Phoma stem canker is a disease of oilseed rape (Brassica napus) caused by closely related plant pathogens Leptosphaeria maculans and L. biglobosa. It is an economically important disease, causing annual yield losses of approximately £770 million worldwide. When colonising oilseed rape, L. maculans and L. biglobosa exist in close proximity on the leaf, competing for resources as they move through the main leaf vein and into the stem. Fungicides are commonly used to decrease severity of phoma stem canker on oilseed rape. However, the efficacy and longevity of active chemicals is under threat from evolution of resistance in pathogen populations and government legalisation. Moreover, it has been suggested that both L. maculans and L. biglobosa differ in their sensitivity to azoles, and important class of fungicides that are used to control the disease through the inhibition of lanosterol 14-α demethylase (erg11, CYP51). This project aims to further understand the role that fungicides have in controlling phoma stem canker by investigating their efficacy against L. maculans and L. biglobosa in crops, in vitro and in planta. In field experiments, established in Cambridgeshire across four cropping seasons, the fungicide mixture penthiopyrad (SDHI) plus picoxystrobin (QoI) was as effective at controlling phoma leaf spotting and phoma stem canker in winter oilseed rape as prothioconazole (DMI), suggesting that both fungicides could be used to reduce phoma stem canker symptoms. The two pathogens differed in their growth rates in vitro, with L. biglobosa growing faster than L. maculans when untreated or treated with lower fungicide concentrations. Fungicide sensitivity assays suggest that L. maculans and L. biglobosa are both sensitive to DMI, SDHI and QoI fungicides and that differences between the species are minor. Prothioconazole and penthiopyrad + picoxystrobin had a similar efficacy on oilseed rape cotyledons colonised with either L. maculans or L. biglobosa. There was no difference between species on prothioconazole treated plants, although there was a difference between L. maculans and L. biglobosa when treated with 20 μg/ml penthiopyrad + picoxystrobin. Heterologous yeast expression of LmCYP51B and LbCYP51B with fungicide sensitivity testing of the yeast transformants suggests that LmCYP51B and LbCYP51B are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. These findings are supported by homology protein modelling, which predicts that LmCYP51B and LbCYP51B are structurally very similar, specifically at the azole-binding site. In conclusion, fungicides are still an effective control method for reducing phoma stem canker symptoms caused by Leptosphaeria species in the UK, and a useful tool to in the sustainable production of oilseed rape.

La lutte contre le Phoma du colza, causé par le champignon Leptosphaeria maculans, repose principalement sur la culture de variétés résistantes dont l'efficacité est peu durable. La reproduction sexuée confère un fort potentiel adaptatif au champignon confronté aux pressions de sélection exercées par les variétés résistantes : elle permet l'acquisition et l'association d'allèles de virulence, alors que les ascospores issues de la reproduction sexuée assurent la transmission de la maladie d'une saison culturale à la suivante. Nous faisons l'hypothèse qu'en début de contournement de résistance, la transmission de la virulence d'une génération à la suivante est limitée d'une part par la faible probabilité de reproduction sexuée entre isolats virulents et d'autre part par une dispersion des ascospores spatialement limitée. Pour tester cette hypothèse nous avons étudié (i) la dispersion des pycnidiospores par la pluie, (ii) la progression systémique du champignon dans la tige de colza et (iii) la structure spatiale des populations de L. maculans pour inférer les distances de dispersion des ascospores. Sous un générateur de gouttes en air calme, les pycnidiospores sont dispersées par les gouttelettes de pluie à moins de 40 cm de la macule source ; des résidus porteurs de pycnides peuvent causer des infections primaires de la maladie. Le succès de la phase systémique détecté par l'expression de nécroses au collet sur des plantules inoculées en conditions contrôlées augmente avec le nombre de points d'infection, mais diminue en présence de la résistance partielle ; la compétition entre individus peut limiter la présence concomitante des types sexuels au collet de la plante. Par conséquent, dans des champs avec des populations de L. maculans à faible densité, la dispersion des pycnidiospores par la pluie peut accroître la taille de la population pathogène et permettre la rencontre d'individus initialement distants, tandis que la progression systémique du champignon peut limiter la rencontre locale d'isolats virulents sexuellement compatibles. Pour 29 populations françaises génotypées à l'aide de marqueurs minisatellites, l'essentiel de la diversité génotypique est détectée à l'échelle d'une population (champ) ; l'absence de différenciation génétique entre populations distantes n'a pas permis de mettre en évidence une limitation des flux de gènes à l'échelle spatiale analysée. Ces résultats suggèrent que la dispersion du Phoma par les ascospores est beaucoup plus élevée que supposée et/ou que les tailles efficaces des populations du champignon dans une parcelle sont très grandes. Ces connaissances épidémiologiques devront être prises en compte pour améliorer les stratégies de déploiement durable des variétés résistantes.

