Isolation, cultivation, identification
polish transl. www.psilosophy.info/kkywpyigaxepaubmcbacbaib
original source: http://website.nbm-mnb.ca/mycologywebpages/Moulds/Moulds.html
Table of Contents:
Moulds, those dusty little spots often found spreading over bread, cheese, books, and other things in the home, cause the loss of millions of dollars to our economy every year and, even worse, may be a menace to your health. To deal with them successfully we must understand what moulds are and exactly what they are doing.
Moulds are microscopic, plant-like organisms, composed of long filaments called hyphae. Mould hyphae grow over the surface and inside nearly all substances of plant or animal origin. Because of their filamentous construction and consistent lack of chlorophyll they are considered by most biologists to be separate from the plant kingdom and members of the kingdom of fungi. They are related to the familiar mushrooms and toadstools,differing only in not having their filaments united into large fruiting structures. For our purposes here, we shall consider as moulds only fungi that are commonly encountered n the home and laboratory and that can be easily grown and studied.
When mould hyphae are numerous enough to be seen by the naked eye they form a cottony mass called a mycelium. It is the hyphae and resulting mycelia that invade things in our homes and cause them to decay.
Moulds reproduce by spores. Spores are like seeds; they germinate to produce a new mould colony when they land in a suitable place. Unlike seeds, they are very simple in structure and never contain an embryo or any sort of preformed offspring. Spores are produced in a variety of ways and occur in a bewildering array of shapes and sizes. In spite of this diversity, spores are quite constant in shape, size, colour and form for any given mould, and are thus very useful for mould identification.
The most basic difference between spores lies in their method of initiation, which can be either sexual or asexual. Sexually initiated spores result from a mating between two different organisms or hyphae, whereas asexual spores result from a simple internal division or external modification of an individual hypha. the recognition of a mating and subsequent spore formation is often difficult for an observer,and is usually reserved for patient specialists. However, for practical purposes one can learn to recognize certain indications of the sexual process, namely, the four kinds of sexually determined spores that appear in mould fungi: (1) oospores, (2) zygospores, (3) ascospores, and (4) basidiospores.
Oospores (Figure 1) are produced when male gametes (reproductive nuclei)enter a large spherical cell (oogonium) and fertilize the eggs within. The result, as seen in routine examination, is numerous oogonia containing one to several spherical and often brownish eggs. The oogonia are usually penetrated by one or more hyphae (antheridia) that give rise to the male nuclei.
Zygospores (Figure 2 and zygospore illustration) do not occur inside any kind of enclosing structure, but are produced by the direct fusion of two hyphal protrusions (suspensors) from neighbouring filaments. Usually zygospores are recognized as large,nearly spherical, often dark brown or black, rough-walled spores with two connecting hyphae, representing the two mating gametangia (figure 2C). Sometimes the zygospore may be surrounded by several finger-like extensions from the two gametangia.
Ascospores (Figure 3) are produced within spherical to cylindrical cells called asci, most often in groups of four or eight (figure 3A). Usually the asci are produced within some kind of enclosing structure and thus are not found exposed on the hyphae. In a few cases the asci may be borne among hyphae and resemble oogonia with eggs, but they will never be penetrated by any sort of fertilizing hypha. Fertilization occurs early in the life cycle and is not evident at the time ascospores are produced.
Basidiospores (Figure 4) are always produced externally on a structure called a basidium. Basidia come in a variety of forms, but those commonly encountered on moulds will be club-shaped and bear four or eight spores on sharp projections at the apex. At first it may be difficult to distinguish between a basidiospores and one of the asexually initiated spore types, but one should always suspect the presence of basidia when externally produced spores consistently occur in groups fo four or eight (figure 4). As with ascospores, basidiospores are the result of an early fertilization that is not easily observed.
Asexual spores usually occur either in sporangia or as conidia. Sporangia are modified hyphae or cells containing numerous spores (sporangiospores). They never have more than a single connecting hypha and the spores do not constantly occur in groups of four or eight as do ascospores.
Conidia are the most difficult group to characterize because of their great diversity of form. The only feature that most conidia have in common is that they occur externally on the cells that produce them. These conidium-bearing (conidiogenous) cells may occur within rather specialized or characteristic structures that resemble those that frequently bear asci, however, and it is often necessary to break them open to confirm that the spores are truly conidia and not ascospores. Conidia that are borne on the hyphae, without any kind of compound fruiting structure, are the most commonly encountered type. Structures that completely enclose the conidium-bearing cells are called pycnidia, and those resulting from a fusion of conidium-bearing cells are called synnemata if they are longer than they are broad or sporodochia if broader than long.
Strictly speaking, all fungi should be classified according to their method of sexual reproduction. In many cases this is possible, allowing us to recognize several groups of fungi. Fungi that are encountered most often or exclusively in the asexual condition create special problems and are discussed separately as anamorphs.
The Chytridiomycota, or "chytrids" as they are commonly called, are a group of mostly unicellular fungi occurring in a variety of habitats ranging from soil and roots to the rumens of cows and deer. Asexual and sexual spores are produced in sporangia and escape as zoospores (swimming cells). Because of their basically non-hyphal nature and often unusual requirements for growth, the Chytridiomycota are seldom encountered as moulds.
The individual in the photograph above right is perched on top a grain of pine pollen. This is in fact an easy way to collect and examine chytrids. Many plants, including pine and other conifers, produce large amounts of pollen over a short period of time. This pollen can be gathered in small bottles and dried for later use. Sprinkle the pollen very sparingly over the surface of some pond water in a dish. Over the next few days examine the pollen under a microscope for evidence of chytrids.
Members of this group all reproduce by oogonia eggs. The hyphae have few or no cross-walls (septa) and thus appear as long, clear tubes. If a hypha is broken, most of the contents run out. Many oomycetes reproduce asexually by zoospores, which are motile and can swim quite rapidly. Because of their motile zoospores, oomycetes commonly require water for reproduction and are often encountered in water or wet soil.
An easy way to see oomycetes is to heat a few sesame seeds until they "pop" and then float three or four on the surface of some pond water in a dish. After a few days the oomycetes with form a thick white growth around the seeds. If you watch there carefully you may be able to observe the release of zoospores.
As their name implies, these fungi all produce zygospores. They resemble the oomycetes in having hyphae that usually lack cross-walls or septa, but differ in lacking motile spores. Asexual reproduction is by sporangia or conidia. The members of this group are usually terrestrial and will be encountered only occasionally in aquatic conditions.
Zygomycetes are easy to observe. They will invariably grow if soil is sprinkled over strawberries or raspberries. Any attempts to isolate fungi from soil (see chapter on cultivation) will reveal numerous zygomycetes.
All ascomycetes have ascospores borne inside asci. The hyphae always bear numerous septa. Asexual reproduction is by conidia that always lack motility. Although most ascomycetes are terrestrial, some occur in freshwater or marine habitats.
Ascomycetes are everywhere and can be collected year around. The minute black dots on rotting wood and leaves or the little cups on lichens will often prove to be perfect examples.
This large group, which includes mushrooms and puffballs, is characterized by the presence of basidia and basidiospores. Like ascomycetes, to which they are related, basidiomycetes have hyphae with septa and lack motile spores. The hyphae of many basidiomycetes bear characteristic swellings, called clamp connections, that play a specialized role in nuclear migration. Asexual spores, when formed, are produced as conidia. Most basidiomycetes are terrestrial.
This group comprises the large number of fungi known to reproduce asexually by conidia and is by far the most important in a study of moulds. The asexually reproducing structures of a fungus are called anamorphs, which, together with the sexually reproducing structures or teleomorphs, make up the holomorph or whole fungus. Until the recent advent of a practical genetic methodology the numerous fungi known only as anamorphs could not be classified confidently with the ascomycetes or basidiomycetes to which they undoubtedly belonged. Mycologists have long used a system of classification that allows anamorphs to be named separately from the holomorph of which they form a part. As a consequence, many fungi could have two different names. For example, the name Eurotium repens pertained to a holomorph with both ascospores and conidia, whereas Aspergillus repens pertained only to the anamorph of the same fungus. Current advances in nucleotide sequencing now allow quite accurate taxonomic placement of species, and as of 1 January 2013 the dual system has come to an end. We now adhere to the more intuitive concept of "One Fungus - One Name". Digging out from more than a century of double names will take time and mycologists temporarily use the old system, but in an admittedly informal sense. Once all the doubly-named species have been sorted out mycologists can breathe a sigh of relief.
Anamorphs show considerable structural diversity and can be classified or described according to three attributes: (1) the method of conidium production (conidiogenesis), (2) the locus of conidiogenesis, and (3) the sequence of conidiogenesis (See Figures 5 and 6).
Production of a conidium involves the transformation of part of a cell into a separable spore. Sometimes a hypha or cell becomes separated into one or more segments by septa. The separate cells then thicken, swell, and finally separate. This kind of conidiogenesis, where the septa appear before the conidium is initiated, is called thallic (Figure 5A,C). When the new conidium is initiated, begins to swell or thicken, and then is cut off by a septum, the conidiogenesis is called blastic (Figures 5B, D;6). When the wall of the conidium is continuous with the cell that produced it, it is called either holothallic (when thallic) (figure 5A) or holoblastic (when blastic) (figure 5B). When only the inner walls of the conidium-bearing cell are involved in conidiogenesis, the prefix "entero-" is used, resulting in the terms enterothallic (figure 5C) and enteroblastic (figure 5D). Although enterothallic types are rare, enteroblastic types are probably the most common of all and are represented by the ubiquitous phialide.
The following genera, illustrated at the end of this document, illustrate features of this section:
Conidiogenesis usually can be traced to a particular point or locus on the conidiogenous cell. If this point remains stationary and gives rise to more conidia it is said to be stable (figure 5D). If subsequent conidia are produced at new points on the cell the conidiogenous locus is said to be dynamic. Dynamic loci may gradually elongate and be called progressive (figure 6C), or move toward the base of the conidiogenesis cell and be retrogressive (figure 6B). Usually it is necessary to examine several cells to "reconstruct" the loci of successive spore productions.
The following demonstrate types of loci:
If only a single conidium is produced on a conidiogenous cell (figure 6A), we can hardly speak of a sequence. If, however, more than one is produced they can occur either simultaneously (figure 5A, B, C) or serially (i.e. one after another) (figures 5D; 6B, C).
The following illustrate sequence types:
In routine work we often encounter organisms that are similar to moulds but do not fit our strict definition of the term. Either the organism is not filamentous or it is filamentous but not strictly a fungus. We can put four groups of organisms into this category; bacteria, actinomycetes, yeasts, and slime moulds.
Bacteria represent a very ancient group of organisms, perhaps as old as four billion years. Colonies of bacteria are composed of minute spore-like cells that together form a slimy mass. Such colonies never contain hyphae and are thus easily distinguished from those of true moulds. Bacterial cells, rarely more than 1 um in diameter, are difficult to examine, even with a good microscope, and are best seen when stained. Many bacteria are motile and swim vigorously.
These organisms are usually classified as bacteria but have filaments like fungi. The filaments are seldom more than 1 um in diameter, however, and are thus considerably narrower than those of moulds. Streptomyces, the only actinomycete genus commonly encountered as a "mould", produces grey to brightly coloured powdery colonies, usually with a soil-like odour.
Yeasts are true fungi, but in lacking hyphae cannot be classed as moulds. They resemble bacteria in forming pasty or slimy colonies of spore-like cells, but differ in having these cells much larger, usually 2 um or more broad. Reproduction of yeasts is usually by budding, a process where a smaller cell appears to bubble slowly out of the parent cell.
Slime moulds normally occur on logs and other natural materials but occasionally occur in the laboratory as "moulds". Cellular slime moulds, the most likely to appear in the laboratory, have creeping amoeba-like cells during part of the life cycle and sporangium-like structures in another. They most closely resemble members of the zygomycete genus Mucor, but differ in their complete lack of hyphae. Myxomycetes, another group of slime moulds, occur on rotting wood and vegetation in a great variety of habitats. The ones in the photograph at right were growing on an old moss-covered log.
Almost any natural material, no matter how small, will support an indigenous population of moulds. Moulds are part of the economy of nature, quickly occupying dead or nearly dead materials and returning them to the basic building components of new organisms. As such, they are essential to what biologists call nutrient cycling, the process whereby nutrients never leave the realm of living things, but simply get used again and again; earth to earth, ashes to ashes, dust to dust.
While acknowledging that moulds are everywhere, it is useful to classify their habitats into a number of categories based on nutritional characteristics. Moulds have specialized nutritional requirements and usually do not range very far from their usual habitats: fungi that naturally decay seaweed on the beach will not be expected to occur on mouldy bread in the kitchen. The competition among moulds having similar nutritional requirements is intense and leaves no room for a mould adapted to another habitat.
Aside from nutritional and competitive barriers, other factors encourage habitat restriction among moulds. Most influential is spore dispersal. Although most moulds seem to produce astronomical numbers of spores, they are, in fact, practising a strict economy. In their highly competitive life there can be little waste; every minute bit of energy must be put to good use or a more efficient organism will prevail. Thus moulds will produce only enough spores to ensure reproduction of their species from year to year. If a mould were to put a disproportionate amount of energy into spore production, it would have to be at the expense of some other activity, such as rapid growth. Economy in spore production is best ensured by mechanisms of spore dispersal conferring a high probability of encountering suitable places to germinate and grow. Many different mechanisms exist and account for the great variety of spores and spore-producing structures found in moulds.
In many instances, moulds living together in particular habitats have similar methods of spore dispersal, even when the moulds themselves are not closely related, illustrating the idea that many structures in organisms are similar because of common ecological pressures rather than common ancestry. Thus we come again to a basis for subdividing habitats, and, as we shall see later, also a basis for applying certain techniques for the isolation and cultivation of moulds.
Most plant diseases are caused by fungi. The largest group of plant parasites are the rusts, a highly specialized group of organisms that grow extremely slowly in the laboratory and are outside the usual definition of moulds. Powdery mildews, like the colonies of Uncinula circinatumparasitzing a maple leaf at right, have never been cultivated away from their plant hosts. Some plant disease fungi are less difficult to grow but are so specialized as parasites that they seldom occur in laboratories other than those of the plant pathologist. Many of these fungi have rather strict nutritional requirements to induce sporulation and are usually seen in cultivation only as sterile hyphae.
The plant disease fungi most commonly encountered as moulds are those having non-parasitic stages in their life-cycles. These fungi usually grow and sporulate well in the laboratory. Examples are species of Phoma, Fusarium, Bipolaris, Graphium, Pestalotiopsis, and Monilia.
Most parasitic fungi do not produce great numbers of spores and it is likely that they are transferred to uninfected plants by fairly specific means. Those having wet spore stages, such as species of Phoma, Fusarium, Pestalotiopsis, and Ustilago (a smut), are probably transmitted by insects or other arthopods, such as mites. Other fungi, such as Cladosporium and Monilia, produce large numbers of dry spores, and appear to be wind-dispersed.
Also conspicuous are fungi attacking plants weakened by senescence or disease. Such invaders are hardly parasites in the strict sense. Many of these fungi are now considered to be "endophytes", organisms that enter plant tissues while they are still healthy and remain there in a dormant state until the tissue is weakened or dies. Some ecologists now view these endophytes not as parasites but as the first arrivals in a long sucession of decay organisms. Many species of Alternaria, Botrytis, Cladosporium, Fusarium, and Ulocladium may belong here.
Moulds occurring on dead herbaceous plants are often the same species attacking dying ones. Thus we can again list Alternaria, Cladosporium, and others, as well as a few new ones such as Epicoccum and Candida. In temperate climates, where many plants die in the fall, there is a tremendous flush of mould activity in autumn. Because the dead plant material is so abundant, finely tuned dispersal mechanisms are not necessary and the moulds simply release their spores in the wind. The likelihood that a spore of Cladosporium, for example, will hit a dead plant part in the autumn is very great.
Nutritional specialization dictates that different plants will support different fungi, and we find, for example, dead grasses yielding a different flora of moulds than dead milkweeds or mustard, at least to a partial extent.
Dead wood is a good source of moulds and provides two distinct habitats, depending on whether it is covered with bark or not. Moulds occurring under the bark of fallen and standing trees cannot disperse their spores into the air and most often utilize insects and mites for their transmission. Although, as we might expect, many have wet spores ideal for sticking to the bodies of their dispersers, some have dry spores that appear to become trapped among the animals' body hairs. I have seen hundreds of the curved, dry ascospores of Fragosphaeria purpurea trapped among the bristles on the backs of mites under bark of maple trees in Ontario. Often the moulds under the bark of dead standing trees are different from those on fallen trees.
The surface of wood not covered with bark often has a distinct community of moulds. Although air dispersal of spores is possible in this situation and apparently often occurs, wet-spored moulds are still abundant. Again standing and fallen trees support different fungi. Freshly cut wood is a good source of moulds causing a phenomenon called blue stain. These moulds, common in lumber-yards as bluish to black discoloration on the wood, produce tall sporulating structures bearing wet drops of insect-transmitted spores. Closely related species occupy the tunnels and galleries formed by beetles under the bark of living and recently dead trees.
