Trichoderma harzianum: Inhibition of mycotoxin producing fungi and toxin biosynthesis.

A quarter of the world-wide crop is spoiled by filamentous fungi and their mycotoxins and weather extremes associated with the climate change lead to further deterioration of the situation. The ingestion of mycotoxins causes several health issues leading in the worst case to cancer in humans and animals. Common intervention strategies against mycotoxin producing fungi, such as the application of fungicides, may result in undesirable residues and in some cases to a stress induction of mycotoxin biosynthesis. Moreover, development of fungicide resistances has greatly impacted pre- and postharvest fungal diseases. Hence there is the need to develop alternative strategies to reduce fungal infestation and thus mycotoxin contamination in the food chain. Such a strategy for natural competition of important plant-pathogenic and mycotoxin producing fungi could be Trichoderma harzianum, a mycoparasitic fungus. Especially in direct comparison to certain tested fungicides, the inhibition of different tested fungal species by T. harzianum was comparable, more sustainable and in some cases more effective, too. Besides substantially reduced growth rates, a transcriptional based inhibition of mycotoxin biosynthesis in the competed Aspergillus species could be shown. Furthermore it could be clearly observed by high-resolution Scanning Electron Microscopy (SEM) that T. harzianum actively attaches to the competitor species followed by subsequent enzymatic lysis of those mycelial filaments. The analyzed isolate of T. harzianum MRI349 is not known to produce mycotoxins. In this study it could be successfully proven that T. harzianum as a biological competitor is an effective complement to the use of fungicides.

Effect of plastic mulching on mycotoxin occurrence and mycobiome abundance in soil samples from asparagus crops.

Plastic mulching (PM) is widely used in modern agriculture because of its advantageous effects on soil temperature and water conservation, factors which strongly influence the microbiology of the soil. The aim of this study was to assess the effect of PM on mycotoxin occurrence in relation with mycobiome abundance/diversity and soil physicochemical properties. Soil samples were collected from green (GA) and white asparagus (WA) crops, the last under PM. Both crops were cultivated in a ridge-furrow-ridge system without irrigation. Samples were analyzed for mycotoxin occurrence via liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Total colony-forming unit was indicative of mycobiome abundance, and analysis of mycobiome diversity was performed by internal transcribed spacer (ITS) sequencing. PM avoided the drop of soil temperature in winter and allowed higher soil temperature in early spring compared to non-covered soil. Moreover, the use of PM provided controlled conditions for water content in soil. This was enough to generate a dissimilar mycotoxin occurrence and mycobiome diversity/abundance in covered and non-covered soil. Mycotoxin soil contamination was confirmed for deoxynivalenol (DON), range LOD to 32.1 ng/g (LOD = 1.1 ng/g). The DON values were higher under PM (average 16.9 ± 10.1 ng/g) than in non-covered soil (9.1 ± 7.9 ng/g); however, this difference was not statically significant (p = 0.09). Mycobiome analysis showed a fungal compartment up to fivefold higher in soil under PM compared to GA. The diversity of the mycobiome varied between crops and also along the soil column, with an important dominance of Fusarium species at the root zone in covered soils.

Biological control as a strategy to reduce the impact of mycotoxins in peanuts, grapes and cereals in Argentina.

Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and ochratoxin A accumulation in grapes.

Taxonomy and important features of probiotic microorganisms in food and nutrition.

Lactic acid bacteria are among the most important probiotic microorganisms typically associated with the human gastrointestinal tract. Traditionally, lactic acid bacteria have been classified on the basis of phenotypic properties, eg, morphology, mode of glucose fermentation, growth at different temperatures, lactic acid configuration, and fermentation of various carbohydrates. Studies based on comparative 16S ribosomal RNA sequencing analysis, however, showed that some taxa generated on the basis of phenotypic features do not correspond with the suggested phylogenetic relations. Thus, some species are not readily distinguishable by phenotypic characteristics. This is especially true for the so-called Lactobacillus acidophilus group, the Lactobacillus casei and Lactobacillus paracasei group, and some bifidobacteria, strains of which have been introduced in many probiotic foods, eg, the novel yogurt-like commodities. Consequently, modern molecular techniques, including polymerase chain reaction-based and other genotyping methods, have become increasingly important for species identification or for the differentiation of probiotic strains. Probiotic strains are selected for potential application on the basis of particular physiologic and functional properties, some of which may be determined in vitro. The classification and identification of a probiotic strain may give a strong indication of its typical habitat and origin. The species, or even genus name, may also indicate the strain's safety and technical applicability for use in probiotic products. Molecular typing methods such as pulsed-field gel electrophoresis, repetitive polymerase chain reaction, and restriction fragment length polymorphism are extremely valuable for specific characterization and detection of such strains selected for application as probiotics.

