International Commission on Food Mycology
International Commision on Food Mycology
Workshop 2010 -“Fungi in food and beverages: new research on spoilage, mycotoxins and prevention”
Freising, Germany,
7 – 9 June, 2010
Abstracts
Session 4: Diagnostic Tools for the Detection and Identification of Food Borne Fungi and Mycotoxins (Chairperson Ludwig Niessen)
PenGuide: the fourth edition of “Laboratory Guide to Common Penicillium Species”
John I. Pitt1*
1CSIRO Food and Nutritional Sciences, P.O. Box 52, North Ryde, NSW 2113, Australia
*Presenter: john.pitt@csiro.au
“Laboratory Guide to Common Penicillium Species” was written for a Workshop in Athens, GA, USA in 1985. Subsequent editions were produced for workshops in Sugadaira, Japan in 1988 and in New Orleans, LA, USA in 2000. One of the drawbacks to those books was that photographs of colonies were in black and white, and the Methuen “Handbook of Colour”, used as a colour dictionary, became increasingly difficult to obtain. Printing methods for colours have improved greatly in recent years, but colour distinctions in Penicillium species are so fine, and need to be so precise, that the printing process remains only just adequate. An alternative is to produce the guide as a computer programme, as modern computer screens provide remarkably accurate colour reproduction. Recently I produced a programme called “FoodMold” that took advantage of this, providing a computer based taxonomy for foodborne fungi. FoodMold was written in “ToolBook” a programme designed for computer based instruction manuals and similar books. ToolBook is now being utilised to produce PenGuide, the fourth edition of the laboratory guide. PenGuide includes the 70 species of Penicillium, Eupenicillium and Talaromyces found in the previous editions, and a small number of additional ones. The taxonomy is being updated. New colour colony photographs and many new photomicrographs will be included, and new keys will be written as appropriate. A demonstration version will be presented at the workshop.
Comparison of Nash and Snyder Medium and Malachite Green Agar 2.5 for the detection of Fusarium infection in conventional and transgenic maize kernels
F. Javier Cabañes1*, Lorena Alborch1, and M. Rosa Bragula1.
1Veterinary Mycology Group, Department of Animal Health and Anatomy , Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
*Presenter: javier.cabanes@uab.es
Some selective culture media have been developed for the specific isolation of Fusarium spp. from natural samples. Nash and Snyder medium (NS) with pentachloronitrobenzene (PCNB) as fungal inhibitor is one of the most widely employed and several modifications of this medium are also recommended for these purposes. However, PCNB has been reported to be carcinogenic and, as an alternative to NS, other antifungal or inhibitory compounds, such malachite green in Malachite Green Agar 2.5 (MGA 2.5). This latter medium was developed in our laboratory and it has been assessed using the spread technique with natural samples, performing as a potent selective medium for Fusarium spp. The aim of this work was to assess differences among these two recommended selective culture media for the detection of Fusarium infection in conventional and transgenic maize kernels. In total, 10,800 kernels from commercial varieties grown in Spain were analyzed. Fusarium verticillioides was predominant in both selective culture media. Mean percentages of Fusarium infected kernels were significantly lower in transgenic maize kernels (12.8%) than in conventional maize kernels (25.1%). These percentages ranged from about 10% to 15% for transgenic maize and from about 20% to 34% for conventional maize. This tendency was detected in both selective culture media used. There were no significant differences in percentage of Fusarium infection detected by the two selective culture media used. MGA 2.5 performed as a selective medium for the detection of Fusarium infection in maize kernels using the direct plating technique. This medium can be used as an alternative to NS in the detection of Fusarium infection in grain samples.
Detection and quantification of ochratoxin A-producing strains of the
Aspergillus niger aggregate by Real-Time PCR
Gemma Castellá1* and F. Javier Cabañes1.
1Veterinary Mycology Group, Department of Animal Health and Anatomy , Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain.
*Presenter: gemma.castella@uab.es
At present, Aspergillus carbonarius, Aspergillus sclerotioniger, a rarely found species, and several members of the A. niger aggregate, are the only ochratoxin A-producing species accepted in the Aspergillus section Nigri. While A. carbonarius consistently produces large amounts of ochratoxin A (OTA), the reported percentages of OTA-producing strains in the A. niger aggregate is much lower. So the detection of ochratoxigenic isolates in this species complex is important to prevent OTA contamination in foodstuffs and biotechnology products. To date, there is limited information about the genes involved in the OTA biosynthesis, but it is well known that a polyketide synthase (PKS) is required. The aim of this study was to develop a real-time PCR procedure for the rapid and specific detection and quantification of OTA-producing strains of the A. niger aggregate. Two specific primers delimiting a 120 bp fragment and a probe were designed and directed to a PKS from the A. niger CBS 513.88 genome. Primer specificity was first evaluated by conventional PCR using as template DNA from 26 reference strains. They included representative OTA producing and non-producing strains of the A niger aggregate, as well as other Aspergillus and Penicillium spp. (A. carbonarius, A. sclerotioniger, A.ibericus, A. westerdijkiae, Penicillium verrucosum and Penicillium nordicum). A PCR fragment was detectable only with OTA-producing strains of the A. niger aggregate. All other analyzed related fungi gave negative results. Melting curve analysis using SYBR® Green I real-time PCR approach showed characteristic Tm values confirming the homogeneity of the target selected. Afterwards, a TaqMan real-time PCR approach was settled. Both real-time PCR systems gave positive results only with strains the A. niger aggregate that were able to produce OTA, indicating that both systems were indeed specific for ochratoxigenic strains of the A. niger aggregate.
