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Influence of ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala
FEMS Microbiology Letters 238 (2004) 133–137 www.fems-microbiology.org
Influence of ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala ¨ del Druvefors, Matilda Nilsson Olstorpe, Elisabeth Fredlund *, Ulrika A Volkmar Passoth, Johan Schnu¨rer Department of Microbiology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7025, SE-750 07 Uppsala, Sweden Received 25 April 2004; received in revised form 17 June 2004; accepted 14 July 2004 First published online 28 July 2004
Abstract A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (aw). At aw 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10–14 lg ml 1 headspace. At aw 0.95, both growth and biocontrol performance of the haploid strain were reduced. Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 lg ml 1. We conclude that ethyl acetate is a major component of the anti-mould activity. The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P. roqueforti. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Pichia anomala; Mode of action; Ethyl acetate; Penicillium roqueforti; Biocontrol; Water-activity
1. Introduction The ability of antagonistic yeasts to control mould infections in stored fruits and cereal grain has been extensively studied during the last decades [1,2]. Several yeast species have now been developed into commercially available biocontrol products (Candida oleophila – Aspire; Cryptococcus albidus – Yield plus; Metschnikowia fructicola – Shemer). Despite extensive research on the biocontrol efficacy of many yeasts, the mode of action by which they inhibit mould growth is less well known. The mechanisms explaining growth inhibition appear to be dependent on the yeast species, the characters of the
*
Corresponding author. Tel.: +46 18 673 212; fax: +46 18 673 392. E-mail address: [email protected] (E. Fredlund).
target mould, and the physical parameters of the biocontrol system. Grain offers a nutrient rich and favourable growth environment for filamentous fungi (mould) as they can profit both from the nutrients secreted on the grain surface, or invade the endosperm of broken kernels and penetrate undamaged grain [3]. Mould growth in grain is influenced by the availability of water (aw), gas composition (O2 and CO2), and by the temperature of the storage environment. Stable grain storage can be obtained by drying the grain to a water content of less than 13–14% [4] or through storage in an airtight container. In an airtight silo, even high moisture cereal grain (>20% water) can be stored due to the rapid production of CO2 (60–95%) and consumption of O2 through grain and microbial respiration [5]. However, the silo constitutes a non-static environment, where feed-out, seasonal
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.07.027
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E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137
temperature variations, and air-leakage can change the growth conditions and eventually enable growth of spoilage moulds. The mould Penicillium roqueforti is a common spoilage organism in mal-functioning airtight stored grain [6]. It is very tolerant to low oxygen availability and is able to grow at 0.14% oxygen tension. Thus, stable airtight storage depends on the rapid CO2 evolvement to levels that reduce the growth of P. roqueforti (>15%). The sensitivity of P. roqueforti to CO2 increases with decreasing aw and temperature [7]. During storage of moist grain at high CO2 and low O2 concentration, Pichia (Hansenula) anomala is the dominating yeast [6]. It has been demonstrated that the addition of P. anomala to cereal grain stored under airtight conditions inhibited the growth of filamentous fungi and made the system more robust and less sensitive to air-leakage [8,9]. During yeast growth, oxygen and available nutrients are consumed and CO2 is produced. However, this is not specific for P. anomala as all aerobic microorganisms consume nutrients and oxygen, as well as produce CO2. Therefore, this cannot solely explain the biocontrol performance of this yeast in comparison to other yeast species [10]. Recently, we found that a product of glucose metabolism, most probably ethyl acetate, is an essential factor in mould inhibition by P. anomala [20]. Additional factors might include the ability to grow rapidly in the silo and to tolerate different levels of water availability. The multiple mechanisms of biocontrol activity might be resolved by genetic analysis. However, the genetics of P. anomala is poorly developed and mutational analysis is hampered by the fact that natural isolates of this yeast are diploid [2,11]. The biocontrol performance of haploid strains of P. anomala has not been previously tested. The aim of this study was to investigate the effect of aw and ethyl acetate production on the biocontrol activity of P. anomala against P. roqueforti. To enable future mutational analysis of the biocontrol process and to test the influence of the ploidy level on biocontrol activity, we included both a diploid and a haploid strain in the study.
2. Material and methods 2.1. Yeast and fungal strains The diploid type strain of P. anomala, CBS 5759 (NRRL-Y-366), and the haploid (+) mating type, CBS 1984 (+; NRRL-Y-366-8) were used in this study (BioloMICS Database, http://www.cbs.knaw.nl/databases/index.htm). Pichia anomala CBS5759 inhibits P. roqueforti in stored grain as efficiently as the described biocontrol ¨ . Druvefors, unpublished strain P. anomala J121 (U. A results). The target mould, P. roqueforti J5 originated from stored cereal grain [10].
