Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Improvement of a dry formulation of Pseudomonas

fluorescens EPS62e for fire blight disease biocontrol by
combination of culture osmoadaptation with a
freeze-drying lyoprotectant

s, E. Montesinos and A. Bonaterra
J. Cabrefiga, J. France
Institute of Food and Agricultural Technology-CIDSAV-XaRTA, University of Girona, Girona, Spain

Keywords
biocontrol, Erwinia amylovora, fire blight,
freeze-drying, osmoadaptation, protective
agents, Pseudomonas fluorescens.
Correspondence
Anna Bonaterra, Institute of Food and Agri-
cultural Technology-CIDSAV-XaRTA, University
of Girona, 17071 Girona, Spain.
E-mail: anna.bonaterra@udg.edu
2014/0619: received 24 March 2014, revised
9 June 2014 and accepted 17 June 2014
doi:10.1111/jam.12582
Abstract
Aims: To study the effect of lyoprotectants and osmoadaptation on viability of
Pseudomonas fluorescens EPS62e during freeze-drying and storage and to
evaluate the formulation in terms of efficacy in biocontrol and fitness on pear
flowers.
Methods and Results: A wettable powder formulation of a biocontrol agent of
fire blight was optimized by means of lyoprotectants and culture
osmoadaptation. Freeze-drying was used to obtain dehydrated cells, and the best
viability (70% of survival) was obtained using lactose as lyoprotectant. Survival
during lyophilization was additionally improved using physiological adaptation
of cells during cultivation under salt-amended medium (osmoadaptation). The
procedure increased the survival of cells after freeze-drying attaining viability
values close to a 100% in the lactose-formulated product (3 9 1011 CFU g 1),
and through the storage period of 1 year at 4°C. The dry formulation showed
also an improved biocontrol efficacy and survival of EPS62e on pear flowers
under low relative humidity conditions.
Conclusions: Cell viability after freeze-drying was improved using lactose as
lyoprotectant combined with a procedure of osmoadaptation during
cultivation. The powder-formulated product remained active for 12 months
and retained biocontrol levels similar to that of fresh cells. The formulation
showed an improved survival of EPS62e on flowers and an increase of the
efficacy of biocontrol of fire blight at low relative humidity.
Significance and Impact of the Study: The results have a potential value for
commercial application in biocontrol agents not only of fire blight but also of
other plant diseases.

Several biocontrol agents (BCA) have been registered

Introduction
Fire blight of apple, pear and ornamental pomaceous
plants is a disease of economic importance across the world
that is caused by Erwinia amylovora (Van der Zwet et al.
2012). Currently, the management of fire blight requires
the combined use of different products like copper com-
pounds, antibiotics and biological control agents (Johnson
and Stockwell 1998; Van der Zwet et al. 2012). However,
the success of these products is variable year-to-year and
location-to-location as has been reported in a meta-analy-
sis of several field trials performed (Sundin et al. 2009).
and commercialized for the management of fire blight,
including the strains Pseudomonas fluorescens A506 (Wil-
son and Lindow 1993), Pantoea vagans C9-1 (Ishimaru
et al. 1988; Smits et al. 2010), Pantoea agglomerans E325
(Pusey 1999), P. agglomerans P10c (Vanneste et al. 2002),
Bacillus subtilis QST713 (Aldwinckle et al. 2002), B. sub-
tilis BD170 and Aureobasidium pullulans strains DSM
14940 and DSM 14941 (Kunz et al. 2011). Ps. fluorescens
EPS62e is a noncommercial fire blight biological control
agent that has been developed in our laboratory for its
high efficacy in controlling fire blight infections in pear

1122 Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
J. Cabrefiga et al.
and apple plants, and a good ecological fitness on pome
fruit tree surfaces, especially on flowers (Cabrefiga et al.
2007; Pujol et al. 2007).
However, the success of BCAs as plant protection
products requires the development of formulations with
high cell viability and storability and an activity similar
to that of the fresh-cultured cells (Powell and Jutsum
1993; Burgues and Jones 1998; Montesinos 2003; Monte-
sinos and Bonaterra 2009). Generally, microbial pesticides
are formulated as liquid products (water oils or emul-
sions of cell suspensions), or dry products (wettable pow-
ders, dusts and granules) (Schisler et al. 2004). Dried
formulations have the advantage of easy handling, trans-
portation and storage, but its production requires dehy-
dration of cell cultures to achieve a viable, stable and
active product.
Microbial cell survival throughout drying and storage
is dependent of many factors related to the process itself,
but also to the drying medium and the growth conditions
(Carvalho et al. 2004). Freeze-drying is the least damag-
ing method for drying micro-organisms, convenient for
gram-negative bacteria that are more sensitive to conven-
tional dehydration processes than other organisms (Pem-
brey et al. 1999; Miyamoto-Shinohara et al. 2000).
Freeze-drying requires the choice of an appropriate dry-
ing medium to increase survival rates during dehydration,
and various groups of substances, such as polyols, disac-
charides and polysaccharides, amino acids, peptides and
glycoproteins are currently used. Unfortunately, the pro-
tection afforded by a given additive during dehydration
will vary with the species of micro-organism, even at
strain level, so conditions should be evaluated and
adjusted for each specific strain (Rhodes 1993; Abadias
et al. 2001; Palmfeldt et al. 2003; Zhan et al. 2011; Mon-
tel Mendoza et al. 2013). The growth medium used for
culture the micro-organism is also a critical parameter,
and several factors like the presence of compatible solutes
can promote survival of bacteria throughout dehydration
and storage in the dried state (Carvalho et al. 2004; Li
and Tian 2006).
Moreover, there are specific problems to solve not
related to the technology of drying and storage. Biopesti-
cides used to control aerial plant pathogens are subjected
to some harsh environmental conditions once applied to
the plant surfaces in the field which could cause significant
declines in survival. In consequence, formulations have to
be improved using strategies to increase field stress toler-
ance and ecological fitness of the BCA. It has been reported
that lyophilized products have better survival rates on tree
surfaces, indicating that the freeze-drying itself induces
some cell tolerance to draught (Stockwell et al. 1998; Pusey
and Wend 2012). In addition, several studies have reported
that the accumulation of compatible solutes under osmotic
Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
A dry formulation of Pseudomonas fluorescens
stress conditions increases efficacy of biocontrol of fire
blight in Ps. fluorescens EPS62e (Bonaterra et al. 2007;
Cabrefiga et al. 2011) and improves survival in P. agglom-
erans E325 (Pusey and Wend 2012). These findings suggest
that the combination of both strategies osmoadaptation
and freeze-drying could provide a beneficial effect to
EPS62e formulation.
In the present work, we studied the effect of a com-
bined strategy of culture osmoadaptation and the addi-
tion of lyoprotectants before the freeze-drying process on
(i) cell viability after freeze-drying, (ii) activity of the
product during storage, (iii) survival on tree surfaces and
(iv) efficacy of fire blight control.
Materials and methods
Bacterial strains and growth conditions
Pseudomonas fluorescens EPS62e was cultured, for routine
use, in Luria-Bertani (LB) broth to obtain standard cells
(SC) or in glucose minimal medium (GMM) plus NaCl
and glycine betaine (GB) to obtain osmoadapted cells
(OC) as previously described (Bonaterra et al. 2007). To
monitor populations of EPS62e on flowers of rosaceous
plants, a spontaneous mutant resistant to nalidixic acid
(EPS62e NAL+) was selected on LB agar supplemented
with 50 mg l 1 of nalidixic acid. Both strains are efficient
in the biocontrol of fire blight and display similar growth
characteristics under laboratory conditions (data not
shown). Erwinia amylovora EPS101 was used in pathogen
inoculation experiments (Cabrefiga and Montesinos
2005). Ultra-freeze-preserved cultures ( 80°C) of the
pathogen and EPS62e were grown overnight at 25°C in
LB agar. Colonies were scraped from the agar surface and
suspended in sterile distilled water. The cell culture was
adjusted to an optical density (OD) corresponding to
1 9 108 CFU ml 1 and diluted with sterile distilled water
to obtain an appropriate concentration.
For experiments, EPS62e was precultured in 300 ml in a
1000-ml baffled Erlenmeyer flask at 100 rev min 1 and
25°C for 12 h in a rotary shaker (Innova 4335, New Bruns-
wick Scientific, Edison, NJ). Precultures were inoculated
into a 5-l Biostat-B bioreactor (B. Braun Biotech, Melsun-
gen, Germany). For inoculation, the preculture was pre-
pared with the same medium composition as the medium
used in the experiment: LB to obtain SC or GMM amended
with NaCl and GB to obtain OC. The operating conditions
of the bioreactor were 25°C and pH = 7. Aeration was car-
ried out by sparging with air at a gas flow of 1
5 l min 1.
Air saturation was controlled to 30% by automatic regula-
tion of the stirring speed, which was in the range 10–
500 rev min 1, depending on oxygen consumption rate.
The pH was adjusted by addition of 2 mol l 1 NaOH and
1123
A dry formulation of Pseudomonas fluorescens
2 mol l 1 HCl. Cells grown in standard conditions (SC) or
osmoadapted (OC) were harvested at the beginning of the
stationary phase (12 h) by centrifugation, 10 000 g, for
10 min at 15°C.
Survival of EPS62e after freeze-drying with different
lyoprotectants
Cells of EPS62e grown in LB, therefore obtained in stan-
dard conditions (SC), were resuspended in a pH 7 physio-
logical buffer (PB) containing per litre 8 g of NaCl, 0
4 g
of NaH2PO4 2H2O and 2
7 g of Na2HPO4 12H2O. The cell
suspension was divided into three replicate aliquots of
10 ml and mixed with the cryoprotectants to obtain a total
amount of cells of 3–5 9 1011 CFU. The protectants used
were lactose (100 and 150 g l 1), skimmed milk (100 and
150 g l 1), sucrose (100 and 150 g l 1), starch 70 g l 1,
trehalose 100 g l 1, lactose 100 g l 1 plus starch 70 g l 1,
skimmed milk 100 g l 1 plus starch 70 g l 1, sucrose
100 g l 1 plus starch 70 g l 1, trehalose 100 g l 1 plus
starch 70 g l 1 and a treatment without cryoprotectant.
The bacterial solution was then placed on a stirrer, and
three replicates of each protectant were placed in an alu-
minium cup of 8 cm diameter and 5 cm depth (the prod-
uct had approx. 1 cm depth). For CFU counting of
samples before freezing, 10-fold serial dilutions of the bac-
terial solutions were plated onto LB agar using a spiral pla-
ter system (Eddy Jet, IUL Instruments, Barcelona, Spain).
Plates were incubated at 25°C for 48 h and the colonies
counted using an automatic counter system (Countermat
Flash, IUL Instruments). The aluminium cups with formu-
lated bacterial solutions were placed on a tray and in a
freezer at 70°C. After overnight at 70°C, samples were
placed in a laboratory scale freeze-dryer (model Unitop
HL, VirTis, Gardiner, NY). Standard freeze-dying condi-
tions were used, which consisted of 10 Pa and a primary
drying for 24 h at 10°C followed by 8 h of secondary
drying at 15°C. Dried samples were weighted and stored in
plastic-coated aluminium bags which were sealed under
vacuum packaging. The number of viable cells after freeze-
drying was determined as colony forming units per g of
lyophilized product. Three samples for each treatment
were used to determine the number of viable cells. Prior to
CFU determination, 0
1 g of freeze-dried samples was re-
hydrated by addition of 9
9 ml of PB followed by 30-min
of incubation at 25°C in a rotary shaker. CFU counting
was performed as described above, and the survival was
calculated as the ratio between CFU of viable cells after
freeze-drying and the CFU of viable cells before freeze-dry-
ing and expressed as a percentage (Hamoudi et al. 2007).
The moisture contents (g H2O/100 g dry weight) of freeze-
dried samples were determined after drying at 105°C until
constant weight. The experiment was carried out in a com-
1124 Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
J. Cabrefiga et al.
pletely randomized design with three replications for each
lyoprotectant, each submitted to lyophilization. The whole
experiment was repeated twice.
Effect of osmoadaptation on EPS62e survival after
freeze-drying and storage
EPS62e SC and OC were obtained from cultures per-
formed in the bioreactor, collected at the beginning of
the stationary phase and harvested by centrifugation as
described above. SC and OC were resuspended in PB and
mixed with three freeze-drying protectants: lactose
150 g l 1 (LA), starch 70 g l 1 (ST) and lactose 150 g l 1
plus starch 70 g l 1 (LS) before freezing and lyophiliza-
tion. Three replicates of each treatment were performed.
The survival rate of OC formulated with either LA, ST or
LS was determined after lyophilization and compared
with the survival rate of SC also formulated with LA, ST
or LS. The experiment was set up as a factorial design in
a completely randomized manner and consisted of two
culture conditions (SC and OC), three lyoprotectants
(LA, ST and LS) and three replicates per lyoprotec-
tant 9 culture condition. The experiment was repeated
twice.
Dried samples of SC and OC formulated with freeze-
drying protectants LA, ST or LS were filled in plastic-
coated aluminium bags which were sealed under vacuum
packaging as described. Aluminium bags were stored for
a year at two different conditions, at 4°C and at 20°C.
Three replicates of each treatment were performed. Dried
samples were removed at different time intervals; at 1, 70,
134 and 346 days, the samples stored at 4°C, and at 1,
90, 202 and 345 days, the samples stored at 20°C. The
cell survival was assessed as described above, using PB as
rehydration medium. The experiment was arranged as a
factorial design in a completely randomized manner and
consisted of two culture conditions (SC and OC), three
lyoprotectants (LA, ST and LS) and three replicates per
lyoprotectant 9 culture condition. Three replicates per
lyoprotectant 9 culture condition were stored at two dif-
ferent temperatures 4C and 20°C.
Biocontrol assays
The effect of different formulations of EPS62e on control
of Erw. amylovora infections was tested on detached flow-
ers and immature fruits of Passe Crassane and Confer-
ence pear under controlled environmental conditions.
Plant material was collected from commercial fields,
taken to the laboratory and prepared individually as
explained previously (Cabrefiga et al. 2011).
For the assays performed at high relative humidity
conditions, individual flowers or immature fruits were
J. Cabrefiga et al.
treated with either suspensions of EPS62e cultured under
standard conditions (SC) or osmoadapted (OC), both
adjusted at 108 CFU ml 1 and PB as a nontreated con-
trol. Both treatments consisting on SC and OC were
applied either as fresh-cultured cells (i.e. nonformulated
and nonfreeze-dried) (NF) or formulated with LA, ST or
LS (i.e. cells were mixed with the protectants and freeze-
dried). In the case of nontreated control, PB was applied
alone (NF) or amended with LA, ST or LS. Three repli-
cates of eight flowers or three immature fruits were used
for each treatment. Flowers were treated by spraying and
immature fruits by immersion. Vials containing treated
flowers were placed in plastic tube racks, into plastic
boxes, and immature fruits were placed on plastic trays.
All plant material was maintained under controlled envi-
ronment chambers at 20°C, at 90% RH in the dark. After
24 h, hypanthia of flowers and wounds of immature
fruits were inoculated with 10 ll of a suspension of
Erw. amylovora EPS101 at 107 CFU ml 1. The inoculated
flowers and fruits were placed again in plastic boxes and
incubated at 20°C and high relative humidity (90%) for
5 days. Incidence of infections was evaluated per each
replicate after 5 days from pathogen inoculation as the
percentage of flowers or wounds of immature fruits with
a partial or total necrosis. The experiment was arranged
as a factorial design in a completely randomized manner
and consisted of two culture conditions (SC and OC)
and a nontreated control using PB, four formulations
(nonformulated nonfreeze-dried (NF), lactose freeze-
dried (LA), starch freeze-dried (ST) and lactose 9 starch
freeze-dried (LS)) and three replications per formula-
tion 9 culture condition, with eight flowers or three
immature fruits per replicate. Two experiments were per-
formed, one in Passe Crassane and another in Conference
pear cultivars.
For the assays performed at low relative humidity con-
ditions, flowers were treated by spraying with fresh-stan-
dard cultured cells (FSC), fresh-osmoadapted cells (FOC),
standard cultured cells formulated with lactose 150 g l 1
and freeze-dried (LASC), or osmoadapted cells formu-
lated with lactose 150 g l 1 and freeze-dried (LAOC).
Also two treatments were included consisting of PB and
PB plus lactose 150 g l 1, as nontreated controls. To
assess the effect of the treatments on control of Erw. am-
ylovora infections, three replicates of eight flowers were
used for each treatment. Vials containing treated flowers
were placed in plastic tube racks in controlled environ-
ment chambers at 20°C, at 50% RH in the dark (Cabrefi-
ga et al. 2011). After 24 h, hypanthia of flowers were
inoculated with Erw. amylovora and incidence of infec-
tions on flowers was evaluated per each replicate after
5 days from pathogen inoculation as explained before.
The experiment was set up in a completely randomized
Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
A dry formulation of Pseudomonas fluorescens
design with three replications for each treatment (non-
treated PB, nontreated PB plus lactose, EPS62e FSC,
EPS62e FOC, EPS62e LASC, EPS62e LAOC). Two experi-
ments were performed, one in Passe Crassane and
another in Conference pear cultivars.
To assess the survival of different formulations consist-
ing on EPS62e (FSC, FOC, LASC and LAOC) on detached
flowers, three replicates of 25 treated flowers per replicate
were used for each treatment. Sampling for monitoring
population levels of EPS62e was performed at 0 and 5 days.
Samples of five flowers were taken from each replicate and
sampling date. Flowers were homogenized in a sterile plas-
tic bag with 20 ml of 0
05 mol l 1 phosphate buffer (pH
7
0) and 0
1% peptone using a stomacher (Masticator, IUL
Instruments). Extracts obtained were serially diluted, and
appropriate dilutions were seeded using a spiral plater sys-
tem (Eddy Jet, IUL Instruments) onto LB agar plates. The
LB plates were supplemented with 50 lg ml 1 of nalidixic
acid to counterselect the strain inoculated and with
50 lg ml 1 of econazole nitrate salt to avoid fungal
growth. Plates were incubated at 25°C for 48 h and the col-
onies counted using an automatic counter system (Count-
ermat flash, IUL Instruments). The population level of
EPS62e was expressed as log10 CFU per flower. The experi-
ment was set up in a completely randomized design with
three replications for each treatment (FSC, FOC, LASC and
LAOC). Two experiments were performed, one in Passe
Crassane and another in Conference pear cultivars.
Data analysis
To test for the effect of formulation on the EPS62e sur-
vival after freeze-drying, and on the biocontrol efficacy
against Erw. amylovora, a one-way ANOVA was performed.
Also, a two-way ANOVA was used to test the effect of for-
mulation and of the cultivation method of EPS62e cells
on survival after freeze-drying and on biocontrol efficacy
against Erw. amylovora. Means were separated according
to the Waller–Duncan test at P < 0
05. Statistical analyses
were performed using the GLM procedure of the PC-
SAS. (Ver. 9.1, SAS institute Inc., Cary, NC).
Results
Effect of lyoprotectants on survival after freeze-drying
Survival and moisture contents of EPS62e freeze-dried
products using different protectants are shown in Table 1.
Survival after the freeze-drying process was significantly
influenced by the type of protectant (P < 0
0001). The high-
est percentage of survival and cell concentration (70
9% and
2 9 1011 CFU g 1, respectively) was obtained with lactose
at 150 g l 1. In contrast, when EPS62e cells were freeze-
1125
A dry formulation of Pseudomonas fluorescens J. Cabrefiga et al.

Table 1 Survival of EPS62e* cells after freeze-drying when different freeze-drying media were used

Survival after freeze-drying†

Concentration
Freeze-drying medium (g l 1) log CFU g 1
%§ Moisture content, %¶

Lactose 150 11
3  0
2 70
9 a 4
4 cde

Sucrose 100 11
0  0
2 50
8 b 7
2 ab
Trehalose 100 11
0  0
4 44
6 b 6
0 bc
Skimmed milk 150 10
7  0
1 36
7 bc 3
7 cde
Lactose 100 11
0  0
5 36
3 bc 6
3 bc
Sucrose 150 10
9  0
3 34
2 bc 8
8 a
Skimmed milk 100 10
8  0
2 24
3 cd 5
1 bcd
Sucrose + starch 100 + 60 10
2  0
1 19
5 cd 3
6 de
Trehalose + starch 100 + 60 9
9  0
1 12
0 de 4
7 cd
Lactose + starch 100 + 60 9
7  0
3 10
0 de 3
2 de
Skimmed milk + starch 100 + 60 9
3  0
3 7
4 de 2
2 ef
Starch 60 7
6  0
4 0
0 e 0
9 f
Control‡ – 6
4  0
5 0
0 e 5
3 bcd

*Initial cell concentrations from an exponential concentrated culture were in the range of 3–5 9 1011 CFU.
†Mean and confidence interval were calculated from three independent samples. Samples were collected immediately after freeze-drying.
‡Control samples contained cells suspended in physiological buffer.
§Different lowercase letters indicate significant differences between freeze-drying medium (P < 0
05) according to the Waller-Duncan test.
¶Different lowercase letters indicate significant differences between freeze-drying medium (P < 0
05) according to the Waller-Duncan test.

dried without protective agents, the viability decreased more
than 4 log units. Freeze-dried products formulated with lac-
tose at 100 g l 1, sucrose (at 100 and 150 g l 1), trehalose
at 100 g l 1 and skimmed milk (100 and 150 g l 1) main-
tained cell concentrations between 6
5 9 1010 and
1
8 9 1011 CFU g 1, provided significantly lower survival
after freeze-drying process that was from 24 to 50%. The
moisture contents in the EPS62e freeze-dried products were
between 0
9 and 8
8%. The lowest moisture content was
obtained in formulations protected with 70 g l 1 starch
(0
9%). In contrast, products formulated with sucrose had
the highest moisture content (7
2 and 8%). The rest of
tested protectants ranged from 2
2 to 6% of moisture con-
tent, without significant differences between them. Globally,
lactose at 150 g l 1 was the best lyoprotectant in terms of
viability and moderate moisture content.
Effect of osmoadaptation and lyoprotectants on EPS62e
survival after freeze-drying
Viability of cells obtained by standard cultivation (SC) or
preadapted by osmoadaptation (OC) after freeze-drying
using lactose (150 g l 1), starch (70 g l 1) or starch plus
lactose (70 and 150 g l 1, respectively) as protectants is
shown in Fig. 1. The ability of EPS62e to survive the
freeze-drying process greatly depended on the method of
cultivation (SC or OC) (P < 0
0001) and the freeze-dry-
ing protectants (P < 0
0001). EPS62e osmoadapted cells
showed greater viability than cells cultured in standard
way. As has already been observed, the highest viability
and cell concentration were obtained using 150 g l 1 of
lactose as lyoprotectant (around 3 9 1011 CFU g 1 and
100% of viability). Neither starch nor the combination of
lactose with starch achieved the levels of cell protection
attained by lactose. For example, OC formulated with lac-
tose showed high survival after freeze-drying with per-
centages of viability around 99% in both experiments
performed. In contrast, freeze-dried products of cells
obtained by culture under standard conditions also for-
mulated using lactose showed lower survival with per-
centages of viability from 58 to 67%.
Shelf-life of freeze-dried EPS62e formulations
Shelf-life of freeze-dried EPS62e-formulated products
stored at 4°C  1 and at 20°C  1 during 12-month
storage is shown in Fig. 2. There were significant differ-
ences between the survival of the formulated products
stored at 4°C  1 (Fig. 2, top panels) and at 20°C  1
(Fig. 2, bottom panels). In general, all formulations
stored at 4°C showed a better survival through the stor-
age period than formulations stored at 20°C. EPS62e for-
mulated with lactose showed higher survival upon storage
than the formulations with starch, and lactose plus
starch. Initial survival of OC with lactose as lyoprotectant
was around 3 9 1011 CFU g 1, and it was slightly
reduced to 9 9 1010 CFU g 1 after 12 months of storage,
whereas SC formulated with lactose had a reduction to
1 9 1010 CFU g 1. The same treatments, SC and OC
formulated with lactose but stored at 20°C showed a
decrease of survival to 1
8 9 105 CFU g 1 and to
1 9 107 CFU g 1, respectively.

1126 Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
J. Cabrefiga et al. A dry formulation of Pseudomonas fluorescens

compared with the nontreated control in both pear culti-

aA' Exp. 1
100 vars, in flowers and in immature fruits (Fig. 3). However,
there were no significant differences in effectiveness
80
bA between SC and OC, neither the incidence of infections
aB'
60 was significantly affected by formulations used as freeze-
40
drying protectants (SC or OC), in flower (P = 0
4733
bB and P = 0
5269) or in fruit assays (P = 0
0578 and
Viability (%)

20 P = 0
6423). EPS62e cells (SC or OC) lyoprotected with

aC aC' lactose showed a similar level of control than the fresh
aA'
Exp. 2 cells in all experiments carried out with flowers or fruits.
100
In contrast, cells formulated with starch or with a combi-
80 bA
nation of lactose and starch showed lower biocontrol in
some experiments (in one out of four experiments in the
60 aB'
case of LS and in two out of four experiments in the case
40 of starch).
20 bB Under low RH conditions, infection incidence signifi-
aC aC' cantly decreased by all EPS62e treatments (FSC, FOC,
LA ST LS LASC and LAOC) in both trials performed (Fig. 4). The
Cryoprotectant treatment consisting of formulated (lyoprotected with
lactose) and osmoadapted cells (LAOC) showed better
Figure 1 Viability of freeze-dried Pseudomonas fluorescens EPS62e efficacy than FSC, FOC and LASC in both experiments.
cells obtained by standard cultivation (SC) ( ) or preadapted by The efficacy of LAOC ranged from 73 to 86%, whereas in
osmoadaptation (OC) ( h ) in the presence of the cryoprotectants lac-
the FSC, FOC and LASC ranged from 36 to 39% in the
tose (LA), starch (ST) and lactose plus starch (LS). Values are the mean
of three replicates. The experiment was repeated two times (1 and
first experiment and from 46 to 53% in the second
2). Different capital letters show significant differences between pro- experiment. In the treatments with fresh-cultured cells,
tectants (P < 0
05), and different lowercase letters indicate significant the efficacy of FOC did not differ significantly from FSC
differences between the method of cultivation (P < 0
05) according in both experiments performed.
to the Waller-Duncan test. The population levels of EPS62e on flowers inoculated
with the pathogen and incubated under low RH condi-
Standard Osmoadapted tions for 5 days were affected by the formulation. There
12 were significant differences between FSC, FOC, LASC and
10 LAOC in both experiments performed. Population levels
4ºC
Survival (Log10 CFU g–1)

