Miriam Etcheverry

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 B(1) (AFB(1)) 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 AFB(1) accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB(1) accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB(1) at 2000 and 3000 microg g(-1). Inhibition of AFB(1) accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.

Detection and quantification of Aspergillus section Flavi spp. in stored peanuts by real-time PCR of nor-1 gene, and effects of storage conditions on aflatoxin production

Aspergillus flavus and A. parasiticus are the main species from section Flavi responsible for aflatoxin accumulation in stored peanuts. A real-time PCR (RT-PCR) system directed against the nor-1 gene of the aflatoxin biosynthetic pathway as target sequence was applied to monitor and quantify Aspergillus section Flavi population in peanuts. Kernels were conditioned at four water activity (a(W)) levels and stored during a 4-month period. The quantification of fungal genomic DNA in naturally contaminated peanut samples was performed using TaqMan fluorescent probe technology. Sensitivity tests demonstrated that DNA amounts accounting for a single conidium of A. parasiticus RCP08300 can be detected. A standard curve relating nor-1 copy numbers to colony forming units (cfu) was constructed. Counts of species of Aspergillus section Flavi from unknown samples obtained by molecular and conventional count (CC) methodologies were compared. A correlation between cfu data obtained by RT-PCR and CC methods was observed (r=0.613; p<0.0001); and the former always showed values higher by 0.5-1 log units. A decrease of fungal density was observed throughout the storage period, regardless of the quantification methodology applied. Total aflatoxin levels ranging from 1.1 to 200.4 ng/g were registered in peanuts conditioned at the higher a(W) values (0.94-0.84 a(W)). The RT-PCR assay developed appears to be a promising tool in the prediction of potential aflatoxigenic risk in stored peanuts, even in case of low-level infections, and suitable for rapid, automated and high throughput analysis.

Occurrence of deoxynivalenol and Fusarium graminearum in Argentinian wheat

During the 1993 harvest period there was a high incidence of Fusarium head blight in wheat in Argentina. Fusarium species that produce trichothecenes in wheat have been reported in several countries including Argentina. Several studies have shown that F. graminerarum and deoxynivalenol (DON) were the main contaminants detected in wheat and by-products in Argentina. The objective of this study was to evaluate the occurrence of Fusarium spp. and DON contamination in wheat from Córdoba, Argentina during the 1993/94 harvest season. F. graminearum was the main Fusarium species isolated. From 40 samples analysed, 80% showed DON contamination. The levels of DON found ranged between 300 and 4500 micrograms/kg. There was good correlation between F. graminearum and DON contamination. Only five samples showed levels of DON higher than those established in the guidelines in Canada and the USA for food and feedstuffs.

Analysis of the Bacterial Diversity Associated with theRoots of Maize (Zeamays L.) through Culture-Dependent and Culture-Independent Methods

The present study investigated bacterial diversity associated with the roots of maize through the use of culture-dependent and culture-independent methods. Bacterial 16S–23S rDNA internal transcribed spacer sequences (ITS) primers were used to amplify sequences obtained directly from the root matrix by Percoll gradient separation. This assay showed that γ-Proteobacteria within Enterobacter, Erwinia, Klebsiella, Pseudomonas ,a ndStenotrophomonas genera were predominant groups. The culturable component of the bacterial community was also assessed, revealing that the predominant group was Firmicutes, mainly of Bacillus genus, while Achromobacter, Lysinibacillus ,a ndPaenibacillus genera were rarely found in association with the roots. Only two genera within γ-Proteobacteria, Enterobacter and Pseudomonas, were found in the culture collection. Differences in richness and diversity between the rhizospheric and endophytic bacterial communities were also evidenced. The spectrum of bacteria naturally associated with maize roots is wide and the magnitude of such diversity will depend on the methods chosen for analysis. The knowledge of this spectrum will facilitate the search of microorganisms capable of exerting antagonism to diverse pathogens or detecting plant growth enhancers.

Occurrence of Aspergillus section Flavi and aflatoxin B1 in corn genotypes and corn meal in Argentina

A study has been carried out in Argentina on samples of corn genotypes from a breeding station as well as in commercially available corn meal. All samples were analyzed for fungal infection and aflatoxin B1.Mycological analysis of corn genotypes showed the presence of three principal genera of filamentous fungi Fusarium (100%), Penicillium (67%) and Aspergillus (60%). In the genus Fusarium three species were identified, F. moniliforme (42%), F. nygamai (56%) andF. proliferatum (1.8%). Eight species ofPenicillium were identified, the predominant species isolated were P. minioluteum, P. funiculosum and P. variabile. In the genus ranked third in isolation frequency, two species were identified, A. flavus and A. parasiticus, the percentage of infection was 78% and 21%, respectively. Only one corn genotype was contaminated with aflatoxin B1 at a level of 5 ppb. The cornmeal samples showed great differences in fungal contamination, the values ranging from 1 × 101 to 7 × 105 cfu g−1. Fusarium (68%), Aspergillus (35%) and Penicillium (21%) were the most frequent genera isolated. Among the genus, Aspergillus, A. parasiticus (38%) was the most frequent species isolated. All the samples of corn meal were negative to aflatoxin B1. These results indicate a low degree of human exposure to aflatoxins in Argentina through the ingestion of maize or corn meal.

