Rozprawy doktorskie na temat „Gaeumannomyces graminis var. tritici”

Gaeumannomyces graminis var. triciti (Ggt), the causal agent of take-all in wheat, is difficult to detect accurately and rapidly due to its similarity to fungi in the Gaeumannomyces-Phialophora complex. My objectives are to detect the fungus in infested plants and soil, and to predict effective combinations of bacteria as biological control agents. Detection was based on avenacinase-based primers and polymerase chain reaction (PCR) conditions specified by earlier research. PCR conditions were modified to effect detection. The annealing temperature was lowered from 68 to 62°C for plant and soil extracts, and the concentration of Taq polymerase was doubled for soil extracts. The lowest detection limit for plant extraction was with plant grown on 4 g Ggt-infested millet seed per kg soil, and that for soil extraction was 16 mg of purified Ggt DNA per g soil. Chemical and cultural control methods are currently inadequate. Biological control using bacteria is an alternative. Combinations of several bacterial strains are expected to work better than a single strain, but they may be less effective if bacteria antagonize each other or compete for the same rhizosphere habitat. Antagonism of potential biological control agents were assessed using a Petri plate assay. To estimate possible habitat competition, nutritional profiles of the strains were evaluated using the BIOLOG system. I hypothesized that bacteria not antagonistic to each other and having low coefficients of nutritional similarity would make better biological control combinations. Six bacterial combinations gave better mean root weight in the greenhouse experiment but not in the field.
Master of Science

Bibliography: leaves 207-220. Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.

Quorum sensing using N-acyl homoserine lactone (N-AHL) signal molecules is a cell density-dependent mechanism which allows bacterial cells to co-ordinate their behaviour in concern with their own population size. Previous studies have indicated that the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) secretes a homoserine lactone that promotes gene expression in the potential biocontrol bacterium Pseudomonas corrugata. The aim of this thesis was therefore to investigate the role of N-AHL signalling in interactions between these two organisms. It was demonstrated that Ggt does not produce an N-AHL signal molecule or similar autoinducer capable of causing the reported increased transcription in P. corrugata. P. corrugata however was confirmed to produce an N-AHL signal molecule, and it was decided to elucidate this system to determine its possible role in expression of virulence/pathogenicity genes. aA gene was identified from the P. corrugata genome with significant homology to the LuxI family of AHL synthases. This was confirmed experimentally to produce multiple N-AHL signal molecules and the gene termed 'pcoI'. A putative transcriptional activator was also identified and termed 'pcoR', but was not required for production of N-AHL. Sequence analysis revealed close homology to Sa1A, a newly identified regulatory required for virulence and toxin production in P. syringae. Both genes were demonstrated to be involved in Ggt suppression in vitro. This work has provided a glimpse into the hierarchy and complex nature of signal pathways regulating virulence/pathogenicity in P. corrugata. Understanding the mechanisms through which the biocontrol of plant disease occurs is critical to the eventual improvement and wider use of biocontrol methods. In addition, the information from this study may prove beneficial for the manipulation of parameters affecting pathogenesis of P. corrugata and for the eventual control of plant disease.

Certaines bactéries contribuent à réduire les maladies racinaires de plantes causées par des champignons pathogènes. La compétence rhizosphérique de ces bactéries antagonistes est un pré-requis à leur efficacité. Cependant, l’influence du champignon pathogène sur le déterminisme génétique de cette compétence n’est pas connue. L’influence d’une part du champignon seul, ou effet hyphosphère et d’autre part de la plante et du champignon, ou effet pathorhizosphère sur la régulation des déterminants génétiques bactérien a été évaluée dans ce travail. Pour cela, des confrontations entre la rhizobactérie antagoniste, Pseudomonas fluorescens Pf29Arp, et le champignon responsable du piétin-échaudage du blé, Gaeumannomyces graminis var. Tritici, ont été conduites in vitro en absence ou présence de racine. La constructin d’une puce génomique de Pf29Arp a permis de mener une approche transcriptomique comparative dans ces dispositifs expérimentaux
Some bacteria reduce root diseases caused by phytopathogenic fungi. Rhizosphere competence is an important prerequisite for the efficacy of biocontrol. However, whether fungal presence alters genetic rhizosphere competence traits remains unknown. Influence of fungus alone, so-called hyposphere effect, and combined fungal-root influence, so-called pathorhizosphere effect, on regulation of bacterial determinants was assessed in this work. In vitro confronations between the antagonistic rhizobacteria Pseudomonas fluorescens PF29Arp and the take-all fungus Gaeumannomyces graminis var. Tritici were conducted in absence or in presence of root. The related bacterial transcriptome were compared thanks to the build-up of a Pf29Arp shotgun DNA icroarray

