Culturing Procedures
Incubation Conditions
Sporulation and pigmentation of fungi are favoured by light, including ultra-violet wavelengths, and fluctuating temperatures. All cultures for identification at the Fusarium Research Laboratory are incubated in an alternating temperature regime, 25°C day/ 20°C night, with a 12 hr photoperiod. Cultures are incubated 40 cm below a light bank (75 cm wide) containing four 40W cool white fluorescent tubes and one 36W black light tube (Philips® TL 36W/80 RS F40 BLB). Two light banks and two shelves for cultures are constructed on a mobile frame including a 20 cm space between the lower light bank and the upper shelf to prevent cultures being heated by the lights beneath.
If near-ultraviolet ('black light') tubes are not available, many fungi will sporulate satisfactorily if illuminated with cool white fluorescent tubes (which emit a significant proportion of near-ultraviolet light) or if placed in diffuse daylight. Direct sunlight may kill cultures. Incandescent light should not be used, since the red wavelengths in it may inhibit sporulation and the heat generated will dry cultures out too quickly.
Single Spore Transfer
Colonies initiated from single conidia or hyphal tips are uniform and consistent in appearance and ensure pure cultures. The single germinated conidium transfer procedure is also valuable for separating mixed cultures encountered in isolations from diseased plant material or from soil. The technique initially requires preparation of a suspension of conidia in 10 mL distilled sterile water in a test tube. A small scrape of macroconidia from a sporodochium is preferred to obtain a concentration equivalent to 1-10 conidia in a drop viewed under a low power objective of a compound microscope. "Dry" WA plates (at least 7 days old) are seeded by pouring the conidial suspension over the surface and the excess shaken off immediately. The plates are incubated in an inclined position (30-40°) in the dark at 25°C for 18-20 hr.
Plates are examined using a dissecting microscope. A single germinated conidium is removed on a small square of agar using a transfer needle or wire with a flattened tip and sharpened edges. The knife edge of the needle is used to cut a grid around the conidium and the flat surface used to remove the block and transfer it to the desired medium.
WA may be amended with antibiotics if the original culture is contaminated with bacteria. Alternatively a drop of 25% lactic acid may be added to the conidial suspension to inhibit bacterial growth. The acidified conidial suspension should be allowed to stand 10 min prior to pouring. Germination of the conidia may be delayed for 24 hr. Some fungi will not germinate without external nutrients and in these cases a small amount of glucose or sucrose in the medium may encourage germination. The most common cause of failure of spores to germinate on single-spore plates is that the needle used to scrape spores from the starting culture was too hot, resulting in the spores being killed.
Hyphal Tip Transfer
This technique is used to initiate cultures of species which frequently develop degenerate cultural variants from germinated conidia, or fungi such as Rhizoctonia or Sclerotinia which do not sporulate in culture. Mycelium from a non-degenerate colony (on CLA for Fusarium species) is used to initiate a colony on WA. Water agar plates should be poured on a slight slope so that the agar is shallower (approximately 1 mm) on one side of the plate where hyphal growth will be more limited. A single hyphal tip is cut out using the procedures outlined for transferring germinated single conidia.
Degenerate Cultures
The use of standard culturing procedures enables degenerate cultural variants to be recognised quickly and discarded. Although cultural degeneration is a major problem in studies on Fusarium and other fungi little is known of the basic mechanisms involved. Such variants do differ from wild-type cultures both morphologically and physiologically. Degenerate cultural variants of pathogenic species are often avirulent and should be avoided in studies on disease. In contrast, mycotoxin production may not be affected in degenerate cultures.
There are two main types of degenerate cultural variants. One tends towards production of spores at the expense of mycelium, while the other is characterised by the loss of ability to sporulate. In the past this was considered to be due to the presence of two types of nuclei, termed 'Mycelial' and 'Conidial' in fungi, and the term 'Dual Phenomenon' was coined to describe this. Although sometimes encountered in the literature, this theory is now discredited and the 'Dual Phenomenon' is recognised as a manifestation of cultural degeneration.
In Fusarium species, conidial degenerates are called pionnotal. The pionnotal type is a flat slimy colony lacking aerial mycelium and consisting of abundant macroconidia which may be distorted. The mycelial type consists of sterile, usually white, mycelium. The wild-type culture cannot be recovered from a degenerate variant.
The occurrence of degenerate cultural variants is more frequent in some species than others. Such variation is promoted by frequent sub-culturing on carbohydrate rich media using mass transfers of mycelium. The occurrence of degenerate cultural variants can be minimised by using natural substrate or low nutrient media such as CLA or WA and by initiating cultures from a single germinated conidium or hyphal tip.
Culture Mites
Culture mites are a chronic problem for mycologists. They can ruin entire culture collections by carrying fungal and bacterial spores between cultures. The best control is hygiene, ensuring that all work spaces, equipment, incubators and culture rooms are kept clean. Old cultures which are no longer required should be discarded promptly.
Culture mites can be excluded from slope cultures by a rice paper cap glued over the mouth of the test tube. The glue consists of 20% gelatine (w/w) with 5% copper sulfate (w/w) which inhibits microbial growth on the glue. The mixture is heated to dissolve the gelatine then poured into Petri plates to set and stored at 5°C. A tube is capped by warming the mouth in a gas flame, inserting it lightly in the glue and then placing the inverted tube onto a small piece of rice paper. The glue is allowed to set and the excess paper is touched to a flame to burn away, leaving a cap over the mouth of the tube.
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