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Scientia Horticulturae 165 (2014) 433438

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Scientia Horticulturae
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Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits

a r t i c l e

i n f o

Keywords:
Imazalil
Biological control
Preventive application
Curative application
Bacillus subtilis
Saccharomyces cerevisiae

a b s t r a c t
This study shows that Saccharomyces cerevisiae (ACB-K1 and ACB-CR1) and Bacillus subtilis (ACB-69 and
ACB-84) isolates perform differently on the control of green mold (Penicillium digitatum), depending on
the citrus variety. In Murcott tangor, the yeast ACB-CR1 resulted in 47% of healthy fruits, which increased
to 67% when combined with imazalil 0.5 mL L1 . In the Hamlin orange, ACB-CR1 (S. cerevisiae) provided
87% control when applied alone. However, when combined with 0.5 and 1.0 mL L1 of fungicide (the
lowest doses), the efciency of ACB-CR1 was decreased, yielding 76 and 78% healthy fruits, respectively.
Both yeasts controlled green mold in the Tahiti acid lime by 40% when used as a curative treatment;
however, the ACB-K1 isolate that was applied as a preventive measure was the best antagonist, yielding
73% healthy fruits. This yeast increased disease control, with healthy fruit percentages ranging from 84
to 89% when the microorganism was combined with the lowest doses of imazalil. In general, B. subtilis
isolates provided only slight disease control when tested in the three citrus fruit varieties during this
study. However, the results of preventive treatments with bacteria on Tahiti acid lime fruits revealed
an improvement in the degree of biocontrol. This study demonstrated the possibility of reducing the
imazalil dose during the post-harvest citrus fruit treatment using a biocontrol agent without losing green
mold control efciency under storage conditions (27 C and 70% RH [relative humidity]). The preventive
application provided the best protection to Tahiti acid lime fruits, suggesting that the mode of action of
these biocontrol agents is through competition, or even resistance induction, considering the specicity
of the antagonisthost relationship within the context of pathogen control. The yeast isolates decreased
their antagonistic activity against P. digitatum under refrigeration (10 C and 95% RH). However, the ACBK1 isolate provided 100% disease control in Tahiti acid lime fruits under these storage conditions when
combined with a quarter dose of imazalil, despite its low activity.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Green mold, which is caused by Penicillium digitatum (Pers.:Fr)
Sacc., is one of the most signicant diseases of post-harvest in citrus fruits causing extensive losses during harvest, transport, and
storage processes (Eckert and Eaks, 1989). Synthetic fungicides,
including imazalil (IMZ) and thiabendazole (TBZ), have traditionally been used to control green mold (Ismail and Zhang, 2004;
Lahlali et al., 2005; Smilanick et al., 2006). However, the efcacy of
chemical treatments has often been limited in the face of pathogen
resistance to these compounds, as well as concerns about environment contamination and public health. Accordingly, a search for
alternative control strategies is needed (Prez et al., 2011).
The use of antagonistic microorganisms has emerged as a potential alternative to synthetic fungicides for controlling diseases.
Previous research showed that the protective activity of the two
selected antagonist yeasts, Rhodosporidium kratochvilovae (LS11)
and Cryptococcus laurentii (LS28), is enhanced by combining them

Abbreviations: IMZ, imazalil; TBZ, thiabendazole; BCA, biological control agent;


PDA, potato dextrose agar; NYDA, nutrient yeast dextrose agar; ANOVA, analysis of
variance; CFU, colony forming unit.
0304-4238/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scienta.2013.11.019

with a low dosage of fungicides and/or natural adjuvants, and that


such strategies can control both resistant and sensitive strains of
fungal pathogens (Lima et al., 2006, 2011). Therefore, selected biocontrol yeasts are very interesting candidates for their utilization
in integrated control strategies aimed at reducing the use of fungicides.
Among the biological control agents, Saccharomyces cerevisiae
and Bacillus subtilis have been used for the biocontrol of pathogens
that occur during the post-harvest period (Coelho et al., 2003;
Leelasuphakul et al., 2008; Sharma et al., 2009; Kupper et al., 2013).
The main mechanisms of action of yeasts during post-harvest
disease biocontrol include competition for space and nutrients, production of volatile metabolites, enzymes that degrade the plant
pathogen cell wall (including -1,3-glucanase and chitinase), host
resistance induction, mycoparasitism, and the killer factor (a toxic
peptide) (Coelho et al., 2003; E.I-Tarabily and Sivasithamparam,
2006). The Bacillus species have been promising in the control
of a variety of fungi that cause plant diseases, and their antagonistic action against pathogens occurs through the production of
antibiotics (iturin, surfactin, and fengycin), by enzymes (chitinase,
-1,3-glucanase) that degrade structural polymers of the fungal
and by production of volatile antifungal agents (Leelasuphakul
et al., 2006; Pinchuk et al., 2002). In certain situations, volatile