Techn. Hochsch., Diss., 2002--Aachen.

A cultura de sorgo granífero no país é amplamente utilizada na alimentação animal. Através deste estudo, decidimos avaliar a micobiota e a ocorrência de ácido tenuazônico em grãos de sorgo cultivados em duas safras. Além disso, através de abordagem polifásica, caracterizamos cepas de E. sorghinum, bem como avaliamos os aspectos ecofisiológicos desta espécie. E. sorghinum foi o fungo mais prevalente nos de grãos de sorgo. O ácido tenuazônico foi detectado na totalidade das amostras avaliadas, com maiores níveis nos grãos cultivados na safra verão. Foi possível verificar considerável variabilidade genética nas cepas de E. sorghinum, apresentando-se como complexo de espécies filogenéticas. Avaliando os fatores abióticos ocorridos no campo, bem como acessando a ecofisiologia de E. sorghinum, foi possível verificar que condições quentes e úmidas são favoráveis a produção de ácido tenuazônico. Estes resultados alertam para presença de compostos tóxicos em grãos de sorgo cultivados no país, bem como contribuem com informações aplicáveis para o manejo desta cultura. / The culture of sorghum in Brazil is widely used in animal feed. The aim of this study was evaluate the mycobiota and occurrence of tenuazonic acid in sorghum grains cultivated in two sowing periods. In addition, through a polyphasic approach, we characterized strains of Epicoccum sorghinum, as well as evaluated the ecophysiological of this species. E. sorghinum was the most prevalent fungus in sorghum grains. Tenuazonic acid was detected in all the samples, with higher levels in the grains grown in the summer crop. It was possible to verify considerable genetic variability in the strains of E. sorghinum, presenting as a complex of phylogenetic species. By evaluating the abiotic factors occurring in the field, as well as the access to an ecophysiology of E. sorghinum, it was possible to verify that hot and wet conditions are favorable for tenuazonic acid production. These results indicate the presence of toxic compounds in sorghum grains cultivated in Brazil, as well as contribute with information for crop management.

Le dessèchement précoce du tournesol résulte d'une infection de bas de tige par Phoma macdonaldii qui provoque une nécrose encerclante et un rétrécissement du diamètre du collet. Cette attaque entraine une sénescence foliaire brutale occasionnant des pertes dommageables pour la culture. Des contaminations artificielles par des spores ou du mycélium de P. macdonaldii ont permis de reproduire artificiellement des pieds secs et confortent l'implication de ce champignon dans ce syndrome. Dans un contexte où la protection chimique et la résistance variétale sont limitées, la maîtrise de P. macdonaldii par le contrôle cultural pourrait constituer une alternative efficace. Des essais au champ et en serre menés de 2006 à 2009 à l'INRA et au Cetiom ont permis de mettre en évidence l'importance de la conduite de culture du tournesol sur la fréquence et la gravité du dessèchement précoce et de mieux comprendre l'étiologie de ce syndrome. Une alimentation azotée importante, un peuplement dense et à un régime hydrique limitant en post floraison forment trois facteurs particulièrement favorables au développement du syndrome de desséchement précoce attribué à P. macdonaldii. L'alimentation azotée, au travers de la fertilisation, est le principal facteur favorisant la maladie ; le régime hydrique limitant et la densité de peuplement amplifient la maladie. Le faible diamètre de bas de tige (sous de fortes densités) associé à la prolifération du champignon dans le xylème (azote), occasionnerait une réduction de la conductance hydraulique par obstruction des vaisseaux. Amplifiée lorsque la disponibilité en eau du sol est limitante, cet arrêt de la transpiration se traduit par une sénescence anticipée de 40 jours par rapport à la maturité physiologique de peuplement sains. / Sunflower premature ripening results from an infection by Phoma macdonaldii causing lesions girdling the stem base and narrowing the diameter of the collar. This attack induces a sudden leaf senescence damaging for the culture. Artificial inoculations with spores or mycelium of P. macdonaldii aimed to reproduce the disease and confirm the involvement of this fungus in this syndrome. In a context where chemical protection and plant resistance are limited, control of P. macdonaldii by cultural control could be an effective alternative. Field and greenhouse trials conducted from 2006 to 2009 at INRA and Cetiom showed the importance of sunflower crop management on the frequency and severity of early drying, and better understand the etiology of this syndrome. A significant nitrogen supply, a dense population and a limited water regime postflowering are particularly conducive to premature ripening. Nitrogen supply is the main factor favoring the disease; limited water regime and density amplify the disease. The small stem base diameter (under high densities) associated with the proliferation of the fungus in the xylem (nitrogen), may reduce hydraulic conductance by vessel blockage. Amplified when soil water availability is limiting, this stop of the transpiration leads to early senescence 40 days before physiological maturity of healthy plants.