8 for LAOC were significantly higher (around 6 to

6
7 9 106 CFU per flower) than those in FOC treatment
4
2 (3 9 106 CFU per flower) in both assays made with
Conference and Passe Crassane pear flowers. In contrast,
12
population levels in LASC (3 9 105 CFU per flower)
10
20ºC

8 were lower than in FSC treatment (1 9 106 CFU per

6 flower).
4
2
0 100 200 300
Days
Figure 2 Effect of lyoprotectants (lactose ( ), starch (○) and lac-
tose-starch ( ) and of the method of cultivation (standard or osmo-
adaptated) on survival of EPS62e after freeze-drying. Samples were
maintained at 4 or 20°C.
Effect of culture osmoadaptation combined with
lyoprotection on the biocontrol efficacy
At high relative humidity conditions, SC and OC
decreased infection incidence by Erw. amylovora
Discussion
0 100 200 300
Exploitation of microbial biocontrol agents requires suit-
able production and formulation to increase shelf-life,
● and to retain biocontrol activity similar to that of freshly
prepared cells (Powell and Jutsum 1993; Burgues and
Jones 1998). Different strategies such as seed coating or
pellet, alginate encapsidated or powder talc have been
used in several studies to formulate biopesticides consist-
ing of Pseudomonas strains to be applied in soil (Russo
et al. 1996; Bashan and Gonzalez 1999; Ali et al. 2001;
Pedersen et al. 2002; Wiyono et al. 2008). As the envi-
ronmental conditions in the agricultural soil are generally

Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology 1127
A dry formulation of Pseudomonas fluorescens J. Cabrefiga et al.

Conference Passe Crassane

100 a a a a
a a a a
80

Flowers
60
b b
b bb b
Incidence of infections (%)

40 b b bb b b
b b b Figure 3 Effect of treatments consisting of
b standard ( ) and osmoadapted (h) cultures
20
of Pseudomonas fluorescens EPS62e and
nontreated (&), on incidence of infections by
100 Erwinia amylovora in flowers and immature
fruits incubated at high relative humidity

Immature fruit
80 a a
a (>90%). Cells were freeze-dried with lactose
a a aa a a (LA), starch (ST), lactose-starch (LS) or
60 a
nonfreeze-dried, and without protectant (NF).
40 ab b Two independent experiments were
b b
b performed. Values are the mean of three
20 bb bb bb b
b b replicates. Bars with the same letter do not
0 differ significantly (P < 0
05) between
NF LA ST LS NF LA ST LS treatments according to the Waller–Duncan
Cryoprotectant test.

Conference
100 A
a B surfaces are exposed to changes in water availability and
C 6
Population level (log CFU flower–1)

80 a
D temperature, UV light and limitation in nutrients, condi-
tions that can be transiently inadequate for bacterial
Incidence of infections (%)

60 b b b
40 4 growth (Stockwell et al. 1998; Lindow and Brandl 2003).
Therefore, the products used to control aerial plant dis-
20 c
eases have to be protected to increase their tolerance to
Passe Crassane the harsh conditions of the phyllosphere and to improve
100 a A
a B their efficiency of control. The majority of biopesticides
C 6
80 D used to control aerial plant diseases and in particular
b b b
60 those used to control fire blight are formulated as wetta-
4 ble powders like Ps. fluorescens A506 at concentrations of
40 c
1 9 1010 to 1 9 1011 CFU g 1 (Stockwell and Stack
20
2007).
In the present study, a formulation of Ps. fluorescens
PB

C
PB

SC

EPS62e was optimized by osmoadaptation during cultiva-

FS

FO

O
LA

LA

LA

tion and by the addition of a lyoprotectant prior to

Figure 4 Effect of treatments of EPS62e cells consisting of osmoad- freeze-drying. The method used to obtain a wettable pow-
aptation during cultivation and/or lyoprotection during freeze-drying, der formulation of EPS62e was freeze-drying. This tech-
on fire blight infections and cell survival in pear flowers under low rel- nology requires the selection of a suitable protectant to
ative humidity conditions. Treatments were fresh-standard cultured
preserve the cells during dehydration to increase their sur-
cells (FSC), fresh-osmoadapted cells (FOC), standard cultured cells for-
vival. We evaluated the efficacy of twelve lyoprotective
mulated with lactose and lyophilized (LASC), and osmoadapted cells
formulated with lactose and lyophilized (LAOC). The treatments were media during the freeze-drying process. Although a total
compared with a nontreated controls consisting of physiological buf- cell survival after dehydration was not attained, the best
fer without lactose (PB) or mixed with lactose (LAPB). Bars for inci- viabilities were obtained using disaccharides, specially lac-
dence with the same lowercase letter do not differ significantly tose but also sucrose and trehalose. The excessive cost of
(P < 0
05) according to Waller–Duncan test. Population levels of trehalose and the high moisture content attained with
EPS62e in blossoms were assessed 5 days after application. Error bars
sucrose (around 8%) limit its use in our case. Therefore,
represent the 95% confidence interval of the mean. Values with the
lactose was the selected lyoprotectant for further studies.
same capital letter do not differ significantly (P < 0
05).
The use of lactose as protectant in the formulation of
favourable to microbial growth, biopesticides can be eas- the BCA may have unexpected effects, like potentiation
ily applied mixed with seeds or with a substrate reaching of the pathogen activity. However, in a previous study
a satisfactory effectiveness. In contrast, aerial plant comparing nutrient use by Ps. fluorescens EPS62e and