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Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits / Scientia Horticulturae 165 (2014) 433438

organic compounds that were secreted by B. subtilis cells were


linked to plant growth and plant systemic resistance induction
(Compant et al., 2005). The efciency of a biological control agent
can be enhanced with the addition of chemical control. Errampalli
and Brubacher (2006) demonstrated the control of P. expansum in
apples by integrating the use of Pseudomonas syringae with cyprodinil, which provided the best results.
Research on post-harvest disease has focused on application of
biocontrol agents after harvest. However, application after harvest
may be too late for the biocontrol agents to effectively compete with
the decay pathogens already established on or in fruit tissues in the
eld. Antagonistic microorganisms that have curative action, controlling pre-existing infections, that prevents subsequent infections
and retards fungal sporulation are desirable. On the other hand, the
ability of the treatments to protect the fruit from future infections
(preventive activity) should be evaluated. In commercial situations
the reinfection of the same fruit or on healthy fruits may occur
during handling and processing within the packing house, where
the surface wounds can be infected by the pathogen for several
days. The application of products based on curative or preventive forms of antagonistic microorganisms and an understanding of
their modes of action against plant pathogens may benet the eld
of biocontrol. Usall et al. (2008) reported that the combination of
sodium carbonate with Pantoea agglomerans (CPA-2) bacteria was
more efcient against P. digitatum.
Considering all these factors, this study was aimed at evaluating the use of S. cerevisiae and B. subtilis to control P. digitatum,
the causal agent of citrus green mold, with or without a chemical
product (imazalil).
2. Material and methods
2.1. Cultivars
The cultivars in this study were Murcott tangor, a hybrid
between tangerine (Citrus reticulata Blanco) and sweet orange
(Citrus sinensis [L.] Osb.). The fruits were harvested from the Agricultural Experimental Station at the Sylvio Moreira APTA (So
Paulo Agribusiness Technology Agency) Citrus Production Center/IAC (Agronomic Institute of Campinas) (Estaco Experimental,
Centro APTA Citros Sylvio Moreira/IAC), Cordeirpolis, So Paulo,
Brazil. A Hamlin orange tree (C. sinensis (L.) Osb.) Cv. and Tahiti
acid lime (Citrus latifolia Tanaka) were purchased from commercial orchards in the state of So Paulo (Brazil). The fruits in these
experiments were not subjected to regular commercial treatment,
which is usually applied post-harvest, and they were used on the
same day or stored up to 2 weeks (5 1 C and 95% RH [relative
humidity]) prior to their use in various assays.
2.2. Pathogen
A highly virulent strain of P. digitatum (PF-1) was obtained from
decayed oranges and used to articially inoculate the fruit. Conidial suspensions for fruit inoculation were obtained as follows: the
pathogen was grown on potato dextrose agar (PDA) for 7 days at
27 C. Ten microliters of sterile distilled water with 0.01% Tween
80 was dispensed into Petri dishes. Conidia were scraped from the
agar using a sterile loop. The suspension was subsequently transferred to a test tube and was sonicated for 5 min to facilitate conidial
suspension, and the concentration was adjusted with the aid of a
hemocytometer.
2.3. Antagonists
B. subtilis strains ACB-84 and ACB-69 were obtained from
the APTA Center Citros Sylvio Moreira IAC, Cordeirpolis, So

