Microbiological Research 182 (2016) 31–39

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Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Antagonistic effects of Bacillus subtilis subsp. subtilis and B.

amyloliquefaciens against Macrophomina phaseolina: SEM study of
fungal changes and UV-MALDI-TOF MS analysis of their bioactive
compounds
M.J. Torres a , C. Pérez Brandan b , G. Petroselli c , R. Erra-Balsells c , M.C. Audisio a,∗
a
Instituto de Investigaciones para la Industria Química (INIQUI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad
Nacional de Salta, Av. Bolivia 5150, 4400 Salta, Argentina
b
Instituto Nacional de Tecnología Agropecuaria-Estación Experimental Salta, Ruta Nacional 68 Km 172, Cerrillos, 4403 Salta, Argentina
c
CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, 3 Ciudad
Universitaria, 1428 Buenos Aires, Argentina

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

Article history: The antifungal effect of Bacillus subtilis subsp. subtilis PGPMori7 and Bacillus amyloliquefaciens PGPBacCA1
Received 12 May 2015 was evaluated against Macrophomina phaseolina (Tassi) Goid. Cell suspension (CS), cell-free supernatant
Received in revised form 1 September 2015 (CFS) and the lipopeptide fraction (LF) of PGPMori7 and PGPBacCA1 were screened against three different
Accepted 26 September 2015
M. phaseolina strains. CS exhibited the highest inhibitory effect (around 50%) when compared to those
Available online 30 September 2015
of CFS and LF, regardless of the fungal strain studied. The synthesis of lipopeptides was studied by UV-
MALDI TOF. Chemical analysis of Bacillus metabolite synthesis revealed that surfactin and iturin were
Keywords:
mainly produced in liquid medium. Potential fengycin was also co-produced when both Bacillus were
Macrophomina phaseolina
Bacillus subtilis subsp. subtilis
cultivated in solid medium. In co-culture assays, the bacterial colony-fungal mycelium interface at the
B. amyloliquefaciens inhibition zone was evaluated by both scanning electron microscopy (SEM) and UV-MALDI TOF, the
Lipopeptides former to determine the structural changes on M. phaseolina cells and the latter to identify the main
UV-MALDI TOF MS bioactive molecules involved in the inhibitory effect. PGPBacCA1 produced surfactin, iturin and fengycin
SEM in the inhibition zone while PGPMori7 only produced these metabolites within its colony and not in the
narrow inhibition zone. Interestingly, SEM revealed that PGPBacCA1 induced damage in M. phaseolina
sclerotia, generating a fungicidal effect as no growth was observed when normal growth conditions were
reestablished. In turn, PGPMori7 inhibited the growth of the Macrophomina mycelium without fungal
injury, resulting only in a fungistatic activity. From these results, it was determined that the two bacilli
significantly inhibited the growth of an important phytopathogenic fungus by at least two different
mechanisms: lipopeptide synthesis and competition among microorganisms.
© 2015 Elsevier GmbH. All rights reserved.

1. Introduction use of antagonistic microorganisms for biological control of plant

Common bean (Phaseolus vulgaris L.) and soybean (Glycine max
L.) are among the most important crops in the northwestern region
of Argentina (Paoli et al., 2013). Charcoal rot in soybean and com-
mon bean, caused by pathogenic fungus Macrophomina phaseolina
(Tassi) Goid, is frequently correlated with viable sclerotia present in
the soil (Iqbal and Mukhtar, 2014). This disease is associated with
periods of drought, reducing crop productivity and incomes. The
∗ Corresponding author. Fax: +54 387 4251006.
E-mail address: audisio@unsa.edu.ar (M.C. Audisio).
pathogens, such as M. phaseolina, could represent a promising and
eco-friendly option in replacing chemical pesticides (Calvo et al.,
2007; Heydari and Pessarakli 2010).
Biological control offers interesting properties, such as: high
specificity against target plant pathogens, low cost, does not pollute
the environment, and presents no problems of waste management.
The mechanisms through which antagonistic microorganisms
affect pathogens populations are not always clear, but are gener-
ally attributed to any of the following effects: (1) direct parasitism
and death of the pathogen; (2) food and space competition with
the pathogen and (3) direct toxic effects on the pathogen by means

