Valerie van Ingen Buijs

Citrus Black Spot, caused by the fungus Phyllosticta citricarpa, causes significant economic losses to global Citrus production. This thesis presents a comprehensive investigation into species of the fungal genus Phyllosticta that colonize Citrus. Through four experimental chapters, it addresses carbon utilization, genomic distinctions related to lifestyle variations, CRISPR/Cas9 applications, and taxonomy.

After a general introduction in Chapter 1, Chapter 2 examines carbon utilization profiles across Phyllosticta species, revealing high similarity regardless of pathogenic or endophytic lifestyles. A notable finding is the selective inhibition of pathogenic species by sugar beet pulp (SBP) at low concentrations (0.25%), even when mixed with other substrates like wheat bran. This effect, absent in non-pathogens, suggests SBP’s potential as a sustainable biocontrol agent. However, the specific inhibitory compound responsible for this effect remains unidentified. In addition, we assessed the CAZyme repertoires of species with different lifestyles but found only little variation.

Chapter 3 explores genomic differences between pathogens and endophytes, identifying minor variations in gene numbers, secreted proteins, and effectors. We found several genomic differences between species of different lifestyles, where unannotated pathogen-specific orthogroups present compelling targets for future virulence studies. We classified Phyllosticta citrichinaensis as having an intermediate lifestyle as it shares genomic and phenotypic attributes with both pathogens and endophytes. We also used CATAstrophy to elucidate CAZyme profiles across 116 Dothideomycetes genomes, and found no clear correlation between CAZyme repertoire and lifestyle reported in literature.

Chapter 4 develops a proof-of-principle CRISPR/Cas9 protocol for Phyllosticta via protoplasting and PEG-mediated transformation. We knocked out the pH-modulation gene palH, which has been well described in several fungi and reported to play a role in pathogenicity. We observed a decreased growth rate in ΔpalH knockout mutants, but found no difference in pH modulation abilities. This tool provides the foundation to effectively study pathogenicity genes in Phyllosticta, though refinements could improve efficiency.

Chapter 5 resolves the P. citricarpa and P. paracitricarpa taxonomic debate. Through phylogenomic analyses using 3,000 single-copy ortholog genes and whole-genome comparisons, we established that the differences between P. citricarpa and P. paracitricarpa constitute infraspecific variation within P. citricarpa. We further assessed variation in mitochondrial genome assemblies across multiple Phyllosticta species and identified only minimal differences between the assemblies of P. citricarpa and P. paracitricarpa. Together with the fact that morphological features overlap, this data indicates that these species should be treated as synonyms.

The discussion (Chapter 6) reviews the previous chapters, notes lifestyle classification challenges, and debates DNA-only types. Overall, this thesis advances our understanding of Phyllosticta by uncovering genomic distinctions between lifestyles, presenting CRISPR tools, and resolving the P. citricarpa/P. paracitricarpa taxonomic debate, paving the way for refined disease strategies.