Biological Control 155 (2021) 104523

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

Role of Bacillus tequilensis EA-CB0015 cells and lipopeptides in the

biological control of black Sigatoka disease
Tatiana Z. Cuellar-Gaviria a, b, Lina M. González-Jaramillo a, b, Valeska Villegas-Escobar a, *
a
CIBIOP Group, Biological Sciences Department, Universidad EAFIT, Carrera 49 No. 7 Sur - 50, Medellin, Colombia
b
Banana Research Center, Augura, Conjunto Residencial Los Almendros, Km 4, Carepa, Colombia

H I G H L I G H T S

• Lipopetides of B. tequilensis EA-CB0015 reduce the severity of black Sigatoka disease.

• B. tequilensis EA-CB0015 cells colonize the leave surface of banana plants.
• B. tequilensis EA-CB0015 cells produce surfactins lipopeptides on the leave surface.
• The mode of action of B. tequilensis product involves competition and antibiosis.

A R T I C L E I N F O A B S T R A C T

Keywords: Black Sigatoka disease is a devastating fungal pathogen that affects banana crops worldwide and is caused by the
Bacillus tequilensis ascomycete fungus Pseudocercospora fijiensis. In an effort to mitigate the environmental impacts of chemical
Pseudocercospora fijiensis fungicides, microbial biological control agents (MBCA) have been developed. However, a comprehensive un­
Banana plants
derstanding of the modes of action of these products are needed to enhance their efficacy under field conditions.
Phyllosphere colonization
Consequently, the functional role of different components of a Bacillus tequilensis EA-CB0015-based product was
Antibiosis
Lipopeptides studied to understand their role in conferring protection to banana plants against the infection of P. fijiensis.
Under this perspective, we evaluated the effect of B. tequilensis EA-CB0015 lipopetides on the severity of black
Sigatoka disease, as well as the ability of the bacterial cells to colonize and produce lipopeptides on the leaf
surface of banana plants (Musa AAA cv. Williams). Our work provides evidence that lipopetides significantly
reduce the disease severity and that the bacterial cells colonize the leaf surface of banana plants inoculated with
P. fijiensis during the first 48 h of incubation. Furthermore, we were able to detect an increased production of
surfactins during colonization using mass spectrometry analysis. Altogether, our results suggest that the bio­
logical control strategies implemented by the MBCA product against P. fijiensis involves a direct interaction via
antibiosis, as well as an indirect interaction via competition of nutrients and space.

1. Introduction
Black Sigatoka disease affects banana crops worldwide and it is
caused by Pseudocercospora fijiensis (formerly known as Mycosphaerella
fijiensis), a Dothideomycetes fungus that produces necrotic lesions on the
leaves, reducing the photosynthetic area, causing alterations in the
quality of the fruit, as well as significantly reducing crop yields
(Arzanlou et al., 2008; Marín et al., 2003). The control of this disease
accounts for more than 25% of the total production costs and relies
mainly on regular applications of chemical fungicides (De Bellaire et al.,
2010); representing a risk to the environment and health of terrestrial
and aquatic ecosystems (Wightwick et al., 2010; Zubrod et al., 2019).
Moreover, due to the rapid development of chemical fungicide resis­
tance, the current strategy represents an imminent threat to banana
production systems (Arango Isaza et al., 2016). As a result of the prob­
lems caused by chemical pesticides, strict regulations have been
imposed and some chemical products, such as chlorothalonil that is
widely used in banana crop systems, have been banned in Europe due to
safety concerns (Regulation No. 2019/677) (The European Comission,
2019). Given these reasons, there has been growing interest in finding
more sustainable alternatives that are safer and more environmentally
friendly. Such strategies include the use of microbial biological control

* Corresponding author.
E-mail address: vvilleg2@eafit.edu.co (V. Villegas-Escobar).

