The need to develop natural alternatives to chemical control strategies has led to the application of various yeast strains as biocontrol agents against various plant pathogenic fungi. There are a large number of reports in the literature detailing that antagonistic yeasts possess effective mechanisms for the control of spoilage microorganisms. These antagonistic properties have been well studied and have been successfully exploited in the biological control of postharvest diseases of fruits [12
]. In addition, yeasts have a long history of proven safe use as fermentative starters in food and beverages [13
]. The aims of this study were: (i) to select wine yeast strains which display antagonistic activity against OTA-
; and (ii) to investigate possible mechanisms of action of an antagonistic strain of Saccharomyces cerevisiae
against both A. carbonarius
and A. ochraceus
During the wine making process, OTA levels are known to decrease, an effect which is believed to be mediated by the activity of the resident microbial flora, particularly of lactic acid bacteria and yeasts [10
]. The reduction in OTA level which is mediated by yeasts has been ascribed to different mechanisms including adsorption onto the yeast cell surface, or due to interactions with yeast metabolites. Moreover, Angioni and co-workers have reported that OTA residues do not affect the fermentative process and that reduction of OTA content is a strain-related peculiarity [6
In the present work, wine strains of S. cerevisiae (five strains) and K. apiculata (two strains) were tested to assess their potential antagonistic effects against A. carbonarius in a co-inoculation assay performed in vitro on agar plates using different culture media. This assay was employed in order to select yeast strains that would be able to inhibit the co-inoculated fungus while colonising a common ecological niche. All seven yeast strains displayed an ability to inhibit fungal growth when co-cultured in CYA and YES media, with levels of inhibition in growth of up to 65% being observed. The yeast strain which exhibited the maximal levels of growth inhibition of A. carbonarius was S. cerevisiae DISAABA1182 following growth on YES medium ().
Growth of Aspergillus carbonarius MPV A566 in YES medium alone or in co-culture with antagonistic Saccharomyces cerevisiae or Kloeckera apiculata strains after seven days at 25 °C.
When A. carbonarius
was cultured alone in liquid media, higher levels of OTA were observed from cultures grown on CYB (0.3 ± 0.1 μg/mL) than on YES (0.2 ± 0.1 μg/mL). Abramson and Clear [19
] have suggested that this could be related to differences in the overall sucrose content of each medium: YES (15% sucrose)/CYB (3% sucrose); while CYB may also represent a less hydrophilic layer making it more permeable to lipophilic solvents used in OTA extraction.
All of the yeast strains tested displayed an ability to completely inhibit OTA contamination in the Aspergillus
culture filtrate (data not shown). This significant decrease in OTA levels in co-cultures with yeast appears to be related to the inhibition of A. carbonarius
growth. Nevertheless, OTA production is not necessarily proportional to the biomass of the mycotoxigenic fungi, as it has been shown for other mycotoxins [20
]. An increase in OTA production in the fungal biomass may in some instances take place as a consequence of competition among microorganisms for essential environmental factors. Inter-microbial competition is a stressful physiological condition and is known to have a dramatic effect on secondary metabolism in both food spoilage and phytopathogenic fungi. In addition nutrient availability is also known to markedly influence OTA production in other Aspergilli
. For example OTA production in A. ochraceus
has been shown to be dependent on nutrient content [21
], as well as a variety of nutritional based factors such as various carbon and nitrogen sources [22
]. In addition Teren et al.
] have suggested that Aspergilli are capable of assimilating the phenylalanine moiety from the OTA molecule, as a nitrogen source in nutrient replete culture medium.
Selected yeast strains were also assessed for their ability to inhibit berry infection by A. carbonarius upon co-inoculation. The wine yeast strains significantly reduced fungal colonisation on artificially inoculated grape berries of two cultivars namely Cannonau (a red cultivar) and Vermentino (a white cultivar) ().
Aspergillus carbonarius MPV A566 infection rate on grape berries (cultivars Vermentino and Cannonau) co-inoculated with antagonistic Saccharomyces cerevisiae or Kloeckera apiculata strains after seven days at 25 °C.