Incidence of aflatoxin, zearalenone, and deoxynivalenol on corn in Argentina

SummaryThe study of the incidence of aflatoxins, zearalenone, and deoxynivalenol was the aim of this work. This investigation was carried out upon recently harvested corn samples collected in the Departamento of Rio Cuarto, Province of Córdoba, Argentina. 150 samples of corn were analized to investigate aflatoxins B1, G1, and zearalenone contamination. Out of these 150 samples 58 were selected and deoxynivalenol was examined.The incidence value for aflatoxin B1 was of 3.3% (5 samples), aflatoxin G1 1.3 % (2 samples), and zearalenone 6 % (9 samples).The analysis of the 58 samples showed that 24% of them were contaminated with deoxynivalenol.

Alternaria mycotoxins in sunflower seeds : incidence and distribution of the toxins in oil and meal

A survey of 150 sunflower-seed samples was carried out to evaluate the contamination from infection with Alternaria alternata with alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA). A high percentage of the samples was contaminated with AOH (85%), AME, (47%), and TA (65%). The average levels detected were 187 μg/kg for AOH, 194 μg/kg for AME, and 6,692, μg/kg for TA. When sunflower seeds fermented by Alternaria alternata were processed under laboratory conditions to obtain the oil and meal, different distributions of Alternaria toxins between the oil and the meal were observed: whereas AOH, AME, and TA were detected in the meal, only AME and TA were detected in the oil, and the latter in a low percentage.

In vitro studies on the potential for biological control on Aspergillus section Flavi by Kluyveromyces spp.

Aims:  Antagonist activity of Kluyveromyces spp. isolates on Aspergillus section Flavi was studied.

Aflatoxin B1 and fumosin B1 in mixed cultures of Aspergillus flavus and Fusarium proliferatum on maize

Production of aflatoxin B1 and fumonisin B1 in pure and mixed cultures of Aspergillus flavus and Fusarium proliferatum were determined on irradiated maize seeds inoculated with different spore concentrations at 0.97 water activity (a(w)) and a temperature of 25 degrees C. The highest levels of aflatoxin B1 were produced by A. flavus at the lowest levels of inoculum (10(3) spore ml(-1)). There was no spore concentration influence on fumonisin B1 production after 10, 20 and 35 days of incubation. When A. flavus was co-inoculated with F. proliferatum, aflatoxin B1 production was inhibited. The higher the inocula levels of Fusarium produced, the higher the inhibition and this inhibition increased during the incubation period. Total inhibition was reached at 35 days of incubation. There was no interaction influence on fumonisin B1 production at all inoculum levels assayed. These results suggest that under optimal environmental conditions of substrate, water activity and temperature, the interaction between A. flavus and F proliferatum could produce inhibition of aflatoxin B1 and stimulation of fumonisin B1.

Evaluation of the control ability of five essential oils against Aspergillus section Nigri growth and ochratoxin A accumulation in peanut meal extract agar conditioned at different water activities levels.

Essential oils (EOs) from boldo [Pëumus boldus Mol.], poleo [Lippia turbinata var. integrifolia (Griseb.)], clove [Syzygium aromaticum L.], anise [Pimpinella anisum] and thyme [Thymus vulgaris]) obtained by hydrodistillation were evaluated for their effectiveness against the growth of Aspergillus niger aggregate and A. carbonarius and accumulation of ochratoxin A (OTA). The evaluation was performed by compound dissolution at the doses of 0, 500, 1500 and 2500μL/L in peanut meal extract agar (PMEA) and exposure to volatiles of boldo, poleo (0, 1000, 2000 and 3000μL/L) and clove oils (0, 1000, 3000 and 5000μL/L), taking into account the levels of the water activity of the medium (a(W) 0.98, 0.95, 0.93). Statistical analyses on growth of Aspergillus strains indicated that the major effect was produced by oil concentrations followed by substrate a(W), and that reductions in antifungal efficiency of the oils tested were observed in vapor exposure assay. At all a(W) levels, complete fungal growth inhibition was achieved with boldo EO at doses of 1500 and 2000μL/L by contact and volatile assays, respectively. Contact exposure by poleo and clove EOs showed total fungal inhibition at the middle level tested of 1500μL/L, regardless of a(W), while their antifungal effects in headspace volatile assay were closely dependent on medium a(W). The fumigant activity of poleo (2000μL/L) and clove oils (3000μL/L) inhibited growth rate by 66.0% and 80.6% at a(W) 0.98 and 0.93, respectively. OTA accumulation was closely dependent on a(W) conditions. The antiochratoxigenic property of the volatile fractions of boldo, poleo and clove EOs (1000μL/L) was more significant at low a(W) levels, inhibition percentages were estimated at 14.7, 41.7 and 78.5% at a(W) 0.98, 0.95 and 0.93, respectively. Our results suggest that boldo, poleo and clove oils affect the OTA biosynthesis pathway of both Aspergillus species. This finding leaves open the possibility of their use by vapor exposure as effective non-toxic biopreservatives against OTA contamination in stored peanuts.