Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity
ABSTRACT The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (aw, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.
- SourceAvailable from: Rachid Lahlali[Show abstract] [Hide abstract]
ABSTRACT: This study investigated the influence of UV-B radiation (280–320 nm) on survival of Candida oleophila strain O, an antagonist yeast that prevents postharvest diseases caused by Botrytis cinerea and Penicillium expansum on apple and pear fruits. Lethal doses (LD50 and LD90) were, respectively, 0·89 and 1·45 Kj m−2 for in vitro exposure and 3·06 and 5·5 Kj m−2 for in vivo exposure. A screening test of UV-B protectants for strain O was also evaluated under in vitro and in vivo conditions. The in vitro results showed that sodium ascorbate (0·1% and 0·01%), riboflavin (0·1%) and uric acid (0·1% and 0·01%) were the most effective and most suitable protectants. However, only riboflavin (0·1%) and uric acid (0·1%) were effective under in vivo conditions. The efficacy obtained with strain O against P. expansum, when subjected to UV-B radiation, was 75·0% and 49·2% for pathogen concentrations of 105 and 106 spores mL−1, respectively. Adding riboflavin to strain O gave a similar efficacy (64·2%). Applying strain O together with uric acid (0·1%) was less active (47·7%). Nonetheless, its efficacy when applied with the antioxidants sodium ascorbate (71·1%) or ascorbic acid (82·5%) was the greatest. Riboflavin and uric acid were the most cost-effective protectants, and could be included in the final formulation of strain O when applied preharvest.Plant Pathology 01/2011; 60:288-295. · 2.73 Impact Factor
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ABSTRACT: When applied preharvest, antagonistic yeasts that act as biocontrol agents of postharvest fruit diseases must survive the environmental conditions in the field. In particular, UV-B radiation (280 to 320 nm) can greatly reduce their survival and effectiveness. The influence of artificial UV-B radiation on Pichia anomala strain K, an antagonistic yeast with potential for control of postharvest fruit diseases, was evaluated in vitro and in vivo. The in vitro 50 and 90% lethal dose values were 0.89 and 1.6 Kj/m2, respectively, whereas lethal values in vivo were 3.2 and 5.76 Kj/m2, respectively. UV protectants tested in combination with strain K included congo red, tryptophan, riboflavin, lignin, casein, gelatine, folic acid, tyrosine, and four mixtures. Riboflavin, folic acid, and the mixtures 1% folic acid + 0.5% tyrosine + 0.5% riboflavin (formula 2), 0.5% folic acid + 1% tyrosine + 0.5% riboflavin (formula 3), and 0.5% folic acid + 0.5% tyrosine + 1% riboflavin (formula 4) reduced yeast mortality caused by UV-B radiation in petri dish assays. Riboflavin, folic acid, gelatine, lignin, and tyrosine reduced yeast mortality caused by UV-B radiation on apple fruit surfaces. With the exception of lignin and folic acid, none of the compounds or mixtures increased significantly the ability of strain K to control the postharvest pathogen Penicillium expansum on wounded apple fruit. In contrast, casein, gelatine, tyrosine, congo red, riboflavin, and formulas 1 to 4 significantly reduced the effectiveness of strain K. Further investigations are justified to verify a potential benefit of lignin and folic acid for UV protection of strain K in preharvest applications.Plant Disease 01/2011; 95:311-316. · 2.74 Impact Factor
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ABSTRACT: Lahlali, R., Brostaux, Y., and Jijakli, M. H. 2011. Control of apple blue mold by the antagonistic yeast Pichia anomala strain K: Screening of UV protectants for preharvest application. Plant Dis. 95:311-316.
Enhancement of the biocontrol agent Candida oleophila (strain O) survival and
control efficiency under extreme conditions of water activity and relative humidity
Rachid Lahlalia,b,*, M. Haïssam Jijaklia
aPlant Pathology Unit, Gembloux Agricultural University, Passage des Déportés 2, 5030 Gembloux, Belgium
bPlant Biology Research Institute, Department of Biological Sciences, Montreal University, 4101 Sherbrooke Est, Montréal, Que., H1X 2B2 Canada
a r t i c l ei n f o
Received 20 May 2008
Accepted 23 July 2009
Available online 29 July 2009
C. oleophila strain O
a b s t r a c t
The objective of this work is to evaluate the ability of some additive substances in protecting the biocon-
trol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water
activity (aw, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various
protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene
glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protect-
ing agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose
and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and
37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried
conditions. The highest strain O density and efficacy was obtained with SM. Under experimental condi-
tions reflecting practical conditions, SM applied in combination with the strain O resulted in improved
biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar pro-
tectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.
