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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 Ó 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 SPME–GC–MS indicated the presence of high quantities of alcohols as well as esters. An artiﬁcial 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 artiﬁcial mixtures
can be a promising control method for citrus black spot or others postharvest diseases. Keywords VOC Á Microbial interaction Á Inhibition Á Alternative control Á Phyllosticta Á 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 signiﬁcant, reports about the emergence of resistant
M. B. Fialho Á L. Toffano Á 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: firstname.lastname@example.org M. B. Fialho e-mail: ﬁalhomb@hotmail.com L. Toffano e-mail: email@example.com M. P. Pedroso Á F. Augusto ´ ´ ´ Departamento de Quımica Analıtica, Instituto de Quımica, Universidade Estadual de Campinas, Caixa Postal 6154, Campinas, SP CEP 13083-970, Brazil e-mail: firstname.lastname@example.org F. Augusto e-mail: email@example.com
it does not produce mycotoxins. To identify the volatile organic compounds (VOCs) produced by S. 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. headspace solid phase micro extraction (HS-SPME) coupled to gas chromatography with mass spectrometric detection (GC–MS) was employed. Franklin Lakes. 20 g l-1 glucose. cerevisiae strains Commercially available polystyrene plates (90 9 15 mm) divided in half by a wall (BD Co. Mycelial growth was evaluated by the average between two opposing measurements when the colony in control plates reached the borders. It is likely that different modes of action occur synergistically during the antagonistic interaction. as well as to elucidate the nature of the inhibitory effect in order to generate information for the development of new control strategies. and PE-2) were isolated from fermentation processes for fuel ethanol production. PDA medium: infusion of 200 g l-1 potato. CR-1. Deising et al. Production of antimicrobial VOCs by S. SPME—a technique introduced in 1990—has been extensively employed in the extraction and pre-concentration steps of analytical procedures applied to a wide range of matrixed compounds. Another advantage of its use in plant disease control is the better acceptance by consumers. in Jaboticabal-SP. 2005. it can be considered as a safe microorganism. 1991). Materials and methods Phytopathogenic fungus Guignardia citricarpa was isolated from orange fruit lesions and maintained in Potato-Dextrose-Agar (PDA) at 26°C. 2006). In order to determine the fungistatic or fungitoxic nature of the volatiles released by the yeast. in Piracicaba-SP. characterized by electrophoresis karyotyping. K-1. Oliveira et al. cerevisiae strains. and 15 g l-1 agar. The yeast Saccharomyces cerevisiae is little studied as a biological control agent..926 World J Microbiol Biotechnol (2010) 26:925–932 isolates to some fungicides in the mid 1980s led to restrictions in their use (Agostini et al. under ﬂuorescent light and 12 h photoperiod. Consequently. 2004. cerevisiae since it is widely used in the production of foods and drinks. 2007). and volatile (Bruce et al. Brazil. This may occur by means of several mechanisms such as parasitism. The volume was completed to 1 l and added glucose and agar. Afterwards. since it is neither a human nor a plant pathogen. the agar plugs containing mycelium that had their growth inhibited were transferred to new plates containing PDA in 123 . 20 g l-1 glucose. production of hydrolytic enzymes (Punja and Utkhede 2003). 200 g of pricked potatoes [cultivar Monalisa] were boiled in 0. and it is classiﬁed as Biosafety level 1 (CDC/OHS 2009). Strobel 2006) as well as non-volatile antibiotic compounds (Wei et al. and maintained in the yeast collection of the Biochemistry and Alcohol Fermentation Laboratory in the Biological Sciences Department at Esalq/ USP. citricarpa. against G. Vinale et al. Lupe et al. 