Ochratoxin A Contamination In Grapes Research Articles

Pre- and post-storage applications of rhamnolipids and cyprodinil + fludioxonil against Aspergillus section Nigri population and ochratoxin A contamination of grapes

Grapevines constitute a very important crop in Greece and ochratoxin A (OTA) contamination of grapes is one of the most serious problems. The main aim of this study was to investigate the fungicidal activity of the fungicide Switch 62.5WG (37.5% v/v cyprodinil + 25% v/v fludioxonil) and Rhapynal (80% rhamnolipids) to reduce the black Aspergillus spp. (section Nigri) population and OTA contamination of grapes grown in Greece. Applications with the above products were conducted both at the harvesting stage and after cold storage. The results showed that pre-harvest applications with Switch 62.5WG or Switch 62.5WG + Rhapynal generally reduced the black Aspergillus spp. population, but not the OTA contamination of grapes. However, a repeated application after cold storage further reduced the black Aspergillus spp. population and significantly reduced the OTA contamination of grapes. Generally, this study showed that it is possible to reduce the black Aspergillus spp. population, but not the ochratoxin A contamination of grapes by spray applications with the fungicide Switch 62.5WG. However, fungicide application to grapes immediately after cold storage (2 months) could reduce ochratoxin A contamination of grapes and further reduce the black Aspergillus spp. population.

Inhibitory effects of Lactobacillus brevis on Aspergillus westerdijkiae and antifungal compounds identification

Ochratoxin A (OTA), a secondary metabolite produced by Aspergillus and Penicillium, seriously contaminates grapes and its products. Inhibiting the growth of OTA-producing fungi is the best approach to reduce OTA contamination in grapes. This study was to reveal the inhibition mechanism of Lactobacillus brevis 8–2B on Aspergillus westerdijkiae. Research has found that L. brevis 8–2B could inhibit the germination, growth, and OTA synthesis of A. westerdijkiae. The antifungal activity of L. brevis 8–2B is attributed to the synergistic effect of acetic acid, lactic acid, citric acid, longifolene, methyl salicylate, 3-phenyl-2-propenal, nystatin, and itraconazole. The inhibitory effect of L. brevis 8–2B on A. westerdijkiae was the synergistic effect of these active substances. Ultrastructure observation and flow cytometry results showed that the mycelia structure, cell membrane and cell wall of A. westerdijkiae were disrupted, the cells showed apoptosis, and the ergosterol content in the cells was reduced. Transcriptome analysis revealed that the expression levels of ribosomal biosynthesis genes and cell wall chitin synthesis genes in inhibited A. westerdijkiae were up-regulated, while the expression of chitin degrading enzyme genes was down-regulated to enhance its primary growth and development. In addition, L. brevis 8–2B also inhibits the expression of amino acid synthesis and glycolysis genes in A. westerdijkiae, reducing the generation of secondary metabolite precursors such as NRPS and PKS, thereby inhibiting OTA biosynthesis. On grapes, L. brevis 8–2B could inhibit the infection of A. westerdijkiae, and prolong its shelf life. This study provides new insights for the research and development of novel agent to control OTA contamination.

Effects of temperature and water activity change on ecophysiology of ochratoxigenic Aspergillus carbonarius in field-simulating conditions

Ochratoxin A (OTA) is the primary mycotoxin threat in wine and dried vine fruits. Its presence in grape and wine is strongly related to climatic conditions and the expected climate change could represent a risk of increasing fungal colonization and OTA contamination in grapes. In this regard, the interacting effect of i) different conditions of water availability (0.93 and 0.99aw) and ii) different 10 h/14 h dark/light alternating temperature conditions simulating a nowadays (18/31 °C) and climate change scenario (20/37 °C) in high OTA risk areas of Apulia region, were studied. Lag phases prior to growth, mycelial growth rate, the expression of biosynthesis, transcription factors and regulatory genes of OTA cluster and OTA production were analysed in Aspergillus carbonarius ITEM 5010 under the combined effect of different climatic factors. At 18/31 °C and under water stress conditions (0.93 aw) the growth rate was slower than at 0.99 aw; on the contrary, at 20/37 °C a higher growth rate was observed at 0.93 aw. An over-expression of OTA genes and genes belonging to the global regulator Velvet complex was observed at 18/31 °C and 0.99 aw, with the specific OTA pathway transcription factor bZIP showing the highest expression level. The up-regulated transcription profile of the genes positively correlated with OTA production higher at 18/31 °C than at 20/37 °C and 0.99 aw; while no OTA production was detected at 0.93 aw at each of the temperature conditions tested. These findings provide preliminary evidence that the possible increase of the temperature, likely to happen in some areas of the Apulia region, may results in a reduction of both A. carbonarius spoilage and OTA production in grapes.