L'accroissement des populations de p. Spp. Fluorescents observes au champ sur le rhizoplan du ble est souvent associe au declin du pietin-echaudage du ble. Le declin du pietin-echaudage constitue un processus naturel de reduction de l'impact au champ de cette maladie causee par le champignon gaeumannomyces graminis var. Tritici (ggt). Cette reduction de maladie est associee a la colonisation racinaire du ble par des souches de p. Spp. Fluorescents antagonistes de ggt. La souche de p. Fluorescens pf29a appartient a un groupe d'homologie rep-pcr dont l'extension est liee a la presence de necroses racinaires. Cette souche est antagoniste de ggt in situ. Le suivi specifique de pf29a avec un marqueur rapd a demontre que la presence de ggt stimulait fortement et specifiquement la colonisation du rhizoplan par la souche pf29a lorsqu'elle est introduite dans un sol non sterile. Pf29a presente egalement un fort potentiel de colonisation du rhizoplan en absence de ggt et de necroses. L'etude du nombre et de la diversite des profils rapd observes pour les communautes indigenes de p. Spp. Fluorescents du rhizoplan nous ont permis d'evaluer l'effet de pf29a et de ggt sur la structure et la diversite de ces communautes. L'introduction de ggt dans le sol entraine une modification des structures et de la diversite des communautes indigenes de p. Spp. Fluorescents. L'introduction de pf29a, uniquement en presence de ggt, accroit fortement cette selection. La souche pf29a selectionne en synergie avec ggt, les p. Spp. Fluorescents indigenes. Une relation atypique est mise en evidence entre la dose de pf29a introduite dans le sol et la reduction de la gravite du pietin-echaudage. Les modeles exponentiels negatifs bases sur des experimentations dose-effet ne peuvent tenir compte d'un plateau et d'une asymptote de reduction de maladie observes pour pf29a. Ce plateau et cette asymptote suggerent l'existence de plusieurs modes d'action de pf29a, soit actives par une dose seuil, soit renforces a partir d'une certaine densite bacterienne. En outre, pf29a stimule la croissance du ble. La dynamique de colonisation de pf29a sur le rhizoplan du ble est similaire pour deux doses correspondant au debut du plateau et de l'asymptote. Un fort accroissement des populations cultivables de la souche coincide avec le developpement des necroses de pietin-echaudage observe entre 20 et 25 jours. L'application de la technique scar a un amplifiat rapd specifique de pf29a a permis la synthese d'un couple d'amorces specifiques de la souche. La mises au point d'une technique de pcr semi-quantitative a confirme la faible proportion de bacteries cultivables dans les populations totales de pf29a. Le role important joue par les necroses de pietin-echaudage dans la mise en place de l'antagonisme des p. Spp. Fluorescents contre ggt est confirme par cette etude. Nos resultats soulignent egalement la complexite de l'activite antagoniste de pf29a. La pcr semi-quantitative associee aux amorces scar fournit un outil de quantification directe de pf29a sur le rhizoplan.

Les maladies d'origine tellurique sont difficilement contrôlables par la lutte chimique ou variétale et se caractérisent par des processus et des échelles spatio-temporelles différents de ceux des maladies aériennes. En particulier, les modes de dispersion des agents pathogènes du sol permettent l'apparition et le maintien d'une forte structure spatiale de ces maladies, qui se développent souvent sous forme de foyers. Cette agrégation influence à la fois la dynamique temporelle des épidémies et la relation dégâts-dommages, ce qui en fait un élément important du raisonnement des méthodes de lutte. L'objectif de cette thèse est de comprendre et de modéliser le développement spatio-temporel des épidémies d'origine tellurique afin d'en déduire des stratégies de gestion susceptibles de limiter les risques associés, en particulier en agissant sur la structure spatiale de ces maladies. Pour ce faire, deux modèles ont été développés. Le premier, spatialement explicite et assez détaillé biologiquement, a été paramétré en conditions contrôlées dans le cas du piétin-échaudage du blé. Le test de ce modèle à l'aide de données issues du champ montre une bonne précision malgré un biais positif et indique des pistes pour améliorer la valeur prédictive du modèle. Le second modèle, plus simple et plus générique, utilise la théorie de la hiérarchie pour simuler le développement d'épidémies à plusieurs échelles spatiales simultanément. Il permet de tester des hypothèses concernant le fonctionnement des épidémies et les liens entre incidences à différentes échelles, et en particulier l'effet de la structure spatiale du peuplement hôte et de l'inoculum primaire sur la dynamique et l'agrégation de la maladie. Dans le cas du piétin-échaudage, pour lequel nous avons montré l'importance de la structure spatiale de l'inoculum primaire, ces simulations conduisent à préconiser des semis différents en fonction du rang dans la succession culturale.