2.2. Assay of yeast growth and antifungal activity in minisilo grain systems Test tubes with grain were prepared as described by Petersson et al. [12] with some modifications. Nonsterile wheat was hydrated with water to aw 0.95 (21% H2O) or 0.98 (29% H2O) and stored at 2 C for 72 h to allow the water content to equilibrate. The yeast inocula were pre-grown in YPD broth (yeast extract 10 g l 1, bacterial peptone 20 g l 1, glucose 20 g l 1) [13] over night. Spores of P. roqueforti were harvested from malt extract agar plates (50 g l 1) and kept in glycerol stock solutions (18 C) until inoculation. All substrates were bought at Oxoid Ltd., Basingstoke, Hampshire, England. The wheat was inoculated by mixing 103 spores of P. roqueforti and 105 cells of P. anomala (strain CBS 1984 or CBS 5759) per g grain. A control, inoculated with mould only (103 spores g 1), was also included. The inoculated grain was poured into test tubes (1.5 cm · 15 cm, 18 g) and sealed with rubber membranes perforated with a needle to simulate air-leakage of a full-scale silo. The tubes were incubated at 25 C, and growth (CFU g 1) of yeast and mould was measured daily during 1 week as previously described [12]. MEAC (50 g l 1malt extract agar with 100 ppm chloramphenicol) was used for the determination of yeast growth and MEACC (50 g l 1 malt extract agar with 100 ppm chloramphenicol and 10 ppm cycloheximide) for the determination of mould growth. Each day during a 7-day cultivation period, three independent tubes for each combination of fungal strains and growth condition were opened and analysed for fungal growth. Results are given as mean values ± standard deviation (SD). Significant differences were evaluated using the StudentÕs ttest. 2.3. Production of ethyl acetate by P. anomala in grain minisilos The volatile ester ethyl acetate can be trapped in the hydrocarbon decane [14]. At each sampling point (see results), 5 ml of 99% decane (Sigma Chemical Co., St. Louis, USA) were added to three independent grainfilled test tubes and shaken for 5 min. Subsequently, a 1-ml extract was filtered through a 0.45 lm Acrodisc syringe filter (Gelman Laboratory, Ann Arbor, USA) and 1 ll was injected in a Hewlett–Packard gas chromatograph with a flame ionisation detector (250 C, Hewlett–Packard Ltd., Cheshire, England) and a capillary column (HP19091S-833, 250 lm · 30.0 m). The carrier gas was H2 at a flow rate of 40.0 ml min 1. The column temperature was programmed from 60 to 250 C at a rise rate of 20 C min 1 and finally held during 2 min at 250 C. Ethyl acetate was identified and quantified by comparison with an external standard. The retention time of ethyl acetate was 1.80 min. The concentration
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137
2.4. Assay of anti-mould activity of ethyl acetate To investigate whether the volatile ester ethyl acetate had an inhibitory effect on mould growth, P. roqueforti was grown in a semi-closed environment with vaporised ethyl acetate. A glass cup attached to a microscope slide was placed on the bottom of one plastic petri dish and 0.1–0.4 ml ethyl acetate was added to the glass cup (Merck Eurolab AB, Stockholm, Sweden). Water was used as the negative control. Spores of P. roqueforti were inoculated in the center of a second petri dish with 30 ml malt extract agar (50 g l 1; Oxoid). The agar plate was placed upside down so that the ethyl acetate containing glass slide was facing the agar surface. The plates were wrapped with at least three layers of parafilm to create a semi-closed environment. Since parafilm is permeable for O2 and CO2, effects of strong oxygen limitation and CO2 inhibition on mould growth were avoided. Complete volatilisation of ethyl acetate occurred within 3 h. Because parafilm is also permeable for ethyl acetate, one cannot directly compare the ethyl acetate concentrations in the in vitro experiments with the levels in the test tubes. The concentrations in the in vitro tests were set according to preliminary experiments. After 5 days of incubation at 25 C the dry weight of the mould colony was measured. To estimate the mycelial dry weight, the mould colony was cut out from the agar and placed in 300 ml distilled water. The water was heated in a microwave oven until the residual agar was melted. The mould colony was washed in distilled water, placed on a pre-weighed filter, and dried at 80 C for 24 h. All treatments were done with three replicates. Results are given as mean values ± SD. Significant differences were evaluated using the StudentÕs t-test.
10 009 8 10 00
Yeast CFU g-1 grain
detected in the decane was recalculated to obtain the headspace concentration in the minisilo. The minisilo headspace volume was approximately 10 ml when filled with 18 g grain.
135
7 10 00
10 006 5 10 00
diploid (0.95)
haploid (0.95)
diploid (0.98)
haploid (0.98)
4
10 00
0
1
2
3 4 Time (Days)
5
6
7
Fig. 1. Growth of diploid (CBS 5759) and haploid (CBS 1984) strains of P. anomala (CFU g 1) inoculated in grain minisilos at aw 0.95 and 0.98 (n = 3, ±SD).
To test the influence of different growth behaviour on the biocontrol performance of P. anomala, we cocultivated yeast and mould in test tubes which were incubated at aw 0.95 and 0.98 for seven days. Growth (CFU g 1) of mould and yeast was analysed. At aw 0.98, the CFU g 1 of P. roqueforti did not increase during the 7-day incubation when co-cultivated with the diploid or haploid strain (Fig. 2), nor did it grow in the tubes inoculated with the diploid strain at aw 0.95. However, when co-cultivated with the haploid strain at aw 0.95, P. roqueforti grew from 102 CFU g 1 to 105 CFU g 1 within 6 days (Fig. 2). The haploid strain slightly inhibited mould growth at aw 0.95 (Fig. 2), but not to the same extent as at aw 0.98. Previous results have shown that the growth of P. anomala is not influenced by the presence or absence of the mould in the grain-filled tubes [10]. To investigate the possible correlation between ethyl acetate production of P. anomala and mould inhibition in the minisilos, we measured ethyl acetate formation
6 000 10
3. Results and discussion
5
000 10
Mould CFU g-1 grain
The diploid and haploid strain of P. anomala, CBS 5759 and CBS 1984, respectively, had similar growth behaviour in the grain-filled test tubes at aw 0.98, showing rapid growth during the first 2–3 days (Fig. 1). The same trend was found for the diploid strain grown at aw 0.95. In contrast, the haploid strain grown at aw 0.95 reached significantly lower (p < 0.05) levels of CFU g 1 during the first two days (Fig. 1). However, after 4 days there was no difference between the haploid strain grown at aw 0.95 (5.8 ± 2 · 106 cells g 1 at day 4) and at aw 0.98 (8.7 ± 1.3 · 106 cells g 1at day 4) (Fig. 1). The different growth curves of the two strains at aw 0.95 was mainly related to a prolongation of the lag phase of the haploid strain.
