Birgitte Andersen

The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi

A secondary metabolite is a chemical compound produced by a limited number of fungal species in a genus, an order, or even phylum. A profile of secondary metabolites consists of all the different compounds a fungus can produce on a given substratum and includes toxins, antibiotics and other outward-directed compounds. Chemotaxonomy is traditionally restricted to comprise fatty acids, proteins, carbohydrates, or secondary metabolites, but has sometimes been defined so broadly that it also includes DNA sequences. It is not yet possible to use secondary metabolites in phylogeny, because of the inconsistent distribution throughout the fungal kingdom. However, this is the very quality that makes secondary metabolites so useful in classification and identification. Four groups of organisms are particularly good producers of secondary metabolites: plants, fungi, lichen fungi, and actinomycetes, whereas yeasts, protozoa, and animals are less efficient producers. Therefore, secondary metabolites have mostly been used in plant and fungal taxonomy, whereas chemotaxonomy has been neglected in bacteriology. Lichen chemotaxonomy has been based on few biosynthetic families (chemosyndromes), whereas filamentous fungi have been analysed for a wide array of terpenes, polyketides, non-ribosomal peptides, and combinations of these. Fungal chemotaxonomy based on secondary metabolites has been used successfully in large ascomycete genera such as Alternaria, Aspergillus, Fusarium, Hypoxylon, Penicillium, Stachybotrys, Xylaria and in few basidiomycete genera, but not in Zygomycota and Chytridiomycota.

Production of mycotoxins on artificially and naturally infested building materials

In this study, the ability to produce mycotoxins during growth on artificially infested building materials was investigated for Penicillium chrysogenum, Pen. polonicum, Pen. brevicompactum, Chaetomium spp., Aspergillus ustus, Asp. niger, Ulocladium spp., Alternaria spp., and Paecilomyces spp., all isolated from water-damaged building materials. Spores from the different isolates of the above mentioned species were inoculated on gypsum board with and without wallpaper and on chipboard with and without wallpaper. Fungal material was scraped off the materials, extracted, and analyzed using high performance liquid chromatography-diode array detection and thin layer chromatography. All six isolates of C. globosum produced the toxic chaetoglobosins A and C, at levels of up to 50 and 7 μg/cm2 respectively. The quantities of secondary metabolites produced by Penicillia were generally low, and no toxin production was detected from any of the five isolates of Pen. chrysogenum. Both isolates of Pen. polonicum produced 3-methoxy-viridicatin, verrucosidin, and verrucofortine. Two of five isolates of Pen. brevicompactum produced mycophenolic acid. From five out of six isolates of Alternaria spp., alternariol and alternariol monomethyl ether were detected. From Ulocladium spp., Paecilomyces spp., and Asp. ustus no known mycotoxins were detected, although the latter two are known mycotoxin producers. Asp. niger produced several naphtho-γ-pyrones and tetra-cyclic compounds. All investigated species, especially Asp. ustus and Asp. niger produced many unknown secondary metabolites on the building materials. Analyses of wallpaper and glass-fibre wallpaper naturally infested with Asp. versicolor revealed sterigmatocystin and 5-methoxysterigmatocystin. Analyses of naturally infested wallpaper showed that C. globosum produced the chaetoglobosins A and C, and Pen. chrysogenum produced the antibiotic meleagrin.

Associations between Fungal Species and Water-Damaged Building Materials

ABSTRACT Fungal growth in damp or water-damaged buildings worldwide is an increasing problem, which has adverse effects on both the occupants and the buildings. Air sampling alone in moldy buildings does not reveal the full diversity of fungal species growing on building materials. One aim of this study was to estimate the qualitative and quantitative diversity of fungi growing on damp or water-damaged building materials. Another was to determine if associations exist between the most commonly found fungal species and different types of materials. More than 5,300 surface samples were taken by means of V8 contact plates from materials with visible fungal growth. Fungal identifications and information on building material components were analyzed using multivariate statistic methods to determine associations between fungi and material components. The results confirmed that Penicillium chrysogenum and Aspergillus versicolor are the most common fungal species in water-damaged buildings. The results also showed Chaetomium spp., Acremonium spp., and Ulocladium spp. to be very common on damp building materials. Analyses show that associated mycobiotas exist on different building materials. Associations were found between (i) Acremonium spp., Penicillium chrysogenum, Stachybotrys spp., Ulocladium spp., and gypsum and wallpaper, (ii) Arthrinium phaeospermum, Aureobasidium pullulans, Cladosporium herbarum, Trichoderma spp., yeasts, and different types of wood and plywood, and (iii) Aspergillus fumigatus, Aspergillus melleus, Aspergillus niger, Aspergillus ochraceus, Chaetomium spp., Mucor racemosus, Mucor spinosus, and concrete and other floor-related materials. These results can be used to develop new and resistant building materials and relevant allergen extracts and to help focus research on relevant mycotoxins, microbial volatile organic compounds (MVOCs), and microparticles released into the indoor environment.

