Team:Calgary/Anti-Fungal
Overview
Investigating pheophorbide's potential as a preventative anti-fungal treatment.
Talking to farmers and agronomists about the green seed problem revealed another issue afflicting Canadian canola farmers: fungus. Dr. Kelly Turkington, a crop pathologist we spoke to, remarked that once fungus has been found in a field, the affected crops cannot be recovered and must be discarded. We set out to find a preventative measure; one that would destroy fungus before it proliferated to that point. We found it in pheophorbide.
Pheophorbide vs Sclerotinia
Empowering farmers against fungal pathogens
The compound pheophorbide has recently received increasing attention in academic circles due to its potential as an anti-cancer and anti-fungal agent. Pheophorbide acts as a photosensitizer, when it is “activated” by light, it creates radical oxygen species that cause harm to cellular structures in adjacent cancer and fungal cells. We were particularly interested in its ability to prevent the fungal infection of canola crops by inhibiting the growth of a pathogenic strain, Sclerotinia sclerotiorum.
Sclerotinia sclerotiorum is a necrotrophic fungal pathogen which can infect over 400 plant species found all over the globe (Bolton, Thomma, & Nelson, 2006). Notably, it occurs in all canola growing areas of Canada and causes stem rot disease, one of the most destructive diseases afflicting canola. According to Dr. Kelly Turkington, most farmers combat fungi through both crop rotation and resistant strains of canola. However, both techniques are ineffective in preventing Sclerotinia infection. Sclerotinia evolves rapidly, making it difficult to create a resistant strain and it can lay dormant in the soil over several growing seasons. As a silent predator, its dormant spores are carried by the wind until they land on leaves and eventually fall into the stem to incubate. Since Sclerotinia's occurence in canola is highly variable, farmers have to make a choice: take a risk and hope their crop is unaffected, or choose to spray with costly fungicides.
We see pheophorbide as a solution to this development. By using a naturally occuring compound to prevent fungal growth, we would effectively be creating an agricultural solution to an agricultural problem.
Experimental Design
How to characterize inhibitory effects on fungal growth
To test pheophorbide for anti-fungal properties, we needed to design an experiment to track the inhibition of mycelial growth on agar plates. We coordinated with Dr. Heather Addy (Mycologist) who helped us design a disc test experiment. This would work by culturing our fungi species in the center of a potato dextrose agar plate and surrounding the fungi culture with pheophorbide a saturated discs of varying concentrations, ultimately allowing us to visualize differential growth rates of the culture.
Figure A. Disc Test Example Plates
Quantitative data was collected by measuring the growth from the epicentre of the original culture to the end of the mycelium in a given direction, based on where the discs were. Pheophorbide a was solubilized in 25% acetone and applied to sterile paper discs (0.6 cm). Several variables were adjusted to optimize our experiments, including distance from the culture to the treatment disc, light or dark treatment, number of discs (number of treatments and dosage of each treatment), and distance of light source to the cultures.
Figure B1 and B2. Light box inside (B1-left) and outside (B2-right)
Inhibition disc tests were done on two species of fungi:
Sclerotinia sclerotiorum, one of the most prevalent fungal pathogens among canola crops (our desired target), and
Pestalotiopsis microspora, a fungus that shares the same genus with Pestalotiopsis neglecta, which was previously used to study the anti-fungal properties of “sodium pheophorbide a” (Jing, Lian-Nan, Xiao-Bo, Yue, Bing, Guo-Cai, & Chuan-Shan, 2019).
Experimentation
The following section dives into the iterative experimentation undertaken to characterize pheophorbide a's capability to inhibit the mycelial growth of Sclerotinia sclerotiorum and Pestalotiopsis microspora. Fungi samples were cultured on potato dextrose agar plates provided by Fran Cusack (University of Calgary, Lab Technician) under varying conditions. Under Fran's recommendations, fungi was incubated at room temperature for all experimentation.
Growth Controls: Light vs Dark
First we had to understand the natural growth of our fungi controls under both dark and light conditions. We sought to determine how long it takes for our organisms to grow and what they qualitatively look like without treatment. The results of a six day incubation are shown below.