4
10000 10
3
1000 10
2 100 10 1 10 10
diploid (0.95) haploid (0.98)
0
1
2
haploid (0.95) mould (0.95)
3 4 Time (Days)
diploid (0.98) mould (0.98)
5
6
7
Fig. 2. Growth of P. roqueforti (CFU g 1) co-inoculated with diploid (CBS 5759) or haploid (CBS 1984) strains of P. anomala in grain minisilos at aw 0.95 and 0.98. Controls without yeast are also shown (n = 3, ±SD).
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137 18.0 18
diploid (0.95) haploid (0.95) diploid (0.98) haploid (0.98)
16.0 16 -1
Ethyl acetate ( µg ml )
Dry weight (%) compared to control
136
14.0 14
12 .0
10 10.0 8.08 6.06 4.04 2.02 0.00
0
1
2
3
4
5
6
7
Time (days)
Fig. 3. Ethyl acetate headspace concentrations in grain minisilos (aw 0.95 and 0.98) inoculated with P. roqueforti and diploid (CBS 5759) or haploid (CBS 1984) strains of P. anomala (n = 3, ±SD).
during one week in test tubes inoculated with the haploid or diploid strain at aw 0.95 and 0.98 (Fig. 3). Ethyl acetate was detected in all test tubes. However, from day 2, the level of ethyl acetate was significantly higher (p < 05) in the test tubes inoculated with the diploid strain (at both aw 0.95 and 0.98) and the haploid at aw 0.98 compared to the haploid strain grown at aw 0.95 (Fig. 3). After 2 days, the detected level from the haploid strain at aw 0.95 was only 10% of that at aw 0.98 and the ethyl acetate level at aw 0.95 never exceeded 40% (day 3) of that at aw 0.98. At aw 0.98, the haploid CBS 1984 continued to produce ethyl acetate during the 7-day incubation period, similar to the diploid CBS 5759 at aw 0.95 and 0.98 (Fig. 3). No ethyl acetate was detected in the mould-only controls (data not shown). The concentration of ethyl acetate in the grain headspace after 3 days was approximately 89 lg ml 1 in all treatments, except for the haploid CBS 1984 at aw 0.95 (3.1 lg ml 1). The maximum headspace concentration of ethyl acetate produced by the haploid strain at aw 0.95 was 3.7 ± 0.6 lg ml 1, while it was 15.6 ± 1.6 lg ml 1 at aw 0.98. This different behaviour suggests an involvement of ethyl acetate in the fungal inhibition. In the test tube system, at ethyl acetate concentrations over 8 lg ml 1, there was a complete inhibition of fungal growth, whereas at ethyl acetate concentrations below 4 lg ml 1, the target mould was able to grow to the same level of CFU g 1 as in the mould-only control. To further confirm the anti-fungal properties of ethyl acetate P. roqueforti was cultivated on agar plates in an atmosphere containing different concentrations of ethyl acetate (Fig. 4). Ethyl acetate concentrations reduced the biomass formation of the mould in a concentration dependent manner (Fig. 4). Because of the fluctuations of CO2, O2 and ethyl acetate in both the in vitro assay and in the grain test tubes, the correlation between fungal growth and ethyl acetate are not directly comparable. The antimould properties of ethyl acetate and the substantial
160 140 120 100 80 60 40 20 0 0
0.1
0.2 0.3 Ethyl acetate (ml)
0.4
0.5
Fig. 4. Inhibition of P. roqueforti grown on malt extract agar and incubated in a semi-closed system with vaporised ethyl acetate (0.1–0.5 ml). The results are given as mycelial dry weight relative to controls with water. The dry weight of the control was 42 ± 2 mg (= 100%; n = 3, ±SD).
amounts produced by the yeast during airtight storage strongly suggests that ethyl acetate is an important part of the biocontrol activity of P. anomala. Acetate esters have been reported to inhibit the conidial germination of filamentous fungi at headspace concentrations of >4 lg ml 1 [15,16]. Mercier and Jime´nez [17] recently showed that volatiles, including esters and alcohols, produced by the yeast Muscodor albus inhibits growth of the spoilage moulds Penicillium expansum and Botrytis cinerea. However, ethyl acetate was not detected from M. albus. Additional factors may also be involved in the mould inhibition. We previously showed that the O2 concentration in 0.2 m3-pilot scale grain silos decreased to 0–4% within the first few days and that the CO2 concentration increased to 90% within the first few weeks [9]. These levels are sufficient to inhibit growth of P. roqueforti [7]. In the present study, the yeast CFU g 1 was significantly lower for the haploid strain at aw 0.95 than in the other treatments during the first 2 days (Fig. 1). Since yeast growth is connected to CO2 formation and O2 consumption, it is possible that the reduced growth of the haploid strain caused a lower CO2 and higher O2 concentration than in the other treatments. This could, in addition to the lower concentration of ethyl acetate, have allowed growth of P. roqueforti in the silos inoculated with the haploid strain at aw 0.95. Nutrient competition has been reported to play a role in the mode of action of other yeast species, for example in Debaryomyces hansenii and Pichia guilliermondi inhibition of Penicillium digitatum when grown together [18,19]. However, this may not influence the anti-mould activity of P. anomala, as addition of various nutrients did not impair its biocontrol activity [20]. Thus, we conclude that ethyl acetate production, possibly together with the increase in CO2 production and O2 consumption, are major components of the anti-mould activity of P. anomala in grain.
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137
We analysed the performance of a haploid P. anomala strain in the cereal grain biocontrol system. No essential differences compared to the diploid type strain were observed regarding growth, biocontrol activity, and ethyl acetate production at aw 0.98 (Figs. 1–3). Thus, this strain provides a tool for genetic analysis of the biocontrol process. Unexpectedly, growth, ethyl acetate production, and anti-fungal activity were impaired at aw 0.95 (Figs. 1–3). This indicates that ploidy plays a role in the handling of environmental stress by P. anomala. A connection between stress tolerance and ploidy has been observed in xylose fermenting yeasts, which raised their ploidy level after cultivation in wood hydrolysate [21]. We have currently no explanation on how ploidy influences stress tolerance of yeasts. Our study demonstrates for the first time a connection between P. anomala biocontrol activity and ethyl acetate production. This will contribute to our general understanding of the mode of action of biocontrol yeasts.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements We are thankful to Inger Ohlsson for her technical support. This work has been financially supported by The Foundation for Strategic Environmental Research (MISTRA) and the EU funded project BIO POSTHARVEST (QoL-PL1999-1065).
[14]
[15]
[16]
References [17] [1] Janisiewicz, W.J. and Korsten, L. (2002) Biological control of postharvest diseases of fruits. Annu. Rev. Phytopathol. 40, 411– 441. [2] Passoth, V. and Schnu¨rer, J. (2003) Non-conventional yeasts in antifungal application In: Functional Genetics of Industrial Yeasts (de Winde, H., Ed.), pp. 297–330. Springer, Berlin, Heidelberg. [3] Chelkowski, J. and Cierniewska, A. (1983) Mycotoxin in cereal grain. Part X. Invasion of fungi mycelium into wheat and barley kernels. Die Nahrung. 27, 533–536. [4] Hoseney, C. (1998) Principles of Cereal, Science and Technology. American Association of Cereal Chemists Inc., St. Paul, Minnesota. [5] Hyde, M.B. and Burell, N.J. (1982) Controlled atmosphere storage In: Storage of Cereal Grains and Their Products (Christensen, C.M., Ed.), third ed, pp. 443–478. American Association of Cereal Chemists, Inc., St. Paul, Minnesota. [6] Lacey, J. and Magan, N. (1991) Fungi in cereal grains: their occurence and water and temperature relations. In: Cereal Grain – Mycotoxins, Fungi and Quality in Drying and Storage
[18]
[19]
[20]
[21]
137
(Chelkowski, J., Ed.), pp. 77–118. Elsevier Science Publishers B.V., Amsterdam, The Netherlands. Magan, N. and Lacey, J. (1984) Effects of gas composition and water activity on growth of field and storage fungi and their interactions. Trans. Brit. Mycol. Soc. 82, 305–314. Petersson, S. and Schnu¨rer, J. (1998) Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture wheat, rye, barley, and oats stored under airtight conditions. Can. J. Microbiol. 44, 471–476. Druvefors, U., Jonsson, N., Boysen, M.E. and Schnu¨rer, J. (2002) Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens. FEMS YEAST Res. 2, 389–394. Petersson, S. and Schnu¨rer, J. (1995) Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae. Appl. Environ. Microbiol. 61, 1027–1032. Naumov, G.I., Naumova, E.S. and Schnu¨rer, J. (2001) Genetic characterization of the non-conventional yeast Hansenula anomala. Res. Microbiol. 152, 551–562. Petersson, S., Jonsson, N. and Schnu¨rer, J. (1999) Pichia anomala as a biocontrol agent during storage of high-moisture feed grain under airtight conditions. Postharv. Biol. Technol. 15, 175–184. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A. and Struhl, K. (1999) In: Current Protocols in Molecular Biology. Wiley, New York. Oda, S., Inada, Y., Kobayashi, A., Kato, A., Matsudomi, N. and Ohta, H. (1996) Coupling of metabolism and bioconversion: microbial esterification of citronellol with acetyl coenzyme A produced via metabolism of glucose in an interface bioreactor. Appl. Environ. Microbiol. 62, 2216–2220. Filonow, A.B. (2003) Germination and adhesion of fungal conidia on polycarbonate membranes and on apple fruit exposed to mycoactive acetate esters. Can. J. Anim. Sci. 49, 130–138. Brown, W. (1922) Studies in the physiology of parasitism. IX. The effect on the germination of fungal spores of volatile substances arising from plant tissue. Ann. Botany 36, 285–300. Mercier, J. and Jime´nez, J.I. (2004) Control of fungal decay of apples and peaches by the biofumigant fungus Muscodor albus. Postharv. Biol. Technol. 31, 1–8. Droby, S., Chalutz, E., Wilson, C.L. and Wisniewski, M. (1989) Characterization of the biocontrol activity of Debaryomyces hansenii in the control of Penicillium digitatum on grapefruit. Can. J. Microbiol. 35, 794–800. Droby, S., Chalutz, E., Cohen, L., Weiss, B., Wilson, C. and Wisniewski, M. (1990). Nutrient competition as a mode of action of postharvest biocontrol agents. In: Biological Control of Postharvest Diseases of Fruits and Vegetables, Workshop Proceedings. Shepherdstown, West Virginia, 12 to 14 September: United States Department of Agriculture, Agricultural Research Service, ARS-92. ˚ . (2004). Yeast biocontrol of grain spoilage Druvefors, U.A moulds, Department of Microbiology. Swedish University of Agricultural Sciences: Uppsala. p. 44. Available from:
Influence of ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala ¨ del Druvefors, Matilda Nilsson Olstorpe, Elisabeth Fredlund *, Ulrika A Volkmar Passoth, Johan Schnu¨rer Department of Microbiology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7025, SE-750 07 Uppsala, Sweden Received 25 April 2004; received in revised form 17 June 2004; accepted 14 July 2004 First published online 28 July 2004
Abstract A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (aw). At aw 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10–14 lg ml 1 headspace. At aw 0.95, both growth and biocontrol performance of the haploid strain were reduced. Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 lg ml 1. We conclude that ethyl acetate is a major component of the anti-mould activity. The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P. roqueforti. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Pichia anomala; Mode of action; Ethyl acetate; Penicillium roqueforti; Biocontrol; Water-activity
1. Introduction The ability of antagonistic yeasts to control mould infections in stored fruits and cereal grain has been extensively studied during the last decades [1,2]. Several yeast species have now been developed into commercially available biocontrol products (Candida oleophila – Aspire; Cryptococcus albidus – Yield plus; Metschnikowia fructicola – Shemer). Despite extensive research on the biocontrol efficacy of many yeasts, the mode of action by which they inhibit mould growth is less well known. The mechanisms explaining growth inhibition appear to be dependent on the yeast species, the characters of the
*
Corresponding author. Tel.: +46 18 673 212; fax: +46 18 673 392. E-mail address: [email protected] (E. Fredlund).
target mould, and the physical parameters of the biocontrol system. Grain offers a nutrient rich and favourable growth environment for filamentous fungi (mould) as they can profit both from the nutrients secreted on the grain surface, or invade the endosperm of broken kernels and penetrate undamaged grain [3]. Mould growth in grain is influenced by the availability of water (aw), gas composition (O2 and CO2), and by the temperature of the storage environment. Stable grain storage can be obtained by drying the grain to a water content of less than 13–14% [4] or through storage in an airtight container. In an airtight silo, even high moisture cereal grain (>20% water) can be stored due to the rapid production of CO2 (60–95%) and consumption of O2 through grain and microbial respiration [5]. However, the silo constitutes a non-static environment, where feed-out, seasonal
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.07.027
134
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137
temperature variations, and air-leakage can change the growth conditions and eventually enable growth of spoilage moulds. The mould Penicillium roqueforti is a common spoilage organism in mal-functioning airtight stored grain [6]. It is very tolerant to low oxygen availability and is able to grow at 0.14% oxygen tension. Thus, stable airtight storage depends on the rapid CO2 evolvement to levels that reduce the growth of P. roqueforti (>15%). The sensitivity of P. roqueforti to CO2 increases with decreasing aw and temperature [7]. During storage of moist grain at high CO2 and low O2 concentration, Pichia (Hansenula) anomala is the dominating yeast [6]. It has been demonstrated that the addition of P. anomala to cereal grain stored under airtight conditions inhibited the growth of filamentous fungi and made the system more robust and less sensitive to air-leakage [8,9]. During yeast growth, oxygen and available nutrients are consumed and CO2 is produced. However, this is not specific for P. anomala as all aerobic microorganisms consume nutrients and oxygen, as well as produce CO2. Therefore, this cannot solely explain the biocontrol performance of this yeast in comparison to other yeast species [10]. Recently, we found that a product of glucose metabolism, most probably ethyl acetate, is an essential factor in mould inhibition by P. anomala [20]. Additional factors might include the ability to grow rapidly in the silo and to tolerate different levels of water availability. The multiple mechanisms of biocontrol activity might be resolved by genetic analysis. However, the genetics of P. anomala is poorly developed and mutational analysis is hampered by the fact that natural isolates of this yeast are diploid [2,11]. The biocontrol performance of haploid strains of P. anomala has not been previously tested. The aim of this study was to investigate the effect of aw and ethyl acetate production on the biocontrol activity of P. anomala against P. roqueforti. To enable future mutational analysis of the biocontrol process and to test the influence of the ploidy level on biocontrol activity, we included both a diploid and a haploid strain in the study.
2. Material and methods 2.1. Yeast and fungal strains The diploid type strain of P. anomala, CBS 5759 (NRRL-Y-366), and the haploid (+) mating type, CBS 1984 (+; NRRL-Y-366-8) were used in this study (BioloMICS Database, http://www.cbs.knaw.nl/databases/index.htm). Pichia anomala CBS5759 inhibits P. roqueforti in stored grain as efficiently as the described biocontrol ¨ . Druvefors, unpublished strain P. anomala J121 (U. A results). The target mould, P. roqueforti J5 originated from stored cereal grain [10].
2.2. Assay of yeast growth and antifungal activity in minisilo grain systems Test tubes with grain were prepared as described by Petersson et al. [12] with some modifications. Nonsterile wheat was hydrated with water to aw 0.95 (21% H2O) or 0.98 (29% H2O) and stored at 2 C for 72 h to allow the water content to equilibrate. The yeast inocula were pre-grown in YPD broth (yeast extract 10 g l 1, bacterial peptone 20 g l 1, glucose 20 g l 1) [13] over night. Spores of P. roqueforti were harvested from malt extract agar plates (50 g l 1) and kept in glycerol stock solutions (18 C) until inoculation. All substrates were bought at Oxoid Ltd., Basingstoke, Hampshire, England. The wheat was inoculated by mixing 103 spores of P. roqueforti and 105 cells of P. anomala (strain CBS 1984 or CBS 5759) per g grain. A control, inoculated with mould only (103 spores g 1), was also included. The inoculated grain was poured into test tubes (1.5 cm · 15 cm, 18 g) and sealed with rubber membranes perforated with a needle to simulate air-leakage of a full-scale silo. The tubes were incubated at 25 C, and growth (CFU g 1) of yeast and mould was measured daily during 1 week as previously described [12]. MEAC (50 g l 1malt extract agar with 100 ppm chloramphenicol) was used for the determination of yeast growth and MEACC (50 g l 1 malt extract agar with 100 ppm chloramphenicol and 10 ppm cycloheximide) for the determination of mould growth. Each day during a 7-day cultivation period, three independent tubes for each combination of fungal strains and growth condition were opened and analysed for fungal growth. Results are given as mean values ± standard deviation (SD). Significant differences were evaluated using the StudentÕs ttest. 2.3. Production of ethyl acetate by P. anomala in grain minisilos The volatile ester ethyl acetate can be trapped in the hydrocarbon decane [14]. At each sampling point (see results), 5 ml of 99% decane (Sigma Chemical Co., St. Louis, USA) were added to three independent grainfilled test tubes and shaken for 5 min. Subsequently, a 1-ml extract was filtered through a 0.45 lm Acrodisc syringe filter (Gelman Laboratory, Ann Arbor, USA) and 1 ll was injected in a Hewlett–Packard gas chromatograph with a flame ionisation detector (250 C, Hewlett–Packard Ltd., Cheshire, England) and a capillary column (HP19091S-833, 250 lm · 30.0 m). The carrier gas was H2 at a flow rate of 40.0 ml min 1. The column temperature was programmed from 60 to 250 C at a rise rate of 20 C min 1 and finally held during 2 min at 250 C. Ethyl acetate was identified and quantified by comparison with an external standard. The retention time of ethyl acetate was 1.80 min. The concentration
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137
2.4. Assay of anti-mould activity of ethyl acetate To investigate whether the volatile ester ethyl acetate had an inhibitory effect on mould growth, P. roqueforti was grown in a semi-closed environment with vaporised ethyl acetate. A glass cup attached to a microscope slide was placed on the bottom of one plastic petri dish and 0.1–0.4 ml ethyl acetate was added to the glass cup (Merck Eurolab AB, Stockholm, Sweden). Water was used as the negative control. Spores of P. roqueforti were inoculated in the center of a second petri dish with 30 ml malt extract agar (50 g l 1; Oxoid). The agar plate was placed upside down so that the ethyl acetate containing glass slide was facing the agar surface. The plates were wrapped with at least three layers of parafilm to create a semi-closed environment. Since parafilm is permeable for O2 and CO2, effects of strong oxygen limitation and CO2 inhibition on mould growth were avoided. Complete volatilisation of ethyl acetate occurred within 3 h. Because parafilm is also permeable for ethyl acetate, one cannot directly compare the ethyl acetate concentrations in the in vitro experiments with the levels in the test tubes. The concentrations in the in vitro tests were set according to preliminary experiments. After 5 days of incubation at 25 C the dry weight of the mould colony was measured. To estimate the mycelial dry weight, the mould colony was cut out from the agar and placed in 300 ml distilled water. The water was heated in a microwave oven until the residual agar was melted. The mould colony was washed in distilled water, placed on a pre-weighed filter, and dried at 80 C for 24 h. All treatments were done with three replicates. Results are given as mean values ± SD. Significant differences were evaluated using the StudentÕs t-test.
10 009 8 10 00
Yeast CFU g-1 grain
detected in the decane was recalculated to obtain the headspace concentration in the minisilo. The minisilo headspace volume was approximately 10 ml when filled with 18 g grain.
135
7 10 00
10 006 5 10 00
diploid (0.95)
haploid (0.95)
diploid (0.98)
haploid (0.98)
4
10 00
0
1
2
3 4 Time (Days)
5
6
7
Fig. 1. Growth of diploid (CBS 5759) and haploid (CBS 1984) strains of P. anomala (CFU g 1) inoculated in grain minisilos at aw 0.95 and 0.98 (n = 3, ±SD).
To test the influence of different growth behaviour on the biocontrol performance of P. anomala, we cocultivated yeast and mould in test tubes which were incubated at aw 0.95 and 0.98 for seven days. Growth (CFU g 1) of mould and yeast was analysed. At aw 0.98, the CFU g 1 of P. roqueforti did not increase during the 7-day incubation when co-cultivated with the diploid or haploid strain (Fig. 2), nor did it grow in the tubes inoculated with the diploid strain at aw 0.95. However, when co-cultivated with the haploid strain at aw 0.95, P. roqueforti grew from 102 CFU g 1 to 105 CFU g 1 within 6 days (Fig. 2). The haploid strain slightly inhibited mould growth at aw 0.95 (Fig. 2), but not to the same extent as at aw 0.98. Previous results have shown that the growth of P. anomala is not influenced by the presence or absence of the mould in the grain-filled tubes [10]. To investigate the possible correlation between ethyl acetate production of P. anomala and mould inhibition in the minisilos, we measured ethyl acetate formation
6 000 10
3. Results and discussion
5
000 10
Mould CFU g-1 grain
The diploid and haploid strain of P. anomala, CBS 5759 and CBS 1984, respectively, had similar growth behaviour in the grain-filled test tubes at aw 0.98, showing rapid growth during the first 2–3 days (Fig. 1). The same trend was found for the diploid strain grown at aw 0.95. In contrast, the haploid strain grown at aw 0.95 reached significantly lower (p < 0.05) levels of CFU g 1 during the first two days (Fig. 1). However, after 4 days there was no difference between the haploid strain grown at aw 0.95 (5.8 ± 2 · 106 cells g 1 at day 4) and at aw 0.98 (8.7 ± 1.3 · 106 cells g 1at day 4) (Fig. 1). The different growth curves of the two strains at aw 0.95 was mainly related to a prolongation of the lag phase of the haploid strain.
4
10000 10
3
1000 10
2 100 10 1 10 10
diploid (0.95) haploid (0.98)
0
1
2
haploid (0.95) mould (0.95)
3 4 Time (Days)
diploid (0.98) mould (0.98)
5
6
7
Fig. 2. Growth of P. roqueforti (CFU g 1) co-inoculated with diploid (CBS 5759) or haploid (CBS 1984) strains of P. anomala in grain minisilos at aw 0.95 and 0.98. Controls without yeast are also shown (n = 3, ±SD).
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137 18.0 18
diploid (0.95) haploid (0.95) diploid (0.98) haploid (0.98)
16.0 16 -1
Ethyl acetate ( µg ml )
Dry weight (%) compared to control
136
14.0 14
12 .0
10 10.0 8.08 6.06 4.04 2.02 0.00
0
1
2
3
4
5
6
7
Time (days)
Fig. 3. Ethyl acetate headspace concentrations in grain minisilos (aw 0.95 and 0.98) inoculated with P. roqueforti and diploid (CBS 5759) or haploid (CBS 1984) strains of P. anomala (n = 3, ±SD).
during one week in test tubes inoculated with the haploid or diploid strain at aw 0.95 and 0.98 (Fig. 3). Ethyl acetate was detected in all test tubes. However, from day 2, the level of ethyl acetate was significantly higher (p < 05) in the test tubes inoculated with the diploid strain (at both aw 0.95 and 0.98) and the haploid at aw 0.98 compared to the haploid strain grown at aw 0.95 (Fig. 3). After 2 days, the detected level from the haploid strain at aw 0.95 was only 10% of that at aw 0.98 and the ethyl acetate level at aw 0.95 never exceeded 40% (day 3) of that at aw 0.98. At aw 0.98, the haploid CBS 1984 continued to produce ethyl acetate during the 7-day incubation period, similar to the diploid CBS 5759 at aw 0.95 and 0.98 (Fig. 3). No ethyl acetate was detected in the mould-only controls (data not shown). The concentration of ethyl acetate in the grain headspace after 3 days was approximately 89 lg ml 1 in all treatments, except for the haploid CBS 1984 at aw 0.95 (3.1 lg ml 1). The maximum headspace concentration of ethyl acetate produced by the haploid strain at aw 0.95 was 3.7 ± 0.6 lg ml 1, while it was 15.6 ± 1.6 lg ml 1 at aw 0.98. This different behaviour suggests an involvement of ethyl acetate in the fungal inhibition. In the test tube system, at ethyl acetate concentrations over 8 lg ml 1, there was a complete inhibition of fungal growth, whereas at ethyl acetate concentrations below 4 lg ml 1, the target mould was able to grow to the same level of CFU g 1 as in the mould-only control. To further confirm the anti-fungal properties of ethyl acetate P. roqueforti was cultivated on agar plates in an atmosphere containing different concentrations of ethyl acetate (Fig. 4). Ethyl acetate concentrations reduced the biomass formation of the mould in a concentration dependent manner (Fig. 4). Because of the fluctuations of CO2, O2 and ethyl acetate in both the in vitro assay and in the grain test tubes, the correlation between fungal growth and ethyl acetate are not directly comparable. The antimould properties of ethyl acetate and the substantial
160 140 120 100 80 60 40 20 0 0
0.1
0.2 0.3 Ethyl acetate (ml)
0.4
0.5
Fig. 4. Inhibition of P. roqueforti grown on malt extract agar and incubated in a semi-closed system with vaporised ethyl acetate (0.1–0.5 ml). The results are given as mycelial dry weight relative to controls with water. The dry weight of the control was 42 ± 2 mg (= 100%; n = 3, ±SD).
amounts produced by the yeast during airtight storage strongly suggests that ethyl acetate is an important part of the biocontrol activity of P. anomala. Acetate esters have been reported to inhibit the conidial germination of filamentous fungi at headspace concentrations of >4 lg ml 1 [15,16]. Mercier and Jime´nez [17] recently showed that volatiles, including esters and alcohols, produced by the yeast Muscodor albus inhibits growth of the spoilage moulds Penicillium expansum and Botrytis cinerea. However, ethyl acetate was not detected from M. albus. Additional factors may also be involved in the mould inhibition. We previously showed that the O2 concentration in 0.2 m3-pilot scale grain silos decreased to 0–4% within the first few days and that the CO2 concentration increased to 90% within the first few weeks [9]. These levels are sufficient to inhibit growth of P. roqueforti [7]. In the present study, the yeast CFU g 1 was significantly lower for the haploid strain at aw 0.95 than in the other treatments during the first 2 days (Fig. 1). Since yeast growth is connected to CO2 formation and O2 consumption, it is possible that the reduced growth of the haploid strain caused a lower CO2 and higher O2 concentration than in the other treatments. This could, in addition to the lower concentration of ethyl acetate, have allowed growth of P. roqueforti in the silos inoculated with the haploid strain at aw 0.95. Nutrient competition has been reported to play a role in the mode of action of other yeast species, for example in Debaryomyces hansenii and Pichia guilliermondi inhibition of Penicillium digitatum when grown together [18,19]. However, this may not influence the anti-mould activity of P. anomala, as addition of various nutrients did not impair its biocontrol activity [20]. Thus, we conclude that ethyl acetate production, possibly together with the increase in CO2 production and O2 consumption, are major components of the anti-mould activity of P. anomala in grain.
E. Fredlund et al. / FEMS Microbiology Letters 238 (2004) 133–137
We analysed the performance of a haploid P. anomala strain in the cereal grain biocontrol system. No essential differences compared to the diploid type strain were observed regarding growth, biocontrol activity, and ethyl acetate production at aw 0.98 (Figs. 1–3). Thus, this strain provides a tool for genetic analysis of the biocontrol process. Unexpectedly, growth, ethyl acetate production, and anti-fungal activity were impaired at aw 0.95 (Figs. 1–3). This indicates that ploidy plays a role in the handling of environmental stress by P. anomala. A connection between stress tolerance and ploidy has been observed in xylose fermenting yeasts, which raised their ploidy level after cultivation in wood hydrolysate [21]. We have currently no explanation on how ploidy influences stress tolerance of yeasts. Our study demonstrates for the first time a connection between P. anomala biocontrol activity and ethyl acetate production. This will contribute to our general understanding of the mode of action of biocontrol yeasts.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements We are thankful to Inger Ohlsson for her technical support. This work has been financially supported by The Foundation for Strategic Environmental Research (MISTRA) and the EU funded project BIO POSTHARVEST (QoL-PL1999-1065).
[14]
[15]
[16]
References [17] [1] Janisiewicz, W.J. and Korsten, L. (2002) Biological control of postharvest diseases of fruits. Annu. Rev. Phytopathol. 40, 411– 441. [2] Passoth, V. and Schnu¨rer, J. (2003) Non-conventional yeasts in antifungal application In: Functional Genetics of Industrial Yeasts (de Winde, H., Ed.), pp. 297–330. Springer, Berlin, Heidelberg. [3] Chelkowski, J. and Cierniewska, A. (1983) Mycotoxin in cereal grain. Part X. Invasion of fungi mycelium into wheat and barley kernels. Die Nahrung. 27, 533–536. [4] Hoseney, C. (1998) Principles of Cereal, Science and Technology. American Association of Cereal Chemists Inc., St. Paul, Minnesota. [5] Hyde, M.B. and Burell, N.J. (1982) Controlled atmosphere storage In: Storage of Cereal Grains and Their Products (Christensen, C.M., Ed.), third ed, pp. 443–478. American Association of Cereal Chemists, Inc., St. Paul, Minnesota. [6] Lacey, J. and Magan, N. (1991) Fungi in cereal grains: their occurence and water and temperature relations. In: Cereal Grain – Mycotoxins, Fungi and Quality in Drying and Storage
[18]
[19]
[20]
[21]
137
(Chelkowski, J., Ed.), pp. 77–118. Elsevier Science Publishers B.V., Amsterdam, The Netherlands. Magan, N. and Lacey, J. (1984) Effects of gas composition and water activity on growth of field and storage fungi and their interactions. Trans. Brit. Mycol. Soc. 82, 305–314. Petersson, S. and Schnu¨rer, J. (1998) Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture wheat, rye, barley, and oats stored under airtight conditions. Can. J. Microbiol. 44, 471–476. Druvefors, U., Jonsson, N., Boysen, M.E. and Schnu¨rer, J. (2002) Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens. FEMS YEAST Res. 2, 389–394. Petersson, S. and Schnu¨rer, J. (1995) Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae. Appl. Environ. Microbiol. 61, 1027–1032. Naumov, G.I., Naumova, E.S. and Schnu¨rer, J. (2001) Genetic characterization of the non-conventional yeast Hansenula anomala. Res. Microbiol. 152, 551–562. Petersson, S., Jonsson, N. and Schnu¨rer, J. (1999) Pichia anomala as a biocontrol agent during storage of high-moisture feed grain under airtight conditions. Postharv. Biol. Technol. 15, 175–184. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A. and Struhl, K. (1999) In: Current Protocols in Molecular Biology. Wiley, New York. Oda, S., Inada, Y., Kobayashi, A., Kato, A., Matsudomi, N. and Ohta, H. (1996) Coupling of metabolism and bioconversion: microbial esterification of citronellol with acetyl coenzyme A produced via metabolism of glucose in an interface bioreactor. Appl. Environ. Microbiol. 62, 2216–2220. Filonow, A.B. (2003) Germination and adhesion of fungal conidia on polycarbonate membranes and on apple fruit exposed to mycoactive acetate esters. Can. J. Anim. Sci. 49, 130–138. Brown, W. (1922) Studies in the physiology of parasitism. IX. The effect on the germination of fungal spores of volatile substances arising from plant tissue. Ann. Botany 36, 285–300. Mercier, J. and Jime´nez, J.I. (2004) Control of fungal decay of apples and peaches by the biofumigant fungus Muscodor albus. Postharv. Biol. Technol. 31, 1–8. Droby, S., Chalutz, E., Wilson, C.L. and Wisniewski, M. (1989) Characterization of the biocontrol activity of Debaryomyces hansenii in the control of Penicillium digitatum on grapefruit. Can. J. Microbiol. 35, 794–800. Droby, S., Chalutz, E., Cohen, L., Weiss, B., Wilson, C. and Wisniewski, M. (1990). Nutrient competition as a mode of action of postharvest biocontrol agents. In: Biological Control of Postharvest Diseases of Fruits and Vegetables, Workshop Proceedings. Shepherdstown, West Virginia, 12 to 14 September: United States Department of Agriculture, Agricultural Research Service, ARS-92. ˚ . (2004). Yeast biocontrol of grain spoilage Druvefors, U.A moulds, Department of Microbiology. Swedish University of Agricultural Sciences: Uppsala. p. 44. Available from: