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Biochemical and molecular characterization of


Bacillus amyloliquefaciens, B. subtilis and B.
pumilus isolates with distinct antagonistic
potential against Xanthomonas campestris
pv...
Article in Plant Pathology October 2002
DOI: 10.1046/j.1365-3059.2002.00753.x

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PPA_753.fm Page 574 Friday, September 27, 2002 10:25 AM

Plant Pathology (2002) 51, 574 584

Biochemical and molecular characterization of Bacillus


amyloliquefaciens, B. subtilis and B. pumilus isolates with
distinct antagonistic potential against Xanthomonas
campestris pv. campestris
Blackwell Science, Ltd

E. G. Wulffa*, C. M. Mgunib, K. Mansfeld-Giesec, J. Felsd, M. Lbecka and J. Hockenhulla


a

The Royal Veterinary and Agricultural University, Institute of Plant Biology, Plant Pathology Section, Thorvaldsensvej 40, DK-1871,
Frederiksberg C, Copenhagen, Denmark; bDepartment of Research and Special Services, Plant Protection Research Institute, PO Box CY
550, Harare, Zimbabwe; cDanish Institute of Agricultural Sciences, Department of Plant Protection, Flakkebjerg DK-4200, Slagelse,
Denmark; and dNovo Nordisk A /S, Novo All, 2880 Bagsvrd, Denmark

Fifty-one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase
chain reaction (UP-PCR) fingerprinting; production of secondary metabolites and antagonistic activity against
Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo. Based on FAME analysis
and /or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens,
B. subtilis and B. pumilus. Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of
black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B. amyloliquefaciens
group contained isolates that were generally the most effective at reducing attack of black rot in vivo. The metabolic
profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates
belonging to the B. subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin,
arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B. pumilus group. No
effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites
produced by the three different Bacillus species was tested in vitro against X. c. pv. campestris. However, inhibition was
seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for
seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to
the species and not to the antagonistic activity of the isolates.
Keywords: Bacillus spp., biological control, brassica black rot, endophytes, fatty acid methyl esters, secondary metabolites

Introduction
The genus Bacillus is characterized by Gram-positive,
aerobic or facultative anaerobic, rod-shaped bacteria
that form spores, and contains more than 60 species that
have quite different phenotypes (Claus & Berkeley, 1986).
Most of the tests conducted for identification of bacteria
have been based on physiological and nutritional tests
(Claus & Berkeley, 1986). Today, methods such as fatty acid,
DNA (including PCR fingerprinting) and RNA analysis
are also useful for identification and classification of
*To whom correspondence should be addressed.
Present address: International Potato Center, PO Box 1558,
Lima 12, Peru. E-mail: e.wulff@cgiar.org
Accepted 2 June 2002

574

bacteria (Sasser, 1990; Ash et al., 1991; Stead et al., 1992;


Alvarez et al., 1994). According to Stead et al. (1992),
bacterial species can easily be identified using cellular fatty
acid profiles, and the accuracy of identification at species
level is often 100%. Bacillus species have been identified
and characterized in a study conducted by Ash et al.
(1991), where the 16S rRNA gene of several Bacillus
spp. was sequenced. The study revealed the presence of five
highly different lines within the genus; based on sequence
homologies, B. amyloliquefaciens, B. subtilis and B.
pumilus belong to the same group (group 1) (Ash et al.,
1991).
Efforts to control plant diseases with antagonistic
bacterial agents have been made successfully (Wei et al.,
1991; Chen et al., 1995). Bacillus species have special
characteristics that make them good candidates as biological control agents. First, they are well known as antibiotic
2002 BSPP

PPA_753.fm Page 575 Friday, September 27, 2002 10:25 AM

Bacillus spp. antagonistic against X. campestris pv. campestris

producers with antagonistic activity against fungal and


some bacterial pathogens (Loeffler et al., 1986; Krebs
et al., 1998). This ability also appears to contribute to
the establishment and persistence of the antagonist in the
plant (Krebs et al., 1993). Second, they form spores that
can be easily formulated, and have high viability compared with vegetative cells (Bochow, 1995). Third, they
are commonly found in soils (Stabb et al., 1994).
Several studies have been performed in an attempt to
elucidate the mechanisms involved in biological control
by Bacillus species. The antagonistic activity has often
been associated with production of secondary metabolites
with antibiotic properties (Silo-Suh et al., 1994; Stabb
et al., 1994; Asaka & Shoda, 1996). Most of the antibiotics
produced by Bacillus spp. have been characterized as
dipeptides or cyclic peptides with low molecular weight
(Loeffler et al., 1986; Nakano & Zuber, 1990). The phytosanitary effects provided by the metabolites appear to
be due to the promotion of growth and resistance of the
host plant and direct antibiotic effects against the pathogen (Dolej & Bochow, 1996). According to Silo-Suh et al.
(1994), the metabolites produced by Bacillus spp. can
also affect the microflora on the rhizosphere, providing
an environment antagonistic to the pathogen, or they can
trigger host defence responses. In addition to antibiotic
production, the ability to colonize plants endophytically
has also been identified as an important feature of biological control agents intended for use against vascular pathogens (Kloepper et al., 1999). Bacillus species are among
the most common bacteria found to colonize plants
endophytically (Lilley et al., 1996; Mahaffee & Kloepper,
1997), and it is likely that their endophytic ability could
play a role in the biocontrol of vascular plant pathogens.
Black rot is one of the most serious diseases of crucifers
(Williams, 1980) and has, for example, become the most
important disease of Brassica spp. in Zimbabwe, causing
yield and quality losses of up to 80% (Page et al., 1985).
The disease is caused by the bacterium Xanthomonas
campestris pv. campestris, which is a vascular pathogen.
Among Brassica crops, cabbage and cauliflower are
the most susceptible species to black rot (Mguni, 1996).
Seed contamination is one of the most important means of
transmission (Cook et al., 1952), but in Zimbabwe seed
health testing of Brassica is not carried out and seed
certification is conducted only by field inspection (Mguni,
1996). Plants need to be protected from attack as soon
as they germinate. Thus seed treatment with biological
control agents may be useful in an integrated control
programme for black rot.
This work was carried out (i) to identify and group 51
Bacillus isolates based on FAME analysis and universal
primer (UP)-PCR fingerprinting profiles; (ii) to characterize the ability of isolates to produce secondary metabolites; (iii) to test the antagonistic activity of isolates
(in vitro and in vivo), their culture filtrate extracts (in vitro),
and three selected metabolites (in vitro) against X. c. pv.
campestris; and (iv) to examine the endophytic ability of
selected isolates (belonging to the three different species)
with different antagonistic potential against black rot.
2002 BSPP Plant Pathology (2002) 51, 574 584

