Covadonga Vázquez

PCR detection assay of fumonisin-producing Fusarium verticillioides strains.

Fusarium verticillioides is considered to be the main source of fumonisins, a group of toxins that contaminate commodities and result in chronic and acute diseases affecting humans and animals. The detection and control of this species is crucial to prevent fumonisins from entering the food chain. The objective of the present research was to develop a specific, sensitive, and robust PCR assay to detect F. verticillioides strains using two pairs of specific primers for F. verticillioides, which have been designed on the basis of the intergenic spacer region of the rDNA units. The first pair of primers was F. verticillioides species specific, whereas the second pair of primers detected fumonisin-producing F. verticillioides strains. This second pair of primers allowed for the discrimination between the major group of F. verticillioides strains, fumonisin-producing strains that are mainly associated with crops, and a minor group of strains, non-fumonisin-producing strains that are associated with bananas. Fifty-four strains of F. verticillioides from different geographical regions and hosts were tested using both sets of primers. Sixteen additional Fusarium species were examined. The specificity of the primer sequences provides the basis for a simple, rapid, accurate, and sensitive detection and identification method of this fungal species that represents a risk for human and animal health.

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.

Differentiation of Fusarium verticillioides from banana fruits by IGS and EF-1α sequence analyses

Fusarium verticillioides(Gibberella moniliformis, G. fujikuroi mating population A) is an important pathogen of maize and produces several mycotoxins, including fumonisins, which cause diseases in humans and animals. The partial sequences of the IGS region (Intergenic Spacer of rDNA units) and the translation elongation factor EF-1α gene of a representative sample (48 strains) of F. verticillioides isolated from diverse hosts, geographical origins and with different levels of fumonisin production were analyzed. A phylogenetic approach by PAUP was used to evaluate the genetic variability in this species and to detect the occurrence of lineages which could be associated with different hosts or produced different toxin profiles within this species. Genetic variability detected by both sequences was high, especially with the IGS sequence which showed a high number of parsimony-informative sites and nucleotide diversity. The results of the phylogenetic analysis indicated that F. verticillioides occurs as (i) a major fumonisin-producing population with a wide geographical distribution, wide host preferences (cereals), showing variability and considerable incidence of sexual reproduction and (ii) a minor fumonisin non-producing population, with restricted host preference (banana), low variability and clonal reproductive strategy.

Genetic markers for the analysis of variability and for production of specific diagnostic sequences in fumonisin-producing strains of Fusarium verticillioides

Fusarium verticillioides(Gibberella moniliformis, Gibberella fujikuroi mating population A) is the main source of fumonisins, a group of toxins which contaminates commodities, causing chronic and acute diseases in humans and animals. Fumonisins are produced during colonisation and infection of host plants even when disease symptoms are not recognisable. Early detection and control of F. verticillioides is crucial to prevent fumonisins from entering the food chain. DNA-based strategies have been used to search for markers to develop sensitive, robust and specific diagnostic assays, mainly based on PCR. The different approaches used, based either on DNA markers unrelated to fumonisin production or on information about the genes involved in fumonisin production, are described and discussed. The ability of these methods to discriminate between the two populations occurring within F. verticillioides, fumonisin-producing and fumonisin non-producing strains, is also addressed.

Differential effect of environmental conditions on the growth and regulation of the fumonisin biosynthetic gene FUM1 in the maize pathogens and fumonisin producers Fusarium verticillioides and Fusarium proliferatum

The effects of ecophysiological factors, temperature and solute potential, on both the growth and the regulation of the fumonisin biosynthetic FUM1 gene were studied and compared in one isolate each of the two closely related fumonisin-producing and maize pathogens Fusarium verticillioides and Fusarium proliferatum. The effect of solute potential and temperature was examined on in vitro mycelia growth and on the expression of the FUM1 gene, quantified by species-specific real-time reverse transcriptase-PCR assays. Although both isolates showed similar two-dimensional profiles of growth, for F. verticillioides, optimal growth conditions were maintained at higher temperatures and lower solute potential values. FUM1 gene expression was markedly induced at 20 degrees C in both isolates, under suboptimal conditions for growth; however, their expression patterns differed in relation to solute potential. Whereas FUM1 expression was induced in response to increasing water stress in the isolate of F. verticillioides, the F. proliferatum one showed a stable expression pattern regardless of water potential conditions. These results suggest a differential regulation of fumonisin biosynthesis in these isolates of the two species that might be related to their different host range, and play an ecological role. Additionally, environmental conditions leading to water stress (drought) might result in increased risk of fumonisin contamination of maize caused by F. verticillioides.

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.

Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 102 spores after 16 h of incubation.

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.