Pernille Skouboe

Technical University of Denmark, København, Capital Region, Denmark

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Publications (11)26.05 Total impact

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    ABSTRACT: Some Alternaria species are able to produce plant pathogenic as well as toxic metabolites. In both agriculture and the food industry it is important know if toxigenic Alternaria are present to rapidly employ the correct corrective actions. The purpose of this work was to establish a real-time PCR method, which can detect and quantify apple pathogenic and toxigenic Alternaria. An AM-toxin I primer set, which could recognize Alternaria DNA only, was designed by using primers complementary to the AM-toxin I gene. The method could detect small amounts of DNA (4 pg) and still obtain a large dynamic range (4 decades) without interference from apple material. Eight Alternaria isolates were analyzed for the presence of AM-toxin I gene and their production of secondary metabolites. Then analyses showed that all eight isolates contained the AM toxin gene and were able to produce the plant pathogenic tentoxin in addition to AM toxin I. The analyses also showed the production of tenuazonic acid, alternariols, Altenuene, altenusin and/or altertoxin I in pure culture. Analyses of inoculated apples showed that both the AM-toxin gene and alternariol monomethyl ether could be detected. Morphological analyses suggested that the eight Alternaria strains, though they all carried the AM toxin genes, probably belong to different but closely related un-described Alternaria taxa in the A. tenuissima species-group based on morphological and chemical differences.
    International Journal of Food Microbiology 10/2006; 111(2):105-11. · 3.43 Impact Factor
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    ABSTRACT: Cereal grain may be infected with a number of Fusarium species some of which are producers of highly toxic compounds such as the trichothecenes. Correct identification of these species is essential for risk assessment of cereal grain for human or animal consumption. Most of the available methods for identification are either time consuming or aimed at only one or a few target species. Microarray technology offers parallel analysis of a high number of DNA targets. In this study 57 capture oligonucleotides (CO) were designed based upon Fusarium ITS2 rDNA sequences, and used for microarray production. From this array COs could be selected that were able to hybridise specifically to labelled PCR products from the ITS region of Fusarium graminearum/Fusarium culmorum, Fusarium pseudograminearum, Fusarium poae, Fusarium sporotrichioides, Fusarium equiseti, Fusarium langsethiae and Fusarium tricinctum/Fusarium avenaceum. A few COs showed some cross hybridisation to non-target species. In a preliminary experiment it was shown that this cross hybridisation could be eliminated by increasing hybridisation stringency. The array could be used to detect individual Fusarium species in mixed samples and in environmental samples. This study demonstrates the feasibility of oligonucleotide microarrays for parallel detection of a number of Fusarium species.
    Journal of Microbiological Methods 08/2005; 62(1):57-69. · 2.16 Impact Factor
  • Jens C Frisvad, Pernille Skouboe, Robert A Samson
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    ABSTRACT: Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded as a synonym of A. nomius, while A. toxicarius resembles A. parasiticus but differs with at least three base pair differences. At least 10 Aspergillus species can be recognized which are able to biosynthesize aflatoxins, and they are placed in three very different clades.
    Systematic and Applied Microbiology 08/2005; 28(5):442-53. · 3.29 Impact Factor
  • Flemming Lund, Anni Bech Nielsen, Pernille Skouboe
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    ABSTRACT: 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.
    Food Microbiology. 01/2003;
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    ABSTRACT: We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatus isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to other species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of A. japonicus and A. aculeatus. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.
    Applied and Environmental Microbiology 03/2001; 67(2):521-7. · 3.95 Impact Factor
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    ABSTRACT: The genetic variability within 600 bp of DNA sequence from the ribosomal internal transcribed spacers (ITS 1 and ITS 2) and the 5.8 S rRNA gene was examined in 52 strains belonging to 29 terverticillate taxa in Penicillium subg. Penicillium from diverse sources and locations. The sequenced region is extremely conserved within the terverticillate penicillia, with only 29 positions differing in one or more taxa. The highest degree of ITS variability was seen among the species close to Penicillium roqueforti (P. roqueforti, P. carneum and P. paneum) whereas the ITS variability was very low between closely related taxa, e.g. among the taxa near P. aurantiogriseum. The relationships among the terverticillate penicillia, and related teleomorphs in Eupenicillium, were analysed from bootstrapped ITS sequence data sets using the neighbour-joining method. The terverticillate penicillia form a well supported clade with Eupenicillium crustaceum. Sequence analysis generally confirmed the overall taxonomy in Penicillium subg. Penicillium but relationships between all the terverticillate taxa could not be clearly established due to the low degree of ITS variability. Nevertheless, clades of Penicillium species sharing environmental characteristics did emerge, e.g. species growing on protein and lipid rich substrates (e.g. P. crustosum and P. commune), species prevalent in dry habitats (e.g. P. chrysogenum and P. nalgiovense), and those prevalent on carbohydrate rich substrates (e.g. P. aurantiogriseum and P. freii).
