Jéssica Gil-Serna

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR.

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10(6)spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.

ITS-based detection and quantification of Aspergillus ochraceus and Aspergillus westerdijkiae in grapes and green coffee beans by real-time quantitative PCR.

Aspergillus ochraceus and A. westerdijkiae are considered the most important Ochratoxin A (OTA) producing species included in Aspergillus section Circumdati which contaminate foodstuffs and beverages for human consumption. In this work a real-time quantitative PCR protocol was developed to detect both species using SYBR Green and primers designed on the basis of the multicopy ITS1 region of the rDNA. The assay had high efficiency (94%) and showed no inhibition by host or fungal DNA other than the target species. The lower detection limit of the target DNA was 2.5 pg/reaction. Accuracy of detection and quantification by qPCR were tested with genomic DNA obtained from green coffee beans and grapes artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/ml could be detected by the assay directly without prior incubation of the samples and a positive relationship was observed between incubation time and qPCR values. The assay developed would allow rapid, specific, accurate and sensitive detection and quantification of A. ochraceus and A. westerdijkiae to be directly used in a critical point of the food chain, before harvesting green coffee and grape berries, to predict and control fungal growth and OTA production.

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp.

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.

Specific detection of Aspergillus carbonarius by SYBR® Green and TaqMan® quantitative PCR assays based on the multicopy ITS2 region of the rRNA gene.

Agroproducts contaminated by ochratoxin A (OTA) represent a risk for human and animal health and, therefore, maximum limits have been established by Food Safety Authorities. Reduction of OTA contamination may be accomplished by early detection of OTA-producing fungal species using rapid, specific and sensitive detection and quantification by PCR-based methods. Aspergillus carbonarius is one of the most important OTA-producing species, in particular in grapes and derivatives from Mediterranean regions. In this work, highly efficient quantitative PCR assays using SYBR Green I and TaqMan methods were developed for specific detection of A. carbonarius to be used in grapes. The primers and the TaqMan probe were based on the internal transcribed region 2 multicopy region (internal 2 sequence of the rRNA gene). The specificity and sensitivity of both assays were tested on genomic DNA mixtures of several A. carbonarius strains and other fungal species frequently present in grapes. Both methods were also compared using grapes inoculated with different spore concentrations of A. carbonarius, detecting up to 0.4 pg DNA g(-1) grape berries. The efficiency and sensitivity of both methods were comparable and only the lower cost of SYBR Green might favour its use in routine screenings.

Discrimination of the main Ochratoxin A-producing species in Aspergillus section Circumdati by specific PCR assays.

Ochratoxin A (OTA) is one of the most important mycotoxins because of its high toxicity to both humans and animals and its occurrence in a number of basic foods and agro-products. Some of the main OTA-producing species belong to Aspergillus section Circumdati, whose taxonomy has been lately revised with the description of new species. The high morphological similarity of these species (Aspergillus ochraceus, Aspergillus steynii and Aspergillus westerdijkiae) makes difficult discrimination among them and with respect to the other species included in the same section unable to produce OTA. In this work, PCR assays specific for the OTA-producing species, A. ochraceus, A. steynii and A. westerdijkiae, were developed on the basis of the multicopy ITS regions of the rDNA. The assays were tested in a wide sample of isolates from diverse origins and culture collections, confirming their specificity, and revealing that the current identification of strains from section Circumdati might need a revision.

Mechanisms involved in reduction of ochratoxin A produced by Aspergillus westerdijkiae using Debaryomyces hansenii CYC 1244

Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within the Section Circumdati contaminating a number of agroproducts. The yeast Debaryomyces hansenii CYC 1244 was previously reported to be able to reduce growth and extracellular OTA produced by A. westerdijkiae. In this work, we examined several mechanisms possibly involved in this OTA reduction in in vitro experiments. OTA biosynthesis was evaluated by quantitation of expression levels of pks (polyketide synthase) and p450-B03 (cytochrome p450 monooxygenase) genes using newly developed and specific real time RT-PCR protocols. Both genes showed significant lower levels in presence of D. hansenii CYC 1244 suggesting an effect on regulation of OTA biosynthesis at transcriptional level. High levels of removal of extracellular OTA were observed by adsorption to yeast cell walls, particularly at low pH (98% at pH 3). On the contrary, no evidences were obtained of absorption of OTA into yeast cells or the production of constitutively expressed enzymes that degrade OTA by D. hansenii CYC 1244. These results described the potential of this yeast strain as a safe and efficient biocontrol agent to decrease OTA in A. westerdijkiae and two important mechanisms involved which may permit its application at different points of the food chain.

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.

Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay.

Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 106 spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.

Aspergillus steynii and Aspergillus westerdijkiae as potential risk of OTA contamination in food products in warm climates.

Aspergillus steynii and Aspergillus westerdijkiae are the main ochratoxin A (OTA) producing species of Aspergillus section Circumdati. Due to its recent description, few data are available about the influence of ecophysiological factors on their growth and OTA production profiles. In this work, the effect of temperature (20, 24 and 28 °C) and water activity (aw) (0.928, 0.964 and 0.995) on growth, sporulation and OTA production by these fungi was examined in CYA and media prepared from paprika, green coffee, anise, grapes, maize and barley. Growth was positively affected by the highest temperature and aw values indicating that both species might be expected in warm climates or storage conditions. However, optimal growth conditions showed differences depending on the medium. OTA production was markedly affected by substrate and showed qualitative and quantitative differences. Both species, especially A. steynii, represent a great potential risk of OTA contamination due to their high production in a variety of conditions and substrates, in particular in barley and paprika-based media. Additionally, neither growth nor sporulation did result good indicators of OTA production by A. steynii or A. westerdijkiae; therefore, specific and highly-sensitive detection methods become essential tools for control strategies to reduce OTA risk by these species.

Fungal diversity, incidence and mycotoxin contamination in grapes from two agro-climatic Spanish regions with emphasis on Aspergillus species.

BACKGROUND Fourteen vineyards from two different agro-climatic regions in Spain were sampled in two consecutive years in order to determinate the grape mycobiota and diversity indexes with the final aim to define the potential mycotoxigenic species from both regions and their relationship. RESULTS The most common fungal genera encountered were Aspergillus (30.0%), Alternaria (53.2%), Cladosporium (11.9%) and Penicillium (2.9%). Black aspergilli presence in the hotter region (south) was significantly higher (P < 0.05) than in the northeast in both years. Among black aspergilli, A. tubingensis seemed to be the better adapted species to environmental conditions, while A. carbonarius was the main potentially ochratoxigenic species in both regions and years, owing to the most relevant percentage of ochratoxigenic isolates. Ochratoxin A (OTA)-positive musts were only detected from southern vineyards, although contamination was always lower than 0.1 µg L(-1) . Finally, none of black aspergilli tested produced fumonisins (FBs) on Czapek yeast extract agar (CYA), while 63% of A. niger tested produced FB2 when inoculated on CYA20S, reaching 100% of isolates from the south. CONCLUSION Climate change scenarios in southern Europe point to an increase in temperature and drought. This could promote particularly adapted species such as A. niger, decreasing OTA risk, but this could lead to an increase in FB2 presence.