575

Materials and methods


Bacterial isolates
Fifty-one Bacillus spp. isolates isolated from Brassica
seeds and plants were kept at 80C in trypticase soy
broth (TSB; Difco Laboratories, Detroit, MI, USA)
amended with 20% glycerol. Isolates W52 (Bacillus
amyloliquefaciens CCUG 28519), W50 and W51 [Bacillus
subtilis CCGU 163B (b) and CCUG 163B (a)], and W43
and W44 [Bacillus pumilus CCUG 26016 (b) and CCUG
26016 (a)] were identified at the University of Gotheburg, Sweden, and used as reference isolates in UP-PCR
fingerprinting.

Fatty acid methyl ester analysis and PCR fingerprinting


Identification and grouping of the Bacillus isolates were
based on fatty acid methyl ester (FAME) analysis results
and UP-PCR fingerprinting profiles for B. amyloliquefaciens and B. subtilis. Bacillus pumilus was identified
and grouped only according to the results obtained with
FAME analysis (first name option).
Fatty acid methyl esters were extracted from each
isolate using standard and recommended procedures for
gas chromatographic (GC) FAME analysis (Sasser, 1990).
Analysis was performed with a Hewlett Packard gas
chromatograph HP5890 CG (Hewlett Packard, Bracknell,
UK) and the sherlock Microbial Identification System
software (MIDI Inc., Newark, DE, USA) using the MIDI
standard method, aerobe Version 39. Identification at
species level was performed by comparing the fatty acid
profiles to the MIDI standard library, tsba version 39.
Isolates with a similarity index of >60% for best match
were considered to have been identified. The fatty acid
profiles were clustered using the dendrogram utilities
included with the sherlock software.
DNA extraction was conducted with the Promega
wizard genomic DNA purification kit for Gram-positive
bacteria (Promega, Madison, WI, USA). UP-PCR (Bulat &
Minorenko, 1990) amplifications were performed using
UP primers that are 1520 bp long and that target intergenic areas of the genome, which are more variable (Bulat
et al., 1998). The primer AS15inv (Lbeck et al., 1998),
which is a 17-mer (5-CATTGCTGGCGAATCGG-3),
was selected from 10 UP primers, based on the ability to
distinguish different Bacillus species according to intense
polymorphic band patterns. UP-PCR was conducted in
20 L volume with 10100 ng DNA, 4 OD units primer,
04 units Dynazyme version 20 (Finnzymes OY, Espoo,
Finland), 10 mm TrisHCl pH 88, 35 mm MgCl2, 50 mm
KCl, 01% Triton x-100, and 04 mm dNTP. PCR was
performed using a MiniCycler, model PTC 150 (MJ
Research, Watertown, MA, USA) and 05 mL standard
PCR tubes (Biozym Diagnostik GmBH, Oldendorf, Germany). The PCR program had an initial DNA denaturation step of 3 min at 94C, followed by 30 cycles of 50 s
at 92C, primer annealing for 70 s at 56C, primer extension for 60 s at 72C, and the final extension step for

PPA_753.fm Page 576 Friday, September 27, 2002 10:25 AM

576

E. G. Wulff et al.

3 min at 72C. The PCR products were analysed by


running 25 40 L of the amplified product on 17%
agarose gels at 300 V for 40 min. The gels were stained
with ethidium bromide and photographed in UV light.

Production and analysis of secondary metabolites


produced by Bacillus isolates
Fermentation
Bacterial cultures 24 h old, growing on trypticase soy agar
(TSA, Difco) at 27C was suspended with 5 mL sterile
saline solution (09%). Bacterial suspension (2 L) was
transferred to 500 mL flasks containing 100 mL boullion
3 (6 g peptone; 4 g pepticase; 3 g yeast extract; 15 g meat
extract; 1000 mL deionized water). The flasks were incubated for 24 h at 30C on a rotary shaker at 250 r.p.m.
Flasks (500 mL) containing 100 mL cabbage broth (50 g
cabbage, 1000 mL deionized water pH 70) or 100 mL
half-strength TSB (15 g TSB, 1000 mL) were inoculated
with 3 mL 24-h-old bacterial cultures growing in boullion
3. After inoculation, flasks were incubated at 30C for
3 days on a rotary shaker (250 r.p.m.).
Extraction of secondary metabolites
1-butanol (2 L) was added to 2 mL of the cultured broth
and shaken in 5 mL vials at 250 r.p.m. for 2 h at room
temperature (23C). Thereafter, the vials were centrifuged
(Varifuge RF Inert, Heraeus Sepatech, Hanau, Germany) for
10 min at 3000 g, 12C and the supernatant (butanol
phase) concentrated until dry. The residue was taken up
with 100 L dimethylsulphoxide (DMSO) and transferred to a plastic vial which was kept in the freezer
(20C) until high performance liquid chromatography
(HPLC) was performed.
HPLC analysis
HPLC analysis was conducted with a Hewlett Packard
1090 m Series II with DAD detector. A Novogram column
[40 mm internal diameter (ID), 60 mm long] was used
together with a precolumn (40 mm ID, 50 mm long)
containing reverse phase material (Grom, Herrenberg,
Germany). Samples were analysed with gradient elution
from 100% solvent A (01% aqueous orthophosphoric
acid) to 100% solvent B (acetonitrile, HPLC grade) in 6 min.
The wavelength of detection was 210 nm. Chromatograms were analysed and identification of the metabolites
was performed with the Hewlett Packard HPLC 3d
chemstation Software (DOS series).

