Biological Control 87 (2015) 39–46

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Effective control of black Sigatoka disease using a microbial fungicide

based on Bacillus subtilis EA-CB0015 culture
Jaime A. Gutierrez-Monsalve a, Sandra Mosquera a, Lina María González-Jaramillo a, John J. Mira b,
Valeska Villegas-Escobar a,⇑
a
Grupo CIBIOP, Departamento Ingeniería de Procesos, Universidad EAFIT, Carrera 49 No. 7 Sur 50, Medellín, Colombia
b
Centro de Investigaciones del Banano ‘‘Cenibanano’’, Carepa (Antioquia), Colombia

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 The microbial fungicide provided

control of black Sigatoka in
greenhouse and field.
 The microbial fungicide was as
effective as chlorothalonil and
mancozeb fungicides.
 The microbial fungicide can be
applied in rotation with systemic
fungicides.
 Bacillus subtilis EA-CB0015 biomass
and metabolites control black
Sigatoka disease.
 The metabolites had a higher control
effect when compared to the bacterial
biomass.

a r t i c l e i n f o a b s t r a c t

Article history: Black Sigatoka disease caused by the fungus Mycosphaerella fijiensis Morelet is the most devastating dis-
Received 1 December 2014 ease of bananas worldwide. Its management is reliant on protectant and systemic fungicides despite their
Accepted 10 April 2015 environmental concerns. This study evaluated the effect of a microbial fungicide (MF) based on Bacillus
Available online 29 April 2015
subtilis EA-CB0015 and its metabolites for the control of black Sigatoka disease on banana plants in green-
house and field conditions. The MF applied at 1.5 L/ha and 3.0 L/ha provided control of the disease com-
Keywords: parable to the protectant fungicide chlorothalonil in greenhouse. In the field, the MF applied in solution
Mycosphaerella fijiensis
with water at 0.15 L/ha and 1.5 L/ha every 11 days during 10 weeks reduced black Sigatoka disease sever-
Microbial fungicide
Metabolites
ity in 20.2% and 28.1% respectively; reductions comparable to those obtained with the protectant fungi-
Biological control cides chlorothalonil (1.5 L/ha) and mancozeb (3.8 L/ha). The MF incorporated into different programs
Banana plants with systemic fungicides reduced disease level up to 42.9% with no significant differences with the con-
ventional program. To determine which component of the MF is responsible for the activity against M.
fijiensis, greenhouse and in vitro tests were set up to evaluate individually the spores, vegetative cells
and secondary metabolites of B. subtilis EA-CB0015. All components reduced the severity of the disease
and the germination of ascospores. For both trials the activity of the metabolites was higher and compa-
rable to the activity obtained with the MF, indicating that the efficacy of the MF depends mainly on the
metabolites and in lesser extent to B. subtilis EA-CB0015 cells.
Ó 2015 Elsevier Inc. All rights reserved.

⇑ Corresponding author.
E-mail addresses: jagutierrez33@gmail.com (J.A. Gutierrez-Monsalve), smosquera@
ucdavis.edu (S. Mosquera), lgonza46@eafit.edu.co (L.M. González-Jaramillo), jmira.
castillo@gmail.com (J.J. Mira), vvilleg2@eafit.edu.co (V. Villegas-Escobar).
1. Introduction
Banana, the fourth most important crop after rice, wheat and
maize, is cultivated in the tropical regions of more than 100