Paulo, Brazil. The S. cerevisiae strains (ACB-CR1 and ACB-K1) were


obtained from the Laboratory for Biochemistry and Plant Pathology at the University of So Paulo (ESALQ), Piracicaba, So Paulo,
Brazil. These strains were selected by assay in vitro and in vivo of
antagonistic action of the BCAs against P. digitatum (Kupper et al.,
2013).
The activated culture was maintained on NYDA (nutrient yeast
dextrose agar) medium at 27 C for 48 h and transferred, and the
cell suspension was used as inoculum for mass production in a
fermentation system at 27 C in the dark.
2.4. Preparation and fermentation of biocontrol agents
B. subtilis was grown in the presence of foliar fertilizer under
glutamic fermentation of molasses to 0.5% with agitation for 72 h
(Bettiol et al., 2005). The residue, which is known as Ajifol , was
used to grow the bacteria because it contains carbon sources, nitrogen, and salts in addition to being low-cost and in common use by
citrus orchards. A liquid medium containing potato dextrose was
used to produce S. cerevisiae with stirring for 72 h.
2.5. In vitro sensitivity of biocontrol agents to imazalil
The aim of this experiment was to evaluate B. subtilis (ACB69 and ACB-84) and S. cerevisiae (ACB-CR1 and ACB-K1) for their
sensitivity to the imazalil (500 g L1 , active ingredient) fungicide
that is used to control green mold with the intention of developing
integrated control methods.
B. subtilis and S. cerevisiae isolates were grown on NYDA (nutrient yeast dextrose agar) with stirring at 250 rpm at 30 C for 48 h.
A 200 L aliquot of the microorganism suspension (108 CFU mL1 )
was subsequently plated on Petri dishes with NYDA medium using a
Drigalski handle. The cells from each biological control agent (BCA)
were subsequently scattered evenly over the culture medium. In
Petri dishes containing the BCAs, two sterilized discs of lter paper
(12.7 mm diameter) were placed and were soaked in the fungicide
imazalil (IMZ) at concentrations of 0.5; 1.0; 2.0 (as recommended
by the manufacturer); 4.0 and 8.0 mL L1 , and ve plates were made
by treatment.
The cultures were incubated at 27 C for 72 h. Five replicates
were used per treatment. An evaluation was performed on the basis
of the presence or absence of an inhibition halo between the lter
paper discs and the bacterial or yeast growth.
2.6. Use of biocontrol agents and imazalil in the integrated
control of citrus fruit green mold
Citrus fruits from the Murcott tangor, Hamlin orange, and
Tahiti acid lime cultivars with no postharvest treatment were
washed using a soft sponge, neutral detergent, and water and were
surface-disinfected with 0.7% (v/v) sodium hypochlorite for 3 min.
The fruits were wounded at two equidistant points on the equatorial region with a sterilized stylus at a depth of 3 mm and inoculated
with 20 L of P. digitatum conidial suspension (1 105 spores mL1 )
24 h prior to starting the different treatments. A preventive biological control effect was only done for Tahiti acid lime fruits under
ambient conditions; therefore, those fruits were treated 24 h prior
to inoculation with the plant pathogen.
The treatments were as follows: (i) each BCA was applied separately, with cell suspensions corresponding to 1 108 CFU mL1
for 2 min; (ii) imazalil 0.5 mL L1 + fermented broth from each BCA
(1 108 CFU mL1 ) for 2 min; (iii) imazalil 1.0 mL L1 + fermented
broth from each BCA (1 108 CFU mL1 ) for 2 min; (iv) imazalil
2.0 mL L1 (as recommended by the manufacturer); (v) the control,
in which fruits were inoculated with P. digitatum (1 105 spores

Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits / Scientia Horticulturae 165 (2014) 433438

n of inhibition halo

Saccharomyces cerevisiae
d

10
9
8
7
6
5
4
3
2
1
0

cd
bc
ab

ab

0.5

1.0

2.0

4.0

8.0

concentration of IMZ (mL L-1)


ACB-CR1

ACB-K1

n of inhibition halo

Bacillus sublis
10
9
8
7
6
5
4
3
2
1
0

2.0

4.0

8.0

bc
b

0.5

1.0

concentration of IMZ (mL L-1)


ACB-84

ACB-69

Fig. 1. Sensitivity of Saccharomyces cerevisiae (ACB-K1 and ACB-CR1) and Bacillus


subtilis (ACB-69 and ACB-84) isolates to various doses of imazalil.

