APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2005, p. 18651869 Vol. 71, No.

4
0099-2240/05/$08.000 doi:10.1128/AEM.71.4.18651869.2005
Copyright 2005, American Society for Microbiology. All Rights Reserved.
Nutrient Effects on Biocontrol of Penicillium roqueforti by
Pichia anomala J121 during Airtight Storage of Wheat
Ulrika A

del Druvefors,* Volkmar Passoth, and Johan Schnurer

Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
Received 1 July 2004/Accepted 2 November 2004
The biocontrol yeast Pichia anomala inhibits the growth of a variety of mold species. We examined the
mechanism underlying the inhibition of the grain spoilage mold Penicillium roqueforti by the biocontrol yeast
P. anomala J121 during airtight storage. The biocontrol effect in a model grain silo with moist wheat (water
activity of 0.96) was enhanced when complex medium, maltose, or glucose was added. Supplementation with
additional nitrogen or vitamin sources did not affect the biocontrol activity of the yeast. The addition of
complex medium or glucose did not signicantly inuence the yeast cell numbers in the silos, whether in the
presence or absence of P. roqueforti. Mold growth was not inuenced by the addition of nutrients, if cultivated
without yeast. The products of glucose metabolism, mainly ethanol and ethyl acetate, increased after glucose
addition to P. anomala-inoculated treatments. Our results suggest that neither competition for nutrients nor
production of a glucose-repressible cell wall lytic enzyme is the main mode of action of biocontrol by P. anomala
in this grain system. Instead, the mold-inhibiting effect probably is due to the antifungal action of metabolites,
most likely a combination of ethyl acetate and ethanol, derived from glycolysis. The discovery that sugar
amendments enhance the biocontrol effect of P. anomala suggests novel ways of formulating biocontrol yeasts.
Mold growth yearly destroys large amounts of vegetables,
fruits, and cereal grain both pre- and postharvest (2). The
growth of molds in food and feed causes reduced nutritional
values and production of allergenic spores and hazardous my-
cotoxins (18). Traditionally, fungicides and chemical preserva-
tives have been used to prevent fungal growth, but concerns for
the environment and the health of the consumer along with
resistance problems and stricter governmental regulations
have generated a demand for alternative control methods.
During the last decade many yeasts and other microorganisms
that provide a biological control alternative have been de-
scribed (3, 4, 8, 17, 21). In contrast to lamentous fungi, bio-
control yeasts have the benet of usually not producing poten-
tially allergenic spores or mycotoxins. Two yeast-based
products, Aspire (Candida oleophila) and Yield plus (Crypto-
coccus albidus), are commercially available for the biocontrol
of molds in apples, citrus, and pome fruits (21). Shemer, a
biological control project registered for use in Israel (www
.agrogreen.co.il), is based on a newly identied yeast Metschni-
kowia fructicola (17) and is effective against a wide range of
pathogens of grape, strawberry, and sweet potato (M. K. Zur et
al., Abstr. Int. Workshop Dev. Biocontrol Agents Dis. Com-
mercial Appl. Food Prod. Syst., abstr. O2-7, 2004).
Pichia anomala (formerly Hansenula anomala [16]) inhibits
the growth of a variety of molds, e.g., Botrytis cinerea, Aspergil-
lus candidus, Penicillium roqueforti, and other plant-pathogenic
or wood decay fungi, in environments as diverse as stored
apple, grapevine plants, grain in airtight storage, and wood (for
a review, see reference 20). P. anomala strain J121 can reduce
the growth of P. roqueforti in vitro, in high-moisture cereal
grain in a test-tube version of a malfunctioning storage system,
and in 0.21-m
3
pilot scale silos for airtight storage of 160-kg
batches of moist grain (3, 7, 2224). Airtight storage of cereal
grain is an energy-saving alternative to hot-air drying. This type
of storage is particularly important for regions of the world
where cereal grain is harvested at moisture contents that allow
the rapid growth of spoilage molds.
Several possible modes of action for biocontrol by yeasts
have been suggested, such as competition for space and nutri-
ents, production of cell wall-degrading enzymes, killer toxins,
antibiotic metabolites, mycoparasitism, and stimulation of host
defense responses (13, 20). For P. anomala the production of
cell wall lytic enzymes and killer toxins has been suggested to
be responsible for antifungal activity (15, 29). Other potential
antifungal mechanisms include competition for nutrients, oxy-
gen, and space; ethanol production (20); and formation of
ethyl acetate (26), a volatile with antifungal activity (27). Iden-
tication of the mode of action of a biocontrol organism is
important both for regulatory approval and for optimizing the
biocontrol system. The objective of this study was to identify
the mode of action of P. anomala J121 responsible for the
inhibition of mold growth in airtight grain storage silos.
