Biological Control 103 (2016) 251260

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Biological Control
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Microbiological characterization for a new wild strain of Paenibacillus

polymyxa with antifungal activity against Botrytis cinerea
Roberto Santiago a, Csar Huiliir b, Luis Cottet a, Antonio Castillo a,
a
Laboratorio de Virologa de Hongos, Departamento de Biologa, Facultad de Qumica y Biologa, Universidad de Santiago de Chile, Alameda 3363, Santiago, Chile
b
Departamento de Ingeniera Qumica, Universidad de Santiago de Chile, Casilla 442, Correo 2, Santiago, Chile

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

A new Paenibacillus polymyxa strain

with antifungal activity was
characterized.

The kinetics of bacterial growth
followed the Monod model.

Antifungal activity was independent
of culture medium nutrients.

Antifungal activity was similar to
Bacillus subtilis QST 713.

The bacterium was able to protect
plant tissue against Botrytis cinerea.

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

Article history: A new bacterial strain with powerful antifungal activity against the phytopathogenic fungus Botrytis
Received 24 August 2015 cinerea was characterized and identified as Paenibacillus polymyxa SCHC33. The kinetics of bacterial
Revised 6 October 2016 growth was fitted to the Monod model, with lmax = 0.218 h1, Ks = 0.087g/L, and YX/S = 0.159 g dry
Accepted 8 October 2016
biomass/g glucose. The antifungal activity of the bacterium was detected in vitro as inhibition halos in
Available online 11 October 2016
antagonism bioassays against B. cinerea, and such activity was unchanged in magnitude when the assays
were done in the same culture media with or without glucose. The ability of the bacterium to protect
Keywords:
grapevine leaves against B. cinerea attack was determined as the percentage of damage to the plant tissue,
Paenibacillus polymyxa
Botrytis cinerea
after inoculation with different ratios of conidia and bacteria. Approximately 88% of the leaf surface was
Antifungal activity protected by the bacteria when a ratio of conidia:bacteria = 1:100 was used. No significant damage was
Monod kinetic model observed in the tissue of the leaves inoculated only with bacteria, whereas the leaves that were treated
Microbial antagonism with solely conidia, the necrotic damage occurred in approximately 100% of their surface. Therefore, the
results indicate that P. polymyxa SCHC33 has great potential to become the active component of a new
biofungicide for control of B. cinerea.
2016 Elsevier Inc. All rights reserved.

1. Introduction mentous fungi to control agricultural pests (Chandler et al.,

Biological control agents (BCAs) are a new generation of fungi-
cides that are based mainly in the use of bacteria, yeasts, or fila-
Corresponding author.
E-mail address: antonio.castillo@usach.cl (A. Castillo).
2011). Synthetic chemical fungicides are recalcitrant molecules of
low biodegradability and potentially toxic. Because their action
mechanisms are restricted to only one molecular target, prolonged
use of these molecules promotes the selection of resistant fungal
strains (Pimentel, 2005; Thind, 2012). For these reasons, there is
currently great interest of the producers and exporters of fresh

