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Pucciniales are fungal pathogens of plants that cause devastating rust diseases in agriculture. Chemically-synthesized pesticides help farmers to control rust epidemics, but governing bodies aim at limiting their use over the next decade. Defense peptides with antimicrobial activities may help to innovate a next generation of phytosanitary products for sustainable crop protection. This review comprehensively inventories the proteins or peptides exhibiting a biochemically-demonstrated antifungal activity toward Pucciniales (i.e., anti-rust proteins or peptides; hereafter ‘ARPs’), and also analyses the bioassays used to characterize them. In total, the review scrutinizes sixteen publications, which collectively report 35 ARPs. These studies used either in vitro or in planta bioassays, or a combination of both, to characterize ARPs; mostly by evaluating their ability to inhibit the spore germination process in vitro or to inhibit fungal growth and rust disease development in planta. Also, the manuscript shows that almost no mode of action against rust fungi was elucidated, although some might be inferred from studies performed on other fungi. This short review may serve as a knowledge and methodological basis to inform future studies addressing ARPs.

Although lupin anthracnose caused by Colletotrichum lupini is a significant threat for spring and winter lupin crops, it has been poorly studied so far. This study aimed at characterizing the (i) phylogenetic, (ii) morphological, and (iii) physiological diversity of collected isolates from anthracnose-affected lupins. The genetic identification of representative isolates (n = 71) revealed that they were all C. lupini species, further confirming that lupin anthracnose is caused by this species. However, multilocus sequencing on these isolates and 16 additional reference strains of C. lupini revealed a separation into two distinct genetic groups, both of them characterized by a very low genetic diversity. The diversity of morphological characteristics of a selected subset of C. lupini isolates was further evaluated. To the best of our knowledge, microsclerotia production observed for some isolates has never been reported so far within the Colletotrichum acutatum species complex. Finally, the modeling of growth responses of a subset of C. lupini strains revealed the capacity of some strains to grow in vitro at 5°C. This ability was also evidenced in planta, because C. lupini DNA was detectable in plants from 14 days postinoculation at 5°C onward, whereas symptoms began to appear a week later, although at a very low level. Since lupin crops are planted during winter or early spring, growth studies in vitro and in planta demonstrated the capability of the species to grow at temperatures ranging from 5 to 30°C, with an optimum close to 25°C. In this study, C. lupini-specific primers were also designed for real-time quantitative PCR on fungal DNA and allowed the detection of C. lupini in asymptomatic field samples. These results open perspectives to detect earlier and limit the development of this pathogen in lupin crops.

The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.

Lupin anthracnose is a major threat caused by the filamentous fungus Colletotrichum lupini. The genus Colletotrichum includes a wide number of phytopathogen species distributed worldwide. They were classified into 11 species complexes, with C. lupini belonging to the C. acutatum species complex. To date, studies performed on C. lupini, aimed at better understanding its phylogenetic classification and infectious lifecycle.But additional studies seemed necessary to increase our knowledge on (i) intraspecific diversity within the C. lupini species and (ii) the molecular determinants of pathogenicity. To address the first objective of thisPhD thesis, a study was performed on strains from official collections and from isolates recently collected in Brittany and Pays de la Loire regions. A poor intraspecific phylogenetic diversity was found compared to the phenotypic diversity, notably characterized by contrasted aggressiveness between strains. The second objective was evaluated by determining fungal gene expression and protein synthesis during lupin infection by C. lupini using, respectively, a RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach with a nLC Q-exactive Orbitrap. Taken together, our results highlighted that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes of unknwown or poorly-described functions were found to be specifically associated to the early or late stages of infection, suggesting that they may be of importance during infection.Functional validation will be required to confirm their role in promoting C. lupini pathogenicity.; L’anthracnose du lupin est une maladie majeure provoquée par le champignon filamenteux Colletotrichum lupini. Le genre Colletotrichum rassemble un grand nombre d’espèces phytopathogènes caractérisées par une distribution mondiale et classées parmi 11 complexes d’espèces,C. lupini appartenant au complexe d’espèces C. acutatum. Jusqu’à aujourd’hui, les travaux de recherche ont permis de mieux comprendre son positionnement phylogénétique ainsi que son cycle infectieux, mais des travaux complémentaires restaient nécessaires afin de mieux connaître (i) la diversité des souches de C. lupini ainsi que (ii) les déterminants moléculaires de sa pathogénicité. Pour répondre au premier objectif de cette thèse, l’étude d’un panel de souches issues de collections mais aussi d’isolement réalisés récemment en région Bretagne et Pays de la Loire a montré que la diversité phylogénétique intra-spécifique est faible par rapport à la diversité phénotypique, notamment marquée par un pouvoir pathogène contrasté selon les souches. L’étude des transcrits et des protéines synthétisées par le champignon, au cours d’une cinétique d’infection sur lupin, a permis de répondre au deuxième objectif via une approche de séquençage RNAseq et de spectrométrie de masse nLC Qexactive Orbitrap MS/MS. Ces études ont mis en évidence que les cinétiques d’apparition des symptômes, d’expression de gènes et de synthèse des protéines semblent caractéristiques des agents hémibiotrophes. Des gènes peu décrits et d’autres de fonction inconnue, spécifiquement associés à des stades précoces ou tardifs de l’infection, ont également été mis en évidence. La détermination de leur rôle dans l’établissement de la pathogénicité de C. lupini nécessitera une validation fonctionnelle.