http://dx.doi.org/10.1016/j.biocontrol.2015.04.012
1049-9644/Ó 2015 Elsevier Inc. All rights reserved.
40 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46
countries and plays a key role in the economies of many develop-
ing countries (Frison et al., 2004). Among the diseases that reduce
banana yields, black Sigatoka disease caused by the hemibiotrophic
fungus Mycosphaerella fijiensis Morelet (Stover, 1980) is the most
damaging. The fungus attacks the leaves decreasing the photosyn-
thetic area of the plant and causes premature ripening of the fruit
resulting in significant production losses (Marín et al., 2003).
Current control of this disease is mostly based in recurrent applica-
tions of protectant and systemic fungicides which are aerially
applied all year around in export banana plantations. In
Colombia, fungicide applications have risen from 19 cycles/year
in 1993 to 32 cycles/year in 2013 and similar increasing trends
have been observed for Costa Rica, Cameroon and other producer
countries (de Lapeyre de Bellaire et al., 2010; Marín et al., 2003).
The increasing number of fungicide applications has brought eco-
nomic and environmental concerns and problems associated to
the emergence of resistant strains of the pathogen (Brent and
Hollomon, 2007). Consequently, more sustainable control strate-
gies such as biological control have become necessary.
Many microorganisms, such as Bacillus spp. have exhibited
promising results against several pathogens in vitro, greenhouse
and field (Raaijmakers et al., 2010; Ongena and Jacques, 2008).
Evidences supporting the activity of different Bacillus strains
in vitro are numerous, and have been related to the production of
different active metabolites such as lipopeptides (Villegas-
Escobar et al., 2013; Yánez-Mendizábal et al., 2012; Romero
et al., 2007). Many other strains have shown potential to control
different diseases in greenhouse and field, some examples are head
smut of corn (Mercado-Flores et al., 2014), Sclerotinia stem rot of
soybean (Alvarez et al., 2012), stem rot of canola (Fernando et al.,
2007), and others. However, the evaluation of biological control
agents against black Sigatoka disease is limited as well as its incor-
poration into conventional fungicides programs. From our knowl-
edge, only two commercial products based on B. subtilis QST 713
(SerenadeÒ) and Bacillus pumilus QST 2808 (SonataÒ) have been
tested in field against black Sigatoka disease (Serrano et al.,
2013) with promising results.
The biological pesticide market have been increasing during the
last decade, although these products only make up a small percent-
age of the total pesticides market (Glare et al., 2012). This might be
explain by the inconsistent field performances and high costs of
biological pesticides and to the lack of knowledge related to persis-
tence, implementation, biology and ecology of their active organ-
isms (Glare et al., 2012; Hynes and Boyetchko, 2006). To
maximizing the potential of a biological product, it is important
to make a rigorous selection of the microbial strain, optimize the
fermentation process and develop a formulation that preserves
shelf life, aids product delivery and enhances biological activity
(Hynes and Boyetchko, 2006).
We previously described the isolation and selection of the strain
B. subtilis EA-CB0015 which produces IAA (Indol Acetic Acid), forms
biofilm, reduces surface tension and produces lipopeptides
(Villegas-Escobar et al., 2013; Ceballos et al., 2012), characteristics
that have been associated to phyllosphere colonizing bacteria
(Lindow and Brandl, 2003). We also optimized a culture medium
for biomass and lipopeptide production (Mosquera et al., 2014)
and developed a liquid formulation that enhances shelf life, activ-
ity and product delivery (Villegas-Escobar et al., 2014).
In the present study we evaluated the efficacy of a microbial
fungicide (MF) based on the biomass and the metabolites of B. sub-
tilis EA-CB0015 for the control of black Sigatoka disease in green-
house and field. We report that this formulation effectively
controls black Sigatoka disease when applied individually or in
rotation with systemic fungicides. We also report evidence that
B. subtilis EA-CB0015 biomass as well as its metabolites reduce
the severity of the disease when tested separately. However we
found that the metabolites had a higher control when compared
to the biomass, suggesting that lipopeptides have a major role in
the control of black Sigatoka disease.
2. Materials and methods
2.1. Microorganisms
Strain B. subtilis EA-CB0015 was previously isolated from the
phyllosphere of a banana plant in Urabá, Colombia and identified
by the analysis of 16s rDNA gene sequencing (Ceballos et al.,
2012). The strain was stored at 80 °C in TSB (Tripticase Soy
Broth, Merck) with 20% glycerol and was activated on half-strength
TSA (Tripticase Soy Agar, Merck) before any experimental use. M.
fijiensis ascospores were isolated from banana leaves cv. Grand
Naine from Urabá, following the methodology of Dupont
(Dupont, 1982). Twenty day-old monosporic cultures obtained in
PDA (Potato Dextrose Agar, Merck) were fragmented and stored
at 23 °C as mycelial suspensions according to Peláez-Montoya
et al. (2006). The identification of these strains was carried out
with the oligonucleotides methodology designed by Liu et al.
(1991) for M. fijiensis, which encode for a preserved region in the
25s subunit of the ribosomal DNA. PCR amplification was con-
ducted in accordance with Romero et al. (1999) and DNA extrac-
tion was carried out with the Ultra Clean Microbial Isolation Kit
(MoBIO). When need it, the stored mycelial suspensions were
grown on PDA complemented with 200 ppm chloramphenicol
and incubated for 10 days at 30 °C for activation.
2.2. Bacillus subtilis EA-CB0015 production and biological fungicide
formulation
The production of metabolites and biomass of B. subtilis EA-
CB0015 was achieved in a 14 L bioreactor (BIOFLO 110, New
Brunswick Scientific Co., Inc., Edison, NJ, USA) by transferring 3%
(v/v) of a 12 h culture into 7 L of optimized culture medium
(Mosquera et al., 2014) and incubated at 30 °C, 500 rpm, 2 vvm,
pH 6.5 for 72 h. After incubation, B. subtilis EA-CB0015 culture
was formulated according to the submitted patent application
number PCT/IB2014/061167 (Villegas-Escobar et al., 2014). The
final mixture was designated microbial fungicide (MF), which con-
tained a concentration of 2.0 ± 1.0 * 109 CFU/mL of B. subtilis EA-
CB0015.
2.3. Preparation of cell-free supernatant, spores and vegetative cells
suspensions of B. subtilis EA-CB0015
Cell-free supernatant (CFS) and suspensions of cells were pre-
pared in order to determine the antifungal activity of B. subtilis
EA-CB0015 metabolites, spore and vegetative cells. Briefly, 20 mL
of an overnight culture of B. subtilis EA-CB0015 was inoculated in
a 1000 mL Erlenmeyer flask containing 180 mL of either optimized
culture medium (Mosquera et al., 2014) for the CFS, Finley medium
(Macagnan et al., 2006) for the spore cell production or TSB med-
ium for the vegetative cell production and incubated at 30 °C and
150 rpm. For the CFS, cells were recovered after 48 h of incubation
by centrifugation (3.289g, 20 min) and filtrated through 0.45-lm
cellulose acetate filters (Sartorius Biolab). For the spore cell sus-
pension, cells were recovered by centrifugation (3.289g,
20 min) after 120 h of incubation and the pellet was washed twice
with distilled water and resuspended in water. Then the spore cell
suspension was subject to heat shock (80 °C, 15 min) and the con-
centration in CFU/mL was adjusted with water. For the vegetative
cell suspension, cells were recovered by centrifugation (3.289g,
20 min) after 12 h of incubation and the pellet was washed twice
 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46 41

