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Effective control of black Sigatoka disease using a microbial fungicide based

on Bacillus subtilis EA-CB0015 culture

Article  in  Biological Control · April 2015

DOI: 10.1016/j.biocontrol.2015.04.012

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Accepted Manuscript

Effective control of black Sigatoka disease using a microbial fungicide based

on Bacillus subtilis EA-CB0015 culture

Jaime A. Gutierrez-Monsalve, Sandra Mosquera, Lina María González-

Jaramillo, John J. Mira, Valeska Villegas-Escobar

PII: S1049-9644(15)00059-6
DOI: http://dx.doi.org/10.1016/j.biocontrol.2015.04.012
Reference: YBCON 3255

To appear in: Biological Control

Received Date: 1 December 2014

Accepted Date: 10 April 2015

Please cite this article as: Gutierrez-Monsalve, J.A., Mosquera, S., González-Jaramillo, L.M., Mira, J.J., Villegas-
Escobar, V., Effective control of black Sigatoka disease using a microbial fungicide based on Bacillus subtilis EA-
CB0015 culture, Biological Control (2015), doi: http://dx.doi.org/10.1016/j.biocontrol.2015.04.012

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 Effective control of black Sigatoka disease using a microbial fungicide based on

Bacillus subtilis EA-CB0015 culture

Jaime A. Gutierrez-Monsalve1, Sandra Mosquera1, Lina María González-Jaramillo, John J.

Mira2, Valeska Villegas-Escobar1,*

1
Grupo CIBIOP, Departamento Ingeniería de Procesos, Universidad EAFIT. Carrera 49 No

7 Sur 50. Medellín, Colombia. E-mail: jagutierrez33@gmail.com (Jaime A. Gutierrez-

Monsalve), smosquera@ucdavis.edu (Sandra Mosquera), lgonza46@eafit.edu.co (Lina

María González-Jaramillo), vvilleg2@eafit.edu.co (Valeska Villegas-Escobar). Phone

number: (57-4)-2619500.

2
Centro de Investigaciones del Banano “Cenibanano”, Carepa (Antioquia), Colombia.

jmira.castillo@gmail.com, Phone number: (57-4) 8236602.

* Corresponding author

1
Abstract

Black sigatoka disease caused by the fungus Mycosphaerella fijiensis Morelet is the most

devastating disease of bananas worldwide. Its management is reliant on protectant and

systemic fungicides despite their environmental concerns. This study evaluated the effect

of a microbial fungicide (MF) based on Bacillus subtilis EA-CB0015 and its metabolites for

the control of black Sigatoka disease on banana plants in greenhouse and field conditions.

The MF applied at 1.5 L/ha and 3.0 L/ha provided control of the disease comparable to the

protectant fungicide chlorothalonil in greenhouse. In the field, the MF applied in solution

with water at 0.15 L/ha and 1.5 L/ha every 11 days during 10 weeks reduced black

Sigatoka disease severity in 20.2% and 28.1% respectively; reductions comparable to

those obtained with the protectant fungicides chlorothalonil (1.5 L/ha) and mancozeb (3.8

L/ha). The MF incorporated into different programs with systemic fungicides reduced

disease level up to 42.9% with no significant differences with the conventional 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 comparable 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.

Key words: Mycosphaerella fijiensis, microbial fungicide, metabolites, biological control,

banana plants.

2
1. Introduction

Banana, the fourth most important crop after rice, wheat and maize, is cultivated in the

tropical regions of more than 100 countries and plays a key role in the economies of many

developing 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 photosynthetic 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 applications 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; Marin et al., 2003). The increasing number of fungicide

applications has brought economic and environmental concerns and problems associated

to the emergence of resistant strains of the pathogen (Brent and Hollomon, 2007).

Consequently, more sustainable control strategies 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

3
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 incorporation into conventional fungicides programs. From

our knowledge, only two commercial products based on B. subtilis QST 713 (Serenade®)

and B. pumilus QST 2808 (Sonata®) 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 percentage 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 persistence, implementation,

biology and ecology of their active organisms (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, activity and product

delivery (Villegas-Escobar et al., 2014).