Take-all is the name given to the disease caused by a soilborne fungus Gaeumannomyces graminis (Sacc.) von Arx and Olivier var. tritici Walker (Ggt), an ascomycete of the family Magnaportheaceae (Cook, 2003). This fungus is an aggressive soil-borne pathogen causing root rot of wheat (primary host), barley and rye crops (secondary host). The flowering, seedling, and vegetative growth stages can be affected by the infection of the whole plant, leaves, roots, and stems. Infections of roots result in losses in crop yield and quality primarily due to a lowering in nutrient uptake. Take-all is most common in regions where wheat is cultivated without adequate crop rotation. Crop rotation allows time between the planting dates of susceptible crops, which causes a decrease in the inoculum potential of soilborne plant pathogens to levels below an economic threshold by resident antagonistic soil microbial communities. Soilborne disease suppressiveness is an inherent characteristic of the physical, chemical, and/or biological structure of a particular soil which might be induced by agricultural practices and activities such as the cultivation of crops, or the addition of organisms or nutritional amendments, causing a change in the microfloral environment. Disturbances of soil ecosystems that impact on the normal functioning of microbial communities are potentially detrimental to soil formation, energy transfers, nutrient cycling, and long-term stability. In this regard, an overview of soil properties and processes indicated that the use of microbiological and biochemical soil properties, such as microbial biomass, the analysis of microbial functional diversity and microbial structural diversity by the quantification of community level physiological profiles and signature lipid biomarkers are useful as indicators of soil ecological stress or restoration properties because they are more responsive to small changes than physical and chemical characteristics. In this study, the relationship between physico-chemical characteristics, and different biological indicators of soil quality of agricultural soils conducive, suppressive, and neutral with respect to take-all disease of wheat as caused by the soilborne fungus Gaeumannomyces graminis var. tritici (Ggt), were investigated using various techniques. The effect of crop rotation on the functional and structural diversity of soils conducive to take-all disease was also investigated. Through the integration of quantitative and qualitative biological data as well as the physico-chemical characteristics of the various soils, the functional and structural diversity of microbial IV communities in the soils during different stadia of take-all disease of wheat were characterised. All results were evaluated statistically and the predominant physical and chemical characteristics that influenced the microbiological and biochemical properties of the agricultural soils during different stadia of take-all disease of wheat were identified using multivariate analyses. Although no significant difference @ > 0.05) could be observed between the various soils using conventional microbiological enumeration techniques, the incidence of Gliocladium spp. in suppressive soils was increased. Significant differences @ < 0.05) were observed between agricultural soils during different stadia of take-all disease of wheat. Although no clear distinction could be made between soils suppressive and neutral to take-all disease of wheat, soils suppressive and conducive to take-all disease of wheat differed substantially in their community level physiological profiles (CLPPs). Soils suppressive / neutral to take-all disease were characterised by enhanced utilisation of carboxylic acids, amino acids, and carbohydrates, while conducive soils were characterised by enhanced utilisation of carbohydrates. Shifts in the functional diversity of the associated microbial communities were possibly caused by the presence of Ggt and associated antagonistic fungal and bacterial populations in the various soils. It was evident that the relationships amongst the functionality of the microbial communities within the various soils had undergone changes through the different stages of development of take-all disease of wheat, thus implying different substrate utilisation capabilities of present soil microbial communities. Diversity indices were calculated as Shannon's diversity index (H') and substrate equitability (J) and were overall within the higher diversity range of 3.6 and 0.8, respectively, indicating the achievement of very high substrate diversity values in the various soils. A substantial percentage of the carbon sources were utilised, which contributed to the very high Shannon-Weaver substrate utilisation indices. Obtained substrate evenness (equitability) (J) indices indicated an existing high functional diversity. The functional diversity as observed during crop rotation, differed significantly (p < 0.05) from each other, implying different substrate utilisation capabilities of present soil microbial communities, which could possibly be ascribed to the excretion of root exudates by sunflowers and soybeans. Using the Sorenson's index, a clear distinction could be made between the degrees of substrate utilisation between microbial populations in soils conducive, suppressive, and neutral to take-all disease of wheat, as well as during crop rotation. Furthermore, the various soils could also be differentiated on the basis of the microbial community structure as determined by phospholipid fatty acid (PLFA) analysis. Soil suppressive to take-all disease of wheat differed significantly (p < 0.05) from soils conducive, and neutral to take-all disease of wheat, implying a shift in relationships amongst the structural diversity of microbial communities within the various soils. A positive association was observed between the microbial phospholipid fatty acid profiles, and dominant environmental variables of soils conducive, suppressive, and neutral to take-all disease of wheat. Soils conducive and neutral to take-all disease of wheat were characterised by high concentrations of manganese, as well as elevated concentrations of monounsaturated fatty acids, terminally branched saturated fatty acids, and polyunsaturated fatty acids which were indicative of Gram-negative bacteria, Gram-positive bacteria and micro eukaryotes (primarily fungi), respectively. These soils were also characterised by low concentrations of phosphorous, potassium, percentage organic carbon, and percentage organic nitrogen, as well as low soil pH. Soil suppressive to take-all disease of wheat was characterised by the elevated levels of estimated of biomass and elevated concentrations of normal saturated fatty acids, which is ubiquitous to micro-organisms. The concentration of normal saturated fatty acids in suppressive soils is indicative of a low structural diversity. This soil was also characterised by high concentrations of phosphorous, potassium, percentage organic carbon, and percentage organic nitrogen, as well as elevated soil pH. The relationship between PLFAs and agricultural soils was investigated using principal component analysis (PCA), redundancy analysis (RDA) and discriminant analysis (DA). Soil suppressive to take-all disease of wheat differed significantly (p < 0.05) from soils conducive, and neutral to take-all disease of wheat, implying a shift in relationships amongst the structural diversity of microbial communities within the various soils. A positive association was observed between the microbial phospholipid fatty acid profiles, and dominant environmental variables of soils conducive, suppressive, and neutral to take-all disease of wheat. Hierarchical cluster analysis of the major phospholipid fatty acid groups indicated that the structural diversity differed significantly between soils conducive, suppressive, and neutral to take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. The results indicate that the microbial community functionality as well as the microbial community structure was significantly influenced by the presence of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici, and that the characterisation of microbial functional and structural diversity by analysis of community level physiological profiles and phospholipid fatty acid analysis, respectively, could be successfully used as an assessment criteria for the evaluation of agricultural soils conducive, suppressive, and neutral to take-all disease of wheat, as well as in crop rotation systems. This methodology might be of significant value in assisting in the management and evaluation of agricultural soils subject to the prevalence of other soilborne diseases.
Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2004.

