World J Microbiol Biotechnol (2010) 26:925932 DOI 10.

1007/s11274-009-0255-4

ORIGINAL PAPER

Volatile organic compounds produced by Saccharomyces cerevisiae inhibit the in vitro development of Guignardia citricarpa, the causal agent of citrus black spot
Mauricio Batista Fialho Leonardo Toffano Marcio Pozzobon Pedroso Fabio Augusto Sergio Florentino Pascholati

Received: 27 August 2009 / Accepted: 13 November 2009 / Published online: 10 December 2009 Springer Science+Business Media B.V. 2009

Abstract Due to the low chemical control effectiveness of citrus black spot, caused by the fungus Guignardia citricarpa at postharvest, and to the search for alternative control methods, this study aimed to evaluate the in vitro effect of volatile organic compounds (VOCs), produced by yeast Saccharomyces cerevisiae, on G. citricarpa. It was observed that the yeast strains evaluated acted as antagonists by VOC production, whose maximum inhibitory capacity was as high as 87.2%. The presence of fermentable carbon sources in the medium was essential for the bioactive VOC production by the yeast. The analysis of VOCs produced in PDA medium by SPMEGCMS indicated the presence of high quantities of alcohols as well as esters. An articial VOC mixture prepared on the basis of the composition of the VOCs mimicked the inhibitory effects of the natural VOCs released by S. cerevisiae. Thus, the VOCs produced by the yeast or the articial mixtures

can be a promising control method for citrus black spot or others postharvest diseases. Keywords VOC Microbial interaction Inhibition Alternative control Phyllosticta Citrus

Introduction Black spot is a disease that occurs in most citrus species. Its causal agent is the fungus Guignardia citricarpa Kiely (anamorphic stage: Phyllosticta citricarpa McAlpine) [Ascomycetes: Dothideales]. It has high economic importance and affects the most important commercial citrus varieties in many producing areas of Africa, Asia, Australia and South America. Symptoms on fruit include hard spot lesions (raised black edges and tan centers that often bear pycnidia), false melanose (small, raised, dark lesions that often coalesce and have been attributed to conidial infection), freckle spots (sunken reddish lesions that may coalesce to form large areas called virulent spot), and cracked spots (raised dark lesions with irregular margins) (Goes et al. 2000; Kotze 2000). Although not showing apparent symptoms, the infected fruits can develop them at postharvest during transport or storage. In spite of the lesions are restricted to the fruit rind, the fruits become aesthetically damaged, making them undesirable to the fresh fruit market. In addition, the fruit cannot be exported especially to the European Community due to phytosanitary restrictions (OEPP/EPPO 2003, 2008). Even though their effectiveness is limited, the use of fungicides is the main control method used at pre- and postharvest. Besides the fact that the chemical control costs are signicant, reports about the emergence of resistant

M. B. Fialho L. Toffano S. F. Pascholati (&) Departamento de Nematologia e Fitopatologia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo, Caixa Postal 09, Piracicaba, SP CEP 13418-900, Brazil e-mail: sfpascho@esalq.usp.br M. B. Fialho e-mail: alhomb@hotmail.com L. Toffano e-mail: toffano@esalq.usp.br M. P. Pedroso F. Augusto Departamento de Qumica Analtica, Instituto de Qumica, Universidade Estadual de Campinas, Caixa Postal 6154, Campinas, SP CEP 13083-970, Brazil e-mail: mpedroso@iqm.unicamp.br F. Augusto e-mail: augusto@iqm.unicamp.br