Dead or dying leaves of trees support moulds similar to those from herbaceous plants, with some notable exceptions. However, tree leaves appear to support fewer Alternaria and Cladosporium colonies, thus often allowing a better chance to observe the more slowly growing or rarer fungi. For example, in Ontario fallen leaves of ash support a pin-sized mushroom called Marasmiusminutus, that never occurs on herbaceous plants.
Leaves submerged in water support a number of moulds with unusual spore forms. The spores may be needle-like, coiled like a watch-spring, or coiled into barrel-shaped structures that bob to the surface of the water and float away. Others have three or four narrow arms and look something like jacks with two or three of the arms missing. These moulds may be examined by submerging leaves in a bowl of water and stirring them vigorously. After a few minutes the spores will float to the surface, where they can be skimmed off in a drop of water. Best results are obtained with leaves likely to become submerged later in the natural habitat but have not yet become so.
A number of diseases of humans and animals are caused by mould and yeast-like fungi (Figure 7). Many are known only from this habitat and are quite specialized. Notable are the moulds called dermatophytes, the cause of a number of skin diseases such as ringworm and athletes' foot. The fungus grows on the outermost layer of skin, causing reddening of the surrounding tissues (zoophilic types) and sometimes scaliness (anthropophilic types). Dermatophytes do not normally attack deeper tissues; the symptoms are usually due to an allergic reaction. Because of their essentially non-parasitic nature dermatophytes are usually easy to grow in the laboratory. Many closely related species occur on substances similar to human skin, such as leather, feathers, hair, and horn, and are unable to grow on living animals or man. Probably the most commonly isolated dermatophytes are species of Microsporum (Figure 7A), Trichophyton, and Epidermophyton.
Several fungi can occur on living tissues and cause serious disease and even death. Many such organisms are known only from individuals who have low disease resistance, due to prior infection, AIDS, old age, or other factors. Most commonly, infections by these fungi occur when the normal bacterial populations of the body are eliminated by the use of antibiotics. Without competition from bacteria these fungi occupy the tissues and grow rapidly, often causing considerable damage.
Other fungi attack healthy living tissues without the aid of antibiotics, causing more localized but very serious infections. Histoplasmosis, a disease with symptoms similar to those of tuberculosis, is caused by Histoplasma capsulatum (Figure 7C), a mould associated with bird nests in nature. It is frequently reported in perfectly healthy people who have been exposed to the dust from nesting materials, such as while tearing down old barns.
The study of medically important fungi is beyond the scope of this book and will not be discussed further. There are several good books dealing with medical fungi from a mycological (rather than medical) point of view. Especially useful are Hoog and Guarro, 1995; McGinnis (1980) and St-Germain and Summerbell, 1996.
One of the most devastating animal diseases to have appeared in recent years is the "White Nose Syndrome" in hibernating bats. This disease, caused by Geomyces destructans is now known from a large part of eastern North America where it has drastically reduced the numbers of bats. Some scientists have suggested that species such as the little brown bat may become almost extirpated in much of its geographical range. The fungus appears on the hibernating bats during the winter, causing them to awaken several times. It is thought that when they wake up they burn of valuable fat reserves necessary to carry them through until the return of flying insects in the spring. Infected bats attempt to leave their caves to feed in late winter and freeze or starve to death. The photo at right shows two infected bats photographed in an old mine in New Brunswick, Canada. The fungus can be seen at white circles on each side of the animals' noses.
Some moulds actually trap small (usually microscopic) animals. Best known are those that trap eelworms or nematodes (see Barron  for an excellent account of these fungi). The traps produced by these fungi are composed of loops, networks of filaments, or knobs and branches. Some are reminiscent of the well known Venus' fly-trap, and are sprung upon contact with an eelworm. When the worm crawls into a ring of one of these fungi, the ring rapidly constricts, holding the eelworm tightly. At first the worm struggles violently, but it soon becomes still and is invaded by the filaments of the mould. In other species, the loops and branches are sticky and hold the eelworm fast as soon as it touches them.
A second group of eelworm catchers produces spores that are swallowed by the worm, germinating later in the gullet. At least one species has an elaborate injection mechanism that, when sprung, forces a small portion of the spore right into the tissues of the side of the eelworm.
Soil is one of the most commonly studied mould habitats. It is source of plant disease fungi, some human disease fungi, predacious fungi, and a host of forms that bring about the transformation of dead plant and animal material into soil.
Soil is not a uniform substance; in fact, it is so complex that a study of soil is a science in itself. Scientists recognize several layers in the soil, starting at the surface and working down. These are:
L layer: the layer at the soil surface composed of undecayed plant litter. The origin of the litter is clearly identifiable.
F layer: the layer with identifiable but partially shredded or decayed plant material.
H layer: the layer containing much material of plant origin, but with individual structures no longer recognizable.
A layer: the layer with plant or organic matter present but unrecognizable as such or well mixed with loam (a mixture of sand, silt, and clay).
B layer: a mineral layer containing little organic matter.
C layer: the parent rock from which the mineral layer is derived.
Naturally these layers are not always distinct, and grade more or less evenly on into the other. Some soils, especially agricultural ones, lack stratification altogether. Soils differ from one another in the kind of plant material falling into them; the relative proportions of sand, silt, and clay in the mineral components; the moisture content; the relative acidity or alkalinity (pH); and in many other characteristics.
Fungi, because they are highly specialized users of organic substances, vary from one soil to another and with depth in the same soil. They are entirely dependent upon the kind of material falling into the soil: a fungus specializing in oak leaves will probably not colonize a pine needle. This factor alone will account for many differences between the mould populations of two soils. In addition fungi may be sensitive to moisture levels, pH, competition from other organisms, and many other influences.
As a leaf falls into the soil it is shredded by arthropods and digested by microorganisms. Gradually it becomes buried under newer generations of leaves and thus moves down into the lower layers of soil. It undergoes continuous decay by moulds, bacteria and other organisms until it finally disappears (somewhere in the B layer). Fungi that first invade the leaf, often while it is still on the tree, will grow on it until the particular nutrient they need is exhausted and then die back, to be replaced by new fungi extracting what they need from the leaf. In this way, a leaf moving down through the layers of soil will have a series of moulds on it, each replacing a previous population. This transition of populations as the habitat ages and is modified is called succession and is the object of much study and discussion among ecologists.
Although we tend to think of soil fungi as decomposers of litter, they play a number of other roles as well. Many are associated with animals and animal products and may trap eelworms and other soil animals, or decay the dead bodies of insects and earthworms. Some invade cast-off feathers and hair, and at least one group specializes in old hooves and horns.
Through their roots, living plants offer an attractive habitat for soil-inhabiting fungi. Some live around the root and decay away the dead root layers or substances exuded by the root. Others invade the living root tissues and either cause plant disease or live in harmony with them. The latter situation involves a form of symbiosis called mycorrhizae, where the root and the fungi within each benefit the other, In fact, most mycorrhizal fungi and plants cannot live apart. Only a few species of plants are consistently free of this association.
Air, of course, is not a habitat for moulds, though many disperse their spores by air currents and are encountered in routine work.
Fungi have a variety of mechanisms for getting their spores into the air. Simplest is that of exposing dry spore masses to air currents. Many moulds use this method, especially those that colonize exposed leaves and stems. Species of Alternaria, Cladosporium, and some of the basidiomycetes called smuts are conspicuous for this kind of dispersal. These species usually produce large numbers of spores, a necessary expenditure if at least a few randomly dispersed spores are to land on a suitable place to grow.
Ascomycetes and basidiomycetes are usually capable of shooting their spores away into the air. Asci are like popguns: the water pressure inside the ascus builds up higher and higher until the spores are fired out the end with considerable velocity. Ascospores can, in fact, be shot a distance of several centimetres from the end of the ascus, a prodigious feat considering the microscopic size of these mechanisms. The equivalent at our own scale would be to fire a 30-cm projectile a distance of 6 km, a feat necessitating a 105-mm howitzer! With such a mechanism, ascomycetes need not be exposed directly to the wind but may grow in places allowing entry to the air of their discharged spores. Thus ascomycetes are often found growing closer to the ground than other air-dispersed organisms.
Basidia discharge their spores from the tips of small projections called sterigmata. Such spores, sometimes called ballistospores, are not shot very far, only far enough to clear the surface of the basidium and the fruiting structure. The fruiting structures of basidiomycetes may be microscopic in size and bear only a few basidia or they may be very large, as in mushrooms and bracket fungi. These last fungi often continue to produce spores throughout the growing season and can produce a staggering number. One of the most prolific of these, the "artist's fungus", Ganodermaapplanatum, may disperse its airborne spores many kilometres from the woods where it grows. Related to the basidiomycetes are the mirror yeasts, a group of yeasts that produce ballistospores on solitary sterigmata. Most basidiomycetes are not normally encountered as moulds, and are really outside our interest here, but a few, such as Sistotrema brinkmanii may be encountered in the laboratory.
Some spores become airborne by a mechanism known as droplet adhesion, a process dependent upon the presence of tiny water droplets in the air. When one of these droplets, as on a foggy day, encounters a mould spore attached to a leaf, for example, the spore adheres to the droplet by surface tension and is carried off on it. It is then either deposited when the drop comes to earth, or becomes truly airborne when the drop dries out.
There are a few other mechanisms whereby spores become airborne, but these are rather more specific to one or a few species. Most are forcible discharge mechanisms and may involve various kinds of spring, water jet, or bellows devices. A discussion of these and other common mechanisms is found in the excellent book on dispersal in fungi by C.T. Ingold (1953).
Although people expect dung to be a rather disgusting material to study, many become so intrigued with the moulds and other organisms found there that they soon overcome their initial objections.
Many fungi found on dung are highly specialized for growth there and never occur anywhere else. They do not produce large numbers of spores and thus require a high probability of success in getting their spores from one dung pile to another.
The most commonly cited life-cycle in dung-inhabiting or coprophilous fungi is that shown by many ascomycetes. The spores of these ascomycetes are very heavy, sticky, and dark, and when they are shot from the ascus their weight allow them to travel a relatively great distance. Because of this weight, however, they do not usually remain in the air long, but follow a parabolic trajectory and land near the dung pile. Because they are sticky, often with rather elaborate slimy coverings or tails, they adhere to whatever they fall on, and because of their dark colours they are not greatly affected by the ultraviolet radiation in the sunlight to which they are exposed for an often considerable length of time. Eventually they may be eaten, if they have fallen on vegetation, by a grazing animal, ultimately to be packaged up in a new pile of dung. Many of these spores have a dormancy mechanism that prevents them from germinating on the vegetation but that can be broken when subjected to the processes of the animal's digestive tract. Most coprophilous ascomycetes are light-sensitive and aim their spore shots away from the dark-coloured dung, a first step in their journey to a new dropping.
Other coprophilous moulds present their spores to the environment in wet drops at the ends of stalks. When one of the many insects or mites attracted to dung brushes against these stalked spore drops they carry with them a few of the sticky spores. Later, perhaps on a new dung pile, the spores rub off and germinate to produce a new colony. Most insect-transmitted fungi lack dormancy mechanisms in their spores, and are unlikely to be away from dung for more than a few hours.
The variety of coprophilous fungi is large, and many occur in the laboratory as moulds. Included are several kinds of ascomycetes, some small mushrooms, slime moulds, zygomycetes, and many anamorphs. It is a particularly rewarding group for study by the beginning mycologist.
One warning must be issued. Dung may contain the eggs, larvae or adults of parasitic organisms. While most of these are of danger only to the species of animal producing the dung, some are able to parasitize humans. Because of this dung should never be handled with bare hands.
Aside from their role in plant, animal, and human disease, many moulds enter directly into human affairs, in either a harmful or beneficial way. On the plus side is the involvement of moulds in the greatest contribution to medicine of all time, that of antibiotics. The discovery of penicillin by Sir Alexander Fleming in 1928 probably resulted in the saving of more lives than all other medical discoveries combined. Penicillin, a product of the common mould Penicillium chrysogenum, is still one of the safest and most widely used of antibiotics, in spite of a more than fifty-year search for others.
The foods we eat are as nutritional to many moulds as they are to us, a fact often put to use in the preparation of food products. For example, several types of cheese, such as Roquefort, Danish blue, Camembert, and Brie, owe their distinctive flavour to the presence of mould growing on them. If the mould were absent, these cheese would not ripen properly. Yeasts, although not really moulds, are among the most important fungi in food preparation. Their value, at least for some Saccharomyces species, lies in their ability to produce carbon dioxide and grain alcohol. In wine-making, where the production of alcohol is valued, yeast is added to the grape juice to bring this about. In bread-making, the important product is carbon dioxide, which is necessary in the rising process, and, again, yeast is added to the dough. In the production of beer, both alcohol and carbon dioxide, to produce carbonation, may be necessary, although today the carbon dioxide may be added later artificially.
In the Far East, a number of moulds are used in food preparation that remain unexploited in most of the world. Among these are species of Aspergillus, Monascus, and Rhizopus, used to process various rice, bean and soybean products.
To most of us, the negative aspects of moulds in foods are more noticeable than the positive. Few of us have failed to notice the pink, black, and green moulds growing on bread or the green or blue-green Penicillium rots of citrus fruits. Moulds are one of the reasons food manufacturers include preservatives in their products. The picture at right shows a species of Penicillium, growing over the surface of some tomato sauce left too long in the refrigerator. The yellowish drops of liquid, called exudate, resting on the surface of the mould, and its overall blue-green colour suggest it is P. chrysogenum. Microscopic examination revealed it had smooth conidiophores and conidia, also indicating it to be P. chrysogenum.
One of the most destructive activities of moulds in foodstuffs occurs in stored seeds and grains. Certain species of Aspergillus, Penicillium, and Eurotium are able to grow under particularly dry conditions and attack stored grains. To guard against this, the grain must be dried to very low moisture levels. Keeping grains dry in the humid tropics is particularly difficult if not, at times, nearly impossible. Not only do such fungi damage the grains or render them unpalatable, they may also excrete toxins that can cause illness or even death. Most famous of these are the aflatoxins - produced by Aspergillus flavus and other moulds - which are not only immediately toxic but are also known to be carcinogenic. Aspergillus flavus often grows on peanuts and was first discovered in peanut feed given to turkeys in Great Britain.
Some of the same species of Eurotium that attack stored grain commonly appear on the surface of jams, jellies, and syrups. These very sugary substances tend to prevent water from entering the cells of most fungi and thus create what amounts to a microbiological desert. Eurotium species, with their ability to grow under dry conditions, are ideally suited to such habitats. Some products, such as prunes and dried apricots, are so dry and so sugary that even Eurotium species are unable to cope with them. They still can be decayed by certain specialized moulds, however. Probably the most remarkable to these is Xeromyces bisporus, which cannot even grow at the sugar concentration used to grow Eurotium species. Laboratory cultures of this species are rather difficult to establish and require very special conditions.
Cellulose is probably the most abundant material of biological origin on the earth and is a prime source of energy for many fungi. We have met some cellulose-decomposing fungi already in our discussion of moulds inhabiting dead and dying plant parts, notably Alternaria, Cladosporium and Epicoccum. While these fungi are common enough, there are several others that seem to become especially common on man-made cellulose products, such as paper, cotton, cardboard, and wood products. Many mould growths on paper and cotton are commonly called mildews, but this term really has little meaning. These fungi are able to dissolve the cellulose fibres in cotton and paper and thus cause the materials to disintegrate. The process is especially rapid under moist conditions, as occur in damp basements or in the tropics. Many species of moulds are involved in cellulose decomposition, but none are more widespread than species of the ascomycete genus Chaetomium. During the Second World War, countries fighting in the South Pacific and Southeast Asian areas lost a great deal of equipment to these species. The resulting increased interest in Chaetomium is reflected most notably in Ames's (1963) monograph on the Chaetomiaceae, published by the United States Army.
Sawdust contains many cellulose-utilizing moulds that may cause unwanted decomposition. Particularly undesirable are members of a group of moulds able to grow at high temperatures (up to 60? C). The growth of these moulds may actually cause the temperature of the sawdust to rise. Combined with bacteria having an even higher temperature tolerance and natural chemical activities these moulds may eventually lead to spontaneous combustion, a common cause of fire in lumber-yards.
Many other manufactured products are susceptible to mould attack. Painted walls, particularly in humid places such as showers, can become overgrown by certain fungi, notably species of Phoma and Exophiala. Wallpapers also serve as a source of nutrition for some moulds. Commonly cited are the Scopulariopsis species which have been reported to grow on wallpapers containing arsenic pigments and to release very poisonous gases. With modern wallpaper pigments, which contain no arsenic, this cannot happen.
Leather is mainly protein and serves as a convenient source of food for moulds. Some of the moulds that occur here are related to the dermatophytes that attack the outer layer of human skin; others belong to quite different groups.
Some substances that seem to be entirely unlikely to support fungal growth may be found to be well colonized by these organisms. Exophiala species, for example, are found in syrupy solutions of polyvinyl alcohol. Cladosporium (Amorphotheca) resiniae often occurs on the surface of aircraft fuels in their tanks and can damage jet engines. Penicillium ochrochloron can be found in electroplating solutions that are extremely acid and contain very high levels of toxic copper salts. I was once involved in a search for the organism responsible for decaying inflatable life-rafts periodically soaked in seawater. The culprit eventually turned out to be a species of Aspergillus.