Take-all, caused by the soilborne fungus, Gaeumannomyces graminis var. tritici (Ggt), is an important root disease of wheat that can be reduced by take-all decline (TAD) in successive wheat crops, due to general and/or specific suppression. A study of 112 New Zealand wheat soils in 2003 had shown that Ggt DNA concentrations (analysed using real-time PCR) increased with successive years of wheat crops (1-3 y) and generally reflected take-all severity in subsequent crops. However, some wheat soils with high Ggt DNA concentrations had low take-all, suggesting presence of TAD. This study investigated 26 such soils for presence of TAD and possible suppressive mechanisms, and characterised the microorganisms from wheat roots and rhizosphere using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A preliminary pot trial of 29 soils (including three from ryegrass fields) amended with 12.5% w/w Ggt inoculum, screened their suppressiveness against take-all in a growth chamber. Results indicated that the inoculum level was too high to detect the differences between soils and that the environmental conditions used were unsuitable. Comparison between the Ggt DNA concentrations of the same soils collected in 2003 and in 2004 (collected for the pot trial), showed that most soils cropped with 2, 3 and 4 y of successive wheat had reduced Ggt DNA concentrations (by 195-2911 pg g-1 soil), and their disease incidences revealed 11 of the 29 test soils with potential take-all suppressiveness. Further pot trials improved the protocols, such that they were able to differentiate the magnitudes of suppressiveness among the soils. The first of the subsequent trials, using 4% w/w Ggt inoculum level, controlled conditions at 16°C, 80% RH with alternate 12 h light/dark conditions, and watering the plants twice weekly to field capacity (FC), screened 13 soils for their suppressiveness against take-all. The 13 soils consisted of 11 from the preliminary trial, one wheat soil that had been cropped with 9 y of wheat (considered likely to be suppressive), and a conducive ryegrass soil. The results revealed that 10 of these soils were suppressive to take-all. However, in only four of them were the effects related to high levels of microbial/biological involvement in the suppression, which were assessed in an experiment that first sterilised the soils. In a repeat trial using five of the soils H1, H3, M2, P7 (previously cropped with 3, 3, 4 and 9 y successive wheat, respectively) and H15 (previously cropped with 5 y of ryegrass), three of them (H1, H3 and M2) had reduced Ggt DNA concentrations (>1000 pg g-1 soil reductions), and were confirmed to be suppressive to take-all. A pot trial, in which 1% of each soil was transferred into a γ-irradiated base soil amended with 0.1% Ggt inoculum, indicated that soils H1 and H3 (3 y wheat) were specific in their suppressiveness, and M2 (4 y wheat) was general in its suppressiveness. The microbial communities within the rhizosphere and roots of plants grown in the soils, which demonstrated conduciveness, specific or general suppressiveness to take-all, were characterised using PCR-DGGE, and identities of the distinguishing microorganisms (which differentiated the soils) identified by sequence analysis. Results showed similar clusters of microorganisms associated with conducive and suppressive soils, both for specific and general suppression. Further excision, re-amplification, cloning and sequencing of the distinguishing bands showed that some actinomycetes (Streptomyces bingchengensis, Terrabacter sp. and Nocardioides sp.), ascomycetes (Fusarium lateritium and Microdochium bolleyi) and an unidentified fungus, were associated with the suppressive soils (specific and general). Others, such as the proteobacteria (Pseudomonas putida and P. fluorescens), an actinomycete (Nocardioides oleivorans), ascomycete (Gibberella zeae), and basidiomycete (Penicillium allii), were unique in the specific suppressiveness. This indicated commonality of some microorganisms in the take-all suppressive soils, with a selected distinguishing group responsible for specific suppressiveness. General suppressiveness was considered to be due to no specific microorganisms, as seen in soil M2. An attempt to induce TAD by growing successive wheat crops in pots of Ggt-infested soils was unsuccessful with no TAD effects shown, possibly due to variable Ggt DNA concentrations in the soils and addition of nutrients during the experiment. Increasing numbers of Pseudomonas fluorescens CFU in the rhizosphere of plants, during successive wheat crops was independent of the Ggt DNA concentrations and disease incidence, suggesting that increases in P. fluorescens numbers were associated with wheat monoculture. This study has demonstrated that TAD in New Zealand was due to both specific and general suppressiveness, and has identified the distinguishing microorganisms associated with the suppression. Since most of these distinguishing microorganisms are known to show antagonistic activities against Ggt or other soilborne pathogens, they are likely to act as antagonists of Ggt in the field. Future work should focus on validating their effects either individually, or interactively, on Ggt in plate and pot assays and under field conditions.

