Growth rate and colony morphology of <i>F. verticillioides</i> wild-type (WT) and Δsda1 strains on various liquid and solid media.

<p>Strains were grown on defined liquid medium (DM) amended with 2% (A) sorbitol or (B) glucose, mannitol and no carbon. DM with 2% of glucose is the standard medium. Strains were inoculated in DM and incubated with constant shaking (100 rpm) for 6 days at 25°C. (C) Colony morphology of wild-type (WT) and Δsda1 strains on DM agar plates amended with ammonium phosphate (AP: NH₄H₂PO₄) and sodium nitrate (SN: NaNO₃). Strains were point inoculated with an agar plug and grown for 10 days at 25°C.</p

Accumulation of polyols and trehalose in maize kernels and liquid media.

<p>Accumulation of indicated metabolite in wild-type and Δsda1 grown for 7 days (A) on maize kernels and, (B) in liquid media containing glucose as the sole carbon source. Analyses were performed with at least four biological replicates. Results are the means of three and four biological replications with standard errors shown as error bars.</p

Schematic representation of the <i>SDA1</i> disruption and complementation strategies in <i>F. verticillioides</i>.

<p>(A) Target replacement of <i>SDA1</i> with the hygromycin phosphotransferase gene <i>(HPH)</i> by split marker technique through homologous recombination. Solid bar indicates DNA fragment used as the probe for Southern hybridization. Lanes: 1, wild-type; 2, Δsda1-7; 3, ectopic integration; 4, Δsda1-9. The wild-type strain produced a 7 kb band, and the Δsda1 knock out mutant produced a 3.5 kb band. (B-C) Schematic representations of constructs used to complement the Δsda1 strain. (B) <i>F. verticillioides SDA1</i> wild-type copy construct. Lanes 1, wild-type; 2, Δsda1; 3 and 4, Δsda1-complements (sdaC). The wild-type strain produced a 1.5 kb band and the Δsda1 knock out mutant produced a 6.2 kb band. The complemented sdaC strains produced a 6.2 kb and a random size band due to ectopic integration of the complementation construct. (C) <i>T. reesei ACE1</i> coding region fused to <i>A. nidulans</i> GPD promoter construct. Lanes 1, wild-type; 2, Δsda1; 3, Δsda-complement (sdaT). The complemented sdaT strain produced a single a random size band due to ectopic integration of the complementation construct.</p

Amino-acid alignment of <i>F. verticillioides</i> (Fv) Sda1 and its comparison with predicted protein sequences of homologous proteins.

<p>Sda1 shares 52%, 50% and 65% identity with homologs in <i>Magnaporthe oryzae</i> (Mo) (GeneBank accession no. XP_003713522), <i>Neurospora crassa</i> (Nc) (GeneBank accession no. XP_963927) and <i>Trichoderma reesei</i> (Tr) (GeneBank accession no. Q9P8W3), respectively. The conserved residues were indicated by white letters on a black background. The regions corresponding to the three zinc fingers were indicated with boxes. Asterisks indicate the zinc coordinating Cys and His residues.</p

Quantification of microconidia and fumonisin B₁ (FB₁) production in <i>F. verticillioides</i> strains.

<p>Wild-type (WT), Δsda1, sdaC, and sdaT strains were point inoculated with an agar plug (0.5 cm in diameter) on nonviable autoclaved maize kernels and incubated for 14 days at 25°C under a 14-h light/10-h dark cycle. (A) Microconidia were harvested and quantified with a haemocytometer. (B) FB₁ production was quantified by high-performance liquid chromatography (HPLC) analysis. FB₁ biosynthesis was normalized to growth with ergosterol contents. All values represent the means of three biological replications with standard errors shown as error bars, and two independent experiments showing similar results.</p

Germination and growth of <i>F. verticillioides</i> wild-type (WT), Δsda1, sdaC, and sdaT strains.

<p>A total of 10⁵ spores were inoculated and incubated in DL media with sorbitol at 25°C for (A) 16 h and (B) 40 h. (A) Note the lack of germination of the Δsda1 strain. (B) After 24 h of incubation in DL media with sorbitol, 2% w/v of glucose was added to the cultures. Note the germination of the Δsda1 strain in the presence of glucose vs. sorbitol. Scale bar = 5 µm.</p

Biomass quantification of <i>F. verticillioides</i> strains.

<p>Wild-type (WT), Δsda1, sdaC, and sdaT strains were grown on DL media amended with 2% of glucose, sorbitol or fructose. The mycelia of each strain were harvested after 6 days of incubation in DL medium and dried at 100°C for 24 hrs. Results are the means of three and four biological replications with standard errors shown as error bars, and repeated at least twice.</p

Proposed schematic model of Sda1 regulation in <i>F. verticillioides</i>.

<p>In mammals, the polyol pathway is composed by two enzymatic reactions, where glucose is reduced to sorbitol by aldose reductase (AR), and subsequently oxidized to fructose by sorbitol dehydrogenase (SDH). The model visualizes the putative transcriptional regulation of SDH by Sda1 in <i>F. verticillioides</i>.</p

Transcription levels of genes corresponding to <i>SDA1</i> and putative sorbitol dehydrogenases in DL medium containing either glucose or sorbitol as the sole source of carbon^a,c.

a<p>Total RNA samples were prepared from <i>F. verticillioides</i> wild-type strain grown on DL media +2% w/v glucose or sorbitol. Mycelia were collected after 60 hours post inoculation. Real time quantitative reverse transcription (qRT)-PCR analysis of gene expression was performed with SYBR-Green as the fluorescent reporter. Gene expression was normalized to endogenous β-tubulin gene expression.</p>b<p>The gene expression was calibrated using 2^−ΔΔCt method. Data represent the relative expression, where gene expression in glucose is standardized to 1.00± the standard error of dCT values (<i>n = 3</i>).</p>c<p>Each value is the mean of 3–4 technical replicates from one biological experiment. A biological replication was performed with no statistically different results.</p

Homogeneous Fluorescence Resonance Energy Transfer Immunoassay for the Determination of Zearalenone

<div><p>This study demonstrates the use of antigen-antibody binding for the detection of zearalenone. Based on the principle of the fluorescence resonance energy transfer (FRET) phenomenon between antibody and antigen, an immunoassay, in which zearalenone coupled with the anti-zearalenone antibody, was developed, optimized, and applied. Owing to intrinsic fluorescence properties in basic pH conditions with the optimal cationic surfactant, anti-zearalenone and zearalenone played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of analyte increased, the antigen/antibody emission intensity ratio (<i>I</i>_430 nm/<i>I</i>_350 nm) was enhanced due to larger amounts of zearalenone/anti-zearalenone complexes. This assay, based on the ratio of intensities (<i>I</i>_430 nm/<i>I</i>_350 nm), displayed high specificity and sensitivity with a detection limit of 0.8 ng mL⁻¹ for zearalenone. The results obtained from analysis of spiked wheat grain samples were found to be in good agreement with those obtained by employing a direct competitive enzyme-linked immunosorbent assay. The label-free, noncompetitive, and homogeneous FRET immunoassay strategy served as a powerful tool for the simple, rapid, and sensitive quantitative determination of zearalenone in food and feed matrices.</p> </div