Penicillium frequentans - Monilinia

Effects of different biological formulations of Penicillium frequentans
on brown rot of peaches
Abstract
B. Guijarro a , P. Melgarejo a, *, R. Torres b , N. Lamarca b , J. Usall b , A. De Cal a
a Department of Plant Protection, INIA, Ctra. de La Coruña km. 7, 28040 Madrid, Spain
b Area de Postcosecha, IRTA, Avda, Alcalde Rovira Roure 177, 25198 Lleida, Spain
Received 26 January 2007; accepted 30 March 2007
Available online 7 April 2007
Four wettable powder formulations of Penicillium frequentans conidia with measurable viability of one year and an improved adherence
to peach surfaces were produced by the addition of various ingredients, in two separate steps of the production and drying processes,
to conidia. These formulations were then evaluated against brown rot of peach fruit caused by Monilinia spp. Formulations
were applied to fruit either as postharvest treatments or before harvest in field treatments to peach trees. In the case of postharvest treatments
to fruit, reductions of brown rot were obtained with all P. frequentans formulations. Treatments applied before harvest were tested
in six field experiments in peach orchards in Spain. A total of 100 fruits, randomly selected in each orchard, were used as the sample unit
and every treatment was repeated four times. Results showed that P. frequentans formulations significantly reduced the inoculum density
of the pathogen (measured as the number of conidia on peach surface) in five trials out of the six tested, better than a chemical fungicide
that only showed a reduction of the pathogen conidial in two of the six trials. The relationship between the number of conidia of the
pathogen and the incidence of brown rot disease is discussed.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Biocontrol; Formulation; Monilinia laxa; M. fructigena; M. fructicola; Penicillium frequentans; Peach; Postharvest; Prunus persica
1. Introduction
Brown rot of peaches (Prunus persica (L.) Batch) is
caused in the European Mediterranean areas by two fungi,
Monilinia laxa (Aderh et Rulh) Honey and Monilinia fructigena
(Aderhold et Ruhl) Honey ex Whetzel (De Cal and
Melgarejo, 1999). M. laxa is the most common species isolated
from brown rot peaches and nectarines (isolated from
85–90% brown rot fruit) in Spain and Italy (Tian and Bertolini,
1999; Larena et al., 2005), followed by M. fructigena
(isolated from 10–15% of fruit affected by brown rot)
(Larena et al., 2005). Ripening fruits are the most susceptible
developmental stage (Xu et al., 2007), but the infections
may also be numerous in opening flowers (Watson et al.,
2002; Fourie and Holz, 2003). Conidia produced on over-
* Corresponding author. Fax: +34 1 3475722.
E-mail address: melgar@inia.es (P. Melgarejo).
1049-9644/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2007.03.014
Biological Control 42 (2007) 86–96
www.elsevier.com/locate/ybcon
wintering fruit mummies, fruit stalks, scars, and buds, as
well as in cankerous lesions (Byrde and Willetts, 1977;
Batra, 1985) infected by Monilinia spp. serve as primary
inoculum sources and cause blossom blight in the spring
(Byrde and Willetts, 1977). Infection of fruit by conidia
of these two fungi on infected blossoms, mummies, aborted
fruit, or other diseased tissues occurs secondarily during
the growing season, depending on weather conditions
(Byrde and Willetts, 1977; Borve and Stensvand, 2004).
When microclimatic conditions are unfavourable, infections
may remain latent until conditions become favourable
for disease expression, which finally lead to fruit rot
(Byrde and Willetts, 1977). Postharvest losses are typically
more severe than preharvest losses and routinely occur during
storage and transport (Ogawa and English, 1991).
When conditions are favourable for disease development,
postharvest losses may be high, reaching in some cases
values of 80–90% (Hong et al., 1997, 1998).

Management of postharvest brown rot decay of peaches
and nectarines begins with preharvest disease control programs.
Currently, the most effective programs rely on the
use of fungicides applied during bloom and preharvest, as
well as postharvest fungicide treatments in those countries
where they are allowed. Chemical control of brown rot in
peach growing Mediterranean areas is done by applying
benzimidazole, dicarboximide, or IBS fungicides (Hong
et al., 1998) as preharvest treatments. With the development
of integrated production systems in the various European
peach growing regions (as in Spain or Italy),
postharvest fungicide applications are no longer allowed
(Malavolta et al., 2003). In addition, resistance to benzimidazole
and dicarboximide fungicides is well documented
in Monilinia spp., especially in M. fructicola populations
(Penrose et al., 1979; Penrose, 1990; Elmer and Gaunt,
1993; Sanoamuang and Gaunt, 1995). New environmentally
friendly alternative control methods are being sought
based on the demands of consumers and environmental
protection agencies.
Biological control of plant disease is currently receiving
increased research effort in order to enhance the sustainability
of agricultural production systems and to reduce
the use of chemical pesticides in these systems (Johnson,
1994). Postharvest inoculants are among the market leaders
of plant disease biocontrol agents, since storage conditions
tend to be near constant and well defined, facilitating
the selection of adapted antagonists for consistent performance.
However, in the case of preharvest applications
many microorganisms with good potential fail to be developed
for practical use, primarily due to inconsistent performance
associated with storage and, often, with adverse
environmental conditions (Cartwright and Benson, 1995).