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). and is signiﬁcantly suitable for in situ isolation of biogenic compounds (Augusto and Sartoratto 2003. and 15 g l-1 agar) at 32°C. The control consisted of plates containing the pathogen in the absence of yeast.5 l of distillated water for 15 min and ﬁltered through cheesecloth. who are more familiar with S. cerevisiae strains were spread using 50 ll of suspension (108 cells ml-1) on one side of the plate containing PDA. 2008). both farmers and researchers started to consider the use of alternative methods to control diseases (Punja and Utkhede 2003). such as fruits intended to be consumed fresh (Droby et al. There are no reports in the literature concerning the inhibition of plant pathogens by volatile substances produced by yeasts. Brazil. sensitivity and speed. In view of the damages caused by the citrus black spot and a high interest for alternative control methods. KD-1. CAT-1. antibiotics. The knowledge about how this mechanisms works is essential to improve the biocontrol effectiveness as well as to develop innovative control strategies. Due to its simplicity. To prepare the potato infusion. cerevisiae. or other molecules whose presence is unacceptable in human foods. a 5 mm diameter agar plug with mycelium of G. Antagonistic yeast Saccharomyces cerevisiae strains (BG-1. The plates were wrapped with paraﬁlm and maintained at 26°C under a 12 h photoperiod. citricarpa was placed onto the opposite side of the plate. In addition. used in fermentative processes for fuel ethanol production. this study aimed to evaluating the in vitro antagonistic capacity of S. The yeast strains were maintained in Yeast Extract Peptone Dextrose Agar (YEPDA medium: 10 g l-1 yeast extract. The antagonistic S. Nevertheless. competition for nutrients and colonization niches. The use of antagonistic microorganisms may interrupt some stage of the disease or the pathogen’s life cycle. NJ— USA) were used. 2006. 10 g l-1 peptone. 2004.
a SPME ﬁber was inserted into the tube through the silicone septa. 1. Wilmington. PA—USA) coated either with 50/30 lm divinylbenzene/polydimethylsiloxane/ carboxen (DVB/PDMS/CAR) or 85 lm polyacrylate (PA) were used. 40 ll of the artiﬁcial mixture were added on a piece Table 1 Identiﬁcation of VOCs produced by S.736 611 181 362 Molecular formula C2H6O C4H8O2 C4H8O2 C5H12O C5H12O C8H10O C10H20O2 MW 44 88 88 88 88 122 172 123 . The injector temperature was kept at 250°C and the detector at 280°C. cerevisiae headspace analyses. and grade 5. maltose or galactose. and the sample headspace. The plates were wrapped with paraﬁlm and maintained at 26°C under a 12 h photoperiod. cerevisiae CR-1 strain was inoculated by spreading 100 ll of cell suspension (108 ml-1) on PDA.478 376 450 1. USA). USA). potato agar supplemented with different glucose concentrations (0–4%) was tested. an artiﬁcial mixture was made by using authentic standard chemicals (Sigma/Aldrich Chemical Co. sucrose. A 5 mm diameter agar plug with G. then increased 3°C/min up to 100°C and then increased 20°C/min to 250°C. cerevisiae strain CR-1 grown on PDA using HS–SPME–GC–MS Peak 1 2 3 4 5 6 7 Analyte Ethanol Unidentiﬁed Ethyl acetate 3-methyl-1-butanol 2-methyl-1-butanol Phenylethyl alcohol Ethyl octanoate Retention time (min) 2.World J Microbiol Biotechnol (2010) 26:925–932 927 the absence of yeast. The injector was operated in splitless mode for all chromatographic runs. the yeast was grown on the various media by streaking 50 ll of the cell suspension (108 cells ml-1).. In addition. Immediately after that. citricarpa mycelium was placed on one side of the divided plate containing PDA. equipped with a HP-5973 massselective detector. Afterwards. 