全 文 ：Received 19 Feb. 2004 Accepted 17 Jul. 2004
Supported by the National Science Fund for Distinguished Young Scholars of China (30225030) and the National Natural Science Foundation
of China (30170663).
* Author for correspondence. Fax: +86 (0)10 82594675; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1324-1330
Application of Antagonistic Yeasts Under Field Conditions and Their
Biocontrol Ability Against Postharvest Diseases of Sweet Cherry
TIAN Shi-Ping*, QIN Guo-Zheng, XU Yong, WANG You-Sheng
(Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,
The Chinese Academy of Sciences, Beijing 100093, China)
Abstract: The yeasts Trichosporon pullulans (Lindner.) Diddens et Lodder, Cryptococcus laurentii
(Kuffer.) Skinner and Rhodotorula glutinis (Fresenius) Harrison were sprayed at concentration of 1×108
CFU/mL onto sweet cherry (Prunus avivum L. cv. Hongdeng) fruit in two orchards prior to harvest. Survival
of these species on fruit surfaces under field conditions was investigated. Also, their biocontrol efficacy
against postharvest decay of cherry fruit stored under various conditions was assessed. All three yeasts
colonized the surface of sweet cherry fruit. However, only C. laurentii and R. glutinis maintained popula-
tions at high and stable levels throughout the 4-d experimental period. C. laurentii was the most effective
and promising of the three antagonists. It had strong survival ability on fruit surfaces under field conditions
and adaptability to postharvest storage conditions of low temperature, low-O2 and high-CO2 concentrations.
Key words: biocontrol efficacy; postharvest decay; preharvest application; sweet cherry fruit; yeast
antagonists
As use of high levels of chemical fungicide in fruit or-
chards results in the development of resistance of fungal
pathogens to fungicides and the growing public concern
over the health and environmental hazards, the need to
develop other methods to control postharvest diseases has
been encouraged in resent years (Wilson and Pusey, 1985;
Wisniewski and Wilson, 1992; Lima et al., 1998). Biological
control using microbial antagonists has emerged as one of
the most promising alternatives, either alone or as part of
an integrated control strategy to reduce synthetic fungi-
cide inputs (Mercier and Wilson, 1994; Chand-Goyal and
Spotts, 1996; Benbow and Sugar, 1999; Fan and Tian, 2000).
The efficacy of several postharvest biocontrol agents has
been evaluated in pilot tests under semi-commercial condi-
tions (Chand-Goyal and Spotts, 1997; Janisiewicz et al.,
1998; Tian et al., 2002a; Spotts et al., 2002).
In the postharvest environments, yeasts appear to be
particularly promising because production of antibiotics
does not seem to be involved in their activity (Droby and
Chalutz, 1994). Antagonistic yeasts have been selected
mainly for their capability to rapidly colonize and grow in
surface wounds and subsequently out compete pathogens
for nutrients and space (Wisniewski et al., 1991; Arras, 1996;
Spadaro et al., 2002) and parasitize postharvest pathogens
directly through strong attachment to their hyphae (Droby
et al., 2002; Wan and Tian, 2002). As the yeasts used in the
experiment were originally isolated from fruit surfaces after
or near harvest, they might be tolerant of the field condi-
tions and adversely affected by preharvest application of
fungicides (Fan and Tian, 2001; Tian et al., 2002a). Some
yeasts can colonize plant surfaces or wounds for long pe-
riods under dry conditions, and can produce extracellular
polysaccharides that enhance their survival and restrict
pathogen colonization sites (Wisniewski and Wilson, 1992;
Chand-Goyal and Spotts, 1996).
Infection of fruit by postharvest pathogens often oc-
curs in the field prior to harvest (Roberts, 1994; Biggs, 1995).
Therefore, it would be advantageous to apply antagonists
before harvest. Preharvest application could reduce initial
infection and the agents then remain active and suppress
the pathogens in storage (Teixidó et al., 1998). Biocontrol
activity of antagonists may also be influenced by the spe-
cific pathogen, host commodity and particularly by envi-
ronmental conditions (Spotts et al., 1998; Tian et al., 2002a).
The success of commercialization of an antagonist depends
on its broad spectrum of action. When applied before har-
vest in the field, antagonistic yeasts need tolerance to en-
vironmental stresses, especially of high temperature, low
water activity, low nutrient conditions, and UV light for
effective establishment and disease control (Deacon, 1991;
Conway et al., 1999). If an antagonist has potential for prac-
tical use, its ability to colonize the surface of fruit both in
the field and in storage, and to persist for as long as pos-
sible is vitally important (Wisniewski and Wilson, 1992).
TIAN Shi-Ping et al.: Application of Antagonistic Yeasts Under Field Conditions and Their Biocontrol Ability Against Postharvest
Diseases of Sweet Cherry 1325
Although many studies have indicated the potential for
biocontrol of postharvest decay when agents were applied
after harvest (Wilson and Pusey, 1985; Chand-Goyal and
Spotts, 1996; Lima et al., 1998; Tian et al., 2002b), relatively
few studies have tried to improve the competence, survival
and activity of antagonistic microbial agents in the field for
optimizing subsequent disease control (Teixidó et al., 1998;
Benbow and Sugar, 1999).
Trichosporon pullulans, Cryptococcus laurentii and
Rhodotorula glutinis were isolated selectively from the
surface of apple fruit. We found previously that these
yeasts had biocontrol ability against postharvest decay of
apple and grape fruits when applied after harvest (Liu et
al., 2002; Qin et al., 2003). The objectives of the study were
(i) to investigate the survival of these antagonistic yeasts,
(ii) to compare their establishment and population dynam-
ics on the fruit surface under field conditions when applied
4 d prior to harvest, and (iii) to determine their biocontrol
efficiency on sweet cherry fruit under different storage con-
ditions
1 Materials and Methods
1.1 Antagonistic yeasts
Trichosporon pullulans (Lindner.) Diddens et Lodder,
Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula
glutinis (Fresenius) Harrison were isolated selectively from
the surface of apple fruit following the method of Wilson
and Chalutz (1989). They were identified by CABI Bio-
science Identification Services (International Mycological
Institute, UK). The yeasts were cultured in 250 mL conical
flasks containing 50 mL of nutrient yeast dextrose broth
(NYDB: 8 g of nutrient broth, 5 g of yeast extract, and 10 g
of dextrose in 1 000 mL water) on a rotary shaker at 200 r/
min for 48 h at 28 °C. Yeast cells were pelleted by centrifu-
gation at 6 000 r/min (about 2 500g) for 10 min, resuspended
in sterile distilled water, and adjusted to a concentration 1
×108 CFU/mL with a haemocytometer.
1.2 Fruit and orchards
Sweet cherry (Prunus avivum L. cv. Hongdeng) fruit
were used in trials conducted in two orchards. One was the
experiment orchard of the Institute of Forest and Fruits,
Beijing Academy of Agricultural Sciences in Beijing. The
other was a commercial orchard in the Jinzhou district of
Liaoning Province, a major production area in China for
sweet cherry. The trees in both orchards were 6-8 years
old. They were grown under standard cultural practices.
Application of fungicides (iprodione and thiabendazole)
was stopped 1 month before the treatments.
1.3 Preharvest treatments
The experiments were carried out on 21-25 May, 2002
in Beijing orchard, in June, 2002 in the Jinzhou orchard 4 d
before harvest. The yeast suspensions of T. pullulans, C.
laurentii and R. glutinis at 1×108 CFU/mL were sprayed
uniformly onto fruit using a hand-held sprayer. Fruits
sprayed with distilled water were used as the control (CK).
There were three replicates of two trees per treatment in a
completely randomized block design. Fruits were harvested
carefully and put into different boxes according to the ex-
perimental design. Fruits harvested from the Beijing orchard
were directly transported to our laboratory. Fruits from the
Jinzhou orchard were immediately precooled after harvest
and transported on the day of harvest to Beijing in a refrig-
erated van at 2-5 °C. Climatic data for each orchard were
obtained during the field trial from nearby weather stations.
1.4 Storage conditions
Storage conditions were room temperature (25 °C), and
in air and under a controlled atmosphere (CA) condition of
10% O2 + 10 % CO2 at 0 °C, respectively. Fruit stored in air
were put into plastic bag to maintain a relative humidity of
about 95%. There were three boxes of 5 kg fruit in each
treatment. Three kg fruits from each of three replicates in
each treatment were used to determine disease incidence at
various times of 10 and 20 d at 25 °C, 30 d in air at 0 °C and
60 d in CA at 0 °C. After 30 and 60 d, fruit stored at low
temperature and under CA condition were raised to 25 °C for
3 d to monitor decay development under shelf life conditions.
