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A technique for rapid demonstration of the production of antifungal substances by fungi or other microorganisms
[ 246 ] A TECHNIQUE FOR RAPID DEMONSTRATION OF THE PRODUCTION OF ANTIFUNGAL SUBSTANCES BY FUNGI OR OTHER MICROORGANISMS By E. G. ]EFFER YS, Imperial Chemical Industries Limited, Butterwick Research Laboratories, The Fry the, Welwyn INTRODUCTION
Most techniques previously described for the determination of the production of antifungal substances by fungi or other organisms have involved growth of the fungi on liquid media, serial dilutions of the culture filtrate being then assayed. Weindling (1934) produced culture filtrates of Trichoderma lignorum and observed microscopically the effect of serial dilutions on young hyphae of Rhizoctonia solani. Brian and Hemming (1945) again used serial dilutions of culture filtrates. These solutions were tested to determine the highest dilution at which they prevented germination of the spores of the test organisms. Irving, Fontaine and Doolittle (1945), using solutions of an antifungal substance produced by tomatoes, employed an assay technique analogous to the cylinder plate technique used in penicillin assay, the activity being determined by the diameter of a zone of inhibition. These techniques are more in the nature of quantitative assays rather than preliminary sorting tests. The use of direct inhibition of the growth of one fungus by another in a streak test was shown by Brian, Curtis and Hemming (1946) to be possible, provided that the test fungus is a yeast-like, rapidly growing form. They used Endomycopsis albicans. This type of test, widely used for demonstration of antibacterial substances, cannot be satisfactorily used with mycelial fungi as the test organism. Where a large number of fungi are to be tested for antifungal activity, the above tests (except the streak test which has other disadvantages noted later) are liable to become very cumbersome, and it was considered desirable to develop a rapid technique using as little time, labour and material as possible. After a number of experiments on various lines, the following technique was developed. TECHNIQUE
A 7 rnm, diameter glass rod is bent to form a flat rectangular cell of I by 2 in. internal dimensions. This cell is sterilized by flaming and immediately placed in a sterile Petri dish. A small quantity of molten, sterile CzapekDox agar is poured into the cell, the quantity being only sufficient to flow round the edge of the cell, sealing it to the base of the dish on cooling. Other agar media can of course be used for fungi which do not grow satisfactorily on Czapek-Dox. When the agar has set, the cell is completely
Production of Antifungal Substances. E. G. Jefferys
247
filled with agar and again left to cool. Some agar usually flows over the edge of the cell or through the gap left between the two ends of the rod and this is removed with a sterile scalpel or spatula. The upper agar surface is then inoculated with a streak of the fungus under test. After two to three days incubation at 25° C. (or when good growth has taken place) the cell is gently prised off the bottom of the dish with a scalpel, picked up with forceps, turned over and transferred to a second sterile Petri dish, sterile precautions being observed throughout. The newly exposed surface is then inoculated with a spore suspension of the test fungus (Botrytis Allii Munn being frequently used in these laboratories). After a further sixteen to eighteen hours incubation the upper surface is examined with the low power of the microscope to determine the degree of germination. RESULTS
In the following table, germination is classified on a o No germination (I) Up to 5 % germination I 5-20 % germination
2
3 4
0-4
basis as follows:
% germination % germination
20-50 50-80 80-100
% germination
It should be noted that little significance is: attached to the difference between class 3 germination and that in class 4. It is often possible to note a gradation of germination between the spores of Botrytis directly over the streak of the fungus under test (column (i)) and those away from the streak (column (ii)).
