The rust fungi (Uredinales, Basidiomycota) are an expansive and diverse group of fungal species, consisting of approximately 7000 different species in over 160 different genera. Fungi of the genus Puccinia represent a large proportion of these rust fungi, many species of which are well known for their role in causing massive yield and subsequent economic losses in agricultural crops worldwide. Puccinia boroniae is one such rust fungus and is a significant pathogen of several species of Boronia (Rutaceae), a native Australian wildflower grown commercially in Western Australia as a cutflower. Complete control of the rust pathogen is rarely achieved using chemical fungicides. Improving the level of disease control is vital for the long-term sustainability and future growth of the Boronia industry, and requires an understanding of the pathogen. The objectives of this thesis were to investigate aspects of the epidemiology, the biology and the diversity of P. boroniae in Western Australia, providing a broad understanding of the pathogen, which in turn could be employed to improve disease control. The lifecycle of P. boroniae was conclusively shown to be microcyclic by artificial inoculation of Boronia heterophylla with basidiospores released from germinating teliospores suspended over the host plant. Telia developed on the leaves within 21 days, with no intermediate rust spore stages (pycnial, uredial or aecial) observed. Rarely, low numbers of pycnia of P. boroniae were observed on field specimens collected from leaves of B. megastigma cultivated at one commercial floriculture plantation. This was the first record of pycnia of P. boroniae; however, as pycnia were not observed on other host species or plantations, or formed during controlled inoculation trials, their functional role in the lifecycle is currently unresolved. Telia were subepidermal, erumpent and pulvinate, amphigenous on leaves, stems and parts of developing flower buds, and generally persistent year round. Intracellular hyphae resembling monokaryotic haustoria (M-haustoria) were observed in leaf mesophyll cells beneath and adjacent of telia. Occasionally Sphaerellopsis filum (teleomorph Eudarluca caricis), a known mycoparasite of rust fungi, was observed on the telia. Under favourable conditions, teliospores germinated immediately without a period of dormancy, with fully mature basidiospores formed within 3–4 h after telia were exposed to moisture. Basidial development in P. boroniae was unusual, in that only one basidiospore was formed from each germinating teliospore cell. Immature teliospores were initially binucleate undergoing karyogamy to form a single large (presumably diploid) nucleus that migrated into the developing metabasidium. Both binucleate and tetranucleate metabasidia were observed, with mature uninucleate, binucleate and tetranucleate basidiospores present. At this stage, more research is required to understand the complete nuclear behaviour during teliospore germination. The morphology of the pycnial stage was similar to other Puccinia species, being ampulliform, subepidermal, amphigenous and arranged in small clusters on leaves of B. megastigma. However, the spine-like periphyses protruded through stomata as apposed to penetrating the leaf epidermis. Environmental conditions favouring the formation and dispersal of basidiospores were assessed in vitro and under field conditions with a spore catcher. Under field conditions, basidiospores were captured from February–August 2004, with peak numbers and daily incidence occurring during autumn (April/May) when the average temperature range was 9.1–22.6 °C. Daily basidiospore numbers were positively correlated with minimum daily temperature and total daily rainfall. A distinct diurnal periodicity of release was observed, with numbers peaking on average between 02:00 and 05:00 hrs. The hourly release of basidiospores was positively correlated with relative humidity and negatively correlated with temperature and evaporation. This data was in agreement with the in vitro experimentation, which showed that basidiospore formation occurred between 10–25 ± 1 °C (apparent optimal temperature of 15–20 ± 1 °C) with telia incubated in continuous darkness promoting a greater number of basidiospores. The level of genetic variation of P. boroniae in Western Australia was assessed by PCR-RFLP of the nuclear ribosomal intergenic spacer 2 (IGS2) region. Two RFLP profiles were observed, separating three specimens (Group 1) from the remaining population (Group 2). Sequence analysis indicated that point mutations at endonuclease recognition sites were responsible for the changes in RFLP profile. Group 2 specimens had been collected from the same host species (B. megastigma) and plantation, and it is suggested that the variant specimens may constitute a subspecies of P. boroniae, isolated by geographic location and possibly host (cultivar) specificity. Further analysis, primarily pathogenicity trials, is needed to confirm this. This study has improved our knowledge regarding the rust fungus P. boroniae and has laid strong foundations for future research into several aspects of the biology, epidemiology and population variation. The implications of the key findings of this research, with an emphasis on the management of P. boroniae in commercial situations, are discussed.

