The effect of the fungicide captan on Saccharomyces cerevisiae and wine fermentation

Fungicides, particularly those used during grape maturation, as captan, can affect the natural yeast population of grapes, and can reach grape must affecting wine fermentation. The objective of the present work was to study the effect of captan on the viability and fermentative behavior of S. cerevisiae. S. cerevisiae (BY4741) on exponential phase was treated with captan (0 to 40 μM) for different periods, and their cell viability analyzed. Cell membrane integrity, thiols concentration, and reactive oxygen species (ROS) accumulation was determined. The fermentation experiments were conducted in synthetic must using wine yeast strain Y904. The results showed that under aerobic conditions, 20 μM of captan reduce 90% of yeast viability in 6 hours. Captan treated cells exhibited alteration of membrane integrity, reduction of thiol compounds and increase in intracellular ROS concentration, suggesting a necrotic and pro-oxidant activity of the fungicide. Fermentative experiments showed that concentrations above 2.5 μM captan completely inhibited fermentation, while a dose dependent fermentation delay associated with the reduction of yeast viability was detected in sub-inhibitory concentrations. Petit mutants increase was also observed. In conclusion, the captan induces yeast necrotic cell death on both aerobic and anaerobic conditions causing fermentation delay and/or sucking fermentations.


Introduction
Captan (N-cyclohex (trichloromethylthio)-4-ene-1, 2-di carboximide) is a broad spectrum fungicide of the phtalimide class, widely used to control several grapevine diseases, as downy mildew, phomopsis, black rot, ripe rot, and bitter rot.With a pre-harvest interval of 24 h to 30 days depending on specific national legislations, experimental data showed that Captan residue in grapes varies between 0.74 to 22 mg/kg [1], and can reach grape must, and decline but persist during fermentation [2].
Captan reaction with thiol groups has been pointed as the main mode of action on phytopathogenic fungi, been responsible for the reduction of enzymatic activities, respiration, physiological changes, and fungal death [3].In the presence of exposed thiol groups, captan oxidize thiols and is hydrolyze to its reactive thiophosgene (SCCl 3 ) moiety, and the 1,2,3,6-tetrahydrophthalimide ring [4].In vitro genotoxicity studies indicated that phtalimide fungicides were associated to point mutations, and gene conversion [5,6], been classified as potential human carcinogen.However, based on in vivo and molecular analysis in mammals they were re-classified as nongenotoxic [7].
As other broad-spectrum fungicides, captan can affect non-target microorganisms, among which epiphytic and wine yeasts [8][9][10][11].A comparison of the toxicity of 25 vineyard pesticides on the growth of wine yeast (Saccharomyces cerevisiae) showed that captan one of the more toxics [8].Moreover, the presence of captan in grape must can drastically reduce yeast viability causing fermentation delay, wine cloudiness, and stuck fermentations [12].Although used for more than 60 years, the mechanism of action and the effect of captan on both target and non-target organisms is poor and most studies just describe its toxicity on mammals.
Considering that captan is used to control grape diseases with a pre-harvest interval as low as 24 h, depending on specific national legislations, and consequently reach wine must, the aim of the present study was to determine captan minimal inhibitory concentration, and evaluate the effect of captan on S. cerevisiae under actively aerobic growth and wine fermentation.

Yeast growth and fermentations
For aerobic experiments, yeasts from overnight cultures were inoculated in YPD broth, and grown to midexponential phase (OD 600 ∼0.7) at 28 • C with orbital shaking (150 rpm).Cells were harvest by centrifugation, washed twice with 0.9% NaCl, and cell density adjusted to 10 7 cells/ml in minimal medium (0.67% Yeast Nitrogen Base without aminoacids, 2% glucose, with 20 mg/L histidine, methionine, and uracil, and 60 mg/L leucine, pH 6.5).Control and captan-treated cultures were incubated for 6 hours at 28 • C with shaking (150 rpm).
For fermentation experiments, yeasts were grown to stationary phase on YEPD broth for 48 hours at 28 • C with shaking (150 rpm), collected by centrifugation, washed with 0.9% NaCl.Yeast inoculation was standardized at 5 × 10 6 cells/ml in 100 ml of MS300 medium [13] in 250 ml Duran flasks with Müller valves.Fermentation were conducted at 24 • C with periodical agitation, and monitored by CO 2 release.

