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1.
Int J Food Microbiol ; 280: 10-16, 2018 Sep 02.
Article in English | MEDLINE | ID: mdl-29754002

ABSTRACT

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.


Subject(s)
Antibiosis/physiology , Biological Control Agents/metabolism , Crops, Agricultural/microbiology , Plant Diseases/prevention & control , Trichoderma/metabolism , Alternaria/growth & development , Alternaria/metabolism , Animals , Aspergillus/growth & development , Aspergillus/metabolism , Fungicides, Industrial/pharmacology , Fusarium/growth & development , Fusarium/metabolism , Humans , Microscopy, Electron, Scanning , Mycelium/metabolism , Mycotoxins/biosynthesis , Penicillium/growth & development , Penicillium/metabolism , Plant Diseases/microbiology
2.
Mycotoxin Res ; 31(4): 191-201, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26412448

ABSTRACT

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.


Subject(s)
Agriculture/methods , Biodiversity , Fungi/isolation & purification , Liliaceae/growth & development , Mycotoxins/analysis , Soil Microbiology , Soil/chemistry , Chromatography, Liquid , Cluster Analysis , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Fungi/classification , Mass Spectrometry , Molecular Sequence Data , Phylogeny , Plastics , Sequence Analysis, DNA
3.
Article in English | MEDLINE | ID: mdl-25427716

ABSTRACT

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.


Subject(s)
Arachis/microbiology , Biological Control Agents , Edible Grain/microbiology , Mycotoxins/analysis , Vitis/microbiology , Aflatoxins/analysis , Argentina , Aspergillus flavus/isolation & purification , Bacillus subtilis/physiology , Brevibacillus/physiology , Food Contamination/analysis , Food Microbiology , Fumonisins/analysis , Fusarium/isolation & purification , Ochratoxins/analysis , Zea mays/microbiology
4.
J Food Prot ; 77(4): 647-52, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24680079

ABSTRACT

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.


Subject(s)
Antifungal Agents/pharmacology , Aspergillus/drug effects , Coumarins/pharmacology , Food Preservation/methods , Ochratoxins/biosynthesis , Penicillium/drug effects , Aspergillus/metabolism , Dose-Response Relationship, Drug , Food Microbiology , Ochratoxins/antagonists & inhibitors , Penicillium/metabolism
5.
Int J Food Microbiol ; 166(3): 407-12, 2013 Sep 16.
Article in English | MEDLINE | ID: mdl-24036489

ABSTRACT

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.


Subject(s)
Food Microbiology , Fungi/drug effects , Fungicides, Industrial/pharmacology , Gene Expression Regulation, Fungal/drug effects , Mycotoxins/biosynthesis , Food Preservation , Fungi/genetics , Fungi/metabolism , Mycotoxins/genetics
6.
J R Soc Interface ; 8(54): 117-26, 2011 Jan 06.
Article in English | MEDLINE | ID: mdl-20462881

ABSTRACT

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.


Subject(s)
Environment , Fungal Proteins/metabolism , Fusarium/physiology , Models, Biological , Transcription Factors/metabolism , Trichothecenes/biosynthesis , Fungal Proteins/genetics , Fungal Proteins/physiology , Fusarium/genetics , Fusarium/metabolism , Oligonucleotide Array Sequence Analysis , Regression Analysis , Temperature , Transcription Factors/genetics , Transcription Factors/physiology
7.
Article in English | MEDLINE | ID: mdl-18608504

ABSTRACT

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.


Subject(s)
Food Microbiology , Ochratoxins/biosynthesis , Penicillium/metabolism , Triticum/microbiology , DNA, Fungal/genetics , Food Handling/methods , Gene Expression , Gene Expression Profiling/methods , Humans , Ochratoxins/analysis , Oligonucleotide Array Sequence Analysis , Penicillium/genetics , Penicillium/growth & development , Polymerase Chain Reaction/methods , Time
8.
Mycotoxin Res ; 24(2): 105-10, 2008 Jun.
Article in English | MEDLINE | ID: mdl-23604687

ABSTRACT

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.

9.
Mycotoxin Res ; 23(1): 13-21, 2007 Mar.
Article in English | MEDLINE | ID: mdl-23605811

ABSTRACT

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).

10.
Mycotoxin Res ; 23(3): 138-46, 2007 Sep.
Article in English | MEDLINE | ID: mdl-23605992

ABSTRACT

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.

11.
Mycotoxin Res ; 22(2): 134-41, 2006 Jun.
Article in English | MEDLINE | ID: mdl-23605586

ABSTRACT

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.

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