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). Identiﬁcation of the detected species was performed by comparing them against the mass spectra library and conﬁrmed by spiking the samples with pure standards (Sigma/Aldrich Chemical Co. A HP-5890 gas chromatograph (Hewlett-Packard.25 mm 9 0. The oven temperature was programmed as follows: 40°C for 5 min. exposed to the headspace for 15 min and them taken to the GC–MS for analyte desorption (desorption time = 3 min). On one side of the plate. on the opposite side of the plate.25 mm 9 0.7 software.15 5. the culture medium. Assay of artiﬁcial mixture of VOCs identiﬁed in S. The MS scan range was set between 35 and 350. separation and detection.10 2. a 5 mm diameter agar plug of the plant pathogen G. citricarpa was placed at the opposite side onto PDA. As in the previous experiment. Analyses of S. USA). and the data subtracted from the analyses carried out on tubes containing the yeast..0 helium was used as carrier gas. Wilmington. all operational conditions were the same as in the GC–MS runs. The proportion of each compound positively identiﬁed was calculated from the relative peak areas (Table 1). Peak identiﬁcation was performed using the automated mass spectral deconvolution and identiﬁcation system (AMDIS) v. and incubated at 26°C for 6 days.25 lm) and a split–splitless injector. USA).. the sealed tubes were kept for 5 min at 30°C to achieve equilibrium among the yeast. DE. Bellefonte.91 3.15 28.63 software and the NIST mass spectral search v. Effect of carbon sources on the production of VOCs Four different carbon sources were tested to grow S.23 27. The polyethylene tubes containing only PDA were employed as blanks. For the extraction of the volatiles. equipped with a ﬂame ionization detector (FID) and a HP-5 fusedsilica capillary column (30 m 9 0. The control consisted of plates containing the pathogen in the absence of yeast. After the culture growth period. USA).05 5. SPME ﬁbers (Supelco Inc. Four replicates were used for each growth medium evaluated. After 5 days of growth. DE. Except for FID temperature (300°C). both programs supplied by NIST (Washington—DC. Preliminary runs were also performed in an HP-6850 gas chromatograph (Agilent. cerevisiae VOCs The S.25 lm) and a split–splitless injector were employed. cerevisiae strain CR-1. polystyrene plates divided in half by a wall were used. a HP-5MS fused-silica capillary column (30 m 9 0. Mycelial growth was evaluated when the colony in control plates reached the borders. Four replicates were used per yeast strain evaluated. 2. inside 50 ml septum-sealed polyethylene centrifuge tubes.30 Area (units) 21. cerevisiae After the S.
Tukey’s test 123 . 1 Mycelial growth inhibition of G.5 1. Tukey’s test Glucose (%) Fig.0 0. cerevisiae strain Fig. The mycelial growth was evaluated daily until 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. observed. The control consisted of plates containing the pathogen in the absence of the artiﬁcial mixture. The plates were immediately wrapped with paraﬁlm and maintained at 26°C under a 12 h photoperiod. citricarpa exposed to VOCs from different S. 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.928 World J Microbiol Biotechnol (2010) 26:925–932 Inibition (%) of sterile cotton.01.0 3. The growth on potato agar supplemented with glucose. The yeast S. 2).6. cerevisiae strains grown on PDA. citricarpa exposed to VOCs from S.01. S. sucrose and maltose provided the highest inhibition levels (85. containing mycelium that showed growth inhibition. cerevisiae strain CR-1 was also evaluated (Fig.0 2. No apparent morphological modiﬁcations were detected by light microscopy analysis (Fialho 2004). In another experiment. however. 2 Mycelial growth inhibition of G. 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. respectively). 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. the agar plugs with G. The control consisted of plates containing the pathogen in the absence of the artiﬁcial mixture. resumed normal development when they were transferred to plates containing PDA in the absence of yeast. 85. in one side of the divided plate containing PDA. cerevisiae (strain CR-1) grown on different carbon sources. In order to determine the fungistatic or fungitoxic nature of the VOCs. citricarpa mycelium that had their growth inhibited were transferred to new plates containing PDA in the absence of VOCs. cerevisiae (strain CR-1) grown on different concentrations of glucose. Tukey’s test 100 Inhibition (%) 80 60 40 20 0 CR-1 ** ** ** ** ** 80 ** ** ** Inhibition (%) 60 40 20 0 ** 0. Afterwards.9%. 