with distilled water and the concentration in CFU/mL was adjusted
with water.
2.4. Antifungal activity of B. subtilis EA-CB0015 and its metabolites
in vitro
To evaluate the antifungal activity of B. subtilis EA-CB0015 and
its metabolites, the percentage of inhibition of M. fijiensis ascos-
pores germination was assessed using the varnish technique mod-
ified by Talavera et al. (1998). Briefly, disinfected discs of banana
leaves cv. Williams were submerged for 30 s in the different treat-
ments and subjected to M. fijiensis ascospores discharge. After 48 h,
leaves were varnished and allowed to dry. Then the varnish was
removed, stained with safranin and observed under bright field
microscope. The length of the germ tube was determined by calcu-
lating the average length of 30 randomly selected ascospores per
disc using the AxioVisionÒ 4.2 microscope software (Carl ZeissÒ).
The percent inhibition of the germinative tube growth was deter-
mined by considering ascospore germ in the absolute control to
be 100%. In this evaluation three replicates were preformed per
treatment and the experiment was independently repeated twice.
The treatments evaluated were: the CFS (treatment T1); B. sub-
tilis EA-CB0015 spore suspension (treatment T2); and B. subtilis EA-
CB0015 vegetative cell suspension (treatment T3). A whole culture
of B. subtilis EA-CB0015 obtained after 48 h of incubation in opti-
mized culture medium (2.0 ± 1.0  109 CFU/mL) was used as the
positive control (C+) and deionized water was used as a negative
control (C).
2.5. Greenhouse trials design
and non-treated application were used as positive and negative
controls, respectively. For the second experiment the treatments
evaluated consisted of CFS (Treatment T1); B. subtilis EA-CB0015
spore suspension (3.1  108 CFU/mL) (Treatment T2); B. subtilis
EA-CB0015 vegetative cell suspension (4.5  107 CFU/mL)
(Treatment T3). The MF and non-inoculated plants were used as
positive and negative controls, respectively. All treatments were
formulated as described for the MF.
In all greenhouse trials the plants were arranged in a complete
randomize design with three replicates per treatment and 6 plants
per replicate. The greenhouse conditions were maintained at 90%
humidity and 28 ± 4 °C and after 45 days the disease severity was
assessed measuring the necrotic area of the treated leaf using a
digital camera with 12 mega-pixels and the processing imaging
software Axio VisionÒ 4.2 (ZeissÒ). Each experiment was indepen-
dently repeated twice.
2.6. Field trials design
During this work two field trials were conducted from March
22nd until May 27th of 2013. The first trial (trial 1) was performed
to determine the best dosage of the MF in field and to compare its
efficacy against black Sigatoka disease with the efficacy of the con-
ventional fungicides chlorothalonil (Bravonil 720Ò) and mancozeb
(Dithane F-MBÒ). The second trial (trial 2) was implemented to
evaluate the efficacy of MF when applied as part of a fungicide pro-
gram. Both experiments were evaluated during 10 weeks and were
achieved in the experimental field of AUGURA (Colombian Banana
Growers Association) in Carepa – Antioquia (7°450 2900 N, 76
390 1900 W) under production conditions with nonflowered banana
plants (cv. Williams) that had 1.5–1.8 m long.

