François Van Hove

Birth, death and horizontal transfer of the fumonisin biosynthetic gene cluster during the evolutionary diversification of Fusarium

Fumonisins are a family of carcinogenic secondary metabolites produced by members of the Fusarium fujikuroi species complex (FFSC) and rare strains of Fusarium oxysporum. In Fusarium, fumonisin biosynthetic genes (FUM) are clustered, and the cluster is uniform in gene organization. Here, sequence analyses indicated that the cluster exists in five different genomic contexts, defining five cluster types. In FUM gene genealogies, evolutionary relationships between fusaria with different cluster types were largely incongruent with species relationships inferred from primary‐metabolism (PM) gene genealogies, and FUM cluster types are not trans‐specific. In addition, synonymous site divergence analyses indicated that three FUM cluster types predate diversification of FFSC. The data are not consistent with balancing selection or interspecific hybridization, but they are consistent with two competing hypotheses: (i) multiple horizontal transfers of the cluster from unknown donors to FFSC recipients and (ii) cluster duplication and loss (birth and death). Furthermore, low levels of FUM gene divergence in F. bulbicola, an FFSC species, and F. oxysporum provide evidence for horizontal transfer of the cluster from the former, or a closely related species, to the latter. Thus, uniform gene organization within the FUM cluster belies a complex evolutionary history that has not always paralleled the evolution of Fusarium.

Gibberella musae (Fusarium musae) sp. nov., a recently discovered species from banana is sister to F. verticillioides

Several strains of Fusarium isolated from banana were identified previously as F. verticillioides (Sacc.) Nirenberg but described as unable to produce fumonisin. Here we report biochemical and morphological evidence, as well as multilocus phylogenetic analyses based on elongation factor (EF-1α), calmodulin, β-tubulin, and the second largest subunit of RNA polymerase II (RPB2) sequences, indicating that these isolates represent a unique lineage in the Gibberella fujikuroi species complex related to but distinct from F. verticillioides. Together with previous results of molecular studies, as well as with results of metabolite analyses, crossing experiments, pathogenicity tests and morphological characterization, these new data indicate that these strains isolated from banana represent a new species, Gibberella musae Van Hove et al. sp. nov. (anamorph: Fusarium musae Van Hove et al. sp. nov.), which is described herein.

Genetic diversity and mycotoxin production of Fusarium lactis species complex isolates from sweet pepper.

An internal fruit rot disease of sweet peppers was first detected in Belgium in 2003. Research conducted mostly in Canada indicates that this disease is primarily caused by Fusarium lactis Pirotta. Ninety-eight Fusarium isolates obtained from diseased sweet peppers from Belgium, as well as from other countries (Canada, the Netherlands and the United Kingdom) were identified by sequencing the translation elongation factor 1α (EF). Of these 98 isolates, 13 were identified as F. oxysporum Schltdl., nine as F. proliferatum (Matsush.) Nirenberg and two belonged to clade 3 of the F. solani species complex. Of the 74 remaining isolates, the EF sequence showed 97% to 98% similarity to F. lactis. Of these isolates, the β-tubulin (TUB), calmodulin (CAM) and the second largest subunit of RNA polymerase II (RPB2) genes were also sequenced. Analysis of the combined sequences revealed that the 74 isolates share nine combined sequences that correspond to nine multilocus sequence types (STs), while the F. lactis neotype strain and one other strain, both isolated from figs, form a separate ST. Together, these 10 STs represent a monophyletic F. lactis species complex (FLASC). An unusually high level of genetic diversity was observed between (groups of) these STs. Two of them (ST5 and ST6) fulfilled the criteria for species recognition based on genealogical exclusivity and together represent a new monophyletic species lineage (FLASC-1). The seven other STs, together with the F. lactis neotype ST, form a paraphyletic species lineage in the African clade of the Gibberella fujikuroi species complex (GFSC). From each of the 10 STs, the mycotoxin production was assessed using a multi-mycotoxin liquid chromatography mass spectrometry method. Out of the 27 analyzed mycotoxins, beauvericin and fumonisins were detected in sweet pepper tissue and in maize kernels. The 10 STs clearly differed in the amount of mycotoxin produced, but there was only limited congruence between the production profile and the phylogenetic analysis. Furthermore, the morphological characterization (based on mycelial growth rate and the length of macroconidia) showed distinct differences between the 10 STs, but again there was limited congruence with the phylogenetic results. In conclusion, the data presented in this study demonstrate that 75% of the isolates obtained from sweet pepper with internal fruit rot belong to a F. lactis species complex (FLASC), including a new FLASC-1 monophyletic species, and that the members of this complex display great genetic and phenotypic diversity.