Increasing attention is becoming focused on fungi in indoor environments. Although most of the moulds occurring in human environments occur indoors, the interior of buildings are themselves a special habitat. It has long been known that indoor fungi cause allergies in sensitive persons, but only relatively recently have indoor moulds been linked to other health problems. While allergy sufferers generally react to substances in fungal spores, other health problems may be caused by voltile substances released by moulds into the air.
Virtually all buildings contain moulds, but some are mouldier than others. Indoor moulds can be remarkably tolerant of dry conditions but none can live without some moisture. Excessively mouldy buildings generally have a source of moisture leading to unusually heavy mould growth. The source of the moisture may be a leaky basement, a dripping pipe, a roof in need of repair or some other fairly obvious cause. In most cases the mould can be seen growing on walls or other materials in contact with the moisture. Sometimes the moisture can occur inside walls and not be apparent. A common but not obvious cause of moisture in cold climates is condensation inside north-facing walls. Severely mouldy buildings may have a musty smell, but not necessarily. Sometimes the only sign of a problem is persistant poor health of the occupants, such as headaches, nausea, respiratory symptoms, etc. It is now known that mouldy buildings can present a serious health risk to occupants. In fact, some infant deaths have been convincingly linked to indoor moulds. Any building with an apparent mould problem should be thoroughly investigated by qualified people.
Not all indoor moulds present a risk to human health, but an abundance of any mould is likely to be accompanied by others, including toxic ones. Species of Stachybotrys are particularly toxic. Obvious occurrences of Stachybotrys may be sufficient cause for a major "decontamination" by qualified technicians wearing special protective clothing. Stachybotrys species produce black colonies on dry wall, ceiling tiles and other materials containing cellulose. The easiest way to confirm the presence of Stachybotrys is to press a piece of cellulose tape against the mouldy spot and then examine it sticky side up on a slide under a microscope for the characteristic spore-bearing structures. The image at right illustrates conidiophores and conidia of a Stachybotrys species collected on tape from a basement in southern Quebec. Not all moulds are identifiable using this technique, but it works well for many, including Stachybotrys.
One of the joys of mycology is being able to go out into the field and isolate from among thousands of moulds a single interesting species. Almost any natural substance will have not one mould but a whole community of moulds growing on it. Each mould is distinct from all others and produces a characteristic colony when grown in pure culture. The isolation, or we might say domestication, of each is a challenge that never seems to lose its fascination.
A most difficult thing for the beginner is to come to terms with the small scale of mould communities. Even an area only 1 millimetre square can support more than one species of mould when nutrients are available. The only really practical way to get down to this scale is to work with a binocular dissecting microscope of sufficient magnification to allow spore-bearing structures to be seen. When this is possible, a sterile needle can be used to remove the spores of the particular mould that is to be isolated.
Isolation of fungi from natural sources is one of the basic skills in mycology that must be mastered by almost everyone concerned with moulds. Isolation techniques are numerous and often complex but can be quite effective in yielding just the mould one wants, while excluding all the others. Isolation techniques can be divided into two broad categories: (1) direct methods and (2) selective methods. Both are routinely used in mycology laboratories and can be further divided into a number of subtypes.
The term "direct is applied to techniques involving the simple transfer of a mould from its natural habitat to a pure culture situation in the laboratory. At its simplest this is done by putting a piece of the habitat or substrate under a dissecting microscope so that the mould growth is easily visible. An inoculating needle is then heated until red hot, cooled, and used to transfer some of the spores into a sterile plate of culture medium. I find this easier to do if I first get a tiny piece of agar on the end of the needle for the spores to stick to. The piece of agar must be very small because it is often necessary to manoeuvre it into rather tight places where other moulds might easily contaminate it. An alternative is to moisten the needle with a slight amount of glycerine. Once the transfer has been made, it is only necessary to wait a few days for the mould to grow and form a colony.
The direct transfer technique is also used to obtain pure cultures of mushrooms and other large fungi. The fruiting body is broken open, without touching the newly exposed flesh, and some of the tissue is transferred to a sterile culture medium. If this has been done carefully, the tissue may give rise to a colony after a few days. For the beginner, the easiest large fungi to culture are those found inhabiting decaying wood. Most wood-inhabiting mushrooms and bracket fungi grow well on the semi-synthetic media listed in chapter 4, although few will form their large fruiting bodies in culture.
Direct isolation of fungi is often more effective if the natural substrate has been kept moist for one to several weeks to allow moulds to grow and sporulate. The easiest method involves a container called a moist chamber (Figure 11). Moist chambers can take any number of forms, but are basically containers holding a material such as cotton, paper, cloth, sterile sand or soil, or peat moss that can be kept moist for several weeks. The specimen is placed on top of the moist material and left until moulds begin to grow on it. In my classes we use glass containers resembling very deep Petri dishes, and like Petri dishes they have loosely fitting lids. For the packing material we nearly always use peat moss, collected in the field and dried for later use. The container is filled with dry moss and then water is added. Peat moss is like a sponge in that it can absorb and hold a tremendous amount of water. When water is added to the moist chamber it soaks up into the moss. The excess can be poured off and the moss squashed down until it forms a moist layer no deeper than about one-quarter of the height of the container. We then place a single or double layer of filter-paper or paper towel over the moss layer so that the specimen does not come in contact with it. Thus prepared, the moist chamber is ready for the specimen.
In many areas peat moss, in its fresh form, is hard to obtain and it is necessary to find a substitute. Although many people use cotton for this purpose, I think that it is best avoided as it is a good substrate for mould growth itself. Instead, try using a layer of fine soil or very fine sand. Soil and sand will not hold water as well as peat, so watch carefully to make sure it does not dry out. But don't make it too wet or all you will get is mud!
When you are ready to add the specimen, moisten it slightly (unless it is already damp) and place it on top of the filter-paper. Cover the dish and leave it in a place where the temperature is reasonably constant.
Within a few days moulds will begin to appear on the specimen. Most beginners are unprepared for the extremely small size of many moulds and tend to overlook them completely. Invariably students using moist chambers for the first time complain that nothing is growing on their specimens, only to have an instructor point out at least half a dozen different moulds! Be sure to examine the material with a magnification of at least 15-20 times and with good bright illumination. Illumination is especially important and should be focused on the area of the specimen that is under examination. When something interesting is found, it can be removed for microscopic examination; but more about that part later.
In order to obtain a pure culture of something in a moist chamber it is only necessary to follow the direct transfer procedure given above, making sure that the sterile needle picks up only the spores that are wanted. If the needle inadvertently touches the surface of the material in the moist chamber it will pick up all sorts of undesirable contaminants.
Moist chambers can be used for all kinds of materials. We have used them to incubate dung, wood, leaves, old stems, corn stalks, bark, seeds, fruits, old fungi, dead insects, and numerous other things. Natural materials are usually more productive than man-made ones, but sometimes old pieces of cloth or leather products can be good. You are only limited by your imagination. Moist chambers are also useful in mycological "detective work", to discover the cause of a particular decay. Placing the decayed material in a moist chamber often will result in abundant sporulation of the guilty organism in the area affected. This technique works well with diseased plant material as well as manufactural products.
With very small specimens, such as insect parts or seeds, it may be easier to use a Petri dish as a moist chamber. We sometimes simply put a few layers of filter-paper in a Petri dish, moisten them, and put the specimen on top. Such moist chambers may dry out very easily, however, and have to be tended closely. A better method is to make up a batch of water agar (a solution of 20 grams of agar in a litre of water), sterilize it, and pour it into sterile Petri dishes. Because the agar solution contains almost no nutrients it will support little mould growth and thus serves only as a water reservoir. The specimen can be placed on the agar surface and examined as in any other moist chamber. Agar media containing nutrients can be used here, but often they only become overrun by the fastest growing moulds at the expense of everything else.
Whether using a moist chamber or working with freshly collected material, one often discovers that moulds produce spores inside some kind of structure, such as a pycnidium. Direct transfer of these structures invariably leads to contamination by other moulds. To avoid the problem it is necessary to clean off the surface of the structure sufficiently that all foreign spores and bacteria are removed. I do this either by pushing the structure over, around, and through an agar medium for 10 minutes or more until it is clean, or by surface sterilizing it for about 60 minutes in a 10 per cent commercial chlorine bleach solution. The required times and concentrations of bleach in the latter technique will differ some what from one specimen to another; the technique is most effective if the treatment can be varied with several samples. The agar technique demands more dexterity but can be applied to a single specimen with predictable results.
Often it is most convenient to place materials that are of interest directly on a nutrient agar medium. As already noted, this technique encourages rapidly spreading moulds at the expense of other fungi but is nevertheless widely used. It is a simple technique, requiring the placing of small bits of the substance on the surface of the agar or the pouring of melted but cooled agar over the fragments. After a few days' incubation mould colonies appear on the surface, and can be transferred into pure culture.
The amount of material placed in the dish varies, depending upon how heavily it is infected with mould spores. This technique is commonly used in soil studies, requiring only a pinch of soil, evenly dispersed over the surface of the agar. It is difficult to apply any rules here, however, because a small amount of material may yield a different set of moulds from a large amount. If the amount of material is large, the result may be a combination of a plating technique and a moist chamber.
We find that Martin's Rose Bengal Medium is a good choice for direct plating, as the rose bengal dye and antibiotics in it slow down colony growth and keep the colonies from growing together at first.
In this technique 1 gram (dry weight) of the material to be studied is ground up (if necessary) and dispersed in 9 ml of sterile water. One millilitre of this solution is transferred to a second tube containing 9 ml of sterile water, resulting in a 0.01 dilution of the spore mass in the original material. The process is repeated to yield dilutions of 0.001, 0.0001, and 0.00001 or even further if necessary. A 1-ml portion from each dilution is pipetted to a separate Petri dish, and cooled, melted agar medium poured over it. The plate should be moved gently on the table in a figure-of-eight motion to effect proper dispersion. Alternatively, the solution can be put on the surface of solidified medium and spread evenly throughout.
After a few days' incubation, colonies will appear in varying densities, depending upon the amount of dilution from the original material. The number of spores present in the original sample can be calculated roughly by selecting the plates showing 40-100 colonies and writing down the colony count. With this information the following calculation can be performed:
The accuracy of this technique is low when only one plate is counted. There are numerous contributing factors, including improper dispersion of spores during dilution, failure to break up spore masses, or mutual inhibition of growth by certain fungi. Greater accuracy is attained by doing several plates at the most desirable dilution, perhaps ten or more.
Dilutions are frequently used in studies on soil fungi. Although the technique has been criticized as not reflecting the "true" soil flora, it is probably the most commonly used method in this area of study. Even if the fungi isolated are not very representative of the whole flora, the technique does allow scientists to compare one soil with another on a statistical basis.
We have already discussed the subject of airborne fungi and have noted that certain moulds are more likely to get their spores into the air than others. Airborne mould spores can serve as an infective agent of plant disease and may also be allergenic. It is well known that allergy sufferers are sensitive to the spores of some moulds but not to others, and it is thus often helpful to know which mould species are present in the air at a particular time or place.
The sampling of airborne fungi is a subject that has attracted much attention in the last twenty years, resulting in the publication of several books. For a thorough treatment of this topic, see Gregory (1973). There are two approaches to air sampling, one that yields spores for microscopic examination and one that yields cultures. The former results in a lot of unidentifiable spores while the latter senses only those that can be cultivated (and many cannot).
It is well beyond my task here to go into all the different sampling techniques that are now in use. We can ignore the spore examination methods completely, as they deal only with spores, and concentrate on the cultural ones. The simplest culture techniques for airborne moulds involve the horizontal or vertical placement of Petri dishes containing a nutrient agar so that they trap any spores that fall on or blow into them. Usually 30 minutes to 3 hours out of doors is required for a good sample using this technique. Indoors the situation is more complicated and depends upon several factors, including the amount of traffic, the type of building, and its cleanliness. A good exposure time for an open Petri dish might be 1-2 hours, but only after testing can one be sure.
It has long been known that open Petri plates and other static spore traps have a rather low efficiency because of the layer of dead air on their surfaces. Any moving air, and its load of spores, will tend to pass over this layer and not reach the surface. To overcome the problem a number of devices have been invented that suck in air and force it against a sticky surface; one device, called an Anderson sampler, accommodates several Petri plates and will even sort spores out according to their size or mass. Such a machine is far more efficient than an open Petri plate but is bulky and is normally used only by specialists.
Many moulds have quite specific nutrient requirements and are specialized to use materials that other fungi use with difficulty or not at all. We can take advantage of this for the isolation of fungi by presenting a particular substance to the environment for colonization and then later recovering it for isolation of the fungi that occupied it. An example of a fungus sometimes recovered by baiting is the creosote fungus, Amorphotheca resiniae. To isolate it, some scientists coat matchsticks with creosote and place them on soil in Petri dishes for two or three weeks. Amorphotheca, a fungus particularly able to utilize creosote, grows from spores in the soil and invades the matchsticks, where it produces abundant hyphae and conidia. From here it is transferred into pure culture. Man inadvertently baits for this same fungus by allowing small amounts of water condensation to get into tanks containing jet fuel. The fungus grows in the water, utilizing the kerosene for energy, and ultimately forms mats of mycelium that can cause engine failure.
Other kinds of baits might be pieces of wood, insects, carrot chunks, plastics, hair, or anything else one can name. The bait can be submerged in a particular habitat in nature or in a moist chamber. To isolate dermatophytes, for example, it is customary to place hair on moist soil in a moist chamber and examine it periodically for sporulating moulds.
The most commonly baited habitat is water, both fresh and marine. Again, almost anything can serve as a bait and the water can be either naturally occurring or in a Petri dish. Good results can be obtained by putting some pond water in a Petri dish and floating on it a few sesame seeds that have been heated until they have popped. Within three or four days the seeds will be covered with oomycetes producing zoospores. Dead files, pollen, bits of apple or carrot, cellophane, and other materials are also productive baits. Since most moulds attracted this way are true aquatics it is necessary to purify them by transferring them to successive changes of distilled water containing new sesame seeds or other baits. When completely isolated into pure culture they can be transferred to solid media, but they may never sporulate there.
In the natural habitat it is interesting to submerge pieces of wood, whole carrots, apples, etc. on a string for several days or weeks and then bring them in for examination. Submerged wood blocks are one of the most commonly used materials for obtaining marine ascomycetes.
Aside from the rather broadly applicable techniques discussed above, there are many others that are quite specific in their effects. It is well beyond my purpose here to go into even a portion of these techniques, but a few are of particular interest and demonstrate some of the kinds of things that can be done to isolate a new set of moulds from a familiar habitat.
All moulds are capable of withstanding environmental stresses, but eventually, when the stress is great enough, they will be killed. Not all moulds have the same tolerance to stress, however, and we can take advantage of this property by subjecting a material to just enough stress to kill some moulds but not others. The application of such techniques has turned up another interesting fact: that some moulds will not germinate until they have been subjected to conditions that kill most others. I have already mentioned one such group in my discussion of the dung-inhabiting or coprophilous fungi that produce spores that do not germinate until subjected to the rigours of the digestive tract of animals. Other fungi produce spores that germinate only after they are exposed to fires or freezing.
To explore these interesting adaptations we need only take a sample of soil, dung, wood, etc. and subject it to some kind ot treatment that we kill most fungi. We can steam the substance in an autoclave or steamer (without pressure), soak it in alcohol, acids, bases, or other chemicals, or alternately freeze and thaw it for several weeks. Almost any drastic treatment will yield a few fungal "holdouts" that might not otherwise appear. After treatment, the substance can be handled in any of the normal ways, such as plating, or moist chambers. Dr. De B. Scott (Scott 1968) outlined a method of this type for isolating species of Eupenicillium (an ascomycete) from soil. About 2 grams of soil were added to 18 ml of sterile water and heated in a water bath at 80°C for 30 minutes. After removal from the bath the soil suspension was treated according to the dilution plating technique described above.
In the section on direct isolation techniques I mentioned the method of sterilizing the surface of fungi with 10 per cent commercial bleach to kill adhering spores. Surface sterilization can also be used as a selective technique on a slightly larger scale. If we see a living leaf that has a dead circular spot on it we might guess that this is caused by a fungus. If we put the spot directly on the agar medium we will probably get some fast-growing fungus but not the causative agent. What we want is the fungus inside the leaf, not the surface adherents, and a good way to obtain it is to apply bleach long enough to kill the external organisms but not the internal ones. Although materials differ considerably, a sterilization time of about 1 hour in a 10% commercial bleach or 3% hydrogen peroxide will often work. If one is unsure, some longer and shorter times should be tried. Surface sterilization will also work for seeds, large fungi, wood, many man-made materials, dead insects, and other things.
This technique is essentially the same as the baiting methods discussed above. It differs only in that the substance to be sampled is added to the "bait" rather than vice versa. We might, for example. wish to isolate those moulds that can utilize cellulose. To select for these fungi we make a medium such as Czapek's, but using cellulose in place of sucrose. If a fungus is to grow well here it must make use of the cellulose; those that cannot will be excluded or grow only very poorly. The possibilities here are almost unlimited, except that when using agar media it is usually necessary to use a "synthetic medium" so that the nutrients that are being modified are known. Because of impurities in the agar, at least some growth of fungi not utilizing the specific ingredient will usually be obtained. These can often be recognized by their sparse growth.
