RICERCA ITALIANA     

Biological and integrated control of Aspergillus carbonarius: effectiveness on ochratoxin A content and grape-wine chain

Università Cattolica del Sacro Cuore

Abstract

This research is related to the EU project "WINE-OCHRA RISK" (www.ochra-wine.com) and goes deeper into an issue included in that project. The coordinator of "WINE-OCHRA RISK" and of this project is the same. Control of A. carbonarius, the key fungus for OTA production in vineyard, is a small part in "WINE-OCHRA RISK".
In this proposed project, the main purpose is minimize OTA content in grapes. Biocontrol agents active against A. carbonarius are studied for their application in organic and integrated farming.
The project has a "farm to fork" approach. Therefore, the project aims are also: the possible effect of BCAs on vinification, the identification of OTA degradation products and their toxicity, compared to OTA toxicity.
This is a multidisciplinary project where each research unit, involved in 1-2 research phases, interacts with the others, contributing with knowledge coming from different scientific areas such as plant pathology, with particular attention to mycotoxigen fungi and their biological control, food technology, especially regarding vinification, analytical chemistry, applied to food products, and applied medical science, to evaluate citotoxicity.
The project will be developed in 5 phases, not successive, but with activities that produce input for one or more further activities.
Phase 1 - Selection of BCAs, on the basis of:
- High antagonistic activity against A. carbonarius;
- Compatibility with fungicides and insecticides commonly used on grapevines in organic and integrated agriculture;
- Survival in the presence of OTA in vitro and on berries infected by OTA-producing A. carbonarius and ability to degrade OTA;
Phase 2 - Effectiveness of biocontrol agents in vineyards and on drying grapes
- Development of innovative strategies for the control of A. carbonarius on grapevine and OTA contamination, using BCAs and reduced rate of fungicides
- Evaluate the efficacy of applying bio-control agents that can control A. carbonarius and OTA contamination in organic or integrated farming
Phase 3 - Micro-vinification of grapes from biological and integrated farming
- Evaluation of the kinetics of alcoholic fermentation and the presence of OTA in the different stages of wine-making and the chemical, physical and sensorial characteristics of wines
Phase 4 - Identification of pesticide residues and degradation products
- Characterization with chromatographic methods of secondary metabolites produced by biocontrol agents
- Chemical identification by spectrometric techniques of OTA sub-products
- Persistence evaluation of the main agro-chemical compounds used in integrated agricultural practices
Phase 5 – Toxicity of ochratoxin A and its degradation products
- Definition of the toxic potential risk of OTA to different cell lines, including time- and dose-dependency and lethal dose 50
- Selection of three cell line models to be used as biosensors in cytotoxicity experiments with complex matrices
- Gaining insight into ochratoxin A pro-apoptotic mechanisms
The project is a "farm to fork" approach, focussed on protecting consumers health which, in future, could form guidelines for applications in other "farm to fork" areas. <<<

Principal Investigator

Paola BATTILANI Università Cattolica del Sacro Cuore

Research Objectives

This research is related to the EU project "WINE-OCHRA RISK" (www.ochra-wine.com) and goes deeper into an issue included in that project. The coordinator of "WINE-OCHRA RISK" and of this project is the same. Control of A. carbonarius, the key fungus for OTA production in vineyard, is a small part In "WINE-OCHRA RISK".
In this project, the main purpose is the vineyard management in organic and integrated farming to minimize OTA content in grapes. Biocontrol agents active against A. carbonarius are studied. Therefore, the project aims are: the possible effect of BCAs on vinification, the identification of OTA degradation products and their toxicity, compared to OTA toxicity.
This is a multidisciplinary project where each research unit, involved in 1-2 research phases, interacts with the others according to the following sheme.



Research Units involved in the project:
- Research Unit Università di Piacenza, responsible Paola Battilani RU_PC
- Research Unit Università di Campobasso, responsible Vincenzo De Cicco RU_CB
- Research Unit Università di Napoli, responsible Alberto Ritieni RU_NA
- Research Unit Università di Bari, responsible Luigi Macchia RU_BA





Therefore, the main program objectives can be summarised as follows:

• Development of innovative strategies for the control of Aspergillus carbonarius on grapevine, through the utilisation of BCAs and/or reduced rate of fungicides
• Development of innovative procedures for reducing OTA contamination in grapes and in wine

Following a "farm to fork" approach and taking into account food safety, further objectives are:

• Evaluation in vineyard of bio-control agents active against A. carbonarius, on bunches during drying
• Evaluation of the kinetic of the alcoholic fermentation and the presence of ochratoxin A in the different stages of wine-making and chemical, physical and sensorial characters of wines
• Identification of secondary metabolites produced by biocontrol agents and by OTA degradation in must
• Chemical identification by spectrometric techniques of OTA subproducts
Persistence evaluation of the main agro-chemical compounds used in integrated agricultural practices.
• Evaluation of OTA toxicity in different mammalian and invertebrate cell lines
• Definition of the cellular and molecular mechanisms of OTA toxicity, with emphasis on apoptosis
• Setting conditions for citotoxicity analysis of real matrices
• Definition of the potential cytotoxic effects of OTA degradation products <<<