NANODETECT – Development of biosensors for the quantification of
contaminants in milk: Development of a detection unit for Aflatoxin
Andrea Mückl1*, Maria Eden1, Caroline Hennigs1, Hauke Hilz1
1Technologie-Transfer-Zentrum Bremerhaven, Fischkai 1,
D-27572 Bremerhaven, Germany
*Presenter: amueckl@ttz-bremerhaven.de
The EC funded project NANODETECT aims to develop on-line monitoring systems (sensors) which combine the expertise of sensitive and specific molecular biological processes with the potency of nanotechnology for application in liquid process streams. Detection systems for following contaminants will be developed: pathogenic microorganisms (Listeria monocytogenes), mycotoxins (Aflatoxin M1), drug residues (sulphonamides and aminoglycosides) and fraud (high value goat milk blended with cheaper cow milk).
The detection unit for Aflatoxin M1 is based on a direct inhibition Fluorescence Immunoassay (FIA) using specific anti-AFM1 antibodies labelled with the fluorophore R-phycoerythrin. The FIA allows quantification of Aflatoxin M1 levels between 1.0 and 100 µg/l in untreated milk samples. Enrichment of samples will improve sensitivity to be able to detect the EC limit of 0.05 µg/l. The established FIA will be transferred to a microfluidic device, where the signal can be read and directly quantified with a laser diode and suitable software. The user friendly system will enable direct online monitoring of contaminants in milk.
Infrared spectroscopy - a simple and rapid tool for species identification and strain typing of yeasts
Mareike Wenning1
1Zentralinstitut für Ernährungs- und Lebensmittelforschung (Z I E L) Weihenstephan, Abteilung Mikrobiologie, Technische Universität München
Presenter: mareike.wenning@wzw.tum.de
Fourier-transform infrared (FTIR) spectroscopy is a whole-organism fingerprinting technique which is based on the absorption of infrared light by proteins, lipids, nucleic acids, polysaccharides and other molecules that make up intact microbial cells. Infrared spectra of cells grown under standardised conditions provide specific fingerprints, which can be used for identification by comparison to reference spectra of known species.
The technique has several advantages over many other methods used for identification. Sample preparation is simple and fast with little hands-on time, running cost are very low and the resulting digital data can be stored, which enables the compilation of large reference databases that can be easily expanded and adapted to specific applications. Over the past 15 years, the Department of Microbiology at the Technical University of Munich has established an extensive reference databases for the identification of yeasts comprising 2.400 strains from 250 different species [1, 2].
However, the application of FTIR spectroscopy is not restricted to the identification of microbes at the species level. FTIR spectra are strain-specific to a certain extent and therefore allow the differentiation of organisms below the species level. This may be useful for the analysis of contamination routes, where contaminants have to be matched with isolates of potential contamination sources or the differentiation of probiotic and environmental isolates [3].
- Kümmerle, M., Scherer, S. and Seiler, H. (1998) Rapid and reliable identification of food-borne yeasts by Fourier-transform infrared spectroscopy. Appl. Environ. Microbiol. 64: 2207-14.
2. Büchl N.R., Wenning M., Seiler H., Mietke-Hofmann H. and Scherer S. (2008) Reliable identification of closely related Issatchenkia and Pichia species using artificial neural network analysis of Fourier-transform infrared spectra. Yeast 25:787-98.
3. Büchl N.R., Hutzler, M., Mietke-Hofmann H., Wenning M. and Scherer S. (2010) Differentiation of probiotic and environmental Saccharomyces cerevisiae strains in animal feed. J. Appl. Microbiol., in press.
Loop-mediated amplification as a novel concept in fungal diagnosis
Ludwig Niessen1*, Daniela Buchner1, Karolina Müller1, Rudi F. Vogel1
Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, 85350 Freising, Germany
*Presenter: niessen@wzw.tum.de
Loop-mediated isothermal amplification (LAMP) of DNA is a simple, cost effective, and rapid novel method for the specific detection of genomic DNA using a set of 8 primers binding specifically to different regions of a target gene, and a thermophilic DNA polymerase (Bst polymerase) from Geobacillus stearothermophilus for DNA amplification. The method has been applied in various assays for the diagnosis of bacterial and viral infections of humans and animals, sexing of bovine and swine embryos, and in the detection of bacteria from environmental samples. Only recently the first applications for fungal organisms (Phytophthora ramorum, Brettanomyces spp.) were published.
We have established LAMP assays for the species specific detection of Fusarium graminearum as one of the major producers of mycotoxins like DON and ZEA. The assay was based on the gaoA gene (galactose oxidase) of the fungus. Another LAMP assay and for F. tricinctum, a producer of emerging toxins like beauvericin, enniatin and moniliformin was based on the sequence of the acl1 gene (ATP citrate lyase 1). Amplification of DNA during the reaction was detected in situ using calcein fluorescence as a marker. No agarose gel electrophoresis was necessary for product visualization. Assays were optimized for rapidness, specificity, and sensitivity with a detection limit of 2 pg and 40 pg of DNA purified from F. graminearum and F. tricinctum, respectively within 30-40 minutes. Within 132 fungal species tested, only DNA isolated from F. graminearum (lineages 1-9) isolates resulted in a fluorescent signal after amplification with the specific LAMP assay. With the F. tricinctum specific assay all target isolates and 3 out of 12 F. avenaceum samples tested positive.
The method was demonstrated to be useful in the analysis of fungal isolates by direct analysis of surface scrapings from agar plate cultures, direct testing of single infected barley grains, and detection of the fungi intotal genomic DNA isolated from samples of ground wheat grains. Results obtained indicate that LAMP offers an interesting new assay format for the rapid and specific DNA-based detection and identification of agriculturally important toxigenic fungi in pure cultures and in contaminated sample materials. Due to its rapidness and ease of use it may offer an alternative to PCR assays in the future.