Germination ofBotrytis Allii in glass cell activity trial on Czapek-Dox Fungus tested Absidia glauco Hagem Botrytis Allii Munn Fusarium caeruleum (Lib.) Sacco Fusarium graminearum Schwabe Metarrhizium glutinosum S. Pope Penicillium Gladioli McCull. & Thorn Penicillium Janc;:;ewskii Zal. Stachybotrys atra Corda Stemphylium sp. Stereum purpureum Pers. Thamnidium elegans Link Trichoderma viride Pers, ex Fries Trichoderma viride Pers. ex Fries Uninoculated controls (2)
(i) 4 4 4 4
(ii) 4 4 4 4 (1)
(I) 2 I
4 3 3
4 4 3
4
4
1
Antibiotic produced None None None None Glutinosin Gladiolic acid ' Curling-factor' Not yet isolated None None None Viridin Gliotoxin
It will be seen that germination of the spores of Botrytis after sixteen hours incubation is always suppressed or absent where an antibiotic is known to be produced. The results are consistently repeatable. DISCUSSION
This technique has several advantages over those previously reported. It is quick, taking only about four days to complete. The glass cells are simple to make and easy to handle though it should be noted that care must be
248
Transactions British Mycological Society
taken when removing the agar-filled cell to insert the scalpel under the agar as well as the glass in order that both are transferred together. This process is quite easy after a little practice and is facilitated by having cells no wider than I in. Finally the technique is very useful with quickly growing fungi which, in the streak test, are very liable to overgrow the test organism before any result can be obtained. Although this technique has been used only for demonstrating the production by fungi of antifungal substances, it could probably equally well be used to investigate the production by bacteria and actinomycetes of substances antagonistic to fungi, bacteria or actinomycetes. SUMMARY
A new technique is described for rapidly determining whether a mould or other micro-organism produces antifungal metabolic products. This requires the use of a small rectangular glass cell filled with agar of which one side is inoculated with the fungus under investigation and the other side is later inoculated with a standard test fungus. The author wishes to thank Dr P. W. Brian for much valuable help and advice. REFERENCES P. W., CURTIS, P. J. & HEMMING, H. G. (1946). A substance causing abnormal development of fungal hyphae, produced by Penicillium janczetoskii ZaI. Trans. Brit. myc. Soc. XXIX, 174-82. BRIAN, P. W. & HEMMING, H. G. (1945). Gliotoxin, a fungistatic product of Trichoderma uiride. Ann. appl. Biol. XXXII, 214-20. IRVING, G. W., FONTAINE, T. D. & DOOLITTLE, S. P. (1945). Lycopersicin, a fungistatic agent from the tomato plant. Science, ell (2636), 9-11. WEINDLING, R. (1934). Studies on a lethal principle effective in the parasitic action of Trichoderma /ignorum on Rhiroctonia solani and other fungi. Phytopathology, XXIV (II), 1153-79·
BRIAN,
(Accepted for publication
20
February 1947)
Most techniques previously described for the determination of the production of antifungal substances by fungi or other organisms have involved growth of the fungi on liquid media, serial dilutions of the culture filtrate being then assayed. Weindling (1934) produced culture filtrates of Trichoderma lignorum and observed microscopically the effect of serial dilutions on young hyphae of Rhizoctonia solani. Brian and Hemming (1945) again used serial dilutions of culture filtrates. These solutions were tested to determine the highest dilution at which they prevented germination of the spores of the test organisms. Irving, Fontaine and Doolittle (1945), using solutions of an antifungal substance produced by tomatoes, employed an assay technique analogous to the cylinder plate technique used in penicillin assay, the activity being determined by the diameter of a zone of inhibition. These techniques are more in the nature of quantitative assays rather than preliminary sorting tests. The use of direct inhibition of the growth of one fungus by another in a streak test was shown by Brian, Curtis and Hemming (1946) to be possible, provided that the test fungus is a yeast-like, rapidly growing form. They used Endomycopsis albicans. This type of test, widely used for demonstration of antibacterial substances, cannot be satisfactorily used with mycelial fungi as the test organism. Where a large number of fungi are to be tested for antifungal activity, the above tests (except the streak test which has other disadvantages noted later) are liable to become very cumbersome, and it was considered desirable to develop a rapid technique using as little time, labour and material as possible. After a number of experiments on various lines, the following technique was developed. TECHNIQUE
A 7 rnm, diameter glass rod is bent to form a flat rectangular cell of I by 2 in. internal dimensions. This cell is sterilized by flaming and immediately placed in a sterile Petri dish. A small quantity of molten, sterile CzapekDox agar is poured into the cell, the quantity being only sufficient to flow round the edge of the cell, sealing it to the base of the dish on cooling. Other agar media can of course be used for fungi which do not grow satisfactorily on Czapek-Dox. When the agar has set, the cell is completely
Production of Antifungal Substances. E. G. Jefferys
247
filled with agar and again left to cool. Some agar usually flows over the edge of the cell or through the gap left between the two ends of the rod and this is removed with a sterile scalpel or spatula. The upper agar surface is then inoculated with a streak of the fungus under test. After two to three days incubation at 25° C. (or when good growth has taken place) the cell is gently prised off the bottom of the dish with a scalpel, picked up with forceps, turned over and transferred to a second sterile Petri dish, sterile precautions being observed throughout. The newly exposed surface is then inoculated with a spore suspension of the test fungus (Botrytis Allii Munn being frequently used in these laboratories). After a further sixteen to eighteen hours incubation the upper surface is examined with the low power of the microscope to determine the degree of germination. RESULTS
In the following table, germination is classified on a o No germination (I) Up to 5 % germination I 5-20 % germination
2
3 4
0-4
basis as follows:
% germination % germination
20-50 50-80 80-100
% germination
It should be noted that little significance is: attached to the difference between class 3 germination and that in class 4. It is often possible to note a gradation of germination between the spores of Botrytis directly over the streak of the fungus under test (column (i)) and those away from the streak (column (ii)).
