Heidrun Anke

Isolation and characterisation of fungi growing on volatile aromatic hydrocarbons as their sole carbon and energy source

Five fungal strains that are able to grow on toluene were isolated from enrichment cultures. Three different techniques were used: solid state-like batches, air biofilters and liquid cultures. Fungal growth in the latter systems was favoured by combining low pH and low water activity. Soil and groundwater samples from gasoline-polluted environments were used as inocula. The isolates were identified as deuteromycetes belonging to the genera Cladophialophora, Exophiala and Leptodontium and the ascomycete Pseudeurotium zonatum. The previously isolated toluene-degrading fungus Cladosporium sphaerospermum was included in the present study. Results showed that fungi grew on toluene with doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. The apparent half-saturation constant ( K m ) for toluene oxidation ranged from 5 to 22 μM. Degradation activity was inhibited by 50% at toluene concentrations ranging from 2.4 to 4.7 mM. These kinetic parameters are comparable to analogous data reported for toluene-degrading bacteria. The ability of fungi to grow at low water activities and low pH suggest that they may be used for the purification of gas streams containing aromatic hydrocarbons in air biofilters.

Assays of the biological activities of guaiane sesquiterpenoids isolated from the fruit bodies of edible lactarius species

Three sesquiterpenoids that are found in the edible mushrooms Lactarius deliciosus, L. deterrimus and L. sanguifluus, have been assayed for biological activity. The compounds tested were a stearic acid ester of a sesquiterpene (I) and a sesquiterpene aldehyde (lactaroviolin, II) and alcohol (deterrol, III). The assays used were for mutagenic activity in the Ames Salmonella assay, for antimicrobial activity against bacterial fungi and algae, for cytotoxicity against Ehrlich ascitic tumour cells and L 1210 cells, and for phytotoxic activity against Lepidium sativum and Seteria italica. All three compounds showed weak mutagenic activity in the Ames assay. Two compounds (II and III) were found to have moderate cytotoxic activity and one (III) exhibited weak antibacterial activity. No compound revealed phytotoxic, algicidal or antifungal activity.

The transcription factor Con7p is a central regulator of infection‐related morphogenesis in the rice blast fungus Magnaporthe grisea

A strain harbouring an insertion within the promoter of the CON7 gene of Magnaporthe grisea was isolated. This gene was previously shown to be essential for appressorium formation and growth in planta and is predicted to encode a transcription factor. Microarray‐based gene expression analysis was used to identify several genes whose transcription during germination depends on Con7p. These include the pathogenicity factor‐encoding gene PTH11 and several other genes which like PTH11 are predicted to encode G protein‐coupled receptors. Microarray analysis also revealed several Con7p‐dependent genes which may encode factors determining cell wall structure or function, either through the synthesis/degradation of cell wall components or by association with the cell exterior. One Con7p‐dependent gene predicted to encode a class VII chitin synthase was deleted, leading to dramatic consequences on the pathogenic development of the resultant strain. Within the con7– mutant, a 29% reduction in chitin content of germinated spores was found and the mutant was hypersensitive to the chitin synthase inhibitor nikkomycin Z. A green fluorescent protein‐tagged Con7p was found to have nuclear localization within spores. Taken together, these observations suggest that Con7p encodes a transcription factor required for the transcription of several genes which participate in disease‐related morphogenesis in M. grisea.

Metabolism of the polycyclic aromatic hydrocarbon pyrene by Aspergillus niger SK 9317

The metabolism of pyrene, a polycyclic aromatic hydrocarbon consisting of four rings, by Aspergillus niger SK 9317 was investigated. The metabolites formed were isolated and identified as 1-hydroxypyrene, 1,6- and 1,8-pyrenequinone, 1,6- and 1,8-dihydroxypyrene, 1-pyrenyl sulphate and 1-hydroxy-8-pyrenyl sulphate. This is the first report of 1-hydroxy-8-pyrenyl as a metabolite in the microbial metabolism of pyrene. The results suggest that A. niger metabolizes pyrene by cytochrome P-450 monooxygenase enzyme systems.

Metabolic products of microorganisms. 185. The anthraquinones of the Aspergillus glaucus group. I. Occurrence, isolation, identification and antimicrobial activity.

