Flemming Lund

Penicillium verrucosum in wheat and barley indicates presence of ochratoxin A

Aims: The aims of this study were to isolate and identify ochratoxin A (OTA) producing fungi in cereals containing OTA and to determine the best selective and indicative medium for recovery of OTA producing fungi.

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.

Czapek‐Dox agar containing iprodione and dicloran as a selective medium for the isolation of Fusarium species

A new selective medium for Fusarium species has been developed using Czapek‐Dox agar (CZ) containing the fungicides iprodione (3 mg/1) and dicloran (2 mg/1). This new medium (CZID) is selective against numerous species of Alternaria, Epicoccum, Penicillium and mucoraceous fungi. CZID was compared with CZ using samples of barley, malt, sorghum, bean and pea. Fusarium species produced large and easily recognizable colonies on CZID while isolates of Alternaria, Epicoccum and Rhizopus were significantly restricted on CZID compared with their growth on CZ. The use of CZID thus facilitates the isolation and subculturing of Fusarium species.

Chemotaxonomy of Penicillium aurantiogriseum and related species

Penicillium aurantiogriseum sensu lato is the most common cereal-borne Penicillium species of worldwide occurrence and it has a complex taxonomy. A total of 519 isolates, including most of the NRRL cultures used by Raper & Thom (1949) in their P. cyclopium, P. viridicatum and P. ochraceum series, was examined for expressions of differentiation, especially of secondary metabolites. The fungi were micromorphologically quite similar. Isolates previously allocated to P. cyclopium and P. viridicatum were separated into nine species based on biosynthetic families of secondary metabolites and from several less conspicuous morphological and physiological differences. The species accepted were P. aurantiogriseum, P. aurantiovirens, P. cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. tricolor and P. viridicatum . These species produced characteristic combinations of the following mycotoxins: xanthomegnin, viomellein, vioxanthin, penicillic acid, terrestric acid, viridic acid, verrucosidin, penitrem A and the other secondary metabolites: viridamine, aurantiamine, auranthine, verrucofortine, puberuline, cyclopeptin, dehydrocyclopeptin, cyclopenin, cyclopenol, 3-methoxyviridicatin, viridicatol, brevianamide A & B, meleagrin and oxaline. All the known secondary metabolites could be allocated to biosynthetic families based on standards and liquid chromatography — diode array detection. Some isolates of P. aurantiogriseum and P. polonicum are known to produce nephrotoxic glycopeptides possibly associated with Balkan endemic nephropathy. It is recommended that isolates should be identified to the species level using a combination of morphological, physiological and secondary metabolite data.

Differentiating Penicillium species by detection of indole metabolites using a filter paper method

The indole secondary metabolites chaetoglobosin C, cyclopiazonic acid, isofumigaclavine A and rugulovasine A and B produced by several Penicillium species growing on Czapek yeast autolysate agar were detected directly in the culture using filter paper wetted with Ehrlich reagent dissolved in ethanol. The filter paper was placed on the mycelial side of an agar plug and the metabolites were visualized as a violet zone on the paper within 10 min. It was shown that the combined characters of the violet reaction on filter paper and the ability to grow on creatine sucrose agar occurred in 5 out of 16 species of Penicillium examined. A few additional simple morphological and physiological criteria were then sufficient for identification of P. camemberti, P. commune, P. discolor, P. expansum and P. roqueforti var. roqueforti.

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

Aims:  To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A‐producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP).

Distribution of Penicillium commune isolates in cheese dairies mapped using secondary metabolite profiles, morphotypes, RAPD and AFLP fingerprinting

In an 8-year study of the diversity and distribution of Penicillium commune contaminants in two different cheese dairies, swab and air samples were taken from the production plants, the processing environment and contaminated cheeses. A total of 321 Penicillium commune isolates were characterized using morphotypes (colony morphology and colours) and secondary metabolite profiles. Based on production of secondary metabolites the P. commune isolates were classified into 6 groups. The genetic diversity of the P. commune isolates was assessed using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). For a sub-set of 272 P. commune isolates RAPD analysis generated 33 RAPD groups whereas AFLP profiling revealed 55 AFLP groups. This study conclusively showed that the discriminatory power of AFLP was high compared to RAPD and that AFLP fingerprinting matched morphotyping. P. commune isolates with identical profiles using all four typing techniques were interpreted as closely related isolates with a common origin and the distribution of these isolates in the processing environment indicated possible contamination points in the cheese dairies. The coating process and unpacking of cheeses with growth of P. commune seemed to cause the contamination problems. Several identical P. commune isolates remained present in the processing environment for more than 7 years in both dairies.

Diagnostic characterization of Penicillium palitans, P. commune and P. solitum

A total of 98 isolates of Penicillium commune and P. solitum were analysed and it was shown that these isolates produced three unique combinations of secondary metabolites. Penicillium commune produced cyclopiazonic acid, cyclopaldic acid and rugulovasine A and B; P. solitum produced compactin and a new group including the ex‐type culture of P. palitans produced cyclopiazonic acid and fumigaclavine A. Isolates in all three groups were able to produce cyclopenin, cyclopenol and viridicatin. An optimal thin layer chromatography system to detect the secondary metabolites from P. commune, P. palitans and P. solitum was developed using an agar plug method. The results were confirmed by high performance liquid chromatography with diode array detection. The chemotaxonomic allocations were backed up by differences in conidial colour on Czapek yeast autolysate agar, reverse colour on yeast extract sucrose agar and origin of the isolates. Even though P. palitans previously has been considered synonymous with P. commune or P. solitum it was concluded that P. palitans is a distinct species.

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.

Direct identification of the common cheese contaminant Penicillium commune in factory air samples as an aid to factory hygiene

F. LUND. 1996. Creatine sucrose dichloran agar (CREAD) was used as a selective medium for Penicillium commune and related species found in air samples in a cheese factory. Using growth and simple colony characters on CREAD together with detection of indole metabolites with a filter paper method, it was possible to identify all 22 P. commune isolates from a total of 43 Penicillium isolates. Penicillium commune numbers on CREAD were compared with those found on a general isolation medium, dichloran 18% glycerol agar.