Several factors determine the success of an antagonist
including the ability to compete with other microorganisms,
unfavourable or fluctuating moisture levels, availability
of nutrients, shelf-life, storage methods, the
concentration of antagonist used, and timing and method
of application (Baker and Cook, 1974; Campbell, 1989).
The potential of Penicillium frequentans (Westling), a
component of the resident mycoflora of peach twigs and
flowers (Melgarejo et al., 1985), for biological control of
peach twig blight caused by M. laxa, has been demonstrated
in the field in experimental orchards after artificial
inoculation of the pathogen (Melgarejo et al., 1986; De
Cal et al., 1990; Madrigal et al., 1994). P. frequentans has
also been used experimentally for biocontrol of other diseases
(Roberti et al., 2002; Yamaji et al., 2005). Recently,
De Cal et al. (2002) developed a mass production method
for P. frequentans conidia by solid-state fermentation.
The conidia that were dried by fluid bed-drying, maintained
28% viability after 180 days of storage at room temperature,
and were as efficient as fresh conidia in
controlling brown rot of peaches (Guijarro et al., 2006).
A statistically significant relationship between biocontrol
efficacy of P. frequentans and conidial viability was demonstrated
by Guijarro et al. (2007): conidial viability of P. fre-
B. Guijarro et al. / Biological Control 42 (2007) 86–96 87
quentans greater than 40% was needed for obtaining more
than 50% biocontrol (Guijarro et al., 2007). It is necessary
to add additives to conidia produced in solid-state fermentation
and dried afterwards to improve the shelf-life of P.
frequentans conidia and to obtain a high levels of effectiveness
in controlling brown rot of stone fruits (Guijarro
et al., 2007). Initially, adherents were added to conidia in
order to improve P. frequentans adhesion to fruit surfaces,
and effectiveness in controlling brown rot of stone fruit was
also improved (Guijarro et al., unpublished). Additional
improvements in formulation and methods of application
of formulated P. frequentans conidia will be needed before
this organism can be developed into a commercial product,
nevertheless, P. frequentans is a promising biological control
agent for brown rot and peach twig blight (Melgarejo
et al., 1986; De Cal et al., 1990, 2002; Guijarro et al., 2006,
2007).
The main objective of this study was to investigate the
viability, stability, and adhesion of spores in several formulations
of P. frequentans to achieve control brown rot.
Reductions in the incidence of brown rot were obtained
after postharvest application of several formulations.
Reduction of inoculum density (the number of conidia on
fruit surfaces) was also obtained after preharvest
applications.
2. Materials and methods
2.1. Cultures
The isolate of P. frequentans (ATCC number 66108) was
obtained from the mycoflora of peach twigs and flowers in
Spain (Melgarejo et al., 1985). The fungi were stored on
potato dextrose agar (PDA) (Difco, Detroit, MI, USA)
slants at 4 °C and grown in darkness at 22 ± 2 °C for 5
days for conidial inocula production.
An isolate of M. laxa (ATCC number 66106) collected
from a commercial apricot orchard in Almonacid de la
Sierra (Zaragoza, Spain), was used in in vivo experiments.
This isolate was cultured on Petri dishes containing potato
dextrose agar (Difco, Detroit, MI, USA) amended with 1%
v/v acetone as described (Pascual et al., 1990). The dishes
were incubated at 22 ± 2 °C for 7–15 days in the dark.
Ten millilitres of sterile distilled water were poured into
each Petri dish to harvest conidia. Conidia suspensions
were filtered through glass wool before inoculations and
were adjusted to 10 3 conidia ml 1 .
2.2. Formulations of P. frequentans
Conidia of P. frequentans were produced in a solid fermentation
system as previously described (De Cal et al.,
2002). The fungus was grown on a mixture of peat (Gebr.
BRILL substrate GmbH&Co. KG, Germany): vermiculite
(Termita, Asfaltex, S.A., Barcelona, Spain): and lentil meal
(2:2:1, w:w:w). Five hundred grams of the substrate (40%
moisture content) were placed in a plastic bag (600 cm 3 )

88 B. Guijarro et al. / Biological Control 42 (2007) 86–96
especially designed for solid fermentation (Valmic R Spawn
Bag, Sacherei de Pont-Audemer S.A., Pont-Audemer
Cedex, France), sealed, and sterilized by autoclaving at
1.2 kg cm 2 and 120 °C for 1 h on three consecutive days.
Bags were then inoculated with a conidial suspension of
P. frequentans in sterile distilled water, to obtain 10 5 conidia
g 1 dry substrate, sealed again and incubated in darkness
at 20–25 °C for five days. Conidia were obtained from
solid fermentation bags by suspending the infested substrate
in sterile water. Suspensions of conidia from solid
fermentation were then shaken in a rotary shaker (Lab-
Line Instruments, Inc., model 3527, Melrose Park, IL,
USA) at 160 rpm for 30 min, filtered through glass wool,
washed with sterile distilled water, and concentrated by
centrifugation at 10,000 rpm (Sorvall RC5C Plus, GMI,
MN, USA) for 10 min, giving pellets of fresh conidia.
