Journal of Applied Microbiology 2005, 99, 213–221 doi:10.1111/j.1365-2672.2005.02614.

Biological control agent of common scab disease by

antagonistic strain Bacillus sp. sunhua

J.S. Han1*, J.H. Cheng1*, T.M. Yoon1, J. Song1,2, A. Rajkarnikar1, W.G. Kim3, I.D. Yoo3, Y.Y. Yang1
and J.W. Suh1
1
Department of Biological Science, Institute of Bioscience and Biotechnology, Myongji University, Yongin, Korea, 2Korean Agricultural
Culture Collection, National Institute of Agricultural Technology, Suwon, Korea, and 3Korea Research Institute of Bioscience and
Biotechnology, Yusong, Taejon, Korea

2004/0204: received 24 February 2004, revised 22 September 2004 and accepted 25 September 2004

ABSTRACT
J.S. HAN, J.H. CHENG, T.M. YOON, J. SONG, A. RAJKARNIKAR, W.G. KIM, I.D. YOO, Y.Y. YANG AND
J . W . S U H . 2005.
Aims: To identify an antagonistic strain against Streptomyces scabiei and to characterize the antibiotic agent.
The efficacy of the isolated strain in controlling common scab disease was also evaluated.
Methods and Results: A bacterial strain antagonistic against S. scabiei was isolated from the soil of a potato-
cultivating area. This bacterium was identified as a Bacillus species by 16S rRNA gene sequence analysis and
was designated Bacillus sp. sunhua. Antibiotics produced by this strain were proven to be stable within a broad
pH range and at high temperatures. The culture broth was extracted with ethyl acetate, and then the crude extract
was applied to HPLC. Two compounds were isolated and identified as iturin A and macrolactin A by 1H-NMR,
13
C-NMR, HMBC, HMQC and mass spectrometer. The culture broth of Bacillus sp. sunhua had a suppressive
effect on common scab disease in a pot assay, decreasing the infection rate from 75 to 35%. This strain also
suppressed Fusarium oxysporum, the pathogen of potato dry rot disease.
Conclusions: Bacillus sp. sunhua was shown to inhibit S. scabiei effectively.
Significance and Impact of the Study: This is the first report demonstrating that macrolactin A and iturin
A inhibit S. scabiei. This study demonstrated the possibility of controlling potato scab disease using Bacillus sp.
sunhua.

Keywords: biological control, common scab disease, iturin A, macrolactin A, Streptomyces scabiei.

