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Axenic Culture of Fungi

Focus

Q1. As a focus for this section, I suggest you examine the material with the following goal in mind. If you were to culture a spore-forming fungus found in soil, how would you establish it in culture to study it further? What media would you use, how would you isolate the fungus and how would you store the isolate once you had the fungus in culture?

Q2. The second goal is to write a detailed protocol for isolation of an endophyte from leaves of a native plant, Banksia ericifolia. Note that this plant has a very hairy abaxial leaf surface. Indicate the checks that you would include to ensure you isolated endophytes and not epiphytes.

Q3. The third goal is to determine how you would confirm that a specific fungus is a pathogen which causes a leaf spot on Banksia ericifolia. Before you start, check Koch’s postulates.

Q4. The fourth, and much more difficult goal, is to determine how you would confirm that a biotroph is causing a specific change to a host.

Introduction

The study of fungi relies, in part, on the axenic culture of isolates. Because so many fungi are found in nature in close proximity to other, quite different organisms, study of their structure and function relies on the capacity to grow the fungi in isolation. This chapter is about general aspects of culturing fungi.

We will deal with two groups of fungi, those that are easily isolated and grown in artificial media in some detail, and one group of specialised biotrophs, arbuscular mycorrhizal fungi, where systems of co-culture with the biotrophic host have been developed.

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Isolation of Fungi

Isolation is the process where a single genotype is taken from its natural habitat and then grown without contaminants. The resultant pure culture is referred to as an 'isolate'. In general, either a spore or hypha is extracted from the substrate. Spores of saprotrophic fungi are commonly found in highly localised concentrations. In soil, for instance, the spores will have formed in association with existing mycelium, which is likely to be limited in spread. Thus, if you take a soil sample, it will contain up to 106 spores per gram dry weight. The sample will commonly include 20 different species of fungi. To isolate a single fungus, you need to use a method to reduce the density of propagules, or specifically select the fungus for further fungal growth.

Soil Dilutions

One common method is to take soil from the specific position, and place a measured quantity in sterile water. Shaking the solution will distribute propagules through the water. By taking increasing dilutions, using water as the diluent, you will arrive a suitable concentration to isolate your target fungus. If you do not know which dilution is suitable, then several need to be plated out. Place the dilution in a Petri dish, and pour warm, molten agar, containing anitbacterial agents, over the dilution. Disperse the solution by gently rotating the dish. After a short period, propagules will germinate and colonies will grow. You then subculture from each separate colony: a large number of isolates will be obtained, many from the same fungus. Soil dilution selects for fungi that form large numbers of spores that germinate rapidly under the specific conditions.

Soil Plating

Alternatively, Warcup showed that you can place a small fragment of soil on an agar plate and hyphae will grow from the fragment. By subculturing from emergent hyphal tips, single isolates can be obtained. This process enables you to isolate fungi that exist as hypha in soil, as well as the spore formers.

Hyphal Extraction

Finally, Warcup developed a method where he removed hyphal fragments from soil and along root surfaces using forceps. The fragments were plated out on agar with low concentrations of carbohydrate and antibacterial agents. Some of the hyphae regenerated and grew. The fungi were subcultured from hyphal tips to obtain pure isolates. The fungi recovered from hyphal extraction were rarely, if ever, found by dilution of soil. LINK

Endophytes and Epiphytes

Endophytes exist in living plant parts. You can isolate fungi from solid substrates such as bark and leaves using similar methods. For example, endophytic fungi growing in leaves can be isolated by surface sterilising the leaves with a common household bleach, cutting the leaves into small fragments and plating out the fragments. Instead of cutting the leaves,they can be ground with a mortar and pestle, separating single cells. The slurry can be diluted with sterile water, and the suspension plated onto or mixed with warm molten agar.

Epiphytes or surface inhabiting fungi can be isolated by either washing the leaf surface with water, and plating out the water, or by pressing the leaf onto an agar plate. Pure cultures can be obtained by subculturing fungi from hyphal tips. LINK

Airborne Fungi

Isolation of fungi from air relies on gathering in the desired spores. In principle, air spora can be sampled by exposing an agar plate for a short length of time and then subculturing the fungi that germinate on the agar. Alternatively, air can be bubbled through a water container, and the water diluted and then plated out on agar. Again, pure cultures arise from culturing hyphal tips. LINK

Selective Media

Some fungi have specific requirements for growth, or the capacity to tolerate inhibitors of potential competitors. This factor is used in formulating selective media. One such medium may enable a range of fungi to grow, but the target fungus will take on a particular colour or form enabling it to be subcultured to pure culture. Other formulations specifically inhibit fungi though the target fungus less than competitors. Inhibitory processes include presence of antibiotics, vitamins, high concentrations of organic carbon, absence of specific nutrients such as amino acids or vitamins, high or low pH, water availability etc.