http://dx.doi.org/10.1016/j.biocontrol.2016.10.002
1049-9644/ 2016 Elsevier Inc. All rights reserved.
252 R. Santiago et al. / Biological Control 103 (2016) 251260
fruit to reduce the use of chemical fungicides and increase the use
of BCAs based on living organisms.
Botrytis cinerea is one of the most important phytopathogenic
fungi worldwide. This fungus is responsible for gray rot, a disease
that affects numerous fruits of great commercial importance,
including the table grape (Choquer et al., 2007). The current control
of B. cinerea is performed mostly through synthetic chemical fungi-
cides, but in recent years some BCAs based on micro-organisms
with antifungal activity have been described. Among them is
Trichodex, a commercial product whose active ingredients are
spores and propagules of the mycoparasitic fungus Trichoderma
harzianum T39 (Restuccia et al., 2006). However, one of the most
promising products is Serenade, whose active component is the
bacterial strain Bacillus subtilis QST 713 that was discovered in soil
samples from an organic peach orchard by AgraQuest Inc. in Davis,
California. This biofungicide is registered in several countries, has
low toxicity, and is used commercially in grapes for the control
of diseases such as sour rot, gray rot, and powdery mildew, which
is caused by Erysiphe necator (Restuccia et al., 2006).
Currently there is a constant search for microorganisms with
antifungal activity that are useful for the design of new bio-
fungicides to control phytopathogenic fungi. Among them, the bac-
teria are very important and have been described species of some
genera such as Bacillus, Pseudomonas, Burkholderia, Serratia and
Paenibacillus that show antagonism against plant pathogenic fungi
(Paul et al., 2013). In this context, it has been showed that Bacillus
cereus, associated with entomopathogenic nematode, produces
compounds identified as diketopiperazines, which have a signifi-
cant activity in reducing postharvest decay of peanut kernels
caused by Aspergillus species (Kumar et al., 2014). Likewise, Bacillus
subtilis strain G-1 presented high effectivity for inhibiting the
mycelial growth of Sclerotium rolfsii, the causal agent of stem rot
disease of groundnut (Hassen et al., 2015). Also, the Bacillus sp.
RMB7 strain, exhibited potent antifungal activities against a vari-
ety of fungal pathogens, such as Aspergillus niger, Aspergillus flavus,
Colletotrichum gloeosporioides, Colletotrichum falcatum, Fusarium
oxysporum, Fusarium moniliforme, Rhizoctonia solani, Pythium ulti-
matum and Pythium irregulare, causing various diseases to a wide
range of crops (Ali et al., 2014). In addition to producing two types
of cyclic lipopeptides, surfactins and iturins, Bacillus sp. RMB7 pro-
duces cellulase, chitinase, siderophores and antibiotics in vitro, and
efficiently protects arugula plants against Pythium irregulare in vivo
(Ali et al., 2014). On the other hand, Pseudomonas sp. strain P2,
drastically inhibited the growth of Rhizoctonia solani mycelia and
strongly reduced fungal infection on potato. In addition to produc-
ing proteases and siderophores, the P2 strain produces two volatile
organic compounds, dimethyl disulfide and dimethyl trisulfide,
that could be related with its antifungal activity (Elkahoui et al.,
2015). In a similar way, the antifungal activity of a group of wild
strains of Burkholderia cepacia against several phytopathogenic
fungi was related with the secretion of two types of molecules
from the family of 2-alkylquinolones (Kilani-Feki et al., 2012).
Therefore, B. cepacia strains that secrete 2-alkylquinolones could
be potential candidates for the development of new biofungicides.
Similarly, the endophytic bacterium Serratia proteamaculans 336x
secretes a protein with chitinase activity that showed antifungal
activity against Gaeumannomyces graminis var. tritici, the causal
agent of wheat take-all (Wang et al., 2014). Therefore, bacteria that
secrete chitinolytic enzymes may be useful in developing strate-
gies for biological control of plant pathogenic fungi.
Species of the Paenibacillus genus are Gram-positive rhizobacte-
ria that generally inhabit soils near crops and colonize plant roots
through biofilms formed by exopolysaccharides (De Vos et al.,
2009). These species secrete various types of antibacterial
compounds against Gram-positive and Gram-negative bacteria, in
addition to producing molecules with antifungal activity, among
which are fusaricidins, a group of cyclic depsipeptides, and the
peptide gavaserin (Raza et al., 2008). Moreover, some Paenibacil-
laceae members have the ability to solubilize phosphorous present
in the soil, fix nitrogen, synthesize the hormones auxin and cytoki-
nin, which can promote the growth of plants. For these reasons,
Paenibacillus spp. are classified as plant growth-promoting
rhizobacteria (Lal and Tabacchioni, 2009).
Recent publications have shown the ability of Paenibacillus
genus bacteria for biocontrol of plant pathogens and protection
of commercially important crops. For example, Paenibacillus
polymyxa M-1 secretes polymyxin P, a molecule that suppresses
the growth of the bacteria Erwinia amylovora and Erwinia
carotovora, causal agents of the blight and soft rot in wheat plants,
respectively (Niu et al., 2013). Moreover, some strains of
Paenibacillus polymyxa produce hydrolytic enzymes, such as
b-1,3-glucanases, chitinases, cellulases, xylanases, lipases, amy-
lases, and proteases (Raza et al., 2008), which could play major
roles in the biocontrol of plant pathogens. Indeed, P. polymyxa
