Accumulation of type A trichothecenes in maize, wheat and rice by Fusarium sporotrichioides isolates under diverse culture conditions

Toxigenic isolates of Fusarium sporotrichioides were tested for the production of type A trichothecenes (T-2 toxin, HT-2 toxin, diacetoxyscirpenol and neosolaniol) when grown on three substrates (maize, rice and wheat) under various conditions of temperature and water activity in the laboratory for 3 weeks. Trichothecenes were determined by high-performance liquid chromatography with fluorescence detection, after derivatisation with coumarin-3-carbonyl chloride. This is the first time this analytical method has been applied to an extensive study of trichothecene accumulation. With minor exceptions, greater trichothecene production occurred when samples were incubated at 20 degrees C and moistened with 35% water (water activity 0.990) although incubation conditions affected the substrates studied in different ways. No correlation between the different pairs of trichothecenes was found except for neosolaniol and diacetoxyscirpenol (r=0.56). Principal component analysis results show that the data points can be grouped in three rough clusters related to cereal type, which points out that the composition of these cereals can influence the production of type A trichothecenes.     

Production of trichothecenes and zearalenone by Fusarium species isolated from wheat

Fusaria isolates from wheat were tested for ability to produce trichothecenes and zearalenone. Four isolates of F. culmorum out of 13 produced vomitoxin (DON) and 3 Ac-DON, one produced diacetoxysirpenol and 12 zearalenone. Particularly high yield of zearalenone was observed in cultures of sever pathogenic isolates. Higher temperature (20 °C) during first week of incubation favoured yield of zearalenone. About 50% of zearalenone was produced by surface mycelium.     

Genotyping and phenotyping of Fusarium graminearum isolates from Germany related to their mycotoxin biosynthesis

Fusarium graminearum is the most important pathogen causing Fusarium head blight (FHB) of small cereal grains worldwide responsible for quantitative and qualitative yield losses. The presence in crops is often associated with mycotoxin contamination of foodstuff limiting its use for human and animal consumption. A collection of isolates of F. graminearum from Germany was characterized genetically and chemically for their potential to produce the B trichothecenes deoxynivalenol (DON) and nivalenol (NIV). Molecular methods with eight PCR assays were implemented based on functional Tri7 and Tri13 genes and on the tri5-tri6 intergenic region to differentiate between chemotaxonomic groups DON and NIV, resulting in a marked majority (61/63) of DON chemotypes. Mycotoxins produced on rice kernels were quantified by means of LC-MSMS including DON, NIV, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON), DON-3-glucoside, fusarenon X, as well as zearalenone; all of them proving to be present in high concentration among the isolates. All DON-chemotype isolates also produced lower amounts of NIV with the amount being positively correlated (R² = 0.89) to the DON amount. 15-ADON and 3-ADON are reported to be produced simultaneously by the isolates, the former dominating over the latter in all but one isolate. Fungal biomass, was quantified via ergosterol amount on rice. It was used to calculate specific mycotoxin production per biomass of isolates, ranging from 0.104 to 1.815 mg DON mg-1 ergosterol, presenting a Gaussian distribution. Genotype and phenotype characterization revealed discrepancies with respect to mycotoxin production potential of the fungi, i.e. isolates from one chemotype were able to produce mycotoxins from other chemotypes in considerable amounts.     

A rapid multiresidual determination of type A and type B trichothecenes in wheat flour by HPLC-ESI-MS

A new, rapid and sensitive method is reported for the multiresidual determination of type A (diacetoxyscirpenol, HT-2 toxin, T-2 toxin) and type B (nivalenol, deoxynivalenol, fusarenon X, 15-O-acetyl-4-deoxynivalenol) trichothecenes in wheat flour samples. Sample extraction was performed with acetonitrile/water mixtures. Mycosep^© columns were used for a fast and effective clean-up procedure. The analytes were separated by HPLC with a RP C18 column by means of a gradient elution and detected in an ESI-interfaced single quadrupole mass spectrometer. Type B and type A trichothecenes were monitored in the negative and in the positive ion mode, respectively. The method performance is reported in terms of linearity (r² = 0.999), specificity, accuracy (recoveries from 70-120%) and precision (CV% = 5), the LOQs are in the range 10-20 µg/Kg.     

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes (CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.     

Control of growth and fumonisin B1 production by Fusarium verticillioides and Fusarium proliferatum isolates in moist maize with propionate preservatives.

