Ochratoxin A Biosynthesis Research Articles

Early detection of Aspergillus carbonarius and A. niger on table grapes: a tool for quality improvement

Aspergillus carbonarius and A. niger aggregate are the main fungal contaminants of table grapes. Besides their ability to cause black rot, they can produce ochratoxin A (OTA), a mycotoxin that has attracted increasing attention worldwide. The objective of this work was to set up a simple and rapid molecular method for the early detection of both fungi in table grapes before fungal development becomes evident. Polymerase chain reaction (PCR)-based assays were developed by designing species-specific primers based on the polyketide synthases (PKSS) sequences of A. carbonarius and A. niger that have recently been demonstrated to be involved in OTA biosynthesis. Three table grape varieties (Red globe, Crimson seedless, and Italia) were inoculated with A. carbonarius and A. niger aggregate strains producing OTA. The extracted DNA from control (non-inoculated) and inoculated grapes was amplified by PCR using ACPKS2F-ACPKS2R for A. carbonarius and ANPKS5-ANPKS6 for A. niger aggregate. Both primers allowed a clear detection, even in symptomless samples. PCR-based methods are considered to be a good alternative to traditional diagnostic means for the early detection of fungi in complex matrix for their high specificity and sensitivity. The results obtained could be useful for the definition of a ‘quality label’ for tested grapes to improve the safety measures taken to guarantee the production of fresh table grapes.

Ochratoxin a: general overview and actual molecular status.

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi that structurally consists of a para-chlorophenolic group containing a dihydroisocoumarin moiety that is amide-linked to L-phenylalanine. OTA is detected worldwide in various food and feed sources. Studies show that this molecule can have several toxicological effects such as nephrotoxic, hepatotoxic, neurotoxic, teratogenic and immunotoxic. A role in the etiology of Balkan endemic nephropathy and its association to urinary tract tumors has been also proved. In this review, we will explore the general aspect of OTA: physico-chemical properties, toxicological profile, OTA producing fungi, contaminated food, regulation, legislation and analytical methods. Due to lack of sufficient information related to the molecular background, this paper will discuss in detail the recent advances in molecular biology of OTA biosynthesis, based on information and on new data about identification and characterization of ochratoxin biosynthetic genes in both Penicillium and Aspergillus species. This review will also cover the development of the molecular methods for the detection and quantification of OTA producing fungi in various foodstuffs.

Amplification of polyketide synthase gene fragments in ochratoxigenic and nonochratoxigenic black aspergilli in grapevine

Members of the Aspergillus section Nigri , also known as black aspergilli produce Ochratoxin A (OTA), a contaminant of wine. Despite potentially severe health effects and economic losses caused by OTA in wine, almost nothing is known about the genetics that lies behind its biosynthesis in black aspergilli in grapevine. In this work, degenerate primer sets were used to amplify 49 Polyketide Synthase (PKS) gene fragments in reference strains of A. carbonarius , A. niger and A. tubingensis . Deduced amino acid sequences were then compared with those of aolc35-12 and aoks1 , two PKS genes involved in OTA biosynthesis in A . westerdijkiae. A putative homologue of aolc35-12 was found in A. carbonarius (63% amino acid identity), the main OTA producer on grapes and in an ochratoxigenic A. niger strain (58%). In A. niger this fragment corresponded to an15g07920, a PKS already annotated in the sequenced A. niger CBS 513.88 genome as putatively involved in OTA biosynthesis. No aolc35-12 candidates were found in atoxigenic A. tubingensis isolates and no putative homologues of aoks1 were found in any of the screened strains. A screening of A. niger field isolates using specific primers for an15g07920 indicated that the absence of this gene is apparently related to a failure to produce OTA. The present work gives a first insight into the genetics of OTA biosynthesis in black aspergilli in grapevine and represents a starting point for further investigation of the OTA biosynthesis pathway and the development of molecular methods to detect the producers in vineyards.

Lipoperoxidation affects ochratoxin A biosynthesis in Aspergillus ochraceus and its interaction with wheat seeds

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a DeltaAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the DeltaAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the DeltaAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the DeltaAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host-pathogen interaction between wheat seeds and A. ochraceus.

Ochratoxin A 'in vitro' biosynthesis by the Aspergillus ochraceus E'G isolate

This paper deals with the biosynthetic capacity for ochratoxin A (OTA) production by Aspergillus ochraceus E'G isolate derived from A. ochraceus CBS 108.08 strain, during 2007. Preliminary analysis of fungal potential for the production of OTA were performed according to the modified method of Filtenborg et al. (1983). Toxin production was tested in the following liquid media: (i) glucose-peptone-yeast extract broth (GPY - pH 5.6), (ii) potato-dextrose broth (PDB - pH 6.9), (iii) yeast extract-sucrose broth (YES - pH 6.5), and (iv) YES broth supplemented with 0.23 mg/l ZnSO4 x 5 H2O (YESZn - pH 6.5) after stationary and submerged cultivation. Dynamics of OTA biosynthesis was tested after the cultivation of A. ochraceus E'G on natural solid substrates, such as wet sterilized rice, corn and wheat grain. Cultivations were performed during different time periods (ranging from four days to few weeks) at different temperatures (ranging from 21?C to 30?C). The presence of OTA was determined as follows: (i) in liquid media according to the method of Balzer et al. (1978) modified by Bocarov-Stancic et al. (2003), and (ii) in the solid substrates according to the Serbian official methods for sampling and analyzing of fodder (Official Gazette of SFRY, No. 15/87). After the cultivation of A. ochraceus E'G isolate in liquid media, the highest yield of OTA (6.4 mg/l) was obtained after submerged cultivation in PDB (4 days, 128 rpm, 21-23?C). In the case of cultivation on solid substrates, the highest amount of OTA (800.0 mg/kg of dry matter) was recorded after several week long cultivation on wheat grain at 30?1?C.