Dr. Barron, in his highly interesting book on eelworm-trapping fungi (Barron 1977), describes a novel medium. He starts a culture of eelworms growing by feeding them dried pea soup and, when they became abundant, adds a little soil. Those fungi that can trap eelworms start to grow and finally dominate the plate. Here the eelworms are the selective nutrient upon which the soil is plated.
There are few organic substances that cannot be metabolized by at least one mould. We can thus prepare media with unusual substances and expect to isolate moulds. Several years ago I made a medium composed of little more than the amino acid tyrosine and isolated a fungus for which I had to describe a new genus and species! The substance one is interested in can be added directly to the medium or a water infusion of a particular substance can be made and agar then dissolved in it.
One commonly used infusion medium is prepared with hay. First 2.5 grams of dry hay are added to 1 litre of water and boiled for 15-20 minutes; then the mixture is strained through cheesecloth, 20 grams of agar are dissolved in the mixture, and it is sterilized as usual. The resulting medium is fairly weak and discourages many fungi that need a rich sugary medium, while encouraging those that are often reluctant to sporulate. Dung infusion agar is prepared in a similar way, but is instead made with 20 grams of dried horse or cow dung. Some materials, such as dead oak leaves or pine needles, will yield an infusion that is toxic to some fungi and not others, resulting in a medium that selects for fungi occupying those materials naturally.
Moulds that grow easily at room temperature are said to be mesophilic. Most fungi are mesophilic and are inhibited or killed at unusually high or low temperatures. There are some, however, that actually require unusual temperatures. Moulds requiring high temperatures (40°C or more) are said to be thermophilic, and those requiring low temperatures (15°C or less) are psychrophilic.
In searching for these fungi we simply incubate our plates or moist chambers at the appropriate temperature and isolate the moulds that develop; all isolates and subsequent transfers must be grown at their optimum temperature as well. Bird nests, for example, yield abundant thermophiles when incubated in a moist chamber at 45-50°C, while rabbit dung or compost materials produce several psychrophiles in moist chambers incubated at 0-5°C. The thermophilic forms are amazing for their rapid growth and are, in fact, the fastest growing of all fungi. Some psychrophiles can also grow quickly, however, proving that temperature is not the only factor contributing to rapid growth. Some thermophilic fungi, or at least those that can grow at 37°C, may cause serious infections in man and animals. In working with them, care should be taken to see that they are not exposed to air currents. I do not recommend the isolation of thermophilic fungi in situations involving large groups of students.
Many stored products, such as grain, museum specimens, and hides, undergo degradation by moulds. Most of us have also experienced mould growth on materials stored in damp places at home. Both phenomena are caused by fungi that can withstand unusually dry conditions. Such fungi are said to be osmophilic, a term that refers to their prevalence in environments of high osmotic potential. I have already discussed some of the osmophilic fungi in the section dealing with moulds on foodstuffs and will concentrate here on their isolation.
Osmophiles occur on relatively dry or sugary substances in the home. Most of the fungi encountered on old cloth or leather materials in the basement are osmophiles as are the moulds growing on the surface of jams and jellies. They are abundant enough in the home that they can be isolated simply by opening a Petri plate for 2 or 3 hours in the basement or kitchen. The critical thing is the medium within the Petri plate. Osmophilic fungi grow poorly on normal culture media and often sporulate abnormally or not at all. To isolate them the water actvities of media must be drastically decreased. This means increasing the sucrose in Czapek's Agar from 20 g per litre to 200-500 g per litre, or adding 200-500 g of maltose to each litre in Leonian's Agar instead of the usual 6.25 g. For the identification of osmophilic Eurotium species, most books will require them to be grown on Czapek's or malt agar with 40% sucrose or on media containing large quantities of glycerine.
Another excellent source of osmophilic fungi is dried fruit. Many producers, however, treat these products with non-toxic fungicides and effectively exclude most moulds. A sampling of dried fruit from several sources may be rewarding, since some may still be productive. As far as is known, the fungi on dried fruits do not produce toxins.
It appears that the seed caches of certain rodents may be good sources of osmophilic fungi, but this habitat has not yet been adequately explored.
Most ascomycetes and basidiomyces and some other moulds are able to eject their spores forcibly. We can take advantage of this property to isolate these fungi by suspending them over an agar surface and allowing the spores to be shot down on it. For mushrooms and bracket fungi a piece of the gill or tube tissue can be attached, perpendicular to the agar, on the lid of a Petri dish. The spores will then float down on to the agar surface and later germinate (if one is lucky). With large mushrooms it is sufficient to attach a single gill, flat side down, to the lid; with small ones the whole cap can be stuck on. We usually petroleum jelly as an adhesive in our laboratory, but other things, such as masking tape or an agar block, may work as well.
Ascomycetes, such as cup fungi, can also be suspended above an agar surface for spore printing. Some of them, however, are so small that they have to left attached to the material they are growing on and that stuck down as well. The problem of contamination from loose particles dropping down then arises, however, which can be avoided by turning the plate over and letting the spores shoot up. Most ascomycetes can shoot their spores far enough to reach the lid of a Petri dish, but basiomycetes cannot.
Suspending an open Petri dish over a moist chamber can be a good way to trap ascomycetes, particularly from dung. Be sure, though, that the agar does not touch the sides of the chamber of the material within. It is also wise to exclude any air currents that might carry spores upward to the agar.
Interesting isolations can be made from Petri plates that have been opened and inverted over soil, logs, and other materials in the field, if proper care is taken to exclude air currents.
Anyone who has to deal with moulds will sooner or later want to grow them. Identification of moulds most often depends upon observing their methods of spore production, which are not always obvious in the natural habitat. Often identification becomes so difficult that it is necessary to send a specimen to a distant university or government laboratory. Such specimens should preferably be sent as living colonies that can be grown out at the identifier's convenience. Even when the mould can be identified in its natural habitat one may still want to cultivate it - to get more material for study perhaps, or to learn something about its physiology.
Whatever one's reason for growing moulds, one needs to understand a few things about their nutritional requirements before starting. Moulds, like people, need a source of energy, a source of nitrogen, a few minerals, and sometimes vitamins. Some moulds are rather specific as to the source of these materials, and, like people, must have them in rather complete forms. In contrast, many fungi can supply all their nutritional needs from very simple materials and construct bit by bit all the highly complex molecules they need. If man has such abilities, he would be able to drink a paper cup full of a simple seawater solution, eat the cup, and go off to work confident that he had eaten a square meal!
Whatever a particular mould needs, it must always be supplied with some form of organic carbon for energy, a source of nitrogen for protein and vitamin synthesis, and several minerals. The substance on which a mould is grown in the laboratory is called a medium and the mould growing on it, a culture. Culture media can be solid or liquid, depending on the sort of information one wishes to obtain. For the purposes of identification, solid culture media are usually more useful, as they allow the mould to sporulate more easily.
Most culture media are prepared by dissolving the necessary nutrients in water, to obtain a balanced solution supplying everything the mould needs for growth. Preparation of solid media involves dissolving a solidifying agent in the solution that will harden to a gel upon cooling. In the past gelatin was used for this purpose, but is was soon found that gelatin, a protein, itself could serve as a nutrient for some fungi. As they grew on the gelatin, these fungi would cause it to liquefy, thus destroying the solidity of the medium.
Since the 1880s, a substance called agar-agar (or, more commonly, just agar) has been the solidifying agent of choice. Agar has the property of dissolving at a fairly high temperature (nearly that of boiling water) but solidifying at about 45°C. Thus, it can be poured over living fungi without killing them, yet can be used for organisms that grow at high temperature.
Agar is relatively stable and cannot be consumed by most organisms, aside from a few specialized bacteria. It is extracted from certain marine algae or kelp by a complex industrial process. It is fairly expensive these days, but extensively used in various food and industrial products, as an emulsifier, thickener, or jelling agent.
Most culture media fit into one of three categories: (1) synthetic, (2) semi-synthetic, and (3) natural. Synthetic media are composed of ingredients of known chemical composition and concentration. These media are useful in physiological or descriptive studies when it is necessary to duplicate exactly a previous batch of medium or to record the effects of the deletion or addition of a particular substance. Few fungi show their best growth on synthetic media; they must sacrifice speed in order to build their necessary cell components from relatively simple materials. However, many produce the sporulating structures necessary for identification more easily on synthetic media than on other kinds.
Semi-synthetic media resemble synthetic media in containing a known set of ingredients, but differ in that at least some of the ingredients are of unknown or variable composition. A synthetic medium, in which all ingredients are chemically defined, can be made semi-synthetic by adding a substance such as yeast extract. We know that the yeast extract contains thiamine and other vitamins, but we do not know the exact amounts or what else might be present. The result is a medium of quite predictable composition but one not completely known chemically. Semi-synthetic media are widely used in routine work and offer something of a compromise between synthetic and natural media.
Natural media are so called because they are partly or completely composed of natural materials, such as ground-up (or whole) plants or animals. A slice of potato is a natural culture medium, as is a piece of meat or bread. Some natural media may consist of a synthetic medium augmented by tomato juice, carrot strips, or plant stems. Natural media are often very good and allow sporulation in fungi that may otherwise remain sterile. Their major disadvantage is that they may differ considerably from batch to batch and thus not yield reliable experimental results. Nevertheless, natural media are widely used in laboratory work and cannot be replaced by any other kind.
Liquid media are employed in laboratory work when the entire colony must be recovered for weighing or chemical extraction. They are also useful when the culture medium itself is to be analysed for chemical changes. For identification purposes, liquid media are seldom chosen because few moulds sporulate well on them. The exception is in work with yeasts, a group of fungi especially adapted to liquid environments.
It has often been pointed out that any medium containing agar is at best a semi-synthetic medium. Agar contains numerous mineral elements and cannot conveniently be purified, even by repeated washings. Thus, for physiological studies, liquid media should be used.
The media described below have been chosen because they represent a sample of the variety of types used by mycologists. There are, however, many additional media; persons interested in pursuing this matter further should consult the more specialized literature.
Czapek's Solution Agar is a synthetic medium widely used in mycological laboratories, particularly for the identification of species of Aspergillus and Penicillium. Many moulds produce very characteristic colonies on it and may also exude pigmented substances. Aerial growth is often suppressed and sporulation may be enhanced. Some moulds, however, grow poorly on this medium and may even fail to sporulate altogether, often because of their inability to synthesize vitamins. Many members of the Zygomycota are unable to process sucrose or nitrates and will do very poorly on Czapek's. The high glucose level may also cause problems. As noted above, the addition of agar to this medium makes it, in reality, a semi-synthetic one.
The medium is prepared as three stock solutions that can be stored and later combined to form the final mixture. The solutions are:
1. Major salts stock: 4.85 g KCl, 4.93 g MgSO4.7H2O, 0.441 g CaCl2.2H2O, 0.88 g NaCl in 1 litre
2. Buffer stock : 60.6 g/l Tris adjusted to pH 7.8 with HCl.
3. Micronutrients + vitamins stock: One litre stock prepared by mixing 5 ml each of 54.4 g/l NaH2PO4.H2O, 12.6 g/l FeCl3.6H2O chelated with 17 g/l of disodium salt of EDTA, 12.2 g/l H3BO3, 732 mg/l MnCl.6H2O, 922 mg/l ZnSO4.7H2O, 29 mg/l Na2MoO4.2H2O, 47.6 mg/l CoCl2.6H2O, 40 mg CuSO4.5H2O, 40 mg/l thiamine chloride, 2 mg/l biotin, 2 mg/l vitamin B12.
The complete mixture contains in a final volume of l litre: 15 g agar, 100 ml each of major salts and micronutrient stocks, and 10 ml of buffer stock.
This medium was formulated by Dr. I. Ahmad for the cultivation of Penicillium species. Although it appears at first glance to be a complicated and difficult medium it is usually prepared from three stock solutions and does not take as long as one might expect. It can be modified in several ways to assess the physiological activities of moulds. Most commonly different sources of carbon are substituted for glucose and different nitrogen sources for nitrate. As formulated above,with nitrate as a nitrogen source, it may be unsuitable for certain fungi, such as many Zygomycota, unable to utilize nitrates. In such cases it may be better to use ammonium salts, although this may result in dramatic plunges in pH. Again, to be truly "synthetic", the medium should be used without agar.
Leonian's medium was devised by the American mycologist L.H. Leonian, and was designed to promote sporulation in certain moulds. Later, at the University of Toronto, R.F. Cain found it to be more suitable for ascomycetes if it contained a little yeast extract; hence the term "modified" in its name. It is a good general purpose medium that will yield good growth with most fungi. Certain fungi, such as many mycorrhizal forms, will not grow on Leonian's Agar because they are unable to use maltose as an energy source. For these fungi we use Modified Melin-Norkran's medium, which has a glucose energy component.
Heat potatoes at 60°C for 1 hour and filter through cheesecloth. Make up volume to 1000 ml and add other ingredients. Cook 1 hour and then sterilize.
Potato Dextrose Agar, or PDA, as it is usually called, is an old formula used by plant pathologists and many mycologists for general laboratory use.
This is the standard medium used in medical mycology. It is probably no better for these moulds than Leonian's or PDA but is simply the traditional choice and thus the medium that must be used if colonies are to be compared with those described by medical workers. It can be used as a general laboratory medium in place of Leonian's or PDA, although the amount of glucose is rather high and may suppress sporulation in some fungi.
Ten milliltres of a 3.3 g/l stock solution of rose bengal is added to the medium after the other ingredients (except the streptomycin) have been dissolved. After sterilization, the streptomycin is added to the cooled medium.
This medium is useful in plating techniques (see below) when the aim is to slow down the growth of colonies in a mixed culture. It discourages the growth of bacteria and certain other organisms so that they will not swamp isolates from natural materials.
DPYA is an excellent medium for isolating fungi from soil and other natural substrates. The oxgall and sodium propionate restrict the growth of some rapidly spreading fungi, while the chlortetracycline and streptomycin discourage bacteria. It can also be prepared as a more general-use medium by omitting these four inhibitory substances.
The V-8 juice used in this medium probably contains many nutrients that fungi can use, but we have little idea what they may be. It is a medium that is used routinely in plant pathology and seems to be a good complement to Leonian's or PDA. Moulds that fail to sporulate on those media often sporulate heavily on V-8, or vice versa.
Weitzman and Silva-Hutner's medium was designed to enhance sporulation in certain medically important fungi, but is useful for a great many other moulds as well. I have found it to be a good (and often better) substitute for V-8 Agar and use it routinely in my laboratory. I grow most of my moulds on both Weitzman and Silva-Hutner's and Leonian's Agar and find very few that fail to sporulate on one or the other. Although the original formula calls for a final pH adjustment, I seldom do this and yet get good results.
Culture media are not hard to prepare, even under relatively primitive conditions. It is often easiest to dissolve the agar separately in hot water and then add the other ingredients. The agar burns easily when it is heated, so it is best to avoid putting it directly on the stove or hotplate. I usually put the agar in a flask of water and then immerse this in a boiling water bath. After an hour or so the agar will be dissolved and the solution will be clear. Some people do not worry very much about whether the agar is completely dissolved or not, but to avoid an uneven batch it is best to be patient.
Once the agar is dissolved the rest of the ingredients can be stirred in; they must be completely dissolved as well. Some ingredients, of course, do not dissolve and must be mixed in suspension as thoroughly as possible.
When the medium is completely mixed it must then be prepared for sterilization. Here one must stop and decide what use is to be made of the finished product. If fresh isolates are to be made from natural materials or some mould we already have is to be studied, we will probably want to use Petri dishes (Figure 9A). Petri dishes are small, round dishes, most commonly 9 cm in diameter, that have a bottom and a loosely fitting top that will admit air but discourage entry of foreign spores. They are the most commonly used vessels for growing moulds and are available either in permanent glass or disposable plastic form. The plastic plates are already sterile when bought, but the glass ones must be heated in the oven at 230°C (450°F) for 60 minutes, to kill all foreign organisms and spores, before they can be used. But more about Petri dishes later.
It is often necessary to save cultures for later use, or for mailing, in a way that they are less vulnerable to contamination by airborne spores. The conventional method is to use test-tube or bottle cultures that have only a narrow opening (Figure 9B). The culture medium is poured into the tubes, sterilized, and allowed to solidify at about a 30° angle. When cool, the agar inside has a smooth slanting surface for the colony to occupy. The tube or bottle is capped with a cotton plug that is just tight enough for the tube to be lifted without it coming out. In place of cotton a screw cap or one of several tube-capping devices can be used.
With the use of medium in mind, one can now proceed to prepare it for sterilization. If it is to be used in Petri plates it should be transferred into smaller containers that will make it easy to pour. It is impractical to sterilize filled Petri plates, so we pour them after sterilization. If the medium is to be used in tubes, they should be filled before sterilization and slanted afterwards. Sometimes it is advantageous to save sterilized medium in larger bottles and melt it down later to pour plates.
