Fungi of the genus Fusarium are important pathogens of numerous crops including cereals. Infection of the grain with Fusarium spp. often results in the accumulation of various mycotoxins which poses a significant risk to food safety. The Fusarium graminearum species complex, together with F. culmorum, are the main source of trichothecenes in cereal grains. They are comprised of fungal strains producing one of three specific trichothecenes: either 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol) (not present in F. culmorum) or NIV (nivalenol). Recently, qualitative PCR approaches have been developed for rapid determination of 3ADON, 15ADON and NIV genotypes within F. graminearum/F. culmorum. This paper presents the development of three TaqMan genotyping assays for quantitative diagnostics of 3ADON, 15ADON and NIV genotypes within F. graminearum/F. culmorum. Primers and probes were designed on the basis of 95 nucleotide site within the Tri12 gene which allowed for specific determination of trichothecene producers. Additionally, to address the problem of detecting inhibitors, an internal positive control (IPC) was developed employing primers and a probe derived from conserved fungal sequence of 5.8S rDNA. The assays developed were successfully applied for direct determination of trichothecene genotypes from Fusarium-damaged wheat kernels (FDK).
Beyer, M., Klix, M.B., Verreet, J.A. 2007. Estimating mycotoxin contents of Fusarium-damaged winter wheat kernels. Int. J. Food Microbiol. 119:153–158.
Verreet J.A. , 'Estimating mycotoxin contents of Fusarium-damaged winter wheat kernels ' (2007 ) 119 Int. J. Food Microbiol. : 153 -158.
Chandler, E.A., Simpson, D.R., Thomsett, M.A., Nicholson, P. 2003. Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis. Physiol. Mol. Plant Pathol. 62:355–367.
Nicholson P. , 'Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis ' (2003 ) 62 Physiol. Mol. Plant Pathol. : 355 -367.
Desjardins, A.E. 2006. Fusarium Mycotoxins: Chemistry, Genetics, and Biology. Am. Phytopathol. Soc. Press, St. Paul, MN, USA, 260 pp.
Desjardins A.E. , '', in Fusarium Mycotoxins: Chemistry, Genetics, and Biology , (2006 ) -.
Edwards, S.G., Pirgozliev, S.R., Hare, M.C., Jenkinson, P. 2001. Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against fusarium head blight of winter wheat. Appl. Environ. Microbiol. 67:1575–1580.
Jenkinson P. , 'Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against fusarium head blight of winter wheat ' (2001 ) 67 Appl. Environ. Microbiol. : 1575 -1580.
Foroud, N.A., Eudes, F. 2009. Trichothecenes in cereal grains. Int. J. Mol. Sci. 10:147–173.
Eudes F. , 'Trichothecenes in cereal grains ' (2009 ) 10 Int. J. Mol. Sci. : 147 -173.
Hogg, R.H., Johnston, A.T., Dyer, A.C. 2007. Applying real-time quantitative PCR to Fusarium crown rot of wheat. Plant Dis. 91:1021–102.
Dyer A.C. , 'Applying real-time quantitative PCR to Fusarium crown rot of wheat ' (2007 ) 91 Plant Dis. : 1021 -102.
Jennings, P., Coates, M.E., Turner, J.A., Chandler, E.A., Nicholson, P. 2004. Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assays. Plant Pathol. 53:182–190.
Nicholson P. , 'Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assays ' (2004 ) 53 Plant Pathol. : 182 -190.
Kulik, T., Fordomski, G., Pszczólkowska, A., Plodziem, K., Lapimski, M. 2004. Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides. FEMS Microbiol. Lett. 239:181–186.
Lapimski M. , 'Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides ' (2004 ) 239 FEMS Microbiol. Lett. : 181 -186.
Kulik, T., Pszczólkowska, A., Fordomski, G., Olszewski, J. 2007. PCR approach based on the esyn1 gene for the detection of potential enniatin-producing Fusarium species. Int. J. Food Microbiol. 116:319–324.
Olszewski J. , 'PCR approach based on the esyn1 gene for the detection of potential enniatin-producing Fusarium species ' (2007 ) 116 Int. J. Food Microbiol. : 319 -324.
Lee, T., Oh, D.W., Kim, H.S., Lee, J., Kim, Y.H., Yun, S.H., Lee, Y.W. 2001. Identification of deoxynivalenoland nivalenol-producing chemotypes of Gibberella zeae by using PCR. Appl. Environ. Microbiol. 67:2966–2972.
Lee Y.W. , 'Identification of deoxynivalenoland nivalenol-producing chemotypes of Gibberella zeae by using PCR ' (2001 ) 67 Appl. Environ. Microbiol. : 2966 -2972.
Lee, T., Han, Y.K., Kim, K.H., Yun, S.H., Lee, Y.W. 2002. Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae. Appl. Environ. Microbiol. 68:2148–2154.
Lee Y.W. , 'Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae ' (2002 ) 68 Appl. Environ. Microbiol. : 2148 -2154.
Leisova, L., Kucera, L., Chrpova, J., Sykorova, S., Sip, V., Ovesna, J. 2006. Quantification of Fusarium culmorum in wheat and barley tissues using real-time PCR in comparison with DON content. J. Phytopathol. 154:603–611.
Ovesna J. , 'Quantification of Fusarium culmorum in wheat and barley tissues using real-time PCR in comparison with DON content ' (2006 ) 154 J. Phytopathol. : 603 -611.
Li, H.P., Wu, A.B., Zhao, C.S., Scholten, O., Löffler, H. and Liao, Y.C. 2005. Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum. FEMS Microbiol. Lett. 243:505–511.
Liao Y.C. , 'Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum ' (2005 ) 243 FEMS Microbiol. Lett. : 505 -511.
Nicholson, P., Simpson, D.R., Weston, G., Rezanoor, H.N., Lees, A.K., Parry, D.W., Joyce, D. 1998. Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol. Mol. Plant Pathol. 53:17–37.
Joyce D. , 'Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays ' (1998 ) 53 Physiol. Mol. Plant Pathol. : 17 -37.
Nicolaisen, M., Suproniene, S., Nielsen, L.K., Lazzar, I., Spliid, N.H., Justesen, A.F. 2009. Real-time PCR for quantification of eleven individual Fusarium species in cereals. J. Microbio. Methods 76:234–40.
Justesen A.F. , 'Real-time PCR for quantification of eleven individual Fusarium species in cereals ' (2009 ) 76 J. Microbio. Methods : 234 -40.
O’Donnell, K., Ward, T.J., Aberra, D., Kistler, H.C., Aoki, T., Orwig, N., Kimura, M., Bjørnstad, Å., Klemsdal, S.S. 2008. Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia. Fungal Genet. Biol. 45:1514–1522.
Klemsdal S.S. , 'Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia ' (2008 ) 45 Fungal Genet. Biol. : 1514 -1522.
Pasquali, M., Giraud, F., Brochot, C., Cocco, E., Hoffmann, L., Bohn, T. 2010. Genetic Fusarium chemotyping as a useful tool for predicting nivalenol contamination in winter wheat. Int. J. Food Microbiol. 137:246–253.
Bohn T. , 'Genetic Fusarium chemotyping as a useful tool for predicting nivalenol contamination in winter wheat ' (2010 ) 137 Int. J. Food Microbiol. : 246 -253.
Quarta, A., Mita, G., Haidukowski, M., Logrieco, A., Mulè, G., Visconti, A. 2006. Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium. FEMS Microbiol. Lett. 259:7–13.
Visconti A. , 'Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium ' (2006 ) 259 FEMS Microbiol. Lett. : 7 -13.
Schnerr, H., Vogel, R.F., Niessen, L. 2002. Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat samples. Lett. Appl. Microbiol. 35:121–125.
Niessen L. , 'Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat samples ' (2002 ) 35 Lett. Appl. Microbiol. : 121 -125.
Starkey, D.E., Ward, T.J., Aoki, T., Gale, L.R., Kistler, A.C., Oeise, A.D.M., Suga, H., Tóth, B., Varga, J., O’Donnell, K. 2007. Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genet. Biol. 44:1191–1204.
O’Donnell K. , 'Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity ' (2007 ) 44 Fungal Genet. Biol. : 1191 -1204.
Tóth, B., Mesterházy, Á., Nicholson, P., Téren, J., Varga, J. 2004. Mycotoxin production and molecular variability of European and American isolates of Fusarium culmorum. Eur. J. Plant Pathol. 110:587–599.
Varga J. , 'Mycotoxin production and molecular variability of European and American isolates of Fusarium culmorum ' (2004 ) 110 Eur. J. Plant Pathol. : 587 -599.
Waalwijk, C., Kastelein, P., de Vries, I., Kerényi, Z., van der Lee, T., Hesselink, T., Köhl, J., Kema, G. 2003. Major changes in Fusarium spp. in wheat in the Netherlands. Eur. J. Plant Pathol. 109:743–754.
Kema G. , 'Major changes in Fusarium spp. in wheat in the Netherlands ' (2003 ) 109 Eur. J. Plant Pathol. : 743 -754.
Waalwijk, C., Heide, R., van der Vries, P.M., de Lee, T.A.J., van der Schoen, C.D., Costrel-Decorainville, G., Kastelein, P., Köhl, J., Kema, G.H.J. 2004. Quantitative detection of Fusarium species in wheat using TaqMan. Eur. J. Plant Pathol. 110:481–494.
Kema G.H.J. , 'Quantitative detection of Fusarium species in wheat using TaqMan ' (2004 ) 110 Eur. J. Plant Pathol. : 481 -494.
Ward, T.J., Bielawski, J.P., Kistler, H.C., Sullivan, E., O’Donnell, K. 2002. Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proc. of the Nat. Acad. of Sci., USA. 99:9278–9283.
O’Donnell K. , 'Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium ' (2002 ) 99 Proc. of the Nat. Acad. of Sci., USA. : 9278 -9283.
Ward, T.J., Clear, R.M., Rooney, A.P., O’Donnell, K., Gaba, D., Patrick, S., Starkey, D.E., Gilbert, J., Geiser, D.M., Nowicki, T.W. 2008. An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America. Fungal Genet. Biol. 45:473–484.
Nowicki T.W. , 'An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America ' (2008 ) 45 Fungal Genet. Biol. : 473 -484.
Wasielewska, J. 2010. Gas exchange parameters and molecular diagnostics of winter wheat diseases treated and not treated by fungicides. Dissertation, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland.
Wasielewska J. , '', in Gas exchange parameters and molecular diagnostics of winter wheat diseases treated and not treated by fungicides , (2010 ) -.
Zhang, J.B., Li, H.P., Dang, F.J., Qu, B., Xu, Y.B., Zhao, C.S., Liao, Y.C. 2007. Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China. Mycol. Res. 111:967–975.
Liao Y.C. , 'Determination of the trichothecene mycotoxin chemotypes and associated geographical distribution and phylogenetic species of the Fusarium graminearum clade from China ' (2007 ) 111 Mycol. Res. : 967 -975.