Species and ecological diversity within the Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales)

The genus Cladosporium is one of the largest genera of dematiaceous hyphomycetes, and is characterised by a coronate scar structure, conidia in acropetal chains and Davidiella teleomorphs. Based on morphology and DNA phylogeny, the species complexes of C. herbarum and C. sphaerospermum have been resolved, resulting in the elucidation of numerous new taxa. In the present study, more than 200 isolates belonging to the C. cladosporioides complex were examined and phylogenetically analysed on the basis of DNA sequences of the nuclear ribosomal RNA gene operon, including the internal transcribed spacer regions ITS1 and ITS2, the 5.8S nrDNA, as well as partial actin and translation elongation factor 1-α gene sequences. For the saprobic, widely distributed species Cladosporium cladosporioides, both a neotype and epitype are designated in order to specify a well established circumscription and concept of this species. Cladosporium tenuissimum and C. oxysporum, two saprobes abundant in the tropics, are epitypified and shown to be allied to, but distinct from C. cladosporioides. Twenty-two species are newly described on the basis of phylogenetic characters and cryptic morphological differences. The most important phenotypic characters for distinguishing species within the C. cladosporioides complex, which represents a monophyletic subclade within the genus, are shape, width, length, septation and surface ornamentation of conidia and conidiophores; length and branching patterns of conidial chains and hyphal shape, width and arrangement. Many of the treated species, e.g., C. acalyphae, C. angustisporum, C. australiense, C. basiinflatum, C. chalastosporoides, C. colocasiae, C. cucumerinum, C. exasperatum, C. exile, C. flabelliforme, C. gamsianum, and C. globisporum are currently known only from specific hosts, or have a restricted geographical distribution. A key to all species recognised within the C. cladosporioides complex is provided.

Characterization of Stachybotrys from water-damaged buildings based on morphology, growth, and metabolite production

Stachybotrys was found to be associated with idiopathic pulmonary hemorrhage in infants in Cleveland, Ohio. Since that time, considerable effort has been put into finding the toxic components responsible for the disease. The name Stachybotrys chartarum has been applied to most of these isolates, but inconsistent toxicity results and taxonomic confusion prompted the present study. In this study, 122 Stachybotrys isolates, mainly from water-damaged buildings, were characterized and identified by combining three different approaches: morphology, colony characteristics, and metabolite production. Two different Stachybotrys taxa, S. chartarum and one undescribed species, were found in water-damaged buildings regardless of whether the buildings were in Denmark, Finland, or the USA. Furthermore, two chemotypes could be distinguished in S. chartarum. One chemotype produced atranones, whereas the other was a macrocyclic trichothecene-producer. The second undescribed taxon produced atranones and could be differentiated from S. chartarum by its growth characteristics and pigment production. Our results correlate with different inflammatory and toxicological properties reported for these same isolates and show that the three taxa/chemotypes should be treated separately. The co-occurrence of these three taxa/chemotypes in water-damaged buildings explains the inconsistent results in the literature concerning toxicity of Stachybotrys isolated from that environment.

Chemical and morphological segregation of Alternaria arborescens , A. infectoria and A. tenuissima species-groups

Correct morphological identification of Alternaria is important and demands a combination of modern standardised methods and up-to-date literature. The production of secondary metabolites has previously been used as a means of identification and classification. In this study, 153 fungal isolates belonging to the genus Alternaria were examined. They were grown under standardised conditions and subjected to morphological and chemical examination. All isolates were grouped according to their three-dimensional sporulation pattern on potato carrot agar and their colony colour on dichloran rose bengal yeast extract sucrose agar (DRYES). After extraction, all isolates were analysed by a high performance liquid chromatograph equipped with a diode array detector and the resulting metabolite profiles were subjected to multivariate statistic analyses. The analyses of metabolite profiles showed that the isolates could be divided into three major species-groups that were morphologically identifiable as the A. infectoria species-group, the A. arborescens species-group and the A. tenuissima species-group. The A. infectoria species-group is chemically very different from both the A. arborescens and the A. tenuissima species-groups with only a few metabolites in common. None of the 35 A. infectoria species-group isolates produced any known metabolites and all had white or greyish white colonies on DRYES. The A. arborescens species-group and the A. tenuissima species-group, shared most of the known metabolites and had colonies of various shades of green on DRYES. One cluster of isolates belonging to the A. tenuissima species-group was able to produce tentoxin, which has not been reported previously from any A. tenuissima isolate. The results suggest that each species-group contains several taxa and these taxa need to be formally described before species specific metabolite profiles can be established.