La maladie de la rouille foliaire du peuplier, causée par le basidiomycète Melampsora larici-populina (Mlp) cause des dégâts importants dans les peupleraies européennes. Le séquençage du génome de la souche 98AG31 de Mlp a ouvert de nouvelles perspectives pour l'identification de déterminants géniques impliqués dans le processus infectieux du champignon et notamment ceux codant des effecteurs fongiques capables de manipuler la structure et le fonctionnement de la cellule hôte pour assurer le succès de l'infection. L'analyse du transcriptome du champignon au cours des différentes phases du processus infectieux, basée sur l'utilisation de puces à oligonucléotides NimbleGen ou le séquençage massifs d'ESTs, a permis d?identifier des gènes marqueurs de la germination, de la phase de croissance biotrophe et de la sporulation du champignon. Nous avons notamment pu montrer l'induction importante de nombreux gènes codant des petites protéines sécrétées (SSPs) au cours de la phase biotrophe à 96 hpi heures post-inoculation (hpi) ainsi qu'au sein du parenchyme lacuneux à 168 hpi par microdissection à capture laser. L?analyse fine du sécrétome de Mlp, basée sur l'annotation, l'évolution et l'expression des gènes codant des SSPs a permis de mettre à jour des effecteurs candidats. Certains, spécifiquement exprimés in planta ou présentant des homologies de séquence avec des effecteurs de rouilles ont été localisés au niveau de l'haustorium. De manière intéressante, d'autre gènes candidats appartenant à des familles multigéniques sous pression de sélection positive, sont riches en cystéines, spécifiquement exprimés in planta et possèdent un motif de translocation potentiellement impliqué dans l'export de l'effecteur dans la cellule hôte. Ce travail d'analyse fine des effecteurs potentiels d'un agent de rouille à l'échelle génomique va contribuer à l'amélioration des connaissances sur la biologie de ces champignons biotrophes et contribuera à faciliter la recherche de nouvelles méthodes de lutte contre la maladie / The leaf rust disease caused by Melampsora larici-populina (Mlp) is the main disease affecting poplar plantations in Europe with severe economic losses. The recent sequencing of the genome of Mlp (strain 98AG31) opens new perspectives to identify key genes involved in the fungal infection process and particularly those encoding fungal effectors that could manipulate host cell structure and function to facilitate host colonization. Analysis of the rust transcriptome during time course infection of poplar leaves, based on NimbleGen oligoarrays and massive EST sequencing led to the identification of genes related to fungal germination, biotrophy and sporulation. A consistant induction of genes encoding small-secreted proteins (SSPs) was observed during the biotrophic growth at 96 hours post-inoculation (hpi) but also at 168 hpi in the palisade mesophyll using laser capture microdissection. Mlp Secretome analysis, based on annotation, evolution and expression of genes encoding SSPs helped in identifying candidate poplar rust effectors. Some, specifically expressed in planta or showing homologies with known rust effectors were localized around the haustorium. Interestingly, other candidate genes, belonging to multigenic families under diversifying selection are cystein-rich, specifically expressed in planta and harbour a translocation signal potentially involved in effector export inside host cell. This genome-wide analysis of putative fungal effectors will contribute to the general knowledge of rust biology and will help to set new approaches to prevent and control the disease

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Plants and microorganisms interact with each other constantly, with some interactions being mutually beneficial and others being detrimental to the plants. The features of the organisms involved in such interactions will determine the characteristics of individual pathosystems. Plants respond readily to pathogen attacks, regardless of the pathosystem; furthermore, variation in the resistance to pathogens within species is common and well documented in many plant species. The variability in pathogen resistance is at the core of genetic improvement programs for disease resistance. True resistance to pathogens in plants is a genetically determined and complex trait that can involve both constitutive and induced mechanisms at different levels of organization. The complexity of this phenomenon makes the study of compatible plant - pathogen interactions challenging, and typically, disease resistance studies focus on specific aspects of a pathosystem, such as field resistance, anatomical or physiological features of