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Weeds are one of the major problems in the agricultural products cultivation and the main strategy of control is the use of chemical herbicides. Although is efficient, the herbicides bring direct and indirect consequences that overcome its benefits and this opened the way for bioherbicides. A significant barrier in the bioherbicide production is the development of an economically viable fermentation process. This way the aim of this work was to assess the production of a bioherbicide from the Phoma sp. fungus by submerged fermentation and solid-state fermentation, optimizing the fermentation medium in both cases and evaluating the herbicidal activity from the fermentation extracts in the control of the target plants. The culture medium was optimized and the cell growing was monitoring as response in the submerged fermentation. The optimum composition for the synthetic medium was glucose and peptone at 20 gL-1, yeast extract 7.5 gL-1 and an initial pH of 6.0. For the industrial medium, the ideal composition was 20 gL-1 of sucrose, 8 % CSL and initial pH of 6.0. Under these conditions, the maximum biomass obtained was 22 and 33 gL-1, for the synthetic and industrial medium, respectively. Therefore it was possible to obtain a 50 % higher biomass production in the industrial medium, reducing the cost of this fermentation medium. In the solid fermentation the composition of the culture medium was evaluated for obtaining the maximum herbicide action on target plants (cucumber). Bioassays were realized and the plant height, fresh weight, dry weight and phytotocixity mainly were the responses evaluated. The results obtained in the bioassays demonstrate that the bioherbicide presented activity towards the target plant and the intensity of the effect was influenced by the formulation of the fermentation medium. The optimized condition for bioherbicide production was moisture at 70 %, soybean bran of 30 wt% and CSL of 20 wt%, being possible to obtain a moderate injury, usually with recovery. The bioherbicide produced showed a mode of action based on the inhibition of carotenoids biosynthesis. / As plantas daninhas são um dos principais problemas no cultivo de produtos agrícolas e o principal método de controle é o uso de herbicidas químicos. Embora eficiente, os herbicidas trazem consequências diretas e indiretas que superam os benefícios em muitos casos, abrindo assim caminho para o desenvolvimento de bioherbicidas. Uma barreira significativa na produção de muitos bioherbicidas é o desenvolvimento de um processo economicamente viável, logo, os objetivos deste trabalho foram produzir um bioherbicida a partir do fungo Phoma sp. por fermentação submersa e fermentação em estado sólido, otimizar o meio de fermentação em ambos processos e avaliar a atividade herbicida dos extratos das fermentações no controle de plantas-teste. Na fermentação submersa foi otimizado o meio de cultivo e monitorado o crescimento celular como variável resposta. A composição otimizada do meio sintético foi 20 gL-1 de glicose e peptona, 7,5 gL-1 de extrato de levedura e pH inicial de 6.0. Para o meio industrial, a composição otimizada foi 20 gL-1 de sacarose, 8 % de água de maceração de milho (AMM) e pH inicial de 6.0. Nessas condições a máxima biomassa obtida foi de 22 e 33 gL-1, para o meio sintético e industrial, respectivamente, com uma produção de biomassa 50 % maior para o meio industrial e ainda possibilitou a redução do custo deste meio de fermentação. Na fermentação sólida foi otimizada a composição do meio de cultivo para obter a máxima ação do bioherbicida em plantas de pepino. Bioensaios foram realizados e o teste de Tukey mostrou que a altura da planta, massa verde da parte aérea, massa seca da parte aérea e principalmente os sinais de fitotoxidade foram significativos, entretanto, o comprimento do sistema radicular, a massa verde do sistema radicular, massa seca do sistema radicular e número de flores não apresentam resultados significativos. Os resultados obtidos nos bioensaios demonstraram que o bioherbicida apresenta atividade em plantas de pepino e a intensidade do efeito foi influenciado pela formulação do meio de fermentação. A condição otimizada para produção do bioherbicida foi 70 % de umidade, 30 % (p/v) de farelo de soja e 20 % (p/v) de AMM, nessas condições o nível de injúria obtida foi moderada com recuperação. O bioherbicida produzido mostrou um possível modo de ação baseado na inibição de biossíntese de carotenóides.