The effect of propionic acid, its sodium salt or a commercial formulation of propionates (0.03, 0.05 and 0.07%), on growth and fumonisin B1 production by Fusarium verticillioides and F. proliferatum isolated was evaluated on irradiated maize at different water activities (aw, 0.93, 0.95, 0.98) and temperatures (15, 25 degrees C). The four isolates grew at all aw x temperature treatments in the absence of propionates. At the highest propionate concentration tested (0.07%), however, growth was restricted to 0.98 aw, for F. proliferatum isolates but not for those of F. verticillioides. Inhibition of growth was maximum when propionates were added in the acid form. In the presence of low propionate concentrations (0.03%), growth was sometimes enhanced probably due to assimilation of these compounds by the fungus. Water activity, temperature, concentration and source of propionate, as well as most two-, three-, four-, and five-way interactions had a significant influence on growth of Fusarium isolates. None of the assayed treatments had any effect on fumonisin B1 production by F. verticillioides isolates. For F. proliferatum, higher fumonisin B1 production occurred in the absence of propionates, and in general concentration decreased with increasing doses of preservatives. Single factors (aw, propionate concentrations and temperature) and temperature x aw and propionate concentration x temperature interactions had a significant effect on fumonisin production (p < 0.01). Moreover, propionate concentration was the single most important factor, besides temperature, which affected fumonisin B1 production.     

Effects of the volatile organic compounds produced by Enterococcus spp. strains isolated from maize grain silos on Fusarium verticillioides growth and fumonisin B1 production

Fusarium verticillioides is a phytopathogenic fungus that can contaminate maize grain silos and result in important losses in the post-harvest product. The objective of this work was to investigate the effects of volatile organic compounds produced by four lactic acid bacterial strains isolated from maize grain silos on F. verticillioides M3125 growth and fumonisin B₁ (FB₁) production. The bacterial isolates 55 and 49 were identified as Enterococcus faecium and M4A and M4G as Enterococcus casseliflavus. The fungal growth was inhibited by 33.33% by the volatiles released by the M4A strain and by approximately 10% by the volatiles emitted by the 55 and 49 strains. The volatiles produced by the M4A strain also significantly reduced (88.75%) FB₁ biosynthesis. The gas chromatography–mass spectrometer analysis identified 21 volatile organic compounds, with diacetyl, acetic acid and acetoin being the main volatiles emitted by the four bacterial strains. Acetoin was the volatile produced in the highest proportion by the four strains, with M4A generating the highest proportion of diacetyl (35.11%). Diacetyl and acetic acid completely inhibited fungal growth at concentrations of 0.3 and 1 mM, respectively, while acetoin promoted fungal growth. Only acetoin significantly reduced FB₁ production. These results showed that diacetyl was the main compound involved in fungal inhibition, while the effect on FB₁ production could have been due to the combination of the volatile organic compounds produced by the M4A strain. In conclusion, the volatiles emitted by the E. casseliflavus M4A strain could be a promising tool for the biocontrol of F. verticillioides in storage maize grain silos.     

底物和环境因子对鱼源腐败希瓦氏菌和假单胞菌生长动力学的影响

水产品在贮运和加工过程中极易腐败,微生物是引起腐败的主要原因,腐败希瓦氏菌和假单胞菌分别是低温有氧贮藏海水鱼和淡水鱼时的特定腐败菌.为探讨环境因子和底物对特定腐败菌的影响,以源自大黄鱼和罗非鱼中的腐败希瓦氏菌和假单胞菌为对象,探究不同底物、温度、pH和NaCl影响下的菌体生长动态,采用Gompertz方程拟合生长曲线,...     

Production of mycotoxins by selected Fusarium graminearum and F. crookwellense isolates.

Corn cultures (five isolates each of Fusarium graminearum Group 1 from wheat crowns, Group 2 from scabby wheat grains and from ear rot of corn and five isolates of F. crookwellense) were screened for their ability to produce deoxynivalenol (DON), nivalenol (NIV), fusarenon-x (FUS-X) and zearalenone (ZEA). Nine of the ten F. graminearum isolates from wheat produced DON (5-165 micrograms g-1) but none produced either NIV or FUS-X. Conversely, 3/5 and 2/5 of the F. graminearum isolates from corn produced NIV (5-40 micrograms g-1) and FUS-X (5-7 micrograms g-1), respectively, while none produced DON. All but one of the F. graminearum isolates produced ZEA (2-1160 micrograms g-1). None of the F. crookwellense isolates produced DON, but 5/5 and 4/5 produced NIV (6-170 micrograms g-1) and FUS-X (3-90 micrograms g-1), respectively, and all produced ZEA (605-1030 micrograms g-1). The results confirmed previous findings on the presence of two distinct F. graminearum chemotypes.     