resistant plants, or molecular mechanisms of resistance. The Thuja sp. - Didymascella thujina (E.J. Durand) Maire interaction is an important pathosystem in western North America, which has been studied for more than five decades. Western redcedar (Thuja plicata Donn ex D. Don) is very susceptible to cedar leaf blight (D. thujina), a biotroph that affects the tree at all stages, although seedlings are the most sensitive to the pathogen. The characteristics of the Thuja sp. - D. thujina interaction, the wealth of information on the pathosystem and the excellent Thuja sp. genetic resources available from the British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development make this interaction an ideal system to advance the study of disease resistance mechanisms in conifers. This Doctoral project presents a comprehensive investigation of the constitutive and induced resistance mechanisms against D. thujina in T. plicata, Thuja standishii (Gord.) Carrière and a Thuja standishii x plicata hybrid at the phenotypic and gene expression levels, undertaken with the objective of exploring the resistance mechanisms against the biotroph in these conifers. The project also aimed to establish base knowledge for the future development of markers for marker-assisted breeding of T. plicata. The investigations included a combination of histological, chemical and next generation sequencing (NGS) methodologies. NGS data were analyzed, in addition to the traditional clustering analyses, with cutting edge machine learning methods, including grade of membership analysis, dynamic topic modelling and stability selection analysis. The studies were progressively more controlled to narrow the focus on the resistance mechanisms to D. thujina in Thuja sp. Histological characteristics related to D. thujina resistance in Thuja sp. were studied first, along with the relationship between climate of origin and disease resistance. The virulence of D. thujina was also documented early in this project. Chemical and gene expression constitutive and induced responses to D. thujina infection in T. plicata seedlings were studied next. T. plicata clonal lines were then comprehensively studied to shed light on the mechanisms behind known physiologically determined resistance. A holistic investigation of the resistance mechanisms to D. thujina in T. standishii, T. plicata and a T. standishii x plicata hybrid explored the possibility of a gene-for-gene resistance model. Thirty-five T. plicata families were screened during the four field seasons carried out between 2012 and 2015, totalling more than 1,400 seedlings scored for D. thujina severity. Thirteen of those families were used in the five studies performed during the program, along with two T. plicata seedling lines self-pollinated for five generations and three T. plicata clonal lines. One T. standishii clonal line, and one T. standishii x plicata clone were also investigated during the program. A total of 16 histological and anatomical characteristics were studied in more than 750 samples, and more than 270 foliar samples were analyzed for 60 chemical and nutritional compounds. Almost one million transcriptomic sequences in four individually assembled reference transcriptomes were examined during the program. The results of the project support the variability in the resistance to D. thujina in T. plicata, as well as the higher resistance to the pathogen in plants originating from cooler and wetter environments. The data collected also depicted the existence of age-related resistance in T. plicata, and confirmed the full resistance to the disease in T. standishii. Western redcedar plants resistant and susceptible to D. thujina showed constitutive differences at the phenotypic and gene expression levels. Resistant T. plicata seedlings had thicker cuticles, constitutively higher concentrations of sabinene, alpha-thujene, and higher levels of expression of NBS-LRR disease resistance proteins. Resistant clones of T. plicata and T. standishii had higher expression levels of bark storage proteins and of dirigent proteins. Plants from all ages, species and resistance classes studied that were infected with D. thujina showed the accumulation of aluminum in the foliage, and increased levels of sequences involved in cell wall reinforcement. Additional responses to D. thujina infection in T. plicata seedlings included the downregulation of some secondary metabolic pathways, whereas pathogenesis-related proteins were upregulated in clonal lines of T. plicata. The comprehensive approach used here to study the Thuja sp. - D. thujina pathosystem could be applied to other compatible plant-pathogen interactions. / Graduate / 2020-08-31