Physiological and Molecular Plant Pathology 73 (2008) 25–32

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier.com/locate/pmpp

An inhibitory effect of a new Bacillus subtilis strain (EU07) against Fusarium

oxysporum f. sp. radicis-lycopersici
Ömür Baysal a, b, *, Mikail Çalışkan c, Özlem Yeşilova a, b
a
Department of Turkish Ministry of Agriculture and Rural Affairs, West Mediterranean Agricultural Research Institute (BATEM), P.O. Box 35, 07100 Antalya, Turkey
b
Department of Plant Pathology and Molecular Biology, P.O. Box 35, 07100 Antalya, Turkey
c
Turkish Ministry of Agriculture and Rural Affairs, Central Research Institute for Field Crops, Genetic and Breeding Department Biotechnology Laboratory,
Ankara, P.O. Box 51, 06171 Turkey

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

Article history: Fusarium oxysporum f. sp. radicis-lycopersici (FORL) is a destructive disease on tomato (Lycopersicon
Accepted 12 November 2008 esculentum Mill.) transplant seedlings and the causal organism of crown and root rot of tomato plants
growing in southern coast greenhouses of Turkey. An isolate of Bacillus subtilis (EU07) identified by the
Keywords: 16s RNA region code gene was selected as the best antagonist and evaluated against FORL in vitro studies.
Bacillus Strain EU07 at 106 CFU ml1 was able to reduce disease incidence by 75%, when applied as an inoculant.
HLS
It efficiently inhibited FORL compared to the control and QST 713 (AgraQuest, Davis, CA) whose inhibition
YrvN
ratio was only 52% in vivo. Random amplified polymorphic DNA analyses showed banding (wgenetic)
Fusarium
Biocontrol differences between EU07 and QST 713 whereas there were no differences between DNAs of strains that
Antibiotics have high homology to genes involved in the synthesis of antibiotics fengycin, bacillomycin and iturin
when screened by oligonucleotide primers designed based on sequence information obtained from the
NCBI database. Furthermore, one specific fragment in the EU07 genome showed the highest similarity to
YrvN protein by 99% and AAA ATPase domain protein (72.2%) after amplifying oligonucleotide primers
that are specific to the N-acyl-homoserine lactonase (HLS) gene as a biocontrol activity marker. These
results suggested an effect of EU07 on control FORL by YrvN protein as subunit of protease enzyme.
Furthermore, this fragment associated with HLS gene may be a potential molecular marker for selecting
effective biological control agent belonging to Bacillus in order to control soilborne pathogens such as
Fusarium, suggesting impairment in FORL invasion by signaling in the plant rhizophere.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Although some chemicals used as soil fumigants such as

Crown and root rot disease caused by Fusarium oxysporum
(Schlechtendahl:Fries) f. sp. radicis-lycopersici (Jarvis and Shoe-
maker). FORL is the most frequent disease observed in Turkey’s
southwest tomato production areas and the pathogen seriously
threatens greenhouse tomato production. The fungus can attack both
tomato seedlings in the transplant house and mature plants in the
field. Early symptoms of the disease in seedlings include stunting,
yellowing, and premature loss of cotyledons and lower true leaves. A
pronounced brown lesion that girdles the root/shoot junction
(crown) and root rot, wilting and, death is an advanced symptom.
Occurrence of FORL has been reported from the Mediterranean
region, Europe (UK, the Netherlands, Belgium and France), where the
tomato is intensively grown, USA, Japan [26] and Turkey [10].
* Corresponding author. Department of Turkish Ministry of Agriculture and Rural
Affairs, West Mediterranean Agricultural Research Institute (BATEM), P.O. Box 35,
07100 Antalya, Turkey. Tel.: þ90 242 3452884; fax: þ90 242 3211512.
E-mail address: obaysal@ttmail.com (Ö. Baysal).
metham sodium, dicholoro-propen are effective and recommended
to alleviate disease severity in infested areas against soilborne
pathogens before planting, unconscious chemical application
against soilborne pathogens leads to environmental pollution and
toxic effects on human health and give possibility to pathogens for
building-up resistance to chemicals. On the other hand, these
treatments, except for soil fumigations, are only effective for a short
time in the growing season. In many cases, however severe infec-
tions occur in problematic fields where soil fumigation has been
applied before the growing season. These infections usually result
from a contamination source originating from growers. Chemical
control of FORL using methyl bromide (MeBr) has sometimes
provided good control in the field, but rapid disease dissemination
can occur if the disinfected soil is re-contaminated by the fungus
[38]. MeBr, an ozone-depleting chemical for soil disinfestations,
was phased out in industrialized countries by 2005 and will be
banned by law by the beginning of 2008 in Turkey [1]. Therefore, to
apply effective biological control agents creating a more long-
lasting effect is a necessity besides their antiphytopathogenic