Gaeumannomyces graminis var. Tritici est un champignon tellurique pathogène responsable du piétin-échaudage des céréales à paille qui peut être à l'origine de pertes de rendement importantes. Le développement de ce champignon dépend de nombreux facteurs liés à la succession de culture, àl'itinéraire technique, au climat et au sol. L'agriculteur peut contrôler le piétin échaudage et réduire son impact sur le rendement en modifiant le système de clture (succession culturale, semis tardif, fertilisation azotée, traitement fongicide etc. ). A ce jour aucun outil d'aide à la décision n'a été développé pour définir de nouvelles méthodes de protection intégrée contre le piétin-échaudage du blé. L'objectif de ce travail est de développer un modèle simulant l'effet des systèmes de culture sur le piétin-échaudage en fonction de variables d'entrée facilement accessibles. Préalablement à ce travail de modèlisation, des expérimentations ont été conduites en 2001 – 2003 afin d'étudier l'effet du mode de gestion de l'interculture sur le développement du champignon. En effet, la succession culturale joue un rôle très important. Cependant elle a jusqu'ici été analysée sans prise en compte de la période d'interculture. Cinq couverts végétaux (colza, moutarde, ray-grass, repousses de blé, avoine) et un maintien du " sol nu " entre deux cultures de blé ont été comparés. En monoculture de blé aucun effet lié à la gestion de l'interculture n'a été observé sur le piétin-échaudage. Le niveau faible de maladie causé par le déclin explique cette absence d'effet. Entre une première et une deuxième année de culture de blé, le risque le plus élevé a été observé après les repousses de blé, le lus faible après un sol maintenu " nu " sans couvert. Le colza (cv Lutin) est sans effet alors que moutarde (cv Carnaval) et avoine (cv Ourasi) donnent les meilleurs résultats (après "sol-nu "), mais principalement lorsque leurs résidus sont enfouis avant le semis de blé
Gaeumannomyces graminis var. Tritici is a soiborne fungus responsible of the take-all of cereals. This disease causes I mportant yield losses. The development of the fungus depends on many factors : crop rotation, crop management, climate and soil conditions. Farmers can control take-all and reduce its impact on yield by modifying their cropping systems (crop succession, late sowing, nitrogen fertilization, seed treatment, etc). So far no decision-support tool has been developed for the design of new methods of integrated crop protection against take-all of winter wheat. This work aims at developing a model to simulate cropping systems and climate effects on take-all using easy-to-measure input variables. Preliminary to the modelling work, however, several field experiments were conducted I the years 2000-2003 to estimate the effect of the summer-fallow period management on take-all. This factor was not studied in detail in previous studies. Five summer-crops (oilseed rape, mustard, ryegrass, oats, wheat volunteers) plus a bare soil situation were compared. In wheat monoculture, summer-fallow management had no effect on take-all. This was explained by reduced levels of disease related to take-all decline. Between first and second wheat crops, the highest risk was observed following wheat volunteers, and the lowest following the bare soil. Oilseed rape (cv Lutin) did not show any significant effect on take all incidence, whereas mustard (cv Carnaval) and oast (cv Ourasi) were the best candidates. These summer-crops decreased disease incidence and severity particularly when their residues were buried before wheat sowing