Timescale

24 months

National and international background

Mycotoxins
In the developed countries, food quality is one of the most important problems for consumers (FAO, 1999). Mycotoxins (MTs) are considered very dangerous molecules for human health and they are a matter of concern in the food safety field.
MT are chemically unrelated molecules and this group of secondary metabolites shows many biological toxic activities. MTs are biosynthesised by microscopic aerobic fungi belonging to the genera Aspergillus, Fusarium and Penicillium.
MT contamination can occur either in plant foods as a result of direct mould spoilage or in animal products as a consequence of indirect transmission into edible tissues from animals exposed to natural contaminated feed. The carry-over causes excretion of MT and detoxification metabolites in milk, meat, eggs and sometimes these metabolites are very dangerous for human health.
MT contamination of food is a very actual problem and some international organisations like FAO and WHO, assert that more than 25% of crops are contaminated by MTs (WHO, 1992).
Among MTs, ochratoxin A (OTA) is one of the most relevant. It was isolated for the first time in 1965 by van der Merwe et al (1965) from A. ochraceus; chemically, OTA is a dihydro-isocoumarin coupled to L-β-phenylalanine (Rizzo et al., 2002).
In Europe, OTA is considered the most ubiquitary MT; in fact, in Central and Northern Europe (Sweden, German, Denmark and United Kingdom) it is found in 90% of human blood samples at 0.1 mg/kg level (Petzinger e Weidenbach 2002).

The toxicity of ochratoxin A
OTA is a potent nephrotoxin to all animal species tested, with the exception of mature ruminants. Regarding the possible carcinogenicity towards humans, the International Agency for the Research on Cancer (IARC) classified OTA into group 2B. OTA can be transferred to milk and constitutes a risk also to infants, through nursing.
Intensive research has been carried out over the last ten years, aimed at addressing the question of OTA impact on human health; a number of in vivo and in vitro models have been studied and proposed (Fink-Gremmels, 1995; Mantle, 1998; Schaaf, 2002). In particular, it has been suggested that the potential cellular damage at renal level may be supported, at least partially, by apoptotic mechanisms (Mantle, 1998).
However, in spite of the abundant data collected to date, the spectrum of cytotoxicity of OTA remains patchy and the mechanisms underlying the observed toxic effects at cellular and molecular level and possibly determining the relevance of this MT to the human health, remain elusive.

Food sources of ochratoxin A
The world-wide occurrence of OTA contamination of raw agricultural products has been amply documented; it occurs in a variety of plant products such as cereals, coffee beans, beans and pulses (Kuiper-Goodman and Scott, 1989; Pohland et al., 1992; Jørgensen, 1998). OTA has been detected also in beverages such as beer and also in wine and grape juice (Majerus and Otteneder, 1996; Zimmerli and Dick, 1996).
After the first detection of OTA in wine in 1996, several surveys were managed, mainly in Europe. They confirmed the presence of this MT and wine is supposed to be the second main source of OTA for human after cereals.
A gradient in OTA concentration in wines, red > rosé > white, was pointed out by several authors (Zimmerli and Dick, 1996; Visconti et al.,1999; Rosari et al., 2000; Filali et al., 2001), while Stefanaki et al. (2003) showed that OTA concentration in red dry wines was not significantly different from that found in white and rosé wines in Greece.
The amount of OTA was dependent on the latitude of the production region: the lower the latitude, the more frequent the occurrence and the greater the concentration. The considerable climatic differences, related to geographic regions, influenced mould contamination and OTA production in a conclusive manner (Zimmerli and Dick, 1996; Otteneder and Majerus, 2000; Rosari et al., 2000; Pietri et al., 2001; Eder et al., 2002; Stefanaki et al. 2003). Differences in OTA level, probably due to different weather conditions, were also reported by Pietri et al. (2001) and Lopez de Cerain et al. (2002) between samples collected in the same regions but in different years.
Dried vine fruit can be a further important dietary source of OTA. Surveys managed by MacDonald et al. (1999), Joint FAO/WHO Expert Committee on Food Additives, JECFA (2001) and Stefanaki et al. (2003) showed a high frequency of positive samples, sometimes with high content of OTA (max 53.6 µg/kg). Due to this, the Commission of the European Communities (2002) fixed a limit for OTA in dried vine fruit of 10 µg/kg.
The limit for wine is not yet fixed but, probably it will be 2 ppb and it will be applied from vintage 2005, also based on OIV recommendation (Office International de la Vigne e du Vin).

Ochratoxin A in grapes and wine
OTA is a problem that originates in the vineyard. Black aspergilli, Aspergillus carbonarius in particular, are responsible for OTA synthesis. They are always present in vineyards and fungi can be isolated from bunches starting from the early stages of the berries development even if their incidence is relevant from early veraison. At early veraison, a low content/absence of OTA has been also detected in the case of relevant contamination at harvesting. Consequently, the period between early veraison and harvesting can be considered as crucial for OTA accumulation and factors able to influence fungal growth and OTA synthesis, in particular meteorological conditions, need to be monitored carefully during this time.
Regarding cropping system, several parameters are deemed to be relevant, such as grape variety, training system or fungicides, but until now only preliminary in vitro data have been available regarding the effect of fungicides on OTA production (Battilani et al., 2003).

