I have been asked before what my process is for identifying plant pathogens – fungi and bacteria that cause disease on plants. Although there are different techniques to identify different pathogens, often the most straightforward approach is to culture them until morphological characteristics are obvious enough to identify using keys. In other words, I grow the pathogens until they produce structures that can be identified!

You may ask, what does it mean for the pathogens to be “cultured”? While I may not be playing Beethoven’s fifth for the pathogens, I am providing them an enriching environment in the form of proper nutrition and a favorable environment. Diseased tissue can be isolated from healthy tissue, placed on a nutritious media, and allowed to grow in a warm protected environment until identifiable reproductive structures are produced.

First, diseased tissue is photographed and examined to see if identifiable signs of the pathogen are already present. An example of such would be a fungus that has grown enough on the submitted plant tissue to be actively producing reproductive spores (see fig.1 and fig. 2). For samples with minor, ambiguous, or fastidious fungal growth, some extra encouragement for growth may be necessary.

Fig. 1. Alternaria fruit rot and spot of pepper is caused by Alternaria solani, a common fungal pathogen.
Fig. 2. A magnified photo of Alternaria solani spores pulled from the pepper lesions in Fig. 1.

In instances where the identity of the pathogen is not immediately obvious, the sample is dissected. Some tissue that was surrounding the symptomatic area is excised and (usually) surface sterilized using a diluted bleach rinse. The remainder of the diseased tissue is placed in a sealed “humid chamber” to allow the pathogen to continue to grow on the submitted tissue. Humid chambers can easily be made by putting a clean, damp paper towel in a plastic deli container, placing the sample inside, then sealing and labeling the container. The sample can be checked each day to see how the pathogen is (or isn’t) developing.

The excised tissue then be placed on a substance called “agar” for the pathogen to incubate and grow. Agar is a product derived from red algae, commonly Gelidium amansii. It is a dense, jelly-like substance and is a common replacement for gelatin in vegan recipes. It is also a common ingredient in many East Asian desserts, such as boba teas. The agar is mixed with nutrients and certain chemicals to discourage non-target organisms from growing, heated and sterilized in an autoclave or pressure cooker, then poured into petri dishes to solidify (see fig. 4).

The tissue is placed on the agar using sterilized tools under a biological safety cabinet, a machine that maintains a sterile environment and protects the user from exposure to harmful agents or accidental contamination (see fig. 3). This process is often called “plating” as the tissue is being placed on petri plates. Once finished, the petri plates are labeled and may be sealed with a film to further reduce the risk of contamination. The finished plates are placed in a warm chamber, like an incubator, to allow the pathogen to grow until spores or other desired structures are produced.

Fig. 3. A biological safety cabinet with a HEPA filtration system and unidirectional airflow protects the user from contaminating samples or exposure to harmful agents.
Fig. 4. A sealed, labeled petri plate with significant fungal colonization.

Once the spores or other desired morphological characteristics have been produced, they may be photographed under a microscope and compared to references or subjected to further testing (this is usually only necessary for bacteria) for identification. When the pathogen has been identified, the used plate is securely stored until it can be sterilized via autoclave and discarded appropriately.

Until next time!

– Nick

Nick Goltz, DPM – Director of the UConn Plant Diagnostic Lab