Isolation Procedures

Isolation from Plants

The choice of isolation procedure will depend on the nature and number of plant samples and the fungal species involved. If there are only a few samples to assess then a range of procedures can be selected to maximise recovery of the target fungi. In contrast, the large number of samples involved in extensive surveys usually precludes the use of a range of procedures.

The isolation of fungi from plants is affected by the nature of the diseased tissues, the method of surface sterilisation, the plating procedures, the medium and the incubation conditions.

The tissue selected for plating should be typical of the diseased material being studied. The oldest necrotic tissue should be avoided as it is likely to be colonised extensively by saprophytes. Toxic metabolites present in very necrotic tissues may also inhibit the recovery of the pathogen. Similarly tissue that has been damaged by implements or insects should be avoided as saprophytes readily colonise wound sites. Ideally, recently infected tissue should be selected for isolation studies. Note that samples should be maintained in a cool dry state during transit to the laboratory to minimise growth of saprophytic fungi and bacteria.

The procedure adopted for surface sterilisation depends largely on the nature of the tissue. One of the most common procedures involves immersion of the sample in 1% sodium hypochlorite in 10% ethanol, the latter acting as a wetting agent, and subsequent rinsing in sterile water. However some tissues are quite porous and absorb the surface sterilant which eliminates the pathogen as well as contaminants on the surface. It may be more appropriate to swab porous tissues, such as sorghum stalks affected by stalk rot, with 95% ethanol. Swabbing with 95% ethanol is a preferred procedure for surface sterilising diseased woody plant parts.

Surface sterilisation may not be suitable for use with fine roots. Partial disinfestation of such roots can be achieved by washing in a fine spray of filtered tap water for 30-120 min, and subsequent rinsing in sterile water. A fine nozzle is recommended for use in washing roots, soil debris, and plant tissue in general.

Whichever technique is selected it is recommended that samples be 'dried' on sterile paper tissues, under a filtered air flow after surface sterilisation or washing. Drying inhibits the growth of bacteria from the tissue.

The plating of small pieces (1-2 x 1-2 mm) of tissue reduces the number of fungi developing from each piece. This simplifies subculturing and there is less chance that slow-growing pathogens will be overgrown by fast-growing saprophytes. However in some studies large sections of tissue can be plated if a selective medium is used. Entire subcrown internodes and crowns of wheat plants, for example, are plated on MPDA to assess the incidence of infection by F. pseudograminearum, at the Fusarium Research Laboratory.

The isolation of pathogenic species from diseased roots is difficult because of the wide range of saprophytes which normally colonise necrotic root tissue, particularly the cortex. Fusarium root rot pathogens normally colonise the cortex and stele (vascular tissues) of the root. The frequency of isolation of these species may be enhanced by plating segments of the stele after removal of part or all of the cortex.

The choice of media depends largely on the nature of the tissue involved in the isolation exercise. Low nutrient media such as CLA, WA or quarter-strength PDA are suitable for isolation from larger roots or stem bases. Antibiotics can be added to these media if bacteria interfere with the recovery of fungi. Carbohydrate rich media are generally avoided in isolation studies because they favour fast-growing saprophytes such as the mucoraceous fungi and Trichoderma. In addition, some pathogens degenerate rapidly to avirulent forms on these media.

Richer media such as PDA may be more appropriate for the isolation of slow-growing fungi or isolation from very fine roots.

Selective media are normally used for the isolation of Fusarium species from diseased crown or root samples. A range of selective media are described here. It should be emphasised that all media are selective as they will favour the growth of certain fungi over others. In these web pages the term selective medium is used only for media containing anti-microbial agents.

Isolation plates are incubated under standard conditions of light and temperature. The colonies which develop from segments of tissue should be subcultured to CLA and the conidia produced on these plates can then be used to initiate cultures for identification as described under culturing - single spore transfer. It may be preferable to subculture from very young colonies developing from tissue segments with the aid of a dissecting microscope. This procedure may reduce the difficulties caused by fast-growing species overgrowing slow-growing species.

There are several other techniques for recovering fungi, directly or indirectly, from plant samples, which do not involve plating tissue segments on agar media. Some species sporulate on the surface of the diseased tissue. Conidia can be taken from these and used to prepare a conidial suspension which is plated on WA containing antibiotics. Germinated single conidia are later taken to initiate pure cultures for identification.

Ascospores can be used to initiate pure cultures from samples on which the target fungus is producing fertile perithecia. A small piece of tissue with perithecia is washed, and after excess water is removed it is placed on the inner side of the lid of an inverted shallow Petri dish containing WA or CLA. The tissue is held in place with petroleum jelly. The inverted plate is incubated at 25°C inside a plastic bag to maintain high humidity. Ascospores are released after 24-48 hr and are impacted on the agar surface where they germinate and develop colonies. The fungus can then be subcultured onto other media by hyphal tip or single-spore transfer for identification.

For large-scale screening for crown rot in cereals, isolations may be made on to selective media poured into trays. At the Fusarium Research Laboratory, stainless steel catering trays approximately 30 x 40 x 2 cm deep are used. Lids are cut from Perspex sheeting to the same outside dimensions and held on with spring (bulldog) clips. The tray and lid are thoroughly swabbed with alcohol before use. Wheat crowns with subcrown internodes are washed and surface sterilised, then placed on the medium, 50 per tray. The lids are replaced and the trays incubated under light for 4-5 days. Fusarium graminearum Group 1 may be recognised by its distinctive colony morphology on MPDA. Best results are obtained if the agar concentration is 1.5% or lower so that the crowns and subcrown internodes can be pushed into the surface of the medium.