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isolates to some fungicides in the mid 1980s led to restrictions in their use (Agostini et al. 2006; Deising et al. 2008). Problems associated with the acquisition of resistance by the pathogen and the public perception in general about the potential impact of traditional control practices on health and on environment led to an increased demand for residue-free chemical products (Gullino and Kuijpers 1994). Consequently, both farmers and researchers started to consider the use of alternative methods to control diseases (Punja and Utkhede 2003). The use of antagonistic microorganisms may interrupt some stage of the disease or the pathogens life cycle. This may occur by means of several mechanisms such as parasitism, competition for nutrients and colonization niches, production of hydrolytic enzymes (Punja and Utkhede 2003), and volatile (Bruce et al. 2004; Strobel 2006) as well as non-volatile antibiotic compounds (Wei et al. 2005; Vinale et al. 2006). It is likely that different modes of action occur synergistically during the antagonistic interaction. The knowledge about how this mechanisms works is essential to improve the biocontrol effectiveness as well as to develop innovative control strategies. There are no reports in the literature concerning the inhibition of plant pathogens by volatile substances produced by yeasts. The yeast Saccharomyces cerevisiae is little studied as a biological control agent. Nevertheless, it can be considered as a safe microorganism, and it is classied as Biosafety level 1 (CDC/OHS 2009), since it is neither a human nor a plant pathogen. In addition, it does not produce mycotoxins, antibiotics, or other molecules whose presence is unacceptable in human foods, such as fruits intended to be consumed fresh (Droby et al. 1991). Another advantage of its use in plant disease control is the better acceptance by consumers, who are more familiar with S. cerevisiae since it is widely used in the production of foods and drinks. To identify the volatile organic compounds (VOCs) produced by S. cerevisiae, headspace solid phase micro extraction (HS-SPME) coupled to gas chromatography with mass spectrometric detection (GCMS) was employed. Due to its simplicity, sensitivity and speed, SPMEa technique introduced in 1990has been extensively employed in the extraction and pre-concentration steps of analytical procedures applied to a wide range of matrixed compounds, and is signicantly suitable for in situ isolation of biogenic compounds (Augusto and Sartoratto 2003; Oliveira et al. 2004; Lupe et al. 2007). In view of the damages caused by the citrus black spot and a high interest for alternative control methods, this study aimed to evaluating the in vitro antagonistic capacity of S. cerevisiae strains, used in fermentative processes for fuel ethanol production, against G. citricarpa, as well as to elucidate the nature of the inhibitory effect in order to

generate information for the development of new control strategies.

Materials and methods Phytopathogenic fungus Guignardia citricarpa was isolated from orange fruit lesions and maintained in Potato-Dextrose-Agar (PDA) at 26C, under uorescent light and 12 h photoperiod. PDA medium: infusion of 200 g l-1 potato, 20 g l-1 glucose, and 15 g l-1 agar. To prepare the potato infusion, 200 g of pricked potatoes [cultivar Monalisa] were boiled in 0.5 l of distillated water for 15 min and ltered through cheesecloth. The volume was completed to 1 l and added glucose and agar. The fungus is deposited as isolate IP-92 in the mycological culture collection of the Laboratory of Plant Pathology in the Department of Phytossanity at FCAV/ UNESP, in Jaboticabal-SP, Brazil. Antagonistic yeast Saccharomyces cerevisiae strains (BG-1, CR-1, CAT-1, KD-1, K-1, and PE-2) were isolated from fermentation processes for fuel ethanol production, characterized by electrophoresis karyotyping, and maintained in the yeast collection of the Biochemistry and Alcohol Fermentation Laboratory in the Biological Sciences Department at Esalq/ USP, in Piracicaba-SP, Brazil. The yeast strains were maintained in Yeast Extract Peptone Dextrose Agar (YEPDA medium: 10 g l-1 yeast extract, 10 g l-1 peptone, 20 g l-1 glucose, and 15 g l-1 agar) at 32C. Production of antimicrobial VOCs by S. cerevisiae strains Commercially available polystyrene plates (90 9 15 mm) divided in half by a wall (BD Co., Franklin Lakes, NJ USA) were used. The antagonistic S. cerevisiae strains were spread using 50 ll of suspension (108 cells ml-1) on one side of the plate containing PDA. Afterwards, a 5 mm diameter agar plug with mycelium of G. citricarpa was placed onto the opposite side of the plate. The control consisted of plates containing the pathogen in the absence of yeast. The plates were wrapped with paralm and maintained at 26C under a 12 h photoperiod. Mycelial growth was evaluated by the average between two opposing measurements when the colony in control plates reached the borders. In order to determine the fungistatic or fungitoxic nature of the volatiles released by the yeast, the agar plugs containing mycelium that had their growth inhibited were transferred to new plates containing PDA in