Le phoma (Phoma macdonaldii / Leptosphaeria linquistii) et le phomopsis (Phomopsis / Diaporthe helianthi) sont deux champignons pathogènes majeurs du tournesol. Dans un contexte d’objectif de réduction de la lutte chimique, une meilleure compréhension des interactions peuplement-agents pathogènes-environnement-conduite de culture est nécessaire. Des essais menés au champ et en serre à Auzeville en 2010 et 2011 ont permis de décomposer les effets de la conduite de culture du tournesol sur l’apparition et le développement des deux maladies. Les variables clés de l’architecture des plantes en peuplement et du microclimat, modifiées par la conduite de culture (choix variétal, densité de semis, fertilisation azoté, irrigation) ont été décrites et mises en relation avec les composantes de la maladie. Ainsi, pour le phomopsis, le rôle déterminant du microclimat résultant du niveau de développement de la couverture foliaire lors des phases initiales d’infection a été confirmé. La taille des feuilles et le diamètre des tiges sont des caractères déterminants de la progression des dégâts sur feuille et tige. Les conséquences des attaques de phomopsis sur feuille en termes de mécanismes de nuisibilité ont été analysées finement et modélisées. Pour le phoma, le rôle du statut nutritionnel azoté dans la progression des attaques sur tige et collet a été confirmé. La fonction régulatrice du phoma vis-à-vis des attaques de phomopsis sur feuille a été quantifiée. Sur le plan de la modélisation, plusieurs approches ont été menées : (i) Evaluation de la qualité prédictive d’Asphodel, modèle épidémiologique prévoyant les émissions de ascospores de Phomopsis ; application à différentes conduites de culture et extension au phoma pour la partie inoculum primaire ; (ii) développement d’un modèle prédictif de l’incidence du phoma prenant en compte les effets climatiques, microclimatiques et agronomiques ; (iii) proposition d’une modélisation conceptuelle des interactions plante-agent(s) pathogène(s)-environnement-conduite de culture. Ce travail pourra, à terme, contribuer à la conception de stratégies de gestion des principales maladies du tournesol, à l’échelle de la parcelle ou du territoire. / Phoma (Phoma macdonaldii / Leptosphaeria linquistii) and phomopsis (Phomopsis / Diaporthe helianthi) are two major sunflower fungal diseases. In a context of reduction of pesticides, a better understanding of the interactions between crop canopy, pathogens, environment and cropping practices is needed. Field and greenhouse experiments set up at Auzeville in 2010 and 2011 allowed to dissect the impact of sunflower crop management on disease appearance and development. Key variables of plant architecture and microclimate, modified by cropping practices (variety choice, sowing density, nitrogen fertilization, irrigation) were described and related to disease components. Thereby, for phomopsis, the key role of microclimate resulting from the level of development of leaf area during the initial stages of infection was confirmed. Leaf length and stem diameter are key characters for necrosis progression on leaf and stem. The impact of phomopsis leaf infection in term of damage mechanism was analysed and modelled. For phoma, the effect of nitrogen nutrition status on necrosis progression on stem and stem base was confirmed. The regulatory function of phoma towards phomopsis leaf infection was quantified. In terms of modelling, several approaches were developed: (i) evaluation of the predictive quality of Asphodel, an epidemiological model predicting Phomopsis ascospore release; application to different cropping practices and extension to phoma for primary inoculum; (ii) development of a predictive model for phoma incidence taking into account the climatic, microclimatic and agronomic effects; (iii) proposal of a conceptual model of the interactions between crop canopy, pathogens, environment and cropping practices. This study could contribute to the design of integrated management strategies for the main sunflower diseases, at field or at territory scale.