Soil Dilution Plates

Fungi can be isolated directly from soil using the dilution plate technique, by plating debris on selective media, by direct physical separation, or indirectly using living root or sterile straw baiting techniques. The most appropriate technique for the isolation of a particular fungus will depend on its mode of persistence in soil. Species such as Fusarium oxysporum and F. solani which form abundant chlamydospores in soil can be isolated consistently from soil using the dilution plate technique. In contrast, species which mainly persist in soil as hyphae in plant residues such as F. graminearum and F. avenaceum may be isolated more consistently by plating debris on selective media.

The spectrum of fungi isolated and the frequency of isolation is influenced by soil sampling procedures in the field, transit and storage conditions of samples, and the isolation technique used. Fungi, particularly plant pathogens, are distributed irregularly in soil . A pathogen is normally more abundant in the vicinity of diseased plant remains. Indeed, infested residues may be concentrated on the surface or in the upper 1-5 cm of the soil profile in fields where residues are retained on the soil surface. The spatial distribution of pathogens, either in residues or as chlamydospores, will also be affected by the frequency and type of cultivation (inversion tillage or sub-tillage). These factors should be taken into account when designing a soil sampling procedure.

It is preferable to transport and store soil samples in paper bags. Plastic containers should be avoided as they prevent drying and encourage an increase in bacterial numbers. Soil samples should be stored at low temperature (2-5°C) to prevent microbial activity.

Debris Isolation

This technique involves plating washed pieces of soil debris on selective media. A soil sample is suspended in water which is poured through a nest of sieves, in order, 4.0 mm, 2.0 mm and 0.5 mm in mesh aperture. The first sieve retains gravel and large pieces of plant residue. This residue can be surface sterilised and plated on selective media to recover the target fungus. The small pieces of debris retained on the other two sieves cannot be surface sterilised because they are too small and porous. This debris is retained on the sieves and washed to remove soil particles under a fine spray of filtered tap water for 2 hr. The washed debris is damp-dried on sterile paper tissues or towels, and then dried over silica-gel for 24 to 48 hr before plating on a selective medium. The authors have isolated a wide range of Fusarium species from pasture and grassland soils using this technique.

Physical Separation of Particles

Many pathogens produce large propagules, such as sclerotia, which can be extracted directly from soil by physical means. For sclerotia, the soil is washed through a stack of sieves using a water spray. The size of the sieves should be appropriate to the size of the sclerotia. For example, sclerotia of Sclerotium rolfsii should pass through a 2 mm sieve and be retained on a 0.6 mm sieve. The material trapped on the smaller sieve is washed into a large beaker using a small amount of water. A 50% w/w sucrose solution is added to the beaker and the material suspended by stirring. The organic material, including sclerotia, floats to the surface. This is decanted back onto the smaller sieve, and the sucrose washed off. Sclerotia are than separated from other organic debris by visual inspection under a dissecting microscope. It may be helpful to transfer the organic fraction to a piece of filter paper first.

Baiting

Baiting techniques use plant material to selectively isolate the pathogen from soil or diseased roots. There are many possible ways that this can be done, and a few typical examples are given here.

Slow-growing fungi may be difficult to isolate directly from necrotic root tissue which is extensively colonised by other fungi and bacteria. However these slow-growing species can sometimes be isolated indirectly using a combination baiting/plating technique. The necrotic tissue is thoroughly washed and cut into small pieces which are mixed with steam-air treated soil. Surface-sterilised seed of the same cultivar is then sown into this mixture. The bait plants are incubated under conditions which are thought to favour the disease. The roots are recovered as soon as lesions develop and segments are plated on suitable media after thorough washing and, if appropriate, surface sterilisation.

Thielaviopsis basicola can be isolated from roots using carrot disc baits. Carrot roots are surface sterilised by immersing in 1% NaOCl for 20 minutes, or by thorough swabbing with alcohol. The carrots are then cut into discs 3-4 mm thick with a sterile knife and placed on moist, sterile filter paper discs in a Petri plate. Washed and dried diseased roots are placed on top of each carrot disc. The plates are kept moist, but not wet, and incubated for 7-10 days at room temperature in the dark. Characteristic spores of T. basicola should be visible on the carrot, and can be transferred directly onto a suitable medium.

Some Pythium and Phytophthora species can be isolated from soil using apples, or a similar firm fruit, as a bait. The apple is swabbed with alcohol, and a hole approximately 10 mm diameter cut through to the core on one side using a sterile cork borer. The hole is packed with soil, and covered with sticky tape to retain the soil. The apple is incubated at room temperature in the light. Isolations are made after a few days from the margins of fast-spreading, brown lesions. The method is not completely selective since fast-growing Zygomycetes will cause similar lesions.

Phytophthora species can be isolated from soil by floating plant tissue over flooded soil. Soil samples, up to 100 g, are placed in a suitable container such as a beaker. They are covered with distilled or deionised water to a depth of 2-5 cm. Pieces of susceptible plant material are floated on top of the water, and the material incubated for several days. Zoospores of Phytophthora are attracted to the plant tissue, while other fungal spores remain in the soil. Mycelium of Phytophthora can be seen growing from the bait across the water surface, and can be transferred to an agar medium or to a grass blade-water culture. The bait material should be from a species susceptible to the Phytophthora species to be isolated. Baits that have been used include pine needles, Eucalyptus cotyledons, citrus leaves, and seedlings of lucerne (alfalfa) and soybean. If the bait will not float by itself, it may need to be suspended from a lid over the container or from a piece of polystyrene foam or other suitable float.

Isolation of pathogens from plant material