The final yield was 10 8–9 conidia g 1 dry weight of substrate
with viability higher than 80% (De Cal et al., 2002).
Conidia were dried in a fluid bed-dryer to reduce the
moisture content below 15%. Freshly harvested conidia
were resuspended in sterile distilled water and filtered
through 1 lm filter paper in a Büchner funnel (Guijarro
et al., 2006). This conidial paste was introduced in a fluid
bed-dryer (FBD model 350s, Burkard Manufacturing Co.
Ltd., Hertfordshire, UK) at the highest air flow rate at
40 °C (Guijarro et al., 2006). The moisture content of each
final product of conidia was measured using a humidity
analyzer (BOECKEL, GmbH+Co., Hamburg, Germany).
Germination of dried conidia was tested by the bioassay
described in Guijarro et al. (2006), always resulting above
80%. Fresh conidia resuspended in water and dried were
named FCW.
Four formulations of P. frequentans (FOR 3, FOR 4,
FOR7, and FOR8) were produced. Formulations FOR3,
FOR4, and FOR7 were obtained as follows: suspensions
of P. frequentans conidia (10 5 conidia g 1 dry substrate)
were prepared in 10% glycerine (FOR3) and 2% sodium
alginate (FOR4 and FOR7) and added to substrate contained
in fermentation bags. After incubation, fresh conidia
were obtained as described above, and then
resuspended in 2% methyl cellulose. After maintaining
these suspensions for 10 min at room temperature, silica
gel (FOR3 and FOR7) or kaolinite (FOR4) were added to
obtain conidial pastes. In the case of FOR8, fresh conidia
were produced as described above without additives and
resuspended in 2% methyl cellulose + 10% glucose and
incubated for 10 min. Then, the silica gel was added to
obtain a paste. All conidial pastes were introduced into a
fluidized bed-dryer, and dried, as previously described
(Guijarro et al., 2006). As a positive control of adherence
1.3% of a commercial adhesive (96% di-menteno, Nufilm-17
(Miller Chemical and Fertilizer Co., Hanover,
Pennsylvania, USA) was used. Fresh conidia were resuspended
in 5 ml solution of Nu-film-17, and shaken on a
vortex mixer (Reax Top, Heildoph, Rose Scientific Ltd.
Alta., Canada). Conidial suspensions were then filtered
through 1 lm filter paper in a Büchner funnel, giving a
conidial paste. Conidial pastes were dried in a fluid beddryer.
2.3. Viability and adhesion of P. frequentans formulations
under different storage conditions
To monitor the shelf-life of P. frequentans, formulations
were stored in different conditions. Each formulation was
stored in Eppendorff microfuge tubes at 4 °C or at room
temperature (ambient), and under vacuum or without vacuum
(three replicates for each combination). Three formulations
were tested: FOR3, FOR7, and FOR8, and FCW
conidia without additives were used as control. Conidia
were packaged under vacuum using a vacuum chamber
machine (Audionvac Elektro VM 101 H, AJ WEESP,
The Netherlands) (at 0.96 · 10 6 Pa for 90 s). Conidial viability
was estimated by measuring germination according
to the bioassay described by De Cal et al. (1988) immediately
after fluid bed-drying and again after 30, 90, 180,
and 365 days of storage. Conidia were resuspended in sterile
distilled water to obtain concentrations of 1 · 10 6
conidia ml 1 , shaken in a laboratory mixer (MS 2 Minishaker
Ika-Works, Inc., USA) operated at full speed
(2500 rpm) for 1 min and then incubated at room temperature
(25 °C) for 1 h. After 1 h sterile glass slides were
placed in glass 15-mm diameter Petri dishes lined with
moistened Whatman paper. On each slide, a 15 ll droplet
of diluted conidial suspension of P. frequentans (10 6
conidia ml 1 ) was mixed with a 30 ll droplet of sterile Czapek
broth (Difco, Detroit, MI, USA). Slides were incubated
for 16 h at 20–25 °C in darkness, after which the
germination of 50 conidia was assessed in each replicate.
Three drops were made for each sample, and the whole
experiment was repeated twice. A spore was considered
to have germinated when a germ-tube was longer than
the length of the spore. The moisture content of each sample
was measured using a humidity analyzer during the
storage period of all formulations.
An experiment was performed to evaluate the effect of
the age of conidia and shelf-life of different P. frequentans
conidial formulations on adhesion to peach fruit
surfaces. Surface-sterilized peach fruit (as described by
Sauer and Burroughs, 1986) were placed in 100 ml of
the different formulation suspensions at a final concentration
of 10 7 conidia ml 1 (Guijarro et al., unpublished).
Peaches were shaken for 30 min at 130 rpm, and then air
dried at room temperature for 5 min. After drying, each
peach was placed in 100 ml sterile distilled water, sonicated
for 45 s (Ultrason Bath, JP. Selecta, Barcelona,
Spain), and shaken on a vortex mixer (Reax Top, Heildoph,
Rose Scientific Ltd. Alta., Canada) for 10 s. Spores
in the resulting suspensions were concentrated by centrifugation
for 10 min at 10,000 rpm after removing the
peaches. Pellets were resuspended in 5 ml sterile distilled
water, where the number of conidia and colony forming
units (cfu) of P. frequentans were estimated as described
below.