Chemical and morphological segregation of Alternaria alternata, A. gaisen and A. longipes

Correct identification of fungi to species level is important because a specific epithet embodies a set of characters that enables us to predict, for example, the mycotoxin production of a species. Many small-spored Alternaria isolates have been misidentified due to inappropriate growth conditions and the use of spore size as the only identifying character. In this study 39 Alternaria isolates were grown under standardised conditions and subjected to chemical, morphological and physiological analyses. All isolates were extracted and analysed by HPLC-DAD. Analysis showed that both A. gaisen and A. longipes were able to produce altertoxin 1, which has not previously been reported. The resulting metabolite profiles were subjected to cluster analysis and principal component analysis. A subset of the isolates was grown at five different temperatures. Colony colour and diameter were recorded and the diameter measurements were subjected to principal component analysis. Analysis of chemical and physiological data showed that the 39 isolates segregated into the same distinct groups that are morphologically identifiable as A. alternata, A. longipes or A. gaisen. The results showed that A. longipes, A. gaisen and A. alternata are different species that can be distinguished morphologically, physiologically and chemically. Therefore, the continued use of the name Alternaria alternata for A. longipes and A. gaisen is unwarranted and pathotypes should not be used.

RAPD fragment pattern analysis and morphological segregation of small-spored Alternaria species and species groups

Accurate identification of small-spored Altemaria spp. is challenging because of morphological plasticity under non-standard conditions and the common misapplication of the name A. alternata to a variety of morphologically distinct taxa. A system used by some authors of naming phytotoxigenic alternarias as pathotypes of A. alternata has further clouded the meaning and usefulness of this specific epithet. Two hundred and sixty isolates of small-spored Alternaria , primarily from fruit substrates, were segregated into morphological groups and then subjected to RAPD-PCR analysis using total genomic DNA and three different primers. When cultured under defined conditions, the pattern of sporulation observed at 50 × magnification was predictive of genetic relatedhess as determined by cluster analysis of RAPD fragment patterns. In RAPD analyses, morphological groups or species were resolved as distinct branches of the dendrogram: Alternaria gaisen (= A. kikuchiana, A. alternata Japanese pear pathotype, group 2), A. longipes (= A. alternata tobacco pathotype, group 5), the ‘ tenuissima ’ group (group 5), the arborescens group (group 3) and the ‘ infectoria group (group 6). Isolates in groups for which there were only a few representatives clustered as a branch. Analysis of RAPD fragment patterns confirmed that when Altemaria isolates are cultured and observed under defined conditions, their phenotypic plasticity is minimized and valid taxonomic separations can be made upon morphological characteristics. We conclude that A. alternata, A. gaisen, A. longipes and other small-spored saprotrophic or perthotrophic taxa are recognizable as morphologically distinct taxa. We suggest that the ‘pathotype’ system of naming small-spored Altemaria taxa confers no predictive value relative to observable morphological and genetic characters, and should be abandoned.

Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings

Twenty-five Stachybotrys isolates from two previous studies have been examined and compared, using morphological, chemical and phylogenetic methods. The results show that S. chartarum sensu lato can be segregated into two chemotypes and one new species. The new species, S. chlorohalonata, differs morphologically from S. chartarum by having smooth conidia, being more restricted in growth and producing a green extracellular pigment on the medium CYA. S. chlorohalonata and S. chartarum also have different tri5, chs1 and tub1 gene fragment sequences. The two chemotypes of S. chartarum, chemotype S and chemotype A, have similar morphology but differ in production of metabolites. Chemotype S produces macrocyclic trichothecenes, satratoxins and roridins, while chemotype A produces atranones and dolabellanes. There is no difference between the two chemotypes in the tub1 gene fragment, but there is a one nucleotide difference in each of the tri5 and the chs1 gene fragments.

Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages

The metabolite profiles of 20 Stachybotrys spp.isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFα in murine RAW264.7 macrophage cells were studied. The 11isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity ofStachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure.