Phylogenetic analysis and growth profiles of Fusarium incarnatum-equiseti species complex strains isolated from Tunisian cereals.

The Fusarium incarnatum-equiseti species complex (FIESC) is a phylogenetically rich complex. It includes more than 30 cryptic phylogenetic species, making morphological identification problematic. FIESC has previously been detected in Tunisian cereals, but knowledge on the phylogeny and the ecophysiology of their species is lacking. In this work a phylogenetic analysis was performed using partial sequences of the translation elongation factor 1a gene (EF1a) of three FIESC strains isolated from barley and wheat from Tunisia, situated south in the Mediterranean basin, and additional strains from other countries. The results indicated that all Tunisian strains clustered with FIESC 5 group (F. clavum) together with other Spanish FIESC 5 strains also isolated from cereals. Growth rate profiles of the Tunisian strains were also determined on wheat and sorghum based media at a range of temperatures (15, 20, 25, 30, 35 and 40 °C) and water potential values (-0.7, -2.8, -7.0, and -9.8 MPa, corresponding to 0.995, 0.98, 0.95 and 0.93 aw values). Optimal growth was observed at 20-30 °C and between -0.7 and -7.0 MPa on both substrates (wheat and sorghum). The highest growth rate for the three strains was seen at 25 °C combined with -2.8 MPa. The comparison between the growth profiles of Tunisian and Spanish FIESC 5 strains showed similar trends with some interesting differences regarding temperature and water potential factors. Tunisian strains seem to perform better between 15 and 30 °C and, notably, at even lower water potentials included -9.8 Mpa. This might suggest that tolerance to low water potentials might be for Tunisian strains a more important selective clue than to higher temperatures. These results appeared to be consistent with a population well adapted to the present climatic conditions and predicted scenarios for North Africa.

Significance of Aspergillus niger aggregate species as contaminants of food products in Spain regarding their occurrence and their ability to produce mycotoxins.

The Aspergillus niger aggregate contains 15 morphologically indistinguishable species which presence is related to ochratoxin A (OTA) and fumonisin B2 (FB2) contamination of foodstuffs. The taxonomy of this group was recently reevaluated and there is a need of new studies regarding the risk that these species might pose to food security. 258 isolates of A. niger aggregate obtained from a variety of products from Spain were classified by molecular methods being A. tubingensis the most frequently occurring (67.5%) followed by A. welwitschiae (19.4%) and A. niger (11.7%). Their potential ability to produce mycotoxins was evaluated by PCR protocols which allow a rapid detection of OTA and FB2 biosynthetic genes in their genomes. OTA production is not widespread in A. niger aggregate since only 17% of A. niger and 6% of A. welwitschiae isolates presented the complete biosynthetic cluster whereas the lack of the cluster was confirmed in all A. tubingensis isolates. On the other hand, A. niger and A. welwitschiae seem to be important FB2 producers with 97% and 29% of the isolates, respectively, presenting the complete cluster. The genes involved in OTA and FB2 were overexpressed in producing isolates and their expression was related to mycotoxin synthesis.

Mycoflora isolation and molecular characterization of Aspergillus and Fusarium species in Tunisian cereals.

Wheat, barley and maize are the mainly consumed cereals in Tunisia. This study aimed to determine the mycoflora of these cereals with special focus on the mycotoxigenic Aspergillus and Fusarium species. Freshly harvested samples and other stored samples of each type of cereal (31 and 34 samples, respectively) were collected in Tunisia and cultured for fungal isolation and identification. Identification of fungal genera was based on morphological features. Aspergillus and Fusarium species were identified by species specific PCR assays complemented with DNA sequencing. Alternaria (70.83%), Eurotium (62.50%), Aspergillus (54.17%) and Penicillium (41.67%) were the most frequent fungi isolated from wheat. Penicillium (75%), Aspergillus (70%), Eurotium (65%) and Alternaria (65%) were the most frequently recovered genera from barley. The predominant genera in maize were Aspergillus (76.19%), Eurotium (42.86%), and Penicillium (38.09%). Aspergilllus, Penicillium, Fusarium and Alternaria were detected in both stored and freshly harvested grain samples. The frequencies of contamination with Aspergillus, Fusarium and Alternaria were higher in freshly harvested samples, whereas Penicillium species were more frequent in stored samples. The predominant Aspergillus species detected were A. flavus and A. niger. The Fusarium species detected were F. equiseti, F. verticillioides, F. nygamai, and F. oxysporum. This study suggested the potential risk for Aflatoxins and, to a lesser extent, for Ochratoxin A in Tunisian cereals. This is the first survey about mycoflora associated with wheat, barley and maize in Tunisia.

Description of an orthologous cluster of ochratoxin A biosynthetic genes in Aspergillus and Penicillium species. A comparative analysis.