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The morphological and cultural characteristics of G. graminis var. graminis isolates from rice and grasses were studied. The fungus forms two types of mycelia, dark macrohyphae that join laterally to form runner hyphae or rhizomorphs and hyaline or infectious microhyphae, as well as fan shaped hyphae characteristic of the pathogen. Pigmented and lobed hyphopodia on lower leaf sheaths were formed both under natural conditions and artificial inoculations of plants. The perithecia containing asci and ascospores were found on leaf sheaths lesions on field samples. The perithecia were produced on leaf sheaths of inoculated plants as well as on detached sterilized leaf sheaths and on culture medium, potato-dextrose-agar (PDA). Hyphae and hyphopodia were formed from germination tubes of ascospores, and the hyphae under moist stress conditions produced chlamydospores which were initially hyaline and later attained dark color. The culture of Ggg, was characterized by fluffy aerial mycelium, white in the initial stages of growth and later with age, the colony color changed from dirty-white or mouse gray to almost black. The marked diagnostic colony characteristic of whorled appearance was the curling back of marginal hyphae. The amount and time of formation of perithecia varied among the isolates tested. The virulence test conduced with 20 isolates of rice and grasses, showed differences in aggressiveness both on rice seedlings and adult plants. In general, isolates from rice were more aggressive on rice than isolates from grasses. The test with four levels of inoculum (0, 5, 1.0, 2.0, 4.0 g per plant of autoclaved sorghum grains) and two plant ages showed that 60-day old were more susceptible than 35-day old plants. The spontaneous infection of healthy plants was observed in the greenhouse indicating the role of ascospores in the dissemination of black sheath rot in rice. Furthermore, the pathogenicity of ascospores of Ggg on rice plants was confirmed by inoculations tests. Six fields of upland rice were surveyed in the advanced stages of maturation for the incidence of black sheath rot. The disease incidence on tillers, under natural conditions of infection, ranged from 68 to 100%. The pathogenicity of 20 isolates retrieved from rice and grasses were studied. All isolates were pathogenic to rice and grasses such as baranyard grass (Echinochloa crusgalli), fountain grass (Pennisetum setosum) signal grass (Brachiaria sp), crab grass (Digitaria horizontalis), plantain signal grass (Brachiaria plantaginea), indian goose grass (Eleusine indica) and southern sandbur (Cenchrus echinatus). Winter cereals such as wheat, oat, rye, barley and triticale as well as sorghum, corn, and millet exhibited different degrees of susceptibility to the isolate Ggg-a 01. Significant differences were observed in relation to characteristic symptoms on the culm, lesion height, number of tillers or dead plants, presence of characteristic mycelium, fan shaped hyphae, production of hyphopodia and perithecia. The formation of perithecia was not observed on leaf sheaths of inoculated plants of millet, sorghum, southern sandbur and maize. All inoculated wheat plants were killed indicating more susceptibility than other cereals. The resistance of 58 upland rice genotypes were tested in the greenhouse, utilizing rice isolate Ggg-a 01. Of the genotypes assessed, the lesion height of SCIA16 and SCIA08 was significantly shorter compared to the highly susceptible genotype CNAS10351. The progress and dissemination of black sheath rot in rice was studied during two years under field conditions in savanna sensu lato ‘cerrado’. The central line of each plot was inoculated with isolate of Ggg to establish the infection foci. The soil was infested with four levels of inoculum (5.0, 10.0, 20.0 and 40.0 g of autoclaved sorghum grains containing mycelium / 40 cm) and main tiller of plants (4, 8, 16 and 32, tillers per plot/ 40 cm) were inoculated with 2.0 cm-long detached leaf sheaths containing perithecia by insertion between the culm and leaf sheath of the tiller. There was no significant effect of inoculum level on the disease severity obtained by soil infestation with mycelium as well as the plants infected with perithecia. However, the total area under disease progress curve was significantly smaller for plant infection with perithecia than for soil infestation by mycelium, during 2002/2003. The evaluation of disease incidence for the analysis of gradients was based on infected tillers in 1.6 square meter area, five lines on either side of the inoculated 40 cm-long central line. The analysis according models of Gregory (1968) and Kiyosawa & Shiyomi (1972) showed the existence of gradients in the first year, both for levels of inoculum of soil infection by mycelium and plant infection with perithecia. In the second year (2004/2005), there was no well defined gradient for all the treatments. The disease progress was not affected by inoculum levels on soil or plant infections. Monomolecular model was found more adequate in tests conduced under greenhouse conditions while the models of Gompertz and monomolecular, better described the disease progress under field conditions.
Foram estudadas características morfológicas e culturais de isolados de Gaeumannomyces graminis var. graminis provenientes de arroz e capins. O fungo se estabelece formando dois tipos de hifas: macrohifas, escuras, superficiais que se juntam lateralmente e formam cordões ou rizomorfas e microhifas, hialinas ou infecciosas, que penetram no hospedeiro. Forma também hifas em leque sobre as bainhas, a partir de macrohifas, que caracterizam o patógeno. Houve a formação de hifopódios lobados e pigmentados em bainhas, tanto em condições naturais como em inoculações. Observou-se peritécios contendo ascas e ascósporos, característicos do fungo, nas bainhas sobre as lesões em amostras coletadas no campo. Através de inoculação artificial, foram produzidos peritécios em bainhas de plantas, em bainhas destacadas e esterilizadas e em meio de cultura de batata-dextrose-ágar (BDA). Foram formadas hifas e hifopódios a partir de tubos germinativos dos ascósporos e as hifas crescidas em condições de estresse hídrico produziram clamidósporos, inicialmente hialinos e, posteriormente, de coloração escura. O micélio de Ggg, geralmente de aspecto aéreo fofo, é branco no início do crescimento, com variação de cor com a idade, do branco cinza ao marrom oliváceo e quase preto. Uma característica marcante é a aparência espiralada das macrohifas escuras nas bordas da colônia. Entre os isolados testados houve variação na quantidade de peritécios bem como na época de formação. Os testes de virulência realizados com vinte isolados provenientes de arroz e capins apresentaram diferenças em agressividade, tanto em plântulas quanto em plantas de arroz. Em geral, os isolados provenientes de arroz foram mais agressivos em arroz que os isolados de capins. O teste com quatro níveis de inóculo (0,5, 1,0, 2,0, e 4,0 g de inóculo por planta, multiplicado em grãos de sorgo autoclavados) e duas idades de plantas mostrou que as plantas inoculadas aos 60 dias após o plantio foram mais suscetíveis do que aquelas inoculadas aos 35 dias, requerendo menor nível de inóculo para a infecção. A patogenicidade de ascósporos de Ggg em plantas de arroz foi comprovada, bem como o papel dos ascósporos na disseminação do mal-do-pé do arroz. A incidência de mal-do-pé em lavouras de arroz de terras altas nas condições naturais de infecção variou de 68 a 100% de perfilhos infectados, entre seis lavouras avaliadas em fase avançada de maturação. Foi estudada também a patogenicidade dos vinte isolados de Ggg obtidos, provenientes de arroz e capins. Todos os isolados foram patogênicos a arroz e aos capins: capim arroz (Echinochloa crusgalli), capim avião (Pennisetum setosum), capim braquiária (Bachiaria sp.), capim digitaria (Digitaria horizontalis), capim marmelada (Brachiaria plantaginea), capim pé-degalinha (Eleusine indica) e capim timbete (Cenchrus echinatus). Os cereais de inverno, trigo, aveia, centeio, cevada e triticale, bem como sorgo, milho, e milheto apresentaram diferentes graus de suscetibilidade ao isolado Ggg-a 01. As diferenças foram significativas quanto a sintomas típicos na base do colmo, altura de lesão escura na bainha, número de perfilhos ou plantas mortas, presença de micélio característico, hifas em leque e produção de hifopódios e peritécios. Não foram observados peritécios em milheto, sorgo, timbete e milho e a maior suscetibilidade foi apresentada pelo trigo, com a morte de todas as plantas inoculadas. Foi testada a resistência de 58 genótipos de arroz de terras altas, utilizando o isolado Ggg-a 01 proveniente de arroz, em casa-de-vegetação. Entre os genótipos avaliados, SCIA16 e SCIA08 apresentaram altura de lesão significativamente menor, sendo considerados resistentes em relação ao genótipo CNAS10351, altamente suscetível. O progresso e disseminação do maldo- pé do arroz foram estudados durante dois anos, em condições de campo em solo de cerrado. Utilizou-se delineamento experimental de blocos completos ao acaso e quatro repetições. Cada parcela foi constituída de dezenove linhas de sete e cinco metros, respectivamente no primeiro e segundo ano, com espaçamento de quarenta centímetros. Foi inoculada a linha central de cada parcela com isolado de Ggg para estabelecer os focos de disseminação da doença. O solo foi infestado com micélio em quatro níveis de inóculo (5,0, 10,0, 20,0 e 40,0 gramas de grãos de sorgo autoclavados e colonizados com micélio / 40 cm da linha) e perfilhos foram inoculados (4, 8, 16 e 32 perfilhos / 40 cm da linha) com pedaços de bainhas de arroz de dois centímetros de comprimento, contendo peritécios e micélio, inseridos entre o colmo e a bainha. Não houve efeito de níveis de inóculo na severidade da doença, tanto para micélio no solo quanto para peritécios na planta, nos dois anos de experimento. Entretanto, a área total sob a curva de progresso da doença na safra 2002/2003 foi significativamente menor nas plantas infectadas com peritécios, do que nas plantas infectadas através de infestação do solo com micélio. A avaliação de incidência da doença para análise do gradiente foi baseada nos perfilhos contados em 1,6 metros quadrados, compostos de cinco linhas de quarenta centímetros de cada lado da fonte de inóculo, na linha central. A análise de gradiente, conforme modelos de Gregory (1968) e Kiyosawa & Shiyomi (1972) mostrou existência de gradiente no primeiro ano, tanto para níveis de inóculo quanto para os focos provenientes dos dois tipos de inóculo. No segundo ano (2004/2005), não houve gradiente definido para os tratamentos testados. O progresso da doença não foi afetado pelos níveis, tanto na infecção do solo com micélio, quanto na planta com peritécios. Em teste de ajuste de modelo matemático para estudos epidemiológicos, o modelo monomolecular foi o mais apropriado para estudos de mal-do-pé do arroz nas condições de casa-de-vegetação e os modelos de Gompertz e monomolecular são os que melhor descrevem o progresso da doença, nas condições de campo.

