This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
I had an opportunity to join the research lab of Prof Pietro Spanu at Imperial College (London, UK) for eight weeks over the summer. My project was built on the previous work, which shows that Fusarium xylarioides (Fx) which causes coffee wilt disease in Africa, gains an effector gene from Fusarium oxysporum (Fo), and this may relate to a re-emergence of the disease in Africa.
My project aims to find whether a hyphal fusion between F. oxysporum and F. xylarioides may be a means of transferring genes horizontally. To start, I set up interaction plates containing Fx and interacting fungi species; Fo. cubense, Fo. pisi, Fo. vasinfectum, Fo. coffea, F. solani (Fs) and F. verticillioides (Fv). I also set up plates that only had Fx. I measured the radial growth of fungal colonies and observed the morphological changes in the interaction plates. The radial growth was measured from the centre of inoculation to the colony’s edge. Overall, the growing distance of Fx towards the interacting fungi is less than that of the interacting fungi toward Fx. The results suggest that Fo inhibits Fx as they expand towards each other. The growth rate of Fx that grew individually is higher than those that grew within interaction plates. The growth rate of the interacting mycelia towards Fx is also higher than that of Fx growing towards them.
To investigate the presence of interacting fungi within the Fx colonies, agar samples from the near-centre of both fungi colonies and in between them were transferred and placed on synthetic-nutrient agar (SNA) plate. Fx spores have a distinct sickle shape which can be used to distinguish between Fx and the interacting fungi growing in the same petri dish. Thus, I used the appearance of spores to investigate if fungi had grown in each other’s mycelium. For each interaction plate, there are three agar cube samples transferred to SNA plates; from between the colonies, from the near-centre of the interacting fungus, and near-centre of Fx. Then I observed the spores formed in each SNA plate. Fx and the interacting fungi were present between colonies for every interaction. Fo. cubense, Fo. pisi, Fo. vasinfectum, Fo. coffea and Fs were found in Fx. However, Fx was not found in any interacting colonies. Additionally, PCR result shows that Fo’s nucleic acids but not Fs are present in Fx. On the contrary, no Fx’s nucleic acids is present in either Fo, except Fo. pisi, or Fs. The result is also consistent with the observation results using fungi spores described previously, with the exception of Fo. pisi and Fs.
Next, I set up slide cultures to observe whether it was possible to see any hyphal fusion in the interactions. I did this by pouring a thin layer of malt extract agar on a slide. Small pieces of mycelium were inoculated on the agar, similar to making interaction plates. Slides were kept in square Petri dishes. Then the Petri dishes were tightly sealed in a box with a damp paper towel to prevent agar from drying out and kept at 25 °C. Each fungi species or strain grows very differently from one another. Therefore, the incubation time ranged from 60 to 75 hours, depending on fungi interactions. After incubation, the slides were observed to detect anastomosis. I detected anastomosis in the cultures of Fx and Fo. cubense, Fo. vasinfectum, Fo. coffea and Fs. The anastomosis observed could be self or non-self. For future work, slide cultures can be improved by, for example, changing of the medium, incubation temperature, and insertion of fluorescence markers upstream the housekeeping genes of each fungi species.
Overall, I enjoyed my time learning in this summer project. I gained knowledge of the project, confidence in lab skills, and several transferable skills such as communication and organising. Furthermore, I’m grateful to Prof Pietro and Lily, who supported me greatly through my project and gave valuable feedback. Lastly, I’d like to thank BSPP for funding this project and enabling me to fulfil my research experience.
Duangta Fah
Imperial College London
IMAGE: Anastomosis observed in the slide cultures of Fusarium xylarioides and Fusarium solani.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