Ochratoxin A (OTA) is one of the most important mycotoxins due to its toxic properties and worldwide distribution which is produced by several Aspergillus and Penicillium species. The knowledge of OTA biosynthetic genes and understanding of the mechanisms involved in their regulation are essential. In this work, we obtained a clear picture of biosynthetic genes organization in the main OTA-producing Aspergillus and Penicillium species (A. steynii, A. westerdijkiae, A. niger, A. carbonarius and P. nordicum) using complete genome sequences obtained in this work or previously available on databases. The results revealed a region containing five ORFs which predicted five proteins: halogenase, bZIP transcription factor, cytochrome P450 monooxygenase, non-ribosomal peptide synthetase and polyketide synthase in all the five species. Genetic synteny was conserved in both Penicillium and Aspergillus species although genomic location seemed to be different since the clusters presented different flanking regions (except for A. steynii and A. westerdijkiae); these observations support the hypothesis of the orthology of this genomic region and that it might have been acquired by horizontal transfer. New real-time RT-PCR assays for quantification of the expression of these OTA biosynthetic genes were developed. In all species, the five genes were consistently expressed in OTA-producing strains in permissive conditions. These protocols might favour futures studies on the regulation of biosynthetic genes in order to develop new efficient control methods to avoid OTA entering the food chain.

Aflatoxins and ochratoxin A and their Aspergillus causal species in Tunisian cereals.

Occurrence of aflatoxins (AFs) AFB1, AFB2, AFG1, AFG2 and ochra toxin A (OTA) was studied in 65 samples of stored and freshly harvested wheat, barley and maize collected in Tunisia. The mycotoxins were simultaneously extracted and quantified by high performance liquid chromatography. Determination of AF-producing (section Flavi) and OTA-producing Aspergillus species (sections Nigri and Circumdati) was conducted in these samples by species-specific polymerase chain reaction (PCR). Results showed that most of maize samples were contaminated with AFs, data after storage showing lower values than those collected at harvest. All contaminated maize samples contained AFG1 and AFG2, among which 27.78% also had AFB1 and AFB2. This AFs pattern was consistent with the A. parasiticus toxin profile. A. flavus however showed the highest frequency in maize but was also found in barley and wheat where no AFs were detected. In contrast, OTA was neither found in maize nor in barley and only one wheat sample contained OTA. A. niger was the only OTA-producing species detected.

Clustered array of ochratoxin A biosynthetic genes in Aspergillus steynii and their expression patterns in permissive conditions.

Aspergillus steynii is probably the most relevant species of section Circumdati producing ochratoxin A (OTA). This mycotoxin contaminates a wide number of commodities and it is highly toxic for humans and animals. Little is known on the biosynthetic genes and their regulation in Aspergillus species. In this work, we identified and analysed three contiguous genes in A. steynii using 5'-RACE and genome walking approaches which predicted a cytochrome P450 monooxygenase (p450ste), a non-ribosomal peptide synthetase (nrpsste) and a polyketide synthase (pksste). These three genes were contiguous within a 20742 bp long genomic DNA fragment. Their corresponding cDNA were sequenced and their expression was analysed in three A. steynii strains using real time RT-PCR specific assays in permissive conditions in in vitro cultures. OTA was also analysed in these cultures. Comparative analyses of predicted genomic, cDNA and amino acid sequences were performed with sequences of similar gene functions. All the results obtained in these analyses were consistent and point out the involvement of these three genes in OTA biosynthesis by A. steynii and showed a co-ordinated expression pattern. This is the first time that a clustered organization OTA biosynthetic genes has been reported in Aspergillus genus. The results also suggested that this situation might be common in Aspergillus OTA-producing species and distinct to the one described for Penicillium species.

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.

Structural variation and dynamics of the nuclear ribosomal intergenic spacer region in key members of the Gibberella fujikuroi species complex.

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium, a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai, these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events.

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.

Effect of solute stress and temperature on growth rate and TRI5 gene expression using real time RT-PCR in Fusarium graminearum from Spanish wheat.

The objective of this work was to study the effect of ecophysiological factors on trichothecene gene expression and growth in Fusarium graminearum. The effect of non-ionic solute water potentials and temperature was examined on in vitro mycelial growth rates and on expression of the TRI5 gene, involved in trichothecene biosynthesis, quantified by real time RT-PCR. This study showed optimal values of 25 degrees C and -2.8MPa (0.982a(w)) for growth. Marginal temperatures such as 15 degrees C and 30-35 degrees C, particularly in combination with water potentials below -2.8MPa, drastically reduced growth. The expression of TRI5 was reasonably constant although some induction was observed between 20 and 30 degrees C, the most favourable temperatures for growth, depending on the water potential imposed, particularly at -7.0MPa. A temporal kinetic experiment at 25 degrees C examined the effect of ionic solute stress on TRI5 gene expression and growth rate. The results indicated independence of growth rate and TRI5 expression, as the fungal biomass increased with time while the gene expression remained constant. This suggested that favourable conditions for growth will result in higher trichothecene production, and that toxin production would always accompany the colonization process at a steady rate while the conditions for growth are permissive. Quantification of key biosynthetic toxin genes by real time RT-PCR was shown to be a valuable tool to gain knowledge of the ecophysiological basis for trichothecene biosynthesis and enable better control strategies to be developed during the life cycle of this important mycotoxigenic pathogen of cereals.

