a completely closed ascocarp called a cleistothecium
3. The perithecium, that is more or less closed, but at maturity is provided with a pore (ostiole) through which the ascospores escape
4. Those that produce their asci in an open ascocarp, called an apothecium; and
5. Those that form their asci directly in a cavity (locule) within the stroma; the stroma, which may be likened to a cushion of closely woven somatic hyphae, forms the wall of the ascocarp. We call such a structure an ascostroma or pseudothecium

(IMAGE p252)

Ascocarps may be formed singly or in groups. They may be superficial, erumpent, or deeply embedded in the substrate. In some cases ascocarps with true walls may form in a stroma. The stroma may be composed of both fungal and host tissue in some plant pathogenic species, or a stroma may be made entirely of fungal tissue

Hamathecium- the sterile cells and hyphae that are interspersed among asci or project into the locule or ostiole of the ascocarp.

1. Interascal pseudoparenchymais the relatively unmodified tissue that may become compressed between developing asci.
2. Paraphysesare elongated, cylindrical or club-shaped hyphae originating at the base of an ascocarp and growing among the asci in a hymenium; branched or unbranched and may be septate or non-septate. Paraphyses remain free at their tips except in some discomycetes in which the tips fuse above the asci forming a solid layer, the epithecium.
3. Periphysesare short unbranched hyphae in the ostiolar canal of an ascocarp. They apparently serve to direct the asci toward the tip of the ostiole prior to ascospore discharge
4. Apical paraphysesor periphysoids are short hyphae that originate above the level of the developing asci and grow down in a palisade layer among the asci
5. Pseudoparaphysesoriginate above the level of the asci of an ascostroma and grow down among the developing asci. They reach the base of the ascocarp where they fuse with the base of the cavity. The upper part may become free. These hyphae may be septate, branched and anastomosing.
6. Paraphysoidsare tissue remnants in ascostromata that remain between the locules as the asci grow upward. Such structures are, of course, attached at both ends from the beginning and often are confused with pseudoparaphyses.
7. A seventh condition, the complete absence of sterile elements, also may occur.

Release, Dispersal and Germination of Ascospores.

In some cases the method of release is passive with physical forces or animals breaking the asci.

Many ascomycetes ascospores are released by forcible ejection and the second event, dispersal, is by another agent.

In species that do not form ascocarps, the release of the spores generally takes place by the breaking or the deliquescence of the asci formed on the substrate. The spores are then free to be dispersed by wind, water, animals, or other agents.

In some groups the ascocarp is completely closed and the ascospores are liberated only on the partial or complete disintegration of the ascocarp. The release process may be hastened by ingestion by animals that also may be dispersal agents; e.g. truffles

In some ascocarps there is a pore through which the ascospores usually are aimed and forcibly ejected.

Ascocarps with the entire hymenia exposed at maturity also have ascospores that are discharged forcibly into the air. In the instances when the spores are shot into the air, some may be carried by air currents for comparatively long dispersal distances, although the majority fall in the vicinity of the place where they were produced.

In a large number of ascomycetes, ascospores are ejected forcibly from the ascus by a puffing action. In some species such as the morels, puffing is accompanied by a hissing sound. Ascospores are ejected either through the bursting of the ascus at the top or through a natural pore, slit, or cap-like operculum hinged at one end.

In certain yeasts and filamentous ascomycetes, ascospores may multiply by budding or conidium formation (repetitive or iterative germination) instead of germinating by germ tubes. Depending upon the environmental conditions, some ascospores have the ability to germinate by either method.

Somatic structures

Sclerotia are considered to be resistant or resting structures and their formation may serve to help a fungus survive conditions that are unfavorable to growth. They often are characterized by thick walls, and this may be the only criterion applied to assess their "resistant" function.

Stromata have a similar function, but rather than initiating mycelial growth directly, they give rise to conidia or ascocarps

Asexual Reproduction

may be carried out by fission, fragmentation, or formation of chlamydospores or conidia according to species and environmental conditions it is in the ascomycetes that conidium development has reached its zenith

Conidia often are important in propagating and disseminating species throughout the spring and summer with several generations being produced in a growing season.

Conidia may arise either directly from the somatic hyphae or from specialized conidiogenous cells, often borne on hyphal branches known as conidiophores.

Conidiophores vary from short hyphal branches to those that are long and intricately branched. In some species the conidiophores may be produced free from each other without any evident organization, while in other species they are joined together to form complex structures

Conidiogenesis

Blastic - growth of conidium occurs after septation; young conidium is recognizable prior to septation

Thallic - growth of conidium occurs prior to septation; young conidium is not recognizable prior to septation

Dehiscence

schizolytic - halves of dle septum split apart by breakdown of middle lamella

rhexolytic - outer wall of cell beneath or between condia breakdown

Development:

blastic acropetal - conidia develop in chains by apical budding;Cladosporium

(IMAGE)

blastic synchronous - conidia develop in chains synchronously;Botrytis

(IMAGE)

blastic retrogressive - conidia develop chains basipetally
blastic sympodial - alternate branching;Beuveria
blastic annelidic - possess annular scars; conidiogenous locus moves;Scopulariopsis
blastic phialidic - produce conidia in rapid succession from a specialized cell, the phialide;Penicillium, Aspergillus, Fusarium, Verticillium
thallic arthric - hyphae stops growing and becomes divided into arthrospores;Geotrichum
thallic solitary - hyphae stops growing and terminal cell becomes conidium;Microsporum

Conidia:

amerospores - aseptate
didymospores - single septum
phragmospores - several horizontal septa
dictyospores - muriform septation
helicospores - coiled spores
staurospores - stellate
scoleospores - curved, filiform

Conidiophores:

simple - complex (penicillate) - synnema - sporodochium

pycnidium - walls of structure are of fungal origin

acervular - walls of structure are of host origin

Classification.

Ascomycetes are taxonomically difficult, and over the last decade mycologists have concentrated on delimiting monophyletic orders rather than grouping orders in higher taxa 45 orders unplaced in higher taxa in the Systema Ascomycetum (Eriksson and Hawksworth, 1993).

Characters that distinguish ascomycetes

• a two layered cell wall containing chitin that does not stain with DBB
• asci
• ascospores delimited by an EMS
• forcible ascospore discharge, and

3 major groups of Ascomycetes based upon molecular data:

Archiascomycetes-Taphrina, Pneumocystis, Schizosaccharomyces

Saccharomycetales are characterized by the loss of simple septal in all except a few taxa, restriction of chitin primarily to bud scars in the cell walls in most; mannans and ß-1,3 glucans are the primary wall polysaccharides; EMS in most yeasts examined appears to be derived from membranes that are associated with individual nuclei, rather than as a cylinder initially enclosing all of the nuclei.

Euascomycetes- the filamentous ascomycetes; mycelium with a simple septal pore, an ascus vesicle, and forcibly discharged ascospores. Woronin bodies are associated with the septal pores; production of ascogenous hyphae with many asci resulting from a single mating and the formation of an ascocarp. This group appears to have diverged rapidly and is marked by a diversity of mycelial types, ascus structure and function, and ascocarp morphology. In addition filamentous ascomycetes are notable for their elaboration of conidium structure and function.

Hymenoascomycetes and Loculoascomycetes

Ascomycetes usually are classified on the basis of sexual reproduction. Forms that do not reproduce sexually have been placed in an artificial group, either Deuteromycota or Fungi Imperfecti; the application of molecular techniques provides a means to incorporate the two.