Figure 1A and 1B. Mycelial growth of Sclerotinia sclerotiorum (1A-left) and Pestalotiopsis microspora (1B-right) in dark or light conditions. Growth was tracked for six days after culturing and measurements were taken once a day. Each point is the average of eight measurements in different (controlled) directions. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 1A and 1B here.
From the results above, we learned that Sclerotinia sclerotiorum goes through a rapid growth phase after 24 hours of incubation, reaching the plate maximum (4.2 cm) after the third day. Pestalotiopsis microspora has a steady growth rate of approximatly 0.6 cm per day. Incubation under light conditions was shown to slightly inhibit Sclerotinia sclerotiorum growth and enhance Pestalotiopsis microspora growth. This information provided us reference for when to add treatment discs.
Testing 8 different pheophorbide a concentrations
Based on the inhibitory action of sodium pheophorbide a characterized in literature, we determined that concentrations around 20 mg/mL would potentially be effective for pheophorbide a, as it was shown to be effective against Pestalotiopsis neglecta. (Jing et al., 2019) In our first treatment experiments we used eight different pheophorbide a concentrations (0, 1, 2.5, 5, 10, 15, 20, 35 mg/mL) to attempt the collection of a broad range of data. Treatment discs were impregnated and set on Day 1 (24 hours of growth) at a distance of 2.5 cm away from the centre of the original fungal culture.
Figure 2A and 2B. Mycelial growth of Sclerotinia sclerotiorum with pheophorbide a in dark (2A-left) and in light (2B-right) conditions. Eight treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 1, 2.5, 5, 10, 15, 20, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Treatment discs were placed 2.5 cm from the epicentre of the fungal culture. Growth was tracked for four days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 2A and 2B here.
Figure 3A and 3B. Mycelial growth of Pestalotiopsis microspora with pheophorbide a in dark (3A-left) and in light (3B-right) conditions. Eight treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 1, 2.5, 5, 10, 15, 20, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Treatment discs were placed 2.5 cm from the epicentre of the fungal culture. Growth was tracked for six days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conduted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 3A and 3B here.
The results above did not show clear significant inhibition by our compound for either of the organisms in dark or light conditions. However, when comparing the light to the dark treatment of the Sclerotinia sclerotiorum experiment there appears to be a small decrease in growth for the 35 mg/mL (pheophorbide a) light treatment at day 2. This showed some promise toward potential inhibition. However, we hypothesized that the treatment discs were not close enough to the fungal culture to take effect when activated by the light at an early stage. Additionally, we hypothesized that the dosage was too low to see a visible effect. Therefore, we decided to decrease the number of different treatments and increase the dosage.
Increasing dosage and decreasing distance
In order to increase the likelihood of inhibiting the growth rate of fungus with pheophorbide a, we changed three variables. First, by decreasing the number of treatments to five (0, 5, 15, 25, 35 mg/mL pheophorbide a), we were able to decrease the distance of the discs from the culture from 2.5 cm to 1.5 cm. Second, we increased the dosage of pheophorbide a by applying two discs of the same concentration, next to each other, on the agar plate. Thirdly, we increased the proximity of the light source to the fungal cultures so as to potentially increase the effect of photo-activation on pheophorbide a.
Figure 4A and 4B. Mycelial growth of Sclerotinia sclerotiorum (4A-left) and Pestalotiopsis microspora (4B-right) in different light conditions. Growth was tracked for six days after culturing and measurements were taken once a day. Each point is the average of eight measurements in different (controlled) directions. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm or 2 cm from the plate. Data points are summarized in Anti-Fungal Table 4A and 4B here.
Figure 5A and 5B. Mycelial growth of Sclerotinia sclerotiorum (5A-left) and Pestalotiopsis microspora (5B-right) with pheophorbide a in close light conditions. Five treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 5, 15, 25, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Two treatment discs for each concentration were placed 1.5 cm from the epicentre of the fungal culture. Growth was tracked for six days after culturing. Measurements were taken once a day (with an additional measurement at day 2.5) from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 2 cm from the plate. Each point is the average of two replicates. Data points are summarized in Anti-Fungal Table 5A and 5B here.