Adhesion of P. frequentans conidia to the surface of peaches
was measured as the number of conidia (no. conidia
cm 2 of fruit) and colony forming units (cfu cm 2 of fruit)
(to estimate conidial viability). Numbers of P. frequentans
conidia were counted in a haematocytometer (Neubauer
improved, 0.100 mm depth, 0.0025 mm 2 , Assistant, Soonheim,
Germany) under a light microscope (100·) (Carl
Zeiss Mikroskopie, D-07740 Jena GmbH, Germany). Four
haematocytometer fields were counted per peach. The cfu
of P. frequentans were estimated on Petri dishes containing
PDA. Five Petri dishes were incubated per peach at
20–22 °C for 4–5 days before counting the colonies. Three
peaches were used for each conidial suspension. Three formulations
were tested (FOR3, FOR7, and FOR8) immediately
after drying and after 365 day of storage at room
temperature. FCW conidia without additives and conidia
with 1.3% v/v Nufilm, were used as controls. The complete
experiment was conducted twice.
2.4. Biocontrol efficacy of P. frequentans formulations at
postharvest
Two experiments were carried out on healthy peaches to
evaluate the biocontrol efficacy of the three conidial formulations
(FOR 3, FOR 7, and FOR 8) following storage for
270 days at room temperature. Healthy peaches, surface
sterilized as recommended by Sauer and Burroughs
(1986), were used. After sterilization, the surfaces of
the peaches were dried by sterile air in a flow-cabinet for
two hours. Three one mm 3 artificial wounds, two cm away
from each other, were made in the fruit surface with a flame
sterilized nail. Fruit were sprayed with 10 ml of a conidial
suspension of each P. frequentans formulation (10 6 conidia
ml 1 ) in sterile distilled water. FCW conidia stored
for 270 days were used as controls. Fruit surfaces were
allowed to air dry for 2 h and 10 ml of a conidial suspension
of M. laxa (10 3 conidia ml 1 ) were sprayed onto each
peach. Control treatments were: C1) fruit inoculated with
M. laxa and not treated with P. frequentans; and C2) fruit
not inoculated with either M. laxa or P. frequentans. To
maintain high humidity, each inoculated fruit was placed
on a dry dish in plastic trays lined with moistened paper.
Trays were covered with a plastic film for 4–7 days at
22 °C in the dark. After incubation, disease incidence (as
percentage of rotten wounds caused by M. laxa in peaches)
was recorded (De Cal et al., 2002). Three wounds were
made per peach and ten peaches were used per treatment.
The experiments were repeated twice. Data are given as
the mean of disease incidence of 20 fruit since data of the
two experiments were pooled and analyzed together.
2.5. Biocontrol efficacy of P. frequentans formulations in
field experiments
Six field experiments were carried out in commercial
peach orchards located in Sudanell and Alfarrás (Lleida,
Spain) over three growing seasons from 2003 to 2005 to
B. Guijarro et al. / Biological Control 42 (2007) 86–96 89
evaluate the effect of P. frequentans treatments on the incidence
of postharvest brown rot; latent infection caused by
Monilinia spp.; and number of conidia of the pathogen on
peach surfaces. Trees were selected at random in each orchard
for each treatment. Three consecutive trees were used
as the sample unit and every treatment was repeated four
times. Two guard trees were used to separate sample units
to avoid spray drift. Cultivars grown in orchards were: nectarine
‘‘Autumn Free’’ in Sudanell 2003 (SU03), 2004
(SU04), and 2005 (SU05), and peach ‘‘Rojo Septiembre’’
in Alfarrás 2003 (ALF03), 2004 (ALF04), and 2005
(ALF05).
Different P. frequentans treatments were applied in each
orchard and in each year at a concentration of 10 7 conidia
ml 1 in a standard schedule as recommended to control
brown rot: at bloom, shuck split, pit hardening and/
or preharvest. Two formulations of P. frequentans (FOR 3
and FOR4) were applied five times (once at pink blossom,
once at pit hardening and three times at preharvest) in
experiments ALF03 and SU03 in 2003. Three formulations
of P. frequentans (FOR3, FOR7, and FOR8) were applied
five times (once at pink blossom, once at shuck split, once
at pit hardening and two times at preharvest) in experiments
ALF04 and SU04 in 2004. Formulation FOR 8 was
applied five times (once at pink blossom, once at shuck
split, once at pit hardening and two times at preharvest)
in experiment ALF05, or (three times at pit hardening
and two times at preharvest) and SU05 in 2005. Application
dates were: 17 March, 15 May and 5, 10, 16 September
2003 in ALF03; 9 March, 15 May, 22 and 28 August, and 2
September 2003 in SU03; 19 March, 23 April, 11 June, and
7, 14 September 2004 in ALF04; 4 March, 15 April, 29
May, 27 August and 3 September 2004 in SU04; 29 March,
27 April, 12 June, and 7, 14 September 2005 in ALF05; and
29 May, 5 and 15 June, and 10, 30 August 2005 in SU05.
Harvest date were 18 September in ALF03, 7 September
in SU03, 22 September in ALF04, 12 September in SU04,
21 September in ALF05, and 5 September in SU05. All
treatments were applied with a backpack sprayer (operating
pressure 10 6 Pa, hollow cone nozzle 1 mm) in the morning.