https://doi.org/10.1016/j.biocontrol.2020.104523
Received 18 June 2020; Received in revised form 15 December 2020; Accepted 18 December 2020
Available online 29 December 2020
1049-9644/© 2020 Elsevier Inc. All rights reserved.
T.Z. Cuellar-Gaviria et al.
agents (MBCAs) to fight plant pathogens. A variety of MBCAs have been
developed for different crop systems, however, low efficacy in field
conditions is a persistent problem, which, in part, could be attributed to
the lack of understanding of the biological basis and modes of action
behind the control of the disease (Ellis, 2017).
In an effort to develop a MBCA product that could be used to control
the severity of black Sigatoka in banana plants, the bacterial strain Ba­
cillus tequilensis EA-CB0015, previously reported as B. subtilis EA-
CB0015, was selected among 648 isolates obtained from the phyllo­
sphere of banana plants, due to its outstanding in vitro ability to inhibit
the growth of P. fijiensis (Ceballos et al., 2012). Subsequent studies led to
the identification of the production of different lipopeptides by the
bacterial strain during culture conditions, including: fengycins, iturins
and surfactins (Mosquera et al., 2014; Villegas-Escobar et al., 2013).
Additional studies also led to the optimization of the culture media used
in the production process to achieve greater amounts of biomass and
metabolites (Mosquera et al., 2014). Moreover, the efficacy of the
formulated MBCA product in reducing the severity of the disease was
corroborated in different greenhouse and field evaluations (Gutierrez-
Monsalve et al., 2015; Villegas-Escobar et al., 2016). More recently, it
was found that both, fengycins and iturins produced by B. tequilensis EA-
CB0015 inhibited the micellar growth and ascospore germination of
P. fijiensis in vitro (González-Jaramillo et al., 2017).
Previous studies have suggested that lipopeptides produced by
different MBCAs play an important role in the control of the disease, not
only in terms of protection against the plant pathogen, but also, in terms
of ecological competence and population establishment (Debois et al.,
2014). In accordance with this idea, there is strong evidence that the
colonization of MBCAs, containing living microorganisms, is an impor­
tant trait for the control of different diseases (Hao et al., 2017; Honeker
et al., 2017; Xu et al., 2019). When MBCAs are applied to crops, they can
act via different modes of action to control plant disease, involving
either direct or indirect interactions with the pathogen. Among the in­
direct modes of action, there is the induction of systemic resistance or
priming, as well as the competition for nutrients and space, while direct
mechanisms can include hyperparasitisms or antibiosis (Köhl et al.,
2019). Toward this end, previous research performed with B. tequilensis
EA-CB0015 suggest that the mode of action could be associated with
antibiosis (Ceballos et al., 2012; González-Jaramillo et al., 2017;
Gutierrez-Monsalve et al., 2015; Mosquera et al., 2014; Villegas-Escobar
et al., 2013). However, studies to understand the mode of action
implemented by the MBCA, based on B. tequilensis EA-CB0015, and the
interactions of the bacterial strain with the infected plant were lacking.
In order to gain a better understanding of the mode of action of the
MBCA product to control black Sigatoka disease in Musa AAA cv. Wil­
liams, we aimed at investigating three main points: i) the ability of the
lipopeptides produced by the bacterial strain to reduce the severity of
the disease in greenhouse conditions; ii) the colonization ability of
B. tequilensis EA-CB0015 using catalyzed reporter deposition–­
fluorescent in situ hybridization (CARD-FISH) as well as scanning elec­
tron microscopy (SEM); and iii) the in situ production of lipopeptides of
B. tequilensis EA-CB0015 on the leaf surface of banana plants using high
performance liquid chromatography - mass spectrometry (HPLC-MS/
MS). By investigating the mode of action of the MBCA product, we
gained important insights about the interaction between the plant,
pathogen, and MBCA; knowledge that is essential to potentially enhance
the efficacy of the MBCA against the disease.
2. Materials and methods
2.1. Microorganisms and growth conditions
Fresh bacterial cultures of B. tequilensis EA-CB0015 wild-type (WT)
(GenBank accession no. KC006063) (Ceballos et al., 2012), were
routinely obtain by activation of frozen cultures on 50% Tryptic Soy
Agar (TSA; Oxoid) and incubation at 30 ◦ C for 48 h prior any
2
Biological Control 155 (2021) 104523
experimental use. A spontaneous and recurrent morphological variant
(MV) of B. tequilensis EA-CB0015, was isolated in this study from the
liquid culture of the WT strain after 120 h of incubation at 30 ◦ C and 150
rpm in D media (33.4 g/L glucose, 32.5 g/L yeast extract, 4.0 g/L
MgSO4*7H2O, 1.0 g/L (NH4)2 SO4, 0.5 g/L K2HPO4, 0.5 g/L KH2PO4,
0.042 g/L MnSO4*4H2O, 0.031 g/L CaCl2) (Mosquera et al., 2014). The
purified MV had a flat and mucoid colony morphology that differed
greatly from the small and wrinkled colony morphology of the WT. The
biomass of B. tequilensis EA-CB0015 (MV) was stored at − 80 ◦ C in
Tryptic Soy Broth (TSB, Merck) with 20% (v/v) glycerol (PanReac) and
activated on 50% Tryptic Soy Agar (TSA; Oxoid), at 30 ◦ C for 48 h prior
experimental use.
Cultures of P. fijiensis were obtained according to Gutierrez-Monsalve
et al. (2015). Briefly, isolated ascospores were grown on Potato Dextrose
Agar (PDA; Merck) supplemented with 200 μg/mL chloramphenicol, for
10 to 15 d at 28 ◦ C and then, fragmented using distilled water and glass
beads. Fragmented mycelia were then inoculated in 50 mL of Sabour­
aud-2% dextrose broth (Merck) supplemented with 200 μg/mL chlor­
amphenicol for 8 to 10 d at 30 ◦ C and 150 rpm, time after which the
fungal inoculum was homogenized using an IKA ULTRA-TURRAX®
Tube Drive P control workstation. For greenhouse experiments, the
fragmented mycelia were resuspended in sterile water to a final con­
centration of 2.5 × 105 fragments/mL, and to ensure adherence and
establishment on the leaves, the following components were added: 3%
(w/v) carboxymethyl cellulose (CMC; Protokimica), 0.5% (v/v) Tween®
20 (Amresco), and 2% (w/v) Sabouraud-2% dextrose broth (Merck).
2.2. Identification of the morphological-variant (MV) of B. tequilensis
EA-CB0015
DNA extraction of B. tequilensis EA-CB0015 (MV) was performed
using the UltraClean® Microbial DNA Isolation Kit (MoBio Labs)
following the instructions of the manufacturer. The 16S rRNA gene was
amplified by PCR using the universal primers 8F (5′ - AGAGTTT­
GATCCTGGCTCAG − 3′ ) and 1492R (5′ - CGG TTA CCT TGT TAC GAC TT
− 3′ ) (Hendrickson et al., 2002). The PCR cycles consisted in an initial
denaturation step at 95 ◦ C for 3 min, 35 cycles including a denaturation
step at 95 ◦ C for 1 min, an annealing step at 55 ◦ C for 1 min, followed by
an extension step at 60 ◦ C for 1 min and a final extension cycle at 72 ◦ C
for 10 min. Amplified samples were used for product purification and
