This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Peas (Pisum sativum) are an important crop due to their high protein content (which is of particular interest due to the rise of meat alternatives) and nitrogen fixing capabilities. Root pathogens infecting pea tend to be present together, in what is known as the root rot complex. Members include Fusarium solani and F. oxysporum, which cause rotting and wilting, respectively. Aphanomyces euteiches, an oomycete is also part of this complex.
I spent 8 weeks at the Arora lab investigating which genetic factors make a landrace of pea partially resistant to the soil pathogen F. solani. This is a fungus which causes black lesions and rotting of roots, weakening plants, and reducing yields by up to 15–60%. Resistance to this pathogen can be characterised as partial when taproots are not dominated by dark lesions and lateral roots are less stunted. Fluorescent staining utilising wheat germ agglutinin revealed that the fungus is unable to penetrate the plant vasculature in partially resistant plants, however, the mechanism behind is not known.
Traditional control methods such as fungicides are limited and although crop rotations do prevent outbreaks, spores can overwinter in soil and can survive for years. Using germplasm collection to find lines conferring partial resistance and understanding the genetic mechanisms behind this is a more promising option, especially with the rise of gene editing.
Conducting a screen of the pea lines phylogenetically close to this partially resistant landrace also showed partially resistant phenotypes. We grew these accessions in three different substrates: agar, soil, and vermiculite. We had four replicates of each accession, with each sterilised seed inoculated with 1ml of 104 F. solani spore suspension. We evaluated the plants after growing them for a set period depending on the growth medium. This involved measuring dark lesion length and visually scoring the plants for disease severity based on a scoring scale from one to five, five being the most susceptible. We also took photos of each plant, and I wrote a python script to read a QR code in each image and rename each file to corresponding accession and replicate numbers.
There is a continuum of susceptible phenotypes observed in different accessions indicating involvement of multiple genetic loci contributing to phenotypes. To elucidate which part of the genome makes the landrace of interest more resistant to F. solani than other accessions, we evaluated 150 Recombinant Inbred Lines (RILs) of a partially resistant landrace and a susceptible cultivar, using the same scoring system used in the clade screen. They were grown in vermiculite and agar, with sterilising, germinating, and planting occurring in two batches, with at least six replicates per RIL.
The disease severity scores indicated varying resistance to F. solani across the 150 RILs. We used these scores and genetic markers with the rQTL package to identify genotyped markers which are associated with partial resistance to F. solani. Excitingly, we identified a peak on Linkage Group 2 which has a significant lod score and are now annotating the genes within the mapped interval. Further work will be elucidating their function and role in providing resistance.
The second half of my project was to use Agrobacterium tumefaciens (strain GV3101) mediated transformation to insert the pCAMBgfp plasmid vector into the root rot pathogens F. solani and F. oxysporum. The plasmid contained a hygromycin resistance gene so we could select successfully transformed fungi using the antibiotic. The presence of GFP meant that we could use fluorescence microscopy in the future to monitor host colonisation more effectively. This was successful for F. oxysporum, however, even after trying different protocols with some alterations, it did not work for F. solani. We are trying to insert the plasmid via protoplasting techniques. Thus far, we have successfully digested the cell wall to make protoplasts and further work will be conducted by the lab.
Overall, I enjoyed the hands-on approach of working in a lab and this internship has given me the skills to pursue a master’s or PhD, potentially in plant science. These include presenting, protocol adaptation and time management skills. I like solving challenges and want to do something useful to improve people’s lives and the planet.
I am very grateful to my supervisor Dr Sanu Arora for hosting me in her lab at the John Innes Centre and give thanks to Nicolas Trenk for his day-to-day training and supervision.
Eve Hallett
University of Bath
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