P. nalgiovense carries a gene which is homologous to the paf gene of P. chrysogenum which codes for an antifungal peptide.

Penicillium nalgiovense is related to P. chrysogenum. P. chrysogenum carries the paf gene (Penicillium antifungal peptide) with homology to the afp gene of Aspergillus giganteus. This gene codes for a peptide with antifungal activity. Based on the sequence of the published P. chrysogenum paf gene primers were generated. By the use of these primers a PCR product of the expected length could be isolated from strains of P. nalgiovense. This fragment was sequenced and compared to the sequence of the paf gene of P. chrysogenum. According to the results P. nalgiovense carries a gene (naf = P. nalgiovense antifungal peptide) which is highly homologous to the paf gene of P. chrysogenum. The gene also codes for a preproprotein with the same processing sites as the paf gene, suggesting that the mature product is also a 55 amino acid (aa) peptide. The naf gene has three amino acid exchanges compared to the paf gene, which however do not influence the amino acid sequence of the mature peptide. It also carries two introns at the same positions, however, the sequence differences between the introns are higher than between the coding regions. When P. nalgiovense were grown on plates containing other food-relevant fungi it showed weak antifungal activity.

Karyotype of Penicillium nalgiovense and assignment of the penicillin biosynthetic genes to chromosome IV.

The karyotype of Penicillium nalgiovense was determined by pulsed-field gel electrophoresis and compared to the karyotype of P. chrysogenum. Both species have four chromosomes, but they differ in the size of the chromosomes and in the overall size of the genome. The sizes of the P. nalgiovense chromosomes as determined by pulsed-field gel electrophoresis are: 9.1 Mb, 7.9 Mb, 5.4 Mb and 4.1 Mb which gives in summary a genome size of 26.5 Mb. This compares to 34.1 Mb for P. chrysogenum. The penicillin gene cluster was located by Southern hybridization on chromosome IV, the smallest chromosome of P. nalgiovense compared to chromosome 1, the largest chromosome of P. chrysogenum.

Genetically modified starter and protective cultures.

Modern approaches towards starter and protective culture improvement rely on advances in molecular biology. For most microorganisms used for food production, gene technological methods have been well developed. By recombinant DNA technology, 'tailor-made' starter and protective cultures may be constructed so as to combine technically desirable features. A single strain which normally would fail to accomplish a given 'task' may now be improved so as to meet a set of requirements necessary for a specific production or preservation process (e.g. wholesomeness, no off-flavour production, overproduction of bacteriocins or particular enzymes). In addition, undesirable properties (e.g. mycotoxin or antibiotic production by cheese moulds) may be eliminated by techniques such as 'gene disruption'.

Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes.

A review is presented on the present status of biological preservation of foods. Recent developments are discussed with respect to underlying mechanisms of inhibition by 'protective' cultures, and special reference is made to lactic acid bacteria (LAB) and their 'food-grade' safety. The role of bacteriocins, their limitations and potentiating role in biological systems, is also addressed. The use of enzymes (e.g. lysozyme) for food preservation is mainly restricted by economic factors, their inactivation by endogenous food components and their limited activity spectrum. Practical applications of protective cultures refer to particular food commodities that either constitute novel systems with respect to packaging and/or composition, or represent special hygienic risks. It is concluded that biological preservation cannot substitute GMP; it, however, offers an additional (and acceptable) processing parameter for improving the safety and assuring the quality of a given food.

Detection of aflatoxinogenic fungi in figs by a PCR reaction.