mL1 ) and treated with water only); (vi) imazalil 0.5 mL L1 ; (vii)
imazalil 1.0 mL L1 .
Following inoculation and treatment, fruits were stored under
different conditions, cooling conditions (10 1 C and 95% RH) for
15 days and at 27 C and 70% RH (ambient conditions) for seven
days. During the Hamlin orange fruits test, the mean air temperature was 20 C (70 5% RH), and the evaluation was performed on
the 15th day after the beginning of the test.
2.7. Statistical analysis
A completely randomized design was used with three replicates,
and each replicate consisted of 15 fruits for each cultivar. An evaluation was performed to determine disease incidence, as assessed
by the percentage of healthy fruits, and the data were submitted
to an analysis of variance (ANOVA) and comparison of means using
Tukeys test at 5% probability.
3. Results
3.1. In vitro sensitivity of biocontrol agents to imazalil
Testing in vitro of the sensitivity of strains bacterial (ACB-69
and ACB-84) and yeast (ACB-K1 and ACB-CR1) at different doses of
imazalil under laboratory conditions is shown in Fig. 1. In general,
the highest fungicide doses 4.0 and 8.0 mL L1 affected the growth
of S. cerevisiae, and yet the ACB-K1 isolate had the highest sensitivity
to fungicide, with inhibition halos being present at doses above
0.5 mL L1 . Bacterial isolates were the most sensitive to imazalil,
with growth inhibition being observed in all assessed doses.
3.2. Use of biocontrol agents and imazalil in the integrated
control of citrus fruit green mold
3.2.1. Murcott Tangor
Disease control was 63 and 53% in treatments only with the S.
cerevisiae ACB-K1 and ACB-CR1 isolates, respectively. ACB-K1 and

435

ACB-CR1 treatment resulted in 77 and 67% healthy fruits, respectively, combined with the lowest dose of fungicide (0.5 mL L1 ).
These combinations efciently inhibited disease onset compared
to the fungicide treatment at the recommended dose (2.0 mL L1 )
with no signicant differences between them (Fig. 2A).
There was a slight increase in disease incidence, with 53%
healthy fruits being observed, regardless of the isolate type, when
yeasts were combined with a half dose of imazalil (1.0 mL L1 ).
This result may be partly explained by data found in vitro (Fig. 1),
in which the ACB-K1 yeasts were sensitive to fungicide at doses
higher than 0.5 mL L1 . Following the combination of B. subtilis isolates with imazalil to control green mold in Murcott tangor fruits,
a combination of ACB-69 and fungicide at a dose of 0.5 mL L1
resulted in 73% disease control with no signicant difference from
the imazalil treatment that was applied at the recommended dose.
Conversely, bacterial isolate ACB-84 combined with the same dose
of fungicide did not lead to satisfactory disease control (27% healthy
fruits). Murcott tangor fruits that were treated only with ACB-69
and ACB-84 bacterial isolates had 0 and 47% healthy fruits, respectively.
3.2.2. Hamlin orange
Fruits treated with doses of 0.5, 1.0, and 2.0 mL L1 imazalil had
the highest numbers of healthy fruits (87, 97.8, and 100%, respectively) (Fig. 2B).
We found that ACB-CR1 yeast promoted more antagonistic
activity against P. digitatum, with 87% control, in contrast to the
ACB-K1 isolate, which yielded only 13% healthy fruits. When
the yeasts were combined with imazalil, ACB-CR1 plus 0.5 and
1.0 mL L1 resulted in 76 and 78% disease control, respectively; and
ACB-K1 yielded 56 and 91% asymptomatic fruits with the same
fungicide concentrations.
Bacterial isolates alone showed no potential to control green
mold in Hamlin orange fruits; the percentages of healthy fruits
ranged from 71 to 93% (with half the dose of imazalil) and 47 to 71%
(with a quarter dose), which occurred only when both were combined with different doses of imazalil, and ACB-84 always provided
the highest protection values in the mixture.
3.2.3. Tahiti acid lime
The Tahiti acid lime fruits were effectively protected with
imazalil at a rate of 0.5 mL L1 when stored under ambient conditions (27 C and 70% RH), yielding 93% healthy fruits, regardless
of antagonist addition (Fig. 2C). The percentages of healthy citrus
fruits ranged from 93 to 98% with no difference between the fungicide dose that was recommended by the manufacturer (2.0 mL L1 ),
and when S. cerevisiae isolates were combined with the lowest
doses of fungicide (0.5 and 1.0 mL L1 ).
With respect to the B. subtilis isolates, the ACB-69 yielded
42% healthy fruits when combined with a half dose of imazalil
(1.0 mL L1 ) or with a quarter dose of imazalil (0.5 mL L1 ). When
the same isolate was applied without fungicide, it yielded 33.33%
asymptomatic fruits (Fig. 2C). The ACB-84 isolate (without fungicide) had no efcacy when compared with ACB-69, in combinations
with the different doses of fungicide (0.5 and 1.0 mL L1 ) which
assessed 42 and 27% of healthy citrus fruits, respectively.
Fruit treatments with different doses of imazalil resulted in the
highest percentages of healthy fruits (80 to 95%) when applied to
Tahiti acid lime fruits and in storage under cold room conditions
(10 C and 95% RH). Under these storage conditions, the yeast isolates decreased their antagonistic activity against P. digitatum; that
is, fruits treated only with ACB-K1 and ACB-CR1 had 0 and 13%
healthy fruits, respectively (Fig. 2D).
When combined with a quarter dose of imazalil under cold storage, the ACB-K1 isolate treatment yielded 100% control in Tahiti
acid lime fruits, despite its low activity, whereas the fungicide alone

