Abstract
Aspergillus carbonarius is a potent ochratoxin A producer that has been found in products such as grapes, coffee, spices, and cocoa. Ochratoxin A has nephrotoxic effect, and it has been classified as a possible carcinogenic substance for humans. Here we describe for the first time a transformation system for A. carbonarius, providing an important step towards its genetic manipulation. Conidia were transformed to acquire hygromycin B resistance using the AGL-1 strain of Agrobacterium tumefaciens. Genetic transformation was evaluated growing A. tumefaciens cells in induction medium containing or not acetosyringone prior to co-cultivation. The mean transforming efficiencies in IM+AS and IM-AS conditions were 62.2 and 44.5 transformants per 105 conidia, respectively. The hph gene was random integrated into the genome of A. carbonarius. Fungal sequences flanking the insertion site could be amplified by TAIL-PCR.
Similar content being viewed by others
Literature Cited
Abarca ML, Accensi F, Bragulat MR, Castellá G, Cabañes FJ (2003) Aspergillus carbonarius as the main source of ochratoxin A contamination in dried vine fruits from the Spanish market. J Food Prot 66:504–506
Battilani P, Pietri A, Bertuzzi T, Languasco L, Giorni P, Kozakiewicz Z (2003) Occurrence of ochratoxin A-producing fungi in grapes grown in Italy. J Food Prot 66:633–636
Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ (1995) Trans-Kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214
Cabañes FJ, Accensi F, Bragulat MR, Abarca ML, Castellá G, Mínguez S, Pons A (2002) What is the source of ochratoxin A in wine? Int J Food Microbiol 79:213–215
Case ME, Schweizer M, Kushner SR, Giles NH (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA 76:5259–5263
Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R (2003) Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiol Lett 220:141–148
Covert SF, Kapoor P, Lee M, Briley A, Nairn CJ (2001) Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res 105:259–264
Da Rocha Rosa CA, Palacios V, Combina M, Fraga ME, De Oliveira Rekson A, Magnoli CE, Dalcero AM (2002) Potencial ochratoxin A producers from wine grapes in Argentina and Brazil. Food Addit Contam 19:408–414
De Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839– 842
Fungaro MHP, Rech E, Muhlen GS, Vainstein MH, Pascon RC, Queiroz MV, Pizzirani-Kleiner AA, Azevedo JL (1995) Transformation of Aspergillus nidulans by microprojectile bombardment on intact conidia. FEMS Microbiol Lett 125:293–298
Leclerque A, Wan H, Abschütz A, Chen S, Mitina GV, Zimmermann G, Schairer HU (2004) Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana. Curr Genet 45:111–119
Liu Y-G, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463
Magnoli C, Violante M, Combina M, Palácio G, Dalcero A (2003) Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina. Lett Appl Microbiol 37:179–184
Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet 48:1–17
Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91:173–180
Sage L, Krivobok S, Delbos E, Seigle-Murandi F, Creppy EE (2002) Fungal flora and ochratoxin A production in grapes and musts from France. J Agric Food Chem 50:1306–1311
Sambrook J, Russel DW (2001) Molecular cloning: A laboratory manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
Stevens R (1974) Mycology guidebook. Seattle, WA: University of Washington Press
Tanaka Y, Murata N, Kato H (1979) Behavior of nuclei and chromosomes during ascus development in the matting between either rice – strain or weeping lovegrass – strain and ragi – strain of Pyricularia. Ann Phytopathol Soc Japan 45:182–191
Taniwaki MH, Pitt JI, Teixeira AA, Iamanaka BT (2003) The source of ochratoxina A in Brazilian coffee and its formation in relation to processing methods. Int J Food Microbiol 82:173–179
Tsuji G, Fujii S, Fujihara N, Hirose C, Tsuge S, Shiraishi T, Kubo Y (2003) Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium. J Gen Plant Pathol 69:230–239
van der Merwe KJ, Steyn PS, Fourie L, Scott DB, Theron JJ (1965) Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature 205:1112–1113
Ward ER, Barnes WM (1989) Transformation of Aspergillus awamori and A. niger by electroporation. Exp Mycol 13:289–293
Acknowledgments
Grants and fellowships from the following Brazilian institutions supported this work: Fundação Araucária and Conselho Nacional de Pesquisa (CNPq).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Morioka, L.R.I., Furlaneto, M.C., Bogas, A.C. et al. Efficient Genetic Transformation System for the Ochratoxigenic Fungus Aspergillus carbonarius. Curr Microbiol 52, 469–472 (2006). https://doi.org/10.1007/s00284-005-0402-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-005-0402-6