In vivo screening of Bacillus isolates for antagonistic


activity against X. c. pv. campestris
Seeds of cabbage, Brassica oleracea cv. Copenhagen
Market, were surface-disinfected by immersion in 70%
ethanol for 1 min, transferred to sodium hypochlorite
(1% available chlorine) for 3 min, and rinsed three times
consecutively in sterile distilled water. Seeds were then left
to dry in the flow cabinet in Petri dishes containing sterile
filter paper. Sterility control was conducted after seed

disinfection by plating 100 seeds per disinfected seed lot


on trypticase soy agar (TSA, Difco Laboratories) and
incubating at 25C. If no microbial growth was detected
on the plates, the seed samples were considered surfacedisinfected and used in further experiments.
Inoculum suspensions were prepared with 24-h-old
bacterial cultures growing on TSA. Sterile saline water
(20 mL, 085% NaCl) was added to the bacterial culture,
which was suspended with a glass rod. The bacterial
suspension was homogenized using a vortex mixer, and
the inoculum amount was adjusted with sterile saline to
OD600 = 001 (107 CFU mL1) for X. c. pv. campestris
and 100 (109 CFU mL1) for the Bacillus isolates.
Disinfected cabbage seeds were inoculated with X. c.
pv. campestris for 30 min and left to dry in the flow cabinet.
Next day, 03 g preinoculated seeds were soaked for 18 h
under agitation (150 r.p.m.) in 10 mL of the inoculum
suspension made from the respective Bacillus isolates.
Control plants consisted of seeds preinoculated with X. c.
pv. campestris and untreated seeds, both dipped in 10 mL
sterile saline solution and treated in the same way as those
inoculated with Bacillus isolates. The seeds were then
sown in trays containing pine bark that were watered
twice a day and kept in a glasshouse without controlled
conditions. The experiment was designed as a completely
randomized block experiment with three blocks and 15
repetitions per treatment per block.
Fifteen days after sowing, the effect of the different seed
treatments on germination and the biocontrol effect were
evaluated for the different treatments. The effect on
germination was assessed by comparing the number of
seedlings obtained with the Bacillus-treated seeds, and the
number of seedlings obtained with the untreated control
treatment (100% germination). The effect on black rot
incidence was measured by comparing the number of
diseased seedlings that developed from seeds treated with
Bacillus isolates with those from seeds treated only with
the pathogen (100% black rot incidence). At the time of
assessment, cabbage seedlings showed two cotyledons
and zero to three true leaves.

Effect of Bacillus isolates and selected metabolites on


X. c. pv. campestris in vitro
The activity of Bacillus isolates, filtered culture extracts
and three selected metabolites [surfactin (Sigma Chemical,
St Louis, MO, USA); iturin (kindly provided by Dr
G Winkelmann, University of Tbingen, Germany); and
acivicin (Sigma)] against X. c. pv. campestris was measured
in vitro according to Thornberry (1950), with minor
modifications. Two different media were used in this
experiment. LB medium (yeast extract 5 g; glucose 10 g;
tryptone, 10 g; NaCl, 5 g; agar, 15 g and distilled water,
1000 mL) was chosen because antibiotic activity against
bacteria was previously detected using this medium
(Jenny et al., 1991). PDA [39 g potato dextrose agar
(Difco), 1000 mL distilled water] was chosen because
X. c. pv. campestris grew well on this medium. PDA or LB
medium (15 mL) was poured into 9 cm Petri dishes and
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Bacillus spp. antagonistic against X. campestris pv. campestris

left to dry for 30 min. Four mL of the liquid agar seeded


with bacterial pathogen (107 CFU mL1) was then spread
uniformly on the previously poured, solidified agar layer
and left to dry for 10 min. Inoculum suspensions of Bacillus
spp. (50 L; 109 CFU mL1), filtered culture extracts
(concentrated 20, also used in HPLC analysis) and
antibiotic suspensions (10, 05, 025 and 012 mg mL1)
were transferred to autoclaved filter paper disks 12 mm in
diameter, and left to dry for 20 min. The filter paper disks
(one per plate) were then placed at the centre of the seeded
PDA or LB plates and incubated at 27C. Two days after
incubation, the inhibition zone around the disks was
measured. Control plates contained disks soaked in sterile
water and in 50 L vancomycin (Eli Lilly, Indianapolis,
IN, USA) at concentrations of 10, 05, 025, 012 and
00 mg mL1. Vancomycin was shown to be an active antibiotic against X. c. pv. campestris in vitro. The experiment
was set up with four replicates per treatment.

577

Statistical analysis
anova was conducted using General Linear Models
(GLM) of the SAS package (Statistical Analysis Systems
Institute Inc., Cary, NC, USA). Data on germination and
reduction of black rot incidence were analysed as a complete randomized block experiment using three repetitions
(15 replicates per treatment per block). The data on inhibition zone against X. c. pv. campestris were analysed as
one-way anova with four repetitions per treatment. Data
on endophytic ability were analysed as one-way anova
using seven repetitions. However, before analysis the latter were transformed to log CFU g1 plant fresh weight;
samples where no bacteria were detected were scored as
zero and included in the average. Means of each treatment
in all three experiments were compared using the Student
NewmanKeuls test (P < 005).