http://dx.doi.org/10.1016/j.micres.2015.09.005
0944-5013/© 2015 Elsevier GmbH. All rights reserved.
32 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39
of antibiotic substances released by the antagonist (Heydari and
Pessarakli, 2010; Whipps, 2001).
Among bacteria commonly used as plant biological control,
Bacillus spp. strains are highly promising (Choudhary and Johri,
2009; Hobley et al., 2013; Ongena and Jacques, 2008). In par-
ticular,Bacillus subtilis and Bacillus amyloliquefaciens strains are
considered safe for the environment, with excellent colonization
capacity and versatility to protect plants from phytopathogenic
fungi (Zhao et al., 2013). They also have the capacity to colonize
plant roots, since Bacillus can be considered as a growth pro-
moter rhizobacteria for plants (PGPR) (Turner and Backman, 1991).
Furthermore, the sporulation ability of these bacteria ensures its
prevalence in the environment and ensures future formulation
strategies (Nihorimbere et al., 2013).
It is known that Bacillus species produces a large number of pep-
tide antibiotics (Tabbene et al., 2009), as well as low molecular
weight volatile compounds and several lipopeptides with spe-
cific activities against phytopathogenic fungi (Arrebola et al., 2009;
Chaves-López et al., 2015; Ben Slimene et al., 2012; Zhang et al.,
2013). Among these lipopeptides, surfactin, fengycin, polymyxin,
bacitracin and the group of iturin can elicit relevant properties
(Ongena and Jacques, 2008). The lipopeptides structural differences
are strongly related to their antifungal and antibacterial activities
(Ramkumar et al., 2013). Thus, fengycin and iturin are known for
having antifungal activities (Caldeira et al., 2011; Savadogo et al.,
2011). Interestingly, each type of lipopeptides is indeed a “family”
of compounds, i.e., surfactin really exists at as least five different
homologues or isoforms (Caldeira et al., 2011). For that reason, it is
not enough to just say or express “surfactin”, “iturin”, etc. Also, the
synthesis of different lipopeptides (families and homologues) by
Bacillus strains is associated with several factors, including the type
of nutrient and culture conditions (Zhu et al., 2013). It is reported
that the differences in the physiological changes given by liquid
and solid media can cause differences in the microbial molecular
behaviors that influence product formation (Barrios-González et al.,
2008).
The aim of this work was to study the inhibitory effect of B. sub-
tilis subsp. subtilis PGPMori7 and B. amyloliquefaciens PGPBacCA1
against strains of M. phaseolina Tassi (Goid). The main metabolites
responsible for the activity were identified by mass spectrome-
try UV-MALDI TOF and their primary chemical structures were
proposed. Finally, the estrutural changes induced in the fungal
mycelium were studied by scanning electron microscopy (SEM).
The type of antifungal effect (fungistatic or fungicidal) generated by
the Bacillus strains were also evaluated by determining the recovery
of the treated fungus in fresh and sterile medium.
2. Materials and methods
2.1. Microorganisms and culture conditions
B. subtilis subsp. subtilis Mori7 (GenBank access code JX120518)
and B. amyloliquefaciens PGPBacCa1 (GenBank access code
JX120520), isolated from a honey sample and from an air sample,
respectively, were studied in the Laboratory of Applied Bacteri-
ology of the Research Institute for Chemical Industry (INIQUI).
These strains were cultured in Mueller Hinton broth (MH, Britania,
Argentina) at 37 ◦ C during 24 h, without agitation.
Macrophomina phaesolina Apolinario Saravia, Campichuelo and
Olleros strains were obtained from a stock culture of the Laboratory
of Soil Microbiology (National Institute of Agricultural Technology
INTA-Salta- Cerrillos, Argentina), and were grown on dextrose-
potato-agar (DPA, Britania, Argentina) at 28 ◦ C during 7 days. All
M. phaesolina strains studied were isolated from roots of soybean
from the northwest of Salta province (Argentina).
Fig. 1. Measurement of inhibitory activity (A) PGPMori7 and (B) PGPBacCA1 against
M. phaseolina Apolinario Saravia using dual culture technique. Circles: zones of sam-
ples for scanning electron microscopy. Arrows: zones of samples for analysis, by
mass spectrometry, of diffusion of lipopeptides on solid medium.
2.2. Antifungal activity
2.2.1. Cell suspensions and cell-free supernatants from Bacillus
The inhibitory effect of the two Bacillus strains on the fun-
gal growth of M. phaesolina Apolinario Saravia, Campichuelo and
Olleros strains were assessed in cell suspensions (CS) and cell-free
supernatants (CFS).
To obtain the CS, Mueller Hinton broth was inoculated with a
pure culture of each bacterial strain in a percentage of 1% (v/v)
and incubated for 24 h at 37 ◦ C. After this period, concentrations of
about 1 × 107 cells per mL and 1 × 105 spores per mL were obtained.
For spore concentration measurement we proceeded according
to Soria and Audisio (2014). Briefly, the cellular suspension was
warmed up for 10 min at 80 ◦ C and then kept for 1 h at 8 ◦ C. The
number of cells and spores were determined by the counts of serial
dilutions in 0.1 % (w/v) meat peptone on BHI agar plates.
CFS fractions were obtained by centrifugation (10,000 × g for
10 min at 4 ◦ C) and were subsequently filtrated in sterilized filter
(0.22 ␮m pore-size cellulose acetate membranes) and kept at 4 ◦ C
until further analysis.
2.2.2. Preparation of lipopeptide extracts
The Bacillus strains were incubated in MH for 24 h at 37 ◦ C
without shaking. CFS was obtained as described before. After cen-
trifugation the CFS were acidified with HCl (c) until pH was close
to 2, in order to precipitate the lipopeptides which were then
recovered by centrifugation (14,000 × g for 25 min at 4 ◦ C) and sub-
sequently were extracted with 10 mL of methanol. Then, the solvent
was evaporated in a safe place and the resulting precipitate, called
the lipopeptide fraction (LF), was dissolved in sterile distilled water.
This solution was adjusted to a final pH 8–9 with 0.5 M NaOH.
2.2.3. Fungal inhibition measurements
For these assays, a dual culture technique was used. Thus, the
inhibitory activity of the CFS, LF and CS from the Bacillus strains
PGPMori7 and PGPBacCA1 against the three Macrophomina strains
was evaluated. Each bacterial sample (i.e., CFS, LF and CS) was inde-
pendently tested against each fungal strain as follows: 25 ␮L of
each sample were inoculated in wells made at the same distance in
9 cm-diameter Petri dishes containing DPA medium (Fig. 1). Then
 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39
4 mm plugs, taken from the leading edge of a 7 day-old culture of