Comparison of fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides

Fusarium verticillioides produces a group of mycotoxins known as fumonisins in maize kernels. Fumonisins are associated with a variety of mycotoxicoses in humans and animals; thus, their presence in food is a considerable safety issue. This study addressed fumonisin B1 (FB1) production in two components of the maize kernel, namely the germ tissues and the degermed kernel. Growth of F. verticillioides was similar in colonized germ tissue and degermed kernels, but FB1 production was at least five times higher in degermed maize kernels than in germ tissue. Expression of the fumonisin polyketide synthase gene, FUM1, as measured by beta-glucuronidase (GUS) and Northern blot analysis, followed the same pattern as FB1 production. Also correlated to FB1 was a concomitant drop in pH of the colonized degermed kernels. A time course experiment showed that degermed kernels inoculated with F. verticillioides became acidified over time (from pH 6.4 to 4.7 after 10 days of incubation), whereas colonized germ tissue became alkaline over the same period (from pH 6.5 to 8.5). Because conditions of acidic pH are conducive to FB1 production and alkaline pH is repressive, the observed correlation between the acidification of degermed kernels and the increase in FB1 provides one explanation for the observed differences in FB1 levels.     

Two-dimensional profiles of fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum in relation to environmental factors and potential for modelling toxin formation in maize grain

This study has examined in detail the effect of temperature (7-37 degrees C) and water availability (water activity, a(w), 0.89-0.97) on fumonisin B1 (FB1) production by an isolate of Fusarium moniliforme and F. proliferatum on irradiated maize grain after incubation for 28 days. The optimum conditions for F. moniliforme and F. proliferatum were 30 degrees C at 0.97 a(w) and 15 degrees C at 0.97 a(w), respectively. The maximum concentrations were 2861 mg kg(-1) and 17,628 mg kg(-1) dry wt. maize grain, respectively. At marginal a(w)/temperature conditions for growth (e.g. 0.89-0.91 a(w)) no FB1 was detected (<0.1 mg kg(-1)). A high variability was found between replicates for F. moniliforme, but not for F. proliferatum. These data were used to construct two-dimensional diagrams of all the a(w) x temperature conditions favourable for FB1 production for the first time. The data were also subjected to a polynomical regression, which demonstrated that there was a very good fit for the 15-30 degrees C range of temperature and at 0.97 a(w). However, at marginal environmental conditions this was not possible. This suggests that it may be possible to predict within a limited environmental range the potential for significant FB1 production.     

Incidence and pathogenicity of Yersinia spp. isolates from poultry in Spain

Forty eviscerated and refrigerated chicken carcasses were collected from retail outlets in León (Spain) and investigated for the presence of Yersinia spp. Following a two-step enrichment procedure (phosphate buffer saline was used as primary enrichment and bile oxalate sorbose as secondary enrichment), yersiniae were isolated on cefsulodin-irgasan-novobiocin (CIN) and MacConkey agar. Yersinia spp., Y. enterocolitica and Y. frederiksenii were detected in 26 (65%), 22 (55%) and six (15%) samples, respectively. The incidence ofYersinia and Y. enterocolitica was greater than that obtained in poultry meat by the majority of the consulted authors. Of the 68 Yersinia isolates, 52 were identified as Y. enterocolitica and 16 as Y. frederiksenii. Biotyping of Y. enterocolitica revealed that 45 (86·5%) strains corresponded to biotype 1A, and three (5·8%) to biotype 3. Four (7·7%) strains could not be classified. Two biotype 3 strains were found to be presumptively virulent according to the dye (Congo red and crystal violet) binding, low-calcium response, and pyrazinamidase activity techniques. It is suggested from results that the presence of Yersinia in poultry could represent a health risk for consumers in Spain. The education of people involved in production, processing and final preparation of poultry products are required in order to assure adequate cooking and to avoid cross-contamination.     

