Siegfried Draeger

Endophytic fungi: a source of novel biologically active secondary metabolites*

In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been found to be superior to combinatorial chemistry for discovering novel substances that have the potential to be developed into new industrial products. Since natural products are adapted to a specific function in nature, the search for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes. Endophytic fungi inhabit such a biotope. In the course of the last 12 years, we have isolated ~ 6500 endophytic fungi from herbaceous plants and trees, screened them for biological activities, and have isolated and determined the structures of the biologically active compounds. Correlations were found between biological activity and biotope, e.g. a higher proportion of the fungal endophytes, in contrast to the soil isolates, inhibited at least one of the test organisms for antialgal and herbicidal activities. The substances isolated originated from different biosynthetic pathways: isoprenoid, polyketide, amino acid derivatives, and belonged to diverse structural groups: terpenoids, steroids, xanthones, chinones, phenols, isocumarines, benzopyranones, tetralones, cytochalasines, and enniatines. The potential role of the endophyte and its biologically active metabolites in its association with its host has been investigated. The fungal endophytes possess the exoenzymes necessary to colonize their hosts and they grow well in the apoplastic washing fluid of the host. When the roots of larch are colonized, the association with the host may be mutualistic, improving growth of the host and supplying the mycobiont with enough nourishment to extensively colonize the hosts roots. The concentrations of some plant defence metabolites are lower than in the control when the host is infected with a pathogen than with an endophyte. We hypothesize that the interaction fungal endophyte-plant host is characterized by a finely tuned equilibrium between fungal virulence and plant defence. If this balance is disturbed by either a decrease in plant defence or an increase in fungal virulence, disease develops. Not only must the endophyte synthesize metabolites to compete first with epiphytes and then with pathogens in order to colonize the host, but presumably also to regulate metabolism of the host in their delicately balanced association. The utilization of a biotope such as that of the fungal endophyte is one aspect of intelligent screening, another very important one is the taxonomy of the fungus in order to avoid redundant structural isolations. It is not a random walk through a random forest. Many groups of fungi in different biotopes are waiting to be exploited.

Endophytes from herbaceous plants and shrubs: effectiveness of surface sterilization methods

Seven common methods of surface sterilization were compared for their effectiveness in isolating endophytes from herbaceous plants and shrubs growing in or near Brunswick, Germany. Only one method proved to be ineffective. Up to 16 different endophytic strains were isolated from each of 11 of the 12 plants tested. The role of endophytes as latent pathogens is discussed.

Fungi from marine sponges: diversity, biological activity and secondary metabolites.

The diversity, biological activity and secondary metabolite production of fungi associated with marine sponges were investigated in order to assess the potential of these fungi for the production of novel biologically active secondary metabolites. 681 fungal strains were isolated from 16 sponge species from six different locations, representing 13 genera of Ascomycota, 2 of Zygomycota, 37 of mitosporic fungi, and 37 strains of sterile mycelium. The spectrum of fungi from each location was dominated by a few genera of mainly mitosporic fungi that are also commonly encountered in terrestrial habitats. A high proportion of culture extracts of sponge-associated fungi was biologically active in tests to detect antifungal, antialgal, and antibacterial activity, cytotoxicity and inhibition of reverse-transcriptase and tyrosine kinase. The corresponding chemistry was structurally diverse and related to that of terrestrial fungi. From the biologically active culture extracts of five fungal strains, nine pure compounds were isolated in addition to 18 previously reported.

Dihydroisocoumarins from fungi : Isolation, structure elucidation, circular dichroism and biological activity

Five known and three new dihydroisocoumarins were isolated from different fungi. The new isocoumarins are 5-chloro-6-hydroxymellein, 5-chloro-4,6-dihydroxymellein and 5,6-dihydroxymellein. The absolute configuration of these secondary metabolites was confirmed by CD measurements and in two cases by X-ray structure analysis.

New Mono‐ and Dimeric Members of the Secalonic Acid Family: Blennolides A–G Isolated from the Fungus Blennoria sp.

Blennolides A-G (2-8), seven unusual chromanones, were isolated together with secalonic acid B (1) from Blennoria sp., an endophytic fungus from Carpobrotus edulis. This is the first reported isolation of the blennolides 2 and 3 (hemisecalonic acids B and E), the existence of which as the monomeric units of the dimeric secalonic acids had long been postulated. A compound of the proposed structure 4 (beta-diversonolic ester) will need to be revised, as its reported data do not fit those of the established structure of blennolide C (4). Other monomers, the blennolides D-F (5-7) seem to be derived from blennolides A (2) and B (3) by rearrangement of the hydroaromatic ring. The heterodimer 8, composed of the monomeric blennolide A (2) and the rearranged 11-dehydroxy derivative of blennolide E (6), extends the ergochrome family with an ergoxanthin type of skeleton. The structures of the new compounds were elucidated by detailed spectroscopic analysis and further confirmed by an X-ray diffraction study of a single crystal of 2. The absolute configurations were determined by TDDFT calculations of CD spectra, including the solid-state CD/TDDFT approach. Preliminary studies showed strong antifungal and antibacterial activities of these compounds against Microbotryum violaceum and Bacillus megaterium, respectively. They were also active against the alga Chlorella fusca and the bacterium Escherichia coli.

