Evaluation of ability of ferulic acid to control growth and fumonisin production of Fusarium verticillioides and Fusarium proliferatum on maize based media

The aim of this study was to evaluate the efficacy of ferulic acid (1, 10, 20 and 25 mM) at different water activity (a_w) values (0.99, 0.98, 0.96 and 0.93) at 25 °C on growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on maize based media. For both Fusarium species, the lag phase significantly decreased (p ≤ 0.001), and the growth rates increased (p ≤ 0.001) at the lowest ferulic acid concentration used (1 mM), regardless of the a_w. However, high doses of ferulic acid (10 to 25 mM) significantly reduced (p ≤ 0.001) the growth rate of both Fusarium species, regardless of the a_w. In general, growth rate inhibition increased as ferulic acid doses increased and as media a_w decreased. Fumonisin production profiles of both Fusarium species showed that low ferulic acid concentrations (1–10 mM) significantly increased (p ≤ 0.001) toxin production, regardless of the a_w. High doses of ferulic acid (20–25 mM) reduced fumonisin production, in comparison with the controls, by both Fusarium species but they were not statistically significant in most cases. The results show that the use of ferulic acid as a post-harvest strategy to reduce mycotoxin accumulation on maize needs to be discussed.     

Control of fumonisin B 1 accumulation in naturally contaminated maize inoculated with Fusarium verticillioides and Fusarium proliferatum, by cinnamon, clove, lemongrass, oregano and palmarosa essential oils

The effect of cinnamon, clove, oregano, palmarosa and lemongrass oils on fumonisin B₁ (FB₁) accumulation by one isolate each of Fusarium verticillioides and Fusarium proliferatum in non-sterilised naturally contaminated maize grain at 0.995 and 0.950 a_w and at 20 and 30 °C was evaluated. The concentration used was 500 mg kg^-1 maize. Under these conditions it was shown that antimycotoxigenic ability only took place at the higher water availabilities, and mostly at 20 °C. Only cinnamon, lemongrass and palmarosa oils were somewhat effective. Moreover, it was suggested that competing mycoflora plays an important role in FB₁ accumulation. It was concluded that the efficacy of essential oils in real substrates, such as cereals, may be much lower than in synthetic media; different essential oils may be found to be useful and at different concentrations. Their effectiveness is highly dependent on both abiotic and biotic factors involved.     

Genetic variability and Fumonisin production by Fusarium proliferatum

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize, such as wheat or barley among others, in particular in certain regions (Southern Europe). The phylogenetic study performed in this work using a wide sample of isolates from diverse hosts and origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCR assay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1, which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significant correlation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosis of metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influence on environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widely distributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides. Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact of fumonisin risk in a number of commodities might need a revision.     

Effects of menthol stereoisomers on the growth,sporulation and fumonisin B1 production of Fusarium verticillioides

Menthol is a naturally occurring cyclic terpene alcohol of plant origin from the Lamiaceae family. It has three chiral centres, implying eight possible different stereoisomers, which in turn define four pairs of enantiomers. This is the first work that reports on the stereoselective antifungal and antitoxigenic activities of the menthol stereoisomers on Fusarium verticillioides, with the (−)-menthol and (+)-menthol enantiomers found to be the most active inhibitors of fungal growth and sporulation. The results obtained suggest the importance of the presence of these substituents in the equatorial positions of menthol stereoisomers in the antifungal activity. The stereoisomer (−)-menthol, followed by (+)-menthol, were the most active compounds in the inhibition of fumonisin B₁ (FB₁) biosynthesis. The different antitoxigenic activities of (−)-menthol and (+)-menthol revealed that the molecular requirements to affect the FB₁ production were dependent not only on the presence of the substituents in the equatorial positions, but also on their spatial arrangements.     

Characterization of Fusarium verticillioides strains isolated from maize in Italy: fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.     

