Functional genetics and genomics of the banana black Sigatoka pathogen Pseudocercospora fijiensis



  • Authors : Diaz-Trujillo, C.

  • Document type : Thesis

  • Diploma : PhD thesis

  • Year of publication : 2018

  • Publisher(s) : Wageningen University

  • Place of publication : Wageningen, the Netherlands

  • Pages : 246

  • Language(s) : English

  • Abstract : In this thesis I describe the genome sequence of P. fijiensis, a protocol for its transformation and the application in tool development for functional assays. Together, these should aid the advance of (functional) genetics in this important banana pathogen. My thesis is divided into five chapters. Chapter 1 introduces the crop banana and plantain, presents their importance and describes the major biological threats of the crop with an emphasis on the fungal diseases to eventually focus on black Sigatoka. The biology of its causal agent P. fijiensis and the interaction with banana are briefly described. Furthermore, a critical analysis of the current standing in banana and black Sigatoka research is provided, thereby indicating the major bottlenecks. I considered that resolving the latter would bring banana research to another level. Chapter 2 describes the genome sequencing of P. fijiensis, which resulted in a 74 Mb, genome size, mostly containing repetitive DNA and a remarkable differential GC content, which was also observed in the other Sigatoka complex constituents P. musae and P. eumusae. Characteristics of gene content, gene models, synteny, gene expression, and genome dynamics are compared with the Dothideomycete reference genome of Zymoseptoria tritici, as well as with other Dothideomycetes, and discussed in relation to its life style. An initial functional analysis of PfAvr4 is included, which is indicative for gene-for-gene interaction in the banana -P. fijiensis pathosystem. Finally, the genome information was used to analyze strobilurin fungicide resistance dynamics in natural P. fijiensis populations in various banana plantations in Costa Rica. Chapter 3 focuses on developing a protocol for Agrobacterium-mediated transformation (ATMT) of P. fijiensis. Initially, I developed a protocol for random mutagenesis and generated GFP-and Ds Red-labeled strains. Subsequently, I modified the protocol for targeted mutagenesis and used it for functional analysis of Pfavr4and for developing a Pfku70 knock-out strain. The latter is expected to have an impaired non-homologous end joining pathway, which would increase homologous recombination and thereby enhance the throughput for homologous recombination and hence, developing targeted knock-out strains. Chapter 4 describes the use of the aforementioned transformation protocol to analyze the mechanism of DMI sensitivity in P. fijiensis. Besides abundant mutations in the coding sequence of the target Pfcyp51 gene, we discovered multiple repeated insertions in the Pfcyp51 promoter region that are crucial for reduced sensitivity of P. fijiensis resistance to triazoles. The promoter sequences were detected in various international field strains with reduced sensitivity to propiconazole, difenoconazole and epoxiconazole as well as in field strains from Costa Rican farms, which suggests that these mutants were selected by the frequent fungicide applications in these plantations. ATMT was used to replace the promotor of a sensitive strain by the promotor of a resistant strain to show that both structural variants are required for reduced sensitivity. Finally, Chapter 5 provides a general and critical treatise of the achieved results and brings a wider scope for forthcoming research that is necessary to improve our understanding of the banana -P. fijiensis pathosystem and to develop strategies that contribute to a sustainable banana production.

  • Keywords : MYCOSPHAERELLA FIJIENSIS; ITC CODE; BLACK LEAF STREAK; GENOME SEQUENCE; FUNGICIDE RESISTANCE

  • Open access : Yes

  • Document on publisher's site : open View article on publisher's site

  • Musalit document ID : IN180379


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