Impact of some environmental factors on growth and ochratoxin A production by Aspergillus niger and Aspergillus welwitschiae

Ochratoxin A (OTA) is a nephrotoxic mycotoxin which may contaminate various foods and feed products worldwide. Aspergillus niger is one of the species responsible for OTA contamination in grapes and derived products. This species has recently been split into A. niger and Aspergillus welwitschiae. Both species can not be distinguished by phenotypic or extrolite profiles and to date there is no ecophysiological information of A. welwitschiae.The aim of this study was to determine the effects of water activity (aw) (0.90; 0.95 and 0.98–0.99), culture media (Yeast Extract Sucrose Broth (YESB); Synthetic Grape Juice Medium (SGM); White grape juice (WGJ)) and temperature (15 °C, 25 °C and 35 °C) on the growth and OTA production of four strains of A. niger and six strains of A. welwitschiae. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA at 1, 2, 4 and 10 days.No significant differences were observed in absorbance and OTA values between the two species under study. The highest absorbance values were recorded in YESB, followed by SGM and WGJ. Absorbance values increased with increasing aw and temperature. The highest OTA values were obtained at 0.98–0.99 aw and the best culture media for OTA production was YESB, followed by WGJ and SGM. The studied strains of A. niger produced the highest mean OTA level at 25 °C whereas A. welwitschiae strains produced the highest mean OTA concentration at 15 °C, although not differing significantly from concentration produced at 25 °C.To our knowledge, this is the first report on the impact of some environmental factors on growth and OTA production by A. welwitschiae.

OTA-Grapes: A Mechanistic Model to Predict Ochratoxin A Risk in Grapes, a Step beyond the Systems Approach

Ochratoxin A (OTA) is a fungal metabolite dangerous for human and animal health due to its nephrotoxic, immunotoxic, mutagenic, teratogenic and carcinogenic effects, classified by the International Agency for Research on Cancer in group 2B, possible human carcinogen. This toxin has been stated as a wine contaminant since 1996. The aim of this study was to develop a conceptual model for the dynamic simulation of the A. carbonarius life cycle in grapes along the growing season, including OTA production in berries. Functions describing the role of weather parameters in each step of the infection cycle were developed and organized in a prototype model called OTA-grapes. Modelling the influence of temperature on OTA production, it emerged that fungal strains can be shared in two different clusters, based on the dynamic of OTA production and according to the optimal temperature. Therefore, two functions were developed, and based on statistical data analysis, it was assumed that the two types of strains contribute equally to the population. Model validation was not possible because of poor OTA contamination data, but relevant differences in OTA-I, the output index of the model, were noticed between low and high risk areas. To our knowledge, this is the first attempt to assess/model A. carbonarius in order to predict the risk of OTA contamination in grapes.

Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.

Characterization and disruption of the cipC gene in the ochratoxigenic fungus Aspergillus carbonarius

Aspergillus carbonarius is considered the most important ochratoxin A (OTA) producing fungi among those causing OTA contamination in grapes and grape-derived products. CipC is a small protein with unknown function that was previously found to be highly up-regulated in an OTA producer strain of A. carbonarius in comparison to a non OTA producer strain. In this study, cipC was characterized and disrupted via Agrobacterium tumefaciens-mediated transformation in an ochratoxigenic A. carbonarius strain in order to study whether this gene has a role in OTA production. Sequence analysis indicated that the promoter region of cipC contains putative binding sites for transcription factors that regulate the utilization of nutrients, the stress response and detoxification processes, all factors that can influence mycotoxin biosynthesis. Although the ∆cipC mutant grew similarly to the wild type strain, the null mutant showed a much higher OTA production. Moreover, when A. carbonarius was grown under the oxidative stress conditions imposed by the presence of hydrogen peroxide, cipC gene expression was up-regulated. These results indicate that cipC is not directly involved in OTA biosynthesis, but sequence analysis of the A. carbonarius cipC gene promoter and the phenotype of the ΔcipC disrupted mutant suggests that CipC could be a stress response protein that would be up-regulated concomitantly with OTA production.

Influence of Planococcus ficus on Aspergillus section Nigri and ochratoxin A incidence in vineyards from Argentina

The aim of this work was to evaluate the effect of Planococcus ficus infection in red wine grapes on Aspergillus section Nigri and ochratoxin A (OTA) contamination. During 2006/2007 and 2008/2009 vintages, Merlot, Malbec and Cabernet Sauvignon varieties divided into two categories of grape samples (undamaged and damaged by P. ficus) were evaluated. Regardless of the grape variety and the harvest season evaluated, Aspergillus section Nigri incidence and the mean OTA concentration in damaged berries were significantly higher than that in the undamaged ones (P < 0.05; P < 0.001). The Merlot variety showed the highest level of black aspergilli contamination in damaged grapes during the 2006/2007 vintage (53.5% of infection), whereas Malbec presented the highest incidence during the 2008/2009 vintage (57.6% of infection). The Cabernet Sauvignon variety showed the highest OTA levels, ranging from 0.1 to 140 microg kg(-1). The presence of P. ficus in vineyards increased the risk of OTA occurrence in grapes, suggesting the need to implement insect control at preharvest stage to reduce the entry of OTA in the wine production chain. This study is the first report on the influence of P. ficus on the potential risk of OTA contamination in grapes.

European research on ochratoxin A in grapes and wine

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 μg/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.

Mycobiota and ochratoxin A producing fungi from Spanish wine grapes

Grapes from three different regions with a long winemaking tradition in Spain were analysed at different growth stages in order to identify the ochratoxigenic mycobiota during three consecutive seasons. The correlation between meteorological parameters and ochratoxigenic fungi was studied and revealed a significant positive correlation between black aspergilli infection and temperatures in the month preceding each sampling date. No significant correlation was found with either relative humidity or rainfall. Biodiversity indexes were also calculated in this study. Black aspergilli species were the most abundant in grapes before harvest, and among them, Aspergillus carbonarius was the main ochratoxin A (OTA) producer species and represented 78–100% of the isolates tested. The results obtained support the key role of A. carbonarius as the main source of OTA contamination in grapes.

Skin damage, high temperature and relative humidity as detrimental factors for Aspergillus carbonarius infection and ochratoxin A production in grapes

This study investigated the impact of skin damage on Aspergillus carbonarius colonization and ochratoxin A (OTA) production in grapes at different temperatures and relative humidity. Four ochratoxigenic A. carbonarius strains isolated from wine grapes were used to inoculate artificially damaged and undamaged table grapes. Grapes were stored at three levels of relative humidity (80%, 90% and 100%) and at two temperatures (20 and 30 °C). After seven days, the infection percentage of A. carbonarius was recorded and OTA accumulation in berries was analysed. Damaged grapes were more commonly infected and development of colonies was higher than in undamaged ones; consequently more OTA was detected in the former treatment. Temperature and relative humidity had significant influences on both infection and toxin content. The amount of OTA detected at 30 °C was higher than at 20 °C in most of the treatments. The highest relative humidity (100%) led to maximum amounts of OTA while no significant differences were found between 90% and 80% in the OTA content. The implementation of preventive measures in order to minimise berry damage in the field by controlling pathogenic fungi and insects during grape growing and removing visibly damaged grapes at harvest may significantly reduce OTA contamination in grapes.