Fusarium oxysporum f. sp. cubense (FOC) is an important fungal pathogen causing Fusarium wilt of bananas, known for wiping out the Gros Michel banana in the 1950s. A global soilborne disease of bananas, it is now also found in beds in botanical gardens and has been identified at attractions such as the Eden Project in Cornwall, and threatens production of the Cavendish banana cultivar. It is estimated that FOC Tropical race 4 threatens 80% of global banana production.
Over 10 weeks, I worked in the laboratory at Eden Project Learning with Dr Rachel Warmington to undertake in vitro experiments measuring and observing the inhibitory interactions between the commercial biofungicide PRESTOP, containing Gliocladium catenulatum fungal strain J1446 spores and mycelia, and FOC Tropical race 4. Usually used as a foliar spray or a soil application, in vitro assessment allowed for observation of effectiveness of PRESTOP as an inhibitory growth control of FOC. FOC was grown on potato dextrose agar (PDA) mixed with PRESTOP at 3 different strengths- 50%, 100% and 150% recommended product application strength per 250ml of agar. A negative control was also included. The cultures were kept in an incubator for 5 days at around 20˚C. 10 plates for each treatment were prepared, and each experiment was repeated six times. Mycelial growth was measured each day for five days. Growth was then compared with the negative control from each experiment for comparison of rate of inhibition percentage across the treatments, and significance levels were tested using a two-way ANOVA, with a 0.05 significance level. Soil samples were also taken from the bed containing the infected plants, for soil plating, and identification of Fusarium colonies in the soil. Samples believed to be Fusarium colonies which were obtained from the soil plates were run through the OptiGene Genie III. The Genie III measures samples against a negative control using loop-mediated isothermal amplification (LAMP) to identify the presence of a microbe. Although our results did not conclusively identify FOC in the samples taken, it was a great opportunity to undertake some practical molecular biological work.
This type of experiment is of significant importance to better understanding control and prevention of FOC in the field, and the role that mycoparasitic biofungicides such as PRESTOP could play. Currently there are no satisfactory or affordable control method of FOC, which is known to persist in the soil for several years after the host plant has been removed. The experiments showed that PRESTOP inhibited FOC, and the most inhibition at 100% recommended strength application. However, the most consistent levels of inhibition were presented at 150% concentration. Repeat in vitro experiments should be carried out to test further the effect of these concentrations, and potted plant trials would be the next step in determining the effectiveness of PRESTOP against FOC in the soil.
During my project, I was able to learn important new skills and was introduced to new machinery, techniques and equipment that would otherwise be unavailable to me, such as the OptiGene Genie III for DNA analysis, beginning and maintaining cultures and slopes of inoculum, and soil plating for identification of microbial colonies in the test area. I was familiarised with good lab practice as a new concept. Further development and reinforcing of literature review and writing up of experiments was valuable and has allowed me to gain insight into the professional research world, and how my interest in pathology can help to build my career in horticulture. It has also provided me with a better understanding of, and keener interest in, the prevention of important plant diseases.
Emily Johnston
Plymouth University/Cornwall College/Eden Project Learning
