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.

Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops.

Various species of Fusarium can produce trichothecene mycotoxins that contaminate food commodities and can represent a risk for human and animal health. In this paper, a full factorial design was applied to study the influence of incubation temperature, water activity (a(w)) and type of isolate on the production of deoxynivalenol (DON), nivalenol (NIV) and 3-acetyldeoxynivalenol (3-AcDON) in corn kernel cultures by three isolates of Fusarium graminearum and three isolates of Fusarium culmorum from crops grown in Spain. The tested temperatures were 15, 20, 28 and 32 degrees C. The a(w)-values were 0.960, 0.970 and 0.980. Moisture of cultures (within the studied range) did not affect significantly production of trichothecenes; however, the temperature affected significantly mycotoxin production and the optimal values were 28, 20 and 15 degrees C for DON, NIV and 3-AcDON, respectively. Four additional isolates of F. graminearum and two additional isolates of F. culmorum were examined for production of these mycotoxins at the optimal temperatures. Of the seven isolates of F. graminearum, four produced DON (0.88-3.97 microg/g), seven produced NIV (1.53-124 microg/g), and three produced 3-AcDON (0.65-10.6 microg/g). Of the five isolates of F. culmorum, four produced DON (1.20-4.93 microg/g), four produced NIV (6.94-701 microg/g), and four produced 3-AcDON (0.83-7.70 microg/g). Practically all isolates seem to belong to the NIV-chemotype. This is the first study done with regard to interaction between strain and ecological variables on type B trichothecene production by isolates of these two species from crops grown in Spain.

Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium.

Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a(w) at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED50 and ED90 values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a(w) and the influence of temperature on growth rates was 25>20>15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED50 and ED90 values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a(w), type of fungicide and dose. Levels of T-2 were usually higher than those of HT-2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a(w) T-2 levels increased in cultures containing fenpropimorph while at 0.96 a(w) the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a(w) and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a(w). This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxins in oat-based media.

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium.

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97 a(w). Mancozeb at 40 mg l(-1 )inhibited fungal growth and provided lag phases >24 days at 10-20 mg l(-1) and 15°C. OTA was observed only at 25°C and doses <10 mg l(-1). At 15°C, copper oxychloride proved inhibitory at 800 mg l(-1), while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5-8 g l(-1) (at 15°C) while at 25°C growth took place even at 8 g l(-1), although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb > copper oxychloride > sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.

Mycobiota and co-occurrence of mycotoxins in Capsicum powder.

This study aimed to: (1) determine the mycobiota of Capsicum powder samples, paying a special attention to the mycotoxigenic moulds; (2) evaluate the contamination levels of aflatoxins (AF), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T2 and HT2 toxins in those samples. Thirty-two samples were obtained through the methods of sampling established by the European Union legislation. Aspergillus and Eurotium were the most frequently found genera. Aspergillus section Nigri had the higher relative frequency in the samples, A. niger aggregate being the most representative group of this section. Other potentially mycotoxigenic Aspergillus, Fusarium and Penicillium species were found, but in a lower frequency. Co-occurrence of mycotoxins was confirmed in the 32 Capsicum powder samples. All samples were contaminated with AF and OTA, 27% with ZEA (36% of chilli and 18% of paprika samples), 9% with DON (18% of chilli and 6% of paprika samples), 6% with T2 (18% of chilli samples) and none of the samples contained HT2. Although in the present study the most common genera found (Aspergillus and Eurotium) belong to storage moulds, some field fungi such as Fusarium spp. were also found, and their toxins were sometimes detected. This fact supports the hypothesis that mycotoxin contamination of Capsicum products may occur both in the field and/or during storage.

Changes in ochratoxin A and type B trichothecenes contained in wheat flour during dough fermentation and bread-baking.

Ochratoxin A (OTA) and type B trichothecenes are mycotoxins that occur frequently in cereals and thus can be found in cereal by-products such as bread. The aim of this work was to study the variation of the levels of OTA, deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and nivalenol (NIV) during the bread-making process. This was done by using wheat flour spiked with different levels of toxins. Mycotoxin levels were controlled after fermentation of the dough with yeasts (Saccharomyces cerevisiae) and after further baking at different temperature-time combinations. Analysis of variance (ANOVA) of the results showed a significant reduction in OTA level (p < 0.05) during fermentation of the dough. The reduction ranged between 29.8% and 33.5%, depending on the initial concentration of toxin in the flour. During this period, the level of the other mycotoxins studied was not modified. By contrast, in the baking phase there were significant changes in the levels of the four mycotoxins, although the reduction was similar under all the baking conditions. Considering all the temperature-time conditions tested, it can be concluded that during the baking period the average reduction of OTA, NIV, 3-ADON, and DON was 32.9%, 76.9%, 65.6%, and 47.9%, respectively.