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the absence of yeast. Four replicates were used per yeast strain evaluated. Effect of carbon sources on the production of VOCs Four different carbon sources were tested to grow S. cerevisiae strain CR-1. Potato agar (infusion of 200 g l-1 potato and 15 g l-1 agar) was supplemented with 20 g l-1 (2%) of glucose (PDA), sucrose, maltose or galactose. In addition, potato agar supplemented with different glucose concentrations (04%) was tested. As in the previous experiment, polystyrene plates divided in half by a wall were used. On one side of the plate, the yeast was grown on the various media by streaking 50 ll of the cell suspension (108 cells ml-1). Afterwards, a 5 mm diameter agar plug of the plant pathogen G. citricarpa was placed at the opposite side onto PDA. The control consisted of plates containing the pathogen in the absence of yeast. The plates were wrapped with paralm and maintained at 26C under a 12 h photoperiod. Mycelial growth was evaluated when the colony in control plates reached the borders. Four replicates were used for each growth medium evaluated. Analyses of S. cerevisiae VOCs The S. cerevisiae CR-1 strain was inoculated by spreading 100 ll of cell suspension (108 ml-1) on PDA, inside 50 ml septum-sealed polyethylene centrifuge tubes, and incubated at 26C for 6 days. For the extraction of the volatiles, SPME bers (Supelco Inc., Bellefonte, PAUSA) coated either with 50/30 lm divinylbenzene/polydimethylsiloxane/ carboxen (DVB/PDMS/CAR) or 85 lm polyacrylate (PA) were used. After the culture growth period, the sealed tubes were kept for 5 min at 30C to achieve equilibrium among the yeast, the culture medium, and the sample headspace. Immediately after that, a SPME ber was inserted into the tube through the silicone septa, exposed to the headspace for 15 min and them taken to the GCMS for analyte desorption (desorption time = 3 min), separation and detection.

A HP-5890 gas chromatograph (Hewlett-Packard, Wilmington, DE, USA), equipped with a HP-5973 massselective detector, a HP-5MS fused-silica capillary column (30 m 9 0.25 mm 9 0.25 lm) and a splitsplitless injector were employed. The injector was operated in splitless mode for all chromatographic runs, and grade 5.0 helium was used as carrier gas. The injector temperature was kept at 250C and the detector at 280C. The oven temperature was programmed as follows: 40C for 5 min, then increased 3C/min up to 100C and then increased 20C/min to 250C. The MS scan range was set between 35 and 350. Peak identication was performed using the automated mass spectral deconvolution and identication system (AMDIS) v. 2.63 software and the NIST mass spectral search v. 1.7 software, both programs supplied by NIST (WashingtonDC, USA). Identication of the detected species was performed by comparing them against the mass spectra library and conrmed by spiking the samples with pure standards (Sigma/Aldrich Chemical Co., USA). Preliminary runs were also performed in an HP-6850 gas chromatograph (Agilent, Wilmington, DE, USA), equipped with a ame ionization detector (FID) and a HP-5 fusedsilica capillary column (30 m 9 0.25 mm 9 0.25 lm) and a splitsplitless injector. Except for FID temperature (300C), all operational conditions were the same as in the GCMS runs. The polyethylene tubes containing only PDA were employed as blanks, and the data subtracted from the analyses carried out on tubes containing the yeast. Assay of articial mixture of VOCs identied in S. cerevisiae After the S. cerevisiae headspace analyses, an articial mixture was made by using authentic standard chemicals (Sigma/Aldrich Chemical Co., USA). The proportion of each compound positively identied was calculated from the relative peak areas (Table 1). A 5 mm diameter agar plug with G. citricarpa mycelium was placed on one side of the divided plate containing PDA. After 5 days of growth, on the opposite side of the plate, 40 ll of the articial mixture were added on a piece