Two greenhouse experiments were performed; the first one to

determine the best doses of the MF and to test its efficacy against
black Sigatoka disease in comparison with the conventional fungi-
cides, and the second one to determine which of the components of
the MF reduced the incidence of black Sigatoka disease. For both
experiments, leaf number one of seven months old Banana plants
cv. Williams, were inoculated with fragmented mycelium of M.
fijiensis (2.0  105 fragments/mL) (Donzelli and Churchill, 2007)
and incubated at 90% humidity and 28 ± 4 °C for 24 h in green-
house conditions for pathogen establishment. Then the different
treatments were applied at 3.3 mL/leaf using a MiniSpray gun with
cup K-3Ò calibrated at 60–80 drops/cm2 at an altitude of 30 cm.
In the first experiment the MF was diluted in water to obtain
four different concentrations: 1.0  107 CFU/mL, 5.0  107 CFU/
mL, 1.0  108 CFU/ha, and 2.0  108 CFU/mL, which are equivalent
to apply 0.15, 0.75, 1.5 and 3.0 L/ha of the MF in field. The conven-
tional fungicide chlorothalonil (Bravonil 720Ò) prepared at 10%
(equivalent to 1.5 L/ha) according to the manufacturer instructions
2.6.1. Trial 1
The MF was applied in solution with water at different concen-
trations: 1.0  107 CFU/mL, 1.0  108 CFU/mL and 2.0  108 CFU/
mL which is equivalent to appling the MF at 0.15, 1.5 and 3.0 L/
ha. Chlorothalonil (Bravonil 720Ò) and mancozeb (Dithane F-
MBÒ) were applied at 1.5 L/ha and 3.8 L/ha according the manufac-
turer instructions and non-treated plants were used as negative
control. The applications were performed every 11 days using a
StihlÒ fumigation machine with 15 L capacity calibrated at 60–
80 drops/cm2.
2.6.2. Trial 2
For this experiment four different fungicide programs were
design (Table 1). All programs included the systemic fungicides
CalixínÒ (Tridemorph), BumperÒ (Propiconazole), SicoÒ
(Difeconazole), OpusÒ (Epoxiconazole), VolleyÒ (Fenpropimort),
CumoraÒ (Buscalid) applied in different weeks and differ in the

Table 1
Fungicide programs designs for field trial 2.