Fungi in Sporocarp and Other Compound Structures

As you may have gathered from the above, fungi are totipotent: we can take hyphae or spores and place them on an appropriate agar and realsitically expect the fungus to grow. In many cases, we would expect that the fungi could be subcultured to provide a clean isolate.

Sporocarps have spores surrounded by gleba which includes hyphae of the parent. In many cases, providing the hyphae are not contaminated, hyphae of the specialised reproductive structure can be removed and placed on agar, where they will revert to their vegetative state and grow. Not all specialised hyphae will revert to vegetative forms, so it is wise to choose generative hyphae that have cytoplasm, relatively thin walls, and are located within the structure where contamination is least likely. To access relatively clean tissue you might break apart the structure, or tear away a surface layer under aseptic conditions.

Using this process, hyphae can be removed from sporocarps, hyphal strands, sclerotia, ectomycorrhiza, orchid mycorrhiza, ericoid mycorrhiza, and stromata. In all cases, special care is needed:

1. with nutrients in the agar: you can have too much carbon especially too much glucose.

2. potential contaminants may be carried with the tissue or from the surrounding tissue. Even within the stalk of a mushrom, a diverse array of fungi and bacteria may be growing., and

3. selecting viable hyphae. Some of the cells will be moribund.

An interesting case concerns orchid mycorrhizal fungi. LINK The roots of all plants, especially the velamen of orchids, are colonised by a diversity of fungi. By surface sterilising the root and plating out fragments, a range of fungi can be obtained. These fungi, however, are unlikely to include those that form orchid mycorrhizas. Successful isolation of orchid mycorrhizal fungi requires the removal of hyphal coils from within cortical cells of the orchid root. By plating out turgid coils on low sugar media, some of the mycorrhizal fungi can be recovered.

Arbuscular Mycorrhizal Fungi

AM fungi form obligate biotrophic associations with the roots of plants. LINK Thus fungal propagules will need to be placed under a host plant in sterile soil to culture the fungus.

AM fungi form spores in soil and roots. Isolation of species to pot culture has been achieved from various sources: commonly spores (single spores from soil or sporocarps), from fragments of dried roots, and very small fragments of freshly colonised root. Fresh propagules are removed from soil. Some species require a period in water at 6C to break dormancy (Glomus . It is sometimes necessary to place the propagule in a funnel made from plastic film to increase the chances of a fresh root passing close to elongating hyphae. Viable propagules germinate and can colonise roots slowly. In many cases, the fungi will form spores in the sterilised soil, enabling the purity of the culture to be checked. It is wise to pass the fungi through several generations in different soils and hosts before using the fungi as “clean” cultures. If you have access to a molecular facility, check the DNA for purity and the sequence alignment with known species for identification. The fungi are unusual in that each spore contains many thousands of nuclei. Indeed, the nuclei are not necessarily of the same genotype, leading to some confusion in identification.

The chance of contamination is greater when root fragments are used initially. Usually the fungi are found in mixed communities in roots. A variation of the dilution principle, is used to separate each isolate. Tiny fragments of living or dead roots are placed under a trap plant in sterile soil. Again, subculturing from spores provides greater security, but as many of these fungi sporulate only rarely and then maybe only in hypogeous sporocarps, purification may be slow and tedious.

A second variation on dilution can be used to isolate these fungi. Soil can be dried, passed through a series of different sized meshes (eg 30, 60, 150mm), and the resultant soil diluted and placed under a trap plant in sterile soil.

Finally, individual root fragments or stored spores may be used in monaxenic culture. It is probably best if the propagules are held in the fridge for a few months before using them: spores in particular have a dormancy period. Rigorously surface sterilised roots, spores or vesicles teased from roots are placed against cultured roots of transformed plants. Under conditions of high antibiotic stress, spores of the AM fungi germinate and colonise the roots. These colonies are then transferred to fresh media where they may be used to develop pure cultures of the fungus. This process is slow and time consuming. Contamination is common. However, use of split plates enables the separation of root and fungus, sometimes necessary for many experimental purposes.

Many biotrophic fungi have never been cultured. Presumably, a variation of the above principle of dilution of propagules and working with the plant host will be needed to isolate and 'culture' these fungi.