3 Mycelial growth inhibition of G. 1). Values are means of four replicates (±SD) and ** indicates values that differ signiﬁcantly from the control at P B 0. citricarpa growth through the production of volatiles only when there was at least 0. on the opposite side different amounts of the artiﬁcial mixture from 10 to 120 ll were added on a piece of sterile cotton. Five replicates were used for each treatment. citricarpa exposed to VOCs from S. On the other hand.5% of available glucose 100 ** 80 60 40 20 ** ** Results VOC production by S. cerevisiae strain CR-1 inhibited the G. The agar plugs. cerevisiae was not able to produce antimicrobial volatiles when grown on medium supplemented with galactose. a 5 mm diameter agar plug of the pathogen was placed.0 4. cerevisiae It was observed the production of VOCs by the S.01. indicating the fungistatic nature of the inhibition. Five replicates were used for each growth medium evaluated. citricarpa vegetative growth (Fig. Values are means of four replicates (±SD) and ** indicates values that differ signiﬁcantly from the control at P B 0. cerevisiae strains able to inhibit the in vitro G. The carbon source inﬂuence on the production of antimicrobial VOCs by S.0 ** ** PE-2 K-1 CAT-1 BG-1 KD-1 -20 S. Mycelial growth was evaluated when the colony in the control plates reached the borders. The plates were immediately wrapped with paraﬁlm and maintained at 26°C under a 12 h photoperiod. statistical differences among the tested strains were not 100 ** 0 Glucose Sucrose Maltose Galactose Carbon source Fig.9. and 81.2% was reached with the strain CR-1. Values are means of four replicates (±SD) and ** indicates values that differ signiﬁcantly from the control at P B 0. A maximum mean inhibition of 87.
(3) ethyl acetate. cerevisiae. 5 Inﬂuence of the artiﬁcial mixture of VOCs (0. 6 Mycelial growth inhibition of G. (2) unidentiﬁed ester. grown on medium not supplemented with glucose.8 ll ml-1 air space) identiﬁed in S. In preliminary experiments.392x2 + 129. 4 Chromatogram corresponding to VOCs produced by S. (6) phenylethyl alcohol. cerevisiae strain CR-1 grown on PDA.0 2.67. cerevisiae atmosphere on G.5 Concentration (µL mL-1) Fig. mimicking the inhibitory effects of the S. (7) ethyl octanoate 123 . cerevisiae CR-1 on PDA were analyzed by SPME–GC–MS (Fig.0 0. the present study demonstrated that the production of VOCs plays an essential role in the antagonistic activity of S. Besides. By exposing the fungus to different concentrations of the artiﬁcial mixture of VOCs.5 2. 3). the remainder peaks are from the control sample or the atmosphere. 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. Tukey’s test 100 Inhibition (%) 80 60 40 20 0 0. The inhibitory activity increased proportionally to the increase of the glucose concentration. (5) 2-methyl-1-butanol.84 ll ml-1) values. caused slight growth stimulation of the phytopathogen. with ethanol as the major component of the headspace besides 3-methyl-1-butanol. (1) ethanol. An artiﬁcial mixture containing each of the positively identiﬁed compounds was prepared and tested (Fig.0 1. cerevisiae strains may reﬂect their capacity of quantitatively and qualitatively produce secondary metabolites. In addition. the artiﬁcial mixture as the natural VOCs had not lethal effect at any concentration on the phytopathogen.901x3 .7763 R2 = 0. Inhibition values were calculated as percentage growth inhibition as compared to the control in the absence of the artiﬁcial mixture. diffusible substances produced by S. 5). did not show any antifungal activity against G.World J Microbiol Biotechnol (2010) 26:925–932 929 Mycelial growth (cm) (Fig.01. citricarpa.5 y = 11. citricarpa (Fialho 2004). cerevisiae on the mycelial growth of G.48 ll ml-1) and MIC100 (2. 6). VOC analyses and biological activity of the artiﬁcial mixture The VOCs produced by S. On the other hand. and the maximum inhibition was seen when the yeast was grown on 4% glucose. The insert shows the ﬁrst 5 min of the same chromatogram in a different signal scale. cerevisiae.89x + 1. Discussion The inhibitory capacity of the S. 1995). citricarpa exposed to different concentrations of the artiﬁcial mixture of VOCs identiﬁed in S. citricarpa. Fig. Labeled peaks are those identiﬁed only in the headspace of yeast culture samples by HS– SPME–GC–MS. S. (4) 3-methyl-1-butanol. ethyl octanoate and one whose identity was not conﬁrmed. The presence of esters was also observed. There was the production of compounds belonging mainly to the alcohol group. 