MATERIALS AND METHODS
Fungal isolates. P. roqueforti Thom (J5) and P. anomala (Hansen) Kurtzman
(J121) were originally isolated from stored grain. P. roqueforti J5 was maintained
on silica gel at 4C, and P. anomala J121 was maintained in glycerol stocks
(K
2
HPO
4
, 0.82 g liter
1
; KH
2
PO
4
, 0.18 g liter
1
; sodium citrate, 0.59 g liter
1
;
MgSO
4
7H
2
O 0.25 g liter
1
; glycerol [87%], 212 g liter
1
) at 70C. All
cultures were pregrown on 3% malt extract agar slants (Oxoid Ltd., Basingstoke,
Hampshire, United Kingdom) at 25C. For biocontrol assays, P. anomala was
grown overnight in YPD medium (yeast extract, 10 g liter
1
[Oxoid]; peptone,
20 g liter
1
[Oxoid]; glucose, 20 g liter
1
) and diluted to an appropriate con-
centration in peptone water (2 g liter
1
; Oxoid). Spores of P. roqueforti were
harvested from agar plates and kept in glycerol stocks at 18C until inoculation.
Cultivation in minisilos. Minisilos were prepared as previously described (22)
with some modications. Commercially available heat-dried nonsterilized wheat
* Corresponding author. Mailing address: Department of Microbi-
ology, Swedish University of Agricultural Sciences (SLU), P.O. Box
7025, SE-750 07 Uppsala, Sweden. Phone: 46 18 673213. Fax: 46 18
673392. E-mail: [email protected].
1865
(Kosack, 10% moisture) was moistened alternatively with water, YPD medium,
2 g of maltose solution liter
1
of rehydrating water, YNB medium (yeast nitro-
gen base, 6.67 g liter
1
) (Difco Laboratories, Detroit, Mich.), or glucose solu-
tions (10, 20, 40, or 60 g liter
1
of rehydrating water) to a water activity (a
w
) of
approximately of 0.96 (25% moisture). Since the wheat was not sterilized
before the experiment, we tested the mold and yeast count and found it to be
below the detection limit (10
2
CFU g
1
). The wheat was stored at 2C for 72 h
to allow the water content to equilibrate. The moist wheat was inoculated with
10
4
spores of P. roqueforti g
1
of grain and 10
5
CFU of P. anomala J121 g
1
of
grain. Spores and yeast cells were applied as drops onto the grain and mixed by
shaking to obtain an even distribution. The inoculated grain was poured into
27-ml test tubes that were sealed with a rubber membrane and perforated with
a needle to simulate the air leakage of a full-scale silo. Minisilos inoculated only
with mold or yeast were used as controls. The minisilos were incubated at 25C
for 7 days. At least three minisilos were used for every measurement.
Fungal growth determination. The growth of yeast and mold was measured as
CFU per gram by using selective growth plates (3). Quantication of mold
growth is not as straightforward as that of yeast, but there was a signicant
correlation between mold CFU and hyphal lengths in previous biocontrol exper-
iments with P. anomala and P. roqueforti (3). The statistical signicance of
treatment differences was evaluated with a Students t test.
Determination of ethyl acetate. Ethyl acetate was extracted from the grain by
shaking the total minisilo content with 5 ml of 99% decane (Sigma, St. Louis,
Mo.) for 5 min (26). Although this extraction method does not differentiate
between intracellular and extracellular ethyl acetate, it reects volatile changes in
the atmosphere of the minisilo as ethyl acetate is highly volatile. One milliliter of
the extract was ltered through a 0.45-m-pore-size Acrodisc syringe lter
(Gelman Laboratory, Ann Arbor, Mich.) and 1 l was injected into a Hewlett
Packard gas chromatograph with a ame ionization detector (250C; Cheshire,
United Kingdom) and a capillary column (HP19091S-833; 250 m by 30.0 m).
The carrier gas was H
2
at a ow rate of 40 ml min
1
. The column temperature
was programmed to increase from 60 to 250C at a rate of 20C min
1
and was
nally held for 2 min at 250C. Ethyl acetate was identied and quantied by
comparison with an external standard.
