After the meeting with Dr. Moore, we immediately had to learn more about working with fungi and anti-fungal assays. Through background research, we had the idea to use the “drop method”, applying drops of pheophorbide to different locations on an agar plate, with varying concentrations to see if it inhibited fungal growth. However, we needed to validate our experimental design. So we met with Dr. Heather Addy.

Dr. Heather Addy is a mycologist and plant biologist who specializes in plant-fungal interactions at the University of Calgary. Her expertise in the field meant we absolutely had to talk to her to inform the design of our anti-fungal assays.

Dr. Addy gave us the idea to use the “disc method”, immersing paper discs in solubilized pheophorbide, placing them around a fungal culture placed in the centre of a potato dextrose agar plate. The fungal colony would grow and eventually come into contact with the pheophorbide discs. Over the course of the experiment, we would measure the distance from the centre of the plate to the edge of the fungal colony’s growth. If pheophorbide does in fact have an inhibitory effect on fungal growth, then there would be a decreased rate of growth for the portions of the colony interacting with pheophorbide.

Dr. Addy generously put us in touch with Fran Cusack, a Biological Sciences Technician who prepares fungal samples for classes. Fran was kind enough to provide us with samples of Pestalotiopsis microspora and Sclerotinia sclerotinium, the same fungus which commonly afflicts canola crops.

We now had the information and tools at our disposal, to begin testing pheophorbide’s application as an anti-fungal agent. However, there was still a gap in our knowledge regarding the what the average Albertan farmer goes through when faced with fungi, like Sclerotinia.

Dr. Turkington holds a Masters Degree and Ph.D in Plant Pathology, focusing on the epidemiology of sclerotinia stem rot in canola. For the last 23 years, Kelly has been working as a Research Scientist for Agriculture and Agri-Food Canada at the Lacombe Research Centre in Alberta.

We consulted him to learn more about the progression of Sclerotinia fungus, its impact on farmers and preventative and prescriptive measures to combat it.

According to Kelly, the cost to treat fungi like Sclerotinia can be around $20-$30 per acre, severely reducing a farmer’s bottom line. To assess risk of fungal growth, farmers employ a checklist by assigning point values to certain factors including the plant, the host and the environment. This is a very broad indication of risk and is not an exact science as there is ambiguity in the checklist. Some companies have started using DNA-based chips to quantitatively determine the percentage infestation of a plant.

In determining if Pheophorbide would be an ideal anti-fungal agent, he directed us to consider the full chemical profile of pheophorbide. Not just it’s effects on fungi but also on non-target organisms. Another consideration is the societal aspect of such a product. Members of the farming community and industry only care if the product is cheap and effective, but society as a whole tends to support products which are “organic” or come from the environment in a responsible manner.

During the initial stages of project design, we were contemplating whether to use a biofilm layer (1) containing chlorophyll-binding proteins to bind the chlorophyll molecules, and use this to filter the chlorophyll out of the canola oil. To gain an idea into the feasibility of using a biofilm, team members contacted Dr. Harrison, a microbiology professor at the University of Calgary who focuses on biofilms as a research interest. Dr. Harrison advised us to consider the amount of chlorophyll present in the oil compared to the amount of protein that can be present on a biofilm layer to capture all of the chlorophyll present. Additionally, he cautioned us that a biofilm is composed of bacteria, and thus the biofilm would have to be composed of bacteria which would not degrade the oil, as otherwise this would result in a lower yield of oil. Dr. Harrison spoke of some positive aspects to this project venture including the ability to kill the bacterial communities that comprise the biofilm using UltraViolet light, autoclaving and boiling without destroying the biofilm or denaturing the proteins in the process. Dr. Harrison also offered us the ability to use his equipment such as reactors and pumps to grow up the biofilm. He also told us to do more research into hydrophobic biofilms that would be able to withstand oil being passed through. Through this meeting, Dr. Harrison gave us valuable feedback on using biofilms as a chlorophyll filtration system, and gave us points to conduct further research in.