A PCR reaction was used to detect aflatoxinogenic Aspergillus flavus strains in contaminated figs. The reaction records the presence of three aflatoxin biosynthesis genes, namely the norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1) and sterigmatocystin-o-methyltransferase: (omt-A). The reaction gave a triplet pattern in the presence of DNA from A. flavus isolated from pure cultures. The reaction gave the same PCR products when pure fungal DNA was mixed with pure DNA isolated from figs, but the sensitivity was reduced by a factor of 10. The same set of bands was observed when isolated DNA from infected figs was used as template DNA but no signal was visible when DNA from uninfected figs was used as template.

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing.

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.

Inhibition of food-related pathogenic bacteria by god-transformed Penicillium nalgiovense strains.

Two strains of Penicillium nalgiovense, which carried the god gene of Aspergillus niger and had increased glucose oxidase (GOD) activity compared with the wild-type strain, were tested for their ability to suppress the growth of certain food-related pathogenic bacteria. In contrast to the wild type, which showed no antibacterial effect when grown in mixed culture with different bacteria, the two transformed strains were highly antagonistic. The strain that expressed higher amounts of GOD in general had higher inhibitory activity. Both strains showed antibacterial activity against Listeria monocytogenes, Salmonella Enteritidis, and Staphylococcus aureus. The inhibitory activity was dependent on the glucose concentration in the medium. S. aureus was completely inhibited at 1% glucose in the presence of the higher GOD-producing transformant. In contrast, if arabinose was used as a carbon source, no inhibition occurred. If catalase was added to the medium, the inhibitory activity of the transformants was completely inactivated, indicating that the hydrogen peroxide produced was responsible for the antibacterial activity of the transformants.

Transformation of Penicillium freii and a rapid PCR screening procedure for cotransformation events.

A strain of Penicillium freii was transformed to hygromycin B resistance using the pAN7-1 plasmid. The transformation frequency was approximately 100 transformants per microgram DNA per 1 x 10(7) protoplasts. Southern blotting revealed different types of integration into the genome of P.freii. When non-selectable plasmid DNA was added together with pAN7-1, this resulted in a cotransformation frequency of more than 60%. The cotransformation frequency was estimated using a facile DNA extraction method together with PCR.

Benzopyrone coumarin leads to an inhibition of ochratoxin biosynthesis in representatives of Aspergillus and Penicillium spp. via a type of feedback response mechanism.

Growth and mycotoxin biosynthesis of the ochratoxin-producing fungal strains Aspergillus carbonarius, Aspergillus steynii, Penicillium verrucosum, and Penicillium nordium were analyzed on standard laboratory growth medium supplemented with different amounts of coumarin, an organic compound of the benzopyrone class. Neither the growth nor the phenotypic morphology of the filamentous fungi analyzed was affected by using coumarin concentrations equivalent to 2.5 to 25 µg/ml of medium. In contrast, the ochratoxin biosynthesis was strongly inhibited in both strains of the Aspergillus species and nearly completely inhibited in both Penicillium strains at coumarin concentrations above 8.75 µg/ml. Analyzing the transcriptional activity of the otapksPN polyketide synthase gene in P. nordicum using real-time PCR revealed a strong concentration-dependent decrease in gene expression. Taken together, the data show that ochratoxin biosynthesis in representative strains of the genera Aspergillus and Penicillium could be effectively inhibited by coumarin in a concentration-dependent manner. It could be suggested that the molecular background behind this inhibition is some kind of feedback response mechanism, based on the structural similarity of coumarin to the benzopyrone moiety of the ochratoxin molecule.

Fungicides effectively used for growth inhibition of several fungi could induce mycotoxin biosynthesis in toxigenic species.

Seven different commercial fungicides (Aliette, Rovral, Cantus, Ortiva, Luna Experience, Fenomenal and Mancozeb) were tested for their ability to inhibit the growth of the fungal species Penicillium nordicum, Penicillium verrucosum, Verticillium dahliae and Cladosporium sp. In case of the mycotoxigenic strains P. nordicum and P. verrucosum, the biosynthesis of the mycotoxins ochratoxin and citrinin was determined. Interestingly individual fungicides were only able to inhibit the growth of the analyzed fungi to some extent. In case of P. verrucosum the fungicide "Rovral", an iprodion belonging to the substance class of imidazoles, led to a decrease in the growth rate but to a strong induction of mycotoxin biosynthesis as has been described earlier for the strobilurins. Consequently before using a given fungicide to protect crops and enhance storage life, the applicability of this chemical compound should be tested not only for its ability to inhibit fungal growth but also for its effect on level of secondary metabolite biosynthesis.