Analytical techniques
The viability of Captan-treated and untreated yeasts was determined by spot assay.Cultures were diluted at 10-fold series, and aliquots (10 µl) of each dilution were spotted onto YEPD plates.Colony were enumerated after 48 h incubation at 28 • C, and expressed as percentage of colony forming units (CFU) compared with the control (untreated cells).
Cell membrane integrity and ROS accumulation were determined with LIFE/DEAD FungalLight Yeast Viability kit (Invitorgen), and the dihydroethidium and dihydrorhodamine 123 oxidant sensitive dyes, respectively, using a flow cytometry FACSCalibur (Becton-Dickinson) instrument equipped with an argon-ion laser emitting at 488 nm.

Statistical analyses
All analyses were carried out in triplicate, and the results expressed as mean values ± standard deviation.The SPSS 20.0 software for Windows (Chicago, IL, USA) was used for the analysis of variance (ANOVA), and means comparison.

Results and discussion
Initial experiments showed that captan minimal inhibitory concentration (MIC) for aerobic exponential growing cells of S. cerevisiae on SD medium was 40 µM, but a 6 hours treatment with 20 µM captan resulted in 97.7% reduction in yeast viability (Table 1).This concentration and time exposure was adopted to analyze captan effect on the reference strain S. cerevisiae BY4741.
A comparison between control (untreated) and captantreated cells (Table 1) showed a significant reduction of both cellular protein and non-protein thiol groups.This result confirms captan non-specific thiol reactivity as one of the most important direct effects of the phtalimide fungicides [3].Moreover, captan-treated cells exhibited a drastic loss of membrane integrity, a typical necrotic behavior [16], which can be the result of the modification of membrane protein structures by protein sulfhydryldisulfide transitions, and consequent increase in membrane permeability [17].
As previously observed in another thiol-reactant fungicide Mancozeb [18], captan-treated cells exhibited an important increase in ROS concentration.ROS accumulation can be related to the reduction of glutathione, the most important reducing compound in yeasts [19].Moreover, ROS accumulation can induce apoptotic cell death, which in turns can be responsible for the difference between the percentage of necrotic cells (78.9%) and loos of culture-ability (97.7%).
Captan MIC determined in synthetic must (MD300) on seven Saccharomyces strains was 5 µM, except for BP725 (2.5 µM) and F15 (10 µM).This values are similar to those reported by Cabras et al [2] that observed complete inhibition of S. cerevisiae and Kloeckera apiculata by 6.7 µM Folpet, a sister molecule of captan.
Captan MIC values under fermentation conditions are lower than those defined for aerobic active growing cells.Evaluation of pH and osmotic effect on captan MIC values, showed that where glucose concentration (2-20%) has not effect, the reduction of pH values directly affect captan toxicity varying from 20 µM at pH 6.5 to 5 µM at pH< 5.0.Acidity and pH affects the dissociation of captan on their thiophosgene and tetrahydrophtalic acid moieties [20].
Fermentation experiments with different concentrations of captan were conducted in MD300 medium using the wine strain Y904.The results showed that 10 µM captan completely inhibited fermentation, but in lower dosages the fungicide determined a dose dependent delay effect with an increase of 1, 3 and 9 day in the presence of 1.25, 2.5 and 5 µM of captan, respectively (Fig. 1).Conversely, the maximum fermentation rate increased from 1.04, in the control, to 1.39 gCO 2 /100ml/day in the 2.5 µM captan treatment.Fermentation delay, inhibition of cell development and reproduction, and modification of wild yeasts population dynamics during wine fermentations, by low concentrations of phtalimide fungicides was previously reported [12].