Characterisation of a pks gene which is expressed during ochratoxin A production by Aspergillus carbonarius

Aspergillus carbonarius is considered the main fungus responsible for ochratoxin A (OTA) contamination in grapes. OTA is a potent nephrotoxin and a possible human carcinogen with a polyketide derived structure. Fungal polyketide synthases (PKSs) have recently been demonstrated to be involved in OTA biosynthesis in both Penicillium and Aspergillus species. We report here on the identification and characterisation of part of a novel polyketide synthase gene, ACpks from A. carbonarius. The sequence appears to encode conserved ketosynthase and acyl transferase domains, which are characteristic of previously characterised PKS enzymes. Expression of the ACpks gene is differentially regulated, with transcription being observed when the fungus was grown on synthetic grape medium and on OTA permissive medium (MM) whereas no transcription was detected when the fungus was grown on OTA restrictive medium (YES). ACpks expression was also observed when A. carbonarius was grown at low pH, with concomitant increases in OTA production. This correlation between ACpks gene expression and OTA production suggests the likely involvement for the product of this gene in ochratoxin A biosynthesis in the fungus. From a preliminary screening of Aspergillus isolates with ACpks specific primers, ACpks gene homologues appear to be present in A. sclerotioniger and A. ibericus, two species of section Nigri which are closely related to A. carbonarius.

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

Ochratoxin A (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 synthase gene “ aoks1”, cloned by using gene walking approach. The predicted amino acid sequence of the 2 kb clone display 34–60% similarities to different polyketide synthase genes 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 aoks1 gene 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.

A cDNA-AFLP approach to study ochratoxin A production in Aspergillus carbonarius

Aspergillus carbonarius is responsible for the majority of mycotoxin contaminations in grapes and its derivatives. Most of A. carbonarius strains are ochratoxin A (OTA) producers, even though at very different levels. This broad variability was used to identify genes whose expression is linked with the ability of producing OTA. A cDNA-AFLP differential display screening was performed in two strains of A. carbonarius, antagonists for the ability of producing OTA, allowing the identification of 119 differentially expressed sequences putatively involved in the regulation of OTA biosynthesis. A likely connection was pointed out between the biosynthesis of the toxin, vegetative growth and sexual/asexual developmental progression, along with common signalling pathways involving G protein and Ca 2+/calmodulin dependent phosphorylation and dephoshorylation cascades.

Physiological relationship between food preservatives, environmental factors, ochratoxin and otapksPV gene expression by Penicillium verrucosum

There is significant interest in trying to understand the relationship between environmental factors, preservative concentration and expression of genes involved in mycotoxin production. However, little information is available on the link between physiological stress factors and expression of genes responsible for mycotoxin production. This study has examined the effect of interactions between ionic and non-ionic water availability modified with glycerol or NaCl ( a w, 0.98, 0.95 and 0.93) and sub-optimal concentrations of calcium propionate and potassium sorbate (150, 300 ppm) at 25 °C on growth, ochratoxin A (OTA) and otapksPV gene expression by the mycotoxigenic species Penicillium verrucosum. Growth was inhibited between 25–35% by the preservatives at each a w level. However, OTA production was stimulated by 150 and 300 ppm of both preservatives, especially at 0.95 and 0.93 a w. If water activity as a single stress factor was changed, a typical OTA production and otapksPV expression profile occurred, indicating that OTA biosynthesis is activated under two conditions, i.e., under optimal growth conditions and under weak stress conditions. Temporal analysis of otapksPV expression showed that there was an optimum after 8–9 days incubation. Statistical analyses indicated good correlation between sub-optimal concentrations of preservatives, intermediate a w levels and genotypic and phenotypic gene and toxin production. This is the first time that genotypic information has been linked to phenotypic mycotoxin production in relation to such interacting stress factors.

Ochratoxin A biosynthetic genes in Aspergillus ochraceus are differentially regulated by pH and nutritional stimuli

Expression of the polyketide synthase ( pks) gene which is involved in ochratoxin A (OTA) biosynthesis in Aspergillus ochraceus is linked to production of the mycotoxin, with high levels of pks mRNA accumulation occurring in cultures producing OTA, as assessed by real-time reverse transcription (RT)-PCR. OTA production is regulated by nutrient availability, with supplementation of OTA restrictive potato dextrose broth with yeast extract resulting in a 39-fold increase in production of the mycotoxin. This effect appears to be mediated at the level of gene transcription as there is a concomitant increase in pks mRNA accumulation. OTA production is also strongly influenced by culture pH with large amounts of OTA being produced at pH values <7.0 with reduced amounts being produced at higher pH values. pks transcript levels again mirrored the OTA production profile observed at the different pH values. The transcription of two putative p450 type monooxygenase genes, namely p450-H11 and p450-B03 genes closely mirrored that of the pks gene under all growth conditions tested, suggesting their involvement together with the pks in OTA biosynthesis. The expression profile of the p450-B03 gene in particular is very similar to that of the pks gene, indicating that this gene may be clustered with the pks as part of the OTA biosynthetic gene cluster.