Modelling the relationship between environmental factors, transcriptional genes and deoxynivalenol mycotoxin production by strains of two Fusarium species.

The effect of changes in temperature/water activity (a(w)) on growth, deoxynivalenol (DON) production and trichothecene gene cluster expression (18 genes) for strains of Fusarium culmorum and Fusarium graminearum was studied. The expression data for six key transcription genes (TRI4, TRI5, TRI6, TRI10, TRI12 and TRI13) were analysed using multiple regression analyses to model the relationship between these various factors for the first time. Changes in a(w) and temperature significantly (p = 0.05) affected growth and DON. Microarray data on expression of these genes were significantly related to DON production for both strains. Multi-regression analysis was done and polynomial models found to best fit the relationship between actual/predicted DON production relative to the expression of these TRI genes and environmental factors. This allowed prediction of the amounts of DON produced in two-dimensional contour maps to relate expression of these genes to either a(w) or temperature. These results suggest complex interactions between gene expression (TRI genes), environmental factors and mycotoxin production. This is a powerful tool for understanding the role of these genes in relation to environmental factors and enables more effective targeted control strategies to be developed.

Comprehensive molecular system to study the presence, growth and ochratoxin A biosynthesis of Penicillium verrucosum in wheat.

Based on the sequence of the ochratoxin A polyketide synthase gene (otapksPV), a polymerase chain reaction (PCR) system for the specific detection of Penicillium verrucosum in wheat has been developed. In a further approach, a real-time PCR system has been applied to determine the growth kinetics of P. verrucosum in wheat at cell numbers above 10(3) colony-forming units (cfu) ml(-1). The data obtained by real-time PCR correlated well with the data obtained by the plate count technique. For this purpose, the DNA was isolated directly from contaminated wheat without any further enrichment step. In a reverse transcriptase real-time PCR, the expression of the otapksPV gene in wheat was detected 22 days after inoculation and storage at ambient temperature. Reasonable amounts of ochratoxin A, however, could not be detected before day 30. This early activation of ochratoxin A related genes was confirmed by microarray analysis.

Cowpeas as growth substrate do not support the production of aflatoxin byAspergillus sp.

A number of 21Aspergillus sp. strains isolated from cowpeas from Benin (Africa) were characterized by RAPD methodology. Seven of these strains grouped withA. flavus in the dendrogram generated with the RAPD data. Only three were able to produce aflatoxin in significant amounts. Twelve other isolates grouped withA. parasiticus. All of these strains except 3 produced aflatoxin. Two additional strains neither fit with theA. flavus group, nor theA. parasiticus group according to their RAPD pattern. Both did not produce aflatoxin in measurable amounts.Generally the aflatoxin positive strains produced high amounts of aflatoxin after growth on YES medium. However after growth on cowpea based medium aflatoxin biosynthesis was strongly ceased, albeit the growth of the colony was only partly reduced. This was true for media made either with the whole cowpea seed or with cowpea seed without seed coat. Interestingly when the cowpea medium was heat sterilized the fungus was able to produce high amounts of aflatoxin. This, however, was not the case after the use of gamma irradiation as sterilization method for the medium. The expression of thenor- 1 gene, which is one of the early genes involved in aflatoxin biosynthesis, was significantly repressed after growth on gamma irradiated cowpea medium in contrast to YES medium.

Gene expression as an indication for ochratoxin A biosynthesis inPenicillium nordicum.