Orchards received the recommended cultural and crop
protection practices for each location.
Control treatments were trees treated with fungicides or
untreated in each orchard. Fungicides used were: Caddy
Pepite 10 (ciproconazole 10% WG, Bayer Cropscience,
S.L. Valencia, Spain) in trials ALF03, ALF04, SU03, and
SU04; and Folicur 25RW (tebuconazole 25% WG, Bayer
Hispania Industrial, S.A., Barcelona, Spain) in trials
ALF05 and SU05. Chemical treatments were applied at
the same dates as biologicals at pink blossom, shuck split,
and/or pit hardening, but only once 21 days before harvest.
To compare Monilinia spp. populations on peach
surfaces for each treatment, 10 flowers or five fruits per
sample unit were sampled 10, 11, and 12 times in 2003,
2004, and 2005, respectively, in each orchard. Each sample
(10 flowers or five fruits per treatment and replicate) were
suspended in sterile distilled water (SDW), shaken for

90 B. Guijarro et al. / Biological Control 42 (2007) 86–96
30 min at 150 rpm; concentrated by centrifugation 10 min
at 14,040g, and resuspended in 5 ml SDW. Monilinia spp.
conidia were estimated as the number of Monilinia spp.
conidia per flower or fruit. Numbers of Monilinia spp. conidia
were counted in a haematocytometer under a light
microscope (100·) as described above.
One hundred asymptomatic fruits per sample unit were
randomly picked by hand on the commercial harvest date
for each location. Each sample (100 fruit per treatment
and replicate) were placed in five packing trays, each containing
20 fruits. All of them were placed in a box and
stored at 20 °C and 85% RH for seven days when the percentage
of brown rot was visually assessed and recorded.
At this time cross-infections did not occur because it takes
seven days for conidia of Monilinia spp. to germinate, produce
mycelia, and sporulate (Byrde and Willetts, 1977).
The presence of conidia sporulating on lesions was necessary
to confirm that symptomatic fruits were infected with
Monilinia spp.
2.6. Data analysis
Data were analyzed by analysis of variance. Prior to
analysis, data of number P. frequentans conidia per cm 2 ,
cfu of P. frequentans per cm 2 , and number of Monilinia
spp. conidia per flower or fruit were log10-transformed to
improve homogeneity of variances. When F-test was significant
at P = 0.05, means were compared by Student–Newman–Keul’s
multiple range test (Snedecor and Cochram,
1980), and number of Monilinia spp. conidia per flower
or fruit from field experiments were analyzed independently
by contrast with the F test at significance levels of
0.05 (Snedecor and Cochram, 1980). Conidial viability of
P. frequentans for 0, 30 90 180, and 365 days of storage
gave a viability progress curve, and the area under this
conidial viability progress curve for a year (AUCVPC)
was calculated as described (Campbell and Madden,
1990) using the formula:
AUCVPC ¼
Xn 1
i¼1
ðtiþ1 tiÞðvi þ viþ1Þ
2
where t is days of storage after drying, v is the percentage of
P. frequentans conidial viability at each estimation date (i)
and n is the number of estimations. The AUCVPC was calculated
for each P. frequentans fomulation using an Excel
spreadsheet. The AUCVPC expressed the dynamic of
conidial viability for one year of storage as a single value
and is useful to compare different viability dynamics. All
the experiments were repeated twice. Since, the second repetition
of the experiment supported the results obtained in
the first, the data were pooled and analyzed together.
3. Results
Conidial moisture content was maintained at a level
lower than 15% at all storage conditions for 360 d,
ð1Þ
although it was slightly less at vacuum. These levels of
conidial moisture content did not affect on conidial viability
in any storage conditions throughout a year.
Conidial viability throughout a year of storage at different
conditions is shown in Fig. 1. Conidial viability of
P. frequentans without any additive (FCW) was less than
a
% Viability
b
% Viability
c
% Viability
d
% Viability
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
0 30 90 180 360
0 30 90 180 360
0 30 90 180 360
0 30 90
days after drying
180 360
FOR3 FOR7 FOR8 FCW
Fig. 1. Conidial viability (%) of different P. frequentans conidia formulations,
FOR 3 (.--.--.), FOR 7 (-- --), FOR 8 (...), and FCW (—–), along
stored period of 360 day at different temperatures and oxygen conditions:
(a) 20 °C with oxygen; (b) 20 °C without oxygen; (c) 4 °C with oxygen; and
(d) 4 °C without oxygen.

Table 1
Effect of storage conditions on the viability of Penicillium frequentans
conidia (as AUCVPC) after 365 days of storage A
Formulations 22 °C 4 °C
Oxygen Vacuum Oxygen Vacuum
FCW 15,292a 12,415a 17,237a 14,690a
FOR3 20,845c 18,047b 22,837b 21,382b
FOR7 19,588bc 19,567b 26,060c 20,355b
FOR8 17,760b 23,432c 25,572c 26,407c
B
MSEwithin 3.56 · 10 6
0.78 · 10 6
2.98 · 10 6
1.16 · 10 6
A
AUCVPC is the area under conidial viability progress curve for a year.