Table 1 Identication of VOCs produced by S. cerevisiae strain CR-1 grown on PDA using HSSPMEGCMS

Peak 1 2 3 4 5 6 7

Analyte Ethanol Unidentied Ethyl acetate 3-methyl-1-butanol 2-methyl-1-butanol Phenylethyl alcohol Ethyl octanoate

Retention time (min) 2.10 2.91 3.05 5.15 5.23 27.15 28.30

Area (units) 21,478 376 450 1,736 611 181 362

Molecular formula C2H6O C4H8O2 C4H8O2 C5H12O C5H12O C8H10O C10H20O2

MW 44 88 88 88 88 122 172

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Inibition (%)

of sterile cotton. The control consisted of plates containing the pathogen in the absence of the articial mixture. The plates were immediately wrapped with paralm and maintained at 26C under a 12 h photoperiod. The mycelial growth was evaluated daily until the colony in the control plates reached the borders. Five replicates were used for each growth medium evaluated. In another experiment, in one side of the divided plate containing PDA, a 5 mm diameter agar plug of the pathogen was placed. Afterwards, on the opposite side different amounts of the articial mixture from 10 to 120 ll were added on a piece of sterile cotton. The control consisted of plates containing the pathogen in the absence of the articial mixture. The plates were immediately wrapped with paralm and maintained at 26C under a 12 h photoperiod. Mycelial growth was evaluated when the colony in the control plates reached the borders. The headspace of the plate was 50 ml and this was used to calculate the concentration of the VOCs mixture per ml and to determine the MIC50 and MIC100 values. In order to determine the fungistatic or fungitoxic nature of the VOCs, the agar plugs with G. citricarpa mycelium that had their growth inhibited were transferred to new plates containing PDA in the absence of VOCs. Five replicates were used for each treatment.

observed. No apparent morphological modications were detected by light microscopy analysis (Fialho 2004). The agar plugs, containing mycelium that showed growth inhibition, resumed normal development when they were transferred to plates containing PDA in the absence of yeast, indicating the fungistatic nature of the inhibition. The carbon source inuence on the production of antimicrobial VOCs by S. cerevisiae strain CR-1 was also evaluated (Fig. 2). The growth on potato agar supplemented with glucose, sucrose and maltose provided the highest inhibition levels (85.9, 85.6, and 81.9%, respectively). On the other hand, S. cerevisiae was not able to produce antimicrobial volatiles when grown on medium supplemented with galactose. The yeast S. cerevisiae strain CR-1 inhibited the G. citricarpa growth through the production of volatiles only when there was at least 0.5% of available glucose

100 ** 80 60 40 20 ** **

Results VOC production by S. cerevisiae It was observed the production of VOCs by the S. cerevisiae strains able to inhibit the in vitro G. citricarpa vegetative growth (Fig. 1). A maximum mean inhibition of 87.2% was reached with the strain CR-1, however, statistical differences among the tested strains were not
100 **

0 Glucose Sucrose Maltose Galactose

Carbon source
Fig. 2 Mycelial growth inhibition of G. citricarpa exposed to VOCs from S. cerevisiae (strain CR-1) grown on different carbon sources. Inhibition values were calculated as percentage growth inhibition after exposure to the gas producer yeast as compared to the control in the absence of the antagonist. Values are means of four replicates (SD) and ** indicates values that differ signicantly from the control at P B 0.01, Tukeys test
100

Inhibition (%)

80 60 40 20 0 CR-1

**

**

**

**

**

80

** **

**

Inhibition (%)

60 40 20 0 ** 0.0 0.5 1.0 2.0 3.0 4.0 ** **

PE-2

K-1

CAT-1

BG-1

KD-1

-20

S. cerevisiae strain
Fig. 1 Mycelial growth inhibition of G. citricarpa exposed to VOCs from different S. cerevisiae strains grown on PDA. Inhibition values were calculated as percentage growth inhibition after 6 days exposure to the gas producer yeast as compared to the control in the absence of the antagonist. Values are means of four replicates (SD) and ** indicates values that differ signicantly from the control at P B 0.01, Tukeys test