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Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits / Scientia Horticulturae 165 (2014) 433438

at the same dose had 20% diseased fruits, while the percentage of
healthy fruits found at the dose recommended by the manufacturer
(2.0 mL L1 ) was 91% (Fig. 2D).
B. subtilis isolates yielded a relatively signicant pathogen
control under refrigerated conditions (10 C and 95% RH) when
combined with the reduced dose of imazalil (Fig. 2D). The ACB-69
isolate combined with 0.5 mL L1 imazalil resulted in 85% disease
control, and fruits treated with the combination of ACB-69 and
1.0 mL L1 imazalil or only ACB-69 provided 30 and 2.22% asymptomatic fruits, respectively. The ACB-84 isolate without fungicide
was the least efcient in disease control, and when combined with
doses of 0.5 and 1.0 mL L1 imazalil, the percentage of healthy fruits
reached 75 and 60%, respectively.
The percentage of healthy fruits were 84 and 89% when the
S. cerevisiae ACB-K1 isolate was combined with the lowest doses
of fungicide (0.5 and 1.0 mL L1 imazalil). It is critical to emphasize that the mixture of ACB-K1 yeast with a half dose of imazalil
(1.0 mL L1 ) inhibited disease onset as effectively as treatment with
the recommended fungicide dose (2.0 mL L1 ) for disease control,
with no signicant difference being detected between them. This
treatment yielded 73% asymptomatic fruits when the ACB-K1 isolate was applied preventively with no fungicide. The ACB-CR1
isolate was not efcient for controlling disease.
With respect to the B. subtilis isolates, the ACB-84 yielded 40, 67,
and 80% healthy fruits when applied alone or in combination with
0.5 and 1.0 mL L1 imazalil, respectively. The ACB-69 isolate without fungicide and in combinations with doses of 0.5 and 1.0 mL L1
fungicide yielded 53, 58, and 73% fruits without green mold symptoms, respectively.
4. Discussion
The present study shows that S. cerevisiae (ACB-K1 and ACB-CR1)
and B. subtilis (ACB-69 and ACB-84) isolates have different levels of
antagonistic activity, depending on the variety of citrus fruit, and
also storage conditions.
In the case of the Murcott tangor, the ACB-CR1 yeast was
the biocontrol agent that best controlled the disease, the control
efciency increased when the antagonist was combined with a
quarter dose imazalil (0.5 mL L1 ). S. cerevisiae isolate ACB-CR1 controlled the disease better when applied by itself to Hamlin orange.
However, when ACB-CR1 was combined with the lowest doses of
fungicide, 0.5 and 1.0 mL L1 the control efciency decreased.
In the case of the Tahiti acid lime, the ACB-K1 isolate was the
best antagonist when applied as a preventative agent, increase of
fruits without disease symptoms, when combined with the lowest
imazalil doses (0.5 and 1.0 mL L1 ), this microorganism increased
control efciency (Fig. 2E).
In general, B. subtilis (ACB-69 and ACB-84) isolates provided little disease control when tested in the citrus fruit varieties, despite
having demonstrated its efcacy against P. digitatum in in vitro
tests (Kupper et al., 2013). However, the results of preventive treatments with bacteria in Tahiti acid lime fruits indicated improved
disease control. As shown in Fig. 2E, ACB-84 and ACB-69 yielded
healthy fruits when applied alone or when combined with 0.5 and
1.0 mL L1 imazalil, decrease in the quantity fruits without green
mold symptoms, comparing preventive and curative treatment.
This study demonstrated the possibility of reducing the imazalil
dose during the post-harvest treatment of citrus fruits using biocontrol agents. This green mold control can be accomplished