2-methyl-1-butanol. and present in culture ﬁltrates. citricarpa. it was possible to calculate the MIC50 (0. The arrow indicates the addition of the artiﬁcial mixture. and phenylethyl alcohol.995 1. cerevisiae on G. Values are means of ﬁve replicates (±SD) mycelial growth relative to the control (Fig. Values are means of four replicates (±SD) and ** indicates values that differ signiﬁcantly from the control at P B 0. On the other hand. ethyl acetate. The mycelial growth of the pathogen stopped when the artiﬁcial mixture was added to the plates in the ﬁfth day. cerevisiae. 4) and the identiﬁcation of analytes is shown in Table 1. which correspond to the minimum concentration of the artiﬁcial 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 ** ** 6 7 8 Day Control Artificial mixture Fig.
1996). the growth condition in the present work. and this observation reinforces the fact that the fermentable carbon source is a limiting factor for bioactive volatile production by S.2–2. 2-methyl-1-butanol. and these included: ethyl acetate. yeasts developing in medium that contains a non-fermentable carbon source may produce just a few volatiles. (2003) identiﬁed the VOCs produced by 98 ascomycetous yeasts strains (no-Saccharomyces spp. (2003) observed that volatile compounds produced by S. the yeast produced eight compounds where ethanol and ethyl acetate were also released as seen by S. 3-methyl-1-butanol. In order to understand the nature of the inhibitory effects of the volatile substances produced by S. The antimicrobial properties of alcohols are long known and are used in the formulation of disinfectants and preservatives. It is likely that volatiles act by changing protein expression (Humphris et al. aldehydes and esters. Very little is known about the mode of action of microbial volatiles on the control of fungi. It was also veriﬁed the biological activity of the artiﬁcial mixture containing each of the positively identiﬁed components of S. 2001. the strain used in the present work can not ferment galactose. Thus. S. Bruce et al. Thus. cerevisiae on G. (2004) identiﬁed only two compounds produced by the yeast in liquid tryptone soybean bean medium. As a consequence. cerevisiae. however. 2002) and the activity of speciﬁc enzymes (Wheatley 2002). Nout and Bartelt (1998) stated that the production of volatiles by yeasts is strongly inﬂuenced by their capacity of assimilating and fermenting carbohydrates. Bruce et al. G. Subsequently. inhibiting the development of other microorganisms (Pfeiffer et al. which may reﬂect on growth. 1994). The VOCs produced were found to be alcohols.48 and 2. the permeability increases accelerating the passive diffusion of essential ions and metabolites through the membrane (Ingram and Buttke 1984. and esters (isobutyl acetate. The higher the availability of carbon sources in the medium. The alcohol accumulation in the presence of oxygen was probably an evolutionary strategy used by some yeasts to increase the competitiveness. This suggests that the lack of capacity of fermenting galactose present in the medium were reﬂected by the quality and low production of inhibitory volatiles. the volatile production in media containing fermentable carbon sources is higher.84 ll ml-1. but it can also later consume the ˇ generated alcohol (Piskur et al. isoamyl propionate and phenylmethyl acetate). The same authors also concluded that the simple addition of sucrose to the nutrient-poor medium increased volatile activity by approximately 50%. Besides. cerevisiae. cerevisiae is able to assimilate and to ferment the sugars glucose. this information is in agreement to the results obtained in the present work. 2-pentanone and phenylethyl alcohol.930 World J Microbiol Biotechnol (2010) 26:925–932 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. In the present