Results
Endophytic ability of selected Bacillus isolates with
different antagonistic potentials against black rot in
cabbage
Disinfected seeds were inoculated as described above with
seven selected Bacillus isolates with different antagonistic
potential against black rot. Twenty days after sowing,
plants were collected and examined for endophytic colonization. Root (1 cm segment collected from the main
root just below the soil line); stem (1 cm segment collected
just above the soil line); cotyledon and true leaf (latestformed leaf) samples from inoculated and uninoculated
plants were thoroughly washed with tap water, cut with
a scalpel and transferred to Petri dishes containing 15 mL
sodium hypochlorite (1% available chlorine) for 2 min.
The samples were then washed three times consecutively
in sterile distilled water, transferred to plastic bags
containing 05 mL sterile saline solution (085%) amended
with glycerol (20%), then crushed with a hammer. To
ensure that the sections were completely surface-disinfected,
100 L of the last wash was transferred to vials containing 1 mL TSB and incubated at 27C. If contamination
was detected, the sample was discarded. Plant extracts
(50 L) were plated on TSBA [15 g Bacto agar (Difco),
15 g TSB, 1000 mL distilled H2O]. After sterilization,
cycloheximide (10 g mL1, Sigma) and 2,3,5-triphenyltetrazolium chloride (10 mg mL1, Merck, Darmstadt,
Germany) were added to the agar. Control plates were
prepared with the inoculum suspension of the reference
isolates serially diluted, and plated in the same way on the
same medium. All plates were incubated at 27C. Five
to 7 days after incubation, the plates were examined and
colonies compared with the reference isolate. Colonies
similar in morphology and colour to the reference isolate
were picked up with a sterile needle and placed on the
TSBA by puncturing the agar. The same was done with
the reference isolate. If test and reference colonies grew
similarly and had similar UP-PCR profiles, the test bacterial colony was considered to be of the same isolate as the
inoculated one.
2002 BSPP Plant Pathology (2002) 51, 574 584

Table 1 shows the group of isolates identified by FAME


analysis and UP-PCR profile as B. amyloliquefaciens.
Although the similarity indices were too low for the
species to be considered as identified (similarity indices
<60%), FAME analysis suggested the same species name
for the isolates belonging to this group (clusters A and B).
However, all isolates showed similar UP-PCR profiles
when compared with the reference isolate W52 (Fig. 1a).
The isolates in this group were also generally similar in
relation to their HPLC profiles. Most of the isolates produced surfactin (A), iturin (B), bacillomycine (C) and/or
azalomycin F (D) (Tables 1 and 4). The ability to inhibit
growth of X. c. pv. campestris in vitro varied from a very
weak (+) to a very strong (++++) effect and generally no
relationship was seen between in vitro inhibition and
biocontrol effect in vivo (Table 1). All isolates reduced seed
germination, but no significant differences (except for isolates 69 and 17C) were found between isolates. Isolates
29C, 69, 8, 17A, 101, 103, 15, 17C and 71 reduced black
rot incidence in >60% of seedlings (Table 1).
The group of isolates, shown in Table 2, was identified
by FAME analysis (similarity indices >67%) and UP-PCR
profiles as B. subtilis, with all isolates belonging to the
same FAME cluster (cluster C). With the exception of
isolate 16, all isolates showed UP-PCR profiles very similar
to reference isolates W50 and W51 (Fig. 1b). Most of the
isolates in this group were also generally found to be
similar in their HPLC profiles, and produced surfactin (A)
and arthrobactin (G) (Tables 2 and 4). Isolate 16 was the
only one that produced bacillomycine (Table 2). The
ability to inhibit growth of X. c. pv. campestris in vitro was
variable, and ranged from a very weak effect (+) to a very
strong effect (++++). However, no relationship was seen
between these results and the biocontrol effect in vivo.
Although no significant differences were found in seed
germination or reduction of black rot incidence among
isolates of group 2, the ability to reduce the incidence
of black rot within isolates was generally low, and varied
from 00 to 421% (Table 2).

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578

E. G. Wulff et al.

Table 1 Characterization of Bacillus amyloliquefaciens isolates (group 1) according to FAME analysis, UP-PCR banding profiles, secondary
metabolite detection in culture extracts, inhibition zone against Xanthomonas campestris pv. campestris in vitro, effect on seed germination (%),
and reduction of black rot incidence (%) in vivo

Isolate
number

FAME suggested
identification [similarity
index (%)clustera]

55
76
29C
69
80
29A
8
17A
58
74
101
103
68
15
17C
73
71
84

B. amyloliquefaciens (275 A)
B. amyloliquefaciens (321A)
B. amyloliquefaciens (382A)
B. amyloliquefaciens (385 A)
B. amyloliquefaciens (280 A)
B. amyloliquefaciens (359 A)
B. amyloliquefaciens (373 A)
B. amyloliquefaciens (390 A)
B. amyloliquefaciens (498 A)
B. amyloliquefaciens (437A)
B. amyloliquefaciens (470 A)
B. amyloliquefaciens (500 A)
B. amyloliquefaciens (177A)
B. amyloliquefaciens (206 B)
B. amyloliquefaciens (366 B)
B. amyloliquefaciens (439 B)
B. amyloliquefaciens (567B)
B. amyloliquefaciens (347B)

UP-PCR
profileb

Secondary
metabolite
detectionc

Inhibition
zone v.
Xccd

Seed
germination
(%)e

Reduction of
black rot
incidence
(%)e

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

A, D & G
A, C & D
A, F, G, I & J
A, D & H
C, D & M
C, D, H & L
A, B, C, D & G
B
A, B, C & F
A, D & M
A, B, C, D & H
A&B
A, B & I
A, B, C & D
B, C, D & H
A, B, D & H
A, B & D
A, B, C & D

+
+
+
+
++
++
++
++
++
++
++
++
++++
++
++
++
++
+++

955 a
818 a
886 a
977 a
727 ab
932 a
866 a
909 a
864 a
932 a
977 a
932 a
955 a
795 ab
591 b
932 a
864 a
886 a
1000 a
(control)f

455 ab
409 ab
682 a
682 a
364 ab
311 ab
773 a
682 a
409 ab
273 ab
773 a
655 a
00 b
682 a
682 a
455 ab
773 a
586 ab
00 b
(control)g

Cluster grouping according to FAME analysis: Euclidian distance = A, 760; B, 434.