1128 Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
J. Cabrefiga et al.
Erw. amylovora (Cabrefiga et al. 2007), it was confirmed
that neither EPS62e nor several strains of Erw. amylovora
used D-Lactose as a carbon source. Moreover, in the
present work, we have demonstrated that a dry formula-
tion of EPS62e including lactose was as effective as the
fresh cells without formulation, which is an indication
that the inhibition of infections of Erw. amylovora was
not affected by lactose. In addition, the incidence of
infections by Erw. amylovora in flowers and immature
fruits treated with lactose remained at the same levels
than the nontreated control, supporting that lactose did
not increase the pathogen infection.
In the present study, we confirmed that osmoadapta-
tion increased the survival of EPS62e cells close to a
100% of viability during freeze-drying and also during
storage for 1 year at 4°C, attaining close to a 30% of via-
bility (9 9 1010 CFU g 1) in the lactose-formulated
product, and this could be likely to the accumulation of
osmolytes into the cells (Bonaterra et al. 2007). In previ-
ous reports, a preadaptation methodology of EPS62e
using osmoadaptation was shown to increase drought
stress tolerance and improved the epiphytic survival of
cells on rosaceous plants (Bonaterra et al. 2007). The
hyperosmotic stress (using high NaCl concentrations
amended with glycine betaine (GB) in the media during
cultivation)-induced cells to accumulate GB by transpor-
tation and the disaccharide trehalose, the dipeptide
N-acetylglutaminylglutamine and the heterosid glucosyl-
glycerol by de novo biosynthesis. Other studies performed
with different micro-organisms have reported that osmo-
adaptation allows cells to tolerate adverse conditions such
as drought or salinity, but also freezing and high temper-
atures (Ramos et al. 1997; Welsh and Herbert 1999; La-
mosa et al. 2000). It was hypothesized that this
accumulation of compatible solutes might provide to
EPS62e a protection not only against the harsh condi-
tions of the aerial surfaces of plants (Bonaterra et al.
2007) but also against the freezing and drought stress
during the freeze-drying process (Pusey and Wend 2012).
Although the mechanisms by which compatible solutes
protect cells during drying and subsequent storage have
not yet been elucidated, it has been suggested to operate
through protection of the membrane by direct hydrogen
bonding with phospholipid head groups maintaining a
crystal state (Leslie et al. 1994) and stabilizing proteins by
water replacement (Csonka and Hanson 1991; Potts
1994).
Fire blight infections of flowers and immature fruits of
pear were controlled effectively by the dry wettable pow-
der of Ps. fluorescens EPS62e obtained by osmoadaptation
and formulated with lactose before freeze-drying, with
the same levels of control than fresh cells, activity that is
maintained even after 1 year of storage at 4°C. Moreover,
Journal of Applied Microbiology 117, 1122--1131 © 2014 The Society for Applied Microbiology
A dry formulation of Pseudomonas fluorescens
under low RH conditions, osmoadapted and formulated
EPS62e cells showed better efficacy than fresh cells (either
osmoadapted or under standard cultivation), and also
than formulated cells without osmoadaptation. The pow-
der formulation of EPS62e developed here has an addi-
tional beneficial effect in terms of increasing population
levels on flowers under low RH conditions.
It is finally concluded that the dry formulation devel-
oped in this study increased viability of cells after freeze-
drying and long-term storage, and in addition improved
survival and biocontrol ability of fire blight on flowers at
limiting RH conditions.
Acknowledgements
Funding was partially provided by Spain MINECO
(AGL2009-13255-c02-01 and AGL-2012-39880-C02-01)
and FEDER of the European Union. The research group
is accredited by SGR 2009-0812 and TECNIO net from
Catalonia. This work was conducted within the European
Science Foundation supported research network COST
Action 864.
Conflict of Interest
No conflict of interest declared.
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