? 2009 Elsevier Inc. All rights reserved.
The yeast Candida oleophila strain O was isolated from the sur-
face of Golden Delicious apples and selected for its high and reli-
able biocontrol activity against Botrytis cinerea and Penicillium
expansum, two serious worldwide pathogens of stored apple and
pears (Jijakli et al., 1993). The underlying mechanisms responsible
for its biocontrol activity have been determined using B. cinerea/
apple as a model and using microbiological, biochemical, genetic
and molecular approaches. Competition for nutrients and space
seem to be the main modes of action of strain O (Jijakli et al.,
1993; Massart et al., 2005). In order to specifically track the popu-
lation dynamics of this strain after its application on apples, differ-
ent monitoring systems have been developed (Massart et al.,
2005). Strain O was also found to be very effective for controlling
Penicillium digitatum and Penicillium italicum, two devastating
post-harvest pathogens of citrus (Lahlali et al., 2004; Lahlali
et al., 2005a). Its biocontrol activity has been shown to be very effi-
cient for post-harvest applications where environmental condi-
tions are generally well controlled. For pre-harvest applications,
however, strain O population density and efficacy during a 2-year
trial were largely influenced by meteorological conditions (Jijakli
et al., 2002). In pre-harvest applications, the biocontrol agent
(BCA) will face large changes in temperature, relative humidity,
light intensity, etc. To be successful in such application, the optimal
and limits of environmental conditions in which strain O might de-
velop must be determined. Water availability and temperature are
among the main factors able to alter strain O growth and establish-
ment. Recently, Lahlali et al. (2008) proposed a validated predictive
model for controlling the population density of BCA C. oleophila in
field conditions 48 h after its application on apples fruit according
to temperature, relative humidity and its initial concentration of
application. The effect of initial yeast concentration and relative
humidity appears more significant than that of temperature on fi-
nal yeast density on apple fruit surface.
Commercial formulation of BCAs should possess adequate shelf
life and retain biocontrol activity similar to that of fresh cells of the
agents (Li and Tian, 2006, 2007).
Exogenous protectants play an important role in the conserva-
tion of viability during and after the freeze-drying process. Various
groups of substances, such as sulfoxides, alcohols and their deri-
vates, monosaccharides and polysaccharides, amino acids, pep-
tides, glycoproteins and compounds have shown protective
action. Therefore, numerous researchers reported that the stress
tolerance of microorganisms could be improved by inducing the
accumulation of intracellular sugars (Teixidõ et al., 1998a,b). Tre-
halose, a non-reducing disaccharide and a major reserve carbohy-
drate, has been shown to be effective as a protectant metabolite
1049-9644/$ - see front matter ? 2009 Elsevier Inc. All rights reserved.
* Corresponding author. Address: Plant Biology Research Institute, Department of
Biological Sciences, Montreal University, 4101 Sherbrooke Est, Montréal, Que.,
Canada H1X 2B2.
E-mail addresses: firstname.lastname@example.org, email@example.com (R. Lahlali).
Biological Control 51 (2009) 403–408
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ybcon
against environmental stresses, such as heat, dehydration, freezing
and hyperosmotic stress (Kown et al., 2003).
Sugars including various monosaccharides and disaccharides
are usually used as exogenous protectants prior to the freeze-dry-
ing process. Direct interactions between sugar molecules and
membrane phospholipids, or sugar molecules and proteins, and
nitrification of sugars in a dry state are thought to be the main pro-
tective mechanisms (Crowe et al., 1990). However, there is little
information about the effect of exogenous protective agents on via-
bility and biocontrol efficacy of antagonists subjected to conditions
of low water availability and relative humidity.