Variability and characterization of mycotoxin-producing Fusarium spp isolates by PCR-RFLP analysis of the IGS-rDNA region.

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB1, FB2, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple 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 rDNA. The results indicated that G. fujikuroi complex isolates can be divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.

Fumonisin production in rice cultures of Fusarium verticillioides under different incubation conditions using an optimized analytical method.

Fumonisin B1 (FB1) and fumonisin B2 (FB2) are the main members of a family of mycotoxins produced by various fungal species belonging to the Gibberella fujikuroi complex. The present work shows the results of a comparative study of various clean-up and derivatization procedures for analysis of fumonisins in rice cultures. Fumonisins were extracted from rice with acetonitrile/water (50/50, v/v). For clean-up, three solid-phase extraction procedures were assayed (C18 cartridge, SAX cartridge, and a combination of both). Two reagents (o-phthaldialdehyde and 4-fluoro-7-nitro-benzofurazan) were studied comparatively for formation of fluorescent derivatives. The separation was carried out by LC using a fluorescence detector. The best procedure for analysis of fumonisins in rice involved clean-up with C18 cartridge and derivatization with o-phthaldialdehyde. The limit of detection was 0.010 mg kg(-1) for both toxins. In the 10-500 mg kg(-1) spiking level range, the recovery rates for FB1 and FB2 in rice varied from 94.6% to 103.6% and from 96.3% to 101.9%, respectively. The optimized analytical method for determination of fumonisins in rice was applied to the study of FB1 and FB2 production by four isolates of the G. fujikuroi species complex in rice cultures carried out at different temperatures and water activities to establish the influence of strain and environmental conditions on fumonisin production in this cereal. In general, fumonisin production was the highest at 20 degrees C and lowest at 37 degrees C. Four of the five assayed water activity (aw) values (0.97, 0.98, 0.99, and 1.0) did not affect significantly fumonisin accumulation but fumonisins were not detected in cultures when aw was 0.96.

Application of SEM/EDX and metallographic techniques to the diachronic study (6th-18th century) of metallurgical materials found in archaeological excavations on the island of Ibiza (Spain).

This work is the archaeometric study of the different types of materials recovered in the same excavation or archaeological dig (which makes it possible to accurately date the remains found) on the island of Ibiza (Spain). The samples found belong only to the phase of iron forging and span a very wide historical period from the 6th to the 18th century, including the Islamic period. Scanning Electron Microscopy (SEM) is proposed to observe the images obtained of the surfaces of the samples, in order to study the topographical, morphological and microstructural characteristics providing information on the materials contained in the samples. It also permits chemical analysis of the elements in the sample using X-ray Microanalysis (SEM/EDX), which provides both qualitative and semi-quantitative information on the elements in the sample. The archaeometric results suggest that the sole use of the area studied was as a forge workshop over a long period of time. The slag studied show that no reduction or refining activities took place in the area. However, in this forge, pieces of other metals were also produced, especially bronze alloys, polymetallism being a common characteristic throughout history.

Analytical study by SEM/EDX and metallographic techniques of materials used in the iron production process during the iberian period.

This work is the archaeometric study of different types of materials used in iron production in the Iberian Period (6th-2nd century b.c.). The materials were recovered in several archaeological digs (which makes it possible to date the archaeological remains) on different sites in the Levante area of the Iberian Peninsula. The samples selected for this study belong to different materials used in the ancient iron production process such as ores, slags, finished objects, etc. The use of scanning electron microscopy (SEM) is proposed to observe surface images of the samples to determine their morphological, microstructural and topographic characteristics to obtain valuable information on the materials studied. The study also proposes chemical analysis of the elements in the sample by X-ray microanalysis (SEM/EDX) which provides both qualitative and quantitative information. The archaeometric study suggests that the iron obtained was very good quality, but the process was not economically efficient when viewed from today's perspective. Furthermore, the slag produced in the furnace can be related with the slag or impurities contained in the iron. It has also been possible to relate the ores and slag, some furnace conditions, the use of fluxes and also to differentiate types of slag.