Each pair was given two of the eight possible species. Firstly, the garlic extract was prepared. This was done by grinding 10g of garlic in a sterilised mortar and pestle with 15ml of deionised water. The outer scales of the garlic bulbs were removed and it was chopped into small pieces on a sterilised tile to make it easier to grind. This extract was then filtered through two layers of muslin into a 15ml centrifuge tube, using an alcohol-cleaned filter funnel. The tubes were spun at 4,000rpm for 10 minutes. The supernatant was collected- this is the garlic extract. The dilutions of this extract were prepared as follows;

The group were given Penicillium hirsutum and Botrytis cinerea. Spore suspension of these two fungi was carried out by adding 20ml of sterile water to the plate, scraping the surface mycelium to suspend the spores and transferring this to a sterile universal bottle. This was then filtered through muslin to remove hyphal debris. This step had to be carried out gently to ensure no release of fungal spores into the atmosphere. Agar plates were prepared using a sterilised cork borer. After each removal of an agar plug, the implements were sterilised using alcohol and a Bunsen burner. One improvement made was to be aware of how long the implements were heated for, as this could melt the agar in the plates, making it harder to remove. The dilutions were added to each central well. A control plate with H2O and a plate of original extract was also added for comparison. The plates were then incubated for around 72 hours at 27°C.

Data Set

The group were given Penicillium hirsutum and Botrytis cinerea. The results are as follows;

Radius of zone of inhibition (mean, mm) results for BLGY 2225 Antifungal properties of garlic practical 2013

Fungal isolate

Neat garlic extract

1/2 dilution garlic extract

1/4 dilution garlic extract

1/10 dilution garlic extract

1/20 dilution garlic extract

1/40 dilution garlic extract

1/100 dilution garlic extract

Negative control

Penicillium hirsutum

5

3.33

2.66

1

0

0

0

0

Botrytis cinerea

22.33

17.66

12.66

8.66

4

2

0

0

Class Data

Figure 1 shows fungus Allternaria altanata’s mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Figure 2 shows fungus Penicillium italicum mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Figure 3 shows fungus Penicillium hirsutum mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Figure 4 shows fungus Penicillium isolate 1 mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Figure 5 shows fungus Penicillium isolate 2 mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Figure 6 shows fungus Penicillium isolate 3’s change in mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Figure 7 shows fungus Botrytis cinerea’s change in mean radius of zone of inhibition (cm) with S.E bars for different concentrations of extract.

Interpretation of class data

There was an inhibition of growth for all the species treated with the fungus. At high concentrations of garlic extract, all the fungi had a zone of inhibition of at a few centimetres. This shows that garlic has an inhibitory effect for all species. Botrytis cinerea was the fungus most affected by the garlic, with a radius of the zone of inhibition of 20.5cm for the neat garlic extract, and a 1.5cm zone of inhibition at the 1/100 dilution. The next most affected fungus species was Allternaria altanata, who’s highest and lowest zones of inhibition were 18.5 (for the neat garlic extract) and 2cm (for the 1/100 dilution) respectively.

The fungi least effected by the garlic extract was Penicillium hirsutum. At the neat garlic extract, Penicillium hirsutum had a zone of inhibition of only 4.1cm. At the 1/40 dilution it had a zone of inhibition of 0cm. The 1/100 fold dilution for Penicillium hirsutum showed a 0.75cm zone of inhibition, which does not fit in the trend we are able to see with the rest of the data, which was as the concentration of garlic extract decreased, the zone of inhibition started to decrease. As the standard error bar for this piece of data was so large, it is likely to be anomalous. Penicillium isolates 1 and 2 closely followed behind Penicillium hirsutum, with zones of inhibition for the neat garlic extract measuring 6.05cm and 5cm respectively.

Statistics

Null hypotheses:

The concentration of garlic extract does not have an effect on the radius of the zone of inhibition for the fungus species.

All the species of fungus used will produce the same growth inhibition to the different concentrations of garlic extract.

A 2-way ANOVA was performed but the residuals of the data were not normally distributed (Kolmogorov-Smirnov, p<0.05, Shapiro-Wilk, p<0.05), so the results of the 2-way ANOVA cannot be trusted so this test is not useful and so other statistical tests have to be applied. A 2-way ANOVA is a parametric test, and there is no non-parametric test equivalent, so a Kruskal-Wallis test had to be performed on each separate factor. The p value was less than 0.001 for the effect of the concentration of garlic extract on the zone of inhibition, so therefore the results were significant, so therefore there was enough evidence to reject the null hypothesis in favour of an alternative hypothesis; the effect of the different concentrations of garlic extract had a significant effect on the radius of the zone of inhibition (Kruskal-Wallis, Chi-squared=158.150, df=7, p<0.001). This means that different concentrations of garlic will be shown to inhibit the growth of fungi at the different levels of concentration.

