Fungal enzymes developed in nanoliters of fluid may be a new organic tool in the fight against crop-killers

Fungus is getting a pretty bad rap in pop culture right now thanks to HBO’s hit zombie TV show The Last of Us, in which a fungal mutation spread through the global food supply leads to the collapse of civilization. But its critical role in the health of the planet’s ecosystems is well-known, especially as it applies to humanity’s food supply. Fungus can both protect and kill grains and plants, and the agribusiness industry has long tried to improve pathogen-destroying methods to keep food stores safe.

That’s the focus of a new paper published in the Nature journal Microsystems & Nanoengineering by researchers at the Shih Microfluidics Lab. They have developed a new method of extracting enzymes that degrade the cell walls of harmful micro-organisms such as other fungal pathogens, nematodes or other pathogens that can impact crop health. In effect, it acts as a biological fungicide and pesticide. The researchers also built a device that makes the technique portable, affordable and easy to use.

“While recent decades have seen improvements in growing strains of mammalian or bacterial cell culture, the techniques used are difficult to apply to fungus due to their specific morphology,” says Kenza Samlali, PhD 21, the paper’s primary author. “So there is a lot of interest in automated systems and microfluidics that can screen for micro-organisms.”

PhD student Chiara Leal Alves co-authored the paper with University of Toronto master’s student Mara Jezernik, Gr. Dip 20, and Steve Shih, associate professor of electrical and computer engineering.

A difficult organism to keep small

The researchers studied Clonostachys rosea, a fungus well-known to be toxic to other types of fungi, bacteria and nematodes, to find a way to make it produce more crop-shielding catalytic enzymes. They were especially focused on a way to protect crops from fusarium, a potentially carcinogenic fungus that attacks grain crops like wheat, rye and barley, as well as fruits like bananas, squash and pumpkins.  

However, the properties of all fungi make them difficult to study, the researchers note. Fungi grow rapidly, producing the spindly hyphae that can make them difficult to study in the kinds of nanolitre droplets used in microfluidics. The researchers created a new substrate to grow the c. rosea on within a droplet. This allowed them to analyze a single spore based on the thousands of kinds enzymes that were expressed by it in a solid-state fermentation process. They also developed a device that could gently sort out those fungi that produced useful enzymes in high quantities using a very low electrostatic force, keeping the droplets intact.

They could then identify a few dozen different strains of enzymes they deemed most useful out of tens of thousands that were expressed. They were looking for strains that produced enzymes that degrade the cell walls of pathogenic fungi — a wholly organic lifesaver for countless kinds of crops.

“Studying fungal-based organisms is so hard,” Shih says. “The microfluidic method that Kenza and Chiara have designed is truly a breakthrough in the field, especially for determining fungal antagonists. With a way to culture these in single-cell format, we can now use this technology to screen through thousands of fungal antagonists, which can then be used as biocontrol agents to prevent plant pathogens and diseases. We see immediate use of this in the field of agriculture, but we also see future uses in examining fungal diseases related to human health.”

This study received funding from the National Sciences and Engineering Research Council (NSERC), the Fonds de recherche nature et technologie (FRQNT) and the Canadian Foundation of Innovation (CFI).

Read the cited paper: “Droplet digital microfluidic system for screening filamentous fungi based on enzymatic activity.”