Electron microscopy of infection of nematodes byDactylaria haptotyla

Infection of nematodes byDactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membrane-bound vesicles, 0.2–0.5 µm in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. When the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0.7 µm in diam, was found in serial thin sections of the knob's cytoplasm. At the site facing the vesicle, the peripheral part of the nematode's cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.     

Time-lapse photomicrography and electron microscopy on initiation of infection of nematodes by Dactylella ellipsospora

 Infection of nematodes by two strains of Datylella ellipsospora was observed using video photomicrography and electron microscopy. By light microscopy, each cell with adhesive knobs contained a number of particles that were distributed evenly before capture of a nematode. The cytoplasmic particles moved to and fro at random. At the moment when the knob cell came into contact with a nematode, the particles accumulated at the place where the cell wall of the knob stuck firmly to the nematode cuticle and exuded adhesive at the same time. The adhesive can be seen near the point of contact between the cell wall of the knob and the cuticle of the nematode. At that point, the knob cell produced an infection peg in most cases, and the cell showed a preference to invade the body of the nematode rather than the tail and head. During capture, accumulation of cytoplasmic particles was seen until infection-bulb formation began. In electron micrographs of ultrathin sections, most of the particles could be seen as electron-dense vesicles, 0.2–0.6 μm in diameter. After attachment of the knob cell to the nematode cuticle, the vesicles were found to fuse with plasmalemma one after another to exude adhesive seen as an amorphous electron-dense substance. Received: January 30, 2002 / Accepted: April 25, 2002     

Bacterial decomposition of coral mucus as evaluated by long-term and quantitative observation

Coral mucus released from Acropora formosa and Montipora digitata was incubated with bacteria under dark conditions for 1 year to evaluate the quantitative degradability. All the mucus samples showed a similar decomposition pattern: about 80% of total organic carbon (TOC) in the mucus was mineralized within 1 month, while some mucus was slowly decomposed over the 1 year. Regression analysis using an exponential curve considering three degradability pools (labile, semilabile, and refractory) fitted the changes of the TOC concentrations very well (r ² > 0.99). Compiling the data on the two coral species, the labile organic C in the coral mucus had mineralization rates of 10–18% d⁻¹ and accounted for 79–87% of the initial TOC in the mucus. Semilabile organic C had mineralization rates of 0.3−1.6% d⁻¹ and accounted for 11−18% of the initial TOC. Refractory organic C accounted for 6% at most. These results suggest that not all coral mucus is rapidly decomposed by bacteria but some mucus remains as semilabile and refractory organic matter for several months.     

Antimicrobial activity of an endophytic <Emphasis Type="Italic">Xylaria</Emphasis> sp.YX-28 and identification of its antimicrobial compound 7-amino-4-methylcoumarin

An endophytic Xylaria sp., having broad antimicrobial activity, was isolated and characterized from Ginkgo biloba L. From the culture extracts of this fungus, a bioactive compound P3 was isolated by bioactivity-guided fractionation and identified as 7-amino-4-methylcoumarin by nuclear magnetic resonance, infrared, and mass spectrometry spectral data. The compound showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus [minimal inhibitory concentrations (MIC) 16 μg·ml⁻¹], Escherichia coli (MIC, 10 μg·ml⁻¹), Salmonella typhia (MIC, 20 μg·ml⁻¹), Salmonella typhimurium (MIC, 15 μg·ml⁻¹), Salmonella enteritidis (MIC, 8.5 μg·ml⁻¹), Aeromonas hydrophila (MIC, 4 μg·ml⁻¹), Yersinia sp. (MIC, 12.5 μg·ml⁻¹), Vibrio anguillarum (MIC, 25 μg·ml⁻¹), Shigella sp. (MIC, 6.3 μg·ml⁻¹), Vibrio parahaemolyticus (MIC, 12.5 μg·ml⁻¹), Candida albicans (MIC, 15 μg·ml⁻¹), Penicillium expansum (MIC, 40 μg·ml⁻¹), and Aspergillus niger (MIC, 25 μg·ml⁻¹). This is the first report of 7-amino-4-methylcoumarin in fungus and of the antimicrobial activity of this metabolite. The obtained results provide promising baseline information for the potential use of this unusual endophytic fungus and its components in the control of food spoilage and food-borne diseases.     

Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus

Several entomopathogenic fungi produce toxins that could be used as bioinsecticides in integrated pest management programs. Paecilomyces fumosoroseus is currently used for the biological control of the whiteflies Bemisia tabaci and B. argentifolii. Supernatants from submerged batch culture, where the fungus produced abundant dispersed mycelium, conidia and blastospores, were toxic to the whitefly nymphs. The most abundant metabolite was purified by HPLC and identified by mass spectrometry and NMR as dipicolinic acid. Both the dipicolinic acid produced by the fungus and the chemically synthesized compound had insecticidal activity against third-instar nymphs of the insect. Dipicolinic acid was toxic to the whitefly nymphs in bioassays involving topical applications. In submerged culture, the specific growth rate of P. fumosoroseus was 0.054 h⁻¹, the specific glucose consumption rate was 0.1195 g g⁻¹ h⁻¹ and the specific dipicolinic acid production rate was 0.00012 g g⁻¹ h⁻¹. Dipicolinic acid was detected after 24 h when the fungus started growing; and dipicolinic acid production was directly correlated with fungal growth. Nevertheless, the yield was low and the maximal concentration was only 0.041 g l⁻¹. The maximal concentrations of conidia and blastospores (per milliliter) were 1.4×10⁸ and 7×10⁷, respectively.     

Adhesive knob formation by conidia of the nematophagous fungusDrechmeria coniospora

We studied conidiogenesis and adhesive knob formation (maturation) by newly developed conidia of the nematophagous fungusDrechmeria coniospora. Upon conidiogenesis on infected nematodes or during saprophytic growth of the fungus in axenic cultures compact clusters of conidia developed. Less than 10% of such clustered conidia matured; mature conidia were invariably located on the periphery of the clusters.The kinetics and rate of maturation of conidia were studied inin vitro systems and in soil. In both cases adhesive knobs were formed; the rate at which knobs were formed appeared to be determined by the age of the conidia, the temperature and the soil moisture. In addition, knob formation was suppressed at increasing conidial densities. Under favorable conditions, however, over 90% of the conidia matured within a period of 3 days. The rate of knob formation was neither influenced by the presence of nematodes nor by that of exogenous nutrients, which suggests that maturation is an autonomous process. Electron-microscopical analysis indicated that budding of the conidia at the initial stage of maturation occurred simultaneously with the deposition of the sticky, adhesive layer around the wall of the developing knob.The ecological significance of the time- and spatially separated maturation of conidia after conidiogenesis is discussed with respect to survival of the conidia.     

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The “nonmammary” mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr ^−/− mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr ^−/− mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen’s kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.     

Carbon sources of Antarctic nematodes as revealed by natural carbon isotope ratios and a pulse-chase experiment

δ¹³C of nematode communities in 27 sites was analyzed, spanning a large depth range (from 130 to 2,021 m) in five Antarctic regions, and compared to isotopic signatures of sediment organic matter. Sediment organic matter δ¹³C ranged from −24.4 to −21.9‰ without significant differences between regions, substrate types or depths. Nematode δ¹³C showed a larger range, from −34.6 to −19.3‰, and was more depleted than sediment organic matter typically by 1‰ and by up to 3‰ in silty substrata. These, and the isotopically heavy meiofauna at some stations, suggest substantial selectivity of some meiofauna for specific components of the sedimenting plankton. However, ¹³C-depletion in lipids and a potential contribution of chemoautotrophic carbon in the diet of the abundant genus Sabatieria may confound this interpretation. Carbon sources for Antarctic nematodes were also explored by means of an experiment in which the fate of a fresh pulse of labile carbon to the benthos was followed. This organic carbon was remineralized at a rate (11–20 mg C m⁻² day⁻¹) comparable to mineralization rates in continental slope sediments. There was no lag between sedimentation and mineralization; uptake by nematodes, however, did show such a lag. Nematodes contributed negligibly to benthic carbon mineralization.     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Carbon nutrition of mature green orchid <Emphasis Type="Italic">Serapias strictiflora</Emphasis> and its mycorrhizal fungus <Emphasis Type="Italic">Epulorhiza</Emphasis> sp.

We studied the nutritional modes of the orchid Serapias strictiflora and its mycorrhizal fungus Epulorhiza sp. using the differences in carbon isotopic composition (δ¹³C) of C₃ orchid and C₄ maize tissues. We found that if cultivated in substrate lacking any organic compounds, the mycorrhizal extraradical mycelia (δ¹³C = −26.3 ± 0.2 ‰) developed well, despite being fully dependent on nutrition from orchid roots (δ¹³C = −28.6 ± 0.1 ‰). If the mycorrhizal fungus had additional access to and colonized decaying maize roots (δ¹³C = −14.6 ± 0.1 ‰), its isotopic composition (δ¹³C = −21.6 ± 0.4 ‰) reflected a mixture of biotrophy and saprotrophy. No statistically significant differences in δ¹³C of new storage tubers were found between Epulorhiza-associated orchids with (δ¹³C = -28.2 ± 0.1 ‰) and without access to maize roots (δ¹³C = −28.6 ± 0.2 ‰). We conclude that autotrophy is the predominant nutritional mode of mature S. strictiflora plants and that they supply their mycorrhizal fungus with substantial amount of carbon (69 ± 3 % of the fungus demand), even if the fungus feeds saprotrophically.     