Glucose (%)
Fig. 3 Mycelial growth inhibition of G. citricarpa exposed to VOCs from S. cerevisiae (strain CR-1) grown on different concentrations of glucose. Inhibition values were calculated as percentage growth inhibition after exposure to the gas producer yeast as compared to the control in the absence of the antagonist. Values are means of four replicates (SD) and ** indicates values that differ signicantly from the control at P B 0.01, Tukeys test

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929

Mycelial growth (cm)

(Fig. 3). The inhibitory activity increased proportionally to the increase of the glucose concentration, and the maximum inhibition was seen when the yeast was grown on 4% glucose. On the other hand, S. cerevisiae, grown on medium not supplemented with glucose, caused slight growth stimulation of the phytopathogen. VOC analyses and biological activity of the articial mixture The VOCs produced by S. cerevisiae CR-1 on PDA were analyzed by SPMEGCMS (Fig. 4) and the identication of analytes is shown in Table 1. There was the production of compounds belonging mainly to the alcohol group, with ethanol as the major component of the headspace besides 3-methyl-1-butanol, 2-methyl-1-butanol, and phenylethyl alcohol. The presence of esters was also observed, ethyl acetate, ethyl octanoate and one whose identity was not conrmed. An articial mixture containing each of the positively identied compounds was prepared and tested (Fig. 5). The mycelial growth of the pathogen stopped when the articial mixture was added to the plates in the fth day, mimicking the inhibitory effects of the S. cerevisiae atmosphere on G. citricarpa. By exposing the fungus to different concentrations of the articial mixture of VOCs, it was possible to calculate the MIC50 (0.48 ll ml-1) and MIC100 (2.84 ll ml-1) values, which correspond to the minimum concentration of the articial mixture in microliters per milliliters of air inside the plate required to produce 50 and 100% of reduction in

4
**

3 2 1 0 1 2 3 4 5

** **

Day
Control Artificial mixture

Fig. 5 Inuence of the articial mixture of VOCs (0.8 ll ml-1 air space) identied in S. cerevisiae on the mycelial growth of G. citricarpa. The arrow indicates the addition of the articial mixture. Values are means of four replicates (SD) and ** indicates values that differ signicantly from the control at P B 0.01, Tukeys test

100

Inhibition (%)

80 60 40 20 0 0.0 0.5
y = 11.901x3 - 67.392x2 + 129.89x + 1.7763 R2 = 0.995

1.0

1.5

2.0

2.5

Concentration (L mL-1)
Fig. 6 Mycelial growth inhibition of G. citricarpa exposed to different concentrations of the articial mixture of VOCs identied in S. cerevisiae. Inhibition values were calculated as percentage growth inhibition as compared to the control in the absence of the articial mixture. Values are means of ve replicates (SD)

mycelial growth relative to the control (Fig. 6). Besides, the articial mixture as the natural VOCs had not lethal effect at any concentration on the phytopathogen.

Discussion The inhibitory capacity of the S. cerevisiae strains may reect their capacity of quantitatively and qualitatively produce secondary metabolites. In addition, the susceptibility of a plant pathogen to yeasts may vary according to the chemical nature and mode of action of the antimicrobial compound produced (Walker et al. 1995). In preliminary experiments, diffusible substances produced by S. cerevisiae, and present in culture ltrates, did not show any antifungal activity against G. citricarpa (Fialho 2004). On the other hand, the present study demonstrated that the production of VOCs plays an essential role in the antagonistic activity of S. cerevisiae on G. citricarpa.