The mean disease reduction rate was up to 70% in all strains tested, ranging from between 80%-99% and 75%-100% for the Vermentino and Cannonau cultivars, respectively (). Differences in grape varieties are known to affect fungal invasion, with skin hardness and thickness as well as tannin content known to be a hurdle for penetration by the pathogen [24
]. A. carbonarius
is well known to be a very invasive fungus which is capable of colonising and penetrating berries even without skin damage and to grow at 25-35 °C and 0.95-0.99 aw
, respectively [25
]. It should be emphasised that the experimental conditions employed here to assess the potential in vivo
biocontrol activity of the yeast strains against A. carbonarius
, were highly favourable to the fungus. OTA accumulation is known to mainly occur at ripening, when the fungus preferentially infects berries by entering skin wounds which are induced either by insects and/or injuries caused by meteorological phenomena. High levels of fungal infection and consequent wine contamination by OTA may then take place when high humidity and temperature conditions occur coupled with grape berry damage. Furthermore, the levels of infection by A. carbonarius
and the synthesis of OTA are the highest on wounded berries that are detached and that are subject to conducive temperatures, such as those adopted in our laboratory experiments. Thus, although the experimental conditions employed here should have been highly conducive to fungal infection, almost all yeast strains provided an efficient protection to the wine grape berries against infection by A. carbonarius
for up to seven days (). Such a time lag could be crucial in ensuring that the wine grape harvest is biologically protected during the most critical phase for OTA contamination, i.e.
, between harvesting and pressing [3
General nutrient competition in the grape berry is not per se
sufficient to explain yeast biocontrol activity. Nevertheless, this finding does not exclude the possibility that the antagonistic behavior being exhibited by these yeast strains may be as a result of competition for a specific growth limiting factor, such as—for example—a vitamin or another nutrient. According to related pathosystems, other possible mechanisms may include biofilm formation on the wound surface [26
], the inhibition of fungal secondary metabolism [27
], the production of antifungal enzymes [28
] or the induction of resistance in the fruit tissues [12
In order to explore possible mechanisms of biocontrol activity the most effective strain, DISAABA1182 was selected for further investigations. In a study involving 20 yeast strains S. cerevisiae
DISAABA1182 had previously been reported to exhibit the most potent inhibitory effect in decreasing OTA levels [6
]. These authors have also shown that as OTA residues were not recovered from the yeast cell biomass and due to the absence of OTα and phenylalanine in the must that another degradation pathway may be employed by this yeast strain.
The OTA biosynthetic pathway, while as yet not fully elucidated in any fungal species, is believed to involve the synthesis of an isocoumarin group which is a pentaketide formed from an acetate and malonate requiring the involvement of a polyketide synthase (PKS) enzyme. A number of studies have reported a direct link between pks
gene expression and OTA production [21
]. In order to examine a possible correlation between reduced OTA production and pks
gene expression [30
] two OTA-producing strains namely A. carbonarius
MPV A566 and A. ochraceus
MPV A703 were co-cultured with S. cerevisiae
DISAABA1182 as well as being exposed to supernatant preparations from the yeast.
Following growth of both A. ochraceus MPV A703 and A. carbonarius MPV A566 under OTA inducing conditions (YES medium), OTA production was observed (). With A. ochraceus MPV A703, OTA production was first observed on day 3 with levels increasing to reach their highest levels on day 7, while the highest level of OTA produced by A. carbonarius MPV A566 was detected on day 6 and started to decrease on day 7 ().
Figure 1 Ochratoxin A (OTA) production by Aspergillus ochraceus MPVA703 (upper) and Aspergillus carbonarius MPVA566 (lower) grown alone or co-cultured with S. cerevisiae DISAABA1182 on YES medium for seven days at 25 °C. OTA values are expressed as μg/mL (more ...)