Fig. 2. Incidence of healthy citrus fruit (%) after inoculation with Penicillium
digitatum (1.0 106 conidia mL1 ), treatment with biological control agents (BCAs)
(1 108 CFU mL1 ) alone or in combination with doses of imazalil (0.5, 1.0 and
2.0 mL L1 ); (A) tangor Murcott fruit incubation for 7 days at 27 C and 70% RH; (B)

orange Hamlin fruit incubation for 7 days at 20 C and 70% RH; (C) acid lime Tahiti
fruit incubation for 7 days at 27 C and 70% RH; (D) acid lime Tahiti fruit (curative
treatment) incubation for 15 days at 10 C and 95% RH; (E) acid lime Tahiti fruit
(preventive treatment) incubation for 7 days at 27 C and 70% RH.

Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits / Scientia Horticulturae 165 (2014) 433438

without the loss of efciency under ambient storage conditions


(27 C and 70% RH). The ACB-K1 yeast yielded 77, 93, and 100%
healthy fruits in the Murcott tangor and Tahiti acid lime when
combined with a quarter of treatment of imazalil and preventive
doses, respectively. The same dose of fungicide combined with the
ACB-CR1 isolate yielded 76% healthy fruits in the Hamlin orange
tree. The combination of a biocontrol agent with reduced doses of
imazalil will help to reduce the chemical residue in citrus fruits
in addition to reducing the amount of this product in the packing
house. With respect to the bacterial isolates, ACB-69 promoted disease control in Tahiti acid lime fruits when combined with a half
dose of imazalil. The combination of biocontrol agents with lower
doses of fungicides has already been reported by several authors
(Kinay et al., 2001; Papadopoulou-Mourkidou, 1991; Usall et al.,
2001; Zhou et al., 2002).
This same hypothesis was successfully conrmed by Lima et al.
(2006 and 2011) integration studies of biocontrol yeasts and fungicides thiabendazole (TBZ), boscalid (BOSC), cyprodinil (CYPR), and
fenhexamid (FENH) in the integrated control of sensitive and
resistant isolates of Botrytis cinerea and P. expansum within the protections of stored apples. Despite the fact that research has shown
that integration of BCA with low doses of fungicide can enhance
control of postharvest fungal rots (Ippolito et al., 2004), Lima and
employees (2006 and 2011), demonstrated that such integration
can also manage fungicide resistant isolates of B. cinerea more efciently. The results are in full agreement with Chand-Goyal and
Spotts (1997); they observed an improved control of postharvest
rots on stored apples caused by either isolates of P. expansum sensitive or resistant to benzimidazoles by using biocontrol yeasts and
a reduced rate of TBZ, whereas the fungicide applied alone yielded
unsatisfactory control. In fact, in the presence of a resistant isolate
of B. cinerea, alone or combined with a sensitive isolate, TBZ was
ineffective since it acts by a specic mechanism of action (Delp,
1995), while the activity of the biocontrol yeast isolate C. laurentii LS28, is based on different mechanisms such as competition for
space and nutrients, hyperparasitism with the production of lytic
enzymes and resistance to oxidative stress in fruit wounds (Castoria
et al., 2001, 2003). These mechanisms are complex and not specic,
affecting multiple sites in the pathogen; in addition, it is unlikely
for a fungal pathogen to develop resistance to a biocontrol yeast
(Lima et al., 2006).
The curative application of these antagonists as a disease treatment does not favor biocontrol because the mode of action of the
biological control agents most likely occurs through by competition
for space and nutrients, which could explain the reduced effectiveness of green mold control in the Murcott tangor (0 to 63% healthy
fruits) and Tahiti acid lime (0 to 40%) fruits. Therefore, P. digitatum
was presumably already established at the site of infection at the
time of biocontrol agent application, resulting in disadvantages for
antagonist development. This hypothesis was also reported by Usall
et al. (2008) in P. digitatum biocontrol tests with Pantoea agglomerans (CPA-2), which only enabled limited healing activity of the
antagonist when applied alone. In previous study, Teixid et al.
(2001) showed that the P. agglomerans bacterial isolate was able to
effectively colonize the wound at the citrus fruit peel, thereby interfering with and preventing the pathogen infection, despite having
shown limited growth.
In our study, the preventive application yielded the best protection of Tahiti acid lime citrus fruits, suspecting that both the
B. subtilis (ACB -69 and ACB -84) and S. cerevisiae (ACB -CR1 and
ACB -K1) modes of action may be accomplished by competition or
even the induction of resistance, considering the specicity of the
host-antagonist relationship in relation to pathogen control.
Tests conducted with acid lime fruits allowed us not only to
assess the mode of biocontrol agent application but also to evaluate the performance of fruits under different storage conditions,