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Culture of fungi

Saprotrophic fungi can be subcultured on media containing nutrients appropriate to their growth and development. Several different types of media have been used successfully. The most commonly used in undergraduate classes consists of a fruit or vegetable, or their extracts, mixed with sugars and agar, and set in Petri dishes. The organic and mineral fractions are designed to supply nutrients similar to or commonly found in the environment of the fungus. A few commonly used materials include:

  • Soil [SOIL AGAR]
  • Potato [PDA]
  • Tomato plus other vegetables [V8 JUICE Agar]
  • Malt extract [MA]
  • Dung [DUNG AGAR]

These can be more highly defined by replacing the organic component with known organic materials including:

  • Nutrient Dextrose [NDY]
  • Sabouraud dextrose [SABOURAUD AGAR]

See recipes for further information.

Many media are available from commercial sources. Commercial sources have a more consistent quality than those prepared from the raw materials each time.

Bacteria are suppressed by adding antibiotics to the agar. As most antibiotics are denatured by heat, the antibacterial agents are usually added to sterilised molten agar, that is just above setting temperature (hand warm, but not hot to touch). Penicillin (50 units per ml), Streptomycin (50 unit per ml), Tetracyclin (30 units per ml) are commonly used this way, either alone or more commonly, in combination. Chlomamphenicol (50mg per l) can be autoclaved and is added during preparation.

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Aseptic technique

This is not an attempt to train you in aseptic technique. Several good guides to techniques can be purchased. However, because most fungi appear similar to the untrained eye, it is wise to work using basic aseptic techniques where ever possible, to reduce the chance of contamination. In principle, clean materials, clean surfaces, clean air and clean utensils are essential.

Five percent hypochlorite can be used for surface sterilisation by immersion of leaves and bark. If you have access to an autoclave, you can sterilise agar, soil, glassware etc. A laminar flow, once the surfaces have been washed with 70% ethanol or UV irradiated, is ideal for handling materials. However, remember that your hands, hair and clothing are covered by fungi and bacteria and these can drop onto open plates. Always work from the front, not over the exposed plates.

Further, it is best to restrict, as much as possible, your use of antibiotics. Overuse and misuse will lead to the development of resistance to antibiotics among a range of microbes, which will, in the long term, be enormously difficult to contain.

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Storage of Fungi

It is extremely expensive to continually subculture the fungi you have isolated. Further, the fungi may mutate, and you may unwittingly select the mutated segments for subculturing, thus losing the characteristics you hope to study. Preservation of fungi is essentially reducing the metabolic rate to the slowest possible. This can be achieved in several different ways.

Storage in Water

Plugs of culture of Dueteromycetes (asexual forms of septate fungi) are placed in vials containing only sterile tap water. The vials are lidded and stored on a shelf or at 10 C for up to 5 years. This is a remarkably useful and cheap technique for the inexperienced. It does not work for many Basidiomycota.

Cold Temperature

Place the isolate on a slope or plate and put into a fridge or cool room. Some fungi remain viable for up to 48 months at 5 C. Some Basidiomycetes can be stored at 5 C.

Under Oil

Subculture the isolate onto low sugar media (eg one sixth strength NDY) in a lidded tube, growing the isolate to cover the surface, and then cover the fungus with sterile mineral oil. The tubes are kept at room temperature. You can keep some fungi for many years. The oil slows access of oxygen to the culture. However, it is crucial the culture is covered by at least 1cm of oil at all times and that the lid is kept clean. Common fungi can contaminate the top of the tube if it has nutrients on it. Moderate changes in air pressure lead to the movement of air in and out of the tube; contaminants are present in the air and spores can lodge on the surfaces of the lidded tube. Thus, topping the oil to just below the lid is helpful, flaming the surfaces before subculturing essential.

Freeze Drying or Lyophilisation

Cultures are placed in a lyophisation tube, cooled and then freeze dried by vacuum evacuation. The tube is sealed and the fungus stored for up to 20 years. The process is commonly used for spore-forming Deuteromycetes. It is less successful for non-spore-forming fungi.

Low-temperature Storage

Some fungi store for many years when placed in 10 to 25% glycerol in water in sealed vials in liquid nitrogen or at minus 70 C. The fungi may need to be taken in stages through the cooling process. It is also essential that you test which dilution of glycerol can be used: some fungi will dessicate in 10% glycerol. This process is the only way we have stored some Zygomycetes for several years.

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Koch's Postulates

The German physician Robert Koch studied the microbial causes of disease. He developed a set of postulates which were used to determine the cause of a specific disease in humans. The postulates have been used more widely to determine the relationship between disease and microbes. According to Koch, disease could be attributed to a particular organism if:

  • The microbe was always found in diseased individuals.
  • The microbe taken from the diseased host could be cultured in isolation in pure culture.
  • A sample of the isolate when placed back in a healthy individual produced the same disease.
  • The newly diseased individual yielded a new, pure culture of the microbe, identical to the one used to inoculate the host.

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