Cycle Week 2013 Base + Systemic fungicidea + Protectants

Conventional program (P) Program M1 Program M2 Program M1–2
1 13 Oil + Tridemorph + Mancozeb MF Mancozeb MF
2 15 Oil + Propiconazole + Mancozeb MF Mancozeb MF
3 16 Water + – + Chlorothalonil Chlorothalonil MF MF
4 17 Oil + Difeconazole + Mancozeb MF Mancozeb MF
5 19 Oil + Epoxiconazole + Mancozeb MF Mancozeb MF
6 20 Water + – + Chlorothalonil Chlorothalonil MF MF
7 22 Oil + Tridemorph + Mancozeb MF Mancozeb MF
8 23 Oil + Fenpropimort + Mancozeb MF MF MF
9 25 Oil + Buscalid + Mancozeb MF MF MF
a
MF: microbial fungicide (1.5 L/ha); Chlorothalonil (BravonilÒ 720Ò, 1.5 L/ha); mancozeb (DithaneÒ, 1.5 L/ha); Tridemorph (CalixínÒ, 0.4 L/ha); Propiconazole (BumperÒ,
0.4 L/ha); Difeconazole (SicoÒ, 0.3 L/ha); Epoxiconazole (OpusÒ, 0.8 L/ha); Fenpropimort (VolleyÒ, 0.4 L/ha); Buscalid (CumoraÒ, 0.4 L/ha).
42 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46
protectant fungicide used. Conventional program (P) included pro-
tectant fungicides chlorothalonil and mancozeb, program M1
included protectant fungicide chlorothalonil and MF, program M2
included protectant fungicide mancozeb and MF, and program
M1–2 included only the MF. All mixtures containing systemic
fungicides were prepared by adding 40.9% v/v of mineral oil;
2.2% VolleyÒ, 1.76% SicoÒ, 4.4% OpusÒ, 2.2% BumperÒ, 2.2%
CalixinÒ or 2.2% CumoraÒ; 10% of protectant fungicide (mancozeb
or MF); 0.41% of emulsifying PegalÒ and was complemented with
water. The mixture containing the protectant fungicide chlorotha-
lonil (BravonilÒ 720) was prepared by adding 10% of the fungicide
and 90% of tap water. All fungicides mixtures were applied at
18.95 L/ha every 11 days with a StihlÒ fumigation machine with
15 L capacity calibrated at 60–80 drops/cm2. Additionally non-
treated plants were used as negative control.
Both trials where conducted using a completely randomized
design with six plants as experimental unit and three replicates
per treatment. The six plants were located in the middle of 24
plants plots that have 18 plants as buffer roads and a road of plants
as border. The border plants were not sprayed to avoid mixing
effects of the treatments and each plot consisted of 220 m2 without
the border roads.
Every 8 days the stage of evolution of the disease (SED) (Foure
and Ganry, 2008) and disease severity (Gauhl, 1989) were mea-
sured. The disease severity was determined using the Stover
(1971) scale modified by Gauhl (1989) for all the leaves. In this
scale grade 0: no symptoms, grade 1: less than 10 spots/leaf, grade
2: until 5% of the leaf area necrotic; grade 3: from 6% to 15% of leaf
area necrotic, grade 4: from 16% to 33% of leaf area necrotic; grade
5: from 34% to 50% of leaf area necrotic; grade 6: more than 50% of
leaf area necrotic. Afterwards an infection index (II) was deter-
mined with Eq. (1):
P
an
 3.0 L/ha no significant reduction of the black Sigatoka disease
II ¼
ðN  1ÞT
where a is the value of severity of the scale, n the number of leaves
in each value and N the total number of grades in the scale, and T is
the total number of leaves assessed.
2.7. Statistical analysis
Statistical software StatGraphics Centurion XVI Version 16.1.18
(Statpoint Technologies Inc., Virginia; USA) was used to analyze the
data obtained. Analysis of variance (ANOVA) and LSD multiple
Fig. 1. Effect of different doses of the microbial fungicide based on B. subtilis EA-CB0015 culture on the percentage of necrotic area of banana leaves inoculated with M. fijiensis
in greenhouse. Cl donates chlorothalonil (BravonilÒ), MF microbial fungicide and C negative control. Different letters indicate significant difference between the treatments
(P < 0.0001; LSD). This experiment was independently repeated twice with reproducible results.
comparison tests were used to analyze the efficacy of B. subtilis
EA-CB0015 and its metabolites in vitro, the greenhouse and field
trials. The confidence level used for ANOVA analysis was 95%.
3. Results
3.1. The microbial fungicide reduce black Sigatoka disease in a dose
dependent manner
To determine the efficacy of different doses of the MF on the
reduction of black Sigatoka disease in greenhouse, leaf number
one of banana plants were inoculated with M. fijiensis and treated
with different MF doses. After 45 days, all treatments significantly
reduced the necrosis caused by M. fijiensis when compared to the
negative control. At 3.0 and 1.5 L/ha the MF showed percentages
of necrosis area of 0.71 ± 0.57% and 2.72 ± 1.60% respectively, com-
parable to the one obtained for the positive control chlorothalonil
(1.26 ± 0.26%). The doses 0.75 and 0.15 L/ha were not as effective
and presented percentages of necrosis area of 10.50 ± 1.68% and
11.84 ± 2.05%, respectively (Fig. 1). These results suggest that the
MF applied at doses around 0.15 and 3.0 L/ha could be effective
on reducing the disease severity and stage of evolution of black
Sigatoka disease in field. To test this hypothesis the field trial
Trial 1 was set up.
The infection index curves (Fig. 2A) and the accumulative infec-
tion index (Fig. 2B) for trial 1 show that the MF at doses of 0.15 and
1.5 L/ha, but not at 3.0 L/ha, delayed the development of the black
Sigatoka disease in field. The MF at 0.15 and 1.5 L/ha reduced the
accumulative infection index in a 20.2% and a 28.1%, respectively
when compared to the negative control (C), reductions that were
comparable to that obtained for the positive controls chlorothalo-
nil (24.4%) and mancozeb (24.0%). For the plants treated with MF at
ð1Þ severity was observed; however, these plants showed chlorotic
spots similar to those associated with fungicide phytotoxicity
and different from black Sigatoka disease symptoms, generating
difficulties during the severity evaluations. These chlorotic spots
were not observed during the greenhouse evaluations possibly
because reduced light intensity, and might explain why we
detected reduction in the severity under this dose application.
Analyzing the stage of evolution of the disease (SED) in the
same field trial, all the doses reduced significantly the disease in
comparison with C and had no significant differences with the
positive controls chlorothalonil and mancozeb (Supplementary
 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46 43

