MALDI-TOF MS fingerprinting for identification and differentiation of species within the Fusarium fujikuroi species complex.

Members of the Fusarium fujikuroi species complex (FFSC) are commonly involved in devastating diseases of many economically important plants. They invade developing seeds and other plant tissues in the field causing significant annual losses. In addition, fungal spoilage can also affect human and animal health because some species in this group, especially F. proliferatum and F. verticillioides, are mycotoxin producers occurring in food/feed worldwide. Since morphology-based species identification is of limited value in the FFSC, the development of new methods is fundamental for accurate identification of the molds to species level. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of subproteomes has been applied as a promising tool for the discrimination of closely related species in many microorganisms. In the present study, MALDI-TOF MS was applied to distinguish closely related species in the FFSC and to validate the effectiveness of a standardized protocol by identifying field isolates that fulfilled the morphological characteristics of FFSC species. Forty-nine of the currently described 61 species were identified by DNA sequencing analysis and their mass spectra were included as reference in a supplementary MALDI-TOF MS database. The discriminative potential of the database was evaluated with more than 80 non-reference FFSC isolates and resulted in 94.61% of correct identifications at the species level. We demonstrate that MALDI-TOF MS is a suitable and accurate technology for the identification and differentiation of species within the FFSC as well as an innovative, time-efficient alternative to multilocus sequencing technology (MLST).

A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp.

Fumonisins are mycotoxins that contaminate maize and maize-based food products, and feed. They have been associated with nerve system disorders in horses, pulmonary edema in swine as well as neural tube defects and esophageal cancer in humans. The fum1 gene codes for a polyketide synthase involved in the biosynthesis of fumonisins. It is present in the genomes of all fumonisin producing Fusarium spp. Reliable detection of fum1 can provide an estimate of the toxicological potential of cultures and food sources. Therefore, a fum1 specific LAMP assay was developed and tested with purified DNA of 48 different species from the Fusarium fujikuroi species complex (FFSC). The fum1 gene was detected in 22 species among which F. fujikuroi, F. globosum, F. nygamai, F. proliferatum, F. subglutinans and F. verticillioides were the most prominent fumonisin producers. None out of 92 tested non-Fusarium species showed cross reactions with the new assay. The lowest limit of detection (LOD) was 5 pg of genomic DNA per reaction for F. fujikuroi, F. nygamai and F. verticillioides. Higher LODs were found for other LAMP positive species. Apart from pure genomic DNA, the LAMP assay detected fumonisin-producers when 103 conidia/reaction were used as template after mechanical lysis. LAMP-results were well correlated with FB1 production. This is the first report on fumonisin production in strains of F. annanatum, F. coicis, F. mundagurra, F. newnesense, F. pininemorale, F. sororula, F. tjataeba, F. udum and F. werrikimbe. Usefulness of the LAMP assay was demonstrated by analyzing fumonisin contaminated maize grains. The new LAMP assay is rapid, sensitive and reliable for the diagnosis of typical fumonisin producers and can be a versatile tool in HACCP concepts that target the reduction of fumonisins in the food and feed chain.