Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

Species Composition and Toxigenic Potential of Fusarium Isolates Causing Fruit Rot of Sweet Pepper in China.

Apart from causing serious yield losses, various kinds of mycotoxins may be accumulated in plant tissues infected by Fusarium strains. Fusarium mycotoxin contamination is one of the most important concerns in the food safety field nowadays. However, limited information on the causal agents, etiology, and mycotoxin production of this disease is available on pepper in China. This research was conducted to identify the Fusarium species causing pepper fruit rot and analyze their toxigenic potential in China. Forty-two Fusarium strains obtained from diseased pepper from six provinces were identified as F. equiseti (27 strains), F. solani (10 strains), F.fujikuroi (five strains). This is the first report of F. equiseti, F. solani and F. fujikuroi associated with pepper fruit rot in China, which revealed that the population structure of Fusarium species in this study was quite different from those surveyed in other countries, such as Canada and Belgium. The mycotoxin production capabilities were assessed using a well-established liquid chromatography mass spectrometry method. Out of the thirty-six target mycotoxins, fumonisins B1 and B2, fusaric acid, beauvericin, moniliformin, and nivalenol were detected in pepper tissues. Furthermore, some mycotoxins were found in non-colonized parts of sweet pepper fruit, implying migration from colonized to non-colonized parts of pepper tissues, which implied the risk of mycotoxin contamination in non-infected parts of food products.

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.

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.

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 ß-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.

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, ß-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.