437

after treatment with antagonists and in combination or not with the


most common fungicide for disease control under typical packinghouse conditions in Brazil.
Under refrigerated conditions (10 C and 95% RH) the yeast isolates decreased their antagonistic activity against P. digitatum; that
is, the fruits treated with ACB-K1 and ACB-CR1 (Fig. 2D). However, the ACB-K1 isolate yielded 100% disease control in Tahiti acid
lime fruits when combined with a quarter dose of imazalil under
these fruit storage conditions, despite its low activity, whereas
the fungicide alone at the same dose resulted in the reduction of
healthy fruits (Fig. 2D) at the dose recommended by the manufacturer (2.0 mL L1 ). In contrast to the present study, Kinay et al.
(2001) found that combination of the antagonist and imazalil or
thiabendazole fungicides (200 g mL1 ) inhibited disease onset
as effectively as the 1000 g mL1 fungicide concentration when
testing the A15/1 yeast isolate for the control of green and blue
molds in grapefruit under refrigeration (10 1 C). The combination of different control methods favored disease control because
the pathogen has its infection and disease onset activity reduced
under low temperature conditions.
Considering that discrepancies in biocontrol efcacy between
controlled laboratory conditions and commercial circumstances
can occur and have various causes, the aim of this study was to
provide the optimal conditions for pathogen development, including the concentration of inoculum used, wounding and favorable
temperature and humidity conditions, especially when studies
were used to assess the effectiveness of biocontrol agents when
combined with low doses of fungicide during citrus fruit processing
under ambient conditions.
Above all, this study demonstrated that it is possible to manipulate ambient conditions and day-to-day practices, which, in this
case, refers to the packing house, to favor the biocontrol agent and
to reduce the use of fungicides, thereby protecting not only the
environment but also the consumers of citrus fruits. Further investigations are needed to test this integrated strategy under large-scale
conditions.
Acknowledgements
The nancial support of the Fapesp (The State of So Paulo
Research Foundation) (Proceeding no. 2007/03692-0), which
provided a scholarship to Cristiane Moretto (Proceeding no.
2007/02045-0), is gratefully acknowledged.
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Cristiane Moretto a
Antonio Lucas Lima Cervantes b
Antonio Batista Filho a
Katia Cristina Kupper b,
a Instituto Biolgico, Avenida Conselheiro Rodrigues
Alves, So Paulo, SP, 1.252-CEP 04014-002, Brazil
b Centro de Citricultura Sylvio Moreira, Instituto
Agronmico, Rod. Anhanguera, Km 158,
Cordeirpolis, SP, CEP 13490-970 Brazil
Corresponding

author. Tel.: +55 19 3546 1399;


fax: +55 19 3546 1399.
E-mail address: katia@centrodecitricultura.br
(K.C. Kupper)
8 August 2013
21 October 2013
13 November 2013
Available online 15 December 2013