    Mycological Research. 07/1999;
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    ABSTRACT: In the present study, the use of aqueous polymer two-phase systems for separation of pathogenic bacteria from a complex food sample was investigated. Three different two-phase systems, a polyethylene glycol 3350/dextran T 500, a methoxy polyethylene glycol 5000/dextran T 500 and a polyethylene glycol 3350/hydroxypropyl starch system, were compared at pH 3 and pH 6 for their capacity to separate the pathogenic bacteria Listeria monocytogenes and Salmonella berta from a Cumberland sausage. In all three phase systems, the food particles partitioned to the lower phase. Best performance was obtained by the polymer combinations, polyethylene glycol 3350/dextran T 500 and polyethylene glycol 3350/hydroxypropyl starch. In these systems, Salmonella berta partitioned to the hydrophobic upper phase both at pH 3 and pH 6 with an average partitioning ratio of 80% and a recovery of 56%. Listeria monocytogenes partitioned to the upper phase at pH 3 only with an average partitioning ratio of 72% and a recovery of 45%. This method may become a valuable tool for separation of bacteria from complex food matrices.
    Letters in Applied Microbiology 02/1998; 26(1):47-50. · 1.63 Impact Factor
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    ABSTRACT: Rapid identification of filamentous fungi is becoming increasingly important in food mycology both for monitoring the production process and for the identification of food spoilers. This paper describes the development and trial of two specific PCR primer sets. A 336 bp fragment from species belonging to Penicillium subgenus Penicillium was amplified by the primers ITS 212d and ITS 549. The other primer set, ITS 183 and ITS 401 specifically identified two species, Penicillium roqueforti and P. carneum, both known as spoilers in the bread industry, by amplification of a 300 bp fragment. The future perspectives of PCR based identification of filamentous fungi in food are discussed.
    International Journal of Food Microbiology 05/1997; 35(2):169-77. · 3.43 Impact Factor
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    M Boysen, P Skouboe, J Frisvad, L Rossen
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    ABSTRACT: Penicillium roqueforti is currently divided into two varieties, one used for cheese starter cultures, P. roqueforti var. roqueforti, and one ubiquitous patulin-producing variety, P. roqueforti var. carneum. The ribosomal regions comprising the 5.8S gene and the internal transcribed spacers, ITS I and ITS II, have been analysed from 10 isolates belonging to each variety. The 10 P. roqueforti var. carneum isolates were separated into two groups of five on the basis of 12 base-pair differences in the ITS regions. One of the groups of P. roqueforti var. carneum, in the following designated P. carneum, differed from P. roqueforti var. roqueforti, here designated P. roqueforti, in just two positions, while the other group, here called P. paneum, differed from P. roqueforti in 12 positions. Random Amplified Polymorphic DNA (RAPD) analysis substantiated these findings, and a comparison of secondary metabolites produced by the three groups showed that the P. roqueforti isolates all produce Penicillium Roqueforti (PR) toxin, marcfortines and fumigaclavine A, while the P. carneum isolates produce patulin, penitrem A and mycophenolic acid, as well as unidentified metabolites. P. paneum produces secondary metabolites in five chromophore families including the known mycotoxins patulin and botryodiploidin. On the basis of these findings it is proposed that P. roqueforti is reclassified into three species named P. roqueforti, P. carneum and P. paneum.
    Microbiology 04/1996; 142 ( Pt 3):541-9. · 2.85 Impact Factor
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    ABSTRACT: Ichthyophonus hoferi Plehn and Mulsow, 1911 is thought to be one of the few pathogenic fungal infections of marine fish. The result of an attack is severe epizootics in herring stocks with drastic reduction in the population as a consequence. The exact phylogenetic position of the genusIchthyophonus is not known. In the present study, a combination of molecular data, ultrastructure and biochemical characters were utilized to investigate the phylogeny ofI.hoferi. The genomic DNA encoding the small subunit ribosomal RNA (18S rRNA) was amplified and sequenced. Comparisons with other eukaryotic 18S rRNA sequences indicate thatI. hoferi is not a member of the Fungi. In both the parsimony and the neighborjoining trees,I. hoferi is the sister taxon to the rosette agent. The clade encompassingI.hoferi and the rosette organism is the sister group to the chanoflagellate clade in the neighbor-joining tree, while in the parsimony tree theI. hoferi/rosette clade is equally distant to both the choanoflagellate and animal clades. Transmission electron microscopy showed thatI. hoferi has a defined cell wall, an endoplasm that consists of a fine granulated matrix with numerous ribosomes, several nuclei, vacuoles of varying density distributed throughout the cell, and mitochondria with tubular cristae. The cell wall ofI. hoferi contains chitin.
    Marine Biology 02/1996; 126(1):109-115. · 2.47 Impact Factor
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    ABSTRACT: Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.
    Microbiology 10/1995; 141 ( Pt 9):2053-61. · 2.85 Impact Factor