Characterisation of the melanin biosynthesis gene cluster and its application in species identification

Abstract

Elsinoë necatrix, the causal agent of a severe leaf and shoot disease of Eucalypts, is considered an emerging fungal pathogen. It was first discovered in 2014 in North Sumatra, Indonesia, affecting a large number of cultivated Eucalyptus plantations. The disease symptoms were unlike any other previously noted on these trees, with signature scab-like lesions on the stems and leaves that, for highly susceptible Eucalyptus variants, resulted in distorted shoots with feathered leaves. The disease was subsequently described as Eucalyptus scab and shoot malformation and the causal agent was confirmed to be Elsinoë necatrix, a member of the genus Elsinoë (Elsinoaceae, Myriangiales). The threat of this disease to commercial Eucalyptus plantations highlighted the need for an early detection method that would assist in monitoring the spread of this pathogen, which can ultimately inform disease control measures. Therefore, this study aimed to develop a rapid Loop-mediated isothermal amplification (LAMP) assay for the sensitive and specific detection of Elsinoë necatrix. The first chapter in the dissertation is a literature review highlighting different types of diagnostic technologies that has the potential for in-field detection of fungal plant pathogens. The review discusses both immunological and nucleic acid-based techniques, with particular focus on isothermal amplification technologies. The principles of each detection method and current equipment available for in-field use is discussed, while an overview of Eucalyptus scab and shoot malformation is also provided. Chapter two aimed to identify and characterize all type I polyketide synthase (PKS) genes present in the genomes of seven Elsinoë species. Using a phylogenetics approach, putative functions were assigned to each identified PKS gene. Seven different PKS cluster types were predicted from these species, with interspecific variation present for the most part in the type and number of PKS genes. The exception was the Melanin and Elsinochrome PKS genes which were present in all species. Further annotation of the extended cluster showed high levels of synteny across both clusters, even between diverse Elsinoë species. A unique region was identified in the Elsinoë necatrix melanin cluster that appeared to be suitable as a target for a species-specific diagnostic assay. The final chapter of this dissertation describes the development and validation of a rapid LAMP assay for the detection of Elsinoë necatrix. The LAMP assay targeted a region in the Melanin biosynthesis cluster that was unique to E. necatrix, and proved to be both highly specific and sensitive, capable of detecting this pathogen directly from infected leaf material within 40 minutes. The detection limits of the assay were established, and the specificity was tested against both Eucalypt-associated Elsinoë species and other fungi commonly present on these trees. When combined with a simple DNA extraction method, it was shown that this LAMP assay has the potential to directly detect E. necatrix from infected leaf material. This assay shows great potential for commercial use to rapidly detect E. necatrix in field and nursery conditions, and could prove to be a valuable tool to prevent further spread of this pathogen. The data presented in this dissertation was produced at the Forestry and Agricultural Biotechnology Institute (FABI) and the Department of Biochemistry, Genetics and Microbiology at the University of Pretoria. This research was performed under the supervision of Dr Markus Wilken, Prof Brenda Wingfield, Prof Mike Wingfield and Mr Nam Pham. The dissertation is written as three standalone articles, where the first is a review of the literature, while the remaining two chapters are written as independent research articles. This format necessitates some overlap of information, including references, between the different chapters that could not be avoided. Although a standardized format was used for the first two chapters, Chapter 3 has been edited in the format of the journal Plant Disease where this manuscript is currently under review.