Aptamer-Based Molecular Recognition of Lysergamine, Metergoline and Small Ergot Alkaloids

Ergot alkaloids are mycotoxins produced by fungi of the genus Claviceps, which infect cereal crops and grasses. The uptake of ergot alkaloid contaminated cereal products can be lethal to humans and animals. For food safety assessment, analytical techniques are currently used to determine the presence of ergot alkaloids in food and feed samples. However, the number of samples which can be analyzed is limited, due to the cost of the equipment and the need for skilled personnel. In order to compensate for the lack of rapid tests for the detection of ergot alkaloids, the aim of this study was to develop a specific recognition element for ergot alkaloids, which could be further applied to produce a colorimetric reaction in the presence of these toxins. As recognition elements, single-stranded DNA ligands were selected by using an iterative selection procedure named SELEX, i.e., Systematic Evolution of Ligands by EXponential enrichment. After several selection cycles, the resulting aptamers were cloned and sequenced. A surface plasmon resonance analysis enabled determination of the dissociation constants of the complexes of aptamers and lysergamine. Dissociation constants in the nanomolar range were obtained with three selected aptamers. One of the selected aptamers, having a dissociation constant of 44 nM, was linked to gold nanoparticles and it was possible to produce a colorimetric reaction in the presence of lysergamine. This system could also be applied to small ergot alkaloids in an ergot contaminated flour sample.

SYNTHESIS AND REARRANGEMENT OF POTENTIAL ZINC BETA -LACTAMASE INHIBITORS

On alkylation of the p-toluene sulfonic salt of benzyl 6-aminopenicillanate (6-APA) with benzyl 2-bromoacetate, the benzyl esters of N-(benzyloxycarbonylmethyl)-6-APA (1) and of N,N-di(benzyloxycarbonylmethyl)-6-APA (3) were obtained. Hydrogenolysis of 1 on Pd/C afforded the N (carboxymethyl)-6-APA (2) while hydrogenolysis of 3 gave a rearranged product (4). Both 2 and 4 are inhibitors of the zinc beta-lactamase II of B. cereus.

Molecular identification of mycotoxigenic fungi in food and feedstuffs

Abstract: Classical culture-based methods for detection and identification of the main toxigenic fungi occurring on food and feedstuffs (belonging to Aspergillus, Fusarium and Penicillium genera, as well as to Alternaria, Claviceps, Monascus, Phoma, Phomopsis, Pithomyces, Stachybotrys genera) present numerous limitations, such as their time-consuming and labour-intensive aspects. Therefore, most detection procedures are now DNA-based and, by using polymerase chain reaction (PCR) based-methods, range from protocols allowing identification of a single species, or of multiple species belonging to the same genus or to mixed populations of different genera. In the present context, the PCR approach has been considered under two main complementary approaches: by targeting conserved functional genes or regions of taxonomical interest, or by focusing on the mycotoxigenic genes. The use of real-time PCR technology, which monitors the DNA (or RNA) amplification products in real time and allows fast detection and identification of the fungal content of food and feedstuffs in a single assay, will be described. Moreover, although less practicable for quantitative purposes, some applications that combine different technologies from ‘basic’ PCR, such as PCR-restriction fragment length polymorphism and PCR and enzyme immunoassay, denaturing gradient gel electrophoresis PCR-based, PCR and single strand conformational polymorphism, microsatellite length polymorphism, loop-mediated-isothermal amplification assay, will also be shown for their applicability to toxigenic fungi detection. Finally, since novel molecular technologies for sequencing the whole genome provide a mine of important information for either detection or characterization of fungal species, their benefit for qualitative and quantitative molecular technologies will be illustrated.