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

• Published in: Journal of applied microbiology 2005-01, Vol.98 (3), p.684-692
• Main Authors: Frisvad, J.C., Lund, F., Elmholt, S.
• Format: Article
• Language: English
• Subjects: AFLP fingerprinting; Avena; Biological and medical sciences; cereals; Denmark; DNA Fingerprinting; Edible Grain; Fundamental and applied biological sciences. Psychology; Genes, Fungal; Genetic Variation; Hordeum; Industrial Microbiology; Microbiology; ochratoxin A; Ochratoxins - biosynthesis; Penicillium - genetics; Penicillium - metabolism; Penicillium verrucosum; Polymorphism, Genetic; recombination; Sweden; Triticum; Triticum aestivum; United Kingdom
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2004.02509.x

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). Methods and Results:  A total of 321 isolates of P. verrucosum were isolated from ochratoxin A‐contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A‐producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP‐identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. Conclusions:  Among 184 ochratoxin A‐producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. Significance and Impact of the Study:  AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.