Sanger sequencing. The obtained consensus sequence was deposited in
the GenBank (accession no. MT527175) and compared to the 16S rRNA
gene sequence of B. tequilensis EA-CB0015 (WT) using a pairwise
alignment with Clustal Omega 1.2.3 (Fig. S1).
2.3. Biofilm formation assay
The (WT) and (MV) of B. tequilensis EA-CB0015 were assessed for
pellicle biofilm formation in static culture conditions. For this purpose,
LBGM media composed of LB broth (10 g/L tryptone (Oxoid), 5 g/L
yeast extract (Oxoid) and 10 g/L NaCl (Merck)), supplemented with 1%
glycerol (v/v) (PanReac) and 0.1 mM MnSO4 (Merck) was used, as
previously described by Shemesh and Chai, 2013. In brief, the bacterial
morphotypes were grown in LB broth at 37 ◦ C and 200 rpm to mid-log
phase (OD600 = 0.5). Then, 4 µL of culture were transferred to 4 mL of
LBGM in 12-well plates (NEST). Plates were incubated in static condi­
tions for 48 h at 30 ◦ C. Images were taken using a 7.1 MP digital camera
(Kodak EasyShare P712).
2.4. Purification of lipopeptides
To purify the active metabolites produced by B. tequilensis EA-
CB0015 (WT) and (MV), a solid phase extraction protocol, was imple­
mented (Arroyave-Toro et al., 2017; Villegas-Escobar et al., 2013).
Briefly, both morphotypes of B. tequilensis EA-CB0015 were cultivated
separately in D media under the aforementioned conditions, and
T.Z. Cuellar-Gaviria et al.
Amberlite® XAD16N 20–60 mesh (Sigma) was added after 12 h of
inoculation. The amberlite resin was recovered from the culture broth
after 108 h and washed three times with distilled water. The adsorbed
components were eluted using 200 mL of 100% methanol (MeOH) and
evaporated using a rotary evaporator (Yamato RE-300) at − 50 psig and
50 ◦ C. The obtained solid residue was resuspended in 10 mL of distilled
water and the resulting sample was fractioned through solid-phase
extraction (SPE), using a C18 column (HF Mega BE-C18, 10 g; Agi­
lent). The column was preconditioned using 100% MeOH, followed by
water and fractions were eluted by consecutive 80 mL solutions of:
water, 50% MeOH, and 100% MeOH. The solvent of the last fraction,
referred to as SPE100, was evaporated at reduced pressure (-50 psig,
50 ◦ C). The solid residue of the (WT) was resuspended in the original
volume of the liquid culture (200 mL of 0.1 M phosphate buffer pH 7.5)
(55.0 μg/mL). The solid residue of the morphological variant was used
only for detection purposes under the reverse phase RP-HPLC. To
calculate the relative concentration of lipopeptides produced by the
(WT) and (MV) morphotypes, we used the area under the curve
(measured in mAU*min) of the corresponding peaks obtained from the
HPLC’s chromatogram. Fig. S2 shows the HPLC chromatograms of lip­
opeptides produced by B. tequilensis EA-CB0015 (WT) and (MV).
2.5. Production of treatments based on B. tequilensis EA-CB0015
For the production of B. tequilensis EA-CB0015 based treatments
(MBCA-WT, CFS-WT, biomass-WT, and MBCA-MV), a single colony of
B. tequilensis EA-CB0015 (WT) or (MV) was transferred to 20 mL D media
(Mosquera et al., 2014) and incubated at 30 ◦ C, 150 rpm overnight. The
seed culture (20 mL) was then transferred to a 1000 mL flask containing
180 mL of D media and incubated for 108 h, at 30 ◦ C and 150 rpm. The
final composition of the MBCA (WT or MV) contained the bacterial
biomass as well as the supernatant containing the metabolites produced
by the corresponding bacterial morphotype. To obtain the CFS-WT, the
aforementioned (WT) bacterial culture was centrifuged twice at 4500
rpm for 15 min and the supernatant was filtered twice through a 0.2 μm
cellulose membrane (Advantec MFS, Inc) to remove the remaining
bacterial cells. Lastly, the bacterial biomass of B. tequilensis EA-CB0015
(WT) was obtained under the same conditions and the recovery of the
bacterial cells was performed by centrifugation at 4500 rpm for 15 min,
followed by the resuspension of the pellet in sterile-distilled water to a
final concentration of 8.6 X 108 CFU/mL.
2.6. Greenhouse trials
2.6.1. Trial 1: Biocontrol efficacy of the metabolites produced by
B. tequilensis EA-CB0015 (WT)
A greenhouse evaluation was performed to evaluate the disease
reduction obtained by the SPE100 fraction of B. tequilensis EA-CB0015
(WT), containing the lipopeptides (Lipopeptides (WT)). For this, six-
months-old banana plants (Musa AAA cv. Williams, Cavendish sub­
group), having between 5 and 6 true leaves, were inoculated with
fragmented mycelia of P. fijiensis on the axial and abaxial sides of leaf
number one. Inoculated plants were incubated for 48 h for pathogen
establishment. Then, using a MiniSpray gun (Discover®), calibrated at
60–80 drops/cm2, different treatments were sprayed, keeping a 30 cm
distance during application. Two treatments were included: (i) lip­
opeptides produced by B. tequilensis EA-CB0015 (WT) and (ii) MBCA-
MV. Additionally, two positive controls were included: (iii) MBCA-
WT, (iv) CFS-WT, and a negative control (v) water. Each treatment
was mixed with CMC 3% (w/v) and Tween 20 0.5% (v/v) prior inocu­
lation to ensure adherence to the leaf surface.
Banana plants were arranged in the greenhouse with natural light
source of 12 h photoperiod; maintaining humidity and temperature
conditions at 90% and 28 ± 4 ◦ C, respectively. Plants were distributed
using a complete randomized design (CRD), using 7 replicates per
treatment and 3 plants per replicate. The experiment was conducted
3
Biological Control 155 (2021) 104523
until the negative control treatment reached a necrotic area of about
45% of the leaf, which occurred 34 dpi. Disease severity was assessed by
measuring the necrotic area of each leaf. Necrotic area (%) was calcu­
lated by dividing the measured necrotic area by the total area of the leaf.
To calculate this area, each harvested leaf was photographed on the
abaxial side using a 7.1 MP digital camera (Kodak EasyShare P712).
Necrotic area calculation was performed by processing the images using
ImageJ software (https://imagej.nih.gov/ij/).
2.6.2. Trial 2: Colonization ability of B. tequilensis EA-CB0015 (WT) and
detection of lipopeptides
For this trial, we used five-months-old banana plants cv. Williams
and inoculated leaves number one and two of each plant with P. fijiensis
on the axial and abaxial sides. After 48 h of incubation with the path­
ogen, the corresponding treatment was applied to both sides of the
leaves: (i) biomass-WT, (ii) MBCA-WT, or (iii) water as negative control.
Treatments were mixed with CMC and Tween 20, and applied using the
MiniSpray gun, as mentioned above. A plant from each treatment was
harvested for analysis at the following time points: 0, 12, 24, 48 h, and
20 dpi. The last timepoint was dictated by the day at which the negative
control reached stage 1 of the disease according to the scale proposed by
Gauhl (1994). We inoculated five plants per treatment and placed them
in the greenhouse at 90% humidity, 28 ± 4 ◦ C and natural light source
with a 12 h photoperiod. Plants were arranged using a CRD.
2.7. CARD-FISH hybridization and scanning electron microscopy