UP-PCR profile 1, see Fig. 1(a).
c
Secondary metabolite identification matches: A, surfactin; B, iturin; C, bacillomycine; D, azalomycin F; F, acivicin; G, arthrobactin; H, rhodutorola
acid; I, valinomycin; J, stenothricin; L, enterochelin; M, nocardamin.
d
Inhibition zone (IZ): + (very weak), 0 2 mm; ++ (weak), 2 < IZ 5 mm; +++ (strong), 5 < IZ 7 mm; ++++ (very strong), 7 < IZ 10 mm.
e
Means were statistically compared within the bacterial isolates belonging to group 1. Values followed by the same letters were not significantly
different from each other.
f
Seeds dipped in sterile saline water.
g
Preinoculated seeds with Xanthomonas campestris pv. campestris and dipped in sterile saline.
b

Isolates identified as B. pumilus by FAME analysis


(similarity indices >60%) were shown to belong to the
same cluster (cluster D) (Table 3). However, the isolates
were shown to be very heterogeneous molecularly, with a
range of different UP-PCR profiles that were distinct from
the reference isolates W43 and W44 (Fig. 1c). Thus the
identification of B. pumilus isolates was based only on
FAME results (first-named option). Figure 1(c) shows the
UP-PCR profiles obtained for B. pumilus isolates (seven
different UP-PCR profiles). Isolates 52, 56, 62 and 64
had a similar UP-PCR banding profile (profile 4); while
BF2 and BF3 had profile 5; and isolates 163, 163B and 11B
had profile 8. Isolates 102, 199, 60, 70 and B7D showed
yet other UP-PCR profiles compared with the other
isolates in this group (Fig. 1c). Considering their HPLC
profiles, most isolates still produced surfactin (A), amphomycin (E), arthrobactin (G) and valinomycin (Is) (Tables 3
and 4). The ability to inhibit growth of X. c. pv. campestris
in vitro varied from very weak to a very strong effect,
although most isolates showed a weak effect (Table 3).
As with the other two Bacillus species, this ability was

generally not related to the ability to reduce the incidence


of black rot in vivo. With the exception of isolate BF3, no
B. pumilus isolate reduced the germination rate compared
with the untreated control. Four isolates (60, 64, 102 and
70) were not able to reduce the incidence of black rot
compared with the untreated control (Table 3).
The general ability of all Bacillus isolates to produce
secondary metabolites is shown in Table 4. All three
species were shown to be efficient metabolic producers
when growing in half-strength trypticase soy broth (TSB)
or in cabbage broth (CB). Generally, TSB stimulated
greater production of metabolites compared with CB.
Surfactin appeared to be a very common metabolite
produced by Bacillus spp., being detected in culture
extracts of all three species when growing in TSB and CB.
Iturin was detected in culture extracts of various isolates
of B. amyloliquefaciens and two isolates of B. subtilis. CB
was shown to be a better substrate for iturin production
compared with TSB (data not shown). Just as iturin was
detected in several isolates of B. amyloliquefaciens, bacillomycine was detected in culture extracts of nine isolates
2002 BSPP Plant Pathology (2002) 51, 574 584

PPA_753.fm Page 579 Friday, September 27, 2002 10:25 AM

Bacillus spp. antagonistic against X. campestris pv. campestris

Figure 1 (a) UP-PCR banding profiles for some Bacillus


amyloliquefaciens isolates (group 1) generated with universal primer
AS15inv. Lanes 217: isolates 8, 15, 17A, 17C, 29A, 29C, 55, 58, 68, 73,
74, 76, 80, 84, 101 and 103, respectively. Lane 18 corresponds to
UP-PCR banding profile of the reference isolate W52. Lanes 1 and 19,
molecular weight marker (lambda phage DNA digested with PstI).
(b) UP-PCR banding profiles (group 2) for some Bacillus subtilis
isolates generated with universal primer AS15inv. Lanes 218: isolates
7A (profile 2), 7B (profile 2), 7D (profile 2), 16 (profile 3), 20 (profile 2),
21(profile 2), 28 (profile 2), 77 (profile 2), 78 (profile 2), 89 (profile 2),
1EF3 (profile 2), 1EL1 (profile 2), 1EL3 (profile 2), 2EL1 (profile 2), 2EL2
(profile 2), BF4 (profile 2) and BB (profile 2), respectively. Lanes 19 and
20 correspond to UP-PCR banding profile of reference isolates W50
and W51, respectively. Lane 1, molecular weight marker (lambda
phage DNA digested with PstI). (c) UP-PCR banding profiles (group 3)
for Bacillus pumilus isolates generated with universal primer AS15inv.