work. no antimicrobial volatiles were produced by the yeast in the medium containing galactose. cerevisiae strain Y1001 on tryptone soya agar were able of inhibit (between 76 and 33%) the growth of fungi related to sapstain in wood. cerevisiae strain CR-1 on PDA in the present work. The endophytic fungus Muscodor albus produced maximum lethality against plant pathogens when grown in nutrient-rich media (Ezra and Strobel 2003). sucrose. the alcoholic fermentation overcomes the respiration when in the presence of high glucose concentrations even when under aerobiosis. The primary site of action seems to be the plasma membrane. 123 . Maclean and Gudelj 2006). However. The inhibition of G. 2006). representative of 40 species belonging to 12 genera) isolated from several tropical habitats. cerevisiae atmosphere. According to Nout and Bartelt (1998). Saccharomyces not only can inhibit the competitor through the alcohol production. the major component of the headspace. maltose and galactose. The inhibitory action of alcohols is complex. isoamyl acetate. phenylethyl alcohol.4 ll ml-1 of air) and the MIC50 and MIC100 values were estimated in 0. such as acetaldehyde and acetic acid. where the accumulation of solvents could affect the organization and stability of the lipid bilayer. In malt extract medium. the identiﬁcation of seven out of eight detected compounds was carried out. 2-methylbutyl acetate. Buzzini et al. On the other hand. since the growth of the yeast on fermentable carbon sources led to the production of high quantities of alcohols. In S. yeasts. but in general it seems to be more related to their physicochemical properties than to speciﬁc receptors. ethyl octanoate and ethanol. In malt extract and minimum media the inhibition was very low or null. it was necessary to identify the components of the atmosphere produced by the yeast. The main VOCs produced by the isolates were alcohols (3-methyl-1butanol and 2-methyl-1-butanol). citricarpa development. the more complex the composition of the headspace produced. citricarpa was exposed to different concentrations of the components (0. respectively. 2004). citricarpa was related to the concentration of glucose in the medium. which comprised the majority of the headspace. Seward et al. Lipophilic compounds such as 3-methyl-1-butanol and 2-methyl-1-butanol have high afﬁnity 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. particularly for alcohols (especially ethanol).
heptanal. Rhizoctonia solani. in the control of plant pathogens had not been previously reported. Braz J Microbiol 39: 286–295 Droby S. a Brazilian foundation associated to the Ministry of Science and Technology (MCT). Thus. who provided the S. Bruce A. causal agent of citrus black spot.D. Baldassari RB. Sposito MB. gov/od/ohs/biosfty/biosfty. Reimann S. and octanal. released by Trichoderma pseudokoningii and T. a new symptom of citrus black spot (Guignardia citricarpa) in Brazil. De Bont JAM (1994) Mechanisms of resistance of whole cells to toxic organic solvents. Available from http://www. University of Sao Paulo Gullino ML. Finally. The MIC50 varied between 0. Pagnoni UM. The volatiles 2-propanone. Cappelli F. albus. Hackett CA. Buttke TM (1984) Effects of alcohols on microorganisms.cdc. Kuijpers LAM (1994) Social and political implications of managing plant diseases with restricted fungicides in Europe. Weber FJ. viride were responsible for the antimicrobial activity against wood decay fungi (Wheatley et al. Verrall S. ˜ Dissertation. the biological fumigation with S. albus on the plant pathogens Pythium ultimum. Pascholati SF (2008) Mechanisms and signiﬁcance of fungicide resistance. Adaskaveg JE. Annu Rev Phytopathol 32:559–579 Heipieper HJ. Sartoratto A (2003) Application of headspace solid phase microextraction and gas chromatography to the screening of volatile compounds from some brazilian aromatic plants. Castillo U. on the growth of wood decay basidiomycetes. Douglas S. this study shows the S. the production of antimicrobial VOCs by yeasts. Thus.3s. Acknowledgments The authors thank Prof. it was demonstrated that those three compounds were required to maintain the inhibitory activity. In addition. Wheatley RE (2002) The effects of volatile secondary metabolites on protein synthesis in Serpula lacrymans.). as action mechanism. cerevisiae strains. and 3-methyl-1-butanol were associated to the biological activity of M. propanoic acid. Holzforschung 58:193–198 Buzzini P. Trends Biotechnol 12:409–415 Humphris SN. Later.htm. This research was supported by CNPq (Conselho Nacional de Desenvolvimento Cientı´ﬁco e References Agostini JP. Humphris et al. Hess WM.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. (Abstr. Feichtenberger E. Biological Sciences Department (‘‘Luiz de Queiroz’’ Agri˜ culture School. Strobel GA (2003) Effect of substrate on the bioactivity of volatile antimicrobials produced by Muscodor albus. indicating a synergy among the different components (Ezra et al. cerevisiae volatiles or artiﬁcial mixtures could be used to treat fruits in closed chambers during storage and transport. Wheatley RE (2003) Effect of volatiles from bacteria and yeast on the growth and pigmentation of sapstain fungi. University of Sao Paulo. Wheatley RE (2004) Identiﬁcation of volatile organic compounds (VOCs) from bacteria and yeast causing growth inhibition of sapstain fungi. Atmosukarto et al. FEMS Microbiol Lett 210:215–219 Ingram LO. By using the artiﬁcial mixtures. These values represent the response of the fungus to the artiﬁcial mixture of VOCs. Buultjens TEJ. Hess WM. Proc Int Soc Citric 1:145 Deising HB. since it ´ minimizes product handling (Mercier and Jimenez 2004). since the yeast does not have to be in direct contact with the fruits and the fumigation is compatible to packing house systems. (2005) studied the susceptibility of human and plant fungi pathogens belonging to several taxonomic groups to artiﬁcial mixture of M. Accessed 12 Jul 2009 de Goes A. Strobel GA (2004) New endophytic isolates of Muscodor albus. Bruce A (2001) The effects of speciﬁc volatile organic compounds produced by Trichoderma spp. (2001) conﬁrmed that several basidiomycetes were completely inhibited by heptanal and octanal at low concentrations (2. Plant Sci 165:1229–1238 Ezra D. decanal. Wilson CL (1991) Antagonistic microorganisms as biological control agents of postharvest diseases of fruits and vegetables. Ph.. Plant Dis 90:1419–1424 Atmosukarto I. The compounds naphthalene. and Sclerotinia sclerotiorum. 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. 2004). Timmer LW (2006) Effect of fungicides and storage conditions on postharvest development of citrus black spot and survival of Guignardia citricarpa in fruit tissues. Davoli P (2003) A study on volatile organic compounds (VOCs) produced by tropical ascomycetous yeasts. Martini A. Mackenzie SJ. Sears J.World J Microbiol Biotechnol (2010) 26:925–932 931 ´gico—National Council for Scientiﬁc and Technological Tecnolo Development). Piracicaba. Plant Sci 169:854–861 Augusto F. 1997). Peres NA. Antonie van Leeuwenhoek 84:301–311 CDC/OHS (2009) US ofﬁce of health and safety and centers for disease control and prevention. Brazil). 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. 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.01 (Verticilium dahliae) and 0.3 ll ml-1 (Colletotrichum gloeosporioides and Aspergillus fumigatus). a volatile antibiotic producing fungus. Holzforschung 55:233–237 Humphris SN. cerevisiae potential for the biocontrol of citrus black spot. 2-methyl-1-butanol. Chalutz E. Strobel G (2005) Isolation and characterization of Muscodor albus I-41. Sikkema J. Luiz Carlos Basso. Postharvest News Inf 2:169–173 Ezra D. Vildosa CIA (2000) Cracked spot. Keweloh H. The artiﬁcial mixture at the concentration of 1. a volatile-antibiotic-producing fungus. SP. Adv Microb Physiol 25:253–300 123 . Wheatley RE. Chromatographia 57:351–356 Bruce A. gloeosporioides and Fusarium culmorum. this process would be an environmentally safer alternative to control diseases at postharvest. Susan V.5 lg ml-1). Microbiology 150:4023–4031 Fialho MB (2004) In vitro effect of Saccharomyces cerevisiae on Guignardia citricarpa. Int Biodeterior Biodegrad 51:101–108 Bruce A.
