7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties. The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site. Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE. 10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8. LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity. LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

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.

<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</p>

Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases). Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones. Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases. Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”). Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols. The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.

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 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.

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

The dioxygenase-cytochrome P450 fusion proteins (DOX-CYP) comprise a heme-containing enzyme family that shares structural and catalytic properties with mammalian prostaglandin H (PGH) synthases. 7,8-Linoleate diol synthase (7,8-LDS) of Gaeumannomyces graminis was first characterized, and DOX-CYP enzymes are of mechanistic and biological interest. The growing number of fungal genome sequences has revealed DOX-CYP homologues in medically and economically important species. The aim of this thesis was to identify novel members of the DOX-CYP fusion protein family. The devastating rice pathogen Magnaporthe oryzae contains two DOX-CYP genes. The fungus synthesizes 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by dioxygenation of linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8R-HPODE), and subsequent isomerisation to the diol. 7,8-LDS of M. oryzae was identified by gene deletion, but the infection and reproduction processes of the Δ7,8-LDS strain were not altered. A mutant with constitutive protein kinase A activity profoundly changed the oxygenation profile, possibly due to post-translational modification. The human pathogens Aspergillus fumigatus and A. clavatus contain three DOX-CYP, designated psi producing oxygenase A (ppoA), ppoB, and ppoC, and form three oxylipins: 5S,8R-DiHODE, 8R,11S-DiHODE, and 10R-hydroxyoctadecadienoic acid.  PpoA was identified as 5,8-LDS, and ppoC as 10R-DOX. The 8,11-linoleate hydroperoxide isomerase activity was reduced by two imidazole-containing P450 inhibitors, miconazole and 1-benzylimidazole. PpoB could not be linked to the biosynthesis of 8,11-DiHODE for the following reasons: First, the 8,11-hydroperoxide isomerase activity was retained in A. fumigatus ΔppoB strains. Second, the P450 domain of the deduced ppoB of A. clavatus lacks a heme-thiolate cysteine ligand, presumably essential for hydroperoxide isomerase activity. Linoleate 9R-DOX activities of Aspergillus terreus and Lasiodiplodia theobromae were discovered. 9R-HPODE was further converted into unstable allene oxides, as judged by the accumulation of their hydrolysis products, α- and γ-ketols. These allene oxide synthase activities were specific for 9R-hydroperoxides. The 9R-DOX and AOS were found to have unique characteristics. In conclusion, novel DOX-CYP enzymes were identified in human and plant pathogenic fungi. These enzymes might be involved in biological processes, and show interesting catalytic similarities to human PGH synthase and thromboxane synthase (CYP5A).

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.