4
In the present study we evaluated the efficacy of a microbial fungicide (MF) based on the

biomass and the metabolites of B. subtilis EA-CB0015 for the control of black Sigatoka

disease in greenhouse 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 ºC 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 conducted in

accordance with Romero et al. (1999) and DNA extraction was carried out with the Ultra

Clean Microbial Isolation Kit (MoBIO). When need it, the stored mycelial suspensions were

5
grown on PDA complemented with 200 ppm chloramphenicol and incubated for 10 days at

30ºC 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 contained 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 Bacillus subtilis EA-CB0015

Cell-free supernatant (CFS) and suspensions of cells were prepared 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 medium 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 (3.289xg, 20 min)

and filtrated through 0.45 -µm cellulose acetate filters (Sartorius Biolab). For the spore cell

suspension, cells were recovered by centrifugation (3.289xg, 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

6
concentration in CFU/mL was adjusted with water. For the vegetative cell suspension,

cells were recovered by centrifugation (3.289xg, 20 min) after 12 h of incubation and the

pellet was washed twice with distilled water and the concentration in CFU/mL was

adjusted with water.

2.4. Antifungal activity of Bacillus 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 ascospores germination was assessed using the

varnish technique modified by Talavera et al. (1998). Briefly, disinfected discs of banana

leaves cv. Williams were submerged for 30 s in the different treatments 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 calculating the average

length of 30 randomly selected ascospores per disc using the AxioVision® 4.2 microscope

software (Carl Zeiss®). The percent inhibition of the germinative tube growth was

determined 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. subtilis 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 optimized culture medium (2.0 ± 1.0x109 CFU/mL) was used as the positive control (C+)

and deionized water was used as a negative control (C-).

7
2.5. Greenhouse trials design

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 fungicides, 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 (2.0x105 fragments/mL) (Donzelli and Churchill, 2007) and

incubated at 90% humidity and 28 ± 4°C for 24 h in greenhouse conditions for pathogen

establishment. Then the different treatments were applied at 3.3 mL/leaf using a MiniSpray

gun with cup K-3® calibrated at 60 to 80 drops/cm2 at an altitude of 30 cm.

In the first experiment the MF was diluted in water to obtain four different concentrations:

1.0x107 CFU/mL, 5.0x107 CFU/mL, 1.0x108 CFU/ha, and 2.0x108 CFU/mL, which are

equivalent to apply 0.15, 0.75, 1.5 and 3.0 L/ha of the MF in field. The conventional

fungicide chlororothalonil (Bravonil 720®) prepared at 10% (equivalent to 1.5 L/ha)

according to the manufacturer instructions 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

(3.1x108 CFU/mL) (Treatment T2); B. subtilis EA-CB0015 vegetative cell suspension

(4.5x107 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

8
mega-pixels and the processing imaging software Axio Vision® 4.2 (Zeiss®). Each

experiment was independently 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 conventional

fungicides chlororothalonil (Bravonil 720®) and mancozeb (Dithane F-MB®). The second

trial (trial 2) was implemented to evaluate the efficacy of MF when applied as part of a

fungicide program. Both experiments were evaluated during 10 weeks and were achieved

in the experimental field of AUGURA (Colombian Banana Growers Association) in Carepa

– Antioquia (7°45′29″N, 76 39′19″W) under production conditions with nonflowered banana

plants (cv. Williams) that had 1.5 to 1.8 m long.

2.6.1. Trial 1:

The MF was applied in solution with water at different concentrations: 1.0x107 CFU/mL,

1.0x108 CFU/mL and 2.0x108 CFU/mL which is equivalent to appling the MF at 0.15, 1.5

and 3.0 L/ha. Chlororothalonil (Bravonil 720®) and mancozeb (Dithane F-MB®) were

applied at 1.5 L/ha and 3.8 L/ha according the manufacturer instructions and non-treated

plants were used as negative control. The applications were performed every 11 days

using a Stihl® fumigation machine with 15 L capacity calibrated at 60 to 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 Calixín® (Tridemorph), Bumper® (Propiconazole), Sico®

(Difeconazole), Opus® (Epoxiconazole), Volley® (Fenpropimort), Cumora® (Buscalid)

9
applied in different weeks and differ in the protectant fungicide used. Conventional

program (P) included protectant 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%

Volley®, 1.76% Sico®, 4.4% Opus®, 2.2% Bumper®, 2.2% Calixin® or 2.2% Cumora®;

10% of protectant fungicide (mancozeb or MF); 0.41% of emulsifying Pegal® and was

complemented with water. The mixture containing the protectant fungicide chlororothalonil

(Bravonil® 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 Stihl® fumigation

machine with 15 L capacity calibrated at 60 to 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 measured. 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 determined with equation 1:

10
Where is the value of severity of the scale, the number of leaves in each value and

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 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, comparable 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.

