Making disease inoculum for field trials

Every growing season, new crop varieties and crop protection products are tested in field trials across Canada. To score disease resistance in new varieties and to test the efficacy of new crop protection products, researchers often introduce the diseases of interest to their test plots using lab-produced disease inoculum. At Insight Plant Health, we manufacture hundreds of kilograms of disease inoculum every spring for customers across Canada. This is how we do it.

Isolating the pathogens

During the summer, our lab receives various crop samples for disease identification. In the course of identification, we often isolate the pathogen in pure culture by incubating parts of the diseased plant on agar followed by successive subculturing until a single fungal or bacterial strain is isolated. We supplement these in-house isolates with disease isolates from other labs or from the customers themselves if they have a particular strain they’re interested in. In practice, all of the strains we produce for field trials have come from our own isolations or other companies because governments and universities needlessly prohibit most uses of their strains with restrictive material transfer agreements. Because the pathogens will be released back into the environment, we follow the Code of Practice for Plant Pathogen Trials, which is a set of rules developed by the Canadian Phytopathological Society and CropLife that outlines how to safely produce and use disease inoculum in field and greenhouse trials. All of the strains we use have been identified by DNA barcoding, which is a standard method for identifying and classifying living things.

Various fungi isolated from diseased plants at Insight Plant Health

Preparing the substrate

Once we have the isolate in hand and a customer orders the inoculum, we prepare growth substrates to produce the pathogen at scale. At Insight Plant Health, we produce both liquid and solid disease inoculums, but in this post, I’ll describe the production of solid inoculum. 

For field scale trials, we generally use grain as the growth substrate. For most of the pathogens we grow, we use a blend of wheat and corn, although some prefer corn or wheat alone, while others grow better on rye or barley. The first step in preparation of the substrate is to soak the grain in water. The soaking time depends on the grain, but overnight soaking works in most cases. Once soaked, the excess water is removed from the grain by sieving and the grain is transferred into autoclavable growth bags.

Soaked grains in autoclavable growth bags

We have tried a number of different growth bags for inoculum production, but have had the best success with Unicorn brand mushroom spawn bags. These bags are fitted with a filter patch that allows for gas exchange without contaminating the grain inside. Once the bags are filled with grain, they are transferred to an autoclave for sterilization. The grain bags are autoclaved for 40 minutes at 121°C and 15 psi to ensure that the grain is free from any potential contaminants ahead of inoculation with the pathogen. Autoclaving also has the advantage of killing all of the seeds, which prevents the inoculum from making a big mess of the customers’ plots.

Growth bags after autoclaving

Inoculation and growth

Once the grain has cooled to room temperature, it is ready for inoculation with the pathogen. Depending on the isolate, the grain can either be inoculated using a liquid culture or several petri dishes chopped up into small pieces. The petri dish method tends to lead to less contamination. The inoculated growth bags are sealed with a heat sealer.

Chopped up agar containing the pathogen spread into the sterilized grain

The growth bags inoculated with the pathogen are left to grow either in the lab or growth chamber, depending on whether growth or development of the pathogen is encouraged by light. We track the colonization of the grain over time, and generally let the growth go until the starch from the seed is thoroughly colonized by the pathogen when a seed is split open. This complete colonization usually takes 2-4 weeks.

Drying and packaging

The colonized grain has to be dried before it can be used by the customer. To get the excess water from the grain, we cut the bags open and leave them in the biosafety cabinet overnight with the fan running. We then transfer the colonized grain into trays and store them in a portable enclosure with a dehumidifier running until they are <15% moisture.

Colonized grain in drying trays

Once dry, the grain is packaged in vacuum bags or pails with silica gel packs to keep it dry and then shipped out to customers for field application.

Whole and ground grain field trial inoculum