Pseudomonas fluorescens Pf29A réduit l'impact du piétin-échaudage, maladie tellurique du blé causée par le champignon Gaeumannomyces graminis var. Tritici (Ggt). Nous avons recherché les modes d'action de la souche Pf29A impliqués dans l'antagonisme. Une antibiose a été révélée in vitro sur le milieu GCY. Le glucose et le galactose entraînaient une forte activité antifongique alors qu'aucune antibiose n'était observée en présence de saccharose ou de fructose. Les sources de carbone responsables de l'activité antagoniste entraînaient également une acidification du milieu (pH 4). Ce pH était responsable de l'antagonisme in vitro car il affectait la croissance fongique. Trois mutants d'antibiose, NA2, NA3, NA4, ont perdu leur capacité d'inhibition. Le gène muté a été caractérisé par la technique de PCR-inverse. Le mutant NA3 est affecté dans le gène gcd codant l'enzyme glucose-déshydrogénase (GDH). La GDH est une enzyme de la voie oxydative qui permet la conversion du glucose en gluconate entraînant l'acidification du milieu. Les séquences d'acides aminés déduites pour les mutants NA2 et NA4 montrent de fortes homologies avec des protéines impliquées dans la biosynthèse de la pyrroloquinoline quinone (PQQ). PQQ est le cofacteur essentiel à l'activité de la GDH. L'implication de la voie oxydative et du pH dans l'antagonisme in vitro a été suggéré par l'incapacité de ces mutants à acidifier le milieu. Des expériences complémentaires ont été effectuées pour évaluer la capacité antagoniste par les mutants in vivo. Le mutant NA3 entraîne que moindre capacité à réduire le piétin-échaudage. La résistance systémique induite (ISR) par la souche Pf29A a été recherchée sur deux génotypes de blé, Talent et L193. Une ISR a été mise en évidence chez la lignée L193 contre le Ggt distant de la souche bactérienne grâce à un test de séparation de racines. Aucune ISR n'a été observée sur les feuilles inoculées par Blumeria graminis, l'agent du mildiou. L'ISR joue un rôle dans la diminution du piétin-échaudage. Deux modes d'action sont impliqués dans l'antibiose de Pf29A. Ces résultats démontrent le rôle de la voie oxydative et ils indiquent que l'acide gluconique et le pH peuvent avoir une action directe dans l'activité antagoniste de la souche Pf29A in situ. Ce travail établit pour la première fois l'existence d'ISR chez les Monocotylédones
In soil, Pseudomonas fluorescens Pf29A reduces the severity of take-all, a wheat root disease caused by the fingus Gaeumannomyces graminis var. Tritici (Ggt). We investigated the modes of action of Pf29A involved in biocontrol. An in vitro antagonistic activity was revealed on GCY medium. Antagonistic activity was influenced by nutritional conditions. Glucose and galactose enhanced the antifungal activity whereas no antibiosis was shown with sucrose or fructose. The high concentration of carbon sources that enhanced antibiosis also lead to a medium acidification (pH 4). This low pH was directly involved in Pf29A antagonism because it affected fungal growth. Three mutants, NA2, NA3, NA4, lose their ability to inhibit fungal growth on GCY. The mutated gene was characterised by inverse-PCR techniques. For NA3, gcd encoding glucose dehydrogenase (GDH) is affected. GDH initiates a direct oxidation of glucose to gluconate by the periplasmic oxidative pathway which leads to a medium acidification. The deduced amino acids sequences of NA2 and NA4 share regions highly homologous with proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ). PQQ is the cofactor required for GDH activity. The involvement of the direct oxidative pathways and of pH in in vitro biocontrol was suggested by the failure of these mutants to acidify the medium. Additional experiments were conducted in vivo to evaluate the biocontrol activity of the mutant inoculated in soil or in a gnotobiotic system. Mutant NA3 showed a reduced capacity to suppress take-all compared with the parental strain. Systemic resistance (ISR) induced by a root colonisation of Pf29A has been investigated on two wheat genotypes, L193 and Talent. Pf29A applied to roots could induce resistance in L193 against spatially distant Ggt in a split-root system. No induce resistance in leaves inoculated by B. Graminis, the powdery mildew agent, have been shown. ISR takes part in the suppression of take-all by Pf29A. This study reveals two modes of action involved in Pf29A antibiosis toward Ggt. The results demonstrate the role of the oxidative pathway in biocontrol and they indicate that gluconate acid and pH could have a direct effect in suppression of take-all by Pf29A in situ. This work establishes for the first time the existence of ISR in Monocotyledons