Sterilization is most often done in some kind of apparatus that will allow steaming at high pressures. In the laboratory we usually use a large sterilizer called an autoclave, but a simple pressure cooker works well, provided it has a pressure gauge. Most fungi and other organisms can be killed by boiling water, but there are a few, notably bacteria, that have highly resistant spores requiring sterner means. To be sure of killing everything, we sterilize at 121°C (250°F) for 20 minutes. To obtain this temperature the pressure must be raised to 1 kg/cm2 (15 lb per in.2 or 1 atmosphere).
When medium is sterilized in an autoclave it should not completely fill its container. Attempting to sterilize 1 litre of medium in a 1-litre flask will usually result in sudden and rather explosive boiling when it is removed from the autoclave. It is best not to fill the container more than half full. Inexperienced mycologists often assume that the 121°C for 20 minutes rule applies in all situations. However, not all materials will heat up enough to be sterilized in 20 minutes. It is an unpleasant surprise when a litre of medium becomes contaminated with bacteria because the person doing the sterilization attempted to do the whole batch in one container and barely managed to heat it to 100°C before the time was up.
After sterilization the medium is allowed to cool and solidify. Petri plates must be poured before the medium solidifies but while it is fairly cool. We usually test the medium by holding it against the inner side of the forearm; if it is cool enough to be poured it will not feel too hot. Beware, though of letting it cool too far, lest it solidify in the bottle! Pouring the medium when it is too hot results in unwanted condensation on the lid of the Petri dish.
Tubes, as stated above, should be left to solidify on a slant. Any agar medium that has solidified can be melted by heating in a boiling water bath. Melting of a 1/2-litre bottle of solidified medium will take about an hour if it is submerged well in boiling water. Petri plates dry up rather quickly, so it is often convenient to keep a number of bottles of solid sterilized medium that can be melted and poured when needed. When Petri plates are poured from melted agar, the neck of the bottle should always be heated over a flame first, either alcohol or gas, to kill any stray spore that may be on it.
Once some Petri plates or tubes of sterile agar have been prepared, cultivation of one's moulds can begin. For the sake of simplicity, let us first suppose that a mould colony is already growing in a Petri dish and must be transferred to another. The essential thing to remember is that the air and all implements exposed to it are contaminated with mould spores and that these would germinate and grow if they got into the sterile plate. The steps we take to avoid this contamination constitute what is called sterile technique.
To transfer the culture we do the following:
Transferring from plates to tubes, tubes to plates, or tubes to tubes is done in a similar manner. When using tubes, always flame the mouth to kill any spores of airborne moulds. Never put the cotton plugs or lids of tubes on the table as they will pick up contamination.
The table itself should be clean and can be washed with water, alcohol, chlorine bleach, or other disinfectants. Some spores can survive long immersion in these substances, however, so one cannot thus expect to kill all spores on the table.
Many laboratories are now equipped with special inoculation chambers. Some, such as laminar flow chambers, have a layer of sterile filtered air flowing over the culture that excludes contaminating spores. Some models of such chambers, however, carry the air over the culture and into the worker's face. Such a device is a health hazard and should not be used. Other culture chambers consist only of an enclosed box, open at the front for the worker's hands, containing an ultraviolet light that kills all spores within the box. It is important here to be certain that the ultraviolet light is off while work is under way to avoid the risk of eye damage.
Since some moulds can cause human disease it is unwise to discard or wash tubes and dishes containing living cultures. It is best to sterilize them as before and then deal with them. Plastic Petri plates will melt in the sterilizer and it is necessary to keep them in a pan or bucket to prevent them from flowing over everything else. It is possible to buy non-melting plastic bags for this purpose, but these are only a convenience, not a necessity.
The most exasperating problem confronting attempts to cultivate moulds is contamination by other moulds. Most mould spores are very light and easily transported by air. Even opening the lid of a Petri dish for a few seconds may allow the entry of contaminating organisms.
The worst contaminants are usually moulds, but bacteria and yeasts can also be a problem, especially when attempting to isolate from natural materials. The slimy or pasty bacterial and yeast colonies grow over the top of young cultures and simply smother them. Many bacteria are motile and "swim" along the mould hyphae, congregating at the end where they can share in the nutrients produced by the enzymatically active hyphal tips.
Actinomycetes may also invade cultures from time to time. In older cultures they may even grow on top of the mould colonies. Actinomycetes usually produce large numbers of spores and can be difficult to eliminate.
One of the worst causes of contamination is mites. Mites are small relatives of spiders and ticks that often feed on fungi. They crawl from one source of moulds to another and in the process carry spores of moulds and actinomycetes with them. Once they get into a culture collection they are extremely difficult to get rid of; usually one must go back and re-isolate all of one's cultures to purify them. The techniques for doing this may be difficult and extremely slow. Sometimes cultures may be lost altogether.
Mites are quite small, sometimes less than 1/10 mm long, and can come in a number of colours. The ones that most commonly get into cultures are white to cream-coloured and move rather slowly. Their eggs, also white, resemble tiny, smooth footballs scattered over the colony surface. All mites have eight legs, are usually covered with scattered bristles, and are often covered with spores.
If you find mites in one of your plates or tubes, separate it from the rest immediately and try to isolate the mould into a pure, mite-free culture. Setting the culture in an airtight box containing a dish of mothballs or paradichlorobenzene for several days will often kill the mites and spare the fungi, although the contaminants brought in with the mites will survive as well. If you have a duplicate culture or do not want the old one, sterilize the contaminated plate or tube, mites and all. I cannot emphasize strongly enough the necessity of keeping mites out of a collection.
The best way to avoid contamination is by cleanliness. Keep the work area as clean as possible, wiping down all surfaces with a 10% commercial bleach solution before starting to work. The major source of contaminants in pure cultures is airborne dust. While cleanliness is essential, it will not completely stop contamination; the cleanest of laboratories will still have problems. The number of airborne spores can be reduced drastically by eliminating as many air currents as possible. Fans, air-conditioners, and all kinds of traffic stir up dust continuously and should be avoided. At least an hour before beginning to work, shut the windows and doors, turn off fans and air-conditioners, and stay out of the room. Such precautions can be effective, even under difficult field conditions.
While transferring plate or tube cultures, open their lids for as short a time as possible. The less time allowed for entry of foreign spores the better. It is also wise to avoid opening Petri dish lids any wider than is needed for the accomplishment of the work in progress. I have seen many students open a Petri dish and put the lid down on the table. This habit is unnecessary and only invites contamination.
Spores sometimes stick to necks of tube cultures and can fall into the tube. For this reason it is wise to flame-sterilize the mouths of all tubes and bottles that one opens.
When a piece of natural material is put in a Petri dish a mixed culture of two to several organisms will often be obtained. Where one of these is a bacterium the culture can usually be purified by means of antibiotics. Media such as Martin's Rose Bengal Agar are especially designed for this purpose and nearly always inhibit the bacteria.
If moulds are a cause of contamination one of the selective isolation techniques discussed earlier will have to be followed or one of the "decontamination" procedures that follow.
In the initial isolation of fungi, the most important thing is to watch cultures closely as they first develop. Frequently the mould that is wanted will be accessible at first and easily transferred, but will be entirely overgrown by undesirable moulds later. In isolation from fresh material the first 12-72 hours are especially critical; cultures should not go unexamined for more than 15 hours at a time during this period.
The ability to purify mixed cultures is a skill that must be developed by everyone working with moulds. It is also one of the most difficult things to learn, as it requires a good knowledge of both the fungus one wants and those contaminating it. Although I will not be able to cover this subject in the detail required to purify all cultures, I can at least lay down some general guidelines.
Purifying cultures can be done in one of two ways: (1) mechanically or (2) chemically. In the first the organism one wants is physically separated from the rest while the second depends on the physiology of the organism. Often it may be useful to use both techniques simultaneously.
The simplest means of separating moulds is to spread their apart far enough that the colonies resulting from their germination will not grow together for a time. A common method is to place a spore mixture on the agar on one side of a Petri dish and streak it up and down that side until the spores are evenly spread along a line. The streaking is done with an inoculating wire having its tip bent into a loop so that it will not cut the surface of the medium. Sterilize the loop and do a second streak at right angles to the first along another side of the dish. This results in a second line bearing far fewer spores than the first. Repeat this a third time to yield another line. If it seems necessary, do it a fourth time, but be careful not to streak into the first line. After two or more days one of the streaks will be seen to contain several separate colonies that can be used for transferring. The previous streak will be too crowded and the subsequent one too sparse. This simple technique is the one I use most commonly for purification of mixed cultures (Figure 12).
A similar result can be obtained by using dilutions as described in the section dealing with isolation techniques. It will prove more useful than the streaking technique when the mould that is wanted is greatly outnumbered. Just take a mixture of spores and stir it up in about 10 ml of sterile water. Dilute this down several times and plate out each dilution separately. The ideal dilution will produce a plate containing about 60 colonies, of which at least some will be the desired mould.
When a mould is contaminated with yeasts or bacteria, dilution techniques usually work well. At times, though, yeast or bacterial cells so outnumber the spores of the desired fungus that dilutions are useless. Then it may be helpful to take advantage of the tunnelling or bridging abilities of fungus filaments. A technique sometimes used is to embed glass rings in the medium and surround the contaminated colony. A sterile glass ring, about 10 mm high and 15 mm in diameter, with three "feet" 2-3 mm in diameter glued or welded to the rim on the bottom, is placed on the bottom of a Petri dish. Sterile medium is then poured in to the usual depth, being careful not to overtop or wet the glass ring. After the medium has solidified, the mixed colony is inoculated into the glass ring. The mould filaments will be able to tunnel down under the ring and back up to the surface, while the bacteria or yeast, lacking the forward pressure of hyphae, will be confined to the ring.
The apical growth of mould hyphae can be used to bridge gaps in the medium insurmountable to bacteria and yeasts. A good method to follow is to make a small moist chamber by putting a few layers of wet filter-paper in a Petri plate. Then flame-sterilize a microscope slide, put a square of agar medium near the edge of it, inoculate the agar with the mixed culture, and put the whole thing in the moist chamber. After a few days the hyphae of the desired mould may extend out into the air from the agar block. If they do, place another agar block on a second sterile slide and move it close enough to the first block to contact the aerial hyphae but not the block itself. If one is successful, the hyphae will grow into the new block, leaving the yeast or bacteria behind.
In some cases, mechanical purification techniques just don't seem to work. If they don't one can take advantage of the chemical or nutritional differences between micro-organisms. The most basic technique is to use antibiotics to discourage bacteria. Most bacteria are sensitive to antibiotics, although no antibiotic will work for all of them. In routine isolation work we use a mixture of antibiotics, most often streptomycin and chlortetracycline at a concentration of 30 and 2 mg/l respectively. Few bacteria are able to grow in such a medium, while most fungi are unaffected. In fact, fungi are so tolerant of these substances that we sometimes just sprinkle a little of the powdered antibiotic right on the agar surface in a Petri plate.
In the formula for Martin's Rose Bengal medium, given in the previous chapter, the rose bengal dye is added because it acts as an inhibitor to the growth of bacteria and many moulds. It can be added to any medium and is often useful in helping to separate otherwise rapidly spreading colonies. Oxgall, used in the formulation of DPYA, serves a similar function.
Most moulds have at least some nutritional preferences that distinguish them from other moulds. These preferences can be used in the purification of cultures if the physiology of the organisms involved is known. Zygomycetes, for example, grow rapidly on glucose or maltose media such as Leonian's and will overgrow everything, but cannot grow on some complex compounds like cellulose. Thus a medium such as Czapek's, with cellulose substituted for sucrose, will put zygomycetes at a distinct disadvantage.
The use of selective media for purification of cultures is so dependent on experience with moulds that it is difficult to generalize. A beginner should experiment with several media to see what will happen. The section on selective nutrients in chapter 5 offers suggestions about specific substances.
There are many good texts on the theory and use of the microscope and I am thus going to assume the reader either has some knowledge of microscopy or can find it. My main interest here is with the particular skills necessary for microscopic examination of moulds.
Most beginners find moulds difficult to prepare for microscopic examination. Often preparations seem to contain only spores or mycelium, or structures that are so unlike any of the illustrations available that they are unidentifiable. Most of these problems can be overcome with a little practice and will, in time, seem trivial.
The first rule to remember in mould studies is to examine young, actively growing material. Older parts of colonies or moulds on natural material will often be partially decomposed or so covered with spores as to be unrecognizable. The best way to begin is to examine the growth from the margin of the colony where spores are being actively produced. It may require two or three attempts before the area of active sporulation is located, but when it is all the features of spore production necessary for identification can be seen. If the colony has been in Petri plate with other moulds for several weeks and is no longer actively spreading it will not yield good material for examination.
To make a good microscopic preparation from a mould colony, start by putting a small drop of mounting medium on a microscope slide. Microscope mounting media come in a variety of types and will be discussed separately; at the beginning, use water. Then, using a sterilized dissecting or inoculating needle remove a small (no more than 2 mm square) portion of the colony near the margin, taking with it a very thin layer of the agar surface. If the colony is thick and woolly, it may not be necessary to take the agar, but in the more appressed type it is essential. Place the piece of colony in the mounting medium, and, with a second needle, tease it out so that the filaments are well spread. A mount that has not been teased out will appear as an opaque lump yielding little information. Place a cover-slip over the mount, lowering one edge to the slide before the other so that air bubbles can escape. The remaining air bubbles can be removed from the mount by gently heating it over an alcohol flame. If it is heated too vigorously, the cover-slip will pop off explosively, splattering fungus and medium into one's face, so it is essential to heat it only until its steams slightly, not until it boils. The picture of Harzia verrucosa above was made from a simple water mount and photographed through the eyepiece of a microscope using a point-and-shoot digital camera. This is usually all that is needed to see what you want.
An interesting technique used by some mycologists for preparing microscope mounts involves sticking the mould to a bit of cellophane tape. The tape is pressed lightly against the colony so that some of the hyphae and spores stick to it. This is then placed sticky side up in a drop of mounting fluid (see below) on a microscope slide and covered with a cover slip. This technique is not restricted to colonies in Petri dishes; it works well on naturally occurring colonies in most habitats. The cellophane tape technique is commonly used for sampling moulds in indoor environments.
Most moulds will yield good results if prepared as outlined above, but a few present extra difficulties. The most problematic are those that tend to disintegrate as soon as they are mounted. Species of Cladosporium, Monilia, and Alternaria have spores connected in very fragile chains that can fall apart at the slightest movement of air. Mounts of these fungi invariably reveal only loose spores and a network of hyphae. To overcome this problem it is useful to set up slide cultures (figure 13). Slide cultures are made by setting up a small Petri dish moist chamber containing a V-shaped piece of glass tubing resting on several layers of moistened filter-paper. A sterile block of agar medium about 1 cm square is placed on a flame-sterilized microscope slide and the slide is then set in the moist chamber on the tubing. The fungus is inoculated near the four edges of the agar block and a sterile cover-slip is put over it. After a few days the slide can be mounted on a microscope, and the undisturbed mould structures viewed as they are growing. Later, if desired, the agar block can be removed from slide and cover-slip and two conventional slide mounts made from them. If they are allowed to dry before this is done, the mould structures are less likely to break apart.
Using water as a mounting medium is easy and is often sufficient for satisfactory preparations. Water mounts dry up quickly, however, and do not allow any particular structures to be seen better than others. To overcome these problems, many different mounting media have been devised. Although the preparation and use of mounting media is a specialized and rather personal matter, there are a few that are in routine use in most laboratories because they offer distinct and well-known advantages. I offer below formulas and comments on some of these.
Several products intended to be used as a photographic wetting agents can be used, eg. Kodak Photo-Flow or Edwal Kwik-wet.
This mounting medium has the advantage of preventing air bubbles from sticking to many fungus structures. I sometimes use it in place of water for making mounts of particularly dry moulds. Many fungi are difficult to "wet", even when a wetting agent is used. For these, I suggest putting them in a drop of 95% ethyl alcohol for a few seconds and then, before the alcohol is completely dried out, adding a drop of the required mounting medium. This often works wonders with the dryest of moulds.
The two solutions are kept in separate dropper bottles. A drop of each is put on a microscope slide and mixed with a needle just before use.
This is a useful staining medium for basidiomycetes and other fungi with compact and difficult-to-spread tissues. The mould hyphae are stained a bright pink colour and are thus more easily seen than in water mounts. It is best to draw as much liquid out from under the cover-slip as possible so that the background colour will be much paler. The picture at right is of basidiospores of Dacrymyces lacrymalis mounted in KOH + phloxine.
Lacto-fuchsin will not dry up on the slide for several weeks and is thus useful if a slide must be saved for awhile. Drying can be prolonged even further by sealing the edges of the cover-slip with clear nail polish. Lacto-fuchsin is a strong stain that is especially useful for mounts of anamorphs and other easily spread structures.
This mounting medium is used extensively in mycology. Certain tissues turn blue to blackish in it and are said to be amyloid; others stain red or dark orange and are called dextrinoid. In the illustration at right the asci of Ustulina vulgaris were treated with Melzer's Solution. The ordinarily clear ascal plugs have stained blue. Melzer's Solution is a good general mounting medium that clears the material somewhat and allows particularly brilliant resolution with a microscope. For good resolution and colour in photographs through the microscope, I make up Melzer's Solution without the iodine, which yields a very clear solution. WARNING: Chloral hydrate should be handled with care as it is toxic. In addition it may be listed as a controlled substance in some jurisdictions, requiring special permission for purchase and possession.
