Ole Filtenborg

TERVERTICILLATE PENICILLIA: CHEMOTAXONOMY AND MYCOTOXIN PRODUCTION

A total of 4346 isolates of terverticillate Penicillium species was examined for production of my? cotoxins and other important secondary metabolites. Nearly all taxa and chemotypes (38 in all) produced one or more known mycotoxins consistently. Profiles of secondary metabolites were specific for each taxon, but several mycotoxins were produced by more than one species, suggesting a high degree of relatedness in the terverticillate penicillia. A high number of misidentified Penicillium isolates were reidentified, clarifying connections between taxa and profiles of mycotoxins and other secondary me? tabolites. Confirmed production of several mycotoxins in different taxa is reported for the first time. Penicillium chrysogenum, P. atramentosum, P. coprophilum, P. crustosum, P. expansum, P. glandicola var. glandicola and var. glaucovenetum, P. griseofulvum, P. hirsutum var. hirsutum, var. albocoremium, var. allii and var. hordei, and P. vulpinum are reported for the first time to be consistent producers of roquefortine C. Griseofulvin is reported from P. coprophilum. Tremorgen penitrem A was consistently produced by P. crustosum, P. glandicola var. glandicola and P. clavigerum. Terrestric acid is reported from P. hirsutum and its varieties, and P. crustosum. P. hirsutum var. hirsutum and P. solitum produce compactin. Penicillium mononematosum produces cyclopaldic acid and isochromantoxin. P. expansum was found to produce the mycotoxin chaetoglobosin C and P. atramentosum produces rugulovasine A. Several new varieties, chemotypes and combinations are proposed, based on profiles of secondary metabolites, morphology and physiological characters. Living ex type and authentic cultures of taxa believed to be synonyms of important species were examined and revised lists of synonyms are pre? sented.

Associated mycoflora of cheese

In this study of hard, semi-hard and semi-soft cheeses from Denmark, France, Greece, UK and other countries, 371 fungal isolates were identified of which 91% were Penicillium species. Penicillium commune was the most widespread and most frequently occurring (42%) species. Most of the isolates (88%) found on cheese belonged to the following species: P. commune, P. nalgiovense, P. verrucosum, P. solitum, P. roqueforti, Aspergillus versicolor, P. crustosum, P. atramentosum, P. chrysogenum and P. echinulatum. Mycological investigations in cheese factories showed that control of cheese smear contamination was important in an attempt to prevent mould growth on cheese. Some species showed a consistent ability to produce mycotoxins: P. commune produced cyclopiazonic acid, P. verrucosum produced ochratoxin A, A. versicolor produced sterigmatocystin and P. crustosum produced penitrem A and roquefortine C.

Analysis and screening for mycotoxins and other secondary metabolites in fungal cultures by thin-layer chromatography and high-performance liquid chromatography.

Methods for the screening of fungal cultures for toxic secondary metabolites are reviewed. Thin layer Chromatography (TLC) and high-performance liquid Chromatography (HPLC) are good general analytical methods for secondary metabolites in unpurified extracts. The combination of normal phase TLC using different chemical spray reagents with reversed phase HPLC, using alkylphenone retention indices and diode array detection, is a powerful technique for identifying the individual mycotoxins detected. The results of the screening methods are very dependent of the growth media and incubation conditions and a general method for the detection ofPenicillium, Aspergillus, Fusarium, Alternaria andCladosporium toxins is suggested.

The Significance of Yeast Extract Composition on Metabolite Production in Penicillium

In the literature and in our own experience, significant variations occur in morphological characteristics and production of secondary metabolites by cultures grown on YES (2% yeast extract and 15% sucrose) agar, a substrate which is often used in screening for mycotoxins in moulds. In this investigation we have demonstrated a very significant influence of yeast extract brand (Difco, Sigma Y4000 and Y0325, Oxoid, Merck, Lab M and Gibco) on the production of mycotoxins in YES by some important Penicillia. Using a TLC screening method the variation in mycotoxin production due to the use of different brands of yeast extract ranged between detection in 5 days and none detected in 4 weeks. The difference in mycotoxin production was often accompanied by differences in several other characteristics like pH changes of the substrate, sporulation, colony diameter and reverse colour. We have been unable to find which components in the yeast extracts were responsible for the observed changes, but the addition of MgSO4 appeared to be a satisfactory compensation in most respects. So it is suggested that this compound in general is added to the YES formula, along with previously suggested compounds like ZnSO4 and CuSO4, thus making this substrate a very valuable and reliable tool in screening for production of secondary metabolites and in mould taxonomy. Further it is suggested to use pH registration monitoring in the cultures parallel to screening for secondary metabolites, since pH differences proved to be a useful indication of significant changes in the detected profiles.

The Systematics of the Terverticillate Penicillia

The species of the terverticillate Penicillia are re-investigated and delimited on the basis of the morphology of the conidiophores, phialides and conidia. In addition, growth characters and profiles of secondary metabolites were taken into account for definition of the taxa. In general, the terverticillate Penicillia represent a biologically homogenous group, but on the basis of their morphology subdivision into series is proposed. The series and the accepted taxa are discussed briefly and keyed out dichotomously. A list of the principal mycotoxins produced by each taxon is presented. Most species can be identified using morphological and cultural characters as observed on Czapek and 2% malt extract agar, while a more detailed speciation requires the use of a standardized medium regime including Czapek yeast extract agar, creatine sucrose agar and 5% acetic acid agar.

Associated mycoflora of rye bread

F. Lund, O. Filtenborg, S. Westall And J.C. Frisvad. 1996. Penicillium roqueforti (27%), P. corylophilum (20%) and Eurotium (15%) made up the important mycoflora associated with rye bread on 3425 identified fungi isolates. These fungi were dominant as spoilers of packaged rye bread in almost every month of a 4 year investigation. Penicillium decumbens (3%), Paecilomyces variotii (8%) and Aspergillus flavus (5%) were found more rarely, but were the major species found over periods of a few months. Penicillium commune (5%), P. Solitum (4%), A. niger (4%) and Mucor species (2%) were a constant, but small, part of the mycoflora of rye bread. Identification of the fungi in the production environmentl in a rye bread factory showed the locality of potential contamination sources. Eliminationl of the contmination sources lby improved cleaning and disinfection procedures quickly resulted in a significant reduction in the frequency of mould growth in the packaged rye bread.