Consumption of glucose and production of metabolites. Concentrations of
glucose and ethanol in grain extracts were measured at appropriate times (see
Results). The compounds were extracted by shaking the contents of each minisilo
with 5 ml of distilled water. Samples (10-l) were analyzed by high-pressure
liquid chromatography (Agilent Technologies, Waldbronn, Germany) by using a
Hamilton (Ha gersten, Sweden) HC-75 column for separation. Analysis was per-
formed at 60C with 5 mM H
2
SO
4
at a ow rate of 0.6 ml min
1
as the mobile
phase. The eluate was monitored with an Agilent 1100 refractive index detector.
The metabolites were identied and quantied by comparison with chromato-
grams from authentic reference compounds (glucose [Oxoid] and ethanol [Pri-
malco Oy, Finland]).
Antimold activity of pure ethyl acetate and ethanol. To determine if there is a
synergistic antimold effect due to ethanol and ethyl acetate, P. roqueforti was
grown on agar containing ethanol in a closed environment with vaporized ethyl
acetate. A glass cup attached to a microscope slide was placed on the bottom of
a plastic petri dish, and 0.6 ml of ethyl acetate (99.8%; Merck) was added to the
glass cup. The amount of ethyl acetate in the in vitro assay was chosen after
preliminary experiments since it is not possible to directly compare headspace
concentrations in the minisilos with the plate assay. Water was used as the
negative control. Spores of P. roqueforti were inoculated in the center of a malt
extract agar plate, with or without 1g of ethanol liter
1
of culture medium. The
plate was placed facing down on the petri dish with the ethyl acetate-containing
glass slide. The ethanol concentration represents an intermediate concentration
detected in the grain (approximately 400 g/g of grain). To reduce ethyl acetate
evaporation, the plates were wrapped with three layers of paralm. After 7 days
of incubation at 25C, the mycelial dry weight of the Penicillium colony was
measured. The colony was placed in distilled water and heated in a microwave
oven until the agar melted. The mold colony was then washed in distilled water,
placed on a preweighed lter, and dried at 80C for 24 h. All treatments were
done with three replicates.
RESULTS
Effect of nutrient addition on mold and yeast growth. The
addition of complex medium (YPD) to a minisilo enhanced the
ability of the yeast to inhibit mold growth. In minisilos con-
taining YPD, mold, and yeast, 15 7 P. roqueforti CFU g
1
of
grain were present after 7 days of growth compared to 340
260 CFU g
1
of grain in the mold, yeast, and water control.
The addition of YPD to control cultivations with yeast or mold
alone did not alter the number of CFU per gram for either
organism (data not shown). Adding maltose gave results with
respect to mold reduction similar to those obtained with YPD
(data not shown). Adding nitrogen and vitamins (YNB) did
not alter the number of either yeast or mold CFU per gram.
Glucose effects on the growth dynamics of P. anomala J121
and P. roqueforti J5 in minisilos were monitored for the 7 days
following inoculation. For the rst 2 days, the number of mold
CFU per gram decreased 10-fold in all treatments (Fig. 1). P.
roqueforti growth was observed in treatments without inocu-
lated yeast beginning on day 3, and this growth continued until
day 7, reaching a nal level of 1.6 10
5
1.0 10
5
CFU g
1
of grain. Final P. roqueforti CFU levels were not inuenced by
glucose addition when cultivated without yeasts (Fig. 1). In
treatments that contained P. anomala but no additional glu-
cose, weak mold growth was observed beginning on day 4 and
continued until the end of cultivation, yielding a nal value of
1.1 10
3
0.1 10
3
CFU g
1
of grain. In contrast, no
signicant increase in mold CFU was observed, when P.
anomala and glucose (10 g liter
1
) were added to the cultures.
Final mold CFU values were signicantly different between
yeast treatments with or without a glucose supplement of 10 g
liter
1
of rehydrating water (P 0.05) (Fig. 1). Higher sugar
amendments (20, 40, an 60 g liter
1
of grain moistening solu-
tion) resulted in the same inhibition of mold growth (P 0.05;
data not shown). Final yeast CFU levels were not signicantly
affected by glucose addition either in the presence or in the
absence of mold (Fig. 1).