Some mycologists prefer to use iodine-containing mounting media without chloral hydrate. Lugol's Solution (0.5 g iodine, 1.5 g potassium iodide, 100 ml distilled water) and IKI (1.0 g iodine, 3.0 g potassium iodide, 100 ml distilled water) are both substitutes for Melzer's Solution and will produce an amyloid or dextrinoid reaction in many fungi. In fact lichenologists (people who study lichens) routinely use Lugol's and only rarely use Melzer's. The amount of iodine can be varied according to the sensitivity of the group of fungi being studied. Although arguments have been made in favour of all of these media, using them in combination will probably yield the most information.
The photo above shows three asci of Agyrium rufum, a fungus commonly encountered on dry branches in sunny locations. The one at left was mounted in Melzer's Solution and yielded no colour changes. The one in the middle was placed in heated 5% KOH solution and then transferred to Melzer's, giving a brown reaction to the ascus tip (perhaps due more to the KOH than to the Melzer's). The one at right was mounted in Lugol's Solution and shows an amyloid apex and dextrinoid contents. This illustration seems to indicate that Lugol's Solution is the best treatment to use, but it's not that simple: many structures can be strongly amyloid in Melzer's alone, as seen in the picture of Ustulina vulgaris, or in Melzer's with KOH pretreatment, so only using all three techniques will give a complete profile.
A very good, all-purpose mounting medium that does not dry upon the slide for several weeks. As with Lacto-fuchsin, it can be sealed in with nail polish for even longer life. Ink Blue was not a part of the original formula but is useful as a stain for the walls of certain fungi. The picture at right shows an ascus of a Claussenomyces species mounted in Shear's Mounting Medium with Ink Blue. The ascospores and ascus contents have become stained blue while the walls of the ascus remain unstained.
Certain mounting media, such as lacto-phenol, are widely used in mycological laboratories but are very toxic and offer no particular advantages over those listed above.
Identification of moulds is based almost entirely on the structures bearing spores and on the spores themselves. Therefore it may be useful for the reader to go back to the beginning and reread the sections in chapters 1 and 2 describing the different kinds of moulds and their anatomy. The process of identification usually involves keys, specialized flow-charts leading to the name of the organism at hand. There are many kinds of keys in use; we present two here, dichotomous keys and picture keys.
The most common means of identifying moulds is by the use of a dichotomous key, a very clever device presenting a series of alternatives for consideration. A glance at any of the keys that follow will serve as a demonstration. In the key for Group I, for example, there are two choices at number 1, two at number 2, two at number 3, etc., on up to number 14. Each pair of choices represents a decision to be made about the mould which is under examination. In number 1 one has to decide whether the mould's spores are composed of one cell or divided by cross-walls into two or more cells. If they are one-celled, the key sends one on to consider the choice in number 2; if they are more than one-celled, one must go to 12. Eventually the series of decisions will lead to a name.
Most books the reader may wish to use will have dichotomous keys that work in the same way as the ones here. But beware; some authors introduce a third or even fourth, fifth, or sixth choice in their keys that may not be noticed as first!
It is difficult to recommend one or even a few books on identification. The best I can do is direct the reader to a few texts as a good starting points. Ainsworth, Sparrow, and Sussman (1973) and Arx (1981) offer keys to most of the groups of fungi you are likely to encounter. If the mould under examination appears not to be an ascomycete, basidiomycete, or zygomycete, try starting with Barron (1968), Barnett and Hunter (1987), or Carmichael et al. (1980). Fungi forming spores in pycnidia can be identified in Nag Raj (1993) and Sutton (1980). For zygomycetes, start with O'Donnell (1979). Domsch et al. (1980), a monumental work treating all fungi known to occur in soil, includes keys, illustrations and extensive literature citations. It is an indispensible book for anyone doing serious work with moulds. De Hoog and Guarro (1995), Gravesen et al. (1994), Samson, et al. (1995), and St-Germain and Summerbell (1996) are beautifully illustrated books, the first and last dealing with fungi of medical interest and the others with fungi found on foods and other human-associated materials. Wang and Zabel (1990), dealing with fungi isolated from utility poles, is a very useful reference for wood-inhabiting fungi. It contains extensive keys and illustrations.
Two approaches are taken to identification in this section: a set of dichotomous keys and a set of picture keys. Which you choose depends upon your individual preference. Some people are verbal in nature and do best when everything is written out; these people usually prefer dichotomous or other types of textual keys. Others are visual and prefer to match what they see to an image. You may even find you do best with a combination of the two.
The dichotomous keys are designed to work like the mind of an experienced mycologist, eliminating the most common or most expected fungi first and relegating the less common ones to the end. These are composed of keys to several groups of genera. Group I contains the most commonly encountered genera, group II those that are a little less common, and group III those that are less common yet, and so on down to the end. To use the key, start with the key to group I. If you are satisfied that the fungus you are trying to identify is not there, try the key to group II. If that does not work, go to Group III, and so on. The chances are greatest, of course, that the mould you want to identify is in group I, since these are the commonest of all moulds. If your fungus is not among the 60 genera in the whole key, you will have to turn to more complete or specialized books, such as those listed above or in the bibliography.
It should be pointed out that all identifications should be checked against the appropriate description and illustration following the key. It may also be necessary to turn to the references given there. Fungi from one group may be identified incorrectly in the key to another group and only the description and illustration will reveal the mistake. For example, Paecilomyces of group II will key out to Penicillium in group I, but this problem will only be discovered when checking the descriptions and illustrations.
These keys are arranged in the same way as the dichotomous keys: that is, the first set of pictures illustrates the most comonly encountered moulds. Those in the second set are also common, but not quite as common as those in the first group. To use the keys, browse over the first group to see if your unknown specimen matches one of the pictures. If you think it fits one of them, click on the picture for further information. If the fit is not very good, go back and try again. If the unknown is not in Group I, go on to Group II.
These are the most common moulds you will encounter
These are the second most common moulds you will encounter
These are the third most common moulds you will encounter
These are the fourth most common moulds you will encounter
These are the fifth most common moulds you will encounter
Species of Acremonium are recognized by solitary to weakly branched, tapering phialides arising from vegetative filaments and bearing a wet cluster of mostly 1-celled spores (conidia). The filaments are sometimes bound together into "ropes" several cells in diameter. The illustrations here present Acremonium in its narrower sense, but a great variety of species have been included in the genus at one time or another. Almost any species bearing colourless or brightly-coloured conidia on rather simple unpigmented phialides might be referred to Acremonium. Seifert et al. (2011) present a key to no fewer than 50 genera that might be confused with Acremonium. Even when taken in its narrowest sense, species of Acremonium are nearly impossible to identify without the use of molecular methods.
Common in soil, plant debris, rotting mushrooms, etc. Some appear to be parasitic on living fungi.
Classification: in a narrow sense of Acremonium the species are mostly members of the Hypocreomycetidae.
The dark brown spores are borne in simple or branched chains from the tips of simple dark conidiophores and are divided into several cells by transverse and vertical walls. New spores are produced by the extrusion of wall material through a pore at the tip of the previous spore. Commonly isolated from decaying plant materials; also causing plant diseases. Spores of Alternaria species are dispersed by air currents and are usually abundant in outdoor air.
The spores (conidia) are dark brown and usually occur in grape-like masses on white woolly colonies. The spores are flattened and have a colourless line at the edge. When germinating they break along the line in the manner of a clam shell. In some species the filaments have dark cross-walls. Common on dead plants, especially grasses and sedges, and often isolated from air near grassy places in the autumn. The drawing at right illustrates several species. The photographs are of Arthrinium cuspidatum found growing on dead grass stems in a salt marsh in St. Andrews, New Brunswick.
Characterized by tall, colourless conidiophores bearing clusters of spores (conidia) at the tip and often at several swollen points below. The conidia are pale, 2- to 3-celled, and leave conspicuous scars on the conidiophore when released.
Species of Arhrobotrys are probably all predators on eelworms (nematodes). They employ several devices to capture these microscopic worms, including (a) constricting rings and (b) sticky networks of loops. Constricting rings work something like the well-known Venus' fly trap. When a nematode, intent upon searching for bacteria and other small edible particles, enters one of the rings, the cells of the ring suddenly inflate and the nematode becomes entrapped. The constricting action of these rings is so powerful that the nematode is almost cut in half. Sticky networks are less theatrical than constricting rings, yet they have their own brand of terror. When a nematode enters the network of loops it is held by a very strong "glue" and is unable to escape. In both cases, the captured worm first struggles wildly and then seems to become comatose. The fungus then sends its filaments into the worm and digests it. When it has obtained sufficient nutrients from its prey the fungus reproduces by producing clusters of conidia at the tops of long conidiophores.
Thanks to the groundbreaking research of Drs. Donald Pfister and Michael Liftik (Pfister, 1994, 1997; Pfister and Liftik, 1995) we now have a much better understanding of the life histories of Arthrobotrys species. These authors provided convincing evidence that Arthrobotrys species are the asexual expression of the well-known discomycete genus Orbilia, a group of ascomycetes commonly found on dead wood, decomposing plant materials, soil and dung. Although species of Orbilia were previously thought to be simple decomposers we now realize they can be active predators.
Arthrobotrys species are undoubtedly important in controlling numbers of nematodes, including those causing damage to agricultural crops. To study them it is necessary to first have a culture of nematodes. We maintain them on a weak medium spread with yeast or bacteria. Dehydrated pea soup mix also seems to be good food for nematodes. To see Arthrobotrys, a pinch of soil is introduced into the nematode culture. After a few days the traps will become abundant.
Classification: Orbiliaceae. Although the sexual stages resemble one of the inoperculate discomycetes, they may not be closely related. Dr Joey Spatafora, writing on The Tree of Life website assigns them to the Class Orbiliomycetes, a group of discomycetes distinct from both the Leotiomycetes and Pezizomycetes.
Recognized by its distinct conidiophores arising from a well-defined "foot cell" and terminated by a swollen vesicle bearing flask-shaped phialides. The phialides may be borne directly on the vesicle (a) or on intervening metulae (b). Some species may form masses of thick-walled cells called "hülle cells" (c). The spores come in several colours, depending upon the species, and are produced in long chains from the ends of the phialides. Commonly isolated from soil, plant debris, and house dust; sometimes pathogenic to man.
Classification: Trichocomaceae (Eurotiales).
This is one of several genera of "black yeasts", characterized by mostly slow-growing, black, pasty colonies. The spores are produced in great masses along the filaments and occur on short lateral branches or pegs. When the spores are released they leave minute roughened scars. Exophiala species are similar but the spores do not leave rough scars as in Aureobasidium. Black yeasts occur in many habitats; some species of Exophiala may even cause human disease. Aureobasidium pullulans, the most frequently reported and possibly only species of Aureobasidium is a common inhabitant of the phylloplane (leaf surfaces) but is also frequently reported from soil, wood, rocks stone monuments and human skin. Stone buildings that have become black over the years often have abundant growth of A. pullulans.
Classification: Dothioraceae (Dothideales).
Bacteria are not fungi and are included here only because they will always be found where fungi occur. They form wet or slimy colonies, often with rather bright colours and unpleasant odours. The cells are nearly always very small and take a variety of shapes including the well-known rods (bacilli), spheres (cocci) and helices (spirilla). Some species, such as those of Streptomyces and related genera can form multicellular filaments. Many bacteria are motile, swimming freely around a microscope slide or wet Petri plate. The photographs above illustrate several features of bacteria. At left is a colony of a species of Bacillus, photographed in a sample of pond water. Each of the rod-shaped cells contains a thick-walled endospore. At top-center is a group of rods of Bacillus aureus, a species that can be isolated from certain foods such as fried rice and which can cause diarrhea in humans. Escherichia coli, at bottom-center, is a normal inhabitant of the human intestinal tract that occurs in both benign and beneficial forms. Because E. coli is always in the intestinal tract, its presence in water and other materials is a good indicator of fecal contamination. The organism at bottom-left is a species of Spirillum, common in nutrient-rich ponds. Note the curved shape that will become a loose helix in larger cells and the fasicle of flagella at the end of each cell. Flagella, which may occur singly or in clusters, are the main means of bacterial locomotion. Bacteria must be handled with great care as some are pathogenic to humans.
The routine identification of bacteria requires specialized facilities not normally available to mycologists. Although morphology can play a role in identification it is also necessary to utilize a battery of nutritional and physiological tests as well. Taxonomic studies based on nucleic acid homologies have shown that the traditional methods for classifying bacteria are often at odds with genetic evidence. Present-day bacteriologists still rely on traditional methods, but these are quickly being replaced by more reliable methods using genetic sequences. The basic reference for traditional methods is the 9th edition of Bergey's Manual of Determinative Bacteriology (Holt, 1994).
Species of Beauveria produce rather slow-growing white cottony colonies. Reproduction is copious and the conidia are easily detached, a condition often leading to scattered colonies when you thought you had carefully transferred only one. The one celled colourless conidia are borne along a thin filament that elongates in a zig-zag fashion as the conidia are produced. The conidia are produced on short spikes or denticles, giving the conidiogenous cells a spiny appearance. Species of Tritirachium are similar to some Beauveria species in having zig-zag conidiogenous cells, but differ in lacking denticles on their conidiogenous cells and in producing yellow brown to purple colonies. Beauveria species are commonly found associated with insects or habitats supporting insects. They commonly occur in private dwellings, where they probably decompose the dead bodies of insects, spiders and mites. They may also attack living insects and cause small epidemics. Beauveria bassiana, the best-known member of its genus, has been investigated widely as a potential agent for the biological control of harmful insects.
Glare and Inwood (1998) and Rehner and Buckley (2005) carefully examined cultures of B. bassiana, B. amorpha, B. brongniartii, B. caledonica and B. vermiconia as well as certain strains of B. bassiana that did not fit the concept of that species very well. They utilized traditional as well as molecular tools in their analysis and concluded that all of these species fall into distinct clades. This suggests that all are therefore "good" species. Unfortunately neither of these papers supplies identification keys, although Glare and Inwood did provide a chart of spore measurements that might be some aid in determination.
In species of Bipolaris the large, dark, multicellular spores are produced from pores along a gradually elongating, dark conidiophore. Often the cells of the spores appear to be very thick walled. Germination occurs only through the two end cells (and hence the generic name). Drechslera species differ in having spores that germinate through any cell. Species of Curvularia are similar, but the spores never appear very thick walled. Common on dead or dying plant material, especially grasses.
Species of Botrytis produce coarse, brown conidiophores with branched tops. The spores (conidia) cover the ultimate branches and are produced synchronously. As the colonies begin to dry, the cells of the conidiophore often flatten in alternate planes from each other. Many isolates produce black stone-like sclerotia. Often isolated from dead or living plants and frequently causing plant disease. Rotting of stored fruits and vegetables by Botrytis species is a major problem. Jarvis (1977) wrote an important overview of Botrytis that discusses many aspects of this economically important genus, although it cannot be used for identification of species.
Conidiophores tall and bearing colourless 2- to 5-celled spores (conidia) at the tips of short apical branches. As the name implies, the branching is like that of a candelabrum. The species are predacious on eelworms and catch them in constricting rings (a) or sticky networks (b). Arthrobotrys species are similar but do not have conidiophores with branched tips. Some authors consider the two genera to be indistinguishable.
Classification: Orbiliaceae. Although the sexual stages resemble one of the inoperculate discomycetes, they may not be closely related. Dr Joey Spatafora, writing on The Tree of Life website assigns them to the Class Orbiliomycetes, a group of discomycetes distinct from both the Leotiomycetes and Pezizomycetes.
Although often appearing to be mould-like, species of Candida are really yeast fungi, differing from filamentous fungi in some rather fundamental ways. The cells of their hyphae are usually rather loosely attached and will fragment when disturbed. The spores are produced along the "hyphae" and become so numerous that the filaments can be entirely obscured. The spores can reproduce themselves by "budding". The abundance of spores can give the colony a pasty or slimy appearance. Species of Candida are common in soil and organic debris and can also cause human disease. Although these organisms often live on the bodies of humans and other mammals causing little harm, if the immune system becomes compromised by immunosuppresant drugs or disease they can cause serious infections or even death. People infected with HIV are often at risk of contracting candidiasis, the name of the disease caused by C. albicans. Not all species of Candida cause disease in humans, many occur in association with plants or insects and never affect humans at all.
Classification: Saccharomycetales, one of the so-called Hemiascomycetes or yeasts.
The dark brown conidiophores are united in a fascicle to form a long compound fruiting structure (synnema). The upper half of the synnema is covered with flask-shaped annellides giving rise to chains of grey-brown spores (conidia). In C. stemonitis there is an accessory Echinobotryum spore form arising from the stalks of the synnemata and on the surrounding hyphae. Other species, such as C. microsporum, pictured at right, have only one spore form. Trichurus species are similar, differing in having straight to coiled hairs arising among the annellides. Young synnemata of Trichurus species may not have well developed hairs and may be mistaken for a species of Cephalotrichum. Related to Scopulariopsis. Many of the species were placed in Doratomyces in the older literature. Common in soil, dung, and decaying plant materials.
Characterized by densely hairy, egg-shaped fruiting bodies (perithecia) containing asci, which in turn enclose 4-8 brown spores (ascospores). The perithecial hairs can take a variety of forms, depending upon the species. The ascospores collect in a dense mass outside the perithecium. Most species are strong decomposers of cellulose and occur wherever this substrate is abundant, such as in soil, dung, or rotting plants.
Recognized by its branched chains of light or brightly coloured spores (conidia) arising from inconspicuous conidiophores. Spores are produced by "budding", with the youngest ones at the tips of the chains.
Species of Chrysonilia, including C. sitophila, the red bread mould, produce very fast growing pink colonies and numerous spores. The spores are dry and easily detached from one another. Because they combine rapid growth and prolific reproduction, species of Chrysonilia can contaminate other laboratory cultures very rapidly. In the school or university laboratory where many students may be cultivating fungi at the same time these fungi can cause lab-wide epidemics, ruining every culture they contact. The best strategy is to discard all rapidly growing, pink, powdery cultures without opening them.
Neurospora, the holomorph of Chrysonilia species, is widely used as an experimental organism. More is known about the physiology and genetics of Neurospora than any other fungus. The ascospores of Neurospora species resist germination until they encounter certain kinds of physical or chemical stimulant. In nature, the ascospores occur in soil and are activated by heat. Thus the species Neurospora and their associated Chrysonilia anamorphs can often be found on the surface of soil following forest or grass fires. They can also appear on the surface of soil that has been partially sterilized in the greenhouse. They can be isolated from soil by first treating the sample with alcohol or heat (see discussion of stress techniques in Chapter 4.
Many texts still treat Chrysonilia species in the genus Monilia. However, Monilia species are plant parasites, commonly causing a soft brown rot of stone fruits (peaches, apricots, etc.), and are not closely related to Chrysonilia. Common in soil, homes, fruits, and as a laboratory pest.
Spores (conidia) are produced along the vegetative filaments by a swelling of the wall and subsequent isolating by a cross-wall. The conidia can be terminal on the filament or at various sites along its length. Chains of conidia may occur and are sometimes separated by empty cells. Most species are entirely colourless to yellow.
Species of Chrysosporium have been shown to be anamorphs of many different fungi (see below), suggesting that they are not a group with a unique ancestor but are instead polyphyletic. Their ecology reflects these diverse origins; some species are psychrotolerant (able to grow at low temperatures) while others, in contrast, are thermotolerant (able to grow at high temperatures). As well, some are osmotolerant (tolerating dryness or osmotic stress) while others are not. Although the species are often not physically complex or highly characteristic, their physiological diversity is a great help in their identification.
Growing in soil, dung, and decaying plant material. Some are able to decompose hair, hoofs, and leather.
Classification: Onygenaceae (Onygenales).
Colonies rather coarse and fast-growing. Spores produced within dark spherical sporangia that are borne along the length of sporangiophores on strongly curved or hooked branches. The filaments have few cross-walls and thus resemble long, hollow tubes. The most characteristic feature is the strongly hooked sporangial branch. Occurring in soil, dung, and decaying nuts.
Colonies dark greenish to black, black in reverse, and relatively slow-growing. The dark spores are 1- or 2-celled and occur in long, branching chains that arise from a dark conidiophore. The youngest spore is at the top of the chain. The slightest movement will disrupt the chains, making microscope mounts of the whole structure nearly impossible. The best way to recognize the genus is by the prominent scars on the spores where the adjacent ones were attached. Very common on decaying plants and fungi; the fungus most commonly isolated from air, both indoors and outdoors.
It has always been difficult to identify species of Cladosporium. Most isolates from air and from habitats associated with humans have been referred to one of three species, distinguished by the following key:
1. Spores usually ellipsoidal in shape, only rarely spherical - 2.
1. Spores often nearly spherical -» Cladosporium sphaerospermum.
2. Spores smooth -» Cladosporium cladosporioides.
2. Spores warty -» Cladosporium herbarum.
Unfortunately the taxonomic reality is that there are many more species of Cladosporium, perhaps hundreds more, than these three. Many are known from specific plants or particular geographic areas. Many have not yet been grown in culture and many remain undescribed. The recent book on Cladosporium edited by Crous et al (2007) addresses this difficult genus in several ways and provides keys, descriptions and illustrations to aid in identification. It is a major advance in the taxonomy of Cladosporium and one that will undoubtedly stimilate further research.
Dendryphiella species are not among the most common of fungi but any isolations from ocean beaches will turn them up in abundance. Two species are known in this habitat, D. arenaria, at left, and D. salina, at right. Both are common but often not overlapping in their ranges, with D. salina found in cool climates and D. arenaria in warm ones. Other species, including the type, occur on decomposing plants in terrestrial situations. Species of Dendryphiella are recognized by their sympodially developing conidiogenous cells producing dark poroconidia with several transverse septa.
Mycologists have long debated whether the two marine species belong in the genus Dendryphiella or Scolecobasidium. They are very similar morphologically, so the distinction really boils down to some very obscure features. Dela Cruz et al (2006) finally laid the controversy to rest by presenting molecular evidence that D. arenaria and D. salina are closely related to one another and only very distantly related to species of Scolecobasidium. Of course there is one futher issue left to resolve; the genus Dendryphiella was typified (created specifically) for Dendryphiella interseminata (Berk. & Ravenel) Bubák, a species orginally described from Phytolacca decandra (=Phytolacca americana), American pokeweed and Cicuta maculata, Spotted cowbane. It is not yet known whether D. interseminata is closely related to the two marine species or not. If not, our two beach inhabitants will have to find a home in yet another genus.
Habitat: decaying stems and marine algae.
The spores (conidia) are brown, 2-celled, and are borne within more or less round fruiting structures (pycnidia). Conidial production occurs (probably holoblastically) on short cells that line the inner walls of the pycnidium. There are a number of similar genera that are distinguished on the basis of pycnidial and spore structure. Most species occur on living or dead plants.
Characterized by small clusters of pear-shaped, beaked, roughened, brown spores (conidia) borne on short, branched or simple conidiophores. Usually occurring as an "accessory form" at the synnematal bases and surrounding hyphae of Cephalotrichum stemonitis. Fairly common on dung, soil, and plant debris.
Easily recognized by its nearly spherical spores (conidia) that are several-celled and strongly roughened. The conidia are usually produced in dense masses from clustered conidiophores (sporodochia). Identification may be difficult at first because isolates often fail to produce conidia in culture. Experienced mycologists easily recognize it by its fast-growing, red, orange or yellow colonies. Heavily sporulating colonies are sometimes encountered but these are less common than the more lightly sporulating or non-sporulating strains. Very common on dead or dying plants; frequently isolated from both outdoor and indoor air.
Classification: Pleosporaceae (Pleosporales).
An ascomycete characterized by whitish to bright yellow spherical fruiting bodies (cleistothecia) containing spherical asci which in turn each enclose eight colourless ascospores. The ascospores are oblate (like a flattened sphere) and may have equatorial ridges, thus resembling pulleys. Species of Eurotium grow best in dry situations and are usually cultivated on media high in sucrose or glycerine. They are common in homes, stored grains, and rodent dwellings.
The three photographs at top are of a strain of Eurotium rubrum found growing on an old pita bread in a New Brunswick home. The bright yellow-orange colony is typical of Eurotium species where the bright yellow cleistothecia greatly outnumber the green masses of conidia. The green colony at left is a species of Penicillium, probably P. chrysogenum. The middle panel is an enlargement of the colony showing the yellow cleistothecia nestled among the red hyphae characteristic of E. rubrum. The right panel shows a group of spores. The darker, roughened spores at the right and top of the picture are conidia of the Aspergillus anamorph of the fungus. The rest of the spores are ascospores, which are shaped like minute hamburgers. In common with hamburgers, ascospores of Eurotium species tend to come to rest on a top or bottom rather than on a side. Thus most of the ascospores look circular. However, two of these are lying on their side and thus have the elliptical outline of a hamburger as viewed by someone about to bite into it. The group of ascospores that are slightly out of focus are still inside an ascus, although the ascal wall cannot be seen. All of these features can be seen more clearly in the drawing at right. The coiled structure in the drawing is an ascogonial coil, discussed in greater in the discussion of the Sordariomycetes.
Exophiala species are usually included among the fungi called "black yeasts". Conidia are typically borne at the tips of short annellides produced along the vegetative hyphae. The annellides are often difficult to see and to determine that they actually are annellides. Species of Phialophora are similar but produce their conidia on phialides rather than annellides. Species of Aureobasidium, another genus of black yeasts, produce conidia holoblastically on minute peg-like extensions of short hyphal branches or directly along the hyphae themselves.
Some species of Exophiala are known to cause a subcuaneous disease in humans and other vertebrates. Although not normally life-threatening, these infections must be removed surgically or they may continue to grow for years. In handling these fungi, care must be taken not to accidentally inoculate oneself with contaminated instruments.
The natural habitats of Exophiala species are hard to pin down. They can be isolated from decaying plant material, wood, sewage sludge, soil, tree exudates and many other sources. They sometimes appear in unlikely places, such as in syrup-like solutions of polyvinyl alcohol. They are often most easily found by locating the small perithecia of the Exophiala holomorphs.
Classification: Herpotrichiellaceae (Eurotiomycetes).
Most characteristic are the colourless spores (conidia), which are canoe-shaped in side view, have a distinct "foot cell" at the lower end, and are divided by several cross-walls. The conidiophores are often clustered to form sporodochia and produce large pasty masses of spores from tapered phialides. Two other spore forms may occur, microconidia (a) resembling spores and phialides of Acremonium, and chlamydospores (b), thick-walled swellings along the filaments. Cultures may be brightly coloured. Common in soil and dead or living plants; often causing plant disease.
The 2- to 4-celled colourless spores (conidia) are produced at the "joints" of more or less zigzag or geniculate conidiophores. The youngest conidium is at the top and new growth of the conidiophore then originates just below it and pushes it to one side. Considered by some authors to be indistinguishable from Arthrobotrys. Predacious on eelworms, which they trap in sticky networks of loops. Occurring in soils high in organic material.
Classification: see Arthrobotrys.
Characterized by short but distinct conidiophores that are branched above and which bear chains of spores formed directly from the cells of the branches. Often only the tips of the branches become converted into spores. The spores (conidia) are 1-celled and may be white or yellow.
Species of Geomyces are generally found in relatively cool habitats, although many can grow well at room temperature. Some species are psychrophilic, that is they are adapted to cold conditions and cannot tolerate temperatures above the mid-teens celsius. These psychrophiles are often abundant in soils at high latitudes or in cold habitats such as mines and caves. Geomyces destructans, cause of white-nose syndrome in bats, thrives on hibernating bats in caves where the year-around temperature remains below 10 degrees C. The picture at right-middle shows the curved conidia characteristic of G. destructans. Occurring in soil, leaf litter and animal materials such as dung and decomposing tissues.
A very simple genus characterized by the formation of chains of colourless, slimy spores (conidia) through the segmentation of vegetative filaments. Some with strong odours. Common in dairy products, fruits, slime fluxes, and sometimes soil. Suh and Blackwell (2006) described three new species from the guts of living insects.
Classification: Dipodascaceae (Saccharomycetales), one of the so-called Hemiascomycetes or yeasts.
Conidiophores erect, terminated by a dense brush-like branching system bearing tapered phialides. The spores (conidia) are colourless, or green and are produced in a dense wet mass from the phialides. The spore-bearing structures are fairly large and complex as in the illustration of G. penicillioides at left. Species of Gliocladium are similar to those of Penicillium but with conidia collecting in wet rather than dry masses.
For many years Clonostachys rosea (two photos at right) was maintained in Gliocladium as G. rosum but is now known to be a member of the Bionectriaceae as an anamorph of Bionectria and Roumegueriella species.
Species of Gliocladium occur in soil or decaying plant matter where they frequently are reported as parasites of other fungi. Some can become pests in petri dish cultures, overrunning the colonies of other fungi.
Consisting of an erect conidiophore with several roughened swellings along its length. The colourless 1-celled spores (conidia) are borne in grape-like clusters around each swelling. Similar to Arthrobotrys but the spores are never more than 1-celled. Parasitic on other fungi by means of small sucker-like structures called contact cells. Common on dead or dying plant parts, especially those not yet fallen to the ground.
Fruiting structure a synnema, composed of fused dark conidiophores terminating in a droplet of liquid containing conidia. The colourless 1-celled spores are produced continuously from annellides, phialides or sympodially occurrring holoblastic loci, and collect in a large drop of fluid. The synnemata can be quite stout and up to 5 mm high.
The two panels in the leftmost photo were taken from a culture isolated from seeds of canola, an edible rapeseed. It is a typical Graphium anamorph of Kernia, Microascus, Petriella and Pseudallescheria, all members of the ascomycete family Microascaceae. A few non-synnematous (mononematous) structures can be seen in the photograph at left; strictly speaking, these should be called Scedosporium because they are not synnematous. However, many strains are highly synnematous when first isolated but become mononematous after a few transfers. Some species of Scedosporium cause disease in humans and should be handled with great care. The second photo is the Graphium anamorph of Kernia pachypleura. This species produces distinct but much smaller synnemata with more compact annellides. The picture at right-middle is a macrophoto of synnemata growing on an agar surface.
Occurring on wood, dung, seeds, and plant debris.
An ascomycete characterized by loose, light-coloured, bramble-like fruiting structures (gymnothecia) containing spherical asci which in turn each enclose eight spores (ascospores). The ascospores are colourless to yellow, smooth, and oblate ( a flattened sphere). The gymnothecia usually bear some sort of hooked or recurved spines.
Several other genera of ascomycetes, all treated and discussed by Currah (1985, 1988), are similar to Gymnoascus. Gymnoascus species differ from all of these in having a combination of oblate, smooth ascospores and gymnothecia composed of distinctive hyphae forming a complete network around the mass of ascospores.
Occurring in soil, dung, or other habitats where hair or feathers are decaying.
Conidiophores very dark and stout, bearing a brush-like apical branching structure that is terminated by tapered annellides. The colourless 1-celled spores (conidia) are produced continuously from the tips of the annelides and collect in a large drop of fluid. Phialocephala species are related but differ in having phialides. Species of Thysanophora are similar but have conidiophores with short swollen branches bearing phialides in a manner strongly reminiscent of Penicillium species. On wood and conifer needles.
The dark spores (conidia) are borne within cellular, more or less round fruiting structures (pycnidia) and are produced on small flask- or peg-like cells (annellides). Usually the spores are extruded through an opening at the top of the pycnidium and collect in a droplet of fluid. The species occur on living or dead plants.
Conidiophores colourless, erect, bearing a single terminal spore (conidium). The conidia have 2 to several cells, are colourless, and have one cell larger than the others. Because of the large cell, the conidia are strongly spindle-shaped. Rubner (1996) recognized 39 species. Predacious on eelworms, which they catch by means of sticky knobs (a), rings (b), or adhesive networks (c). Closely related or possibly synonymous with Arthrobotrys. Occurring in soil, dung, and decaying plant matter.
Mortierella colonies are relatively fast-growing and often spread in overlapping "waves" or lobes. Some have a peculiar odour suggesting garlic. Many species produce a white oily substance in large drops among the aerial hyphae. One group of species, characterized by velvety, odourless colonies, is included with Mortierella in the older literature but is now segregated into the separate and unrelated genus Micromucor. The spores (sporangiospores) of Mortierella species are produced inside spherical sporangia at the tips of sporangiophores and are colourless to brownish. A columella is usually lacking or very poorly developed (compare with Mucor for this feature).The photograph at left illustrates a species of Mortierella, possibly M. verticillata, isolated by museum technician Karen Vanderwolf from the surface of a hibernating bat in a New Brunswick cave. The leftmost panel shows a sporangiophore with a single spore at its apex. In fact, the sporangial wall had already broken and the spore is one of three or four that were originally there. The middle panel depicts several sporangiospores from broken sporangia. Typical of Mortierella species the spores are irregular in size and shape. The rightmost panel illustrates two pairs of gametangia in the midst of mating. The contents of one will move into the other, where the nuclei of the two will fuse, producing temporarily diploid nucei (zygotes). The gametangium containing the diploid nuclei will dvelop into a zygosporangium.
Mortierella species often fail to reproduce under normal laboratory conditions. We have obtained good sporulation by growing the colonies on water agar (agar media containing no nutrients) and incubating at 5-10°C. Growth is slow but sporangia are often abundant. Apparently cold and starvation triggers reproduction.
Some species, such as M. polycephala, produce spherical "chlamydospores". These spores can be large in some species and may be variously roughened. In culture, the chlamydospores may be more conspicuous than the sporangia; in fact sporangia may be absent at first. Similar spores are produced by Histoplasma capsulatum, cause of a serious disease in humans, and by species of Sepedonium.
Common in soil and dung.
Classification: Mortierellaceae (Mucorales, Zygomycota).
Colonies fast-growing, whitish to greyish, usually thick owing to the abundant upright sporangiophores. Spores (sporangiospores) produced inside spherical sporangia at the tips of the sporangiophores, brownish. Always with a large columella that remains after the sporangial wall is broken (a). Large dark zygospores may be produced. Common almost everywhere fungi occur.