Isolation and characterization of a fungus <Emphasis Type="Italic">Aspergillus</Emphasis> sp. strain F-3 capable of degrading alkali lignin

A fungus strain F-3 was selected from fungal strains isolated from forest soil in Dalian of China. It was identified as one Aspergillus sp. stain F-3 with its morphologic, cultural characteristics and high homology to the genus of rDNA sequence. The budges or thickened node-like structures are peculiar structures of hyphae of the strain. The fungus degraded 65% of alkali lignin (2,000 mg l⁻¹) after day 8 of incubation at 30°C at pH 7. The removal of colority was up to 100% at 8 days. The biodegradation of lignin by Aspergillus sp. F-3 favored initial pH 7.0. Excess acid or alkali conditions were not propitious to lignin decomposing. Addition of ammonium l-tartrate or glucose delayed or repressed biodegradation activities. During lignin degradation, manganese peroxidase (28.2 U l⁻¹) and laccase (3.5 U l⁻¹)activities were detected after day 7 of incubation. GC-MS analysis of biodegraded products showed strain F-3 could convert alkali lignin into small molecules or other utilizable products. Strain F-3 may co-culture with white rot fungus and decompose alkali lignin effectively.     

Biological control of the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> by application of the endoparasitic fungus <Emphasis Type="Italic">Esteya vermicola</Emphasis>

Esteya vermicola (Ophiostomataceae) is the first reported endoparasitic fungus of the pinewood nematode (PWN), Bursaphelenchus xylophilus (Nematoda: Aphelenchoidoidea). It has high in vitro infectivity. In this study, the nematocidal effect of E. vermicola in logs was investigated and evaluated. Two months after inoculation of pine wilt-killed Pinus densiflora logs with E. vermicola conidia suspensions of 3 × 10⁸ and 3 × 10⁶ ml⁻¹, the density of nematodes decreased by approximately 79% and 47%, respectively. When the fungus was sprayed on to four-year-old pine seedlings one month before PWN inoculation, the survival index of seedlings reached 0.67 compared with only 0.067 for control seedlings without fungal spraying. These results suggest that conidia spraying of E. vermicola can, to some extent, protect pine trees from wilt disease. Moreover, infected nematodes and hyphae of E. vermicola were observed in the treated wood sections.     

In situ release of coral mucus by <Emphasis Type="Italic">Acropora</Emphasis> and its influence on the heterotrophic bacteria

In situ mucus release by Acropora nobilis and degradation of mucus from A. nobilis and Acropora formosa, by heterotrophic bacteria were investigated at Bidong and Tioman Island, Malaysia. Mucus release rate for A. nobilis was on average 38.7 ± 35.2 mg C m⁻² h⁻¹, of which ca. 70% consisted of dissolved organic carbon (DOC) and 30% particulate organic carbon (POC). In the mucus degradation experiment, seawater-mucus mixtures were incubated and compared with control runs for 24 h. Bacterial abundance in the seawater-mucus mixture increased significantly and coincided with a decline in DOC concentration. In controls, bacteria and DOC did not significantly change. The coral mucus had a high content of inorganic phosphate. It is suggested that the coral mucus rich in DOC and phosphate can induce the high bacterial growth.     

Heterologous expression of an endo-β-1,4-glucanase gene from the anaerobic fungus <Emphasis Type="Italic">Orpinomyces</Emphasis> PC-2 in <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

The codon modified neutral endo-β-1,4-glucanase gene celEn, originating from the anaerobic fungus Orpinomyces sp. strain PC-2, was inserted between the strong promoter Pcel7A and the terminator Tcel7A from Trichoderma reesei. The resulting expression cassette was ligated to the pCAMBIA1300 Agrobacterium binary vector to construct pCB-hE that also contains a hygromycin B resistance marker. pCB-hE was introduced into T. reesei ZU-02 through an Agrobacterium tumefaciens–mediated transformation procedure that has been modified with an improved transformation efficiency of 12,500 transformants per 10⁷ conidia. Stable integration of the celEn gene into the chromosomal DNA of T. reesei ZU-02 was confirmed by PCR. After 48 h fermentation in shaking flasks, the endo-β-1,4-glucanase activities increased to 55–70 IU ml⁻¹ in transgenic strains, which were about 6–7 times higher than that of the original ZU-02 strain (9.5 IU ml⁻¹). When the avicel was added in fermentation medium, the endo-β-1,4-glucanase activity in the transgenic strains could be further increased to 193.6 IU ml⁻¹ after 84 h fermentation. Transgenic T. reesei strains with high neutral endo-β-1,4-glucanase activity will be particularly suitable for certain applications in textile industry. The improved procedures for overproduction and secretion of heterologous proteins in transgenic T. reesei can also be used to generate similar recombinant proteins for research or industrial purposes.     

Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger

The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h⁻¹ and was constant in the range 0.1–0.2 h⁻¹. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h⁻¹ irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l⁻¹ were obtained even though the strain contained only one copy of the glaA gene. Received: 5 May 1999 / Received revision: 7 September 1999 / Accepted: 17 September 1999     

Use of acephate, benomyl and alginate encapsulation for eliminating culture mites and fungal contamination from in vitro cultures of hardy hibiscus (Hibiscus moscheutos L.)

Summary Shoot cultures of three Hibiscus moscheutos (L.) cultivars were infested with micro-arthropods (mites). Nodal segments (1 cm long) were excised from these cultures and encapsulated in a sodium alginate gelled Driver and Kuniyuki Walnut DKW medium containing 10, 50, or 100 mg l⁻¹ acephate (insecticide) or 10 mg l⁻¹ acephate plus 0, 50, or 100 mg l⁻¹ benomyl (fungicide), then placed in refrigerated (5°C) darkness for 4 wk. Acephate was tested alone if visible fungus was not touching the shoot masses and benomyl was tested if fungus was in contact with the proliferating shoots. Cold-stored encapsulated nodes were then placed on DKW medium with 0.1 μM thidiazuron for 6 wk for subsequent shoot development. The presence of acephate in the encapsulation medium completely eradicated or killed the mites, with 38–69% of cultures fungus-free; 12% of the fungal-contaminated nodes encapsulated with 100 mg l⁻¹ benomyl were fungus-free.     

Cellular signalling of PCH-induced pigment aggregation in the crustacean Macrobrachium potiuna erythrophores

The cellular system responsible for the transduction of the pigment-concentrating hormone (PCH) signal was investigated in erythrophores of the freshwater shrimp, Macrobrachium potiuna. Dose-response curves to the hormone were determined in the absence and in the presence of several drugs that affect sequential steps of the Ca²⁺-dependent signalling pathway. Additionally, the ability of forskolin to induce pigment dispersion was evaluated. Neomycin sulphate (10⁻⁴ and 10⁻³ mol · l⁻¹), trifluoperazine (10⁻⁵ and 10⁻⁴ mol · l⁻¹), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10⁻⁷ and 10⁻⁵ mol · l⁻¹) and okadaic acid (10⁻⁷ mol · l⁻¹) significantly (P<0.05) decreased the responses to PCH. However, okadaic acid at low concentration (10⁻⁹ mol · l⁻¹) and cyclosporin A (10⁻⁶ and 10⁻⁵ mol · l⁻¹) did not significantly (P>0.05) affect PCH activity. Forskolin (10⁻⁴ mol · l⁻¹) was able to half-maximally reverse the hormone-induced aggregation. Our results suggest that the pigment-concentrating hormone induces pigment aggregation through a Ca²⁺-dependent pathway with a posteriori phosphatase activation, probably the serine/threonine phosphatase 1. Accepted: 30 June 1997     

The Actions of Calmodulin Antagonists W-7 and TFP and of Calcium on the Gating Kinetics of the Calcium-activated Large Conductance Potassium Channel of the Chara Protoplasmic Drop: A Substate-sensitive Analysis

The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca²⁺-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte plants. The large-conductance (170 pS), voltage- and Ca²⁺-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the Maxi-K channel from animal cells. This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes of action of- and different bindings sites for these calmodulin antagonists. Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten curve. W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent. In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the closed state at −50 mV but is distinguishable from it at voltages more negative than −100 mV. This substate may represent times that trifluoperazine is bound to the channel. Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect of Ca²⁻. This is good kinetic evidence against the view that the channel is activated by Ca²⁺-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca²⁻-calmodulin less available. Received: 26 August 1996/Revised: 7 October 1996     

Enhancement of a pentacyclic tyrosine kinase inhibitor production in Cladosporium cf. cladosporioides by Cladosporol

The binaphthyl derivative cladosporol 3 was supplied from 60 to 200 mg l⁻¹ to shaken cultures of Cladosporium cf. cladosporioides. Compared to blank, fungal biomass was not affected by adding cladosporol till 100 mg l⁻¹: it rather increased at higher ratios between 150 and 200 mg l⁻¹. The production of the major pentacyclic metabolite 1, a cytokine production and tyrosine kinase inhibitor, was enhanced tenfold when cladosporol was supplied at the highest ratio (200 mg l⁻¹) to shaken growing cultures of the fungus. The bioconversion of cladosporol to cladosporol D through reductive cleavage of the epoxide group was also observed. Interest in this kind of metabolites lies in their potential activity vs DNA topoisomerase I.