Fig. 2. Effect of different doses of the microbial fungicide based on B. subtilis EA-CB0015 culture on the severity of black Sigatoka disease on banana plants in field. (A)
Evolution of the infection index (II) in time, (B) Area Under de Curve (AUC) of the infection index (II). Cl donates chlorothalonil (BravonilÒ), Mc: mancozeb (DithaneÒ), MF:
microbial fungicide and C negative control. Different letters indicate significant difference between the treatments (P = 0.0025; LSD).

material, Fig. S1A and B). These results indicate that the maximum
level of protection for black Sigatoka disease resulted from the
application of the MF at 1.5 L/ha which reduce the severity and
the stage of evolution of the disease with the same efficacy than
the conventional fungicides chlorothalonil and mancozeb.
Therefore our next step was to evaluate effectiveness of the MF
when incorporated as a protectant fungicide in programs with sys-
temic fungicides.
3.2. The microbial fungicide incorporated in fumigation programs with
systemic fungicides reduces black Sigatoka disease
To test the effect of the MF (1.5 L/ha) in the reduction of black
Sigatoka disease when incorporated into a fungicide program with
systemic fungicides, different fungicides programs were design
and evaluated. As shown in the infection index curves (Fig. 3A)
and the accumulative infection index (Fig. 3B), all programs (M1,
M2, M1–2, and CP) were able to delay the development of black
Sigatoka disease in field. As shown in Fig. 3A non-treated plants
(C) showed progressive symptom developments until week 21
and then slowly drops until the end of the evaluation period,
whereas those plants treated with different fungicide programs
resulted in minor disease development. The accumulative disease
levels for programs M1, M2, and M1–2 were equivalent to that of
the CP with reductions of 37.3%, 42.9% and 38.9% respectively
when compared to the C (Fig. 3B). The same effect was obtained
when determining the stage of evolution of the disease (SED), all
the programs applied reduced significantly the disease in compar-
ison to the C and had no difference with the CP (Supplementary
material, Fig. S2A and B).
3.3. Vegetative cells and spores of B. subtilis EA-CB0015 and its
metabolites reduced black Sigatoka disease and M. fijiensis ascospore
germination
To determine which component of the MF reduces the inci-
dence of black Sigatoka disease and inhibits germination of M.
fijiensis ascospores, B. subtilis EA-CB0015 vegetative cells, spores
and the CFS where evaluated in greenhouse and in vitro, respec-
tively. Under greenhouse conditions M. fijiensis caused severe
necrosis on the leaves of non-treated plants (40.2 ± 3.0% necrotic
area) after 45 days (Fig. 4). When the leaves were treated with
the MF or the formulated CFS (T1), the percentage of necrosis area
of the leaf was significantly reduced to 4.0 ± 0.2% and 2.0 ± 0.6%,
respectively. Application of formulated spores (T2) and vegetative
cells (T3) of B. subtilis EA-CB0015 also attenuated the disease
symptoms with reductions of 13.5 ± 4.0% and 19.6 ± 2.5% of necro-
sis area, respectively, but they were not as effective as the CFS or
the MF. These results suggest that the control efficacy of the MF
is mainly dependent on the metabolites present in the CFS and in
less extent to the vegetative and spore cells of B. subtilis EA-
CB0015.
To further confirm this result, the effect of B. subtilis EA-CB0015
vegetative cells, spores and CFS were tested over M. fijiensis ascos-
pores in vitro. The result obtained for this experiment was conse-
quent with the one obtained under greenhouse. All the
treatments were able to inhibit ascospore germination; the CFS
(T1) being the most effective (81.0%) with no significant differences
with the MF; and the spores (T2) and vegetative cells (T3) the less
effective (30.1% and 57.0%, respectively) (Supplementary material,
Fig. S3). Our results suggest that the metabolites contained in the
44 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46