0885-5765/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pmpp.2008.11.002
26 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32
potential of soils. The relatively poor efficacy of chemical control
and the lack of resistance in some commercially important tomato
cultivars have focused attention on the feasibility of biological
control of the pathogen [23]. However, biocontrol agents such as
Pseudomonas spp. and Trichoderma harzianum, which have been
evaluated against FORL do not provide disease control when
applied alone [24,34,43]. The development of biological products
based on beneficial microorganisms can extend the range of
options for maintaining the health and yield of crops. The roles of
plant-associated microorganisms have extensively been examined
in nature and suppression of soilborne diseases by these microor-
ganisms has been reported [20]. Among the many groups of such
organisms are root-associated bacteria, which generally represent
a subset of soil bacteria. Rhizobacteria are a subset of total rhizo-
sphere bacteria having the capacity, upon re-introduction to seeds
or vegetative plant parts to colonise the developing root system in
the presence of competing soil microflora [47]. Targeted research
into the principles of biological control of microbial pathogens
began in the late twentieth century [14]. Bacillus species are the
most common types of bacteria isolated from soil samples and can
account for up to 36% of the bacterial populations [18]. Like the total
number of microorganisms, this amount varies according to envi-
ronmental factors, plantation, and type of fertilization [28]. Endo-
phytic and epiphytic species of Bacillus have the potential to reduce
pathogenicity in planta [49] and mycotoxin accumulation by Fusa-
rium species [5]. In a recent study, although the four Bacillus subtilis
strains have been isolated from avocado, an inhibitory effect to
FORL and R. necatrix by producing hydrolytic enzymes such as
glucanases or proteases and the antibiotic lipopeptides surfactin,
fengycin, and/or iturin has been reported [11]. Furthermore
significant changes in gene transcription in the host plant have
been revealed by cDNA-AFLP analyses in order to determine genes
specifically induced by the Bacillus treatment [33].
On the other hand, another property is the N-acyl-homoserine
lactones (AHLs) which comprise a family of quorum sensing (QS)
signals identified in many Gram-negative bacteria. Different
bacterial species may produce different AHLs, which vary in the
length and substitution of the acyl chain but maintain the same
homoserine lactone moiety [2,4,5]. Acyl-homoserine lactonase
(HLS), a new enzyme from Bacillus sp., inactivates AHL activity by
hydrolysing the lactone bond of AHLs. Biocontrol efficiency was
correlated with the ability of bacterial strains to produce HLS [31].
Our present study shows interesting points to be investigated on
HLS activity role of B. subtilis strain (EU07) in controlling of FORL in
plant rhizophere in view of plant–pathogen–biocontrol agent
interactions.
To our knowledge, the rhizosphere microflora and its impact on
plant growth are in many respects still investigated, and biocontrol
is of importance as a research area. This study presents a B. subtilis
isolate, EU07, showing efficient control against crown rot disease,
and these findings are supported by molecular and pathogenicity
experiments. These findings show the particular effectiveness of
EU07 compared to another B. subtilis strain QST 713 commercial
preperat called Serenade tested in previous study [46], known to
have antagonistic properties against plant pathogens. They also
fortify the importance of biological control and clarify, partly, some
unexplored points of biological control mechanisms with an
effective agent (EU07) for controlling soilborne pathogens.
2. Materials and methods
2.1. Plant material
_
Greenhouse-grown 5-week-old tomato seedlings (cv. Ikram F1,
Syngenta which is susceptible to FORL) with four fully expanded
leaves were used for all experiments. Plants were grown in pots in
a sterilized soil mix, containing sand, perlite, and peat compost and
placed in a glasshouse at 25  5  C with 68–80% RH and natural
light was supplemented by a single 1000 W sodium vapour lamp
during a 16 h photoperiod. The soil mix also contained a slow-
release fertilizer (14–12–14, N–P–K).
2.2. Fungal pathogen and inoculation
A reference isolate, FORL, which was first published in a report
by [10], was used in pathogenicity experiments. The isolate was
grown on potato dextrose agar (PDA) at 25  C for 4 days in a glass
flask and then stored at 4  C. The isolate, recovered as needed from
storage, was grown on PDA at 25  C for 4 days prior to inoculation of
plant tissues. All experiments were conducted at 25  C under
controlled climate conditions.
2.3. Isolation, selection, inoculation and identification of bacteria
Potential bioantagonistic bacteria were isolated from the
rhizosphere of diseased tomato plants as follows: all parts of root
pieces were removed from tomato plants, washed with tap water,
and placed inside tubes containing MgSO4 (0.5%) and shaken for
10 min [36]. The bacterial suspension obtained was diluted to 103,
105 and to 107 for isolation of bacteria. Inhibitory studies were
conducted between the bacterium and the fungi on either nutrient
agar (NA) or PDA, and these inhibition zones were measured as
bioassays for antagonism. FORL was cultured on PDA for 10 days.
From this culture, one mycelial plug was removed using a flame
sterilized borer from the outer margins of each fungal mycelium
mass, and placed on the outer edge (3.5 cm from center) of a fresh
agar plate of both nutrient agar and PDA. The antagonistic bacteria
used for these experiments were grown on nutrient agar for 3 days,
and one inoculation loop was streaked down the other edge of each
plate, immediately after inoculation with the fungal plug. Control
plates consisted of fungi or bacteria placed on plates alone as
described above. All plates were incubated in the dark at 25  2  C,
until the fungi on the control plates had grown together. Bacteria
that completely inhibited the growth of FORL were selected for
further experiments while those that did not inhibit fungal growth
were discarded according to test steps. The isolates showing the
greatest bioantagonistic activity were stored in tubes containing
King’s B medium at 4  C, and in flasks containing TSB (tryptone soy
broth) plus glycerol (20%, v/v) at 20  C. Isolated bacteria were
distinguished from other B. subtilis colonies according to Bergey’s