each fungal strain, were placed in the center of the dishes; plates
without bacterial samples were used as controls. Plates were incu-
bated at 28 ◦ C and the myceliar growth was daily checked. After
7 days, the myceliar growth diameter of each phytopathogen was
measured and the radial growth inhibition (RGI) degree was deter-
mined according to Landa et al. (1997). In this sense, the following
equation was used:
RGI = (C − T )/C; FI (%) = RGI × 100
where T is the average diameter of the myceliar growth in presence
of the CFS, LF and CS. C is the average diameter of the myceliar
growth without bacterial samples. The values were informed as the
percentage of fungal inhibition (FI). In particular, when the bacterial
sample was the cellular suspension, the bacterial colony was not
considered in the fungal inhibition measurement.
2.3. Analysis UV-MALDI TOF
Lipopeptides involved in the antifungal activity were identi-
fied by UV-MALDI TOF. The CFS of both strains were evaluated
and the identification of the lipopeptides on the LF recovered was
carried out, too. Spectra were recorded on a Bruker Ultraflex II
TOF/TOF (Bruker Daltonics, Bremen, Germany). Mass spectra were
acquired in linear positive and negative ion modes and with the
LIFT device on MS/MS mode. Stock solutions of samples were
prepared in water at pH 8. External mass calibration was made
using ␤-cyclodextrin (MW 1134) with 9H-pyrido[3,4-b]indole or
norharmane (nHo) as matrix in positive and negative ion mode.
The matrix signal was used as an additional standard for calibra-
tion in both ion modes. Sample solutions were spotted on a MTP
384 target plate polished steel from Bruker Daltonics (Bremen,
Germany). For UV-MALDI-MS matrix solutions were prepared by
dissolving nHo in acetonitrile/water (1:1, v/v) solution. For UV-
MALDI-MS experiments dry droplet sample preparation (sandwich
method) was used according to Nonami et al. (1997), loading suc-
cessively 0.5 ␮l of matrix solution, 0.5 ␮l of analyte solution, 0.5 ␮l
of matrix and 0.5 ␮l of matrix solution after drying each layer at
normal atmosphere and room temperature. The matrix to analyte
ratio was 3:1 (v/v) and the matrix and analyte solution loading
sequence was: (i) matrix, (ii) analyte, (iii) matrix, (iv) matrix. Des-
orption/ionization was obtained by using the frequency-tripled Nd:
YAG laser (355 nm). Experiments were performed using firstly the
full range setting for laser firing position in order to select the opti-
mal position for data collection, and secondly fixing the laser firing
position in the sample sweet spots. The laser power was adjusted
to obtain high signal-to-noise ratio (S/N) while ensuring minimal
fragmentation of the parent ions and each mass spectrum was
generated by averaging 100 lasers pulses per spot. Spectra were
obtained and analyzed with the programs FlexControl and Flex-
Analysis, respectively.
2.3.1. Synthesis of lipopeptides on solid medium (agar)
The lipopeptides synthesis by these bacteria was analyzed when
they grew on the agar medium DPA. The interaction between B.
amyloliquefaciens PGPBacCA1 and M. phaseolina Apolinario Saravia,
produced an inhibition zone of large diameter; then, a sample of
the bacterial colony (BC) was extracted from the area surrounding
it (called ZBC), and from the area surrounding the fungus (ZFM)
(Fig. 1, arrows). On the other hand, the interaction between PGP-
Mori7 and the fungus produced a large bacterial-colony diameter
and a narrow inhibition zone. To analyse the effect of this strain, a
sample of the BC and the area around it (ZBC) were extracted (Fig. 1,
arrows). In all cases, a piece of 4 mm was removed from each zone
and was resuspended into 0.5 mL of acetonitrile (ACN). Finally, each
33
sample was vigorously shaked for 30 s and kept at −18 ◦ C until the
UV-MALDI TOF studies.
2.4. Scanning electron microscopy (SEM)
For the SEM analyses, a piece of ca. 4 mm area between the fun-
gal mycelium and the zone of inhibition produced by each Bacillus
(Fig. 1, circles) was taken. A similar sample was recovered from
another Petri dish with a fungal mycelium grown without the bac-
terial presence (control).
The samples taken from the culture agar medium with fungal
inhibition and the respective controls were fixed with glutaralde-
hyde 2.5% v/v in a 0.1 M phosphate buffer solution of pH 7.2, for 24 h
at 4 ◦ C. Then, an alcohol dehydration was performed using 10%, 30%,
50%, 70%, 96% and 100% of ethanol 96◦ , subsequently leaving the
samples in contact for 15 min with each alcoholic solution, except
with the last concentrations, in which the samples were left up for
total drying. Finally, the metallization of the dehydrated samples
was carried out and seen at 15 kV on a Joel JMS 6480 LV computer
(Scanning Electron Microscopy and Microanalysis by X-ray Scatter-
ing), LASEM laboratory—INIQUI (Laboratory of Electron Microscopy
and Microanalysis).
2.5. Evaluation of the mode of action of PGPMori7 and
PGPBacCA1 on M. phaseolina
To determine the type of effect produced (fungicide or
fungistatic) by the strains PGPMori7 and PGPBacCA1 on the viabil-
ity of M. phaseolina Apolinario Saravia, samples of fungus–bacteria
dual cultures were used. Briefly, pieces of the DPA medium contain-
ing fungal mycelium next to the areas of bacterial inhibition were
moved to fresh and sterile DPA medium. Then these plates were
incubated at 28 ◦ C for 7 days. After this period, the pathogen growth
and the antifungal effect (fungistatic or fungicidal) produced by the
bacterial strains were determined.
2.6. Statistical analysis
The inhibitory activities of the CS, CFS and LF of PGPBacCA1
and PGPMori7 strains were conducted against three different M.
phaseolina strains (Apolinario Saravia, Campichuelo and Olleros).
All the assays were done in triplicate. The antifungal activities are
indicated with the average of the values determined ±standard
deviation (SD). The statistical analyses of the data were performed
using InfoStat statistical software.
3. Results
3.1. Antifungal activity of Bacillus subtilis subsp. subtilis
PGPMori7 and B. amyloliquefaciens PGPBacCA1 by dual
cultivation
The cell suspension of both Bacillus produced a inhibitory activ-
ity greater than 50% against the three strains of M. phaseolina
(Table 1). While, the antifungal activities of the bacterial CFS
were between 32.5% and 37.5%, respectively. However, the LF of
PGPMori7 and PGPBacCA1 showed no significant inhibitory effect
(Table 1).
As similar results were determined with all the tested M.
phaseolina strains, it was decided to continue with M. phaseolina
Apolinario Saravia for deeper studies.
34 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39