When A. ochraceus and A. carbonarius were grown in co-culture with living cells of S. cerevisiae DISAABA1182 living cells, a decrease of 98% and 95% in overall OTA concentrations was observed, respectively. Similarly, a reduction in fungal biomass of up to 95% was observed for both fungi following six days of incubation ().
Table 3 Fungal growth and OTA production in A. ochraceus MPVA703 and A. carbonarius MPVA566 grown for six days at 25 °C in YES medium amended with living cells of S. cerevisiae strain DISAABA1182, together with crude and autoclaved supernatant preparations. (more ...)
To provide further insights into potential mechanisms which may be involved in OTA reduction caused by the yeast, A. ochraceus
MPV A703 and A. carbonarius
MPV A566 were statically grown for six days at 25 °C in YES medium amended with either crude supernatant from strain DISAABA1182 or with supernatant which had been previously autoclaved for one hour. A reduction in OTA levels was observed for both fungi with these decreases being accompanied by reductions in fungal biomass (). Crude supernatant preparations from strain DISAABA1182 reduced OTA production by 99% and 94%, respectively in A. ochraceus
and A. carbonarius
; and fungal growth was inhibited by up to 96%. When autoclaved supernatant preparations from DISAABA1182 were tested complete suppression of OTA production in both fungi was observed, while fungal biomass levels were reduced by 58% in the case of A. ochraceus
and 96% for A. carbonarius
(). Thus while in the case of A. carbonarius
it is quite clear that fungal biomass affects OTA production, i.e.
, the growth of the fungus corresponds to the production of OTA, this does not seem to apply to A. ochraceus
when co-cultured with autoclaved supernatant preparations from S. cerevisiae
DISAABA1182. Schmidt-Heydt and co-workers previously reported that OTA production does not always directly correlate with fungal biomass, and showed that different environmental factors can affect the production of this mycotoxin in different ways [33
Thus to determine whether the observed effects were being mediated at the level of gene expression, polyketide synthase (pks) gene transcript levels were analysed in A. ochraceus and A. carbonarius during co-culture with strain DISAABA1182 (). Reduced levels of pks gene transcript were evident in A. ochraceus, while in the case of A. carbonarius the pks gene expression was only slightly reduced. The expression levels of β-tubulin and calmodulin genes observed under the test conditions suggest that changes in pks gene expression are specific and not simply occurring as a result of changes in overall gene transcription levels in the fungus.
Figure 2 RT-PCR analysis of pks gene expression in Aspergillus ochraceus MPVA703 and Aspergillus carbonarius MPVA566 grown alone or in presence of Saccharomyces cerevisiae strain DISAABA1182 living cells (A), or its crude and autoclaved supernatants (B). RNA was (more ...)
As previously mentioned, it has been well established that a number of physiochemical parameters, such as nutritional and environmental stimuli, play an important role in the regulation of mycotoxin biosynthesis [22
]. Previous studies have shown that OTA production by A. ochraceus
is dependent on the growth medium [30
] and that production is concomitant with increases in pks
gene transcript levels. A correlation has also been reported between physiological stress factors and the expression of genes responsible for ochratoxin A production in Penicillium verrucosum
]. In our study the strong reduction in pks
gene expression in A. carbonarius
and, to a lesser extent, in A. ochraceus
correlates closely with the reduced OTA levels, suggesting that the observed reduction in OTA production may be mediated at the level of gene transcription (; ).
Thus S. cerevisiae
DISAABA1182 inhibits growth, OTA production and pks
expression in two ochratoxigenic strains of A. carbonarius
and A. ochraceus
and constitutes one of a few reports which have to date described the effects of biocontrol agents on the expression of mycotoxin biosynthetic genes [34
]. One such report involves an antagonistic strain of Debaryomyces hansenii
which has been observed to reduce the expression of OTA biosynthetic genes in A.
]. The mechanisms resulting in reduced OTA production and inhibition of Aspergillus
growth by S. cerevisiae
DISAABA1182 remain to be further elucidated but are likely to involve a number of synergistic mechanisms, including effects mediated at the level of gene transcription.