11
The infection index curves (Fig. 2A) and the accumulative infection 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 chlorothalonil (24.4%) and mancozeb (24.0%). For the plants treated with MF at 3

L/ha no significant reduction of the black Sigatoka disease 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 material, Fig. S1 A

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

systemic fungicides.

3.2. The microbial fungicide incorporated in fumigation programs with systemic

fungicides reduces black Sigatoka disease

12
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 comparison to the C-

and had no difference with the CP (Supplementary material, Fig. S2 A and B).

3.3. Vegetative cells and spores of Bacillus subtilis EA-CB0015 and its metabolites

reduced black Sigatoka disease and Mycosphaerella fijiensis ascospore

germination

To determine which component of the MF reduces the incidence 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, respectively. 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 necrosis area, respectively, but

13
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 ascospores in vitro. The result obtained for this

experiment was consequent 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 CFS, which are mainly lipopeptides

(Villegas et al., 2013; Mosquera et al., 2014), are inhibiting M. fijiensis ascospores

germination and are responsible for most of the control of black Sigatoka observed for the

MF in greenhouse and field. B. subtilis EA-CB0015 cells are also active against M. fijiensis

however in lesser extents; they might need to colonize the leaf, compete for nutrients and

produce active metabolites for inhibiting M. fijiensis ascospores contrary to the active

metabolites present in the CFS.

4. Discussion

Control strategies for black Sigatoka disease in banana crops are mainly based in

recurrent applications of chemical fungicides mixtures all year round. In areas where

bananas are produced, the increases in fungicide application have become a concern

(Marin 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 controlling the disease in greenhouse and field. The MF applied as a water suspension

14
at a dose of 1.5 L/ha was able to reduce the disease 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 combination 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 available 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 developing 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 ability of the MF to control black

Sigatoka disease is mainly based on the effect of the active metabolites produced during

the fermentation 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 antifungal 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 production, which was the

15
same media use in this work for the MF production, 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 responsible 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 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.

colonization, 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

16
phyllosphere. 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) successfully 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 production of fengicin 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 metabolites to control black Sigatoka disease in banana. The public

acceptance 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.

17
Acknowledgements

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 category “Contrato de Acceso a Recursos Genéticos para la Investigación

Científica sin Interés Comercial”.

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Table captions

Table 1. Fungicide programs designs for field trial 2.

24
Figures caption

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 (Bravonil®), 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.

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 (Bravonil®), Mc: Mancozeb (Dithane®), MF: microbial fungicide and C- negative

control. Different letters indicate significant difference between the treatments (P = 0.0025;

LSD).

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 (Dithane®) in the CP;

M2: MF replace chlorothalonil (Bravonil®) 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; ANOVA test).

25
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*10^8 CFU/mL); T3 vegetative cells of B. subtilis EA-CB0015 (4.5*10^7 CFU/mL); C-

negative control. Different letters indicate significant difference between the treatments (P <

0.0001; ANOVA test).

26
Table 1.

Protectants
Week Systemic
Cycle Base + +
2013 fungicide* Conventional Program Program Program
program (P) M1 M2 M1-2

1 13 Oil + Tridemorph + Mancozeb MF Mancozeb MF

2 15 Oil + Propiconazole + Mancozeb MF Mancozeb MF
3 16 Water + --- + Chlorothalonil Chlororothalonil MF MF
4 17 Oil + Difeconazole + Mancozeb MF Mancozeb MF
5 19 Oil + Epoxiconazole + Mancozeb MF Mancozeb MF
6 20 Water + --- + Chlorothalonil Chlororothalonil 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
*MF: Microbial Fungicide (1.5 L/ha); Chlororothalonil (Bravonil® 720®, 1.5 L/ha); Mancozeb

(Dithane®, 1.5 L/ha); Tridemorph (Calixín®, 0.4 L/ha); Propiconazole (Bumper®, 0.4 L/ha);

Difeconazole (Sico®, 0.3 L/ha); Epoxiconazole (Opus®, 0.8 L/ha); Fenpropimort (Volley®, 0.4

L/ha); Buscalid (Cumora®, 0.4 L/ha).

27
 Highlights:
• 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.

28

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