1.5 Colonization of yeasts on cherry fruit surface
Fifteen fruits from each treatment were taken at 0, 1, 2, 3,
and 4 d after application to determine population numbers
of the yeasts on the fruit surface by the method of Benbow
and Sugar (1999). These fruits were placed in beakers con-
taining 100 mL of sterile distilled water and then shaken at
200 r/min for 30 min. Serial (1:10) dilutions were made of the
washing solutions. Four aliquots were taken from each di-
lution and plated on NYDA medium (NYDB plus 15 g of
agar in 1 000 mL water). Colonies were counted after incu-
bation at 28 °C for 48 h and expressed as Log10 CFU/fruit.
The three yeasts used in this experiment could be visually
distinguished because of their different colony color and
morphology. Fruit treated with sterile water was used as
the control and only those resembling the yeasts used in
the treatments were counted.
1.6 Statistical analysis
All data were analyzed by one-way analysis of variance
(ANOVA). The treatment means were separated at the 5%
significant level using Duncan’s multiple range test.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041326
2 Results
2.1 Population dynamics on fruit surface
All three yeasts C. laurentii, R. glutinis and T. pullulans
could colonize the surface of sweet cheery fruit under field
conditions (Fig.1). Only C. laurentii and R. glutinis main-
tained populations at high and stable levels of 5.2×106 to
1.3×106 CFU per fruit throughout the experiment at both
orchards. During the 4-d experimental period, populations
of the yeasts on the surfaces of the sprayed fruit were
significantly higher compared to the control (P = 0.05). C.
laurentii and R. glutinis populations remained similar and
high at 1.5-1.3× 106 and 1.8-1.9× 106 CFU per fruit
throughout the 4 d, but T. pullulans populations rapidly
dropped to 5.9´104-5.7× 105 CUF per fruit on day 4
(Fig.1). There were no significant differences between popu-
lation numbers of the same yeasts across the two orchards
(P = 0.05).
2.2 Climatic data
There were obvious differences during the 5 d prior to
harvest in the minimum and maximum temperatures between
the two orchards (Fig.2). Temperatures ranged from 10.8-
15.8 °C to 28.7-32.2 °C in Beijing and from 18-19.3 °C to
23.1-26.6 °C in Jingzhou. The mean temperatures were not
greatly different, being 22-24 °C in Beijing and 21-23 °C in
Jingzhou during the experimental period. Temperature did
not significantly (P > 0.05) affect populations of the yeasts
on the surface of sweet cherry fruit, although there
were differences in the field temperatures of the two
orchards.
2.3 Storage decay control in different conditions
C. laurentii was the most effective antagonist of the
three yeasts for control of postharvest decay of sweet
cherry over the different storage conditions (Figs.3-5). At
25 °C, fruit sprayed with C. laurentii before harvest had a
significantly (P = 0.05) lower disease incidence than those
treated with other yeasts or control (Fig.3). All fruit har-
vested from the Jingzhuo orchard appeared to have less
decay compared to those from the Beijing orchard.
Low temperature and controlled atmosphere storage lim-
ited decay development. Only 1.3%-3.3% of the fruit
sprayed with C. laurentii decayed after 30 d at 0 °C (Fig.4).
Treated fruit stored in 10% O2 + 10% CO2 CA showed only
0-5.1% decay after 60 d (Fig.5). Transferring fruit stored in
either air or under CA at 0-25 °C for 5 d resulted in a signifi-
cant increase in disease incidence (Figs.4,5). The patho-
gens causing sweet cherry decay were mainly Alternaria
alternata, Monilinia fructicola, Penicillum expansum and
Botrytis cinerea. A. alternata was the major pathogen dur-
ing storage and shelf time.
4 Discussion
Most pathogenic fungi inciting postharvest diseases
of fruit attack wounds and cause decay immediately after
Fig.1. Populations of T. pullulans, C. laurentii, and R. glutinis
on surface of cherry fruit in orchards in Beijing (A) and Jinzhou
(B). Data are from 10 sample fruit for three replicates. Bars rep-
resent standard deviations of the means.
Fig.2. Temperatures and rainfall in sweet cheery orchards in
Beijing (A) and Jinzhou (B) during the periods of the experiments.