To determine the colonization ability of B. tequilensis EA-CB0015
(WT), leaf number one of each plant was cut in half along the midrib,
and each half was fixed for either catalyzed reporter deposition–­
fluorescent in situ hybridization (CARD-FISH) or scanning electron mi­
croscopy (SEM). Samples used for CARD-FISH hybridization were fixed
with NaCl 0.85% and 96% ethanol (1:1 ratio) and stored at − 20 ◦ C until
processing. The protocol used for CARD-FISH hybridization was adapted
according to the protocols presented by Wendeberg (2010) and Teira
et al. (2004). In brief, samples were permeabilized, treated for inacti­
vation of peroxidases, and hybridized using the probe HRP-Bsub-ss-
0463-aA-22A (Posada et al., 2018). Then, samples were treated with
amplification buffer and CF®555 dye (Biotum). Previous to microscope
observation, samples were treated with 4′ ,6-Diamidino-2-Phenylindole
R37606 (DAPI, Invitrogen) and Citifluor AF1 (Electron Miscroscopy
Science). Samples were analyzed under the fluorescence microscope
AxioScope A.1 (Carl Zeiss), using the corresponding filter. Pictures were
taken with Axiocam ICm 1 (Carl Zeiss) and images were processed using
the software ZEN 2 (blue edition) (Carl Zeiss).
Samples used for SEM evaluation were fixed using 2.5% glutaral­
dehyde in phosphate buffer solution pH 7.4 for 4 h; followed by suc­
cessive washes in 25%, 50%, and 70% ethanol for periods of 20 min.
Samples were then stored at 4 ◦ C in 70% ethanol until processing. Before
images were taken, the leaves were processed in 96% ethanol for 24 h
and coated with gold prior visualization under the SEM (FEI QUANTA
250).
2.8. Detection of the in-situ production of lipopeptides
To extract lipopeptides from banana leaves, an extraction protocol
reported by Kinsella et al. (2009) was implemented with minor modi­
fications. We included the following treatments: i) a positive control that
consisted on the application of the MBCA product (WT) containing the
lipopeptides, harvested 0 hpi, ii) the bacterial biomass of B. tequilensis
EA-CB0015 (WT), and iii) a negative control that consisted of infected
leaves treated with water. In brief, the metabolite extraction procedure
was performed to 3 g of the harvested leaf tissue at each time point (0,
12, 24 and 48 hpi). Tissue samples were submerged in 30 mL of aceto­
nitrile containing 0.05% trifluoroacetic acid and placed in an ultrasonic
bath for 10 min. Then, each sample was placed in the rotary evaporator
T.Z. Cuellar-Gaviria et al.
(Yamato RE-300) at − 50 psig and 50 ◦ C, resuspended in 20 mL of
methanol, and filtered through 0.2 µm nylon membrane (Sartorius
Biolab). Samples were evaporated again and the solid fractions were
used for MS analysis.
For MS analysis, samples were resuspended in 5% of a buffer
composed of 0.1% formic acid in acetonitrile and then diluted to reach a
concentration of 1 mg/mL. Samples were analyzed using high-
performance liquid chromatography coupled with quadrupole Orbi­
trap high-resolution mass spectrometry (HPLC-Q-Exactive Orbitrap MS/
MS). A C18 column was used for HPLC separation. The solvent system
consisted of 0.1% formic acid (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). Twenty (20) µL were injected per sample and
metabolites were eluted using a gradient program that went from 20 to
100% B for 29 min. The optimized parameters for the MS spectra con­
sisted of 70,000 of resolution, 3e5 AGC, 80 ms of maximum injection
time, and for the MS2 spectra the parameters were as follows: 17,500
resolution, 2e5 AGC, and 80 ms of maximum injection time. Purified
iturins, fengycin and surfactins produced by B. tequilensis EA-CB0015
(WT) were used as standards. MS raw data obtained in the MS2
4
Biological Control 155 (2021) 104523
fractionation experiments were converted to .mzXML and deposited in
MASSIVE (MSV000086176).
The MS2 analysis was performed by using mass spectral molecular
networking (Aron et al., 2020; Watrous et al., 2012). The molecular
networking analysis was performed in the Global Natural Product Social
Molecular Networking platform (GNPS, http://gnps.ucsd.edu). Analysis
of the data was done by grouping the mzXML files in four groups (G1:
treatments with the biomass of B. tequilensis EA-CB0015; G2: negative
control, G3: standards, G4: positive control). The following parameters
were used for the construction of the network: precursor and fragment
ion mass tolerance of 0.02, minimum cosine score of 0.7, minimum
matched fragment ions of 4, library search minimum matched peaks 6,
score threshold of 0.7. Visualization of the network as nodes and edges
was done using Cytoscape 3.8.0 (https://cytoscape.org/).
2.9. Statistical analysis
The data corresponding to the greenhouse Trial 1 (section 2.6.1) was
analyzed using R v4.0 (R Core Team, 2020). Analysis of variance was
Fig. 1. Effect of lipopeptides of B. tequilensis
EA-CB0015 (WT) on the severity of black
Sigatoka disease and phenotypic differences
between the (WT) and morphological variant
(MV) of B. tequilensis EA-CB0015. (A)
Greenhouse evaluation of the effect of the
lipopeptides produced by B. tequilensis EA-
CB0015 (WT) on the percentage of necrotic
area of banana leaves inoculated with
P. fijiensis. MBCA-WT refers to the microbial
biological control agent product containing
the biomass and supernatant of B. tequilensis
EA-CB0015 (WT). CFS-WT refers to the cell
free supernatant containing metabolites and
spent media components of B. tequilensis EA-
CB0015 (WT). Lipopeptides-WT refers to
lipopeptides extracted from the culture of
B. tequilensis EA-CB0015 (WT), containing
iturins, fengycins and surfactins. MBCA-MV
refers to the microbial biological control
agent product containing the biomass and
supernatant of B. tequilensis EA-CB0015
(MV). Control label refers to the application
of water as negative control. Necrotic area
(%) was calculated by dividing the measured
necrotic area by the total area of the leaf (n
= 7). The presented data correspond to the
average necrotic area per treatment.
Different letters above each bar indicate
significant differences between the mean of
the treatments according to multiple ranges
LSD with 95% confidence (p-value = 0.0009).
Standard error of the mean is presented for
each bar. The pictures below each bar are
representative for each treatment and show
the necrotic area produced by P. fijiensis 20
dpi. (B) This table presents differences on the
production of lipopeptides (iturins, fengy­
cins, and surfactins) of the (WT) and (MV) of
B. tequilensis EA-CB0015. The values shown
in (mAU*min) represent the sum of the areas
under the curve of each peak obtained from
the RP-HPLC chromatogram (Fig. S2), which
is proportional to the concentration of the
lipopeptide in each sample. Reduction (%)
indicates the reduced production of lip­
opeptides in (MV) with respect to (WT) for
each lipopeptide. (C) Pellicle biofilm forma­
tion of B. tequilensis EA-CB0015 (WT) and
(MV) in LBGM media after 48 h.
T.Z. Cuellar-Gaviria et al. Biological Control 155 (2021) 104523