When comparing the closer light treatment (2 cm) with the original dark and light (25 cm) growth controls, it is clear that Sclerotinia sclerotiorum is unable to grow after 2 - 3 days of incubation with close light. Conversely, Pestalotiopsis microspora was able to grow further in less time with the increase in light. Figures 4A and 4B reveal that the mycelial growth of Sclerotinia sclerotiorum is negatively correlated to light, whereas Pestalotiopsis microspora mycelial growth is positively correlated.
The results above (Figure 5A and 5B) did not show clear significant inhibition by our compound for either of the organisms. However, these results are confounded by the obvious negative effect of decreasing the distance of the light source from the culture. As shown in our initial eight treatment tests, the mycelial growth of Sclerotinia sclerotiorum appears to have some kind of recognizable inhibition occurring at day 2 that is positively correlated to increased concentrations of pheophorbide a. Though this is once again not enough to definitively provide confidence for our hypothesis of pheophorbide a as an anti-fungal agent, it did let us know we were on the right track. Therefore, we ascertained that day 2 was an important day to study, likely due to our experimental methods of setting the discs after day 1 measurements and incubating in light between days 1 and 2, activating the pheophorbide within this time-span.
Inhibition via pheophorbide a
Due to the closer light (2 cm distance) confounding our results, we decided to return to a 25 cm distance of light source to culture, while also maintaining the change in distance and dosage of discs applied in our last experiment. We hypothesized that these conditions should allow for the light to activate the pheophorbide a at a distance and time to visualise inhibition on day 2, particularly for Sclerotinia sclerotiorum.
Figure 6A and 6B. Mycelial growth of Sclerotinia sclerotiorum with pheophorbide a in dark (6A-left) and in light (6B-right) conditions. Five treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 5, 15, 25, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Two treatment discs for each concentration were placed 1.5 cm from the epicentre of the fungal culture. Growth was tracked for four days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 6A and 6B here.
Figure 7A and 7B. Mycelial growth of Pestalotiopsis microspora with pheophorbide a in dark (7A-left) and in light (7B-right) conditions. Five treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 5, 15, 25, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Two treatment discs for each concentration were placed 1.5 cm from the epicentre of the fungal culture. Growth was tracked for four (dark) or six (light) days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 7A and 7B here.
Figures 6A and 6B show evidence for the photo-driven activation of pheophorbide a as an anti-fungal agent. Particularly, under light conditions it is apparent that Sclerotinia sclerotiorum mycelial growth is increasingly inhibited at pheophorbide a concentrations greater than 15 mg/mL. The same result is not seen under dark conditions, as pheophorbide a would not have been photo-activated other than unintentional exposure in recording measurements. This light exposure error may explain the slight inhibition positively correlated with increasing pheophorbide a concentrations seen in dark conditions. However, there may also be "leaky" effects from the pheophorbide without photo-activation. Inhibition was shown most directly on day 2 with residual effects on day 3. It is unclear if the trend would have continued longer, as data collection reaches a maximum point due to the size of the agar plate.
Figures 7A and 7B show that Pestalotiopsis microspora mycelial growth is unaffected by pheophorbide a under dark or light conditions. This may indicate that pheophorbide a's mode of function may have some kind of specificity toward cellular structures present in Sclerotinia sclerotiorum and not Pestalotiopsis microspora. However, Pestalotiopsis microspora may also have more defense mechanisms for dealing with environmental hazards, such as radical oxygen species.
Pictures of obvious qualitative inhibition of Sclerotinia sclerotiorum mycelial growth by pheophorbide a on "Day 2" from the above experiment, along with a picture of the corresponding Pestalotiopsis microspora potato dextrose agar plate, are shown below (Figure 8A and 8B).
Figure 8A and 8B. Mycelial growth of Sclerotinia sclerotiorum (8A-left) and Pestalotiopsis microspora (8B-right) with pheophorbide a discs in light conditions 48 hours after culturing. Pictures of potato dextrose agar plates were taken after 24 hours of incubation in light conditions with the pheophorbide a discs set. These pictures correspond to the experimental treatment shown in Figures 6B and 7B (light conditions).