Classification: Mucoraceae (Mucorales, Zygomycota).
The conidiophores are united together to form fruiting structures (sporodochia) which are usually flat but may be on a stalk. The spores (conidia) are borne from the tips of the long phialides which are in turn borne on densely branched and brush-like conidiophores. The mass of dark green conidia collect in large green to blackish wet drops. In soil and decaying plant debris.
Classification: Hypocreales (Hypocreomycetidae), but its family placement is still unclear.
The white woolly colonies grow fairly rapidly. Spores (conidia) are produced singly on swollen urn-shaped conidiophores and are egg-shaped to flattened-spherical, black, and often have an equatorial colourless line or germ slit. Occurring as parasites on living grasses but also present on dead ones; easily isolated from dead lawn grass in the autumn.
Classification: Trichosphaeriales (Sordariomycetidae).
Characterized by erect colourless conidiophores with a terminal swelling or vesicle. The spores (conidia) are colourless, 1-celled, and are borne in a single layer on the surface of the vesicle. Isolated from dung, wood, soil, and decomposing plant matter.
The dark conidiophores are erect, usually tall, and are terminated by a rather irregular system of tree-like branches. The brown to colourless spores (conidia) are produced by the fragmentation of the conidiophore branches. Occurring in soil and organic debris.
Colonies with more or less well developed, colourless, simple or branched conidiophores bearing two to several phialides. The phialides are characteristically swollen at the base and gradually narrowed into a long beak. The spores (conidia) are produced in chains from the tips of the phialides, are colourless or brightly pigmented, and are usually rather narrow. Similar to Penicillium, but the chains of spores tend to be widely divergent in Paecilomyces and more parallel in Penicillium.
Species of Penicillium are recognized by their dense brush-like spore-bearing structures. The conidiophores are simple or branched and are terminated by clusters of flask-shaped phialides. The spores (conidia) are produced in dry chains from the tips of the phialides, with the youngest spore at the base of the chain, and are nearly always green. Branching is an important feature for identifying Penicillium species. Some, such as Penicillium glabrum at left, are unbranched and simply bear a cluster of phialides at the top of the stipe. Others (middle photo) may have a cluster of branches, each bearing a cluster of phialides. Penicillum atramentosum, at right, represents a third type haing branches that bear a second order of branches, bearing in turn a cluster of phialides. These three types of spore bearing systems (penicilli) are called monoverticillate, biverticillate and terverticillate respectively. Penicillium is a large and difficult genus encountered almost everywhere, and usually the most abundant genus of fungi in soils.
The common occurrence of Penicillium species in food is a particular problem. Some species produce toxins and may render food inedible or even dangerous. It is a good practice to discard foods showing the development of any mould. On the other hand some species of Penicillium are beneficial to humans. Cheeses such as Roquefort, Brie, Camembert, Stilton, etc. are ripened with species of Penicillium and are quite safe to eat. The drug penicillin is produced by Penicillium chrysogenum, a commonly occurring mould in most homes.
Classification: Aspergillaceae (Eurotiales).
Conidiophores (annellides) produced within compact fruiting structures (aecervuli or pycnidia). Spores (conidia) 4- to 5-celled, with the two or three central cells dark brown, and with two or more apical appendages or hairs; collecting in a wet mass outside the aecervulus.
Pestalotiopsis is just one of a complex group of fungi. For example the photo at top-center is of Seiridium abietinum, a species found on dead branches of balsam fir in Atlantic Canada. It differs from species of Pestalotiopsis in having conidia with a single apical appendage rather than several. The following key may be some help in distinguishing these difficult genera. However, anyone seriously interested in identifying a member of this group should consult the monumental work on coelomycetes with appendaged conidia by T. R. Nag Raj (Nag Raj, 1993).
Simplified key to Pestalotiopsis and related genera
Parasitic or endophytic on living leaves and twigs but are often isolated from dead plant matter and even soil.
Classification: Pestalotiopsis species, and those of related genera, are anamorphs of members of the ascomycete family Amphisphaeriaceae (Sordariomycetidae). Members of the Amphisphaeriaceae and their anamorphs include Amphisphaeria (Bleptosporium), Blogiascospora (Seiridium), Broomella (Pestalotia and Truncatella), Discostroma (Seimatosporium), Ellurema (Hyalotiopsis), Griphosphaerioma (Labridella), Lepteutypa (Seiridium), Neobroomella (Pestalotia), and Pestalosphaeria (Pestalotiopsis). Doliomyces, Monochaetia, Sarcostroma and Zetiasplozna have not yet been found associated with a holomorph.
Characterized by brown to black colonies bearing phialides directly on the vegetative filaments or on short branches. The phialides are dark, flask-shaped, and have a collar-like or flared apex. The colourless to brown 1-celled spores (conidia) are produced successively from the apex of the phialides. Some species may produce dark brown holoblastic conidia along the vegetative hyphae or at the tips of short branches. Species of Exophiala are similar and are separated from those of Phialophora mainly on the basis of producing annellides rather than phialides. The differences between the two are subtle in some instances. There are numerous other fungi not closely related to Phialophora as strictly defined. Seifert et al. provide a key written by the eminent Dutch mycologist Dr. Walter Gams to 36 genera that could be confused with Phialophora.
Phialophora species have been isolated from soil, water, dung, wood and plant debris.
Classification: Herpotrichiellaceae (Eurotiomycetes).
Recognized by its cellular and more or less round fruiting structures (pycnidia) containing masses of 1-celled colourless to yellow or pink spores (conidia). The conidia are borne from inconspicuous peg-like phialides lining the inner wall of the pycnidium. Species of Phoma having spines or setae on their pycnidia are sometimes confused with those of Pyrenochaeta. However, the two genera can be distinguished on the basis of their conidium-bearing structures: conidia of Phoma species are produced from simple phialides while those of Pyrenochaeta arise from phialides ocurring along the sides of elongated conidiophores.
Phoma is a taxonomically difficult genus and is still under study. Species identification is often difficult. Much of our present knowledge has come from the work of Dr. G.H. Boerema and his colleagues in The Netherlands. Boerema (in Aa, et al., 1990) divided Phoma into five sections, extended in 2004 (Boerema et al., 2004) to nine in a comprehensive identification manual.
Aveskamp et al., (2010) have studied the genetic relationships of fungi referrable to Phoma and have found, as Boerema himself suspected, that the genus is highly polyphyletic; that is, its species belong to a number of distinct families within the Pleosporales., including the Cucurbitaceae, Didymellaceae, Leptosphaeriaceae, Phaeosphaeriaceae, Pleosporaceae, and Sporormiaceae. While these authors agree that Boerema's classification is useful for practical identification the sections do not really reflect natural relationships.
Species of Phoma are frequently encountered in indoor environments. In fact, in an intensive study of the dust in 369 homes in western Ontario, Dr. James Scott found the species Phoma herbarum to be in nearly 30% of the homes and to be the 14th most common mould overall (Scott, 2001). This study is available online at Dr. Scott's Sporometrics website. Phoma herbarum is the type species of the genus Phoma, meaning that it is the species upon which the genus is based. It is therefore undisputedly a 'good' species of Phoma. Beside being found in indoor environments species of Phoma are common in soils, dung, and both living and dead plants.
Classification: In the true genetic sense, based on its type species P. herbarum, Phoma belongs in the Didymellaceae (Pleosporales). In a broader and less evolutionary sense it can be located in all of the families listed above.
Spores (conidia) produced at the apex of short side branches of the vegetative filaments, dark brown, 2- to several-celled. When the spores are released they retain a small portion of the cell that produced them. The spores of P. chartarum, the most commonly isolated species, have both longitudinal and transverse walls. Growing on decaying plants, especially grasses.
Fruiting structures (pycnidia) more or less round and ornamented, at least on the upper part, with stiff spines. The colourless to yellowish 1-celled spores (conidia) are borne from phialides produced along the length of elongated conidiophores lining the inner walls of the pycnidium. Some species of Phoma also have pycnidial spines, but differ in having simple phialides borne dirctly from the walls of the pycnidium. Isolated from soil and plant debris.
Colonies very fast growing and coarse. Characterized by dark sporangia containing dark to pale spores and a large columella. At the base of the sporangiophores are root-like rhizoids. Often spreading by means of aerial, creeping stolons. Rhizopus species are often a pest in the laboratory. Because of their rapid growth and dry, easily airborne spores they can take over all the cultures in the lab in a few days. One species is capable of transforming soy beans into edible products and is much used in some Asian countries. Common on decaying fruits, soil, and house dust.
Spore-bearing cells may be solitary or united into more complex brush-like structures. Conidia are borne at the apices of cells with irregularly elongated necks and widely separated annellations. The conidia are generally flattened at the point of attachment and occur in wet masses. The spore-bearing structures are sometimes united into complex synnemata referrable to the genus Graphium.
Species of Scedosporium can cause a variety of diseases in humans. Mostly they affect people with compromised immune systems, but healthy individuals may also become infected. Cultures of all Scedosporium species should be treated with caution.
The species occur in soil, decaying plant matter or dung. They can become exceedingly abundant in some situations, such as in piles of feed-lot manure.
Conidiophores dark, usually highly branched and terminating in a brush-like complex bearing flask-shaped annellides. The colourless to dark 1- or 2-celled spores (conidia) are borne in chains from the tips of the annellides and usually have a flattened base where they were originally connected. Occurring in soil, dung, decaying plant debris, and house dust.
Most easily recognized by the spores, which are colourless to yellow, spiny, round, 1-celled, and produced singly at the ends of short filaments. Sometimes phialides of the Acremonium or Gabarnaudia type may also occur. A few species of Mortierella, as well as the human pathogen Histoplasma capsulatum, produce spores resembling those of Sepedonium. Isolated from soil, but most commonly parasitized boletes.
The 1-celled colourless spores (conidia) are produced on short roughened or toothed branches of the vegetative filaments. Occasionally more distinct conidiophores may develop.
According to de Hoog (1993), species of Sporothrix have been reported as anamorphs of both ascomycetes and basidiomycetes. However, he believes the name should be restricted to those with ascomycetous relationships and refers the basidiomycetous ones to the genus Cerinosterus. Unfortunately, distinguishing Sporothrix from Cerinosterus with certainty requires an electron microscope or advanced biochemical techniques.
Isolated from soil, decaying plant materials, other fungi, insects, and air. Sometimes causing human disease.
Classification: Ophiostomataceae (Sordariomycetidae) in a strict sense, but found in other families of ascomycetes as well.
Characterized by clusters of colourless to brown swollen phialides at the tips of colourless to brown, sometimes branched, conidiophores. The dark brown 1-celled spores (conidia) are produced successively from the tips of the phialides and collect in wet masses. Species with spores in chains are referred to Memnoniella. A strong decomposer of cellulose and thus usually associated with decaying plant materials.
Species of Stachybotrys have earned a considerable notoriety in recent years due to their production of potent toxins in indoor environments. They have been linked to some cases of infant death in mouldy buildings. Rarely pathogenic for man.
Classification: Chaetosphaeriaceae (Hypocreomycetidae).
The dark conidiophores produce a single apical spore (conidium) through a pore and then continue to grow through the pore, thus dislodging the spore. Successive periods of spore production and regrowth give the conidiophore a noded appearance. The spores are dark brown and are divided by several longitudinal and transverse walls. Occurring on decaying and living plants and in soil.
Classification: Pleosporaceae (Pleosporales).
Streptomyces species are filamentous Gram-positive bacteria (actinobacteria), not fungi, and are thus out of place here. However, they occur in the same habitats as fungi and are superficially similar. The filaments and spores are very small (usually 1 µm or less in diameter). The spores are formed by the fragmentation of the filaments and are borne in straight, wavy, or helical chains. The colonies are slow-growing and often have a soil-like odour. Common in soil, plant debris, dung, house dust, and many other habitats.
Ascomycetes characterized by loose hyphal fruiting bodies (gymnothecia) containing spherical asci, which in turn each enclose 8 ascospores. The ascospores are colourless to yellow, spherical to disc-shaped, and 1-celled. The whole fungus is either colourless or brightly coloured. Anamorphs are strongly like those of Penicillium and are often difficult to distiguish from them. Their most characteristic features are highly symmetrical spore-bearing structures (penicilli) and elongated and rather lance-like phialides. Conidia are often sharply fusoid. In soil, dung, or decaying plant material. The species can be quite aggressive in culture with other fungi, often completely overrunning other colonies, suggesting some level of parasitism may be involved.
The dark brown 2- to several-celled spores (conidia) are produced by the swelling and subsequent delimitation of the terminal portion of short branches of the vegetative filaments. The most common species, T. asperum, has 2-celled, roughened spores. In soil and plant debris, including wood.
Usually recognized by fast-growing colonies producing white, green, or yellow cushions of sporulating filaments. The fertile filaments or conidiophores produce side branches bearing whorls of short phialides. The 1-celled spores (conidia) are produced successively from the tips of the phailides and collect in small wet masses. The photograph at left, and the drawing, show a species of Trichoderma as seen with a microscope, but Trichoderma species are also very conspicuous in the field, especially under the dead bark of trees. The photo at top-center shows a species of Trichoderma producing its green conidia among the nearly mature cushion-shaped stromata of its teleomorph.
Trichoderma species are strongly antagonistic to other fungi. The exact nature of this relationship is still not clear, but it appears that they kill other fungi with a toxin and then consume them using a combination of lytic enzymes. This suggests they are actually microbial predators. This antagonistic behaviour has led to their use as agents of biological control of some fungi causing plant disease. On the other hand, they can be serious pests in cultivated mushroom beds. Species of Trichoderma are common in soil (especially water-logged soil), dung, and decaying plant materials.
Characterized by colourless spores (conidia) that are nearly sessile and usually produced at right angles to the fertile filament, or as terminal swellings. Most conidia are 1- or 2-celled (micro-conidia) but at least a few are more than 2-celled (macroconidia). Similar to Chrysosporium. Usually occurring as a skin parasite on man and animals but occasionally isolated from soil, leather, feathers, etc. The picture at top-left shows hyphae of T. rubrum growing within a flake of dead skin scraped from an infected patient. The picture at top-right shows conidia as they are seen in pure culture.
The 2-celled colourless to pink spores (conidia) are bilaterally symmetrical and are produced retrogressively in long chains from the unbranched conidiophores. The youngest spore is at the bottom of the chain and is always attached at an oblique angle to the conidiophore. Occurring in soil and decaying plant materials, often found as an epiparasite on black knot of cherry.
The brown conidiophores are united to form large complex cylindrical structures (synnemata) bearing a dense layer of annellides on the upper half. Curved to straight sterile hairs project out from among the annellides. The spores (conidia) are colourless to brown and are produced in chains from the tips of the annellides. Similar and possibly congeneric with Cephalotrichum.
Shoemaker and Kokko (1977) discussed four species and Udagawa et al (1985) added a fifth, T. dendrocephalus. These species can be distinguished with the following key:
1. Sterile hairs straight - 2.
Occurring in soil, dung, and plant debris.
Characterized by dark conidiophores bearing conidia through a pore at several points along their length. The conidia are dark brown, more or less egg-shaped to cylindrical, and divided into several cells by transverse and longitudinal walls. Occurring in soil and dead or dying plants.
Classification: Pleosporaceae (Pleosporales).
Characterized by whorls of phialides produced along the length of undifferentiated filaments or on conidiophores. The colourless to brightly coloured 1- or 2-celled spores (conidia) collect in small wet masses. Common in soil and decaying plant matter; also causing plant disease. Some species, such as V. lecanii in the above photos, are parasites on other fungi.
Classification: Plectosphaerellaceae (Sordariomycetidae).
The conidia are the most distinctive feature, being 1- or 2-celled, dark brown, bullet-shaped to egg-shaped, flat at the base, and with a colourless line or germ slit on one side. They are borne on upright, branched conidiophores. Occurring on dung, soil, and meat in cold storage.
A complex group of fungi resembling one another in existing as single cells that "bud" directly to form new cells. The colonies are pasty in appearance. Some yeasts may form ascospores within their cells. Common in moist habitats and often able to grow at reduced oxygen levels. See the discussion of yeasts under the broader topic of fungal growth.
The pictures above left illustrates Saccharomyces cerevisiae, a yeast used for the rising of bread and the brewing of beer. The one at right is probably Pichia membranaefaciens, a species found in a variety of habitats. It often colonizes stored foods where it may form a thin film over the surface. The picture above shows a jar of olives colonized by P. membranaefaciens.
Holomorphs: various and not necessarily related to one another.
These structures represent the sexually reproductive spores of several genera of Mucorales. They are usually dark, roughened, 1-celled, and connected to the filaments by short cells called suspensors. They rarely occur apart from asexual fruiting structures and are not normally used exclusively for identification purposes.
Specialized reference books are often necessary for identifying fungi. The bibliography presented here contains many such references. Unfortunately, many of these cannot be found in smaller community libraries and must be sought in university and government collections. Some of these large libraries can be visited by anyone, but many others are generally inaccessible. The best way to see a specialized publication is to ask your librarian to request an interlibrary loan. Interlibrary loans may take a few weeks but are usually available.