Manual of Systematic Bacteriology notes, including some tests [13].
In this way, a B. subtilis isolate Egem-Utku (EU07) was identified
(Table 1). Afterwards, identification of EU07 was confirmed with
a pair of designed primers 50 -AATGGCGGTGTATTCCTTGACC-30 and
50 -CAGCTCGTGTCGTGAGATGT-30 that were prepared with Primer 3
input software from known B. subtilis beta-glucanase gene (acces-
sion no. Z29076, NCBI) and 16S rDNA sequences (accession no.
EF101729) sequence after genome extraction as described below.
2.4. Screening and comparison of pathogen suppression invoked by
EU07 and QST 713 in vitro
Fungal inhibition tests were performed by a plate assay. In this
procedure, a loop of bacterial culture was streaked over the surface
of a potato dextrose agar plate (Difco) and, after 4 days of incuba-
tion at 28  C, the plates were inoculated with potato dextrose agar
plugs containing mycelia of F. oxysporum f. sp. radicis-lycopersici
(FORL). Plates were then incubated at 25  C for 20 days and
examined for evidence that growth of the fungus was inhibited by
the bacterium. A positive response was the visible zone of inhibi-
tion around the fungus. The widths of cleared zones of antagonism
(distances between the bacterial and fungal growth) were
 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32
Table 1
Biochemical test results for characterization of strain EU 07.
Biochemical test results
Catalase Oxidase Methyl Citrate- Nitrate Indol
reaction reaction red utilizing reduction formation
þ þ  þ þ 
measured after 20 days and classified as previously described [4].
Mycelium diameters were evaluated according to a scale based on
the mycelium diameter: þ, <3 mm; þþ, 3–9 mm; þþþ, >9–
18 mm; þþþþ, >18–20 mm; >20 mm, þþþþþ. The most efficient
isolate EU07 was compared with QST 713 in biological assays to
assess the efficiency to control pathogen growth. Each experiment
was repeated for four times. Results were expressed as the mean
values with standard error deviation in inhibition distance between
the growth of the corresponding FORL isolate and presence of any
bacterial isolate tested. Percent inhibition was calculated using the
following formula: % inhibition ¼ (1  (Fungal growth/Control
growth))  100.
2.5. Pretreatment with EU07 (root application) and QST 713 and
their effects against FORL
Nine days after sowing, each tomato seedling was inoculated
with 50 mL (5  109) CFU mL1 suspensions of either antagonist,
EU07 or QST 713, which is present in the commercial preparat
Serenade after growing in liquid culture containing NA for 2 days and
cells washed before use as inoculant. Inoculum was applied directly
to the compost at the base of the tomato plantlet stem. Twenty-four
hours after incubation, each seedling was similarly treated with
FORL conidial suspension (concentration ¼ 5  107 spores mL1).
During the procedure of inoculation, plant roots were injured by
minor vulnerable cuts. Plants were watered daily and the number of
wilting or dead seedlings were recorded. Disease severity was
recorded on a 0–3 visual scale according to [50], in which: 0 ¼ no
symptoms; 1 ¼ light yellowing of leaves, light or moderate rot on
taproot and secondary roots and crown rot; 2 ¼ moderate or severe
yellowing of leaves with or without wilting, stunting, severe rot on
taproot and secondary roots, crown rot with or without hypocotyls
rot, and vascular discoloration in the stem; and 3 ¼ dead seedlings.
Disease severity percentage was determined using the following
P
formula: ( scale  number of plants infected)/(highest scale  total
number of plants)  100. Twenty plants per treatment were used
and analysis of variance of the treatment effect on measured data
was performed by using the general linear model procedure of SPSS
11.0. Experiments were analysed using standard analysis of variance
(ANOVA) with factorial treatment structure and interactions. Anal-
ysis of the differences between the categories was calculated with
a confidence interval of 95% with LSD analysis.
2.6. Molecular analysis of EU07 and QST 713 and their genetic
comparison
2.6.1. DNA extraction
Isolates were cultured on NA plates for 48 h in order to compare
the inhibitory effect of EU07 and QST 713. From cultured colonies,
DNA was isolated and subjected to RAPD analysis. Genomic
subtraction was performed for two tester strains QST 713 and EU07.
DNA was extracted from bacterial cultures B. subtilis grown in
Nutrient Broth. After 24 h, 50 mL of culture was removed and
centrifuged at 3000  g for 5 min, after which the cells were
washed in 0.85% NaCl solution, re-centrifuged and resuspended in
2 mL of TE buffer (100 mM EDTA; 150 mM NaCl; 100 mM Tris–HCl,
pH ¼ 8.0) containing 4 mg mL1 lysozyme. The suspension was
incubated at 37  C for 45 min and 0.5 mL of 8.5% SDS was added,
27
Hydrogen sulfide Urease Starch Casein Gelatin Arginine
formation reduction hydrolysis hydrolysis hydrolysis degradation
 þ þ þ þ 
followed by incubation at 75  C for 30 min before the addition of
1.5 mL of potassium acetate (5 M, pH ¼ 5.2) and incubated for
20 min at 4  C. The DNA was extracted with chlor-
oform:isoamylalcohol (24:1), precipitated with ice-cold iso-
propanol [41], washed with 70% ethanol, briefly dried and
resuspended in 200 mL of TE buffer.
The concentration of DNA was measured using a Biowave
S2100 Diode Array spectrophotometer and stored at 20  C until
further use.
2.6.2. RAPD-PCR analysis
Ten primers were selected based on their effectiveness
for differentiation of B. subtilis in previous studies [22,45]: OPA-01
(50 -CAGGCCCTTC-30 ), OPA-09 (50 -GGGTAACGCC-30 ), OPA-11
(50 -CAATCGCCGT-30 ), OPE-02 (50 -GGTGCGGGAA-30 ), OPE-04 (50 -
GTGACATGCC-30 ), OPF-06 (50 -GGGAATTCGG-30 ), OPG-19
(50 -GTCAGGGCAA-30 ), OPH-19 (50 -CTGACCAGCC-30 ),OP-T-04
(50 -CACAGAGGGA-30 ), OPT-07 (50 -GGCAGGCTGT-30 ) (Operon Tech-
nologies, Alameda, CA, USA). DNA amplifications were carried out in
total volume of 15 mL (1.5 mM 10 PCR Buffer, 2.5 mM MgCl2, 0.2 mM
(each) dATP, dGTP, dCTP, and dTTP, 50 ng primer, 5 U Taq DNA
polymerase (Gibco-Life Technologies), 20 mM Tris–HCl, pH 8.4
containing 50 mM KCl), 50 ng template DNA, ddH2O according to
[53]. The thermocycler was programmed for 40 cycles at 92  C for
1 min, 4 min initial denaturation at 92  C, annealing temperature of
37  C for 2 min with a final cycle at 72  C for 3 min and 3 min at 72  C
using a Techne (Touchgene-Gradient, model, FTGRAD2D). PCR
products were separated on a 1.5% agarose gel stained with 0.5 mg
ethidium bromide at 165 V for 3 h and then photographed under UV
light. The molecular ladder was 1 kb DNA (Gibco BRL).
2.6.3. Screening gene for antibiotics production in EU07 and QST
713 genes
DNA amplifications were carried out in a total volume of 15 mL
(1.5 mM 10 PCR Buffer, 2.5 mM MgCl2, 0.2 mM (each) dATP, dGTP,
dCTP, and dTTP), and 50 ng oligonucleotide primers were designed
based on sequence information of genes involved in antibiotic
production obtained from [25]. The HLS gene sequence was from
the NCBI database (accession no.: AF397400). Antibiotic, beta-
glucanase, 16s RNA, and the HLS primer sequences are listed in
Table 5. For each RAPD reaction 5 U Taq DNA polymerase (Gibco-
Life Technologies), 20 mM Tris–HCl, pH 8.4 containing 50 mM KCl,
50 ng template DNA, and ddH2O were used according to [51]. The
thermocycler was programmed for 40 cycles at 92  C for 1 min and
5 min initial denaturation at 94  C, annealing temperature of 34  C
for 2 min with a final cycle at 72  C for 2 min and 2 min at 72  C
using a Techne (Touchgene-Gradient, model, FTGRAD2D). PCR
products were separated on a 1.5% agarose gel stained with 0.5 mg
ethidium bromide at 165 V for 3 h and then photographed under
UV light. The molecular ladder was 1 kb DNA (Gibco BRL).
2.6.4. Characterization of subtracted DNA fragment sequences
An Applied Biosystems ABI Prism 3100 Avant Genetic Analyser
automated sequencer (REFGEN laboratory, MTU Ankara, Turkey)
was used for sequencing different DNA fragments of EU07 observed
after amplification of the HLS primer with the bacterial genome.
Another amplification with the same primer was performed on
these diverse fragments in order to obtain a highly enriched pool of
28 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32
sequences. Fragments were purified for sequencing from the gel by
a PCR clean up-kit (Roche nucleic acid purification kit). Sequencing
reactions were carried out with the reagents of the reaction kit
according to the manufacturer’s instructions and with the
following program: 30 cycles of denaturation at 96  C for 10 s,
primer annealing at 50  C for 5 s, and extension at 60  C for 4 min.
Products of sequencing reactions were separated by electropho-
resis on a 0.2-mm 6% polyacrylamide denaturing gel and recorded
with an ABI Prism 3100 Avant Genetic DNA sequencer (Perkin–
Elmer Applied Biosystems) according to the supplier’s standard
protocol. The determined sequence was compared with those in
GenBank using the BLAST N and BLAST X software provided online
by the National Center for Biotechnology Information [3].
2.6.5. Experimental design and statistical analyses
Twenty tomato seedlings were placed per pot in which each
treatment was tested on the same pathogen-inoculated soil. Signifi-
cant differences were assessed from the mean of three different
treatments (control, QST 713 and EU07) evaluated from four inde-
pendent experiments (dpi). Analysis of variance (ANOVA) was carried
out, and the significance of differences among the treatments was
determined using mean values of each repetitive considering stan-
dard error of mean of treatments. Differences were calculated with
LSD analysis (P < 0.05) with SPSS 11.0 between treatments.
2.6.5.1. Data analysis of RAPD-PCR. Polymorphic bands were eval-
uated as being either present (1) or absent (0) and bands were
analysed according to [44]. A similarity index was used to compare
genetic differences. Similarity index ¼ a/a þ b where: a ¼ number
of bands showing homology between two samples, b ¼ number of
bands showing no homology between two samples.
The genetic distance between two genotypes was calculated by
UPGMA cluster analysis [40] of NTSYS-pc (Numerical Taxonomy
and Multivariate Analysis System, Version 1.8) software.
3. Results
3.1. Characterization of antagonistic bacterium EU07
EU07, the B. subtilis strain showing the highest efficiency of
antagonism against the pathogen was identified (Table 1) according
to Bergey’s Manual of Systematic Bacteriology notes, which
includes some biochemical tests [13]. Afterwards, amplification of
Fig. 1. Bacillus subtilis beta-glucanase gene amplification on two strains EU07 (1) QST 713 (2) (A). Bacillus subtilis 16s RNA gene amplification on two strains EU07 (1) QST 713 (2) (B).
specific oligonucleotide primers of the beta-glucanase and 16s RNA
genes confirmed that both tester are B. subtilis isolates (QST 713,
EU07) which had a specific band 344 bp and around 1500 bp in
length, respectively (Fig. 1).
3.2. Suppressive effect of EU07 against FORL in vitro and in vivo
EU07 effectively inhibited FORL growth when streaked on the
edge of Petri dishes. EU07 has showed more suppression on path-
ogen growth compared to QST 713 in same petri plates. Growth of
the mycelium, as measured by the radius, was significantly
different in EU07- and QST 713-inoculated Petri plates compared to
the control. Suppression of FORL mycelial growth clearly appeared
by 10 days post inoculation (dpi), the inhibition zone being
evidence of the suppressive effect of EU07 and QST 713. The
suppressive effect by both EU07 and QST 713 on FORL growth could
be observed until the end of the experimental period at 15 dpi. The
width of the inhibition zone and the radius of the pathogen’s
mycelium examined at 15 dpi are summarized in Table 2. EU07 and
QST 713 suppressed the pathogen’s growth by 64% and 57%,
respectively.
In addition to in vitro experiments carried out in Petri dishes,
experiments done in pots showed that plants treated with EU07
and QST 713 showed a significant reduction in disease progress
compared to the control. EU07 showed a more efficient control of
FORL when applied into soil compared to QST 713. Treatments
affected the disease index (DI) by 30 dpi, which was the beginning
of the evaluation period for pot experiments. DI on plants in EU07
solution-treated plants was 35%, significantly lower than QST 713
(64%) and control (C) plants, which was 98% by 30 dpi. EU07
solution was the most effective treatment, leading to enhanced
plant resistance to FORL, and better than QST 713. The differences in
plant growth related to different treatments on plants were visible
in in vivo experiments conducted in pots. Plant length differed
significantly (Table 3). The highest average value of plant length
was 14.5 cm following the application of EU07, 10.2 cm in QST 713-
treated plants, and 11.5 cm in control plots; these values differ
significantly.
3.3. RAPD-PCR analysis
Ten 10-mer RAPD primers selected for RAPD analysis produced
a total of 56 bands, 30 of which showed polymorphism within
 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32 29

Table 2 Table 4
Differences in radius of pathogen growth, width of inhibition zone, percentage value RAPD analysis of two tester strain EU07 and QST 713 and proportion showing
of inhibition of pathogen growth in vitro by strains of Bacillus at 15 days post polymorphism.
inoculation. These are mean values of independent experiments. Analysis of the
differences between the categories with a confidence interval of 95%: LSD mean Primer name Number of Number of Proportion of
value is 24.0 for width of inhibition zone, 6.338 for radius of pathogen mycelium observed bands polymorphic bands polymorphic bands, %
values and 56.25 for inhibition % values. The values with the different letters OPA-01 5 2 40.0
represent values that are significantly different according to Fisher (LSD) analysis OPA-09 8 6 75.0
(P > 0.05). OPA-11 10 5 50.0
OPE-02 7 4 57.1
Treatments Width of Radius of pathogen Inhibition % OPE-04 5 2 40.0
inhibiton mycelium (cm) OPF-06 5 2 40.0
zone (mm) OPG-19 2 1 50.0
Control (FORL) – 9.8a 0 OPH-19 6 2 33.3
EU07 þ FORL þþþþþ 32a 3.5b 64a OPT-04 4 4 100.0
QST 713 þ FORL þþþþ 20b 4.2c 57b OPT-07 4 2 50.0
Total 56 30 53.6

genotypes (Table 4). The highest band number was produced with
OPA-11 primer, and the least by OPG-19. OPA-09 primer produced
the highest number of polymorphic bands. OPG-19 primer
produced the least polymorphism (one band, Fig. 2).
3.4. Screening of antibiotic and HLS genes in EU07 and QST 713
Antibiotic screening showed no difference when the Fen D, Bmy
A, and Itu C genes were amplified showing that the same three
antibiotics could be produced by these two different isolates except
for HLS screening results (Fig. 3). Fen D gene amplification showed
one specific band at around 220 bp length in both tester genomes.
Bmy A and Itu C gene amplification was also specific and Bmy A
gene showed a specific band at around 344 bp and Itu C gene
amplification was around 506 bp (Fig. 3). HLS primer amplification
showed possible diversity in one band (indicated by a white circle)
at around 600 bp that was missing in the QST 713 strain. Afterwards
sequencing of this band revealed that this corresponded to a gene
sequence encoding an unknown protein. When compared with
GenBank, sequences of different fragments of the EU07 genome
had the highest degree of similarity to YrvN (accession no.:
CP000560.1, NCBI), 99% and 72.2% to AAA ATPase domain protein
(ZP_01173813). A protein BLAST search indicated that this novel
gene show similarity to YrvN protein sequence (Fig. 4) that also
shares high similarities with hypothetical protein and putative
membrane proteins of B. subtilis.
4. Discussion
We present a practical and effective approach using an antag-
onistic microorganism EU07 to control FORL, an economically
important plant pathogen, in tomato. This study showed that
substantial levels of resistance that decreased disease severity –
which is attributed to fungal growth – and inhibited fungal pene-
tration into plants compared to QST 713 – a conventional,
commercial antibacterial agent – and control and could be induced
by EU07 treatment in tomato plants.
Table 3
Seedling height differences, disease severity on the tomato seedlings (Ikram F1)
inoculated with FORL after plants were treated with EU07 and QST 713. Treatments
were applied at one day before inoculation of pathogen by adjusting bacterial
suspension to ca. 107 CFU mL1 and experiments were analysed at 30 dpi. LSD
analysis of the differences between the categories with a confidence interval of 95%:
LSD mean value is 11.413 for seedling height values, 70.625 for disease severity %. The
values with the different letters represent values that are significantly different
according to Fisher (LSD) analysis (P > 0.05).
Treatment Seedling height (cm) Disease severity in pots %
Control 11.5a 98a
EU07 14.5b 35b
QST 713 10.2c 64c
In general, soilborne pathogens such as Fusarium and Pythium
species that infect through mycelial contact are more susceptible to
competition from other soil- and plant-associated microbes than
those pathogens that germinate directly on plant surfaces and
infect through appressoria and infection pegs. Interestingly plants,
under controlled conditions, inoculated with FORL 1 day post EU07
treatment suppressed the pathogen much more efficiently than the
control and QST 713. In vitro results supported the pathogen
suppression and concurrent decrease in pathogen growth observed
ex vitro following the application of EU07 when plants were chal-
lenged with a pathogen. The possible signal, released by EU07, may
play an important role for triggering plant defense when applied as
an inducer. Plants also respond to a variety of chemical stimuli
produced by soil- and plant-associated microbes. Such stimuli can
either induce or condition plant host defenses through biochemical
changes that enhance resistance against subsequent infection by
a variety of pathogens. Induction of host defenses can be local and/
or systemic in nature, depending on the type, source, and amount
of stimuli [53]. Recent studies showed that biocontrol agents, most
of which are plant growth-promoting rhizobacteria (PGPR) and the
initiation of these inducible reactions require specific receptor-
mediated recognition of pathogen or plant cell wall-derived
molecules, termed exogenous or endogenous elicitors [35].
Growth-promoting effect of EU07 was also determined in pot
experiments. EU07 treatment resulted in plant height differences
which were higher than QST 713 compared to control on tomato
plants. Refs. [51,39] demonstrated that some PGPRs can induce
priming by the release of volatiles. For instance, B. subtilis GB03
induces a signaling pathway that is independent of SA, JA, and the
NPR1 gene, yet it requires ethylene [39]. The GB03 strain produces
the C4 carbon compounds 3-hydroxy-2-butanone and (2R,3R)-
()-2,3 butanediol, which can prime plants for augmented defense
responses to attack by herbivores or pathogens [35]. Therefore,
EU07 may possess the similar properties, which may be expected
Table 5
Oligonucleotide primers used to detect antibiotic and HLS genes.
Primer Sequence
16S RNA F 50 -CAGCTCGTGTCGTGAGATGT-30
R 50 -TGTGGGATTGGCTTAACCTC-30
1 b-glucanase F 50 -AATGGCGGTGTATTCCTTGACC-30
R 50 -GCGCGTAGTCACAGTCAAAGTT-30
2 HLS F 50 -TATTTCATCCCAGCAGGTCGTTGC-30
R 50 -GCGAACGGCACTTCATCTTCAA-30
3 Fengycin D F 50 -CCTGCAGAAGGAGGAGAAGTGAAG-30
R 50 -TGCTCATCGTCTTCCGTTTC-30
4 Bacillomycin A F 50 -TGAAACAAAGGCATATGCTC-30
R 50 -AAAAATGCATCTGCCGTTCC-30
5 Itu C F 50 -TTCACTTTTGATCTGGCGAT-30
R 50 -CGTCCGGTACATTTTCAC-30
30 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32

Fig. 2. Comparison of QST 713 (1) with EU07 (2) RAPD patterns by using 10 different Operon 10-mer primers. The same results on RAPD analysis were also confirmed with four
other independent replicates.

from an effective biocontrol agent. The most suppressive effect on
pathogen growth evoked by EU07 compared to QST 713 can be
associated with differences in EU07, probably involving different
genes and gene products. It must be considered that a biocontrol
organism possess genes for a function, this does not follow auto-
matically that the function is part of the mechanism. Therefore
a site-directed mutagenesis study is necessary in order to demon-
strate the function/mechanism relationship. In the present study,
a bacterium from the rhizosphere (EU07) was also compared with
one suggested by its producers as an active biocontrol agent (QST
713) that could be used in controlling of foliage disease control
more than soilborne disease although it displayed inhibitory effect
against FORL in vivo conditions. It should be considered that the
genomic differences shown in EU07 can answer the question why
would we not expect an effect from a phylloplane bacterium on
root disease when applied to the soil.
In addition a sizable database of 16S rRNA gene (rDNA) has been
successfully used in determining phylogenetic relationships or in

Fig. 3. Fen D (A) and HSL (AHL) (B) gene amplification on two strains EU07 (1) QST 713 (2). Bmy A (C) and Itu C gene (D) amplification on two strains EU07 (1) QST 713 (2). The same
results on PCR amplifications were also observed with three other independent replicates.
 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32 31

f35Frame-2 SNRPFSVTLYM-GEMFMKPLAYRMRPANIEDIIGQEHLVKEDKIIGRMVRAKHLSSMILY
YrvN SNRPFSVTLYM-GEMFMKPLAYRMRPANIEDIIGQEHLVKEDKIIGRMVRAKHLSSMILY
*********** ************************************************

f35Frame-2 GP---LEHRIYVHRHRDSRI--K-QTRKQTPSIIIKQTWKKKDKKRKNQPRSF-SKKITH
YrvN GPPGIGKTSIATAIAGSTSI----AFRKLNAVIHNKKDMEIVVQEAKMSGQVILILDEVH
** : * . .: * ** .. * *: : :: * . : : . .*

f35Frame-2 SRTRESMFLCCWVYENGMSI-YA
YrvN - RLDKGKQDFLLPYLENGMII---
: . : **** *

Fig. 4. The sequence (FASTA format) of a DNA fragment nearly 600 bp from gel that was detected only in EU07 genome (shown in Fig. 3B) after amplifying with a specific HLS
primer. The sequence showed peptidase signature according to NCBI database.

identifying bacteria. It is also effective for the classification and
identification of Bacillus species [17]. 1.3 b-glucanase primer,
employed in order to characterization EU07, confirmed the presence
of a b-glucanase gene encoding PR2 protein. PR2 protein expression
alone in EU07 or QST 713 may not be associated with pathogen
suppression evoked by two B. subtilis strains on FORL growth.
In a previous study, RAPD was used to differentiate strains of
B. subtilis strains [45]. Differences in RAPD bands by amplification
with common primers can be associated with genetic characters, in
which potential differences between QST 713 and EU07 may be
related to effective antifungal metabolite production of EU07
against FORL. However, specific agents must compete with other
soil- and root-associated microbes to survive, propagate, and
express their antagonistic potential during those times when the
targeted pathogens pose an active threat to plant health. In
contrast, general suppression frequently invoked can be explained
with the reduced incidence or severity of plant diseases because
the activities of multiple organisms can contribute to a reduction in
disease pressure [29]. Alternatively, antifungal agents produced by
microorganisms may be used and be effective as biocontrol agents.
Most of the known antifungal agents produced by B. subtilis are
polypeptides [30], including iturins A–E, bacillomycins D, F and L
[6–8]. In particular, B. subtilis is known to produce a number of
antifungal compounds including alboleutin, bacitracin, botrycidin,
clorotetain, fengycin, iturins and rhizocticins [54]. These antifungal
peptides inhibit the growth of a large number of fungi, such as
Aspergillus, Penicillium and Fusarium species [30]. Whereas most of
these antifungals have been tested against mycelial growth, very
little information is available about their effect on fungal spore
survival and germination. These antifungal compounds are very
heat stable, their activity is reduced in the presence of cholesterol,
and they are resistant to proteolytic degradation. These character-
istics indicate that antifungal compounds may belong to the iturin
group of antibiotics, which are known to interact with sterols of the
cytoplasmic membrane of fungi describing the antifungal action of
two bacillomycin D variants. Iturins are cyclic lipopeptides char-
acterized by the presence of seven amino acids [12]. For instance:
iturin A has been shown to increase the permeability of lipid
membranes of fungal cells by pore formation, resulting in the loss
of essential macromolecular compounds [48]. It is however, not
clear why a biological agent belonging to the same species would
be superior to others. This case is consistent with our present
findings in which the sequenced DNA fragment showed high
similarity to YrvN and AAA ATPase, a large and functionally diverse
group of enzymes that are able to induce conformational changes in
a wide range of substrate proteins [19]. These proteins are involved
in a range of processes, including protein degradation, membrane
fusion, microtubule severing, peroxisome biogenesis, signal trans-
duction and the regulation of gene expression [19]. Some sections
contain genomic regions that are related to antibiotic genes in the
bacterial genome, biological control and improving plant health by
interfering in bacterial quorum sensing have been suggested as
another potential mechanism in Bacillus spp. [25]. As indicated in
our findings the similarity of AAA ATPase domain protein gene that
was determined after sequenced DNA fragment performed by
amplifying a specific HLS primer showed a strong relation with the
biological control capacity of the agent and HLS. Biochemical
studies using membrane vesicles have provided further evidence
on plasma membrane that in another study, inhibition and acti-
vation of the Hþ-ATPases in tomato suspension cultures that
differentially regulate plant defense response to pathogen and
wounding has been reported [42]. Elucidation of the precise role of
each Hþ-ATPase’s isoform is needed although it has been impli-
cated as a primary transporter and/or as an intermediate in certain
specific signal transduction pathways within the symbiosis. The
effect induced by the mycorrhizal fungus on root Hþ-ATPases has
been compared with control group in which plant has not been
treated with mycorrhizal fungus and the effect of Hþ-ATPases
induced on roots led to suppressive effect on root pathogen
Phytophthora parasitica [16]. Interestingly, fusaric acid (FA) is on
roots and root-hairs as the probable first site of contact between the
fungi and the host. The root and root-hair growth recession by FA
and induction of a rapid transient membrane hyperpolarization,
followed by a large depolarization due to the inhibition of Hþ-
ATPase currents was suggested [9]. Moreover, FA induced only an
early transient membrane hyperpolarization of root-hairs which in
this case shows compatibility with the induction of a signal
transduction pathway and FA failed to induce salicylic acid- and
jasmonic acid/ethylene-dependent defense-related genes [9]. A
gene showing significant similarity to AAA domain (ATPase asso-
ciated with various cellular activities) exhibited ATP hydrolysis
activity as a subunit of proteases [21,27]. Therefore, subtracted gene
showing similarity to ATPase domain protein gene in EU07 genome
may be related to these findings, which can also clarify how EU07
regulates its higher suppressive affect on pathogen growth in soil
and plant defense response to pathogens.
The present study not only shows that EU07 is an original
B. subtilis isolate, which is able to trigger resistance in tomato plants
against FORL, and is more effective than registered strain QST 713 in
commercial preparation SerenadeÒ. In addition, there is the pres-
ence of a strong biocontrol capacity and an HLS gene analogue
showing similarity to protease subunit YrvN protein by its pepti-
dase signature in addition to antibiotic genes. Therefore, this
specific gene sequence may be used as a marker in selection of
antagonistic agents in biological control. Moreover, detailed chro-
matographic studies in order to characterization of compounds
secreted by EU07 and testing of this biological control agent in
other plant–pathogen systems are also necessary. Although
biocontrol organisms still have ecological parameters that must be
determined before their use in disease control and reducing disease
severity the data presented in this paper show positive results
based on tomato. In this paper, EU07 is however, recommended
32 Ö. Baysal et al. / Physiological and Molecular Plant Pathology 73 (2008) 25–32
against FORL as a biocontrol organism having a high potential for
controlling FORL and leads to a suppressive effect on pathogen
growth. We believe that our findings will enhance our insight on
the essentials of biocontrol in achieving perfect harmony to be
success in control of pathogen.
Acknowledgements
The authors wish to thank A.R. Türkyılmaz and E. Karataylı for
assistance in gene sequence analysis that was carried out in the
REFGEN laboratory in METU/Techno-city, Ankara and Dr. Z. Devran
from Nematology Department of Plant Protection Department in
BATEM Antalya, for financial support in order to provide special
primers and B. Nizam for assistance in biochemical tests.
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