carried out by ANOVA, with 95% confidence interval. LSD multiple
comparison test was used to analyze the differences between group
means. Normality of data was checked using Shapiro-Wilk test, inde­
pendence was confirmed by Durbin-Watson test, and homogeneity of
variance was assessed using Leven’s test.
3. Results
is impaired in the production of lipopeptides (Fig. 1B). This result could
also suggest that the colonization ability of the (WT) strain could be
enhancing the biocontrol activity, as B. tequilensis EA-CB0015 (MV) is
impaired in its ability to form pellicle biofilm (Fig. 1C). Therefore, we
next aimed at identifying if the bacterial cells present in the MBCA (WT)
product were capable of colonizing the leaf surface after being applied
on banana plants infected with P. fijiensis.

3.1. Lipopeptides produced by B. tequilensis EA-CB0015 reduce the 3.2. B. tequilensis EA-CB0015 (WT) can colonize the leaves of banana
severity of black Sigatoka disease in banana plants plants infected with P. fijiensis

We had previously evaluated the biocontrol activity of different
components of the biofungicide based on B. tequilensis EA-CB0015 (WT)
and showed that the cell-free supernatant (CFS), containing the me­
tabolites produced by the bacterial strain, reduced the development of
disease in a greater extent than the bacterial biomass alone (Gutierrez-
Monsalve et al., 2015). However, to further characterize the mode of
action employed by the MBCA, we extracted the lipopeptides produced
by B. tequilensis EA-CB0015 (WT) (iturins, fengycins and surfactins) and
evaluated the effect of the combined metabolites on the severity of black
Sigatoka disease (Fig. 1A). Analysis of necrotic areas showed that
treatments with the MBCA-WT and CFS-WT, caused a significant
reduction of necrotic area (73.1% and 60.3%, respectively) when
compared to the negative control. Treatment with the extracted lip­
opeptides produced by B. tequilensis EA-CB0015 (WT) also showed a
significant reduction of the necrotic area, however this occurred in a
lesser extent (40.8%), suggesting that either the concentration of the
extracted lipopeptides (55 μg/mL) was lower than the one found in the
MBCA (WT), or that other metabolites could be contributing to the
reduction of the disease symptoms. On the other hand, the application of
the MBCA-MV, which showed 99.87% identity to 16S B. tequilensis EA-
CB0015 (WT) rRNA gene sequence (Fig. S1), did not cause reduction
of the necrotic area; suggesting that the lipopeptides are a key compo­
nent ingredient of the MBCA (WT) product, as the morphological variant
Inspection of different areas of the treated leaves showed coloniza­
tion of the bacterial biomass under both evaluated conditions, that is
when applying the MBCA-WT product or when applying the bacterial
biomass-WT alone. Progressive increments in bacterial population was
evident as time elapsed (Figs. 2 and 3). At time zero, only a few isolated
cells of B. tequilensis EA-CB0015 (WT) were detected. However, after 24
and 48 h we observed abundant cell populations and formation of
biofilm-like structures. The size of the bacterial structures within the
biofilm corresponded to that of B. tequilensis EA-CB0015 (2.0 ± 0.2 µm).
Also, we detected a reduced population of bacteria colonizing the leaf
surface when we evaluated 20 d after inoculation. Although inspections
of the negative control treatment, revealed the presence of some isolated
bacteria, there was neither indication of abundant bacterial populations
nor formation of bacterial biofilms. The progressive colonization
observed in this evaluation could suggest that once the bacterial cells
land on the leaf surface, they are able to multiply and possibly, actively
produce lipopeptides during phytopathogen interaction. Hence, to test
this hypothesis, we evaluated the ability of the bacterial biomass of
B. tequilensis EA-CB0015 (WT) of producing lipopeptides on the leaf
surface of infected banana plants.