See Section 2 for details. Means in each column, followed by the same
letter are not significantly different (P = 0.05) by Student–Newman–Keuls
multiple range test. Data are the means of six replicates with 50 conidia
per replicate.
B
MSEwithin, mean square error.
15% after 365 d of storage at room temperature (Fig. 1a),
and lower (P = 0.05) than viability of any formulation in
all storage condition (Fig. 1 and Table 1). Conidial viability
after 365 days of storage at 4 °C was higher than at room
temperature independently of formulation (Fig. 1c and
d). The highest conidial viability after one year of storage
(as AUCVPC) was recorded for formulation FOR8 (Fresh
conidia with 2% methyl cellulose + 10% glucose, and silica
gel before drying) stored at 4 °C at vacuum (Table 1).
Adhesion of conidia to peach surfaces was significantly
(P = 0.05) improved (10-fold) with any formulation or
Nu-film-17 (Table 2). Furthermore, adhesion of conidia
to fruit surfaces was maintained for formulations stored
365 days at room temperature (Table 2).
P. frequentans formulations were applied to peaches as
postharvest treatments. Peaches inoculated with M. laxa
showed brown rot symptoms after 4 days of incubation.
The disease incidence on untreated fruit was approximately
62%. All of the formulations, and also FCW, significantly
(P = 0.05) reduced disease incidence by more than 50%
(Table 3).
Postharvest brown rot was very low in all field experiments
(data not shown), ranging from 0.5% in Sudanell
in 2005 to 19% in Alfarras in 2003.
Table 2
Effect of formulations on conidial adhesion of Penicillium frequentans to fruit surface A
B. Guijarro et al. / Biological Control 42 (2007) 86–96 91
Table 3
Effect of different formulations of Penicillium frequentans FORx conidia
on disease incidence caused by Monilinia laxa on peaches at 4 days after
inoculation in Postharvest experiments A
Treatments B
Monilinia laxa
inoculum C
FOR3 1 28b
FOR7 1 32b
FOR8 1 22b
FCW Pf dried storage 1 38b
C1: Pf untreated 1 62c
C2: Pf untreated 0 0a
MSEwithin D
30.0
Disease
incidence (%)
A
Data of disease incidence are the mean of 20 fruit. Means in each
column, followed by the same letter are not significantly different
(P = 0.05) by Student–Newman–Keul’s range test.
B 6
Fruit were sprayed with 10 ml of a conidial suspension (10 conidia
ml 1 ) of each P. frequentans formulation (FCW, FOR3, FOR7, and
FOR8) which had been stored for 270 days at room temperature. C1, P.
frequentans untreated; C2, P. frequentans untreated.
C
(1) Fruit inoculated with 10 ml of a conidial suspension of M. laxa
(10 3 conidia ml -1 ); (0) uninoculated fruit.
D
MSEwithin, error mean square.
The effects of different treatments on the number of
Monilinia spp. conidia on peach surfaces are illustrated in
Fig. 2. For clarity, only representative biological formulations
were represented in each trial. The number of Monilinia
spp. conidia on peach surfaces at harvest is presented in
Table 4. The number of Monilinia spp. conidia on non-treated
peach surfaces followed the same pattern in all orchards.
The highest levels of Monilinia conidia were observed at harvest,
with another peak at pit hardening (Fig. 2).
Table 5 shows the contrast analysis of different methods
of control (biological and chemical made by grouping the
corresponding groups of treatments) tested in the six trials.
When data from biological treatments were compared with
the untreated control, significant differences (P = 0.05)
were observed in four trials (ALF04, SUD04, ALF05,
and SUD05). However, significant differences were
obtained only in two trials (SUD04, and ALF05) when
Formulations Surface fruit adhesion after fluid bed-drying Surface fruit adhesion 365 days after fluid beddrying
cfu B (cm 2 ) Conidia C (cm 2 ) cfu B (cm 2 ) Conidia C (cm 2 )
FOR3 2891 (3.44)c 166797 (5.22)d 1285 (3.07)bc 330961 (5.35)b
FOR7 1526 (3.15)b 53177 (4.72)b 648 (2.72)ab 208160 (5.22)b
FOR8 4554 (3.66)d 86140 (4.93)c 2497 (3.29)c 120125 (5.07)b
Nufilm 1.3% 2057 (3.31)bc 164931 (5.21)d 1466 (3.13)bc 503935 (5.45)b
Control 228 (2.33)a 12325 (4.08)a 397 (2.44)a 24479 (4.23)a
D
MSEwithin (0.022) (0.0046) (0.103) (0.162)
A
Conidia tested were FORx. Data in parentheses are log10-transformed values. Formulation means in each column, followed by the same letter are not
significantly different (P = 0.05) by Student–Newman multiple range test.
B
Cfu of P. frequentans were estimated onto Petri dishes containing potato dextrose agar (PDA). Data are the means of six replicates, with five dishes per
replicate.
C
Number of P. frequentans conidia was counted in a haematocytometer under a light microscope (100·). Data are the means of six replicates with four
drops per replicate.
D
MSEwithin, error mean square.