The p value for the effect of different species of fungi on the zone of inhibition was p<0.001. This means there is enough evidence to reject the null hypothesis, again in favour of an alternative hypothesis; there is a significant difference between the zone of inhibition when different fungi were used (Kruskal-Wallis, Chi-squared= 55.807, df=6, p<0.001). This shows that depending on the fungus used, the garlic concentrations show differing degrees of inhibition, meaning some fungi species used are more resilient to the garlic extract.

To see if there was a significant difference between the results for the most and least effected species (Penicillium hirsutum and Botrytis cinerea), a Mann Whitney U test was carried out. The value of the test was less than the critical value, and so there is a significant difference between the sizes of the zones of inhibition between these two species due to the addition of the garlic extract, meaning when different concentrations of garlic extract, the results for Penicillium hirsutum and Botrytis cinerea differ significantly from each other (Mann Whitney U=9, n1=8, n2=8, CV=13).

Discussion

This practical was to determine whether different concentrations of garlic extract affected the size of the zone of inhibition of fungal growth around the well of the agar plate. Garlic can be easily grown in the garden, but is prone to several disease. A few of these are: Basal Rot (Fusarium culmorum), White Rot (Sclerotium cepivorum), Downy Mildew (Peronospora destructor), Botrytis Rot (Botrytis porri) and Penicillium Decay (Penicillium hirsutum) (Moyer et. al. 2006). When raw garlic is crushed, allicin is produced by a reaction with the enzyme allinase. It is deactivated below pH 3 and because of this; it is not usually produced in the body when garlic is consumed. Allicin is also unstable and can break down within 24 hours. Allicin is an organosulfur compound that can be isolated in the laboratory from garlic. It was first isolated in 1944 by Cavallito et al. Allicin exhibits certain antibacterial and anti-fungal properties and is garlic’s mechanism for protection against pests and fungi. It has been studied in relation to its effects and biochemical interactions. A potential application of allicin is in the treatment of methicillin-resistant Staphylococcus aureus (MRSA), as it becomes an increasing concern, especially in hospitals. When allicin was screened against 30 strains of MRSA, it was found that there were high levels of antimicrobial activity. A water-based formation of allicin is more chemically stable than other preparations, due to the hydrogen bonding of water to the reactive oxygen atom found in allicin. Allicin features a thiosulfinate functional group, which is not present in allicin unless tissue damage occurs.

Extracts of garlic have been shown to have a suppressive effect on fungi, both in this experiment and in other experiments carried out. Botrytis cinerea was the fungus most inhibited by the presence of the garlic extract at varying concentrations. In a paper published in the scientific journal Plant Disease, there are findings that support this. The paper found that certain species of garlic showed antifungal properties against Botrytis cinerea. Plant extracts showing the greatest antifungal activity were those from species of Allium and Capsicum, corresponding to our results. It was shown that a 10% dilution of the extracts taken from these species of garlic and pepper registered under 40.00 OD in fungal growth, and inhibited completely spore germination of Botrytis cinerea after 24 and 48 hours (Wilson et. al. 1997). This corresponds with the results that were found in our experiment.

In a paper produced by Tingting et al. in 2006 showed that the fungus who’s growth was least inhibited was Penicillium hirsutum. Mould diseases in garlic are associated with various Penicillium species, and is attributed to severe annual crop losses in garlic (Valdez et al. 2006) Penicillium decay (also known as seed clove decay) produces stunted and wilting leaves and weak yellow plants which may not survive (Greathead 1978) The disease can spread into the stems and root of plants, affecting their development. Infection with Penicillium also brings about a secondary infection by nematodes, bacteria and maggots, which often mask the primary cause of the decay. This disease is caused by the fungus Penicillium hirsutum. If garlic plants had natural resistance to this, it would not cause diease in the garlic plants. This is not the case, and Penicillium hirsutum is able to thrive in the presence of garlic and the literature on this subject correlates with our findings.

In conclusion, allicin is an incredibly important organosulfur compound found in garlic which contributes greatly to garlic’s antibacterial and antifungal properties against many, but not all fungi species. There are certain fungi that are unaffected by the presence of allicin in garlic, such as Penicillium hirsutum which is able to thrive in the presence of garlic, even when allicin is produced, prompted by tissue damage. Garlic is showing to be a successful antifungal compound, potentially in the future being able to treat methicillin-resistant Staphylococcus aureus.