In our previous works, the effect of water activity, temperature
and relative humidity has been successfully evaluated on the
in vitro and in vivo growth of B. cinerea and (Lahlali et al., 2007a,
2006b), P. expansum (Lahlali et al., 2005b, 2006b), P. digitatum
and P. italicum (Lahlali et al., 2006a) and Pichia anomala strain K
and C. oleophila strain O (Lahlali et al., 2008). The obtained results
showed that both antagonistic yeast P. anomala and C. oleophila are
highly sensitive to water stress as compared to four Pencillium spe-
cies and B. cinerea (Lahlali et al., 2007b). These studies have en-
abled us to determine and compare the ecological niches of these
economically important pathogens. Accordingly, this study has
been undertaken in order to (i) evaluate the ability of protectant
substances to improve strain O viability at low values of water
activity and relative humidity (ii) determine its biocontrol efficacy
against P. expansum at low relative humidity when it is applied in
combination with protectant substances and (iii) determine the
best additive, which could be used as support material in the for-
mulation of strain O as a biocontrol product.
2. Materials and methods
Candida oleophila (strain O) was isolated from the surface of cv.
Golden Delicious apples at the Plant Pathology Unit (Gembloux
Agricultural University, Belgium) and identified by the Industrial
Fungi & Yeast collection (bccMTM/MUCL, Belgium). Stock cultures
were stored at 4 ?C on Potato dextrose Agar (PDA, Merck, Darms-
tadt, Germany) plates. Before an experiment, this strain was grown
on PDA at 25 ?C for three successive subcultures under the same
conditions with an interval of 24 h. Before application to apples,
yeast colonies were flooded with sterile distilled water (SDW)
and scraped from Petri plates. The final concentration of this strain
was adjusted according to optical density measurements with an
UltrospecII spectrophotometer (LKB Biochron Ltd, Uppsala, Swe-
den) at 595 nm (Jijakli and Lepoivre, 1998).
Penicillium expansum (strain vs2) was isolated from decayed ap-
ple fruits (Plant Pathology Unit, FUSAGx, Belgium). For long-term
storage, the strain was placed at ?70 ?C in tubes containing 25%
glycerol. During experiments, the initial conidial inoculum was ta-
ken from Petri-dish cultures on PDA medium, preserved at 4 ?C for
no more than 6 months. The conidial suspension was prepared
from 10 ± 1-day-old culture grown at 25 ?C in SDW containing
0.05% Tween 20.
2.2. Medium and fruits preparation
The basic medium used for the present study was PDA with a
water activity (aw) of 0.995. Water activity is a measure of the en-
ergy status of the water in a system. It is defined as the vapor pres-
sure of water above a sample divided by that of pure water at the
same temperature; therefore, pure distilled water has a water
activity of exactly one (Hallsworth and Magan, 1996). Water activ-
ity (aw) was modified by the addition of increasing amounts of
glycerol to obtain levels of 0.980 and 0.930 at 25 ?C (Lahlali et al.,
2005b, 2006a, 2007a). The awof all media was measured by a CX
3 Aqua Lab device (Decagon Devices, Inc.).
‘Golden Delicious’ apple fruits were disinfected by soaking for
2 min in sodium hypochlorite solution (10%), then rinsed twice in
2.3. Efficacy of protectant substances under low water activity
The protectant substances used in this work were peptone,
skimmed milk (SM), sorbitol, lactose, sucrose, maltose and poly-
ethylene glycol. These protectants were tested in combination with
C. oleophila strain O at three concentrations 1%, 0.5%, and 0.1%. Be-
fore the experiment, all additives were prepared in falcon tubes of
15 ml containing 10 ml of SDW and then autoclaved at 120 ?C dur-
ing 20 min. For each protectant-strain O combination, the final
concentration was adjusted to 104CFU/ml. An aliquot of 100 ll
of serial 10-fold dilutions from each prepared mixture was spread
over the modified PDA medium after incubation 48 h at 25 ?C and
then sealed with the polyethylene bags in order to avoid the water
losses. The control contains only strain O. Petri dishes were incu-
bated at 25 ?C for varying time depending on the awof medium.
For non-modified PDA medium and awof 0.98, dishes were incu-
bated for 72 h, and for 5–6 days for media with awof 0.93. The
experiments were performed in triplicate for each protectant-
strain O-aw combination and the trial was repeated twice over
time. The cell viability was calculated using the following formula:
viability = [(colony number in the presence of protectant)/(colony
number in non-stressed medium) (control)] ? 100.
2.4. Efficacy of protectant substances under low relative humidity
In this experiment, all protectant substances were evaluated at
both extreme concentrations 1% and 0.1%. The concentration of C.
oleophila strain O protectant biocontrol combination was adjusted
to 107CFU/ml. After drying, disinfected apple fruits were treated
by dipping into 400 ml suspension of strain O-protectants combi-
nation for 2 min. Treated fruits were placed in desiccator with dif-
ferent relative humidities (RH, 75% and 98%). The approximate
value of equilibrium relative humidity (98% and 75%) inside the
desiccator was controlled by means of saturated salt solutions:
K2SO4(98%) and NaCl (75%). There were four apples for each des-
iccator and RH treatment. In this experiment, two controls of strain
O without protectant substances were used one each at RH of 75%
and 98%. All treatments were kept at 25 ?C. After 24 h of incuba-
tion, four apples per treatment were washed in 1 L KBP buffer
[KH2PO4(0.05 M), K2HPO4(0.05 M) and 0.05% (wt/v) Tween 80,
pH 6.5] on a rotary shaker for 20 min at 120 rpm. Serial 10-fold
dilutions were prepared from 1 ml washing buffer and were plated
in triplicate on PDA medium. Petri dishes were kept at 25 ?C for 2–
3 days. This experiment was twice repeated. The surface area of the
apple was calculated as follows: [Area (cm2) = 0.488 ? volume of
displaced water (v/v) + 66.1 (r = 0.99)] (Lahlali et al., 2008, 2009).
Population sizes were expressed as cfu/cm2. This step was carried
out in triplicate for each treatment.
2.5. Efficacy of protectant substances against P. expansum
Disinfected apple fruits were wounded at two equidistant
points of equatorial zone (2–3 mm in diameter, 4 mm in depth)
using a cork borer instrument. Each wound was treated with
10 ll of strain O suspension at a concentration of 107CFU/ml with
or without protectant. The control treatment was only inoculated
with the same suspension of SDW. Inoculation with P. expansum
was carried out 24 h after biological treatment. Each wound re-
ceived an aliquot of 10 ll from a pathogen suspension of
R. Lahlali, M.H. Jijakli/Biological Control 51 (2009) 403–408
105spores/ml. Treated fruits were stored at a temperature of 24 ?C
from 11 days. There were four apples per treatment. Two trials
were conducted over time with three replicates per treatment.
2.6. Efficacy of protectant substances against P. expansum under low
Treated apple fruits with strain O alone (108CFU/ml), SM (1%)
or in combination were wounded at two equidistant points in
the equatorial zone (2–3 mm in diameter, 4 mm deep) using a cork
borer 24 h after biological treatment and then followed by patho-
gen inoculation 24 h later. Wounds were inoculated with 10 ll of
pathogen suspension (105spores/ml). These sequences of treat-
ment were designed to reflect practical conditions (Lahlali et al.,
2009). Desiccators containing various treatments were sealed and
kept at 24 ?C. Generally, there are eight treatments. After 11 days
of incubation, the lesion diameters were recorded for each
2.7. Statistical analysis
One way ANOVA procedure using SAS software (SAS Institute,
Inc., Cary, NC, USA) was performed for viability (%), population
density (CFU/cm2) and lesion diameter (mm) and when significant
effects were observed, the Newman–Keuls test was used for the
mean separation (P 6 0.05).
3.1. Efficacy of protectant substances at low water activity
The efficiency of selected protectant substances in protecting C.
oleophila strain O against low water activity was evaluated ‘in vitro’
at 0.1%, 0.5% and 1% application concentrations. At awof 0.98, all
protectants gave higher viability of strain O in comparison with
strain O alone (Table 1). Strain O cells showed the highest viability
when protectants were applied at a concentration of 1%. SM, malt-
ose and sucrose as protectants resulted in the greatest viability for
strain O cells at high aw(0.98). The corresponding viabilities were
200%, 191% and 189% when they were applied at 1% in modified
medium as compared to other treatments tested here (Table 1).
At awof 0.93, SM gave better protection than other sugars when
it was applied at 1%. The results also showed no significant differ-
ence between sucrose and peptone (Table 1). At 0.5%, all protectant
substances improved significantly the strain O viability, except lac-
tose. This result was confirmed at 0.1%. However, all additives
showed to be significantly different from the control and from
the lactose, even if the recorded viability was altogether lower than
100%. Peptone resulted in the nominally highest viability (94%) at
0.1% application rate and an awof 0.93 (Table 1).
3.2. Efficacy of protectant substances at low relative humidity
Trials were carried out on apple fruits in order to evaluate the
ability of various protectant substances in protecting the strain O
against low RH. Strain O mixtures with various additives at 1%
and 0.1% were applied to fruits, placed in desiccators at RH of
75% and 25 ?C and then compared with both controls (strain O
alone at RH of 75% and 98%) (Fig. 1). Variance analysis showed that
only SM (1%), sucrose (1%) and sorbitol (1%) improved significantly
the viability of strain O cells at dried conditions (RH of 75%), but
this increase in strain O population density remains significantly
lower than that observed with strain O alone at wet conditions
(RH of 98%).
3.3. Efficacy of protectant substances against P. expansum
The biocontrol efficacy of strain O applied together with differ-
ent protectant substances was evaluated against P. expansum. Sta-
tistical analysis show insignificant difference between the obtained
efficacy of a strain O-protectant mixtures and strain O alone, ex-
cept at concentration of 1% (Table 2). However, a significant effect
of additive substances used in combination with strain O was ob-
served in comparison with the untreated control with yeast. The
best control efficacy was obtained when strain O was applied to-
gether with SM.
3.4. Efficacy of protectant substances against P. expansum at low
SM was the protectant used in this experiment due to the great
ability to protect strain O against the low water availability in
‘in vitro’ and ‘in vivo’ conditions. Likewise, its combined application
did not influence statistically its effectiveness against apple blue
rot. The average diameter of the lesions caused by P. expansum
was significantly higher at RH of 75% as compared to that of 98%
(Table 3). The addition of SM (1%) alone increased significantly
the apple rot infection under dried conditions (RH of 75%). How-
ever, at high RH (98%), no significant difference was observed be-
combination with strain O significantly improved biocontrol effi-
cacy against P. expansum under dry conditions (low RH), which re-
flects the common natural conditions.
Viability of C. oleophila strain O (applied at 104CFU/ml) cells as affected by the application of different protective compounds at three concentrations (0.1%, 0.5% or 1%) and under
different water activity levels (awof 0.98 and 0.93).
Water activity (aw) 0.98 Water activity (aw) 0.93
0.1%0.5% 1%0.1% 0.5%1%
*In the same column, treatments having the same letter are not significantly different according to Newman and Keuls test (P 6 0.05).
P.E.G., polyethylene glycol.
R. Lahlali, M.H. Jijakli/Biological Control 51 (2009) 403–408
Infection by post-harvest pathogens often arises during crop
harvesting (Teixidõ et al., 1999; Ippolito and Nigro, 2000); there-
fore it would be advantageous to apply the BCA prior the harvest
process in order to colonize wounds before the arrival of the patho-
gens. Such pre-harvest application would have numerous benefits,
such as decreasing the level of damages, which can occur during
the post-harvest treatment. In order to be successful, the inoculum
of BCA should tolerate various environmental stresses of the orch-
ard, including higher temperatures, lower water availability, low
nutrients and ultraviolet radiations (Teixidõ et al., 1999). Bonaterra
et al. (2005), reported that the application of Pantoea agglomerans
to unwounded fruits was practically ineffective for controlling
the blue mold when the biological treatment, subsequent wound-
ing and pathogen inoculation were separated by long periods of
low relative humidity conditions. The authors attributed the lack
of efficacy to the difficulties in colonization and survival of antag-
onistic bacteria in the intact peel surface compared to the rapid
growth observed in fresh wounds. Mercier and Wilson (1995)
demonstrated the lowest growth of C. oleophila in old wounds of
apples. Our previous research evaluated the efficacy of biocontrol
agent P. anomala strain K against P. expansum in the laboratory in
three scenarios designed to mimic practical conditions, with differ-
ent periods of incubation between biological treatment, wounding
of fruit surface, and pathogen inoculation. The results showed the
impact of wetness on apple surfaces on P. anomala growth, and
subsequent biocontrol efficacy against blue mold (Lahlali et al.,
A substantial amount of research has been conducted on
improving the resistance of BCA to abiotic stress by adaptation of
growth to unfavorable osmotic conditions (Bonaterra et al.,
2005). In this study, we were interested in an exogenous approach
focused on the application of some protectant additives in combi-
nation with the BCA to improve its tolerance to environmental
stress and, therefore, promote their establishment on apple fruit
surfaces. To our knowledge, the use of additive substances has
two main functions in the production of viable cells. The first is
to restore damage done to the cytoplasmic membrane. The second
is the biochemical protection of cells against damage caused dur-
ing osmotic stress (Berny and Hennebert, 1991). Our in vitro results
showed that the strain O viability was significantly improved at aw
of 0.93 when it is applied in combination with various protectants.
This viability varies significantly depending on the type of protec-
tant and its applied concentration. The greatest value was obtained
for all protectants at 1%. However, the highest was recorded by SM.
This may be due to its richness in protein, vitamins and sugars,
which contributes effectively to improving the strain O viability.
Moreover, SM contains many solutes, such as phosphates and cit-
rate, which may provide buffering capacity and stabilize pH (Zayed
and Roos, 2004). Champagne et al. (1991) underlines that the pro-
tein contained in SM offers a good protection for the cell mem-
brane toward the adverse environmental factors and restores
cells affected during the dehydration and osmotic stress or the dis-
ruption of hydrogen bonds (Ray et al., 1971). Additionally, increas-
ing the viability of the strain O could also be explained by the
nature of polyol (sorbitol), and some sugars (sucrose, maltose,
and lactose) used in this work. Sugar includes various monosaccha-
rides and disaccharides which are usually used as exogenous pro-
tectants of BCA prior the freeze-drying process. Teixidõ et al., 1999
reported that the intracellular accumulation of certain sugars al-
lows cells to work and survive under conditions of stress, in partic-
ular at low water availability. Similarly, Hanafusa (1985) found
that the addition of certain protectant substances, such as sugars
or glycerol, reduced the amount of bound water on the surface of
proteins. The protectant substances may themselves form hydro-
gen bonds with the protein, thus substituting for water in order
to maintain the stability of the protein (Font de Valdez et al., 1983).
Our in vivo results confirmed the in vitro findings and showed
that, among the protectant substances tested here, only SM, sorbi-
tol and sucrose significantly improved the strain O population den-
sity on apple fruit surface at low RH (75%) as compared with strain
O alone. The highest density was obtained in the presence of SM.
Skimmed milk (0.1%)
Skimmed milk (1%)
Control (RH 98%)
Control (RH 75%)
Fig. 1. Strain O population densities obtained on apple fruit surface at RH of 75%,
after applying C. oleophila strain O (107CFU/ml) in combination with various
protectants at both concentrations 1% and 0.1%. Controls are strain O, applied alone
at RH of 75% and 98%. Bars represent the standard error of the means. Treatments
having the same letters are not significantly different according to Newman and
Keuls test (P 6 0.05).
Efficacy of strain O formulations, applied in mixture with different concentrations of
protectants, against P. expansum inoculated at with 10 ll/wound (105spores/ml).
Lesion diameter (mm)
Sorbitol + C. oleophila (strain O)
Sucrose + C. oleophila (strain O)
P.E.G. + C. oleophila (strain O)
Lactose + C. oleophila (strain O)
Maltose + C. oleophila (strain O)
Skimmed milk + C. oleophila (strain O)
Peptone + C. oleophila (strain O)
C. oleophila (strain O)
*In the same column, treatments having the same letter are not significantly dif-
ferent according to Newman and Keuls test (P 6 0.05).
P.E.G., polyethylene glycol.
Biocontrol efficacy of C. oleophila strain O (108CFU/ml) applied with/out SM (1%)
against P. expansum, applied at 10 ll/wound (105spores/ml), at RH of 75% and 98%.
The pathogen inoculation was made 24 h after wounding. Treatments having the
same letters are not significantly different according to Newman and Keuls test
(P 6 0.05).