92 B. Guijarro et al. / Biological Control 42 (2007) 86–96
Fig. 2. Population dynamics of Monilinia spp. (number of conidia per flower or fruit) on peach surfaces treated with different applications: biological
treatments FOR 3 (d d), FOR 8 (n n); chemical treatment Q (·- ---·) and untreatment NT (s—s) recovered from pink blossom to harvest for
2003–2005 on six orchards of Lleida (Spain) (ALF03, ALF04, ALF05, SUD03, SUD04, and SUD05). Number of Monilinia spp. conidia was counted in a
haematocytometer under a light microscope (100·) and are represented on a logarithmic scale. Data are the mean of four replicates, with 10 flowers or 5
fruits per replicate. Application dates for treatments are represented by vertical arrows.
chemical treatments were compared with untreated controls.
Comparisons between biological treatments and
chemical treatments were significant (P = 0.05) in SUD05.
Contrasts were also made among biological formulations
including silica gel (FOR3) versus kaolinite (FOR4)
in ALF03 and SUD03. Significant differences (P = 0.05)

Table 4
Effect of P. frequentans formulations on the populations of Monilinia spp. (number of conidia per fruit) in different orchards where chemical and biological
treatments were applied along crop during 2003–2005 a
Treatment b
2003 2004 2005
ALF03 SUD03 ALF04 SUD04 ALF05 SUD05
FOR3 42,969 ± 5750 0.0 ± 0.0 2930 ± 1870 4883 ± 1224 — —
FOR4 59,570 ± 6046 6835 ± 3335 — — — —
FOR7 — — 4883 ± 976 — — —
FOR8 — — 0.0 ± 0.0 1953 ± 738 4400 ± 1871 488 ± 488
Chemical 39,062 ± 5289 2930 ± 1870 4639 ± 2008 2930 ± 1224 4394 ± 2497 5859 ± 1476
Untreated 48,828 ± 6478 6836 ± 4331 24,170 ± 7191 7812 ± 1808 23,926 ± 6196 12,207 ± 3417
a
Data are the mean of four replicates with five fruit per replicate ± standard error of the mean.
b
See Section 2 for details of treatments.
Table 5
Contrast analysis of different methods of control of Monilinia spp. in the field experiments of the years 2003, 2004, and 2005 a
Methods of control Trials
2003 2004 2005
ALF03 SUD03 ALF04 SUD04 ALF05 SUD05
Biological versus untreated NS NS * * * *
Chemical versus untreated NS NS NS * * NS
Biological versus chemical NS NS NS NS NS *
Biological with silica gel versus biological with kaolinite ** * — — — —
were obtained in SUD03. Formulations including kaolinite
were not tested in the following years of the study.
4. Discussion
Brown rot of peaches at postharvest can be reduced by
application of fungicides or biological agents either as postharvest
or preharvest treatments. The results of the present
study show that applications of P. frequentans formulations
that maintained conidial viability and good adhesion
to peach surfaces after one year of storage reduced brown
rot of peaches when applied as postharvest treatments and
significantly reduced the inoculum density of Monilinia
spp. at harvest (measured as pathogen conidia on peach
surface) in four field trials out of the six tested when
applied as preharvest treatments. Other fungal biocontrol
agents such as Epicoccum nigrum Link or Metschnikowia
fructicola Kurtzman & Droby also reduced brown rot of
peaches or strawberries, respectively, in storage by preand/or
postharvest applications (Karabulut et al., 2004;
Larena et al., 2005). Infections of stone fruit by Monilinia
spp. occur mainly before harvest in orchards by air-borne
conidia. Smilanick et al. (1993) reported that postharvest
applications of Pseudomanas corrugata Roberts and Scarlett
and P. cepacia (Burkholder) Palleroni and Holmes gave
poor control of naturally occurring infections of brown rot
on nectarine and peach, and they concluded that early
application of biocontrol agents in the field may enable
early colonization and reduction of latent infections.
Establishment of a biocontrol agent before a pathogen
arrives would appear to be a good strategy to manage in
B. Guijarro et al. / Biological Control 42 (2007) 86–96 93
a *F test significant at P = 0.05; ** F test significant at P = 0.10; NS, not significant at P = 0.05; —, not tested.
preventing fruit from these infections. However establishment
of the biocontrol agent in fruit surface is more difficult
in open air conditions than in conditions found in
confined chambers used for postharvest storage of fruit
(Andrews, 1992; Ippolito and Nigro, 2000). Application
and optimization of biocontrol agents before harvest
requires considerable understanding of the crop system,
pathogen epidemiology, the biology, ecology, and populations
dynamics of the antagonists, and the interactions
among these factors (Adams, 1990; Andrews, 1992).
Improvement of inoculum quality was usually necessary
in preharvest applications of antagonist to achieve success
(Teixido et al., 1998). One way to improve performance of
biocontrol agents is by formulating conidia. Formulations
of conidia of P. frequentans to improve fruit adhesion and
conidial viability gave good results in reducing number of
air-borne conidia of Monilinia on fruit surface.