Bibliography: leaves 141-145
vii, 145 leaves, [4] leaves of plates : ill. (4 col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Depts. of Plant Pathology and Agronomy, 1986

Bibliography: leaves 207-220.
220 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Pseudomonas corrigata strain 2140 (Pc2140), isolated from wheat field soil in Australia, antagonises the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt) in vitro and significantly reduces take-all symptoms on wheat in pot trials. This study investigates the mechanisms by which the biocontrol agent reduces the disease symptoms. Biochemical analysis of metabolites of P. corrugata 2140 reveal a number of compounds potentially antagonistic to Ggt and which may play a role in disease control. These include water-soluble antibiotics, siderophores, proteases, peptides and volatiles including hydrogen cyanide.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1996

Bibliography: leaves 82-89. This study is aimed at providing information on control measures against Gaeumannomyces graminis var. tritici (abbreviated to Ggt) in annual pastures across southern Australia. The impact of timing of herbicide application, the impact of variation in timing of rainfall patterns, as well as herbicide application on the control of Ggt are also discussed. Additional experiments examine the ability of grass genera to host and carry over Ggt.

Brassica napus ist eine der wichtigsten Pflanzen in der Landwirtschaft. Pathogene verursachen jährlich große Ertragsverluste. Im Rahmen dieser Arbeit wurden die Interaktionen zwischen der Nutzpflanze Brassica napus bzw. der Modellpflanze Arabidopsis thaliana und dem phytopathogenen Pilz Verticillium longisporum untersucht. Verticillium longisporum ist ein auf Brassica spezialisierter Pilz, der die sogenannte Verticillium-Welke verursacht. Er dringt in die Wurzeln der Pflanze ein und steigt durch die Leitgefäße in den Spross auf. Die Pflanze zeigt in Folge der Infektion vor allem einen stark verkürzten Spross, Chlorosen und eine Notreife. Für die Experimente wurde Brassica napus und Arabidopsis thaliana mit Verticillium longisporum infiziert. Die Emissionen von Volatilen (volatile organic compounds (VOC)) der Pflanzen wurden sowohl ober- als auch unterirdisch bezüglich sich ändernder VOCs analysiert. Bei der Probennahme wurde darauf geachtet, die Pflanzen nicht zu verletzen und durch Messungen bedingten Stress zu minimieren. Auch während der Messungen der Wurzel-emissionen wurden die Pflanzen nicht aus ihrem Substrat entfernt, sondern innerhalb dessen gemessen. Während sich das Duftspektrum der Wurzeln von Brassica napus nicht änderte, emittierten die Sprosse infizierter Brassica Pflanzen signifikant mehr Dimethyldisulfid, ß-Ionon und ß-Cyclocitral. Bei der Infektion von Arabidopsis thaliana mit Verticillium longisporum hingegen spielten diese VOCs keine Rolle. Im zweiten Teil der Arbeit wurde die Vermutung überprüft, dass sich im Infektionsverlauf ändernde VOCs als Abwehrmoleküle dienen könnten. Hierfür wurde ein Bioassay entwickelt und zwei weitere pathogene Pilze, Gaeumannomyces graminis var. triciti und Sclerotinia sclerotiorum, unterschiedlicher Spezialisierung einbezogen. Wir setzten sie Dimethyldisulfid und ß-Ionon in unterschiedlichen Konzentrationen aus. Sowohl Dimethyldisulfid als auch ß-Ionon zeigten nur eine geringe fungizide Wirkung, gemessen am Myzelwachstum, gegen den Brassica-Spezialisten Verticillium longisporum. Gaeumannomyces graminis var. triciti wurde bereits bei geringeren Konzentrationen gehemmt. Sclerotinia sclerotiorum reagierte nicht oder sogar positiv auf die VOCs. Die (Mikro-) Sklerotienbildung wurde nur bei Verticillium longisporum beeinflusst. ß-Ionon unterstützte diese Entwicklung signifikant.