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6 authors, including:
Wulff Ednar
Mette Lbeck
Aalborg University
SEE PROFILE
SEE PROFILE
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
575
576
E. G. Wulff et al.
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
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
579
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).
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
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
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
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
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
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)
+ (13 : 14)f
+ (10 : 14)
+ (2 : 14)
+ (8 : 14)
+ (12 : 14)
+ (5 : 14)
+ (3 : 14)
+ (1 : 14)
582
E. G. Wulff et al.
Surfactin
Acivicina
Iturinb
Media
PDA and LB
PDA and LB
PDA and LB
PDA
PDA
PDA
PDA
LB
PDA and LB
PDA and LB
PDA and LB
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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2002 BSPP Plant Pathology (2002) 51, 574 584
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584
E. G. Wulff et al.