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 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.

Relationship between solute and matric potential stress, temperature, growth, and FUM1 gene expression in two Fusarium verticillioides strains from Spain.

The objective of this work was to study the effect of ecophysiological factors on fumonisin gene expression and growth in Fusarium verticillioides. The effects of ionic and nonionic solute water potentials, matric potential, and temperature on in vitro mycelial growth rates and on expression of the FUM1 gene, involved in fumonisin biosynthesis, were examined. FUM1 transcript levels were quantified using a specific real-time reverse transcription-PCR (RT-PCR) protocol. Low temperature and water stress reduced fungal growth. Water stress increased FUM1 transcript levels, especially in the case of stress caused by nonionic solute. The temporal kinetic assays showed that water stress had opposite effects on fungal growth versus FUM1 expression. These results indicate that water stress may be an important factor for fumonisin accumulation, particularly in the later phases of maize colonization when water availability decreases. The quantitative RT-PCR methods described here provide a valuable tool for investigating the ecophysiological basis for fumonisin gene expression and ultimately may lead to more effective control strategies for this important mycotoxigenic pathogen.

Real-Time RT-PCR assay to quantify the expression of fum1 and fum19 genes from the Fumonisin-producing Fusarium verticillioides.

Fumonisins are a group of mycotoxins produced by Fusarium species of the Gibberella fujikuroi species complex that contaminate food and feed products, and represent a risk for human and animal health. In this work, we have developed a specific real-time reverse transcription-PCR (RT-PCR) assay to quantify the level of expression of two genes of the fumonisin biosynthetic cluster in F. verticillioides: fum1 (that encodes a polyketide synthase enzyme) and the ABC transporter encoding gene fum19. The level of expression of both genes was compared with the amount of fumonisin B(1) (FB(1)), measured by HPLC, produced by several strains of F. verticillioides in liquid culture. The results indicated a good correspondence between the levels of fum1 and fum19 expression and the production of fumonisin B(1). The analysis described provides a good approach for the rapid and specific detection and characterization of the potential ability of F. verticillioides strains to produce fumonisins.

Morphological, chemical and molecular differentiation of Fusarium equiseti isolated from Norwegian cereals.

The morphological variation, secondary metabolite profiles and restriction fragment length polymorphisms (RFLPs) of PCR amplified intergenic spacer (IGS) ribosomal DNA (rDNA) were studied in 27 isolates of Fusarium equiseti, 25 isolated from Norwegian cereals and 2 from soil obtained from the IBT culture collection (BioCentrum, Technical University of Denmark). All 27 isolates were tested for production of fusarochromanone (FUSCHR), zearalenone (ZEA) and the trichothecenes: 15-monoacetoxy-scirpentriol (MAS), diacetoxy-scirpenol (DAS), T-2 and HT-2 toxins, T2-triol, neosolaniol (NEO), deoxynivalenol (DON), nivalenol (NIV) and 4-acetylnivalenol (Fus-X). The trichothecenes were analysed by GC-MS in a selected ion monitoring mode, while FUSCHR was determined by ion pair HPLC with fluorometric detection and production of ZEA by TLC. For amplification of IGS rDNA primers CNL12 and CNS1 were applied. IGS rDNA was digested with the four restriction enzymes: AvaII, CfoI, EcoRI and Sau3A. In addition, we sequenced the IGS rDNA region of three of the Norwegian isolates. There were two morphological types among the Norwegian strains of F. equiseti, type I with short apical cells (dominating) and type II with long apical cells, with four haplotypes identified based on the RFLP data. Variation in secondary metabolite profiles within and between the morphological groups was observed and the levels of produced toxins were: FUSCHR 3000-42,500 and 25-30 ng/g, NIV 20-2500 and 120-700 ng/g, FUS-X 20-15,000 and 0 ng/g, DAS 30-7500 and 0-600 ng/g, and MAS 10-600 and 0-500 ng/g, for strains with short and long apical cells, respectively. NEO was detected in 16/27 strains tested (all morphotype I). All but four strains of type I (these four lacked a restriction site for EcoRI) had identical RFLP profiles. The isolates of type II had two haplotypes. The IGS sequence similarity data indicated differences between these morphotypes corresponding to two separate lineages apparently at the species level.

Differential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA.

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates.

In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B1 and fumonisin B2 by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers. The molecular characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B1 and B2 producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.