Fusarium pallidoroseum in maize samples of three agro-ecological zones of Cameroon

Investigations were conducted to determine the presence of mycotoxigenic fungi in maize samples from Cameroon. The deep freezing blotter method, and the medium DG-18, with and without 1% sodium hypochlorite surface sterilization pre-treatment, were used. The plated grains were incubated for 7 days under a cycle of 12 h (NUV) daylight and 12 h darkness. Fusarium pallidoroseum was found infecting 1-2% of the tested grains. Eleven samples out of 65 tested were found infected, 2 samples from the locality of Melong in the humid forest agro-ecological zone with monomodal rainfall, 1 from Foumbot and 2 from Bamenda in the highlands agro-ecological zone, and 6 from Yaounde, in the humid forest with bimodal rainfall agro-ecological zone. An infection rate of 2% of the grains was found on blotter paper while only 1% was recorded on the reduced water activity medium DG-18, with or without surface sterilization. F. pallidoroseum is reported here for the first time from maize samples of Cameroon. This underlines the need for detailed research on toxigenic fungi and the improvement of common storage practices to avoid mycotoxin contamination and the resulting human and animal health problems.     

Effect of Capsicum carotenoids on growth and ochratoxin A production by chilli and paprika Aspergillus spp. isolates

The aim of this study was to determine the effect of a natural carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and mycotoxin production of ochratoxin A-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates.One isolate of each species, previously isolated from paprika or chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0 to 1%) and incubated at 10, 15 and 25 °C for 21 days. Growth rates and lag phases were obtained, and OTA production was determined at 7, 14 and 21 days.The taxonomically related A. ochraceus and A. westerdijkiae showed the same behavior at 15 °C, but A. ochraceus was able to grow at 10 °C and had higher growth rates at 25 °C. A. tubingensis had the highest growth rates and lowest OTA production capacity of the assayed isolates, and it was not able to grow at 10 °C.Capsantal addition resulted in increased lag phases at 15 °C for all the strains, while growth rates remained rather constant. At 25 °C capsantal reduced growth rates, with rather constant lag phases. However, the effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and mostly was not significant.Low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. Industrial storage temperature for paprika and chilli is approximately 10 °C. If this temperature is maintained, mould growth and OTA production should be reduced.     

Integrated management of insect vectors of Aspergillus flavus in stored maize, using synthetic antioxidants and natural phytochemicals

The purpose of this study was to investigate the insecticidal activity of two benzoic acids 2(3)-tert-butyl-4 hydroxyanisole (BHA) and 2,6-di(tert-butyl)-p-cresol (BHT); two phenolic acids 3-phenyl-2-propenoic acid (CA) and trans-4-hydroxy-3-methoxycinnamic acid (FA) and two essential oils of Eugenia caryophyllata (clove tree) and Thymus vulgaris (thyme) against Sitophilus zeamais, Tribolium confusum and Rhyzopertha dominica, vector carriers of aflatoxigenic fungi in stored maize. The susceptibility of insects, the frequency of isolation of Aspergillus section Flavi in insects and maize, and the analysis of aflatoxin B₁ in maize were determined. BHA, BHT, BHA/BHT mixture and the natural phytochemicals AF and AF/AC mixture showed the highest insecticidal activity against S. zeamais, T. confusum and R. dominica after 120 days of incubation. The insecticidal efficacy of the volatile fraction of essential oils of clove and thyme showed less inhibition. There was no contamination of Aspergillus section Flavi in dead and live insects collected from maize treated with BHA. No aflatoxin B₁ accumulation was detected in the control and treatments. The information obtained shows that these substances have the potential to control pest insect vectors of aflatoxigenic fungi in stored maize in microcosms during 120 days.     