Fig. 2. CARD-FISH hybridization micrographs of banana leaves infected with P. fijiensis and treated with the microbial biological control agent product (WT) or the
biomass of B. tequilensis EA-CB0015 (WT). Detection of the colonization of B. tequilensis EA-CB0015 (WT) at different time-points after the application of the microbial
biological control agent product based on B. tequilensis EA-CB0015 (WT) (MBCA-WT), or the bacterial biomass of B. tequilensis EA-CB0015 (WT) (Biomass-WT).
Infected leaves treated with water are labeled as (Control). CARD-FISH hybridization was performed using the probe HRP-Bsub-ss-0463-aA-22A with CF®555 dye
and reported using fluorescent microscopy. Micrographs correspond to the overlapping images obtained with filters FITC and Cy3. Red cells correspond to hybridized
B. tequilensis EA-CB0015. Thick white arrows point at hybridized bacterial cell structures; white arrow heads indicate biofilm structures (bf); and thin white arrows
show fungal mycelia (fm). White bars indicate 5 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

5
T.Z. Cuellar-Gaviria et al. Biological Control 155 (2021) 104523

Fig. 3. SEM micrographs of banana leaves infected with P. fijiensis and treated the microbial biological control agent product (WT) or the biomass of B. tequilensis EA-
CB0015 (WT). Micrographs showing bacterial colonization at different time-points after the application of the microbial biological control agent product based on
B. tequilensis EA-CB0015 (WT) (MBCA-WT), or the bacterial biomass of B. tequilensis EA-CB0015 (WT) (Biomass-WT). Infected leaves treated with water are labeled as
(Control). Thick white arrows indicate bacterial cell structures; white arrow heads indicate biofilm structures (bf); thin white arrows show fungal mycelia (fm); and
white star indicates an extra cellular matrix-like structure (ECM). The scale bar for each time point in indicated at the bottom right of each micrograph.

3.3. B. tequilensis EA-CB0015 produces surfactins on the surface of
banana leaves infected with P. fijiensis
The ability of B. tequilensis EA-CB0015 (WT) to produce previously
identified lipopeptides (Mosquera et al., 2014; Villegas-Escobar et al.,
2013) when inoculated on the surface of banana leaves infected with
P. fijiensis, was analyzed by mass spectral molecular networking. The
resulting network (Fig. S3) reflects the molecular diversity captured in
mass spectrometry experiments by computing the relationships of the
MS2 spectra as spectral similarity. Therefore, to capture the distribu­
tions of the different lipopeptides families, the network was filtered in
Cytoscape to obtain the nodes that contained the MS data of surfactins
(Fig. 4), iturins (Fig. S4A) and fengycins (Fig. S4B). In the filtered
network (Fig. 4), several nodes had spectral library matches to surfactins
(Table S1, Fig. S5A), and interestingly, that cluster reported a 1.24-fold
increase of spectral counts for the treatment with the biomass of
B. tequilensis EA-CB0015 when compared to the negative control
(Table S2), suggesting that the bacterial strain produces surfactins in the
leaves which could confer advantages in the colonization process. The
other clusters in the network with features of m/z values between
902.393 and 1091.69, although not annotated, also have a 1.18-fold
increase in the number of spectral counts for the B. tequilensis treat­
ment when compared to the negative control (Table S2). When
searching for iturins, the cluster with features at m/z 1043.55 and
1057.57 were manually annotated by analyzing the MS2 spectra and
corresponded to C14 and C15 iturin A (Fig. S5). These features were

Fig. 4. Refined molecular network of the metabolic

profile of different extracts obtained from the leaves
of banana plants. Molecular network after filtering the
complete network with parent masses between 900
and 1200, RTmean from 850 to 1200 s. The nodes
shown in the figure only include those that were
detected in the B. tequilensis EA-CB0015 biomass
treatment. The size of each node is proportional to the
number of spectral counts. The different colors of the
nodes correspond to: cyan: standards (surfactins,
fengycins and iturins); red: extracts of plant leaves
inoculated with the biomass of B. tequilensis EA-
CB0015 and the phytopathogen P. fijiensis; purple:
negative control (extracts of leaves inoculated with
P. fijiensis); and green: positive control (extracts of
leaves inoculated with the MBCA product and
P. fijiensis at time 0 hpi). This figure was created using
Cytoscape Version 3.8.0 (https://cytoscape.org/).
(For interpretation of the references to colour in this
figure legend, the reader is referred to the web version
of this article.)