Fig. 4 Chromatogram corresponding to VOCs produced by S. cerevisiae strain CR-1 grown on PDA. The insert shows the rst 5 min of the same chromatogram in a different signal scale. Labeled peaks are those identied only in the headspace of yeast culture samples by HS SPMEGCMS; the remainder peaks are from the control sample or the atmosphere. (1) ethanol; (2) unidentied ester; (3) ethyl acetate; (4) 3-methyl-1-butanol; (5) 2-methyl-1-butanol; (6) phenylethyl alcohol; (7) ethyl octanoate

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The variety of volatiles as well as the proportions among the various components of the headspace produced may represent the distinct metabolic conversions performed by the microorganism on different substrates (Bruce et al. 2004). Nout and Bartelt (1998) stated that the production of volatiles by yeasts is strongly inuenced by their capacity of assimilating and fermenting carbohydrates. In the present work, no antimicrobial volatiles were produced by the yeast in the medium containing galactose. S. cerevisiae is able to assimilate and to ferment the sugars glucose, sucrose, maltose and galactose, however, the strain used in the present work can not ferment galactose. This suggests that the lack of capacity of fermenting galactose present in the medium were reected by the quality and low production of inhibitory volatiles. The higher the availability of carbon sources in the medium, the more complex the composition of the headspace produced. The endophytic fungus Muscodor albus produced maximum lethality against plant pathogens when grown in nutrient-rich media (Ezra and Strobel 2003). The same authors also concluded that the simple addition of sucrose to the nutrient-poor medium increased volatile activity by approximately 50%. The inhibition of G. citricarpa was related to the concentration of glucose in the medium, and this observation reinforces the fact that the fermentable carbon source is a limiting factor for bioactive volatile production by S. cerevisiae. In order to understand the nature of the inhibitory effects of the volatile substances produced by S. cerevisiae on G. citricarpa development, it was necessary to identify the components of the atmosphere produced by the yeast. Thus, the identication of seven out of eight detected compounds was carried out, and these included: ethyl acetate, 3-methyl-1-butanol, 2-methyl-1-butanol, phenylethyl alcohol, ethyl octanoate and ethanol, the major component of the headspace. Buzzini et al. (2003) identied the VOCs produced by 98 ascomycetous yeasts strains (no-Saccharomyces spp. yeasts, representative of 40 species belonging to 12 genera) isolated from several tropical habitats. The VOCs produced were found to be alcohols, aldehydes and esters. The main VOCs produced by the isolates were alcohols (3-methyl-1butanol and 2-methyl-1-butanol), and esters (isobutyl acetate, isoamyl acetate, 2-methylbutyl acetate, isoamyl propionate and phenylmethyl acetate). On the other hand, Bruce et al. (2003) observed that volatile compounds produced by S. cerevisiae strain Y1001 on tryptone soya agar were able of inhibit (between 76 and 33%) the growth of fungi related to sapstain in wood. In malt extract and minimum media the inhibition was very low or null. Subsequently, Bruce et al. (2004) identied only two compounds produced by the yeast in liquid tryptone soybean bean medium, 2-pentanone and phenylethyl alcohol.

In malt extract medium, the yeast produced eight compounds where ethanol and ethyl acetate were also released as seen by S. cerevisiae strain CR-1 on PDA in the present work. According to Nout and Bartelt (1998), yeasts developing in medium that contains a non-fermentable carbon source may produce just a few volatiles, such as acetaldehyde and acetic acid. However, the volatile production in media containing fermentable carbon sources is higher, particularly for alcohols (especially ethanol). Thus, this information is in agreement to the results obtained in the present work, since the growth of the yeast on fermentable carbon sources led to the production of high quantities of alcohols, which comprised the majority of the headspace. Very little is known about the mode of action of microbial volatiles on the control of fungi. It is likely that volatiles act by changing protein expression (Humphris et al. 2002) and the activity of specic enzymes (Wheatley 2002), which may reect on growth. The antimicrobial properties of alcohols are long known and are used in the formulation of disinfectants and preservatives. The inhibitory action of alcohols is complex, but in general it seems to be more related to their physicochemical properties than to specic receptors. The primary site of action seems to be the plasma membrane, where the accumulation of solvents could affect the organization and stability of the lipid bilayer. As a consequence, the permeability increases accelerating the passive diffusion of essential ions and metabolites through the membrane (Ingram and Buttke 1984; Seward et al. 1996). Lipophilic compounds such as 3-methyl-1-butanol and 2-methyl-1-butanol have high afnity for the plasma membrane and therefore they present higher toxicity when compared to less lipophilic compounds as the ethanol that is toxic for microorganisms only in higher concentrations (Heipieper et al. 1994). In S. cerevisiae, the alcoholic fermentation overcomes the respiration when in the presence of high glucose concentrations even when under aerobiosis, the growth condition in the present work. The alcohol accumulation in the presence of oxygen was probably an evolutionary strategy used by some yeasts to increase the competitiveness, inhibiting the development of other microorganisms (Pfeiffer et al. 2001; Maclean and Gudelj 2006). Besides, Saccharomyces not only can inhibit the competitor through the alcohol production, but it can also later consume the generated alcohol (Piskur et al. 2006). It was also veried the biological activity of the articial mixture containing each of the positively identied components of S. cerevisiae atmosphere. G. citricarpa was exposed to different concentrations of the components (0.22.4 ll ml-1 of air) and the MIC50 and MIC100 values were estimated in 0.48 and 2.84 ll ml-1, respectively.