The occurrence of emodin, erythroglaucin, physcion, physcion-9-anthrone, questin, catenarin, and catenarin-8-methyl ether in different species of the Aspergillus glaucus group (genus Eurotium) was investigated. So far catenarin-8-methyl ether (1, 4, 6-trihydroxy-8-methoxy-3-methylanthraquinone) has not been described as a natural product; it was therefore given the name rubrocristin. The chemical and physical properties of rubrocristin are reported. In addition a new violet pigment (C16H12O5) was isolated and characterized by its MS-, IR- and UV-spectra.The antimicrobial properties of all substances were examined in the agar diffusion assay. Gram-positive bacteria were the most sensitive organisms and catenarin was the most active naturally occurring substance. Synthetically obtained 1, 4, 6, 8-tetrahydroxy-anthraquinone was slightly more active than catenarin, whereas rubrocristin showed no antibacterial activity.

Ferricrocin synthesis in Magnaporthe grisea and its role in pathogenicity in rice

SUMMARY Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host-pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea, the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1, an NRPS gene, and a gene encoding an l-ornithine N5-monooxygenase (OMO1) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N(5) hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea.

Heptemerones A-G, seven novel diterpenoids from Coprinus heptemerus: Producing organism, fermentation, isolation and biological activities

Seven novel diterpenoids, named heptemerones A∼G, were isolated from the broth of submerged cultures of Coprinus heptemerus, a basidiomycete which previously had not been known to produce secondary metabolites. The compounds were purified by solid phase extraction and silica gel chromatography followed by preparative HPLC. Among the biological activities the inhibition of fungal germination was the most potent, and depended highly on the composition of the assay medium. In water, inhibition occurred at 5∼10 fold lower concentrations as compared to complex media. Heptemerone G was the most active compound with MICs starting at 1 µg/ml. Four of the antifungal compounds exhibited plant protective activity in a leaf segment assay using Magnaporthe grisea as the pathogen. Growth of yeasts and bacteria was hardly affected. Cytotoxic activities were moderate and only heptemerone D was phytotoxic.

1-Methoxypyrene and 1,6-dimethoxypyrene: two novel metabolites in fungal metabolism of polycyclic aromatic hydrocarbons.

Abstract The metabolism of pyrene by Penicillium glabrum strain TW 9424, a strain isolated from a site contaminated with polycyclic aromatic hydrocarbons (PAHs) was investigated in submerged cultures. The metabolites formed were identified as 1-hydroxypyrene, 1,6- and 1,8-dihydroxypyrene, 1,6- and 1,8-pyrenequinone, and 1-pyrenyl sulfate. In addition, two new metabolites were isolated and identified by UV, 1H nuclear magnetic resonance, and mass spectroscopy as 1-methoxypyrene and 1,6-dimethoxypyrene. Experiments with [methyl-3H]S-adenosyl-l-methionine (SAM) revealed that SAM is the coenzyme that provides the methyl group for the methyltransferase involved. To our knowledge, this is the first time that methoxylated metabolites of PAHs have been isolated from fungal cultures.

Omphalotin, A New Cyclic Peptide with Potent Nematicidal Activity from Omphalotus Olearius I. Fermentation and Biological Activity

Abstract Omphalotin (1), a new cyclic peptide possessing strong and selective nematicidal activity against the plant pathogenic Meloidogyne incognita, was isolated from cultures of the basidiomycete Omphalotus olearius together with the antibiotic sesquiterpene illudin M (2) and the inactive 3-(3-indolyl)-N−methylpropanamide (3). Omphalotin (1) is only weakly active against the saprophytic nematode Caenorhabditis elegans, it is weakly cytotoxic at 100 μg/ml but shows no phytotoxic, antibacterial or antifungal activities. The fermentation of O, olearius and the production of the metabolites as well as the evaluation of their biological activities are reported.

Fungal secondary metabolites as inhibitors of infection-related morphogenesis in phytopathogenic fungi

The life-cycle of many plant-pathogenic fungi, especially those infecting aerial plant organs, contains several specific developmental stages. If these are sufficiently distinct in their physiology from vegetative hyphal growth, they present potential targets for non-fungitoxic plant protectants. The present review identifies such targets especially in the pre-penetration stages of the infection cycle of Magnaporthe grisea and other fungi infecting from air-borne spores. Examples of non-toxic natural products with activity against spore germination, attachment, appressorium formation, appressorium maturation and penetration of the host surface are given. In contrast, no substances selectively active against in planta growth or sporulation appear to be known. The selective activity of numerous secondary metabolites against specific infection stages without accompanying toxicity against vegetatively growing hyphae indicates a direction for the development of future natural product-derived fungicides which are more easily degraded in the environment and possess fewer non-target effects. Such substances are produced by many saprotrophic and endophytic fungi in pure culture. The paucity of data on the production of biologically active substances in natural situations limits the interpretation of their ecophysiological significance for the producer.