The evaluation of viable yeast population (UFC/ml) and fermentation behavior in control, 0.625 and 2.5 µM captan confirmed a fermentation delay, particularly evident  on the highest captan concentration (Fig. 2).The delay to begin fermentation in 2.5 µM captan treatment was associated to a drastic initial decrease in the overall viable yeast population, which fall from 5 × 10 6 to 2.2× 10 4 CFU/ml (99.6%) in the first 24 hours.However, the cells that remain viable rapidly adapted and grew, reaching 5 × 10 8 CFU/ml, the same population of the control, at the fifth day.Yeast adaptation to stress depends on the mode of action of the stressing agent.Studies on S. cerevisiae adaptation to the thiolreactive and pro-oxidant fungicide Mancozeb led to the identification of 286 genes that provide protection, most of which involved in oxidative stress response, protein degradation, and carbohydrate/energy metabolism, and controlled by the major yeast oxidative stress regulator, Yap1p [21].
A large number (24.5%) of small colonies were observed at the end of fermentations with 2.5 µM of captan.These colonies were unable to grow on glycerol as only carbon source and confirmed as petite (respiratory deficient) mutants.The highest frequency of petite mutants can be attributed to the direct mutagenic action of the tiophosgene moiety of the captan molecule [4], to high ROS accumulation, and the adaptive advantage of mitochondrial defective petite yeast under the stress condition imposed by the fungicide [22].
A comparison of fermentation parameters in the absence and presence of captan showed non-significant differences in wine density and pH (Table 2).However, wines obtained from 2.5 µM captan treatments exhibited significantly higher concentrations of ethanol, glycerol, and total acidity, lower concentration of residual sugar, and a dose dependent decrease of volatile acidity.Yeast cells from 2.5 µM captan treatments recovered at the end of fermentation showed higher concentration of total thiol groups, mainly glutathione.This increase may be part of the adaptation of yeast to glutathione (G-SH) depletion by the captan, as well as a response to ROS accumulation in the presence of the fungicide.In this sense, it is important to note that although progressively degraded in must and wines, phtalimide fungicides and their degradation products can remain at the end fermentation [2], and affect yeast behavior.
In summary, the data reported in this study showed that 20 µM of captan drastically reduce the viability of aerobic active growing cells of S. cerevisiae in just 6 hours treatments.Captan treated cells exhibited loss of membrane integrity, reduction of protein and nonprotein thiol compounds and increase in intracellular ROS concentration, indicating necrotic and pro-oxidant activity.Under wine fermentation condition, pH stimulated fungicide dissociation resulting in a 4× reduction of MIC values.Fermentation delay caused by 2.5 µM captan was associated to the reduction of yeast viability.However, those cells that survived the initial shock adapted to the fungicide, reassumed growth, and finished fermentation.Wines obtained from 2.5 µM captan treatments showed higher concentrations of ethanol, glycerol, and total acidity, and lower volatile acidity.Moreover, yeast cells from 2.5 µM captan treatments recovered at the end of fermentation showed higher concentration of total thiols, and almost 25% of them were respiratory deficient petite mutants.

Figure 1 .
Figure 1.Fermentation behavior in increased concentrations of captan.Values are mean of three replications.

Figure 2 .
Figure 2. Yeast population and fermentation kinetics in subinhibitory concentrations of captan.Values are mean of three replications.

Table 2 .
Wine fermentation parameters, cell mass and cellular thiols in the absence and presence of captan.Values are the means of three replicates, and different letters in each line are significantly different at the 0.05 level according to ANOVA by Tukey's test.1Totalthiol groups (mg cysteine/mg protein); 2 g dry weight/L; 3 g tartaric acid/L; * 4 g acetic acid/L.