Phoma medicaginis is a necrotrophic fungal pathogen, commonly found infecting Medicago truncatula and M. sativa in temperate regions of Australia. To identify, characterize and differentiate eight P. medicaginis isolates from Western Australia, morphological phenotypes and five gene regions (actin, â- tubulin, calmodulin, internal transcribed spacer, translation elongation factor 1-á) were examined. Sequence comparisons showed that specimens isolated from M. truncatula in Western Australia formed a group that was consistently different from, but closely allied to, a P. medicaginis var. medicaginis type specimen. Characterization of three P. medicaginis genotypes showed that all exhibited a narrow host range, causing disease only in M. sativa and M. truncatula among eight commonly cultivated legume species sampled. Infection of 85 M. truncatula accessions showed a continuous distribution in disease phenotypes, with the majority of accessions susceptible. Differences in disease phenotypes suggest that M. truncatula harbours specific and diverse sources of resistance to individual P. medicaginis genotypes. To characterize the genetic basis of resistance to P. medicaginis two F2 populations derived from crosses between the resistant accession SA27063 and the susceptible accessions SA3054 and A17 were phenotyped for disease symptoms. Highly significant recessive QTLs for resistance to P. medicaginis OMT5 were identified in each mapping population. In SA27063 x A17 a QTL named resistance to the necrotroph Phoma medicaginis one (rnpm1) was identified on the short arm of LG4. In SA27063 x SA3054 a QTL (rnpm2) was identified on the long arm of LG8. Further fine mapping of the areas surrounding the QTLs is underway to identify the genes underlying rnpm1 and rnpm2. Examination of the recombination frequencies between genetic markers on the long arms of chromosomes 4 and 8 in the SA27063 x A17 cross revealed an apparent genetic linkage between these chromosomes. Subsequent analysis of other crosses showed this unexpected linkage relationship is characteristic for genetic maps derived from A17. Furthermore F1 individuals derived from crosses involving A17 showed 50% pollen viability or less. This semisterility and the unexpected linkage relationships provide good evidence for a reciprocal translocation in A17 between chromosomes four and eight. The implications of the distinctive chromosomal rearrangement in A17 on genetic mapping, genome sequencing and comparative mapping are discussed. The Mt16kOLI1plus microarray was used to identify transcriptional changes in M. truncatula expressed in defence against P. medicaginis. Three-hundred-and-thirty-four differentially expressed transcripts showed a change of two-fold or more in either the resistant or susceptible interaction, and most of the Phoma-regulated genes could be assigned to functional categories which have been reported to be involved in plant defence responses. RT-qPCR and HPLCUV confirmed involvement of the octadecanoid and phenylpropanoid pathways in response to P. medicaginis infection. Faster induction of lipoxygenase genes and constitutively higher levels of certain phenolic metabolites were observed in resistant plants.

<p>Since the beginning of mycology, studies of species concept in fungi have been mainly based on morphology, partially due to the history of mycology as part of botany. Current advances in biochemical and molecular research have provided mycologists with powerful tools that can be used for delineation of fungal taxa. Recently, an integrated approach to fungal taxonomy involving both morphological and molecular traits has found a wide application for identification of species, especially in anamorphic (mitosporic) fungi.</p><p>In this thesis, I have tried to use this approach for identification of species units in two rather unrelated groups of organisms. One of the case studies concerned <i>Colletotrichum acutatum</i> – a worldwide economically important plant pathogenic anamorphic fungus, which is exhibiting a high level of variation in both morphological and molecular features. This fungus has been intensively studied during the past decades, and several attempts have been made to find reliable markers to separate it from other closely related species of <i>Colletotrichum</i>. The second case studied in this thesis was <i>Mycelia Sterilia</i> – an artificial group of fungi, which are deficient in production of spores of any kind, therefore lacking the main morphological feature used for assigning them to any certain fungal taxon below class level. Due to this peculiarity, <i>Mycelia Sterilia</i> have usually been neglected, and currently there is no working species concept applicable to these fungi. </p><p>In this work, I have tried to clarify the relationships among <i>C. acutatum</i> and several other anamorphic (<i>C. gloeosporioides</i> and <i>C. fructigenum</i>) and teleomorphic (<i>Glomerella acutata</i>, <i>G. cingulata</i> and <i>G. miyabeana</i>) taxa that are closely related to each other. For this purpose, examination of morphological traits was employed in combination with comparison of DNA sequencing data from three loci and subsequent phylogenetic analysis. As a result, re-description of <i>C. acutatum</i> and separation of (at least) two new species was proposed.</p><p>For studies of <i>Mycelia Sterilia</i>, a large collection of sterile strains was screened in search for biologically interesting organisms. One novel pathogen has been found, and two plant growth promoting strains with antifungal properties were selected. Attempt for tentative identification of those fungi was made based on their morphological, physiological and molecular features. Sequencing of several genes and spacers of the ribosomal DNA array revealed that the plant pathogenic strain is closely related to the teleomorphic basidiomycete genus <i>Campanella</i>, and plant growth-promoting isolates were identified as belonging to the anamorphic ascomycete genus <i>Phoma</i>. However, assigning the sterile strains to any existing species was not possible.</p><p>The main conclusion of the thesis is that species in anamorphic fungi should be defined based on a combination of morphological and molecular methods, both equally important, involving as many aspects of fungal biology as is possible at our current state of knowledge. </p>

Reproduction de : Thèse de doctorat : Génétique et biotechnologie végetale : Toulouse, INPT : 2007. / Titre provenant de l'écran-titre. Bibliogr. 200 réf.