Effect of water activity, temperature and incubation time on growth and ochratoxin A production by Aspergillus niger and Aspergillus carbonarius on maize kernels

The aim of this study was to determine the effects of water activity (a_w) (0.92-0.98), temperature (5-45 °C) and incubation time (5-60 days) on growth and ochratoxin A (OTA) production by Aspergillus niger and Aspergillus carbonarius on maize kernels using a simple method. Colony diameters of both strains at 0.92 a_w were significantly lower than those at 0.96 and 0.98 a_w levels. The optimum growth temperature range for A. niger was 25-40 °C and for A. carbonarius 20-35 °C. A. niger produced OTA from 15 to 40 °C, and the highest OTA level was recorded at 15 °C. The concentration of OTA produced at 0.92 a_w was significantly lower than those at 0.96 and 0.98 a_w. A. carbonarius produced OTA from 15 to 35 °C and the maximum concentration was achieved at 15 °C, although not differing statistically from the concentration detected at 20 °C. At 0.98 a_w the OTA concentration was significantly higher than at 0.96 and 0.92 a_w. Our results show that maize supports both growth and OTA production by A. niger and A. carbonarius. The studied strains were able to produce OTA in maize kernels from the fifth day of incubation over a wide range of temperatures and water availabilities. Although the limit of quantification of our method was higher than that required for the analysis of OTA in food commodities, it has proved to be a useful and rapid way to detect OTA production by fungi inoculated onto natural substrates, in a similar way as for pure culture. Both species could be a source of OTA in this cereal in temperate and tropical zones of the world.     

Application of essential oils in maize grain: impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation

The antifungal activity of Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) essential oils (EOs) against Aspergillus section Flavi was evaluated in sterile maize grain under different water activity (a_w) condition (0.982, 0.955, and 0.90). The effect of EOs added to maize grains on growth rate, lag phase, and aflatoxin B₁ (AFB₁) accumulation of Aspergillus section Flavi were evaluated at different water activity conditions. The five EOs analyzed have been shown to influence lag phase and growth rate. Their efficacy depended mainly on the essential oil concentrations and substrate water activity conditions. All EOs showed significant impact on AFB₁ accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB₁ accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB₁ at 2000 and 3000 μg g⁻¹. Inhibition of AFB₁ accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.     

Molecular biodiversity of mycotoxigenic fungi that threaten food safety

Fungal biodiversity is one of the most important contributors to the occurrence and severity of mycotoxin contamination of crop plants. Phenotypic and metabolic plasticity has enabled mycotoxigenic fungi to colonize a broad range of agriculturally important crops and to adapt to a range of environmental conditions. New mycotoxin-commodity combinations provide evidence for the ability of fungi to adapt to changing conditions and the emergence of genotypes that confer enhanced aggressiveness toward plants and/or altered mycotoxin production profiles. Perhaps the most important contributor to qualitative differences in mycotoxin production among fungi is variation in mycotoxin biosynthetic genes. Molecular genetic and biochemical analyses of toxigenic fungi have elucidated specific differences in biosynthetic genes that are responsible for intra- and inter-specific differences in mycotoxin production. For Aspergillus and Fusarium, the mycotoxigenic genera of greatest concern, variation in biosynthetic genes responsible for production of individual families of mycotoxins appears to be the result of evolutionary adaptation. Examples of such variation have been reported for: a) aflatoxin biosynthetic genes in Aspergillus flavus and Aspergillus parasiticus; b) trichothecene biosynthetic genes within and among Fusarium species; and c) fumonisin biosynthetic genes in Aspergillus and Fusarium species. Understanding the variation in these biosynthetic genes and the basis for variation in mycotoxin production is important for accurate assessment of the risks that fungi pose to food safety and for prevention of mycotoxin contamination of crops in the field and in storage.     