Germination ofBotrytis Allii in glass cell activity trial on Czapek-Dox Fungus tested Absidia glauco Hagem Botrytis Allii Munn Fusarium caeruleum (Lib.) Sacco Fusarium graminearum Schwabe Metarrhizium glutinosum S. Pope Penicillium Gladioli McCull. & Thorn Penicillium Janc;:;ewskii Zal. Stachybotrys atra Corda Stemphylium sp. Stereum purpureum Pers. Thamnidium elegans Link Trichoderma viride Pers, ex Fries Trichoderma viride Pers. ex Fries Uninoculated controls (2)
(i) 4 4 4 4
(ii) 4 4 4 4 (1)
(I) 2 I
4 3 3
4 4 3
4
4
1
Antibiotic produced None None None None Glutinosin Gladiolic acid ' Curling-factor' Not yet isolated None None None Viridin Gliotoxin
It will be seen that germination of the spores of Botrytis after sixteen hours incubation is always suppressed or absent where an antibiotic is known to be produced. The results are consistently repeatable. DISCUSSION
This technique has several advantages over those previously reported. It is quick, taking only about four days to complete. The glass cells are simple to make and easy to handle though it should be noted that care must be
248
Transactions British Mycological Society
taken when removing the agar-filled cell to insert the scalpel under the agar as well as the glass in order that both are transferred together. This process is quite easy after a little practice and is facilitated by having cells no wider than I in. Finally the technique is very useful with quickly growing fungi which, in the streak test, are very liable to overgrow the test organism before any result can be obtained. Although this technique has been used only for demonstrating the production by fungi of antifungal substances, it could probably equally well be used to investigate the production by bacteria and actinomycetes of substances antagonistic to fungi, bacteria or actinomycetes. SUMMARY
A new technique is described for rapidly determining whether a mould or other micro-organism produces antifungal metabolic products. This requires the use of a small rectangular glass cell filled with agar of which one side is inoculated with the fungus under investigation and the other side is later inoculated with a standard test fungus. The author wishes to thank Dr P. W. Brian for much valuable help and advice. REFERENCES P. W., CURTIS, P. J. & HEMMING, H. G. (1946). A substance causing abnormal development of fungal hyphae, produced by Penicillium janczetoskii ZaI. Trans. Brit. myc. Soc. XXIX, 174-82. BRIAN, P. W. & HEMMING, H. G. (1945). Gliotoxin, a fungistatic product of Trichoderma uiride. Ann. appl. Biol. XXXII, 214-20. IRVING, G. W., FONTAINE, T. D. & DOOLITTLE, S. P. (1945). Lycopersicin, a fungistatic agent from the tomato plant. Science, ell (2636), 9-11. WEINDLING, R. (1934). Studies on a lethal principle effective in the parasitic action of Trichoderma /ignorum on Rhiroctonia solani and other fungi. Phytopathology, XXIV (II), 1153-79·
BRIAN,
(Accepted for publication
20
February 1947)