Fig. 3. Effect of the microbial fungicide based on B. subtilis EA-CB0015 culture incorporated in different fungicides programs on the severity of black Sigatoka disease on
banana plants in field. (A) Evolution of the infection index (II) in time, (B) Area Under de Curve (AUC) of the infection index (II). CP donates conventional program, M1: MF
replace mancozeb (DithaneÒ) in the CP; M2: MF replace chlorothalonil (BravonilÒ) in the CP; M1–2: MF replace mancozeb and chlorothalonil in the CP; C negative control;
MF: microbial fungicide. The description of each fungicide program CP, M1, M2, and M1–2 are described in Table 1. Different letters indicate significant difference between
the treatments (P = 0.0025; LSD).

Fig. 4. Effect of different components of the microbial fungicide based on B. subtilis EA-CB0015 culture on the percentage of necrotic area of banana leaves inoculated with M.
fijiensis in greenhouse. MF denotes microbial fungicide, T1 the CFS; T2 spores of B. subtilis EA-CB0015 (3.1 * 108 CFU/mL); T3 vegetative cells of B. subtilis EA-CB0015
(4.5 * 107 CFU/mL); C negative control. Different letters indicate significant difference between the treatments (P < 0.0001; LSD).

CFS, which are mainly lipopeptides (Villegas-Escobar et al., 2013; cells are also active against M. fijiensis however in lesser extents;
Mosquera et al., 2014), are inhibiting M. fijiensis ascospores germi- they might need to colonize the leaf, compete for nutrients and
nation and are responsible for most of the control of black Sigatoka produce active metabolites for inhibiting M. fijiensis ascospores
observed for the MF in greenhouse and field. B. subtilis EA-CB0015 contrary to the active metabolites present in the CFS.
 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46
4. Discussion
Control strategies for black Sigatoka disease in banana crops are
mainly based in recurrent applications of chemical fungicides mix-
tures all year round. In areas where bananas are produced, the
increases in fungicide application have become a concern (Marín
et al., 2003) and the biological control appears as an alternative
that could help to alleviate the load of conventional fungicides in
this crop. The present work shows the effectiveness of a biological
fungicide base on B. subtilis EA-CB0015 and its metabolites for con-
trolling the disease in greenhouse and field. The MF applied as a
water suspension at a dose of 1.5 L/ha was able to reduce the dis-
ease severity in the same extent as the fungicides chlorothalonil
and mancozeb, protectants fungicides commonly used in fungicide
programs for the control of black Sigatoka disease in banana. The
MF at 1.5 L/ha also showed to be effective when applied in combi-
nation with systemic fungicides as part of a fungicide program
having a control effectiveness comparable to a fungicide program
commercially used for the control of this disease. These results
showed that the MF could be introduced in the pool of tools avail-
able for the control of black Sigatoka disease in banana crops,
either as a water suspension or in combination with systemic
fungicides, reducing the fungicide load to which the productive
regions are subjected and the risk of fungicide resistance develop-
ing in a pathogen.
During the evaluation of the different components of the MF we
found that the formulated CFS was more effective in inhibiting M.
fijiensis ascospores germination and in controlling the disease
under greenhouse than the formulated spores or vegetative cells,
and was as effective as the MF. These results suggest that the abil-
ity of the MF to control black Sigatoka disease is mainly based on
the effect of the active metabolites produced during the fermenta-
tion process that are present in the CFS. Several B. subtilis strains
are known for their ability to produce antimicrobial compounds
(Raaijmakers et al., 2010; Ongena and Jacques, 2008; Cochrane
and Vederas, 2014). Among these compounds, the lipopeptides
fengycin and iturin have been well characterized for their antifun-
gal activity (Raaijmakers et al., 2010; Ongena and Jacques, 2008;
Cochrane and Vederas, 2014). These lipopeptides have been shown
to inhibit fungi growth by disrupting the cell membrane (Tao et al.,
2011; Romero et al., 2007). In previous works, we demonstrated
the presence of lipopeptides fengycin C and iturin A in B. subtilis
EA-CB0015 CFS and linked them to the antifungal activity of this
strain (Villegas-Escobar et al., 2013; Mosquera et al., 2014). We
also optimized a culture medium for B. subtilis EA-CB0015 produc-
tion, which was the same media use in this work for the MF pro-