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931 gicoNational Council for Scientic and Technological Tecnolo Development), a Brazilian foundation associated to the Ministry of Science and Technology (MCT).

These values represent the response of the fungus to the articial mixture of VOCs. Atmosukarto et al. (2005) studied the susceptibility of human and plant fungi pathogens belonging to several taxonomic groups to articial mixture of M. albus. The MIC50 varied between 0.01 (Verticilium dahliae) and 0.3 ll ml-1 (Colletotrichum gloeosporioides and Aspergillus fumigatus). The articial mixture at the concentration of 1.2 ll ml-1 was able to inhibit in 100% the mycelial growth and it was lethal to all tested plant pathogens except for C. gloeosporioides and Fusarium culmorum. The volatiles 2-propanone, 2-methyl-1-butanol, decanal, heptanal, and octanal, released by Trichoderma pseudokoningii and T. viride were responsible for the antimicrobial activity against wood decay fungi (Wheatley et al. 1997). Later, Humphris et al. (2001) conrmed that several basidiomycetes were completely inhibited by heptanal and octanal at low concentrations (2.5 lg ml-1). The compounds naphthalene, propanoic acid, and 3-methyl-1-butanol were associated to the biological activity of M. albus on the plant pathogens Pythium ultimum, Rhizoctonia solani, and Sclerotinia sclerotiorum. By using the articial mixtures, it was demonstrated that those three compounds were required to maintain the inhibitory activity, indicating a synergy among the different components (Ezra et al. 2004). It is clear that the role of volatiles on the interactions among microorganisms must be known to allow an optimized handling of this characteristic in the alternative control of pathogens. Thus, the biological fumigation with S. cerevisiae volatiles or articial mixtures could be used to treat fruits in closed chambers during storage and transport. In addition, this process would be an environmentally safer alternative to control diseases at postharvest, since the yeast does not have to be in direct contact with the fruits and the fumigation is compatible to packing house systems, since it minimizes product handling (Mercier and Jimenez 2004). Finally, the production of antimicrobial VOCs by yeasts, as action mechanism, in the control of plant pathogens had not been previously reported. Thus, this study shows the S. cerevisiae potential for the biocontrol of citrus black spot, since the chemical control of this disease has been ineffective due to resistance to the limited spectrum of fungicides allowed to be used at postharvest. It also provided background information for future studies and in the moment new experiments has been carried out to evaluate to use of VOC formulations as fumigants to control citrus black spot and other postharvest diseases in fruits.
Acknowledgments The authors thank Prof. Luiz Carlos Basso, Ph.D., Biological Sciences Department (Luiz de Queiroz Agri culture School, University of Sao Paulo, Piracicaba, SP, Brazil), who provided the S. cerevisiae strains. This research was supported by CNPq (Conselho Nacional de Desenvolvimento Cientco e

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