JSA-9 secretes a glycoprotein that has an apparent inhibitory
activity against a broad spectrum of fungi and bacteria (Deng
et al., 2011). In addition, there is an active participation of the
exopolysaccharides secreted by this bacterium, and the biofilms
formed in the plant-bacterium interaction have been directly
associated with the colonization and protection of plant tissue
(Yegorenkova et al., 2011).
In the present study a new bacterial strain, Paenibacillus poly-
myxa SCHC33, with powerful antifungal activity against Botrytis
cinerea was characterized. This bacterial strain has properties that
could enable it to become the active ingredient of a new fungicide
for the control of the fungus responsible for gray rot.
2. Materials and methods
2.1. Isolation of the bacterial strain
Soil samples were obtained from the VII Region of Maule, Chile.
One gram (1 g) of each sample was suspended in 10 mL of sterile
distilled water. A 100 lL sample from each suspension was seeded
onto Petri dishes containing MEGAC culture medium (malt extract
10 g/L, glucose 2 g/L, agar-agar 15 g/L, cycloheximide 50 lg/mL),
and incubated at 20 C until there was development of
microorganisms.
2.2. Determination of antifungal activity
With the obtained bacterial isolates, antagonism bioassays
against B. cinerea were carried out in Petri dishes. The bioassays
were performed in an MEA medium (malt extract 10 g/L, agar-
agar 15 g/L), without or with glucose (2 g/L), and the virulent B.
cinerea CCg149 of our strains collection, was used.
A 5 mm circle of fungal mycelium of Botrytis cinerea was placed
on center of Petri dish that contained the culture medium, and bac-
terial isolates were seeded at four equidistant points in identical
amounts. Antifungal activity was monitored for seven days at
20 C. Controls were performed by replacing the bacterial isolates
with E. coli DH5a. Only the bacterial isolate which had the highest
inhibitory activity, was selected for further characterization. Then,
in order to obtain one pure bacterial strain, serial dilutions were
performed until obtain colonies formed from one cell. Finally, to
confirm the magnitude of antifungal activity with this pure strain,
the antagonism bioassays were repeated. The bacterial strain
obtained was named SCHC33.
Similar tests were conducted using conidia suspensions of B.
cinerea evenly distributed in the Petri dishes, which were incu-
bated for 30 min at 20 C, to ensure the correct adsorption of the
 R. Santiago et al. / Biological Control 103 (2016) 251260
sample in the culture medium. Then 10 lL of the bacterial suspen-
sion with an optical density of 0.9 at 600 nm, was placed in the
center of a Petri dish and incubated for seven days at 20 C to
determine whether an inhibition halo of germination of conidia
of B. cinerea is formed.
2.3. Amplification and sequencing of the 16S rRNA gene for
phylogenetic analysis
Genomic DNA of the bacterial strain SCH33 was purified using
the PureLink Genomic DNA Kit (Invitrogen), and subsequently
amplified by PCR using the universal primers for Eubacteria (8F
and 1392R) to obtain a specific 16S rDNA fragment of approxi-
mately 1400 base pairs (Martnez et al., 2010). The PCR product
was resolved by electrophoresis on 1% (w/v) agarose gel, and
sequencing was performed using primers 27F and 800R, respec-
tively (Kisand and Lettieri, 2013). The obtained sequence was com-
pared with the sequences of the NCBI database, obtaining
preliminary information that SCHC33 corresponded to Paenibacil-
lus polymyxa
For phylogenetic analysis, the sequence of the 16S rDNA from
SCHC33 strain, in addition of the sequences of type strains, repre-
senting to the different species of the Paenibacillus genus (De Vos
et al., 2009), were used. The phylogenetic tree was constructed
using the neighbor-joining method. For this, sequences were
aligned using the CHAOS + DIALIGN software (Cooper et al.,
2004), and the tree was constructed with the MEGA 5.1 Phyloge-
netic Suite (Tamura et al., 2011). A bootstrap value of 1000 was
used to establish statistical significance of the constructed
branches. From this analysis it was confirmed that the bacterial
strain corresponded to Paenibacillus polymyxa and was named
SCHC33. This bacterial strain has been deposited in the Coleccin
Chilena de Recursos Genticos Microbianos (CChRGM), under the
access code RGM2141.
In parallel, the presence of fusC gene, whose expression product is
part of the metabolic pathway involved in the synthesis of the anti-
fungal molecule fusaricidin, was determined. For the PCR reaction,
we used the specific primers described by Li et al., 2013. The
sequence obtained was phylogenetically compared with the
sequences of the fusC genes from another members of the Paenibacil-
lus genus. Said sequences were obtained from NCBI and EBI
databases.
2.4. Electron microscopy
Morphological and structural aspects of the P. polymyxa SCHC33
were determined by electron microscopy. A sample obtained from
a liquid culture was fixed for 24 h in 100 mM sodium phosphate
pH 7.4, containing 2.5% (v/v) glutaraldehyde. Shading with gold
and palladium was applied for examination under a JEOL JSM-
25-SII scanning electron microscope. For transmission electron
microscopy, ultrathin sections were prepared as previously
described (Vilches and Castillo, 1997). The samples were visualized
under a Philips Tecnai 12 Bio Twin microscope at 80 kV.
2.5. Kinetic analysis of microbial growth
To characterize microbial growth, experimental runs were car-
ried out in batch reactors. A 2 L bioreactor supplied with sterile air
from a compressor with 2 lm filters and a 20 L/min flow rate was
used. Glucose was used as an energy source, with initial concentra-
tions of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 g/L in a YEG culture medium
(yeast extract 5 g/L, glucose). For all experimental runs, tempera-
ture, pH, and stirring were maintained constant at 30 C, 5.0, and
200 rpm, respectively (von der Weid et al., 2000; Kahng et al.,
2001). The aeration rate was maintained at 1 vvm in all the assays,
253
as previously recommended, to avoid oxygen limitation (Garcia
et al., 2009). To start an experimental run, 200 mL of bacterial cul-
ture in exponential phase was inoculated in the bioreactor contain-
ing 1.8 L of the culture medium. Zero time was established as the
moment when the bioreactor began agitation and aeration. Every
30 min, increases in biomass were detected as increases in the
optical density of the culture at a wavelength of 600 nm, and
decreases of glucose were measured using the Liquicolor kit
(Human diagnostics). With these data, graphs of bacterial growth
and glucose consumption at each time point were constructed.
To determine the growth kinetics of P. polymyxa, the Monod,
Meser, and Tessier models (Shuler and Kargi, 2002) were evalu-
ated. The kinetic parameters of the three models were obtained
by minimizing the residual sum of squares (RSS) between the
experimental values and values calculated by the model using
Microsoft Office Excel Solver (Huiliir and Villegas, 2014). To
determine which model was the best fit for the experimental data,
the following statistical parameters were used:
Correlation Coefficient
XN
2
V C  V E 2
R Xi1
N
i1
V E  V C 2
where: VC: Values calculated on the basis of the model; VE:
Experimental values; N: Data number.
RSS
Chi-square v2
Nn
where RSS: Residual sum of squares; N: Data number; N: Constants
number.
2.6. Bioassays in plant tissue
Grapevine leaves were washed one time with a solution of
0.5% (w/v) sodium hypochlorite and rinsed three times with
distilled water. Each wash cycle had a duration of one minute.
These leaves were used to evaluate the inhibition of conidia
germination in plant tissue. The leaves were incubated in Petri
dishes with 1.5% (w/v) agar-agar to maintain humidity and
wounded to facilitate infection. Each leaf was inoculated with
B. cinerea CCg149 conidia suspensions and P. polymyxa SCHC33
bacterial cultures in a liquid medium in proportions of 1:10
and 1:100 conidia:bacteria, respectively. As controls, some
grapevine leaves were inoculated only with distilled water and
others only with conidia, while a comparative control used
Bacillus subtilis QST 713, the active ingredient of the commercial
biofungicide Serenade. The results were quantified as the per-
centage of damage produced on the leaf surface.
To determine the inhibition of the fungal spore germination by
P. polymyxa SCHC33, new infections were performed in grapevine
leaves as described above. At 24 and 48 h post-infection were cut
discs of leaves from the area where the fungus and the bacteria
were inoculated, subsequently the fungus spores were fixed and
stained with trypan blue as described by Vogel and Somerville,
2000, and were visualized to the light microscopy.
2.7. Statistics
All assays were performed with n = 5. Differences between the
samples were determined by ANOVA with Bonferroni correction.
254 R. Santiago et al. / Biological Control 103 (2016) 251260
3. Results
3.1. Sequencing, phylogeny, and microscopy
By sequencing the 16S rDNA of the SCHC33 bacterial strain and
comparing this sequence with those deposited in the NCBI database,
it was possible to determine that SCHC33 strain corresponded to the
Paenibacillus genus, presenting a 97% of identity with the strains SC2,
M1, M-1 and WY110 of P. polymyxa, of which only M-1 has not been
described as a possible biological control agent of fungi. To corrobo-
rate the BLAST results, a phylogenetic reconstruction was per-
formed. In addition to the sequence of the 16S rDNA of the
SCHC33 strain, the sequences of type strains of the genus Paenibacil-
lus described in Bergeys Manual were used. The resulting phyloge-
netic tree (Fig. 1) revealed that SCHC33 strain is associated with P.
brasilensis PB172, P. peoriae DSM 8320, P. jamilae CECT 5266 and P.
polymyxa DSM 36, the type strain of the genus (Fig. 1). Furthermore
as complementary tests, we used the API identification system 50
CHB (bioMrieux), in order to investigate the carbohydrate metabo-
lism and confirm the genus and specie of the bacterial strain ana-
lyzed in this study (see Table 1S in supplementary material). The
specific biochemical reactions that allowed differentiate among
the species of Paenibacillus more closely related, were the fermenta-
tions of glycerol, L-arabinose, D-Xylose, b-methyl-xyloside, a-
Methyl-D-mannoside and a-Methyl-D-glucoside (see Table 2S in
supplementary material). Collectively, our results allowed us to
identify to the bacterial specie under study as Paenibacillus polymyxa
y was named SCHC33 strain. The 16S rDNA sequence data of P. poly-
myxa SCHC33 have been deposited in GenBank database under
accession number KT280273.
Electron microscopy of the intact bacterial cells and ultrathin
sections, revealed that P. polymyxa SCHC33 has rod-shaped, with
a length between 2.0 and 5.0 lm, and diameter between 0.7 and
1.0 lm (Figs. 2A and C). Clusters of cells were formed, and an extra-
cellular material, probably exopolysaccharides, was secreted quite
possibly to form biofilms (Fig. 2B) (Timmusk et al., 2005). Observa-
tions also included spheroidal or oval spores with approximate
1 lm diameter, a typical morphology found in the dormant struc-
tures of the Paenibacillus genus (see small arrows in Figs. 2AC)
(Huo et al., 2010), and bacterial appendices composed mainly by
peritrichous flagella approximately 35 lm in length (data not
shown) (Lal and Tabacchioni, 2009). Furthermore, high magnifica-
tion revealed that the cell wall was multilayered, with three highly
electron-dense layers and two internal layers with low electron
density (Fig. 2D). This finding is interesting because it would reveal
that the density of peptidoglycan not be the same throughout the
thickness of the cell wall.
3.2. Bacterial growth and modeling
Using a 2 L bioreactor, curves for P. polymyxa SCHC33 bacterial
growth with different concentrations of glucose were obtained
(Fig. 3). No assays showed a lag phase, and exponential growth
started immediately. This could be attributed to the high volume
of inoculum used in the assays. Glucose consumption occurred
during the entire phase of exponential growth, maintaining a high
concentration of glucose (P1 g/L). However, during the slowdown
period of growth (>7 h), substrate concentration decayed to values
lower than 0.076 g/L (Fig. 3A). There was no bacterial growth inhi-
bition by substrate nor product. Therefore, kinetic analyses were
restricted to models that did not exhibit this type of situation,
which was the case for the Monod, Moser, and Tessier models
(Shuler and Kargi, 2002).
The curve that relates specific growth rate with the initial con-
centration of glucose permitted obtaining the kinetic parameters
intrinsic to this bacterium (Fig. 4). The curve presented behavior
expected by existing literature, where increasing substrate concen-
tration steadily increases the specific rate of bacterial growth until
reaching a peak at which the rate is constant (Kovrov-Kovar and
Egli, 1998).
Evaluation of the three kinetic models based on statistical
parameters indicated that P. polymyxa SCHC33 growth, using
glucose as carbon source, followed the Monod kinetic model; this
model had the highest correlation coefficient (R2) value and the
lowest Chi-square value (Table 1). Thus, the maximum specific
growth rate of P. polymyxa SCHC33 using glucose as the main sub-
strate was lmax = 0.218 h1; the saturation constant of glucose was
Ks = 0.087 g/L; and the yield of biomass production from glucose
was YX/S = 0.159 g of biomass/g of glucose (Table 1). These values
are within the ranges specified for bacteria in previous studies
(Tobajas and Garcia-Calvo, 2000; Okpokwasili and Nweke, 2005;
Kim et al., 2005).
3.3. Antifungal activity against B. cinerea
To determine if the metabolite fusaricidin contribute to the
antifungal activity of P. polymyxa SCHC33, the fusC gene of the
SCHC33 strain, that encodes for the enzyme 3-oxoacyl-acyl carrier
protein synthase, that is part of the enzymatic complex related
with the synthesis of fusaricidin, was cloned and sequenced. By
BLAST analysis, the sequence of this gene showed similarity with
the fusC gene sequences of another P. polymyxa strains, whereas
a phylogenetic analysis by neighbor-joining, using sequences of
members of the Paenibacillus genus, the fusC gene of SCHC33 strain
was associated with the fusC gene of P. polymyxa E618 in a clade
where members of the P. polymyxa specie were mainly found
(see Fig. 1S in Supplementary material).
The antifungal activity of P. polymyxa SCHC33 against B. cinerea
was evaluated by four trials of antagonism. The first test is shown
in Fig. 5A and B and consisted in the measurement of antifungal
activity in culture media with and without glucose, since the pro-
duction and secretion of active molecules in other biocontrol
agents that have antifungal activity, is dependent on the nutrients
present in the culture medium, particularly of the presence of glu-
cose as the main carbon source (Gohel et al., 2006). In both culture
media, the inhibition halos of the fungal mycelium growth were
essentially of the same magnitude.
The second trial compared the inhibitory effect of P. polymyxa
SCHC33 on the growth of B. cinerea mycelium, with the effect
produced by the active agent of the commercial biofungicide
Serenade, B. subtilis QST 713 (Fig. 5C). The inhibition halos of both
were virtually the same in identical growth conditions, even when
using two different culture media.
The third antagonism trial determined if the antifungal activity
of P. polymyxa SCHC33 had the ability to inhibit the germination of
B. cinerea conidia, as compared to B. subtilis QST 713 (Fig. 5D).
Halos of inhibition of conidia germination were more pronounced
for P. polymyxa SCHC33 than for B. subtilis QST 713, with approxi-
mate areas of 7.5 cm2 and 4.0 cm2, respectively. Taken together
with the prior data, this result indicates that P. polymyxa SCHC33
is more effective in preventing spore germination than in inhibit-
ing the growth of the fungal vegetative mycelium.
The fourth trial assessed the protection of plant tissue against
attack by B. cinerea. Two known amounts of bacteria were used
to evaluate the effectiveness of the biocontrol by P. polymyxa
SCHC33 comparatively with B. subtilis QST 713 (Table 2 and
Fig. 6). After seven days, the level of protection for plant tissue
was slightly higher with P. polymyxa SCHC33. The negative controls
showed small areas of necrosis attributed to the injuries caused to
facilitate infection, whereas positive controls suffered an infection
by B. cinerea that spanned almost 100% of the leaf surface. The
 R. Santiago et al. / Biological Control 103 (2016) 251260 255

Fig. 1. Phylogenetic tree constructed with the sequence of the 16S rDNA of P. polymyxa SCHC33 and the sequences of type strains that representing the different species of the
Paenibacillus genus. The tree was constructed using the neighbor-joining method with the program MEGA 5.0 and the branches strength was supported by 1000 bootstrap
replicates. The position of SCHC33 bacterial strain is indicated by a black square.
256 R. Santiago et al. / Biological Control 103 (2016) 251260

Fig. 2. Electron micrographs of P. polymyxa SCHC33. (A, B) Scanning electron microscopy (SEM) of intact cells. (C, D) Transmission electron microscopy (TEM) of ultrathin
sections. (A) Bacilli and ellipsoid spores in liquid culture. The arrows in (A) indicate the position of some spores. (B) Clustering of bacilli associated with an extracellular
material, probably exopolysaccharides secreted by a group of bacteria. (C) Ultrathin section showing cells dividing. (D) Ultrathin section showing the multilayered structure
of cell wall. HD = high density, LD = low density. The bars in A, B, C, and D, correspond to 5 lm, 15 lm, 1 lm, and 100 nm, respectively.

Fig. 4. Experimental growth curve and adjustment to the kinetic models, Monod,
Moser, and Tessier.

leaves protected by either P. polymyxa SCHC33 or B. subtilis QST

Fig. 3. Curves of bacterial growth and consumption of glucose. (A) Biomass increase
and consumption of glucose over time. (B) Curve of the effect of initial glucose
concentration on the specific growth rate of P. polymyxa SCHC33.
713 showed significantly decreased fungal damage as compared
to the positive control. In the case of P. polymyxa SCHC33, when
a 1:10 ratio of conidia:bacteria was used, the damage produced
by the fungus corresponded to 20.5% of the surface area of the leaf,
and this decreased to 11.7% when the ratio was 1:100. For tests
with B. subtilis QST 713, plant tissue necrosis covered 36.9% of
the surface area at a ratio of 1:10, and this decreased to 15.4% when
the ratio was 1:100. These results indicate that P. polymyxa SCHC33
protects more efficiently than B. subtilis QST 713 the live plant tis-
sue of the attack caused by B. cinerea (Table 2 and Fig. 6).
Finally, the inhibition of germination of conidia on plant tissue
by P. polymyxa SCHC33 was observed. 24 and 48 h post infection,
leaf discs of the area where the fungus was inoculated, were ana-
lyzed by optical microscopy to observe conidia after staining with
trypan blue. Using the same proportions of conidia:bacteria
described above, it was observed that after 24 h there is only ger-
mination in the samples that are not in presence of P. polymyxa
 R. Santiago et al. / Biological Control 103 (2016) 251260 257

Table 1
Statistical analysis and obtention of the kinetic parameters of the 3 models evaluated.

Kineticmodel Formula lmax Ks n R2 v2

Monod 0.218 0.087 0.992 0.0000434

S
l lmax 
KS S

Moser 0.216 0.102 0.879 0.988 0.0000744

n
S
l lmax 
K S Sn

Tessier 0.199 0.112 0.981 0.0000996

l lmax  1  eSK S

Fig. 5. Antagonism bioassays of Paenibacillus polymyxa SCHC33 against B. cinerea. (A) Bioassay in MA medium without glucose. (B) Bioassay in MA medium containing
glucose. (C) Graphical representation of bioassays for growth inhibition against B. cinerea, where ( Glu) is in absence of glucose and (+ Glu) is in presence of glucose. (D)
Graphical representation of bioassays for germination inhibition of B. cinerea conidia. All bioassays were performed with n = 5. ANOVA with Bonferroni correction was used.
***
p < 0.0001, **p < 0.001.
258 R. Santiago et al. / Biological Control 103 (2016) 251260

Table 2 allowed us to identify it as Paenibacillus polymyxa, and was named

Damage caused by the attack of B. cinerea on the surface of grapevine leaves. SCHC33. The BLASTn alignment results showed that the four first
Assays Total Area Damaged Infection bacterial strains with a 97% identity, belonged to the Paenibacillus
[cm2] Area [cm2] percentage genus with biocontrol ability of phytopathogenic organisms and
Negative control 6.3 0.6 9.8 for only three of them, SC2, M1, and WY110, it has been reported
Positive control number 1 11.2 10.8 95.5 to possess antifungal activity. In phylogenic terms, P. polymyxa
(1:10 = conidia:H2O) SCHC33 was located in the same clade as the type strain of species
Positive control number 2 6.2 6.1 98.8
(1:100 = conidia:H2O)
of the genus, P. polymyxa DSM-36 (Fig. 1). Accordingly, the SCHC33
B. subtilis QST 713 10.1 4.0 38.2 strain has been classified as a new member of the bacterial species
(1:10 = conidia:bacteria) P. polymyxa, with antifungal activity against Botrytis cinerea.
B. subtilis QST 713 9.3 1.5 17.1 Likewise, the presence of fusC gene is a strong evidence that P.
(1:100 = conidia:bacteria)
polymyxa SCHC33 could have the metabolic machinery necessary
P. polymyxa SCHC33 8.2 1.8 22.9
(1:10 = conidia:bacteria) for the synthesis of fusaricidin, because the primers used for ampli-
P. polymyxa SCH33 10.3 1.2 11.4 fication were designed to recognize this gene in the SQR-21 strain,
(1:100 = conidia:bacteria) in which the fusaricidin synthesis have been demonstrated (Li
For negative controls, the grapevine leaves were inoculated with sterile distilled
et al., 2013), and in addition, the phylogenetic analysis of this gene
water only. revealed that in the generated phylogenetic tree, the SCHC33 strain
was grouped in a clade with strains classified as P. polymyxa,
specifically was associated with the E681 strain (Fig. 1S in Supple-
SCHC33 (Fig. 7). At 48 h, increased the number of germinated coni- mentary material), which has been described to exhibit antifungal
dia in leaves infected only with the fungus, when using a 1:10 ratio activity, conferred by various types of peptide antibiotics of the
of conidia:bacteria was observed a minimum number of germi- fusaricidin family, against Fusarium oxysporum (Choi et al., 2008).
nated spores (Fig. 7E); otherwise, when was used a 1:100 ratio of Cell structure and morphology of P. polymyxa SCHC33 coincides
conidia:bacteria was not observed conidia germination (Fig. 7F). with other strains of P. polymyxa previously described (De Vos
These results confirms that the minor damage observed on the et al., 2009). However, at the level of the ultrastructure of the cell
leaves was due to a decrease in germination of conidia. wall it was possible to visualize five layers of different electron
density in ultrathin sections of P. polymyxa SCHC33. Two low elec-
4. Discussion tron density layers sandwiched by three layers of high electron
density (Fig. 2D). This important finding not described so far, could
The results of the characterization of the bacterial strain that indicate that there are areas of different density of the peptidogly-
showed the most powerful antifungal activity against B. cinerea, can or that could exist different composition of polymers of cell

Fig. 6. Bioassays for germination inhibition of B. cinerea conidia on grapevine leaves. (A) Example of leaves infected with B. cinerea, treated and untreated with P. polymyxa.
Tests were conducted at ratios of 1:10 and 1:100, conidia:bacteria. (C) negative control, (C+) mock infection replacing bacteria with water, (P) P. polymyxa SCHC33, and (S) B.
subtilis QST 713. (B) Graphical representation of the percentage of damage caused by B. cinerea on grapevine leaves treated and untreated with P. polymyxa SCHC33. All
bioassays were performed with n = 5. ANOVA with Bonferroni correction was used. ***p < 0.0001, **p < 0.001.
 R. Santiago et al. / Biological Control 103 (2016) 251260
Fig. 7. Light microscopy of grapevine leaves infected with B. cinerea CCg149 spores, 24 h (AC) and 48 h (DF) post-infection. The assays were performed by applying the
spores (A and D), together with P. polymyxa SCHC33 at ratios 1:10 (B and E) and 1: 100 (C and F), conidia:bacteria.
wall in P. polymyxa SCHC33. Similar but not identical results were
obtained by Matias and Beveridge (2007), who used cryo-electron
microscopy to show that the septum of Staphylococcus aureus con-
tains a multilayer structure with two high electron-dense regions
sandwiched between three low-density zones. The present study
visualized five layers in the cell wall of P. polymyxa SCHC33, but,
in contrast with the results obtained for S. aureus (Matias and
Beveridge, 2007), three high electron-dense regions were found
sandwiched between two low-density zones (Fig. 2D), and this five
layer structure was visualized throughout the cell periphery and
not only in the septum.
Antifungal activity of P. polymyxa SCHC33 against B. cinerea
remained constant in rich and minimal culture media, with and
without glucose as major carbon source, indicating that such activity
does not depend on the type of nutrients present in the culture med-
ium. This result is very important, because it has been described that
the presence of glucose in the culture media inhibits the production
of fungicide molecules by a catabolic repression mechanism
(Suryawanshi et al., 2014). Likewise, the fit of experimental data to
a kinetic model of bacterial growth Monod type, without inhibition
by substrate nor product, along with unaltered biocontrol ability,
independent of the culture medium used, allowed the production
of large quantities of biomass with full antifungal activity, in nutri-
tive medium with glucose as carbon source.
The ability of P. polymyxa SCHC33 to inhibit the growth of the
vegetative mycelium of B. cinerea was of similar magnitude as that
of B. subtilis QST 713. However, this effect was higher for P. polymyxa
SCHC33 when both bacteria were grown in presence of glucose
(Fig. 5C), because a drastic reduction was observed in the inhibitory
ability of B. subtilis QST 713, as compared with the obtained in the
assay without glucose (Fig. 5C). This difference could be attributed
to some type of catabolic repression yet undescribed for the active
component of the Serenade. In addition, P. polymyxa SCHC33 inhib-
ited the conidia germination of B. cinerea more effectively than B.
subtilis QST713. Finally, bioassays of protection against attack of B.
cinerea performed on grapevine leaves, revealed that P. polymyxa
SCHC33 effectively protects the plant from attack by B. cinerea,
and the necrotic damage produced is of minor magnitude in the
259
leaves inoculated with P. polymyxa SCHC33, that on the leaves trea-
ted with B. subtilis QST713. In addition, it was determined that P.
polymyxa SCHC33 is able to inhibit the germination of conidia of B.
cinerea to 48 h postinfection, when is performed an infection on
grapevine leaves and applied in a ratio of 1:100 conidia:bacteria.
These results showed that the minimum damage observed on the
leaves, when a ratio of conidia:bacteria of 1:100 was used for infec-
tion, is due to inhibition of spore germination in a period greater
than 48 h. However, when we used a ratio of conidia:bacteria of
1:10, spore germination was observed at 48 h. Collectively, these
results indicate that P. polymyxa SCHC33 could become the active
component of a new BCA similar to Serenade.
In conclusion, P. polymyxa SCHC33 possesses similar antifungal
activity as the active component of Serenade, in addition to being
able to protect living plant tissue against the fungal attack more
effectively than B. subtilis QST713. Its ability to form biofilms, to
sporulate, to maintain the antifungal activity in different culture
media and the ease of obtaining large quantities of biomass, are
properties that strengthen the possibility of using this bacterial
strain as a commercial biological control agent.
Conflict of interest
None declared.
Acknowledgment
We would like to thank the Direccin de Gestin Tecnolgica of
the Universidad de Santiago de Chile by financial support (DGT-
2014AC project). In addition, this work was partially funded by
the projects FONDEF-IDeA ID15I10589 and FONDEF-VIU 14E011.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biocontrol.2016.
10.002.
260 R. Santiago et al. / Biological Control 103 (2016) 251260

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