Final Results
Pheophorbide a inhibits Sclerotinia sclerotiorum mycelial growth
Ultimately, pheophorbide a was shown to have an inhibitory effect on the mycelial growth rate of Sclerotinia sclerotiorum (Figure 2B, 5A, 6B, 9) and had no visible effect on Pestalotiopsis microspora (Figure 3, 5B, 7). This inhibitory effect was proven to be controlled by photo-activation and was positively correlated with increasing treatment concentrations (Figure 2B, 5A, 6B, 9). Recorded inhibition of Sclerotinia sclerotiorum under varying conditions, as described above in "Experimentation", suggests that the time of pheophorbide a photo-activation relative to the treatment discs' distance to the culture is important. Particularly, the compound may need to be reapplied if there was insufficient pheophorbide a application initially, which may be a result of diffusion. It was also shown that with increasing light exposure there is a significant decrease in Sclerotinia sclerotiorum mycelial growth (Figure 4A) and a marginal increase in Pestalotiopsis microspora mycelial growth rate (Figure 4B). These results provide support for the use of pheophorbide a as an anti-fungal agent and provide preliminary grounding for its use as a preventative measure against Sclerotinia sclerotiorum infection.
Figure 9A, 9B, 9C, and 9D. Mycelial growth of Sclerotinia sclerotiorum with pheophorbide a [(9A - 5 mg/mL), (9B - 15 mg/mL), (9C - 25 mg/mL), (9D - 35 mg/mL)] in light conditions. These graphs represent the same data shown in Figure 6B. Five treatments were applied as treatment discs impregnated with pheophorbide a, solubilized in 25% acetone (0, 5, 15, 25, 35 mg/mL). The 0 mg/mL treatment was 25% acetone. Two treatment discs for each concentration were placed 1.5 cm from the epicentre of the fungal culture. Growth was tracked for four days after culturing. Measurements were taken once a day from the epicentre of the original culture to the edge of the mycelial growth toward the disc. Maximum growth is recorded at 4.2 cm due to the potato dextrose agar plate capacity. Light conditions were conducted using 1400 lumen white LED light at a distance of 25 cm from the plate. Data points are summarized in Anti-Fungal Table 6B here.
Future Directions
From lab bench to canola field
We hope to eventually have pheophorbide a used as a preventative measure for Sclerotinia sclerotiorum infection in agriculture. In order to achieve this goal, more work must be done to further characterize the efficacy of pheophorbide a as an anti-fungal agent. We hope to perform more experiments to determine its minimum inhibitory concentration (MIC) by increasing the range of treatment concentrations used and studying dosage effects. Most presently, we would like to replicate our last experiments and enhance them by comparing the results to a new treatment in which we re-apply pheophorbide a each day. This would test if increased dosage and application would prevent further mycelial growth entirely. Furthermore, we would like to verify the mode of action of pheophorbide a as a potential drug compound using chemical-genetic profiling. This would be done using yeast deletion collections through drug-induced haploinsufficiency profiling and homozygous profiling, whereby we would compare the effects of pheophorbide a to the primary targets of industry characterized anti-fungal agents.
Once more fully characterized we would like to test pheophorbide a on canola crops via application to their leaves in whole organism assays. This would allow us to test potential side effects to plant cells and their vitality. Further testing can be done by applying Sclerotinia sclerotiorum spores to the leaves of the plant along with pheophorbide a to test if our compound does successfully prevent crop infection.
References
Bolton, M. D., Thomma, B. P., & Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Molecular plant pathology, 7(1), 1-16. doi: 10.1111/j.1364-3703.2005.00316.x
Jing, Y., Lian-Nan, L., Xiao-Bo, Z., Yue, W., Bing, B., Guo-Cai, Z., & Chuan-Shan, Z. (2019). Sodium pheophorbide a has photoactivated fungicidal activity against Pestalotiopsis neglecta. Pesticide Biochemistry and Physiology, 158, 25-31. doi: 10.1016/j.pestbp.2019.04.003