Lupin anthracnose is a major threat caused by the filamentous fungus Colletotrichum lupini. The genus Colletotrichum includes a wide number of phytopathogen species distributed worldwide. They were classified into 11 species complexes, with C. lupini belonging to the C. acutatum species complex. To date, studies performed on C. lupini, aimed at better understanding its phylogenetic classification and infectious lifecycle.But additional studies seemed necessary to increase our knowledge on (i) intraspecific diversity within the C. lupini species and (ii) the molecular determinants of pathogenicity. To address the first objective of thisPhD thesis, a study was performed on strains from official collections and from isolates recently collected in Brittany and Pays de la Loire regions. A poor intraspecific phylogenetic diversity was found compared to the phenotypic diversity, notably characterized by contrasted aggressiveness between strains. The second objective was evaluated by determining fungal gene expression and protein synthesis during lupin infection by C. lupini using, respectively, a RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach with a nLC Q-exactive Orbitrap. Taken together, our results highlighted that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes of unknwown or poorly-described functions were found to be specifically associated to the early or late stages of infection, suggesting that they may be of importance during infection.Functional validation will be required to confirm their role in promoting C. lupini pathogenicity., L’anthracnose du lupin est une maladie majeure provoquée par le champignon filamenteux Colletotrichum lupini. Le genre Colletotrichum rassemble un grand nombre d’espèces phytopathogènes caractérisées par une distribution mondiale et classées parmi 11 complexes d’espèces,C. lupini appartenant au complexe d’espèces C. acutatum. Jusqu’à aujourd’hui, les travaux de recherche ont permis de mieux comprendre son positionnement phylogénétique ainsi que son cycle infectieux, mais des travaux complémentaires restaient nécessaires afin de mieux connaître (i) la diversité des souches de C. lupini ainsi que (ii) les déterminants moléculaires de sa pathogénicité. Pour répondre au premier objectif de cette thèse, l’étude d’un panel de souches issues de collections mais aussi d’isolement réalisés récemment en région Bretagne et Pays de la Loire a montré que la diversité phylogénétique intra-spécifique est faible par rapport à la diversité phénotypique, notamment marquée par un pouvoir pathogène contrasté selon les souches. L’étude des transcrits et des protéines synthétisées par le champignon, au cours d’une cinétique d’infection sur lupin, a permis de répondre au deuxième objectif via une approche de séquençage RNAseq et de spectrométrie de masse nLC Qexactive Orbitrap MS/MS. Ces études ont mis en évidence que les cinétiques d’apparition des symptômes, d’expression de gènes et de synthèse des protéines semblent caractéristiques des agents hémibiotrophes. Des gènes peu décrits et d’autres de fonction inconnue, spécifiquement associés à des stades précoces ou tardifs de l’infection, ont également été mis en évidence. La détermination de leur rôle dans l’établissement de la pathogénicité de C. lupini nécessitera une validation fonctionnelle.

International audience; Colletotrichum lupini is a species within the Colletotrichum acutatum species complex (CAsc) causing anthracnose, a serious disease affecting lupin crops worldwide. In addition to its economic impact, C. lupini is a useful model to study host speciation process since it is known to infect only lupins. To date, this pathogenic fungus has been poorly described. In this context, our work aims at getting better insight into the genome, the physiology and the infectious process of the fungus. First, based on the recent annotation of two C. lupini reference genomes, we identified specific regions of C. lupini that are absent in the genome of other species from the CAsc. In order to better understand the infectious process of the fungi, transcriptomic and proteomic analyses were performed on C. lupini infected lupin seeds during transition from the biotrophic to the necrotrophic stage. The results allowed us to identify specific pathogenicity gene markers of both stages. The expression of these markers has been further evaluated using a set of C. lupini strains. These results together with the description of the physiology of C. lupini during the first steps of the infection will significantly increase knowledge in host speciation process and ultimately contribute (i) to the early detection of the fungus in the field and (ii) to the development of new resistant lupin varieties.

International audience

Lupin anthracnose is a major threat caused by the filamentous fungus Colletotrichum lupini. The genus Colletotrichum includes a wide number of phytopathogen species distributed worldwide. They were classified into 14 species complexes, with C. lupini belonging to the C. acutatum species complex. To date, studies performed on C. lupini, aimed at better understanding its phylogenetic classification and infectious lifecycle. But additional studies seemed necessary to increase our knowledge on (i) intraspecific diversity within the C. lupini species and (ii) the molecular determinants of pathogenicity. To address the first objective, a study was performed on strains from official collections and from isolates recently collected in the west of France. A low intra-specific phylogenetic diversity was found compared to the phenotypic diversity, notably characterized by contrasted aggressiveness between strains. The second objective was evaluated by determining fungal gene expression and protein synthesis during lupin infection by C. lupini using, respectively, a RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach with a nLC Q-exactive Orbitrap. Taken together, our results highlighted that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, few genes of unknwown or poorly-described functions were found to be specifically associated to the early or late stages of infection, suggesting that they may be of importance for pathogenicity. Functional validation will be discussed to confirm their role in promoting C. lupini pathogenicity.