6
T.Z. Cuellar-Gaviria et al.
detected in the standard, positive control and negative control, but not
in the B. tequilensis biomass treatment. Other features, with masses and
retention times related to iturins, were found in the network (Fig. S4A),
and the sum of spectral counts of all features were 3.4-fold higher in
B. tequilensis treatment than in the negative control (Table S2). Finally,
fengycins were only detected in the standards, as shown in the network
in Fig. S4B, and other features with masses and retention times related to
this lipopeptide family were also detected with more spectral counts in
B. tequilensis treatment than in the control (Table S2).
4. Discussion
Inconsistent results on the efficacy of MBCAs remains a topic of in­
terest as it will have an impact in the broader acceptance and imple­
mentation of biological based products (Adesemoye et al., 2009; Pertot
et al., 2017). Hence, in-depth understanding of ecological interactions
and modes of action demands consideration. The efficacy of a MBCA is
likely based on the strategy implemented for the application of the
product and thus, understanding the mode of action involved in the
specific pathosystem, may increase the rates of success. In this context,
the present study provides evidence that the colonization ability and
production of lipopeptides by B. tequilensis EA-CB0015 are among the
strategies implemented to reduce the development of black Sigatoka
disease in banana plants (Musa AAA cv. Williams, Cavendish subgroup).
We show that the CFS-WT reduced the development of black Siga­
toka disease to the same extent than the MBCA-WT product, a finding
that was previously described by Gutierrez-Monsalve et al. (2015).
Additionally, we demonstrated that treatment with the extracted lip­
opeptides reduced the necrotic area of the leaf, suggesting that the
metabolites contained in the extracted fraction were capable of inhib­
iting the growth of the fungus. Similar results involving the production
of lipopeptides have been described in other pathosystems and have
been related to the control of different disease (Alvarez et al., 2012;
Gong et al., 2015; Pertot et al., 2017; Romero et al., 2007). Notwith­
standing this observation, when the effect on disease severity reduction
of the extracted lipopeptides-WT was compared to the CFS-WT and the
MBCA-WT, the extent of necrotic area reduction was smaller. This result
could suggest the presence of additional metabolites in the CFS-WT that
have not been identified for this bacterial strain. Amongst the arsenal of
antimicrobial metabolites that are known to be produced by Bacillus spp.
there are non-ribosomally synthesized peptides (NRPs), polyketides
(PKs), and ribosomally synthesized peptides (RPs) (Caulier et al., 2019;
Stein, 2005). Even though the NRPs iturin and fengycin are among best
characterized lipopeptides with antifungal activity, other metabolites
such as bacilysin, rhizocticins, octapeptins, bacillibactin, bacillaene and
marolactin have also been reported for their antifungal activity (Bor­
isova et al., 2010; Caulier et al., 2019; Cochrane and Vederas, 2016; Devi
et al., 2019; Özcengiz and Öğülür, 2015). Therefore, it could be possible
that some of these metabolites are produced by B. tequilensis EA-CB0015.
Thus, further analysis on the potential of B. tequilensis EA-CB0015 (WT)
of producing additional bioactive compounds remains to be evaluated.
To further corroborate the hypothesis that the lipopeptides produced
by B. tequilensis EA-CB0015 (WT) were responsible for the disease con­
trol, we performed multiple attempts to genetically transform our (WT)
isolate; however, neither natural competence, electroporation, proto­
plast electroporation, transfection, nor nanotube-mediated trans­
formation worked for this purpose (data not shown). Reports on the
difficulties to transform undomesticated strains have been previously
known. Given that there are inherent differences regarding the strain
restriction and modification systems, the determination of ideal pa­
rameters for transformation are usually strain specific (Heinze et al.,
2018; Romero et al., 2006). Hence, we decided to evaluate the effect of
the spontaneously generated morphological variant of B. tequilensis EA-
CB0015 (MV) on the reduction of the severity of the disease. The
emergence of such spontaneous mutants has previously been described
for different bacterial species such as P. fluorescence, P. aeruginosa,
7
Biological Control 155 (2021) 104523
B. anthracis, B. subtilis, among others (Kim et al., 2014; Penterman et al.,
2014; Robleto et al., 2007; Sastalla and Leppla, 2012). The inclusion of
B. tequilensis EA-CB0015 (MV) as a treatment was based on the rationale
that the inoculation with this morphological variant, would not have an
effect on the reduction of the disease, given the loss of important
phenotypic traits such as lipopeptides production and biofilm formation.
As expected, we observed that the inoculation with the morphological
variant did not show significant differences when compared to the
control. This finding could possibly suggest that the loss of the above­
mentioned phenotypic traits are important features in the biocontrol
strategy implemented by the MBCA (WT) against P. fijiensis. However,
we need to further explore the genetic/transcriptomic basis for the re­
ported phenotypical differences among these two morphotypes; such
studies are currently being performed in our laboratory.
In the evaluated pathosystem, establishment of P. fijiensis on the leaf
surface and penetration through stomata is crucial for infection and
disease development. Both, ascospores (sexual spores) and conidia
(asexual spores) are infective structures that germinate on the leaf sur­
face during epiphytic growth (Churchill, 2011). Usually, it takes about
two to three hours after spore deposition for germination to take place.
Then, germ tube penetration through stomata takes place after 48–72 h
(Fouré and Moreau, 1992; Stover, 1980). Once in the mesophyll, hyphal
growth occurs and usually, it takes around three to four weeks after first
symptoms emergence for the leaf to die (Gauhl, 1994; Stover, 1972).
Taking into account the dynamics of pathogen establishment, along with
the results obtained from the spatiotemporal colonization of the MBCA,
we hypothesize that the colonization of B. tequilensis EA-CB0015 (WT)
between 24 and 48 h after inoculation is an important part of the mode
of action against P. fijiensis. Our finding of the bacterial biomass being
capable of colonizing the leaf surface after a couple of days, supports the
idea that B. tequilensis EA-CB0015 could be competing for nutrients and
space with P. fijiensis. At the same time, given that we detected a higher
amount of spectral counts of surfactins in the samples treated with the
biomass of strain EA-CB0015, suggests that the bacterial cells produce
surfactins on the leaf surface (Fig. 5), which in turn further favors
colonization and biofilm formation; findings that have been supported
in previous studies with different Bacillus species (Chowdhury et al.,
2015; Zeriouh et al., 2014). Although the production of iturins and
fengycins ions was not supported by our MS/MS data, we identified MS1
features with the same reported masses for these molecules during the
expected retention times. However, is possible that due to the low
abundance of these ions, the additional fragmentation step did not take
place and hence, we cannot ensure that these molecules were produced.
Difficulties in detecting such metabolites have been related to the in­
teractions of these molecules with the surrounding environment, or to
the interference of detection with other plant-derived compounds
(Raaijmakers et al., 2010).
Most of the reports on bacterial colonization of plants that exist to
date correspond to studies done in the rhizosphere and involve different
pathosystems than the one evaluated in this work (Bais et al., 2004;
Cazorla et al., 2007; Hao et al., 2017; Xu et al., 2019). Evaluations done
in the rhizosphere of banana plants by Gamez et al. (2019) and Posada
et al. (2018) report the colonization ability of beneficial Bacillus strains
after 24 hpi and up to 30 dpi. Nonetheless, a significant smaller number
of reports have evaluated the in situ colonization on the phyllosphere. To
our knowledge, this is the first study to present bacterial colonization on
the phyllosphere of banana plants during the interactions with
P. fijiensis. Among the colonization studies that have been done in the
phyllosphere, the strains Pantoea agglomerans and Erwinia herbicola were
evaluated on bean leaves and colonization was traced up to 24 hpi
(Axtell and Beattie, 2002; Leveau and Lindow, 2001; Tecon and Leveau,
2012). Our work provides evidence that bacterial colonization in the
phyllosphere can still be detected to some extent 20 dpi, in spite of the
severe environmental restrictions of this habitat.
Although establishment on the phyllosphere may be limited due to
the harsh environmental conditions, including scarcity of nutrients as
T.Z. Cuellar-Gaviria et al.
Fig. 5. Proposed mode of action implemented by the microbial biological control agent product based on B. tequilensis EA-CB0015 (WT). The MBCA product, based
on B. tequilensis EA-CB0015 (WT) reduces the severity of black Sigatoka disease when inoculated on the leaves banana plants via competition of nutrients and space
and antibiosis. ECM: extracellular matrix surrounding the bacterial cell. Illustration created with BioRender.com.
well as rapid fluctuations in physical conditions such as temperature, UV
radiation, and humidity (Vorholt, 2012), biofilm formation is a strategy
that may allow bacteria to cope with these adverse conditions (Romero,
2013). A successful establishment and bacterial colonization could be
attributed to specific phenotypes of the inoculated bacteria, such as the
production of surfactin, which can act as signaling molecule in the
activation process of biofilm formation (Lopez et al., 2009). Moreover,
the presence of surface active metabolites have also been reported to
allow the movement of bacteria towards nutrient rich locations, influ­
encing the water content dynamics and indirectly contributing to the
limitations of nutrients of the pathogen (Köhl et al., 2019). Another
factor that may influence bacterial establishment on the phyllosphere is
the production of indole-3-acetic-acid (IAA) by the colonizing bacteria.
Production of this plant hormone has been related to the leakage of
nutrients by the host plant, favoring bacterial division (Vorholt, 2012).
In fact, the production of both, surfactin and IAA, have previously been
reported for B. tequilensis EA-CB0015 (Ceballos et al., 2012; Mosquera
et al., 2014). Hence, it is possible that the colonization of B. tequilensis
EA-CB0015 (WT) provides Musa AAA cv. Williams plants with an indi­
rect protection mechanism via competition of nutrients and space,
preventing the expansion of P. fijiensis and its penetration through sto­
mata. A second proposed mode of action of the MBCA product is anti­
biosis, a direct mechanism that involves the antagonist interaction
between the antifungal metabolites produced by B. tequilensis EA-
CB0015 (WT) and P. fijiensis; interaction that can possibly lead to
membrane permeabilization of fungal cell wall as previously suggested
(Dunlap et al., 2019; Gong et al., 2015). Given the complexity of in­
teractions new or unexpected mechanisms for the MBCA may be
revealed in future evaluations. For instance, performing RT-qPCR
analysis could help determine more specific information regarding the
growth dynamics of B. tequilensis EA-CB0015 over the leaf surface and its
interaction with P. fijiensis. Further studies using this technique could
help reveal the time course expression of genes associated with the
production of lipopeptides, biofilm formation, motility, quorum sensing,
among other genes that may relate to biocontrol mechanisms, as well as
it will allow to identify if P. fijiensis mycelia is being reduced upon
8
Biological Control 155 (2021) 104523
treatment with the MBCA. Such evaluations have provided important
insights in other pathosystems (Gerin et al., 2018; Lysøe et al., 2017).
Lastly, performing whole genome sequencing (WGS) of the bacterial
strain could help gain additional understanding of the control
mechanisms.
Our work provides a more in-depth knowledge about the modes of
action of the product. We show evidence that the biomass of
B. tequilensis EA-CB0015 (WT), is capable of colonizing the surface of
Musa AAA cv. Williams leaves infected with P. fijiensis. Moreover, our
findings suggest that the lipopeptides produced by B. tequilensis EA-
CB0015 (WT) significantly reduce the severity of black Sigatoka dis­
ease in greenhouse evaluations and also, that surfactins are actively
produced by the bacterial biomass after the application on the leaf
surface of banana plants infected with P. fijiensis. Given the collected
evidence, we propose that the MBCA product, based on B. tequilensis EA-
CB0015 (WT) reduces the severity of black Sigatoka disease via (i)
competition of nutrients and space and, (ii) antibiosis.
5. Conclusions
Successful development of MBCA to fight plant pathogens relays in a
basic understanding of the modes of action taking place during the
interaction with the host plant as well as the pathogen. Hence, under­
standing the biology of the interactions that occur in planta conditions,
may result in better application strategies and improved efficacy of
MBCA products. Our findings indicate that the lipopetides produced by
B. tequilensis EA-CB0015 (WT) can control the severity of black Sigatoka
disease in Musa AAA cv. Williams. Moreover, it was identified that the
biomass of the MBCA (WT) product is capable of colonizing the leave
surface, while actively producing surfactins. Hence, we propose that the
mechanisms employed by the MBCA product based on B. tequilensis EA-
CB0015 (WT) to reduce the severity of black Sigatoka disease, involves
the synergy of two modes of action that include the competition of space
and nutrients, as well as antibiosis.
T.Z. Cuellar-Gaviria et al.
CRediT authorship contribution statement
Tatiana Z. Cuellar-Gaviria: Conceptualization, Data curation,
Investigation, Formal analysis, Writing - original draft. Lina M. Gon­
zalez-Jaramillo: Conceptualization, Data curation, Investigation,
Formal analysis. Valeska Villegas-Escobar: Conceptualization, Inves­
tigation, Formal analysis, Funding acquisition, Supervision, Project
administration, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We would like to thank Sandra M Correa-Garhwal for her help in
proofreading the manuscript and Gustavo Adolfo Bolaños from Uni­
versidad de Caldas in Manizales, Colombia for the images taken with the
Scanning Electron Microscope. We thank Universidad EAFIT, Associa­
tion of Banana Producers of Colombia (AUGURA) and Colciencias
(contract number 0147-2013) for the financial support for this project.
We thank Colciencias for the financial support provided to T.Z. Cuellar-
Gaviria for her PhD studies (Convocatoria 617 Doctorados Nacionales).
This research was made possible by the subscribed Contract Number 166
and 139 with the Ministerio de Medio Ambiente y Desarrollo Territorial
of Colombia in the categories “Contrato de Acceso a Recursos Genéticos
y Productos Derivados para Investigación Científica” and “Contrato de
Acceso a Recursos Genéticos y Productos Derivados con Fines Comer­
ciales” respectively.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biocontrol.2020.104523.
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