Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize

Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A

OchratoxinA (OTA) is a potential nephrotoxic, teratogenic, immunogenic, hepatotoxic and carcinogenic mycotoxin, produced by Aspergillus westerdijkiae NRRL 3174. Herein we describe the characterization of a putative OTA-polyketide synthasegene “aoks1”, cloned by using gene walking approach. The predicted amino acid sequence of the 2 kb clone display 34–60% similarities to different polyketide synthasegenes including lovastatine biosynthesis gene “lovb” in A. terreus, compactin biosynthesis gene “mlcA” in Penicillium citrinum and OTA biosynthesis gene “otapksPN” in P. nordicum. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aoks1 expression was found to be associated with OTA biosynthesis. Further a mutant, in which the aoks1gene was inactivated by Escherichia coli hygromycin B phosphotransferase gene, lost the capacity to produce OTA, but still producing mellein. To our knowledge this report describes for the first time characterization of a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. This study also suggests that aoks1 may be the second polyketide synthase gene required for OTA biosynthesis in A. westerdijkiae NRRL 3174

Approche de la mycotoxinogénèse chez Aspergillus ochraceus et Aspergillus carbonarius : études moléculaire et physiologique

Aspergillus ochraceus et Aspergillus carbonarius, contaminants fongique fréquents de plusieurs produits agricoles, produisent de nombreux polycétones dont les mycotoxines. Durant ce travail, la diversité des gènes de polycétones synthases (PKSs), enzymes responsables de la biosynthèse des polycétones, présentes chez ces champignons a été étudiée en utilisant des couples d’amorces spécifiques et dégénérées. Au total, neuf PKSs différentes chez Aspergillus ochraceus NRRL 3174 et cinq PKSs différentes chez Aspergillus carbonarius 2Mu134 ont été identifiées. Elles sont distribuées dans les trois principaux groupes de PKS connus chez les champignons. Parmi les gènes identifiés chez Aspergillus ochraceus, AoKS1, est responsable de la biosynthèse de l’ochratoxine A et AoLC35-2 est responsable de la biosynthèse des composés lactoniques. D’autre part, nous avons développé un système de détection et de quantification d’Aspergillus carbonarius par PCR en temps réel en ciblant un gène de polycétone synthase. Le système développé nous a ainsi permis aussi d’avoir une estimation rapide de la contamination en ochratoxine A dans le raisin à partir de la quantification d’ADN d’Aspergillus carbonarius. Enfin, une étude physiologique menée chez Aspergillus carbonarius pour connaître la répartition d’ochratoxine A dans les spores, le mycélium et le substrat a montré que la majorité de cette toxine se trouve accumulée au niveau des spores. Les résultats ont aussi montré que la toxine accumulée se relargue dans le milieu et arrive après 4 heures d’incubation à un niveau plus grand que celui de la valeur initiale. Ceci montre que les spores d’Aspergillus carbonarius peuvent participer à l’augmentation de l’ochratoxine A durant la macération dans les procèdes oenologiques. L’ensemble de ces travaux se situe dans le cadre de la gestion du problème des mycotoxines dans la chaîne alimentaire. ABSTRACT : Aspergillus ochraceus and Aspergillus carbonarius are considered as the most frequent fungal contaminants of several agricultural products. They are able to produce several polyketide-derived secondary metabolites including mycotoxins. Fungal polyketide synthase (PKS) is the key enzyme responsible for the biosynthesis of the polyketide. In this study, the diversity of polyketide synthase (PKS) genes in Aspergillus ochraceus NRRL 3174 and Aspergillus carbonarius 2Mu134 has been investigated using different primers pairs previously developed for the ketosynthase (KS) domain of fungal PKSs. Nine different KS domain sequences in A. ochraceus NRRL 3174 as well as five different KS domain sequences in A. carbonarius 2Mu134 have been identified. The identified KS fragments were distributed in the 3 different groups of the known fungal polyketide synthases. Among the identified genes in Aspergillus ochraceus, AoKS1 was found to be involved in the biosynthesis of ochratoxin A and AoLC35-2 was found to be responsible for the biosynthesis of lactone metabolites using a targeted gene deletion strategy. In Aspergillus carbonarius we have developed a molecular assay for the direct detection and quantification of this fungus in grape by targeting a polyketide synthase gene using real-time PCR. The developed system allow us to have an approximately estimation of the OTA contamination level by the quantification of A. carbonarius DNA in grape. Finally, a physiological study has been conducted in order to examine the partitioning of ochratoxin A in substrate, mycelium and conidia by Aspergillus carbonarius showed that the majority of this toxin was accumulated in the conidia. The OTA in spores was found to be rapidly excreted into the medium during the initial few hours of conidial germination leading to an increase of OTA in must during maceration for wine production

Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification

Robust Automatic Speech recognition System Implemented in a Hybrid Design DSP-FPGA

The aim of this work is to reduce the burden task on the DSP processor by transferring a parallel computation part on a configurable circuits FPGA, in automatic speech recognition module design, signal pre-processing, feature selection and optimization, models construction and finally classification phase are necessary. LMS filter algorithm that contains more parallelism and more MACs (multiply and Accumulate) operations is implemented on FPGA Virtex 5 by Xilings, MFCCs features extraction and DTW ( dynamic time wrapping) method is used as a classifier. Major contribution of this work are hybrid solution DSP and FPGA in real time speech recognition system design, the optimization of number of MAC-core within the FPGA this result is obtained by sharing MAC resources between two operation phases: computation of output filter and updating LMS filter coefficients. The paper also provides a hardware solution of the filter with detailed description of asynchronous interface of FPGA circuit and TMS320C6713-EMIF component. The results of simulation shows an improvement in time computation and by optimizing the implementation on the FPGA a gain in space consumption is obtained

Global warming as a driving factor for cyanobacterial blooms in Lake Karaoun, Lebanon

18 pagesInternational audienceThe Middle East region suffers already from the gradual effects of climate change and will be among the most vulnerable regions in the future. As a result, productivity should undergo losses due to high temperatures, drought, floods and soil degradation which threaten food security of Levantine countries. Since water is the critical factor in the region, even slight changes in air temperature and rainfall patterns will have considerable impact. It has been proven that potential climate change may disrupt, on one hand, most ecosystems through changes in their physicochemical conditions, and on the other hand the species which are living in these ecosystems. Then, the biodiversity can be found challenging. In this study, the effects of climate change on population and phytoplankton communities of Lake Karaoun were investigated since 1992. The climate regime shifts have been shown to alter the lake ecosystem. In the past, Lake Karaoun was characterized by a highly diversified microflora dominated by diatoms and green algae. Recent climatic fluctuations, with culmination in 2008-2011 and temperatures exceeding 40˚C have upset this biodiversity. Blooms of cyanobacteria, specifically Microcystis aeruginosa and Aphanizomenon ovalisporum, have occurred and disturbed both the ecosystem and the functioning of the lake

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in Aspergillus westerdijkiae

Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298 bp polyketide synthase gene ‘‘aomsas” has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40–56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant ‘‘aoDmsas” of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone,but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoDmsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone

False synergy between vancomycin and β-lactams against glycopeptide-intermediate Staphylococcus aureus (GISA) caused by inappropriate testing methods

ABSTRACTThe combination of vancomycin and β-lactams is often considered synergistic and has been recommended for the treatment of glycopeptide-intermediate Staphylococcus aureus (GISA) infections. In this study, the combination of vancomycin or teicoplanin with different β-lactams was tested. When using NaCl 4% w/v, for better expression of heterogeneous resistance to β-lactams, with a longer (48-h) incubation period and a higher (107 CFU/mL) inoculum, the association of vancomycin with β-lactams was antagonistic. However, a synergistic effect was observed for teicoplanin under the same conditions

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation

Essential oils modulate gene expression and ochratoxin a production in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin, mainly produced on grapes by Aspergillus carbonarius, that causes massive health problems for humans. This study aims to reduce the occurrence of OTA by using the ten following essential oils (E.Os): fennel, cardamom, anise, chamomile, celery, cinnamon, thyme, taramira, oregano and rosemary at 1 µL/mL and 5 µL/mL for each E.O. As a matter of fact, their effects on the OTA production and the growth of A. carbonarius S402 cultures were evaluated, after four days at 28 C on a Synthetic Grape Medium (SGM). Results showed that A. carbonarius growth was reduced up to 100%, when cultured with the E.Os of cinnamon, taramira, and oregano at both concentrations and the thyme at 5 µL/mL. As for the other six E.Os, their effect on A. carbonarius growth was insignificant, but highly important on the OTA production. Interestingly, the fennel E.O at 5 µL/mL reduced the OTA production up to 88.9% compared to the control, with only 13.8% of fungal growth reduction. We further investigated the effect of these E.Os on the expression levels of the genes responsible for the OTA biosynthesis (acOTApks and acOTAnrps along with the acpks gene) as well as the two regulatory genes laeA and vea, using the quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) method. The results revealed that these six E.Os reduced the expression of the five studied genes, where the ackps was downregulated by 99.2% (the highest downregulation in this study) with 5 µL/mL of fennel E.O. As for the acOTApks, acOTAnrps, veA and laeA, their reduction levels ranged between 10% and 96% depending on the nature of the E.O and its concentration in the medium