Team:NCTU Formosa/Fungal Experiment
Fungal Experiments
Figure 1: The flow chart of fungi experiments.
Inhibition Zone
- Inhibition Measurement for Mycelia
In order to know whether the peptides are effective in inhibiting mycelia and the accurate concentration of peptides for inhibition, we measured the zone of inhibition.
In order to know whether the peptides are effective in inhibiting mycelia and the accurate concentration of peptides for inhibition, we measured the zone of inhibition.
Dual Cultures on Potato Dextrose Agar
Putting the mycelium on the Petri plates of Potato dextrose agar, we cultivated plates in 20℃ for 2 days. After making sure that the mycelial colony has developed, we dug holes which the different concentrations of peptides and the negative control were added respectively. After waiting about 12~18 hours, we could observe the result.
Observation
The white part was the mycelium. The holes were added with different concentrations of peptides and negative control HEPES respectively.
Figure 2: The white part is mycelium, the holes were added different concentrations of peptides and negative control HEPES respectively.
If the peptides can inhibit the mycelium, then there will be a range around the holes that the mycelium cannot be occupied.
Otherwise, the mycelium will grow ignoring the holes, like the hole containing HEPES. The whole scene will look like a white circle, while the edges of effective peptides concave.
Sample
Table 1: The peptides which we used to conduct the experiments
Each peptide was repeated the experiments for three times to ensure the results.
The HEPES buffer, which has no toxic on cells, is applied to be the negative control.
Spore Germination
- Inhibition Measurement for Spores
In order to know whether the antifungal peptides can inhibit the germination of spores or not and the accurate concentration of antifungal peptides for inhibition, we conducted the spore germination experiment.
In order to know whether the antifungal peptides can inhibit the germination of spores or not and the accurate concentration of antifungal peptides for inhibition, we conducted the spore germination experiment.
Culturing the Spores to germinate
The concentration of the spore suspension solution was calculated and adjusted by using a hemacytometer and a microscope. After adjusting the concentration of the solution, we mixed the spore suspension solution, 2% glucose solution and peptide together into PCR tubes. Then we drew the mixture from the PCR tubes to the double concave slide in the Petri dishes. Incubating for 20℃, 6 hours in an incubator.
Percentage of Spore Germination
After incubating under 20℃ for 6 hours, we observed and classified the spores into four grades according to the lengths of the germination tubes, and calculated the percentage of each germination grade of each sample. Then, we compared all the samples with the control, and we can know the effectiveness of every peptide we predicted for the spore germination.
Observation
The spores were classified into four grades according to the lengths of the germination tubes, 0 means the spores that did not germinate; 1, the lengths of germination tubes were same as the spore itself; 3 represents the germination tubes three times longer than the spores and 5 stands for the germination tubes five times longer than the spores. And then we calculated the percentage of each germination grade of each sample.
Table 2: Illustration of four grades of spores according to the lengths of the germination tubes
Sample
Table 3: Peptides and their sequences.
Each peptide was repeated the experiments for three times to ensure the results.
The HEPES buffer, which has no toxic on cells, is applied to be the negative control.
Botany Experiment
- Observation of Disease Occurrence in Reality
In addition to the experiments that have been conducted in the lab to demonstrate the effect of inhibition, we applied our peptides to plants to know how effective they were and whether they were genuinely useful in reality. So, we infected the plants and sprinkled our peptides to verify whether the peptides can inhibit the diseases on plants.
In addition to the experiments that have been conducted in the lab to demonstrate the effect of inhibition, we applied our peptides to plants to know how effective they were and whether they were genuinely useful in reality. So, we infected the plants and sprinkled our peptides to verify whether the peptides can inhibit the diseases on plants.
Experiment
Draw two points on each side of the flower with a black pen as a marker. Then point two drops of pathogens on each side of Oncidium. One side pipetted with the negative control, and the other side pipetted with our antifungal peptides. Put it into a 20℃ incubator for three days.
Each peptide was repeated the experiments for three times to ensure the results.
Observation
The right side of Oncidium petal was infected by Botrytis cinerea. The left side of Oncidium petal was uninfected. If the Oncidium petal is infected by Botrytis cinerea, it grows black spots on it.
Figure 3: Left: Botrytis cinerea infection on Oncidium petal; Right: uninfected Oncidium petal
Sample
- Pathogen: Botrytis cinerea
- Plant: Oncidium
Table 4: Peptides abd their sequences
Reference
[1]Antifungal Mechanism of a Novel Antifungal Protein from Pumpkin Rinds against Various Fungal Pathogens.J. Agric. Food Chem. 2009, 57, 9299–9304. DOI:10.1021/jf902005g