TreatmentsLesion diameter (mm)
C. oleophila strain O + skimmed milk (1%) +
relative humidity (98%)
C. oleophila strain O + skimmed milk (1%) +
relative humidity (75%)
Skimmed milk (1%) + relative humidity (98%)
Skimmed milk (1%) + relative humidity (75%)
C. oleophila strain O + relative humidity (98%)
C. oleophila strain O + relative humidity (75%)
Relative humidity (98%)
Relative humidity (75%)
R. Lahlali, M.H. Jijakli/Biological Control 51 (2009) 403–408
However, this density remains significantly lower than that ob-
served at RH close to the saturation (98%) without protectant
substances. Additionally, applying C. oleophila strain O (108CFU/
ml) in combination with SM (1%) gave better protection against
P. expansum at low RH under a scenario reflecting practical
conditions. This result could be explained by the direct protective
properties of SM against desiccation, or by improving the nutri-
tional state of strain O, when applied together with SM. As shown
in Fig. 1, SM increased the strain O survival on apple fruit surface
under stress conditions. However, a fast blue mold infection was
observed on apple fruit when SM was applied alone. Abadias
et al. (2001) also reported the best efficacy of Candida sake cells
when it was applied in combination with SM. This product also
provided the freeze-dried product with a porous structure that
caused rehydration. The workers underlined the importance of
using a SM as a support material in mixture with the best protec-
tant substances. It is thought that protein contained in milk pro-
vides a protective coat for the cells (Champagne et al., 1991).
At higher relative humidity, SM (1%) did not affect the strain O
viability. However, a significant difference was observed between
both controls (apples only inoculated with the pathogen) with
the highest lesion diameter of blue decay at RH of 75% in compar-
ison with RH of 98%. This result is in disagreement with those pre-
viously reported by Lahlali et al. (2006b) and with those found
under ‘in vitro’ conditions where the mycelial growth was posi-
tively correlated with the water activity of the medium and incu-
bation temperature. This could be explained by the environment
of the wound, which would seem to serve as a barrier protecting
the fungus from the exogenous effects of RH.
Our ‘in vivo’ results shown in Fig. 1 (SM, sorbitol and sucrose)
are in agreement with those of Bonaterra et al. (2005). These
authors showed that the osmoadaptation of P. agglomerans
EPS125 cells in a medium containing the NaCl or the glycine beta-
ine increased considerably the survival of this antagonistic yeast
on the intact surface of apple fruits. This effect was highly signifi-
cant under low RH and fluctuating (RH) conditions, but was not
significant at high RH. It has been shown that osmoadapted cells
accumulated trehalose and glycine betaïne (GB) intracellularly,
and that they show a higher tolerance to desiccation compared
to non-osmoadapted cells. Furthermore, it also has been reported
that the osmoadaptation significantly improved blue mold control
under conditions where the standard biological control treatments
were ineffective. The rot diameter was significantly reduced in ap-
ple fruits, which were treated with EPS125 and incubated for sev-
eral days under low, high or fluctuating RH, followed by wounding
and inoculation of P. expansum (Bonaterra et al., 2005).
Previous research on the effect of water activity on the intracel-
lular accumulation of endogenous solutes of C. sake showed a sig-
nificant change in ecophysiological parameters that can affect the
endogenous contents of yeast cells (Teixidõ et al., 1998a,b). These
results could be used to develop an efficient and more stable bio-
control inoculum in storage with a higher resistance to desiccation.
The principle of stress tolerance is based on the induction of intra-
cellular accumulation of compatible solutes to make BCAs more
tolerant to environmental stress and to maintain their cells turgor
(Csonka, 1989; Miller and Wood, 1996). Generally, theses compat-
ible solutes including polyols, sugars, aminoacids and their by-
product substances are synthesized or taken directly from the
environment (Potts, 1994; Teixidõ et al., 1998b; Magan, 2001; Aba-
dias et al., 2001). Overall, the combination of intracellular accumu-
lation of compatibles solutes and SM, may improve the resistance
of our BCA to adverse environmental factors, including water
activity and low relative humidity for its eventual application in
pre-harvest conditions. This could be done without using a sugar
together with SM. As a result, further trials will be planned to as-
sess the effectiveness of a formulation based on the strain O in
combination with SM and trehalose under field conditions at least
48 h before harvesting.
This manuscript is a part of my doctoral research performed at
Plant Pathology Unit of Gembloux Agricultural University. The
authors are grateful to my colleagues Drs. M. Sabar and J. Puigagut
(IRBV, Montreal University) for reviewing this manuscript.
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