The wettable powder formulations of P. frequentans
applied in this study contained different additives and
two different carriers: silica gel and kaolinite. Carriers are
inert ingredients in the sense that they do not have disease
control capabilities; however, they can profoundly affect
shelf-life and efficacy of the product (Fravel et al., 1998).
Kaolinite and silica gel are two hygroscopic additives
which when mixed with the hydrophobic conidia of P. frequentans,
followed by drying, usually were sufficient to permit
wetting of the spores when the mixture is resuspended
in water (Boyette et al., 1991). However, when comparing
biological formulations including silica gel (FOR 3) with
that including kaolinite (FOR 4), significant differences
(P = 0.05) were obtained in one of the two trials tested,

94 B. Guijarro et al. / Biological Control 42 (2007) 86–96
showing an advantage of silica gel versus kaolinite. In addition,
peach fruit treated with FOR4 exhibited white spots
caused by kaolinite, reducing their market value.
The shelf-life of a biological product refers to the period
of time during which the propagules of microbial agent
remain viable and effective (Elzein et al., 2004). Shelf-life
of P. frequentans conidia has been correlated with biocontrol
efficacy (Guijarro et al., 2007). There was a statistically
significant relationship between biocontrol efficacy of P.
frequentans and conidial viability (P = 0.01). A conidial
viability of P. frequentans up to 40% was reported necessary
for obtaining more than 50% biocontrol (Guijarro
et al., 2007). An adequate shelf-life of mycopesticidal product
at room temperature is an essential requirement for
their acceptance and commercialization (Rhodes, 1993;
Jones and Burges, 1998). FOR 3, FOR 7, and FOR 8 maintained
more than 40% viability after 365 days of storage
at 4 °C, but only FOR8 (fresh conidia with 2% methyl cellulose
+ 10% glucose, and silica gel before drying) presented
a conidial viability up to 40% after 365 days of
storage at room temperature. Glucose (7.5%) and carboxymethyl
cellulose (1.5%) have been reported as a stabilizer
and sticker, respectively, of P. frequentans conidia
(Guijarro et al., 2007). An enhancement of shelf-life of conidia
was also reported when glucose and methyl cellulose
were added to E. nigrum conidia before drying (Larena
et al., 2007). On the other hand, glycerol, sodium alginate,
and sugars were also added to conidia before drying to
improve P. oxalicum dispersal and to improve wilt reduction
of tomato caused by Fusarium and Verticillium (Sabuquillo
et al., 2005).
Storage temperature is the most important abiotic factor
that affects the shelf-life of biological formulations (Connick
et al., 1997; Elzein et al., 2004) by maintaining them
in a state of low metabolic activity (Elzein et al., 2004). It
is generally postulated that stability for 12–18 month without
refrigeration would be required for general agricultural
markets (Rhodes, 1993), although stability for 3–6 months
would probably be suitable for products produced on contract
for applications at a specific time (Couch and Ignoffo,
1981; McCoy, 1990).
Conidial adhesion to peach surfaces was improved
with each formulation. Adhesion of P. frequentans conidia
has been correlated with biocontrol efficacy (Guijarro
et al., unpublished). The adhesion to surfaces was estimated
by two methods; by counting the number of conidia
under a light microscope and by counting the cfus in
Petri dishes on PDA. The number of conidia was always
greater than the number of cfus. These results suggested
that many of the P. frequentans conidia estimated on any
surface were not able to grow on PDA after ultrasonication
for 45 s. Self inhibition has been observed when a
high concentration of Trichoderma sp. was used (Sreenivasaprasad
and Manibhushanrao, 1993; Adekunle et al.,
2001).
Experiments carried out in the field resulted in low levels
of brown rot. Reductions of pathogen inoculum densities
on fruit surfaces were observed at harvest. Application of
P. frequentans formulations, especially FOR8, significantly
decreased the numbers of Monilinia spp. conidia on fruit
surfaces at harvest, when pathogen conidia levels were at
their highest. This occurred in four out of the six experiments.
Although chemical control usually has been
reported as more consistent than biological control of
brown rot (Larena et al., 2005) and other pathogens
(Campbell, 1989), in the present work, chemical control
was significantly different from untreated control in only
two out of the six trials tested. A reason for this result
could be that chemical treatments were applied 21 days
before harvest due to security regulations and deadlines
while biologicals were applied two or three times before
harvest.
This work demonstrates that wettable powder formulations
of P. frequentans conidia that maintain a high viability
after one year of storage and good adherence to fruit
surfaces, especially FOR8, can be applied to fruit in postharvest
treatments to reduce brown rot in storage. Applications
of formulations at preharvest resulted in a decrease of
pathogen inoculum density. This result is particularly
important since brown rot incidence of stone fruit caused
by Monilinia spp. is mainly dependant on inoculum density
together with climatic factors (temperature and wetness
duration), and fruit maturity (Luo and Michailides, 2001,
Gell et al. unpublished).
Acknowledgments
This work was carried out with financial support from
AGL2002-4396-CO2 (Plan Nacional de I+D+I, Ministerio
de Educación y Ciencia, Spain) and from RTA2005-0077-
CO2. B. Guijarro received a scholarship from MEC (Ministerio
de Educación y Ciencia, Spain). We thank to Y.
Herranz, A. Barrionuevo, and M.T. Clemente for technical
support.
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