Fig. 2. UV-MALDI mass spectra of (I) PGPMori7 and (II) PGPBacCA1 in positive ion mode. (a) cell-free supernatant; (b) lipopeptides fraction. Matrix: nHo; m/z range:
1000–1150. References: circle: surfactins homologues; triangle: iturins homologues.
 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39 35

Table 1 Table 3a
Antifungal activity of two strains of Bacillus against different strains of M. phaseolina; MALDI-TOF mass spectrum: Main mass peaks of lipopeptides produced by B. amy-
samples: cell suspensions (CS), cell-free supernatant (CFS) and lipopeptides fraction loliquefaciens PGPBacCA1. Samples: zone near bacterial colony (ZBC); zone near
(LF). fungal mycelium of M. phaseolina Apolinario Saravia (ZFM).

Bacillus M. phaseolina Lipopeptides Chemical formula Calculated Experimental

Apolinario Saravia Campichuelo Olleros ZBC ZFM

PGPMori7 CS 54.0 ± 5.7 a

50.5 ± 0.6 53.0 ± 7.1 Surfactin [C48 H82 N7 O13 Na]+
987.59 WS a
985.86
CFS 37.5 ± 17.7 37.5 ± 16.6 33.0 ± 0.3 Surfactin [C49 H84 N7 O13 Na]+ 1001.60 1000.48 1000.81
LF n.i.b 4.9 ± 2.1 6.3 ± 5.7 Surfactin [C50 H86 N7 O13 Na]+ 1015.62 1014.59 1015.86
Surfactin [C51 H88 N7 O13 Na]+ 1029.63 1030.38 1031.63
PGPBacCA1 CS 56.5 ± 2.1 54.0 ± 5.7 52.5 ± 3.5
Surfactin [C52 H90 N7 O13 Na]+ 1043.65 1044.46 1044.47
CFS 36.5 ± 2.1 34.0 ± 5.7 32.5 ± 1.5
Surfactin [C53 H92 N7 O13 Na]+ 1057.66 1058.39 1058.71
LF 18.5 ± 2.1 11.3 ± 0.2 n.i.
Iturin [C48 H74 N11 O15 Na]+ 1067.53 1066.31 1066.55
a
Percentage of fungal inhibition ±standard deviation. Surfactin [C53 H92 N7 O13 K]+ 1073.64 1072.36 1072.65
b
n.i. (no-inhibition). Iturin [C49 H76 N11 O15 Na]+ 1081.54 1080.41 1081.48
Iturin [C49 H76 N11 O15 K]+ 1097.51 1097.99 1097.47
Iturin [C52 H82 N11 O15 H]+ 1101.61 WS 1103.39
Table 2 Not assigned WS 1119.16
Main peaks detected by UV-MALDI TOF mass spectrometry analysis of the lipopep- Not assigned 1165.88 1166.14
tides produced by PGPMori7 and PGPBacCA1; samples: cell-free supernatant (CFS) Not assigned 1182.62 1181.62
and lipopeptides fraction (LF). Fengycin [C70 H104 N12 O20 Na]+ 1455.78 1454.37 WS
Fengycin [C71 H106 N12 O20 Na]+ 1469.75 1469.58 1469.86
Lipopeptides Chemical formula Calculated Experimental Fengycin [C72 H110 N12 O20 Na]+ 1485.78 1485.36 1484.88
CFS LF Fengycin [C73 H110 N12 O20 Na]+ 1497.78 1498.29 1497.41
Fengycin [C74 H112 N12 O20 Na]+ 1511.80 1511.88 1512.21
Bacillus subsp subtilis PGPMori7 Fengycin [C75 H114 N12 O20 Na]+ 1525.81 1525.03 WS
Surfactin [C50 H86 N7 O13 Na]+ 1015.62 1016.53 WSa
a
Surfactin [C51 H88 N7 O13 Na]+ 1029.63 1030.51 1030.45 WS: without signal.
Surfactin [C52 H90 N7 O13 Na]+ 1043.65 1044.51 1044.53
Surfactin [C53 H92 N7 O13 Na]+ 1057.66 1058.54 1058.71
Surfactin [C53 H92 N7 O13 K]+ 1073.64 1074.51 1074.63 siated surfactin homologue was detected in LF at m/z = 1088.68
Surfactin [C54 H94 N7 O13 K]+ 1087.65 WS 1088.68
([C54 H94 N7 O13 K]+ ) (Table 2).
Iturin [C49 H76 N11 O15 Na]+ 1081.54 1080.53 1081.05
Iturin [C49 H76 N11 O15 K]+ 1097.51 1096.60 1096.93 PGPBacCA1 showed also higher intensity lipopeptides signals
Iturin [C52 H82 N11 O15 H]+ 1101.61 1102.51 1103.11 in the CFS than in the LF (Fig. 2(II)) as was observed for PGP-
Bacillus amyloliquefaciens PGPBacCA1
Mori7 (Fig. 2(I)). Similarly, 3 homologues of surfactin were detected
Surfactin [C50 H86 N7 O13 Na]+ 1015.62 1015.49 1016.76 in CFS as sodiated adducts ([C50 H86 N7 O13 Na]+ at m/z = 1015.49,
Surfactin [C51 H88 N7 O13 Na]+ 1029.63 1030.67 1030.87 [C51 H88 N7 O13 Na]+ at 1030.67 and [C52 H90 N7 O13 Na]+ at 1043.58)
Surfactin [C52 H90 N7 O13 Na]+ 1043.65 1043.58 1044.87 and the fourth as sodiated ([C53 H92 N7 O13 Na]+ ) and potassi-
Surfactin [C53 H92 N7 O13 Na]+ 1057.66 1057.56 1058.83
ated ([C53 H92 N7 O13 K]+ ) adducts at at m/z = 1057.56 and as
Surfactin [C53 H92 N7 O13 K]+ 1073.64 1073.57 1075.06
Surfactin [C54 H94 N7 O13 K]+ 1087.65 WS 1089.33 m/z = 1073.57, respectively (Table 2). Additionally, one more
Iturin [C49 H76 N11 O15 Na]+ 1081.54 1080.69 1081.30 surfactin homologue was detected in LF at m/z = 1089.33
Iturin [C49 H76 N11 O15 K]+ 1097.51 1096.69 1097.29 ([C54 H94 N7 O13 K]+ ) (Fig. 2(II) and Table 2). Only 2 homologues
Iturin [C52 H82 N11 O15 H]+ 1101.61 1102.90 1103.46 of iturin were observed in both samples [C49 H76 N11 O15 ] and
Not identified WS 1489.57
Not identified 1503.73 1501.95
[C52 H82 N11 O15 ]; in CFS the first was detected as sodiated at
Not identified 1517.30 1519.16 m/z = 1080.69 and potassiated at m/z = 1096.90 and the second
Not identified WS 1535.44 as potonated species at m/z = 1102.90 (Table 2). Similarly as is
a
WS: without signal. shown in Fig. 2(II) and Table 2, for LF the signals observed
were: [C49 H76 N11 O15 Na]+ at m/z = 1081.30, [C49 H76 N11 O15 K]+ at
m/z = 1097.29 and [C52 H82 N11 O15 H]+ at m/z = 1103.46.
3.2. Identification of the bioactive metabolites by mass On the other hand, the MS analysis of lipopeptides syn-
spectrometry UV-MALDI TOF thesized by PGPBacCA1 in solid medium against this fungal
strain showed several signals in both inhibition zones (ZBC and
UV-MALDI TOF analysis in CFS and LF allowed identification of ZFM) (Figs. S1 and S2 in Supplementary data). As is detailed in
those lipopeptides involved in the antifungal activity. As it is shown Table 3a the signals observed were identified as surfactin homo-
in Fig. 2(I) similar number of homologues for both surfactin and logues, majorly as sodiated aducts [C48 H82 N7 O13 ] at m/z = 985.86
iturin in the CFS as in the LF of PGPMori7 were detected. However, (ZFM), [C49 H84 N7 O13 ] at m/z = 1000.48 (ZBC) and at m/z = 1000.81
the intensity of the signals correlated with the lipopeptides present (ZFM), [C50 H86 N7 O13 ] at m/z = 1014.59 (ZBC) and m/z = 1015.86
in the samples was higher in the CFS than in LF. This result is in (ZFM), ([C51 H88 N7 O13 ] at m/z = 1030.38 (ZBC) and m/z = 1031.63
accordance with the degree of inhibition obtained with these two (ZFM), [C52 H90 N7 O13 ] at m/z = 1044.46 (ZBC) and m/z = 1044.47
extracts. (ZFM)) and only one homologue, [C53 H92 N7 O13 ], as sodiated
Three homologues of surfactin were detected in CFS as sodium (m/z = 1058.39 (ZBC) and m/z = 1058.71 (ZFM)) and potassiated
adducts ([C50 H86 N7 O13 Na]+ at m/z = 1016.30; [C51 H88 N7 O13 Na]+ at aducts (m/z = 1072.36 (ZBC) and m/z = 1072.65 (ZFM)). Furthermore
m/z = 1030.51 and [C52 H90 N7 O13 Na]+ at m/z = 1044.51) (Table 2). 3 iturin homologues were detected as follows: (i) [C48 H74 N11 O15 ]
Another one was detected as adducts of sodium and potas- only observed as sodiated adduct at m/z = 1066.31 (ZBC) and
sium ([C53 H92 N7 O13 Na]+ at m/z = 1058.54 and [C53 H92 N7 O13 K]+ m/z = 1066.55 (ZFM), [C49 H76 N11 O15 ] detected as sodiated and
at m/z = 1074.51); Additionally, 2 homologues of iturin in CFS potassiated aducts [m/z = 1080.41 (ZBC) and m/z = 1081.48 (ZFM)]
were identified, one as adduct of sodium and potassium and [m/z = 1097.99 (ZBC) and m/z = 1097.47 (ZFM)] respectively,
([C49 H76 N11 O15 Na]+ at m/z = 1080.53 and [C49 H76 N7 O11 O15 K]+ and (iii) [C52 H82 N11 O15 ] as protanted adduct (m/z = 1103.39
at m/z = 1096.60), and the other one as a protonated form at (ZFM)). Finally, 6 fengycin homologues were detected as sodi-
m/z = 1102.52 ([C52 H82 N11 O15 H]+ ) (Table 2). One additional potas- ated adducts (Table 3a): [C70 H104 N12 O20 ] at m/z = 1454.37 (ZBC),
36 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39

Table 3b The control culture of M. phaseolina Apolinario Saravia pre-

MALDI-TOF mass spectrum: main mass peaks of the lipopeptides produced from
sented branching hyphae that were generally originated as a right
B. subtilis subsp subtilis PGPMori7. BC: bacterial colony; ZFM: zone near fungal
mycelium of M. phaseolina Apolinario Saravia. angle prolongation from the main hyphae. Tipically acute angle
ramifications of this pathogenic strain were also noted (Fig. 3A),
Lipopeptides Chemical formula Calculated Experimental
and regular size sclerotia that followed different development pat-
BC ZFM terns (thickening, branching and main hyphae septation) were also
Surfactin [C49 H84 N7 O13 Na]+
1001.60 1000.48 WSa identified (Fig. 3B).
Surfactin [C50 H86 N7 O13 Na]+ 1015.62 1014.59 WS SEM studies showed structural changes on the intersection zone
Surfactin [C51 H88 N7 O13 Na]+ 1029.63 1030.38 WS between the inhibition area generated by the diffusion of PGPMori7
Surfactin [C52 H90 N7 O13 Na]+ 1043.65 1044.46 WS metabolites and the mycelium of M. phaseolina Apolinario Saravia,
Surfactin [C53 H92 N7 O13 Na]+ 1057.66 1058.53 WS
Iturin [C48 H74 N11 O15 Na]+ 1067.53 1066.31 WS
compared to the fungus control. A greater density of sclerotia with
Surfactin [C53 H92 N7 O13 K]+ 1073.64 1072.36 WS similar sizes were determined and characterized by a repeated
Iturin [C49 H76 N11 O15 Na]+ 1081.54 1080.41 WS branching pattern and hyphae intertwinement, which could be due
Iturin [C49 H76 N11 O15 K]+ 1097.51 1097.99 WS to the compaction and collapse of the outer cells of the periph-
Not assigned 1165.88 WS
eral hyphae (Fig. 3C and D). These hyphal-effect resulted in the
Not assigned 1182.62 WS
Fengycin [C70 H104 N12 O20 Na]+ 1455.78 1454.37 WS formation of sclerotia with more solid and denser tissues layers
Fengycin [C71 H106 N12 O20 Na]+ 1469.75 1469.58 WS (Fig. 3D).
Fengycin [C72 H110 N12 O20 Na]+ 1485.78 1485.36 WS Finally, when this pathogen grew in the presence of PGPBacCA1,
Fengycin [C73 H112 N12 O20 Na]+ 1499.80 1498.29 WS the inhibition zone revaled intertwined hyphal extensions and scle-
Fengycin [C74 H112 N12 O20 Na]+ 1511.80 1511.88 WS
rotia were not formed correctly (Fig. 3D). However, these sclerotia
Fengycin [C75 H114 N12 O20 Na]+ 1525.82 1525.04 WS
were not compacted into solid hyphal masses as observed for the
a
WS: without signal.
controls, indeed cells becomes globular (Fig. 3F).

[C71 H106 N12 O20 ] at m/z = 1469.58 (ZBC) and m/z = 1469.86 (ZFM),
[C72 H110 N12 O20 ] at m/z = 1485.36 (ZBC) and at m/z = 1484.88
(ZFM), [C73 H112 N12 O20 ] at m/z = 1498.29 (ZBC) and m/z = 1497.41
(ZFM), [C74 H112 N12 O20 ] at m/z = 1511.88 and m/z = 1512.21 and
[C75 H114 N12 O20 ] at m/z = 1525.03 (ZBC). As a general result the sig-
nals intensity observed were higher in ZFM spectra than in ZBC
spectra (Figs. S1 and S2).
B. subtilis subsp. subtilis PGPMori7 produced a large colony
diameter and a narrow inhibition zone against M. phaseolina Apoli-
nario Saravia. For that reason, MALDI MS studies were performed
with the area where bacterial growth finished and with the inhibi-
tion zone surrounding the fungal mycelium. In the bacterial colony
(i.e., only bacterial cells) the homologues of surfactin observed
were: (i) only as sodiated adducts: [C49 H84 N7 O13 ] at m/z = 1000.48,
[C50 H86 N7 O13 ] at m/z = 1014.59, [C51 H88 N7 O13 ] at m/z = 1030.38,
[C52 H90 N7 O13 ] at m/z = 1044.46; (ii) [C53 H92 N7 O13 ] detected
as sodiated and potassiated adducts at m/z = 1058.53 and at
m/z = 1073.64, respectively. Two iturin homologues were detected:
(i) as sodiated adduct [C48 H74 N11 O15 ] at m/z = 1066.31 and (ii)
[C49 H76 N11 O15 ] as sodiated at m/z = 1080.41 and potassiated at
m/z = 1097.99 (Table 3b, BC column; Fig. S3 in Supplementary
data). Finally six homologues of fengycin were detected as sodium
adducts: ([C70 H104 N12 O20 ] at m/z = 1454.37, [C71 H106 N12 O20 ] at
m/z = 1469.58, [C72 H110 N12 O20 ] at m/z = 1485.36, [C73 H112 N12 O20 ]
at m/z = 1498.29, [C74 H112 N12 O20 ] at m/z = 1511.88 and
[C75 H114 N12 O20 ] at m/z = 1525.04) (Table 3b; Fig. S4 in Sup-
plementary data). As it is shown in Fig. 2(II) and Fig. S1 in
Supplementary data, PGPBacCA1 yielded higher relative amounts
of the surfactin homologues with m/z 1044.47 and 1058.71 in the
presence of M. phaseolina Apolinario Saravia. The same effect can
be observed in the Fig. 2(I) and Fig. S3 in Supplementary data;
the production of the above mentioned species was higher in the
PGPMori7 bacterial colony. However, in the narrow inhibition
zone analysed, lipopeptides were absent (Fig. S4 in Supplementary
data).
3.3. Evaluation of the lipopeptides effect on Macrophomina cell
structures by SEM
Morphological changes in M. phaseolina Apolinario Saravia
caused by lipopeptides synthesized by PGPMori7 and PGPBacCA1 in
a solid medium were evaluated with scanning electron microscopy
(Fig. 3).
3.4. Mode of action of PGPMori7 and PGPBacCA1 on M.
phaseolina
This analysis determined that PGPBacCA1 exerted a fungicide
effect on M. phaseolina Apolinario Saravia (Fig. S5 in Supplemen-
tary data) because the fungus mycelia that interacted with this B.
amyloliquefaciens cells had not the ability, compared to the control,
to recover and generate a new mycelium in the evaluated time (7
days). On the other hand, PGPMori7 only generated a fungistatic
effect on this pathogen when a similar assay was carried out (Fig.
S5 in Supplementary data).
4. Discussion
M. phaseolina, both a soil-borne and a seed-borne fungus,
induces charcoal rot in different crops including soybean and com-
mon bean. Sclerotia are its principal survival structures; they are
considered an important factor in the spread of fungal propagules in
soil. Also, they have the ability of surviving in soil for years (Almeida
et al., 2001; Pérez Brandan, 2009). Thus, the disease produced by
this fungus is quite important for the agroeconomy of our province,
because Salta is one of the main producers of common bean and
soybean in the Argentinean Republic. Taking into account this situ-
ation, and as chemical control and most of the cultural practices do
not represent effective tools to lower the density of sclerotia in soil,
the use of biocontrol organisms becomes a promising alternative.
Even though potential PGPR bacteria strains are in general iso-
lated from soil (Dimkic et al., 2013; Landa et al., 1997; Liu et al.,
2014; Nihorimbere et al., 2013), the fact that they proceed from
a different origin does not necessarily implicate a barrier to act
as novel PGPR microorganisms. For example, in this work, B. sub-
tilis subsp subtilis PGPMori7 and B. amyloliquefaciens PGPBacCA1
came from honey samples and environmental air, respectively.
Since they have demonstrated high antifungal activity against
phytopathogenic fungi such as Sclerotium, Sclerotinia, Rhizoctonia,
Macrophomina, Penicillium and Fusarium (Torres, 2014), their anti-
fungal effect against different strains of M. phaseolina was evaluated
in more depth. It was found that the inhibitory effect of the cell
suspensions (CS) of both bacterial strains (grown in solid medium)
proved to be independent of the fungal strain, i.e., all the M. phase-
olina strains tested were affected in more than 50%. This result
becomes an important feature when a field application is consid-
 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39
Fig. 3. SEM microphotographs of M. phaseolina Apolinario Saravia growth without the presence of antagonistic bacteria (control, A and B); faced with B. subtilis subsp. subtilis
PGPMori7 (C and D), and B. amyloliquefaciens PGPBacCA1 (E and F). The figures show: dense mycelium with sclerotia at the intersection with the zone of inhibition (C),
sclerotia intertwined with hyphae (D), no sclerotia formed at the intersection with the zone of inhibition (E), sclerotia without defined areas inside tissues (F).
ered for potential PGPR bacteria, because it indicates that they may
be active on crops infected with different strains of M. phaseolina.
Surprinsingly, when the cell-free supernatants (CFS) from both
bacteria were assayed, the antifungal activities were much lower
than those observed with the cell suspensions. This situation may
have at least two feasible explanations: (i) the aggregation state
of the culture medium where the bacterial cells developed, and (ii)
the interaction between the Bacillus cells and the fungus mycelium.
In support of the first statement, Islam et al. (2012) and Price et al.
(2007) have reported that the state of aggregation of the culture
medium can have a significant impact on the type of lipopeptides
produced by a determined strain. As well, Akihiro et al. (1993) and
San-Lang et al. (2002) informed that growth of different Bacillus
strains in a heterogeneous medium enhances both the concen-
tration and the variety of lipopeptide production. Refering to the
second explanation, several researchers have reported that the
expression of certain genes of Pseudomonas and Bacillus strains can
be directed according to which organism they are facing (Garbeva
et al., 2011; Zhu et al., 2013). In this sense, Li et al. (2014) have eval-
uated the production of lipopeptides by B. amyloliquefaciens SQR9
in solid medium when the strain was confronted with six differ-
ent soil-borne fungal pathogens. These authors indicated that such
production was affected by the nature of each pathogen, suggesting
that B. amyloliquefaciens can fine-tune its strategy by using different
antibiotics when faced with a particular fungal pathogen.
The above statements are confirmed in this work, where we
also inform the different families of lipopeptides produced by Bacil-
lus strains, which were detected by mass spectrometry UV-MALDI
TOF. The analysis of the compounds present in the liquid culture
media allowed identification of surfactin and iturin both in the CFS
and in the LF of PGPBacCA1 and PGPMori7. Whereas for solid cul-
ture medium, both bacterial cell growth co-produced a third family
of lipopeptides identified as fengycin, which was also detected in
the zone of bacterial – fungal growth inhibition of PGPBacCA1. To
our knowledge this is the first time that the zone of bacterial –
fungal growth inhibition is studied by MALDI TOF mass spectrom-
etry. This analysis mainly allowed identification of the potential
lipopeptides involved in the antifungal activity of PGPBacCA1: six,
three and four surfactin, iturin and fengycin homologues, respec-
37
tively. Meanwhile, no lipopeptide signals were detected at the
narrow interface between PGPMori7 cells and the Macrophomina
mycelium. It is important to remark that the mass spectrometry
technique detected the different lipopeptides with high sensitiv-
ity and precision, and without the requirement of time-consuming
steps related with the isolation, chromatographic separation and
concentration of the compounds.
Another important result appeared when the antifugal effect of
the lipopeptide fraction (LF) was assayed: its biological activity was
even lower than that determined for the CFS. As described in this
work, the different LF were obtained by acid precipitation and sub-
sequent removal with methanol. This technique was chosen and
followed because it has been commonly used by several authors
to recover the lipopeptides produced by Bacillus spp. (Cheng et al.,
2013; Feignier et al., 1995; Slimene et al., 2012). However, when
mass spectrometry analysis was carried out for the LF, a low recov-
ery was observed compared to the mass spectra of the CFS (less
manipulated sample). This change was detected due to the inten-
sity of the signals corresponding to each lipopeptide homologue:
in the LF, signals were smaller than those obtained for the CFS. This
situation was more remarkable for the LF from PGPMori7, where a
decrease in signal intensity of 70% was detected. Nevertheless, the
lower antifungal activity detected with the LF might also be related
to the enrichment of that sample in surfactin homologues, com-
pounds that have relevant antibacterial effects but poor antifungal
action (Ongena and Jacques, 2008).
Apart from the chemical analyses discussed above, scanning
electron microscopy was applied to evaluate the effects of PGP-
Mori7 and PGPBacCA1 on the cellular structure of M. phaseolina
Apolinario Saravia. This technique proved important to reveal key
information about the effect produced on the fungal morphology,
especially in microsclerotia. For both Bacillus strains, a high density
of sclerotia was observed specifically distributed in the zone of inhi-
bition and intertwined with hyphae. Interestingly, PGPBacCA1 also
generated both surface and inner damages of the sclerotia, situation
that was not observed for PGPMori7. In agreement with our results
for PGPBacCA1, Dubey et al. (2012) observed that the metabolites
produced by Bradyrhizobium sp. VR2, secreted in the area of interac-
tion, caused several changes on the cellular structure of hyphae and
38 M.J. Torres et al. / Microbiological Research 182 (2016) 31–39
sclerotia of M. phaseolina, such as fragmentation, lysis, contraction,
drilling and loss of pigmentation.
Taking into account the types of lipopeptides synthesized, the
morphological changes and the reactivation tests of the treated
mycelium in fresh culture medium, we were able to determine
the mode of action exerted by PGPMori7 and PGPBacCA1 on the
viability of M. phaseolina Apolinario Saravia. B. amyloliquefaciens
PGPBacCA1 generated a fungicidal effect: in solid culture medium,
this strain co-produced different homologues of surfactin, iturin
and fengycin, which had the ability to spread from the bacterial
colony onto the agar. The co-production, by the same bacterial
strain, of surfactin (lipopeptide with surfactant properties) along
with iturin and fengycin (compounds with powerful antifungal
activity), has been reported as an advantageous feature because
surfactin can potentiate the antifungal activity of the other lipopep-
tides (Thimon et al., 1992). It is known that the interaction among
those lipopeptides produces alteration of the structure and perme-
ability of biological membranes (Deleu et al., 2005; Liu et al., 2014).
On the other hand, B. subtilis subsp. subtilis PGPMori7 only gener-
ated a fungistatic effect against M. phaseolina Apolinario Saravia.
A feasible explanation can be related to the low concentration or
even the absence of the active lipopeptides in the narrow interface
between the fungal mycelium and the bacterial colony. Moreover,
the antagonistic effect produced by that Bacillus strain may also be
connected to a competition for space or nutrients, instead of a toxic
effect due to antifungal bacterial metabolites.
In conclusion, this study reveales that two different Bacillus
species, B. amyloliquefaciens PGPBacCA1 and B. subtilis subsp. sub-
tilis PGPMori7, have the ability of inhibiting M. phaseolina by at
least two different mechanisms: lipopeptide synthesis and compe-
tition between microorganisms. In particular, B. amyloliquefaciens
PGPBacCA1 exerted a fungicidal effect on the fungus by generat-
ing lethal damage on its reproductive structures. This result turns
PGPBacCA1 into a potential alternative for the biological control of
Macrophomina under field conditions.
Conflict of interests
All authors declare that they have no competing interests in the
present work
Acknowledgements
The authors would like to thank Consejo de Investigación de la
Universidad Nacional de Salta (CIUNSa, P1974) and National Sci-
ence and Technology Promotion Agency (ANPCyT) of Argentina
for the financial support (PICT2011-0767). M.C. Audisio, R. Erra-
Balsells and G. Petroselli are members of the Research Career of
CONICET. M.J. Torres is a post-doctoral fellowship of CONICET.
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.micres.2015.09.
005.
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