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Submission note: This thesis is submitted in total fulfilment of the requirement of the degree of Doctor of Philosophy to the School of Life Sciences, Department of Animal, Plant and Soil Sciences, La Trobe University, Bundoora.

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.

International audience; Objectives: Species from the Scedosporium apiospermum complex rank the second among the filamentous fungi colonizing the respiratory tract of cystic fibrosis (CF) patients. Nevertheless, the pathogenesis of Scedosporium infections remains poorly understood. Notably, the mechanisms developed by these molds to establish within the bronchial tree are still unknown. Among the species from the S. apiospermum complex, four have been identified as capable to chronically colonize the CF respiratory tract (S. boydii, S. apiospermum, S. aurantiacum ,and S. minutisporum) whereas S. dehoogii , which is common in the environment is unable to establish chronically in the lungs of CF patients. Since several years, we aimed at identifying the key molecular mechanisms underlying the ability of Scedosporium species to establish within the respiratory tract of CF patients and to cause chronic colonization of the airways. Methods: To identify candidate genes potentially involved in the adaptation and pathogenicity of Scedosporium species, we recently performed two complementary omics approaches including transcriptomics and comparative genomics. First, the transcriptome of the S. apiospermum reference strain (IHEM14462) was compared in different growth conditions mimicking the various physio-chemical constraints encountered in the respiratory tract of CF patients (hypercapnia, hypoxia, acid pH, low osmolarity, increased lactate concentration). For the comparative genomic strategy, we sequenced 5 isolates from each of the five species S. boydii, S. apiospermum, S. aurantiacum, S. minutisporum, and S. dehoogii . To identify candidate proteins that may enable Scedosporium species to establish within the respiratory tract of CF patients, we first searched clusters of orthologous proteins with no copy found in S. dehoogi isolates but conserved in all other isolates. In a second time we also detected genes present in all pathogenic species, as well as in S. dehoogii but exhibiting in this last species point mutations potentially leading to a loss of function. Results: As revealed by the transcriptomic approach, while hypercapnia, increased lactate or low osmolarity modified the expression of a limited number of genes, the transcriptome of S. apiospermum was greatly influenced by hypoxia, growth at acid pH or in a cystic fibrosis synthetic medium. In addition, the whole-genome sequencing project of several isolates from the five Scedosporium species allowed to obtain a total of 2.4-8.9 mega-reads per isolate which permitted good quality draft genome assemblies. Finally, comparative genomics allowed to identify a series of 134 genes lost or with a loss of function in S. dehoogii but still functional in all other species. Conclusion: The transcriptomic changes observed during S. apiospermum exposure to the physico-chemical conditions en-countered in the respiratory tract of cystic fibrosis patients as well as the set of candidate genes revealed by the comparative genomic strategy would help deciphering in a near future the crucial mechanisms allowing these opportunistic pathogens to specifically adapt to the CF bronchial mucus.

The opportunistic fungal pathogen Scedosporium boydii is the most common Scedosporium species in French patients with cystic fibrosis. Here we present the first genome report for S. boydii, providing a resource which may enable the elucidation of the pathogenic mechanisms in this species.

Pipeline of programmes used to identify the in silico-predicted secretome of each isolate. (DOCX 147 kb)

The predicted proteins belonging to OrthoMCL clusters present in the secretomes of five Venturia isolates. The coloured horizontal bars represent proportions of predicted proteins (scale at bottom of figure) that have been annotated according to the key at the top of the figure, that are present in the secretomes indicated by grey boxes to the left of the figure (white boxes indicate lack of similar proteins). S: indicates singleton proteins. Numbers in the boxes are numbers of proteins. The order of the categories in the key is the same as that in the horizontal bars. For example: the core secretome is represented by the top-most horizontal bar; there are similar proteins present in each of the secretomes (5 grey boxes), of which 15% (77 proteins) are classified as SSPs between 200 and 500 amino acids in length with 2 or more cysteines. (PPTX 56 kb)

Small, secreted protein (SSP) family 07 members. Sequence logo of members of A. family 07 from Vi1 and B. family 07 from all isolates. (DOCX 221 kb)