Distribution of zearalenone in malted barley fractions dependent on Fusarium graminearum growing conditions

Zearalenone (ZON) distribution was measured in main fractions of malted barley, dependent on incubation time (17, 26, 34 days), water activity (0.95 and 0.98) and temperature (20 °C and 30 °C). Malted samples were sterilised and inoculated with Fusarium graminearum. ZON levels were higher (p < 0.01) in bran and germ than flour under almost all growing conditions. Incubation at 30 °C resulted in generally lower ZON levels in germ and bran, regardless of a_w and incubation time. After 34 days, ZON levels in flour from samples that were incubated at the higher temperature rose significantly. At 20 °C ZON concentration showed a bell-shaped concentration profile with increasing incubation time in bran and germ, whilst ZON levels in flour increased at a_w 0.98 and dropped at the lower a_w. The results indicate the importance of storage conditions for ZON levels in commercially relevant grain fractions of malted barley and help predict existing mycotoxin levels or manipulate storage conditions to reduce ZON content.     

Biodiversity of Aspergillus section Nigri populations in Argentinian vineyards and ochratoxin A contamination

Aspergillus section Nigri are described as the main source of ochratoxin A (OTA) contamination in grapes and wine worldwide. The knowledge of the factors affecting grape contamination by species included in this section and OTA production is essential to be able to reduce their presence, not only to improve wine quality, but also to maintain their safety. Therefore, the aims of this study were to determine the incidence of Aspergillus section Nigri species harvested in different grape-growing regions from Argentina, their ability to produce OTA, to correlate with meteorological conditions and geographical coordinates with their prevalence and to evaluate the OTA natural occurrence in grapes and wines. The morphological identification showed that Aspergillus niger aggregate species were the most prevalent ones, followed by Aspergillus carbonarius and Aspergillus uniseriate. These populations were confirmed through using AFLP markers and sequencing and, Aspergillus tubingensis was separated from A. niger aggregate. Climatic factors, altitude, longitude and latitude have influenced on the distribution of species included in the section. A. carbonarius and A. niger were OTA producers but differed in their OTA producing ability. Temperature was the factor which influenced the most over the highest incidence of A. carbonarius in La Rioja and San Juan regions. The trellis system in vineyards and drip irrigation also influenced the species isolation. The OTA levels detected in grapes and wines were low, but grape variety was more important in susceptibility to fungal infection and OTA levels.     

Modelling the effect of temperature and water activity on the growth of Aspergillus parasiticus on irradiated Argentinian flint maize

A full factorially designed experiment including storage temperature (10, 20, 30 and 37 °C) and water activity (0.88, 0.92 and 0.96) was undertaken to study the growth of Aspergillus parasiticus in maize samples. Kinetic parameters such as specific growth rate (μ), lag phase duration and maximum logarithmic increase were determined by fitting the Modified Gompertz equation to the viable mould count data (N in CFU/g) as a function of time collected in twelve experiments. The average coefficient of determination (R²) was 0.987, being the mean standard deviation of the estimate of 0.216 in units of log₁₀N. In the practical range of 10-30 °C, the relationship of the three kinetic parameters with temperature was described by second order polynomial expressions, whose parameters, in turn, depended on water activity. The combined or full model i.e., the Modified Gompertz model with its parameters expressed as a function of temperature and water activity, was able to predict log₁₀N with an average percentage error of 4.3, so agreement with the experimental data was highly satisfactory.In a simulation exercise, the full model was able to predict the viable mould count, given an initial value and grain temperature and water activity histories, with promising results for maize storage.     

Free radical scavenging activity and phenolic content in achenes and thalamus from Fragaria chiloensis ssp. chiloensis, F. vesca and F. x ananassa cv. Chandler