Consumption of glucose and production of ethanol. When
glucose was added to silos inoculated with yeast, all of the
glucose was consumed within 7 days (Fig. 2A). In control silos
FIG. 1. Effects of glucose addition (10 g glucose liter
1
of moist-
ening water) and yeast-mold cocultivation on the growth of P. anomala
J121 and P. roqueforti J5 in a minisilo system. E, yeast CFU in yeast
control without glucose addition; , yeast CFU in yeast control with
glucose addition; , yeast CFU in yeast-mold cocultivation without
glucose addition; , yeast CFU in yeast-mold cocultivation with glu-
cose addition; F, mold CFU in mold control without glucose addition;
, mold CFU in mold control with glucose addition; , mold CFU in
yeast-mold cocultivation without glucose addition; , mold CFU in
yeast-mold cocultivation with glucose addition. Values are the means
of three experiments standard deviations. Similar results were ob-
served at all glucose concentrations (10, 20, 40, and 60 g liter
1
).
1866 DRUVEFORS ET AL. APPL. ENVIRON. MICROBIOL.
that were inoculated only with mold, glucose consumption was
very low compared to those inoculated with yeast (Fig. 2B). At
the two highest glucose levels, some glucose was consumed
during the rst 3 days, but virtually no glucose consumption
was observed from day 3 to day 7 (Fig. 2B). There is an
unusually high standard deviation on day 3.
Ethanol was detected after 7 days in all minisilos whether or
not they were inoculated with P. anomala. However, in yeast-
inoculated minisilos, more ethanol was produced, and the eth-
anol concentration increased with increasing glucose concen-
tration in the moistening water (Fig. 3). Similar levels of
ethanol production also were observed for silos with yeast only
and silos inoculated with both yeast and mold. However, at the
lowest glucose supplement level (10 g liter
1
of rehydrating
water) no clear difference in ethanol concentration was ob-
served between the two yeast treatments and the silos inocu-
lated with only P. roqueforti. When P. roqueforti was grown
without yeast but with glucose, the ethanol concentration
reached the same levels as in treatments with P. anomala and
10 g of glucose liter
1
.
Ethyl acetate formation. The ethyl acetate concentration in
the grain minisilos increased both over time (data not shown)
and with the amount of added glucose (Fig. 4). Regardless of
the glucose addition, ethyl acetate was never detected in treat-
ments inoculated with only P. roqueforti.
Antifungal activity of ethyl acetate and ethanol. The inhib-
itory action of ethyl acetate and ethanol on fungal growth was
evaluated with agar plate assays. The addition of ethanol to the
agar (1 g per liter, corresponding to the average amount of
ethanol measured in the minisilo grain water) decreased the
fungal colony dry weight from 130 39 mg to 65 5.8 mg.
When 0.6 ml of ethyl acetate was added to the atmosphere
above a plate without ethanol, the mean dry weight of the P.
roqueforti colony decreased to 7.3 2.8 mg. When ethanol-
containing agar was combined with ethyl acetate in the atmo-
sphere, the colony dry weight was further reduced to 4.7 1.5
mg. P. anomala itself is able to grow at 10% (wt/vol) ethanol
(9) and can survive in 0.2 M ethyl acetate (28).
DISCUSSION
Nutrient competition is frequently suggested as an impor-
tant inhibition mechanism in biocontrol systems (20). The ad-
dition of nutrients to the system may diminish the biocontrol
effect if nutrient competition is the mode of action of the
biocontrol agent (4, 6, 8). In the present study, the addition of
extra nitrogen and vitamins had no effect on the biocontrol
ability of P. anomala. Thus, competition for nitrogen is not
responsible for the biocontrol we observed, even though com-
petition for nitrogen may be the mechanism through which
yeast biocontrol of wound-invading pathogens on apples oc-
curs (12).
Surprisingly, the addition of glucose, maltose, or complex
FIG. 2. Glucose concentration in minisilos inoculated with P.
anomala and P. roqueforti (A) or with only P. roqueforti (B). , no
glucose addition; , 10 g of glucose liter
1
of grain moistening water;
, 20 g of glucose liter
1
; F, 40 g of glucose liter
1
; , 60 g of glucose
liter
1
. Values are the means of three experiments standard devia-
tions.
FIG. 3. Ethanol concentration in water extracts from grain minisi-
los after 7 days of incubation with different glucose supplements and
fungal inocula. , P. anomala without P. roqueforti inoculation; , P.
anomala and P. roqueforti cocultivation; , P. roqueforti control with-
out yeast inoculation. Values are the means of three experiments
standard deviations.
