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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
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Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits / Scientia Horticulturae 165 (2014) 433438
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
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
ACB-69
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
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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.
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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