Influence of Listeria monocytogenes and environmental abiotic factors on growth parameters and aflatoxin B1 production by Aspergillus flavus

Aspergillus flavus growth is influenced by a variety of abiotic factors including water activity (a_W), pH, redox potential, relative humidity, temperature and availability of oxygen. Furthermore, biotic factors such as the presence in the same habitat of different microorganisms can develop ecological interactions between them that are positive, negative or indifferent for fungal growth. The aim of the present work was evaluate the effect of Listeria monocytogenes in interaction with a_W, pH and temperature on the lag phase, growth rate and AFB₁ production of A. flavus. Ecophysiological in vitro studies of A. flavus demonstrate that the co-occurrence with L. monocytogenes in culture medium and abiotic factors (a_W, pH and temperature) influence the growth of the fungal strains and the production of AFB₁. The presence of L. monocytogenes generally decreased fungal growth and increased significantly aflatoxin production. These studies are essential in the prediction of contamination risk of food with these microorganisms or their toxins in interaction with several environmental parameters.     

Investigations on Fusarium spp. and their mycotoxins causing Fusarium ear rot of maize in Kosovo

After wheat, maize (Zea mays L.) is the second most important cereal crop in Kosovo and a major component of animal feed. The purpose of this study was to analyse the incidence and identity of the Fusarium species isolated from naturally infected maize kernels in Kosovo in 2009 and 2010, as well as the mycotoxin contamination. The disease incidence of Fusarium ear rot (from 0.7% to 40% diseased ears) on maize in Kosovo is high. The most frequently Fusarium spp. identified on maize kernels were Fusarium subglutinans, F. verticillioides/F. proliferatum and F. graminearum. Maize kernel samples were analysed by LC-MS/MS and found to be contaminated with deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, zearalenone, zearalenone-14-sulphate, moniliformin, fumonisin B₁ and fumonisin B₂. This is the first report on the incidence and identification of Fusarium species isolated from naturally infected maize as well as the mycotoxin contamination in Kosovo.     

Detection of masked mycotoxins derived from type A trichothecenes in corn by high-resolution LC-Orbitrap mass spectrometer

Masked mycotoxins (mycotoxin glucosides) derived from type A trichothecenes were detected in commercially available corn powder reference material. These new glucosides were identified as neosolaniol-glucoside (NESGlc) and diacetoxyscirpenol-glucoside (DASGlc) on the basis of accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography–Orbitrap mass spectrometric (LC-Orbitrap MS) analysis. Although the absolute structure was not clarified, 3-OH glucosylation appeared to be the most probable when considering the structures of neosolaniol and diacetoxyscirpenol and the fragmentation profiles of these masked mycotoxins. Concomitant detection of deoxynivalenol-3-glucoside, the most well-known masked mycotoxin derived from the type B trichothecene, deoxynivalenol, in the identical material further supports this probability.     

Genotypic and phenotypic characteristics of Yersinia spp. isolates from food and man

Yersinia strains collected by the Unit of Enteric Pathogens of the Istituto Superiore di Sanità were analysed for bioserogroup, virulence-related characteristics, DNA plasmid profile and biotype. The isolates were from clinical and food samples, 26 were Y. enterocolitica, four Y. intermedia and four Y. frederiksenii. Seventeen were isolated in Northern Italy, 11 in Southern Italy, five in Central Italy and one from a sample of mussels imported from Greece. Virulence-related characteristics were expressed only by four Y. enterocolitica, bioserogroup 4:O3 associated with the presence of a plasmid weighing 76·8 Kbp. The DNA digested by Spe I and Xba I and analysed by PFGE generated restriction patterns of 12–23 bands, ranging between 400 and 20 Kbp. The similarities of the DNA fingerprintings ofY. enterocolitica strains generated using Spe I and Xba I and calculated by Dice's index were not closely related to bioserogroup.     

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds

The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC₅₀ and MIC₉₀, respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to both compounds. The MIC₅₀ and MIC₉₀ of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae, respectively. The MIC₅₀ and MIC₉₀ of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae, respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC₉₀, whereas an 8-fold increase of the MIC₉₀ was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti-Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows.     

Influence of temperature on zearalenone production by regional strains of Fusarium graminearum and Fusarium oxysporum in culture.

Zearalenone production by Fusarium graminearum and Fusarium oxysporum was studied under two temperature conditions. Incubation at 25 degrees C for 4 weeks enhanced zearalenone synthesis, improving detection of zearalenone-producing strains of Fusarium oxysporum. Zearalenone production was either totally or partially inhibited when temperature was lowered to 12-14 degrees C during the last 2 weeks of incubation.