FIG. 4. Ethyl acetate concentrations in decane extracts from P.
anomala-inoculated grain minisilos with different glucose amendments
after 7 days of incubation. , P. anomala without P. roqueforti inocu-
lation; , P. anomala and P. roqueforti cocultivation. Values are the
means of three experiments standard deviations. No ethyl acetate
was detected from treatments without P. anomala; i.e., P. roqueforti did
not produce ethyl acetate even at high levels of glucose supplementa-
tion.
VOL. 71, 2005 NUTRIENT EFFECTS ON BIOCONTROL BY P. ANOMALA 1867
sugar-rich media increased the biocontrol effect. Thus, compe-
tition for a carbon source or for an energy source is likely not
the major mechanism responsible for the antifungal activity.
Exo--1,3-glucanase, a cell wall lytic enzyme, is glucose re-
pressible in other strains of P. anomala (14). This enzyme is
involved in the inhibition of gray mold by P. anomala on apple
but would be repressed by the glucose added to our system.
The addition of glucose without inoculation of P. anomala did
not alter the growth of the mold, which rules out possible
inhibitory actions by the glucose itself or by glucose stimulation
of the indigenous microorganisms. Since the nal yeast num-
bers (CFU) were not affected by the addition of glucose, spa-
tial crowding by the yeast presumably is not occurring as a
result of sugar addition.
Our results strongly suggest that the enhanced antifungal
effect may be due to the products of sugar metabolism. Possi-
ble candidate products are ethyl acetate, ethanol, acetate, arab-
itol, glycerol, or various combinations of these products (9).
Ethanol has well-known antifungal effects (19, 20, 25). Ethanol
concentration in the minisilos increased only marginally fol-
lowing the addition of the glucose solution at a concentration
of 10 g l
1
of rehydrating water, even though the full inhibitory
effect of the glucose addition on mold growth could be ob-
served at this sugar concentration. Furthermore, in minisilos
supplemented with 10 g of glucose liter
1
and inoculated with
P. roqueforti alone, as much ethanol was produced as in the
control treatment with P. anomala alone. If ethanol alone were
the critical inhibitory component, then Saccharomyces cerevi-
siae would be the best yeast for the biocontrol of grain molds,
since this yeast is an efcient ethanol producer and is particu-
larly tolerant of high ethanol concentrations (25). However, S.
cerevisiae has no known biocontrol activity in this minisilo
system (23).
Ethyl acetate could result in the enhanced biocontrol activity
that follows glucose addition, and this ester demonstrated con-
siderable antifungal activity in our plate assays. We have pre-
viously shown a connection between the biocontrol activity and
ethyl acetate production in a haploid P. anomala strain (11). In
the grain minisilos ethyl acetate concentrations were already
signicantly enhanced after the grain was moistened with water
containing 10 g of glucose l
1
. The volatile nature of ethyl
acetate would enable it to spread easily in the nonhomoge-
neous environment of the grain silo. Ethyl acetate and ethanol
also might have synergistic antifungal effects in grain silos,
similar to the effect observed in our agar plate experiments.
Even without glucose supplementation, i.e., at substantially
lower ethyl acetate levels, the growth of P. anomala reduced
the number of mold CFU per gram by about 100-fold relative
to growth within silos without yeasts. Thus, in addition to ethyl
acetate and perhaps ethanol production, other factors may be
involved in the biocontrol activity of P. anomala on cereal
grain, as is typical of several biocontrol systems (13). In airtight
grain storage systems these additional factors might include
competition for O
2
, inhibitory levels of CO
2
, and possibly pro-
duction of antifungal compounds, e.g., killer toxins (10).
The positive effect of the addition of sugar on the biocontrol
activity of P. anomala could provide an opportunity to increase
the performance of this organism in industrial applications.
Sugars can improve the viability of freeze-dried cultures that
are important components of the formulation of biocontrol
agents (1, 5). Thus, sugar amendments to a potentially com-
mercialized formulation could have a double effect by increas-
ing both the viability and the biocontrol performance of P.
anomala.
ACKNOWLEDGMENTS
We thank Inger Ohlsson for technical assistance, Elisabeth Borjes-
son for assistance with gas chromatography, and Ingvar Sundh for
comments on the manuscript.
This work was supported nancially by the project BIO POSTHAR-
VEST (QoL-PL1999-1065) funded by the European Union and by the
Foundation for Strategic Environmental Research (MISTRA).
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