A. carbonarius control in vineyard
The reduction of fungicide residues and of MT contamination in food commodities, are two important social and scientific issues raised in the 6th UE Framework Programme. The application of antagonistic microorganisms (biocontrol agents: BCA) alone (biological control) or in combination with reduced rates of chemicals (integrated control), is a very promising strategy for reducing fungicide utilisation.
At present, there are no products specifically registered for the control of fungal pathogens responsible for OTA contamination, even if encouraging results were obtained.
Regarding BCAs, they have a complex mode of action against fungal pathogens that makes rare the selection of resistant pathogens. Several researchers studied BCAs mode of action, pointing out the relevance of competition for space and nutrients (Droby e Chalutz, 1994; De Curtis et al., 1996; Castoria et al.,2001, 2003; Janisiewicz e Korsten, 2002)
Several yeasts and yeast-like fungi with high antagonism and a broad range of activity against the main postharvest pathogens (B. cinerea, P.expansum, A.niger, R.stolonifer, M.laxa) of various fruit species were isolated and characterised (De Curtis et al., 1996; Arras et al., 1998; Lima et al., 1998a), and studied for their modes of action (Castoria et al., 2001, 2003). In most cases, biocontrol yeasts are more effective when applied before harvest (Lima et al., 1997; Lima et al., 2002). In particular, very high control levels of grey mould on strawberry and table-grape have been obtained applying BCAs at the flowering stage (Wisniewski et al. 1991; Lima et al., 1997; Ippolito and Nigro 2000).
The use of BCAs against mycotoxigenic fungal pathogens could represent also an innovative solution of the problem of MT contamination, which has not been solved yet by chemical control. The use of fungicides, although it is often effective, cannot set to zero the infection by fungal pathogens and, in case of a mycotoxigenic fungus, a low percentage of diseased plants/ commodities constitutes the source of MT contamination. Once infection by the mycotoxigenic fungus has occurred, the residual fungicide cannot inhibit MT synthesis. On the other hand, BCAs are "active elements", potentially able to interfere with or to inhibit MT synthesis, or to degrade these substances. This is the case of the biocontrol yeast Rhodotorula glutinis LS11. This BCA is able to degrade the MT patulin in vitro and, in the low percentage of BCA-treated apples infected by mycotoxigenic P. expansum, it remains viable, determining a significant decrease of MT accumulation (Castoria et al., 2002; Logrieco et al., 2002). Furthermore, preliminary data suggest that some strains of BCA, apparently degrade OTA in vitro (Castoria et al., 2004). In particular, A. pullulans strain LS30, recently characterised at molecular level (De Curtis et al., 2004, submitted), has shown high levels of grey mould control on grapevine in the field (unpublished data).

Effect of A. carbonarius control methods on winemaking
Studies carried out till now have demonstrated that OTA originates in the vineyard and that winemaking operations allow only a partial OTA reduction (Silva et al. 2003). Each treatment in the vineyard must be evaluated in relation to its effectiveness on OTA presence, but it must be evaluated also in relation to its influence on the winemaking process and on the global quality of the product obtained. It is important to evaluate if control treatments effective on A. carbonarius on grapes are able to modify the natural ecosystem must-wine with negative consequences on the fermentation process.
Yeasts, bacteria and fungi have, in fact, a relevant influence on the chemical characteristics of wines: they influence grapes quality before harvesting and, during fermentation, yeasts metabolise sugars and other compounds producing alcohol, CO2 and many secondary products that affect the elegance and the individuality of wines. Many factors influence growth of yeasts during the alcoholic fermentation, such as the concentration of initial population, the variability of species and strains in must, the use of starters cultures, the chemical composition of the juice, the process parameters and the interactions between different yeast species and strains (Fleet, 2003).
A yeast can influence growth and activity of other micro-organisms through different mechanisms: the availability of nutrients (nitrogenous substances, vitamins, carbonic compounds, etc.) and the production of some metabolites, such as fatty acids and alcohol. These factors modulate the ecology of yeasts during fermentation; for example, Saccharomyces cerevisiae, thanks to its ability to form high amounts of alcohol, even if not frequent on grapes, gets the upper hand of Non-Saccharomyces yeasts during fermentation. Non-Saccharomyces yeasts have a low oenological interest and they support negative characters for wine quality (Fleet, 2003; Loureiro et al., 2003).
Non-Saccharomyces yeasts, growing during the first stages of the alcoholic fermentation, can deduct nutrients to S. cerevisiae with consequent low or incomplete fermentations. Moreover, fatty acids with short or medium chain like esanoic, octanoic and decanoic acid, that are formed during the fermentation in different amounts depending on the species and the strains, greatly influence the sequence of yeasts growth during fermentation. If Non-Saccharomyces yeasts grow during the alcoholic fermentation, high amounts of compounds like acetic acid, hydrogen sulfide and sulphur-containing volatile compounds and ethyl-phenols, that cause a negative sensorial impact on wine, can be formed (Romano et al., 2003). <<<