2002 BSPP Plant Pathology (2002) 51, 574 584

579

of B. amyloliquefaciens and one isolate of B. subtilis when


the isolates were growing in CB and TSB. Azalomycin
F was particularly synthesized by B. amyloliquefaciens
isolates (Table 4). This antibiotic was detected only when
the isolates were growing in TSB. Amphomycin and
stenothricin were mainly detected in culture extracts
of B. pumilus isolates, but only a few isolates of B. amyloliquefaciens and B. subtilis were also able to produce these
metabolites. Valinomycin and acivicin were detected in
culture extracts of some isolates of B. amyloliquefaciens,
B. subtilis and B. pumilus. Valinomycin was found only
in culture extracts of Bacillus spp. growing in TSB, while
acivicin was detected in culture extracts of isolates
growing in both TSB and CB. Bacillus amyloliquefaciens
and B. subtilis were able to synthesize arthobactin and
rhodutorola acid, which are iron chelators (siderophores),
while B. pumilus synthesized only arthrobactin. Colistin
was produced by three isolates of B. pumilus, and enterochelin (siderophore), which was produced only when
Bacillus spp. were cultured in CB, was detected in culture
extracts of B. amyloliquefaciens and B. pumilus (one
isolate of each species). Nocardamin, which is also an iron
chelator, was detected only in culture extracts of two
isolates of B. amyloliquefaciens.
The effects of three selected metabolites tested in vitro
are shown in Table 5. Neither surfactin, iturin nor acivicin
inhibited the growth of X. c. pv. campestris in vitro when
the pathogen was growing on PDA or LB. Surfactin and
acivicin had a slight effect on the growth of the pathogen
on PDA and LB in the form of clump formation (granulated growth), but no growth inhibition was seen on the
plates. Concentrated filtered culture extracts of three
Bacillus isolates (BB, B50A and B7D) were also tested
and showed no inhibition of growth of X. c. pv. campestris
in vitro.
The endophytic ability of seven Bacillus isolates is
shown in Fig. 2. Bacillus pumilus isolates BF3 (high
antagonistic activity) and 60 (no antagonistic activity)
showed the best endophytic ability, as they could be
reisolated from all plant parts 20 days after inoculation.
Bacillus subtilis isolates 77 (high antagonistic activity)
and 7D (low antagonistic activity) colonized mainly roots,
but were also found with less frequency in some stem
sections, and rarely in cotyledons and true leaves. Bacillus
amyloliquefaciens isolates 68 (high antagonistic activity),
8 (no antagonistic activity), and 101 (high antagonistic
activity) showed the poorest endophytic ability to colonize
cabbage seedlings compared with the other Bacillus
species. Thus isolates 8 and 68 were reisolated from roots
and sometimes from stem sections and cotyledons (only

Lanes 215: isolate 52 (profile 4), BF2 (profile 5), BF3 (profile 5), 102
(profile 6), 199 (profile 7), 56 (profile 4), 62 (profile 4), 163B (profile 8),
64 (profile 4), 60 (profile 9), 163 (profile 8), 70 (profile 10), B7D (profile
11) and 11B (profile 8), respectively. Lanes 16 and 17 correspond to
UP-PCR banding profile of reference isolates W43 and W44,
respectively. Lanes 1 and 18, molecular weight marker (lambda phage
DNA digested with PstI).

PPA_753.fm Page 580 Friday, September 27, 2002 10:25 AM

580

E. G. Wulff et al.

Table 2 Characterization of Bacillus subtilis isolates (group 2) according to FAME analysis, UP-PCR banding profiles, secondary metabolite
detection in culture extracts, inhibition zone against Xanthomonas campestris pv. campestris in vitro, effect on seed germination (%), and reduction
of black rot incidence (%) in vivo

Isolate
number

FAME suggested identification


[similarity index (%)clustera]

UP-PCR
profileb

Secondary
metabolite
detectionc

78
2EL1
21
1EK2
2EL2
1EL3
20
77
7A
16
BF4
28
1EL1
1EF3
7B
B7A
7D
BB
89

B. subtilis (674 C)
B. subtilis (854 C)
B. subtilis (851 C)
B. subtilis (781 C)
B. subtilis (829 C)
B. subtilis (869 C)
B. subtilis (874 C)
B. subtilis (881 C)
B. subtilis (884 C)
B. subtilis (678 C)
B. subtilis (846 C)
B. subtilis (875 C)
B. subtilis (854 C)
B. subtilis (901 C)
B. subtilis (775 C)
B. subtilis (815 C)
B. subtilis (817 C)
B. subtilis (870 C)
B. subtilis (890C)

2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2

A, G & H
F&H
A&G
A&F
G
A
A&G
A, G, H & J
A&G
A&C
A, I & J
A, F & G
A
A&G
A, G & H
A, B & G
A, G & H
A&G
A, B, F & G

Inhibition
zone v.
Xccd

Seed
germination
(%)e

Reduction of
black rot
incidence
(%)e

+
+
+
++
++
++
++
++
+++
+++
+++
+++
+++
+++
++++
++++
++++
++++
++++

972 a
1000 a
972 a
972 a
889 a
889 a
861 a
667 a
1000 a
1000 a
1000 a
972 a
1000 a
1000 a
861 a
889 a
972 a
1000 a
833 a
1000 a
(control)f

211 a
00 a
158 a
368 a
53 a
368 a
263 a
421 a
158 a
263 a
53 a
316 a
263 a
316 a
211 a
421 a
00 a
363 a
368 a
00 a
(control)g

Cluster grouping according to FAME analysis: C, Euclidian distance = 669.


UPPCR profiles 2 and 3, see Fig. 1(b).
c
Secondary metabolite identification matches, see Table 1.
d
Inhibition zone (IZ), see Table 1.
e
Means were statistically compared within the bacterial isolates belonging to group 1. Values followed by the same letters were not
significantly different from each other.
f
See Table 1.
g
See Table 1.
b

isolate 68), while 101 was found only in a few root samples
20 days after inoculation.