The expression level of theotapksPN gene ofPenicillium nordicum was determined by Reverse Transcription Real Time PCR in relation to several growth parameters known to have an influence on ochratoxin A biosynthesis, like pH, medium composition and temperature. Generally there was a good correlation between the expression level of this key gene and ochratoxin A biosynthesis. The results, which were obtained by Real Time PCR for a single gene, could generally be confirmed by microarray analysis with a whole set of genes related to ochratoxin A biosynthesis. Growth conditions which favour the expression of the ochratoxin A biosynthetic genes also resulted in the biosynthesis of ochratoxin A by the fungal culture, indicating that the influence of abiotic parameters is mediated via the regulation of transcription of the ochratoxin A biosynthetic genes. The influence of several parameters, like pH, salt concentration and temperature on the expression of the ochratoxin A biosynthetic genes has been analysed. Optimum conditions for the activation of the ochratoxin A biosynthesis genes were: pH 6.0-8.0, 25 °C and a sodium chloride concentration of 20 g/l. This was paralleled by the capacity ofP. nordicum to produce ochratoxin A under the same conditions. After combining the most inhibitory conditions (15 °C, sodium chloride 10 g/l, pH 5.0) nearly a complete deactivation of the ochratoxin A biosynthetic genes could be observed resulting in a complete abolishment of ochratoxin A biosynthesis. In contrast, a high expression and a high production of ochratoxin A could be observed after growth ofP. nordicum under more optimal conditions (25 °C, 20 g/1 sodium chloride, pH 8.0).

Molecular and chemical monitoring of growth and ochratoxin a biosynthesis ofP. verrucosum in wheat stored at different moisture conditions.

The growth and ochratoxin A producing activity at the phenotypical and molecular level ofP. verrucosum in wheat stored at different relative moisture conditions have been followed. Growth has been measured by determination of colony forming units (cfu). The expression of the ochratoxin A polyketide synthase (otapksPV) has been followed over time by reverse transcriptase Real Time PCR, whereas the biosynthesis of ochratoxin A by the fungus has been followed by HPLC. The remoisted and inoculated wheat was kept in perforated plastic bags and stored in tower silos. The bags were adjusted to 24%, 19% and 14% (m/m) relative moisture and stored for 6 month (September 06 - February 06) at ambient temperatures. A high increase of the cfu number up to a level of about lx10(8) cfu/g could be observed in the 24% sample. Interestingly no growth could be detected in the 14% and 19% samples. This result was supported by the quantitative Real Time PCR data. In congruence with this growth behaviour ochratoxin A could be detected from the first to the last time point in the 24% sample. Interestingly the ochratoxin A amounts measured over time did not show increasing or constant values, but varied from higher to lower values. After storage of three month (November 2006) the ochratoxin A concentration in the wheat was highest. The expression of theotapksPV gene mirrors this behaviour. The expression of this gene also fluctuates around a certain value, showing high activity after three month of storage.

A gene cluster of the ochratoxin A biosynthetic genes inPenicillium.

A putative ochratoxin A (OTA) biosynthetic gene cluster inP. nordicum has been identified. The first part of the gene cluster is located on a DNA fragment of 10 kb in length and harbours three genes. A gene with high homology to an alkaline serine protease gene (accession number AY557343), which represents the upstream border of the cluster. Furthermore the fragment carries a large part (about 2 kb) of the 5' end of a polyketide synthase (otapksPN, accession number AY196315) and a complete non-ribosomal peptide synthetase (otanpsPN, accession number AY534879). The second part of the cluster is located on a 4.3 kb fragment that harbours three open reading frames (ORFs) encoding putative OTA biosynthetic proteins: one incomplete ORF at the 5' end of the fragment demonstrated homology to an organic anion transporter from rat kidneys (otatraPN). This transporter has been described to be responsible for the transport of toxic OTA out of the cell. One complete ORF of 951 nucleotides is also located on this fragment. This gene has limited homology to a chloroperoxidase fromGluconobacter oxidans. At the 3' end of this DNA fragment is an incomplete open reading frame of a potential nitrate transporter. The transcription of all putative OTA biosynthetic genes is increased under OTA conducive conditions. The expression kinetics of the genes resembles that of secondary metabolite biosynthetic genes, in which these genes are co-ordinately expressed during the late growth phase.PCR analysis demonstrated that the gene cluster is only present in the two ochratoxin A producingPenicillium species,P. verrucosum andP. nordicum. P. nalgiovense, a species occurring in the same habitat asP. nordicum carries inactive homologues of the genes. All other species proved to be negative for the genes. This was also true for OTA producing Aspergilli.