Undergraduate Student Report 2021

Endophytic Trichoderma sp. as biocontrol for Armillaria mellea

Jude Turner

Jude Turner worked with Dr Andy Bailey, University of Bristol, to investigate biocontrol of honey fungus.

My name is Jude Turner, and I am now in my third year of a Biological Sciences degree at the University of Bristol. As a result of the great generosity of the British Mycological Society, I had the privilege of undertaking research in the University of Bristol plant pathology lab, supervised by Dr Andy Bailey, working with PhD student Morgan Millen.

Figure 1:  Three different isolates of Trichoderma side by side. It is interesting to note the differences in their appearances, all had been cultured for the same amount of time, so we can see the different rates at which they grow.

The project we worked on investigated the biocontrol applications of fungus Trichoderma to prevent infection of plants by honey fungus – Armillaria.  The group had already screened a collection of root-endophytic Trichoderma sp. for efficiency of biocontrol in planta and the aim of my project was to try and find how this control might be working. Initially, we started with Agrobacterium mediated transformations of Trichoderma. The goal of this transformation was to introduce the DsRed gene into the genome of Trichoderma. This would allow us to visually track the endophytic colonisation of plant roots through florescence. However, these transformations were, unfortunately, unsuccessful.

The research then moved to assaying each isolate for any enzyme activity that might correlate with biocontrol ability. The first of which being protease assays of Trichoderma. Media were made to detect the presence of protease enzymes released by the colony, and after 4 days of growth, these plates were dyed with amido black to visualise remaining proteins in the plate. Areas in which the plate was dark still contained protein, and lighter areas contained less or no protein, indicative of protease activity.

Through this it was possible to determine how far a colony sent its protease enzymes into a substrate, a potential mechanism through which Trichoderma could prevent the growth of Armillaria. Figure 2 below shows the stained plates. Similar plate-based assays using dyes were set up to detect different enzymes, such as chitinase, which could digest the cell walls of Armillaria, thus preventing its growth, but there was no clear correlation to biocontrol ability.

Figure 2: a plate used for a protease assay. The lighter blue area in the centre indicates that the colony has sent proteases throughout this area.

In another approach, volatile organic compounds (VOCs) were assessed. We hypothesised that endophytic Trichoderma might release VOCs with the potential to inhibit the growth of Armillaria or to promote the growth of the plant. The set up for these experiments were as follows: Trichoderma and Armillaria cultures were initially established in separate petri dishes, then after 2 days of growth, the bottom halves of these plates were sealed together with parafilm, such that they shared an atmosphere. The plates were then incubated for at least a week. Controls containing Armillaria only were also set up. The diameter of the Armillaria colony was measured and compared against the control plates to determine the percentage growth inhibition caused by the VOCs emitted by Trichoderma.

It was found that the VOCs emitted by Trichoderma caused Armillaria colonies to be around 40% smaller. VOC assays using lettuce seedlings instead of Armillaria were also carried out to determine whether Trichoderma could increase result in faster growth rates for plants. Unfortunately, these experiments took a long time and the results were inconclusive.

Figure 3: the strawberry plants in the greenhouse.

Finally, strawberry plants were potted and inoculated with Armillaria and different isolates of Trichoderma. The plants were checked on weekly, and once one had died, the plant was taken back to the lab and the root collar opened to check for the presence of Armillaria. Samples of mycelium were plated onto JJG agar – this medium is selective for Armillaria, enabling us to confirm that the cause of death of the plant was in fact Armillaria. This experiment also enabled us to determine which isolates of Trichoderma were most efficient at preventing Armillaria infection.

I had a wonderful experience undertaking research in the lab, and I am extremely grateful to Andy Bailey and the BMS for this placement. It was inspiring, challenging and a fascinating insight into life as a scientific researcher. I would like to give a huge thanks to Andy Bailey, the British Mycological Society and Morgan Millen for providing me with the resources and training to make this incredible experience possible.