duction, and showed that during the fermentation fengycin C and
iturin A are produced at high concentrations (Mosquera et al.,
2014). Consequently, our results suggest that fengycin C and iturin
A present in the MF, are the main active ingredient and are respon-
sible for reducing the incidence of black Sigatoka disease in banana
plants.
We also found that the formulated B. subtilis EA-CB0015 cells,
independent of the cell structure (spores or vegetative cells), were
able to inhibit M. fijiensis ascospores germination and reduce the
incidence of black Sigatoka disease in greenhouse, even though
they were less effective than the CFS. These results might indicate
that B. subtilis EA-CB0015 spores and vegetative cells are able to
establish on the leaves of banana plants and to control black
Sigatoka disease development. They could be active either by
out-competing M. fijiensis or by producing antifungal metabolites
that prevents M. fijiensis growth and establishment, however this
needs further study.
In previous work, we found that strain B. subtilis EA-CB0015
originally isolated from leaves of banana plants had different
45
properties associated with phyllosphere colonizing bacteria
(Ceballos et al., 2012). These characteristics can provide the strain
adaptive advantages to survive in this environment. There are
many studies that address the issue of the ecology of phyllosphere
microbiota of plants using metagenomic and culture dependent
approaches (Rastogi et al., 2013; Earl et al., 2008). Nonetheless,
both approaches have a down side, they do not provide knowledge
about temporal and spatial aspect of the microbial colonization.
Therefore, in order to get a better understanding of Bacillus sp. col-
onization, other approaches need to be used such as bioreporters
or biomarkers that allow for the detection of Bacillus sp. on leaves.
These approaches have been used in few studies (Crane and
Bergstrom, 2014; Huang et al., 2012) but never for banana phyllo-
sphere. Once the bacterium is established, there is a high chance
that it will start producing and secreting antimicrobial metabolites
that will help it in the competition and colonization process. In situ
production of antimicrobial metabolites has been studied for few
metabolites. Romero et al. (2007) and Touré et al. (2004) success-
fully determined the in situ production of lipopeptides on melon
leaves and fruits when inoculated with B. subtilis. To prove the
hypothesis that B. subtilis EA-CB0015 is able to establish in the
banana phyllosphere and can produce the antifungal metabolites
that prevents M. fijiensis growth, we are currently evaluating the
colonization capability of B. subtilis EA-CB0015 and the in situ pro-
duction of fengycin C and iturin A on banana leaves.
5. Conclusions
The use of biological products in agriculture is increasing (Glare
et al., 2012) and here we successfully demonstrated the ability of a
biological fungicide base on B. subtilis EA-CB0015 and its metabo-
lites to control black Sigatoka disease in banana. The public accep-
tance and the high cost of biological products are one of the major
barriers that this kind of products needs to overcome before its
introduction to the pesticides market. The lack of acceptance of
biological products is mostly caused by examples of inconsistent
field performances (Glare et al., 2012; Hynes and Boyetchko,
2006), although integrating biological products in a conventional
fungicide rotation strategy could reduce fungicide applications
and a better manage of fungicide resistance. New trials are being
conducted to assure the effectiveness of the MF in more extensive
areas and to identify possible causes of inconsistences if any.
Additionally, there are other aspects that still need to be addressed
related to persistence, implementation, biology and ecology of the
active organisms in the banana system that our research group is
beginning to explore.
Acknowledgments
The authors are thankful to the Universidad EAFIT, the
Association of Banana Producers of Colombia (AUGURA) and
Colciencias (contract number 0836-2012) for the financial support
of this project. This research was made possible by the Subscribe
Contract Number 89 from December 20 of 2013 with the
Ministerio de Medio Ambiente y Desarrollo Territorial in the cate-
gory ‘‘Contrato de Acceso a Recursos Genéticos para la
Investigación Científica sin Interés Comercial’’.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biocontrol.2015.
04.012.
46 J.A. Gutierrez-Monsalve et al. / Biological Control 87 (2015) 39–46

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