Bioactive Isocoumarins Isolated from the Endophytic Fungus Microdochium bolleyi

Three new isocoumarin derivatives ( 2- 4) were isolated together with monocerin ( 1) from Microdochium bolleyi, an endophytic fungus from Fagonia cretica, a herbaceous plant of the semiarid coastal regions of Gomera. Compounds 2 and 3 are both 12-oxo epimers of 1, and 4 is a ring-opened derivative of 1. The structures were elucidated by detailed spectroscopic analysis and comparison with reported data. The absolute configurations were determined by a modified Moshers method. Compounds 1, 3, and 4 showed good antifungal, antibacterial, and antialgal activities against Microbotryum violaceum, Escherichia coli, Bacillus megaterium, and Chlorella fusca. Compound 2 was moderately antifungal and antialgal.

Detection and identification of fungi intimately associated with the brown seaweed Fucus serratus.

ABSTRACT The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.

Sponge-associated fungi and their bioactive compounds - the Suberites case

Specimens of Suberites domuncula that had been cultured in aquaria for 4 weeks were analyzed for their associated fungi. A total of 81 fungal strains belonging to 20 different genera was isolated and identified by morphological and molecular methods. The most frequently isolated taxa were Cladosporium spp., Penicillium spp., Petriella sp., Phialophora spp. and Engyodontium album. Based on chromatographic and mass spectrometric analysis of fungal extracts, as well as on bioassay results, Aspergillus ustus, Penicillium sp., Petriella sp. and Scopulariopsis sp. were selected for in-depth analysis of their natural products. A total of 19 different fungal metabolites, including three new natural products, was isolated and structurally identified. A. ustus yielded two sesquiterpenes, a drimane derivative and deoxyuvidin, as well as a sesterterpene ophiobolin H. The drimane derivative had an ED50 value against L5178Y cells of 1.9 mu g ml(-1) in vitro. The crude extract of Petriella sp. was also strongly cytotoxic against the L5178Y cell line. The cyclic tetrapeptide WF-3161 was primarily responsible for the activity; the ED50 value was <0.1 mu g ml(-1). It was identical to the known compound WF-3161 and had been previously isolated from Petriella guttulata. In addition to WF-3161, three further natural products were obtained and unequivocally identified as new derivatives of infectopyrone by one-and two-dimensional NMR spectroscopy and by mass spectroscopy. Of the new compounds, only dihydroinfectopyrone was active against L5178Y cells; the ED50 value was 0.2 mu g ml(-1). Penicillium sp. yielded the largest number of metabolites. Viridicatin, viridicatol, cyclopenin and cyclopenol suppressed larval growth of the polyphagous pest insect Spodoptera littoralis when incorporated into an artificial diet at an arbitrarily chosen concentration of 237 ppm. Viridicatol was the most active compound and had an ED50 value of ca. 50 ppm. Scopulariopsis sp. yielded three metabolites, including the known acetylcholinesterase inhibitors quinolactacin A1 and A2.

Bioactive nonanolide derivatives isolated from the endophytic fungus Cytospora sp.

Cytospolides F-Q (6-17) and decytospolides A and B (18 and 19), 14 unusual nonanolide derivatives, were isolated from Cytospora sp., an endophytic fungus from Ilex canariensis. The structures were elucidated by means of detailed spectroscopic analysis, chemical interconversion, and X-ray single crystal diffraction. The solution- and solid-state conformers were compared by the combination of experimental methods (X-ray, NMR) supported by DFT calculations of the conformers. Absolute configurations were assigned using the modified Moshers method and solution- and solid-state TDDFT ECD calculations. In an in vitro cytotoxicity assay toward the tumor cell lines of A549, HCT116, QGY, A375, and U973, the γ-lactone 17 demonstrated a potent growth inhibitory activity toward the cell line A-549, while nonanolide 16 with (2S) configuration showed the strongest activity against cell lines A-549, QGY, and U973. A cell cycle analysis indicated that compound 16 can significantly mediate G1 arrest in A549 tumor cells, confirming the important role of the C-2 methyl in the growth inhibition toward the tumor line. The discovery of an array of new nonanolides demonstrates the productivity of the fungus, and it is an example of chemical diversity, extending the nonanolide family by derivatives formed by ring cleavage, oxidation, esterification, and Michael addition.

Xanthones and oxepino[2, 3-b]chromones from three endophytic fungi.

Three new metabolites, microsphaeropsones A-C (1-3) with a unique oxepino[2,3-b]chromen-6-one (ring-enlarged xanthone) skeleton, were isolated from the endophytic fungus Microsphaeropsis species, co-occurring with their putative biogenetic anthraquinoide precursors citreorosein (4) and emodin (5). From another Microsphaeropsis species, large amounts of fusidienol A (8 a), smaller amounts of emodin (5), the known aromatic xanthones 9 a and 9 b, the new 3,4-dihydrofusidienol A (8 b), and the new aromatic xanthone 9 c were isolated. The endophyte Seimatosporium species produced a new aromatic xanthone, seimatoxanthone A (10), and 3,4-dihydroglobosuxanthone A (12), closely related to alpha-diversolonic ester (13) from Microdiplodia sp.. The structures were determined mainly by extensive 1D and 2D NMR experiments and supported by X-ray single-crystal analysis of 1 and the oxidation product 7. The absolute configurations of the microsphaeropsones A-C (1-3) were established by comparison of the electronic and vibrational circular dichroism (ECD and VCD) spectra of 1 with time-dependent DFT (TDDFT) and DFT calculations by using either the solid-state structures or DFT-optimized geometries as inputs. Preliminary studies indicated that 1, 2, and enone 7 showed antibacterial, fungicidal, and algicidal properties.