The total phenolic, flavonoid and anthocyanin content of achenes (true fruit) and thalamus (receptacle) from the native South American Fragaria chiloensis ssp. chiloensis (f. patagonica and f. chiloensis), Fragaria vesca and Fragaria x ananassa cv. Chandler was determined by spectrophotometric means. Highest phenolic content was found in F. vesca while lowest content was measured for white strawberry (F. chiloensis ssp. chiloensis, f. chiloensis). The total anthocyanin and total flavonoid contents in the samples investigated was lower for the white strawberry and higher in F. x ananassa cv. Chandler. Total flavonoid content showed a better correlation than total anthocyanins with the free radical scavenging effect of the extracts measured by means of the DPPH discoloration assay. In the superoxide anion assay all the acetone extracts of strawberries showed similar activity. The data presented in this study demonstrate that the amount of phenolic compounds differ significantly between species and subspecies and determine the free radical scavenging activity of fruits. On a w/w basis, higher total phenolics including flavonoids was found in achenes. The highest total anthocyanin content was found in the achenes of F. chiloensis and F. vesca, while F. ananassa presented higher antocyanin content in thalamus. The main anthocyanin in thalamus of F. ananassa (95%) were pelargonidin derivatives which were also present in F. chiloensis ssp. chiloensis f. patagonica (62.6%) but were not detected in F. vesca and F. chiloensis ssp. chiloensis f. chiloensis. In F. vesca and F. chiloensis ssp. chiloensis f. chiloensis only cyanidin derivatives were found both in thalamus and achenes.     

Development and application of a predictive model of Aspergillus candidus growth as a tool to improve shelf life of bakery products

Molds are responsible for spoilage of bakery products during storage. A modeling approach to predict the effect of water activity (a_w) and temperature on the appearance time of Aspergillus candidus was developed and validated on cakes. The gamma concept of Zwietering was adapted to model fungal growth, taking into account the impact of temperature and a_w. We hypothesized that the same model could be used to calculate the time for mycelium to become visible (t_v), by substituting the matrix parameter by t_v. Cardinal values of A. candidus were determined on potato dextrose agar, and predicted t_v were further validated by challenge-tests run on 51 pastries. Taking into account the a_w dynamics recorded in pastries during reasonable conditions of storage, high correlation was shown between predicted and observed t_v when the a_w at equilibrium (after 14 days of storage) was used for modeling (A_f = 1.072, B_f = 0.979). Validation studies on industrial cakes confirmed the experimental results and demonstrated the suitability of the model to predict t_v in food as a function of a_w and temperature.     

Water activity and temperature effects on mycotoxin production by Alternaria alternata on a synthetic tomato medium

Alternaria spp. have been reported to be the most frequent fungal species invading tomatoes. Certain species, in particular the most common one, A. alternata, are capable of producing several mycotoxins in infected plants and in agricultural commodities. Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA) are some of the main Alternaria mycotoxins that can be found as contaminants of food. The objective of this study was to determine the effect of water activity (a_w, 0.904, 0.922, 0.954, and 0.982) and temperature (6, 15, 21 and 35 °C) on mycotoxin production on a synthetic tomato medium of a cocktail inoculum of five strains of A. alternata isolated from tomato fruits affected by Blackmould. The optimum AOH production occurred at 0.954 a_w after 28 days of incubation at 21 °C. A temperature of 21 °C was the most favourable for AOH synthesis at all a_w levels. The maximum concentration of AME was determined at 0.954 a_w and 35 °C. The optimum conditions for TA accumulation were 0.982 a_w and 21 °C. At the 0.904 a_w no growth or germination was registered at 6 °C and 15 °C over the whole incubation period. At 21 °C and 35 °C growth occurred slowly but none of the toxins were detected at this a_w level. In general, high a_w levels were favourable for mycotoxin production. None of the other toxins was detected at quantifiable levels at 6 °C after the whole incubation period. A storage temperature of 6 °C or below could be considered as safe for tomato fruits and high moisture tomato products (a_w > 0.95), in relation with Alternaria toxins. The results obtained here could be extrapolated to evaluate the risk of spoilage in tomato fruits and tomato products caused by this pathogen.     

Exploiting Catharanthus roseus roots: Source of antioxidants

Catharanthus roseus is among the most important medicinal plants, mainly due to its anticancer alkaloids.     

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B₁ and B₂ production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB₁ and FB₂, respectively, P < 0.0001); r = 0.93 and r = 0.78 (correlation of FUM19 with FB₁ and FB₂, respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity.