The development of a multiplex real-time PCR to quantify Fusarium DNA of trichothecene and fumonisin producing strains in maize

Contamination of cereals with Fusarium species is one of the major sources of mycotoxin contamination in food and feed. Despite great progress in plant breeding, a complete resistance to Fusarium species has not yet been achieved. Visual scoring of disease symptoms combined with the determination of mycotoxins are common approaches to identify new Fusarium tolerant lines, but these methods are only indirect and therefore of limited use to determine the level of resistance against Fusarium spp. Aiming at a rapid and sensitive quantification method for trichothecene and fumonisin producing Fusarium species in maize, a multiplex qPCR assay was developed. This method enables high-throughput screening of a large number of samples for Fusarium infection in a relatively short time due to simultaneous quantification of the mycotoxin-related genes tri5 and fum1. The multiplex method was applied to 24 maize field samples. All these were analyzed for the trichothecenes deoxynivalenol (DON), DON-3-glucoside (D3G), nivalenol (NIV), 3-acetyl-DON (3-ADON), T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and neosolaniol (NEO) and the fumonisins fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) by LC-MS/MS and for the mycotoxin producers by the new qPCR multiplex assay. The assay was found to be specific for fumonisin as well as for trichothecene producing Fusarium species. The limit of quantification was found to be 0.32 pg per μl for both Fusarium strains. To the best of our knowledge, this is the first report of the use of a multiplex qPCR for the quantification of trichothecene and fumonisin producing Fusarium species.