Straney DC (2005) Multiple non-ribosomal peptide synthetase genes determine peptaibol synthesis in Trichoderma virens. hexan-1-ol. Accessed 21 Jul 2009 Oliveira AM. Int Biodeterior Biodegrad 39:199–205 123 . Willetts JM. J Inst Brew 102:439–443 Strobel G (2006) Muscodor albus and its biological promise. Scala F. Schuster S. Utkhede RS (2003) Using fungi and yeasts to manage vegetable crop diseases. J Chromatogr A 1025: 115–124 Pfeiffer T. viability. synergistic inhibition. Postharvest Biol Technol 31:1–8 Nout MJR. and energy status. Kundzewicz A (1997) Effect of substrate composition on the production of volatile organic compounds from Trichoderma spp. Nature 441:498–501 ´ Mercier J. Marra R. Augusto F (2004) Studies on the aroma of cupuassu liquor using headspace solid phase microextraction and gas chromatography. Paul. inhibitory to wood decay fungi. Merico A. Pereira NR. In: Garnsey SM. ex Spreng and analysis by HS-SPME. Hodgson VJ (1995) Interactions between killer yeasts and pathogenic fungi. Lemes AC. Dinsdale MG. Rozpedowska E. Compagno C (2006) How did Saccharomyces evolve to become a good brewer? Trends Genet 22:183–186 Punja ZK. Sivasithamparam K (2006) Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens. Available from: http://archives.htm.org/EPPOStandards/general. Lloyd D (1996) The effects of ethanol. and 2-phenylethanol on cider yeast growth. Lett Appl Microbiol 43:143–148 Walker GM. Yang F. Bruce A. Marsaioli A Jr. Antonie van Leeuwenhoek 81:357–364 Wheatley RE. Bonhoeffer S (2001) Cooperation and competition in the evolution of ATP-producing pathways. Hackett C. Graham JH. Science 292:504–507 World J Microbiol Biotechnol (2010) 26:925–932 ˇ Piskur J. Polakova S. Trends Biotechnol 21:400–407 Seward R. Ghisalberti EL. J Chem Ecol 24:1217– 1239 OEPP/EPPO (2003) Diagnostic protocols for regulated pests: Guignardia citricarpa. St. J Ind Microbiol Biotechnol 33:514–522 Vinale F. OEPP/EPPO Bull 33:271–280 OEPP/EPPO (2008) EPPO standard PM 1/2 A1 and A2 lists of pest recommended for regulation as quarantine pests.932 ´ Kotze JM (2000) Black spot. Augusto F. Mcleod AH. Jimenez JI (2004) Control of fungal decay of apples and peaches by the biofumigant fungus Muscodor albus. Lorito M. Gudelj I (2006) Resource competition and social conﬂict in experimental population of yeast. Timmer LW (eds) Compendium of citrus diseases. FEMS Microbiol Lett 127:213–222 Wei X. Bartelt RJ (1998) Attraction of a ﬂying nitidulid (Carpophilus humeralis) to volatiles produced by yeasts grown on sweet corn and a corn-based medium. pp 23–25 Lupe FA.eppo. Can J Microbiol 51:423–429 Wheatley RE (2002) The consequences of volatile organic compound mediated bacterial and fungal interactions. J Essent Oil Res 19:271–273 Maclean CR. Barata LE (2007) Fragrant lactones in the steam distillation residue of Aeollanthus suaveolens Mart. American Phytopathological Society Press.
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