Schadler, D.L. and A.A. George 2006. Synthesis and Bioassay of a Volatile Fungistatic Agent. The Plant Health Instructor. DOI: 10.1094/PHI-I-2006-0717-02
Daniel L. Schadler and Anna A. George, Department of Biology, Oglethorpe University, Atlanta, Georgia 30319
Acknowledgement: Dr. Monte W. Wolf, Department of Chemistry, Oglethorpe University, provided the protocol for the synthesis of methyl salicylate from aspirin. Dr. Kimberly D. Gwinn, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, provided the procedure for the bioassay of volatile fungistatic oils and the formula for calculating comparative growth inhibition.
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In the natural soil environment, the growth of fungi and the formation and germination of their spores are often inhibited in a nonspecific manner. This phenomenon is called fungistasis. Fungi are not killed in this process, and the inhibition can often be overcome by nutrients released from the roots of plants.
Methyl salicylate (oil of wintergreen) is an essential oil that was originally isolated from an odoriferous plant (wintergreen, Gaultheria procumbens); today, several additional species are known to produce methyl salicylate. It is a highly volatile substance and such volatile oils have been shown to be fungistatic to several species of fungi (1, 3, 4, 6).
In addition to their potential fungistatic activity, volatile essential oils have shown promising results in controlling medically important fungi such as Candida albicans (7, 9, 10, 11). The use of essential oils to control post-harvest diseases of fruits and vegetables has also been studied (2, 8), with the oils replacing traditional synthetic pesticides. Thus, essential oils that are registered food grade materials could be used as alternative anti-fungal treatments for fresh produce.
This exercise provides an easily followed procedure for the synthesis of methyl salicylate (oil of wintergreen). The oil is then tested for its ability to induce fungistasis in fungi in culture.
This procedure should be carried out in a fume hood or ventilated work station. Students should wear safety glasses and follow other routine precautions for working with strong acids and flammable organic liquids. Although used as a remedy in traditional medicine, methyl salicylate is toxic if swallowed and may irritate the eyes, respiratory system and skin. Students should limit their inhalational and skin exposure to the compound.
Place 2 aspirin tablets into a test tube. Add 3 ml of methanol and stir until the tablets begin to dissolve. Using a 1 ml pipet equipped with a mechanical pipet filler/dispenser, add 1 ml of concentrated sulfuric acid to the contents of the test tube drop by drop and with stirring. Heat the test tube in a boiling water bath for 10 minutes (no stirring is necessary). After heating, quickly pour the contents of the test tube into the sodium bicarbonate solution in the 250 ml beaker to neutralize the excess acid. Stir the solution thoroughly to ensure that all the acid is neutralized. The methyl salicylate is an oily, slightly brown liquid that spontaneously floats to the surface of the solution; a strong scent of wintergreen should be apparent but the amount of oil will be very small. This protocol should be followed without any aspirin tablets to provide a control treatment for the assay of fungistatic activity that follows.
Students should follow aseptic techniques when performing this procedure; working in a sterile environment such as a laminar flow hood will minimize contamination.
Cut mycelial discs (0.7 cm diameter) from the edge of an actively growing colony of the fungus using a sterile cork borer. Place one mycelial disc in the center of a layer of solid fungal growth medium in a 100 mm Petri dish; the mycelial side of the disc should be in contact with the surface of the medium. Tape one circle of sterile filter paper to the inside of the top of the Petri plate. Using a micropipettor, apply 50 µL of methyl salicylate to the filter paper and replace the cover on the plate. Seal the plate with sealing film and incubate at room temperature in ambient light or in darkness. The growth rate of the fungus will determine the length of the incubation period; 1 to 5 days is typical for most fungal species. The cultures should be checked daily and the diameter of the colonies measured before hyphae in the control treatment reach the edge of the plate. Experimental and control plates must be prepared for each fungus that is tested.
Determination of Comparative Growth Inhibition When the growth of the colony on the control treatment nears the edge of the plate, measure the diameters of both the experimental and control colonies to the nearest millimeter using a ruler. If a colony is not perfectly round, measure its diameter at several points and use the mean in the calculation below.
Comparative growth inhibition (CGI) is calculated using the following equation (5)
CGI =100[(Dc – Dt)/Dc] – 100
where Dc is the mean colony diameter for the control and Dt is the mean colony diameter for the experimental treatment. This formula determines the growth inhibition as a percent of the control. Thus, a CGI value of 0 indicates that the growth of the fungus in the experimental treatment was completely inhibited, while a CGI of -100 indicates that the colonies on the control and experimental treatments were the same size. To be fully accurate, 0.7 cm should be subtracted from the diameter of each colony because this was the diameter of the mycelial disc at the beginning of the procedure; this mycelium was already present before treatment and was not subject to inhibition by the fungistatic agent.
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Eight fungal species (Alternaria tenuis, Aspergillus flavus, Aspergillus niger, Fusarium oxysporum, Neurospora crassa, Penicillium chrysogenum, Penicillium notatum, and Rhizopus nigricans; Presque Isle Cultures) grown on Potato Dextrose Agar (Difco Laboratories) were tested and shown to work well with the procedures described.
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Figures 1-4 show examples of growth inhibition of several fungi by methyl salicylate
Although most fungi grow from mycelial discs in a uniform manner, some show a tendency to form sectors of more rapidly or more slowly growing hyphae; further, inexperienced students may accidentally touch the mycelial disc to the agar surface creating eccentric growth from the deposition of spores or hyphal fragments. Figure 2 shows the growth of Aspergillus flavus after five days, and illustrates irregular colonies. Pure methyl salicylate completely inhibited the growth of the fungus while methyl salicylate from aspirin showed a CGI of -67.
Rhizopus nigricans grows rapidly but was completely inhibited by both pure methyl salicylate and methyl salicylate prepared from aspirin (Figure 3).
The fungistasis induced by methyl salicylate is long lasting. Figure 4 shows Alternaria tenuis after 7 days of growth. Although the control is overgrown, the experimental treatment with pure methyl salicylate shows no growth and the treatment with methyl salicylate made from aspirin shows significantly decreased growth.
The growth of many species of fungi appears to be inhibited by exposure to methyl salicylate (oil of wintergreen). In our studies, all eight species that were tested showed complete inhibition by pure methyl salicylate and showed variable inhibition by methyl salicylate prepared from aspirin. Since not all fungi would be expected to be equally sensitive to the same concentrations of fungistatic compounds, there will be variations in the results depending on the species or specific fungal isolates used. The lack of further purification of the synthetic material is a likely explanation of the difference in its effects when compared to the pure sample. Using a micropipette to draw off the oil meant that the amount of active compound in each sample was variable. Thus, students performing this procedure should be advised to remove the oil carefully, attempting to get as much of it in the sample as possible. Visible inspection of the contents of the pipette tip for the presence of the brown oil may help to ensure consistent results in the bioassay of fungistatic activity.
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