TIAN Shi-Ping et al.: Application of Antagonistic Yeasts Under Field Conditions and Their Biocontrol Ability Against Postharvest
Diseases of Sweet Cherry 1327
harvest (Sugar and Spotts, 1993; Spotts et al., 1998). Thus,
application of biocontrol agents as close as possible to the
time of wounding should provide the best opportunity to
protect the fruit (Wilson and Pusey, 1985; Robberts, 1994).
Consequently, timing of applications of biocontrol agents
is very important. Benbow and Sugar（1999）empha-
sized the important consideration in the context of
preharvest application of the ability of antagonists to
Fig.3. Effects of preharvest application of Trichosporon pullulans, Cryptococcus laurentii, and Rhodotorula glutinis in orchards in
Beijing (A, B) and Jinzhou (C, D) against postharvest diseases on sweet cherry fruit subsequently stored at 25 °C. There were three
replications in each treatment and the experiment was repeated twice. Bars represented standard deviations of the means. Values followed
by different letters were significantly different according to Duncan’s multiple range test at P = 0.05.
Fig.4. Effects of preharvest application of Trichosporon pullulans, Cryptococcus laurentii and Rhodotorula glutinis in orchards in
Beijing (A, B) and Jinzhou (C, D) against postharvest diseases on sweet cherry fruit subsequently stored at 0 °C. There were three
replications in each treatment and the experiment was repeated twice. Bars represented standard deviations of the means. Values followed
by different letters were significantly different according to Duncan’s multiple range test at P = 0.05.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041328
survive at sufficient populations on the fruit surface after
application. Good survival ability should allow antagonists
to colonize wounds quickly. In the present study, C.
laurentii was the most effective antagonist for control of
postharvest decay of sweet cherry under different storage
conditions and among the three yeasts (Figs.3-5). This
antagonist could survive at high, relatively stable popula-
tion levels on the surface of sweet cherry fruit under field
conditions. In contrast, T. pullulans did not effectively
control decay of sweet cherry under the different storage
conditions. Its lower efficacy may be due to a rapid de-
crease in population numbers after the 4-d of application
(Fig.1). These results agree with the view of Wilson and
Wisniewki (1989), who considered that a major problem in
biocontrol was maintaining a high population of an active
biocontrol agent throughout the entire period of disease
activity.
Effective suppression of plant diseases by biocontrol
agents is largely influenced by environmental conditions.
Environment affects the establishment, survival and activ-
ity of the biocontrol agents (Benbow and Sugar, 1999). Both
C. laurentii and R. glutinis remained at high and stable
population levels on the surface of sweet cherry fruit under
field conditions. However, C. laurentii more significantly
reduced the fruit decay than R. glutinis (Figs.4, 5). The
difference may relate to the adaptability of antagonists to
postharvest storage environments (temperature, O2 and CO2
concentrations). C. laulentii apparently had better adapt-
ability to storage conditions in comparison with R. glutinis.
Antagonistic activity on fruit surfaces or in wounds de-
pend well also on relative biocontrol ability against patho-
genic fungi. Adaptability of the yeasts to storage environ-
mental conditions influence the ability to control
postharvest disease (Droby et al., 2002; Tian et al., 2002a).
The positive characteristics of C. laulentii and R. glutinis
coupled with the apparent evident broad range of activity
and the ability to control decay at storage temperatures
could pave the way for a practical use of these species.
High populations on unwounded fruits under field condi-
tions suggest that these isolates will perform well when
applied before harvest onto fruit surfaces under commer-
cial conditions. Moreover, the use of the yeasts prior to
harvest might also reduce incidence of latent infections by
pathogens that do not require wounds for infection (Biggs,
1995). The results of this study show that preharvest appli-
cation of antagonistic yeasts should provide an alternative
to chemical application.
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Fig.5. Effects of preharvest application of Trichosporon pullulans, Cryptococcus laurentii and Rhodotorula glutinis in orchards in
Beijing (A, B) and Jinzhou (C, D) against postharvest diseases on sweet cherry fruit subsequently stored in controlled atmosphere (CA)
condition at 0 °C. There were three replications in each treatment and the experiment was repeated twice. Bars represented standard
deviations of the means. Values followed by different letters were significantly different according to Duncan’s multiple range test at
P = 0.05.
TIAN Shi-Ping et al.: Application of Antagonistic Yeasts Under Field Conditions and Their Biocontrol Ability Against Postharvest
Diseases of Sweet Cherry 1329
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