Discussion
The use of FAME analysis and UP-PCR fingerprinting
profiles was generally helpful in the identification of
Bacillus species, making these features useful for the
classification of the genus at species level.
Isolates of group 1, mainly identified by UP-PCR analysis
as B. amyloliquefaciens, showed an ability to produce
secondary metabolites characteristic for this group.
However, the ability to inhibit growth of X. c. pv. campestris
in vitro was generally not relatd to the biocontrol effect
in vivo. Utkhede & Gaunce (1983) and Schreiber et al.
(1988) described similar findings. In vitro, antibiosis can
be influenced by the agar used and order of application
of the bacteria (Bell et al., 1995). Schreiber et al. (1988)
found that, despite high inhibition of the Dutch elm pathogen in vitro by metabolites produced by an endophytic
B. subtilis isolate, no biological control was obtained

in vivo. They suggested that Bacillus isolates might not


produce the same quantity or quality of antibiotics in vivo
as in vitro, or that the population of B. subtilis in planta
might not be high enough or located near the pathogenic
cells (Schreiber et al., 1988). Two isolates belonging to
group 1 were also shown to inhibit seed germination, and
their activity against black rot in vivo was generally higher
compared with the isolates of the other Bacillus groups.
Isolates of group 2, identified as B. subtilis, showed the
highest indices of similarity according to FAME analysis,
with all isolates belonging to the same cluster (Table 2).
Furthermore, with the exception of isolate 16, all isolates
showed similar UP-PCR profiles (profile group 2) and
ability to produce surfactin and arthrobactin in culture.
As with B. amyloliquefaciens, the ability to inhibit growth
of X. c. pv. campestris in vitro generally did not relate to
the biocontrol effect found in vivo. Reduction of seed
germination was lower compared with the other groups.
Also, the ability to reduce black rot was generally low,
with no significant differences between the isolates and
the untreated control.
2002 BSPP Plant Pathology (2002) 51, 574 584

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Bacillus spp. antagonistic against X. campestris pv. campestris

581

Table 3 Characterization of Bacillus pumilus isolates (group 3) according to FAME analysis, UP-PCR banding profiles, secondary metabolite
detection in culture extracts, inhibition zone against Xanthomonas campestris pv. campestris in vitro, effect on seed germination (%), and reduction
of black rot incidence (%) in vivo

Isolate
number

FAME suggested identification


[similarity index (%)clustera]

UP-PCR
profileb

Secondary
metabolite
detectionc

163
11B
60

B. pumilus (865 D)
B. pumilus (887 D)
B. pumilus (919 D)
B. pumilus (884 D)
B. pumilus (681 D)
B. pumilus (896 D)
B. pumilus (909 D)
B. pumilus (855 D)
B. pumilus (835 D)
B. pumilus (617 D)
B. pumilus (912 D)
B. pumilus (818 D)
B. pumilus (742 D)
B. pumilus (815 D)

8
8
9
4
4
4
4
5
5
6
7
10
11
8

A, E, I & J
A&G
J&K
A, E, I & J
A, G & I
A, E, G, I & K
A, E, G, I & J
A, E & I
A, E & I
A, E, F, I & K
G
A, E, I & J
A, F, G & I
A, E, G & I

BF3
BF2
102
199
B7D
163B

Inhibition
zone v.
Xccd

Seed
germination
(%)e

Reduction of
black rot
incidence
(%)e

+
+
+
++
++
++
++
++
++
++
++
++
++
++++

841 a
750 a
864 a
955 a
932 a
795 a
750 a
527 b
773 a
818 a
932 a
886 a
864 a
682 a
1000 a
(control)f

379 a
482 a
172 ab
414 a
414 a
311 ab
517 a
551 a
379 a
241 ab
551 a
311 ab
414 a
448 a
00 b
(control)g

Cluster grouping according to FAME analysis: D, Euclidian distance = 939.


UPPCR-profile 4 11, see Fig. 1(c).
c
Secondary metabolite identification matches, see Table 1 except for E = amphomycin; K = colistin.
d
Inhibition zone (IZ), see Table 1.
e
Means were statistically compared within the bacterial isolates belonging to group 1. Values followed by the same letters were not
significantly different from each other.
f
See Table 1.
g
See Table 1.
b

Secondary metabolite
matchesa
b

Surfactin
Iturinb
Bacillomycineb
Azalomycin Fc
Amphomycinc
Acivicinb
Arthrobactinc
Rhodutorola acidc
Valinomycinc
Stenothricinb
Colistinc
Enterochelind
Nocardaminc

Bacillus species
B. amyloliquefaciens
e

+ (12 : 18)
+ (11 : 18)
+ (9 : 18)
+ (15 : 18)
+ (1 : 18)
+ (3 : 18)
+ (8 : 18)
+ (7 : 18)
+ (3 : 18)
+ (1 : 18)
g
+ (1 : 18)
+ (2 : 18)

B. subtilis

B. pumilus
f

+ (17 : 19)
+ (2 : 19)
+ (1 : 19)

+ (1 : 19)
+ (5 : 19)
+ (14 : 19)
+ (5 : 19)
+ (1 : 19)
+ (2 : 19)

Identified substances (>60% match factor: retention time and UV spectrum).


Detected in both half-strength trypticase soy broth and cabbage broth.
c
Detected only in half-strength trypticase soy broth.
d
Detected only in cabbage broth.
e
+, Ability to produce the corresponding metabolite.
f
Numbers of producing isolates : total numbers of tested isolates.
g
, No ability to produce the corresponding metabolite.
b

2002 BSPP Plant Pathology (2002) 51, 574 584

+ (13 : 14)f

+ (10 : 14)
+ (2 : 14)
+ (8 : 14)

+ (12 : 14)
+ (5 : 14)
+ (3 : 14)
+ (1 : 14)

Table 4 General ability of Bacillus species to


produce secondary metabolites when growing
in tryptic soy broth and cabbage broth at 30C

PPA_753.fm Page 582 Friday, September 27, 2002 10:25 AM

582

E. G. Wulff et al.

Table 5 Effect in vitro of surfactin, iturin and acivicin on growth of


Xanthomonas campestris pv. campestris on PDA and LB medium
Secondary metabolite
a

Surfactin
Acivicina
Iturinb

Inhibition zone (mm)


00
00
00

Media
PDA and LB
PDA and LB
PDA and LB

Control (vancomycin) (mg mL1)


10
107
05
87
025
43
012
13
10
98

PDA
PDA
PDA
PDA
LB

Culture extract (20 concentrate)


BB
00
B7D
00
B50A
00

PDA and LB
PDA and LB
PDA and LB

Tested at four concentrations: 10, 05, 025 and 012 mg mL1.


Tested at two concentrations: 10 and 05 mg mL1.

The final group (group 3) consisted of isolates identified


by FAME analysis as B. pumilus. Although the isolates
pertained to the same FAME cluster and showed some
similarity in their HPLC profiles, the group was shown to
be very heterogeneous considering their UP-PCR profiles.
There were eight different UP-PCR profiles for the 14
isolates tested of this group, and their profiles were shown
to be different from those of the two reference isolates
used. As with B. amyloliquefaciens, some isolates in group
3 significantly reduced germination, and the ability to
inhibit black rot in vitro was generally not related to the
ability to reduce incidence of the disease in vivo.
The production of secondary metabolites was a feature
associated with individual Bacillus isolates, but generally
the detection of some metabolites was associated with
a group of Bacillus species. Bacillus amyloliquefaciens
isolates generally produced surfactin, iturin, bacillomycine
and /or azalomycin F, while B. subtilis isolates were
mostly able to synthesize surfactin and arthrobactin.
Surfactin, amphomycin, arthrobactin and valinomycin were
generally found in culture extracts of B. pumilus isolates.
Surfactin was produced by all three Bacillus species. This

antibiotic has generally been found in culture extracts


of B. subtilis (Hiraoka et al., 1992; Asaka & Shoda,
1996). In contrast, azalomycin F, amphomycin, acivicin,
valinomycin and stenothricin have not been described
for Bacillus species before, whereas they have been for
Streptomyces species. None of the iron chelators (arthrobactin, rhodutorola acid, enterochelin and nocardamin)
detected in the present study has been previously reported
in the literature on Bacillus spp. However, the identification of the secondary metabolites in this study should be
interpreted with caution, as they are based only on HPLC
match profiles suggested by a reference HPLC library.
Mass spectophotometry is still recommended to confirm
the identification of the metabolites detected.
No antibiotic activity was found in vitro when testing
filtered culture extracts of three Bacillus isolates (representative of the three species), or when surfactin, iturin
and acivicin were tested in different concentrations
against X. c. pv. campestris. In the literature, the activity
of antibiotics produced by Bacillus spp. is more commonly
found against fungal than against bacterial pathogens. In
the work conducted by Ebata et al. (1969), three types of
subsporins, which are peptide metabolites produced by
B. subtilis, were active against many filamentous fungi
and yeasts, but no activity was found against most of the
bacteria tested. Gatavalin, another metabolite produced
by Bacillus, was active against Gram-positive bacteria,
mycobacteria, yeasts and moulds, but no activity was
found for Gram-negative bacteria (Nakajima et al., 1972).
Swinburne et al. (1975) tested the activity of antibacterial
components found in culture extracts of B. subtilis, and
proved that they were rapidly deactivated following
maximum production. However, Loeffler et al. (1986)
reported that bacilysin, an antibiotic produced by B.
subtilis, was active against some isolates of B. subtilis at
the rate of 1 mg mL1, and against the Gram-negative
Escherichia coli at the rate of 10 mg mL1. The antagonistic activity found for metabolites produced by Bacillus
spp. has also been associated with the synergistic effect
caused by the combination of antibiotics (Asaka &
Shoda, 1996). Growth inhibition of X. c. pv. campestris
was seen in the study reported here when using bacterial
liquid cultures (containing living cells) of most of the
Bacillus isolates tested. Perhaps the antibiotic effect is

Figure 2 Endophytic ability of seven Bacillus


isolates with high (BF3, 77 and 101) and low
(60, 7D and 68) antagonistic potential against
Xanthomonas campestris pv. campestris,
20 days after inoculation. Isolates BF3 and 60
(B. pumilus); 77 and 7D (B. subtilis); and 8, 68
and 101 (B. amyloliquefaciens) were reisolated
from different plant parts of cabbage. Error
bars represent standard deviations.

2002 BSPP Plant Pathology (2002) 51, 574 584

PPA_753.fm Page 583 Friday, September 27, 2002 10:25 AM

Bacillus spp. antagonistic against X. campestris pv. campestris

transmitted only from bacterial cell to cell; or perhaps


the Bacillus cells changed the agar composition at the
inhibition zone, thereby affecting the growth of X. c. pv.
campestris.
Considering the endophytic ability of seven isolates
with distinct antagonistic potential against black rot, the
ability to colonize cabbage tissues internally appears to be
related to the Bacillus species. No relationship was found
between their endophytic ability and antagonistic potential. Both isolates BF3 (highest antagonistic activity
against black rot in vivo in group 3) and 60 (lowest antagonistic activity) were found in root, stem, cotyledon and
true leaf. Bacillus subtilis isolates differing in antagonistic
activity were reisolated with lower frequency compared
with the isolates of group 3, but at 20 days after sowing,
the isolates could be reisolated from all four parts of the
plant. Bacillus amyloliquefaciens isolates with (isolates 8
and 101) and without (isolate 68) antagonistic potential
against black rot showed the poorest ability to colonize
cabbage internally. Isolates 8 and 68 were sometimes
reisolated from root and stem sections, while isolate 101
was found almost nowhere in any plant part. The results
presented here suggest that the mechanisms involved
in biological control of black rot may be complex, and
appear to be dependent on the individual isolate rather
than the species.

Acknowledgements
The present work was funded by the Danish Council
of Development Research, project number 90842. We
would like to thank Mr Lovemore Mukwicho for excellent technical assistance during glasshouse experiments
and part of the laboratory work conducted in Zimbabwe.
We thank Dr G Winkelmann from the University of
Tbingen in Germany for providing the antibiotic iturin
used in the in vitro tests.

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