most prevalent in neutral or slightly alkaline soils, especially

INTRODUCTION
Potatoes are widely cultivated, and could contribute to
reducing worldwide food shortages. However, potato plants
are susceptible to devastation by various diseases, such as
soft rot disease, bacterial wilt disease, scab disease and dry
rot disease (Agrios 1997). Common scab disease occurs
throughout the potato-cultivating regions of the world and is
*These authors contributed equally to this work.
Correspondence to: Joo Won Suh, Department of Biological Science,
Myongji University, San 38-2, Nam-dong, Yongin 449-728, Korea
(e-mail: jwsuh@mju.ac.kr).
ª 2005 The Society for Applied Microbiology
during dry years (Bouchek-Mechiche et al. 2000). Common
scab disease has little or limited effect on tuber yield, but it
can greatly affect tuber quality and, therefore, can severely
reduce its market value (Liu et al. 1996). At least four
Streptomyces species have been described to cause scab
disease, including Streptomyces scabiei, Streptomyces acidisca-
bies, Streptomyces turgidiscabies and Streptomyces ipomoeae.
(Healy et al. 2000). Of these four species, S. scabiei is the
most important pathogen of potato crops (Keinath and Loria
1989).
Streptomyces scabiei penetrates tissues through lenticels and
occasionally through wounds or young tubers, causing scab
214 J . S . H A N ET AL.
disease by producing a family of cyclic dipeptides, thaxto-
mins (Lawrence et al. 1990). Control of this disease has been
achieved by soil treatment with the fungicide pentachloro-
nitrobenzene (Potter et al. 1958), by using potato seeds
treated with maneb-zinc dust when planting in scab-condu-
cive soil (Agrios 1997), by regulating the pH of the soil
(Doyle and Maclean 1960) or by irrigation (Hooker 1981;
Loria et al. 1997). However, agrichemicals used for crop
protection adversely affect the quality of crops and the
environment, so studies on alternatives to synthetic chemicals
are necessary (Liu et al. 1995; Neeno-Eckwall et al. 2001).
Recently, biological control has been recognized as a
viable approach for the treatment of plant disease. Control of
pathogen by antibiotic producing micro-organisms is now
considered a viable disease control technology (Ferreira
et al. 1991; Pietro et al. 1992; Kim and Kim 1994;
Munimbazi and Bullerman 1998). Derived from micro-
organisms, validamycin (Iwasa et al. 1971) and kasugamycin
(Tominaga and Kobayashi 1978) are widely used agricultural
antibiotics. In the case of common scab disease, it has also
been reported that S. scabiei can be inhibited by an antibiotic
isolated from Streptomyces diastatochromogenes strain PonS-
SII, although the structure of this antibiotic has not been
distinctly characterized (Eckwall and Schottel 1997).
Due to the high economic losses caused by potato scab
disease, studies on biocontrol mechanisms are of import-
ance. In this study, an antagonistic Bacillus strain effective
against scab pathogen was isolated and named sunhua. The
biologically active compounds from the cultural filtrate of
this strain were isolated and characterized as macrolactin A
and iturin A. Furthermore, a pot assay proved the
suppressive effects of this strain against scab disease caused
by S. scabiei. Our results demonstrated the prospect of
Bacillus sp. sunhua as a potentially promising biocontrol
agent for common scab disease.
M A T E R I A LS A N D M E T H O D S
Selection of antagonistic bacterium
Streptomyces scabiei KACC 20101 was provided by Korean
Agricultural Culture Collection, National Institute of Agri-
cultural Biotechnology, Korea. The strain was cultured in
GYM broth (glucose 4 g l)1, yeast extract 4 g l)1, malt
extract 10 g l)1, calcium carbonate 2 g l)1, pH 7Æ2) at 28
C
for 7 d. Soil samples were taken from a spot of low infection
of scab disease in a area of heavy infection on Jeju Island in
the southern part of South Korea. 207 strains were isolated
from soil samples and were incubated at 28
C (for actino-
mycetes) and 37
C (for other bacteria) in tryptic soya broth
(TSB; Bacto, Becton Dickinson, Sparks, MD, USA). Each
isolated strain was inoculated into potato dextrose broth
(PDB; Difco) plates by toothpick on which S. scabiei had
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
been previously spread. After incubation for 5 d, strains that
inhibited the mycelium growth of S. scabiei were considered
suppressive strains. The antagonistic strain with the biggest
inhibition zone was identified by analysis of 16S rRNA
sequence using primers fD1, rP2 (Chun and Goodfellow
1995). The amplified 16S rRNA gene fragment was ligated
into pGEM T-Easy vector and then transformed into E. coli
DH5a. The plasmids, purified with a Wizard plasmid prep
kit (Promega, Madison, WI, USA), were sequenced using an
automated DNA Sequencer (Model ABI 3100; Applied
Biosystems, Foster City, CA, USA). The sequence was
aligned using CLUSTAL W software (Thompson et al.
1994) and a phylogenic tree was constructed using the
neighbour-joining method (Saitou and Nei 1987).
Production of secondary metabolites and
antimicrobial activity
The isolated strain was cultured in TSB liquid medium
until the final OD600 reached 1Æ0. Then, 100 ll of inoculum
was transferred to a 500-ml flask containing 100 ml of GYM
broth and incubated at 37
C for 24 h in a rotary shaker
(150 rev min)1).
Antimicrobial activity against S. scabiei was detected by an
agar disc diffusion assay (Kimura et al. 1998). Plates were
prepared with S. scabiei spread on the surface of the GYM
agar. Paper discs with a diameter of 8 mm were laid on the
agar. Then, 50 ll of cell-free culture supernatant was added
to the discs and allowed to diffuse into the agar, followed by
incubation at 28
C for 2–4 d, when the diameter of the
inhibition zone was measured.
Effects of pH and temperature on antimicrobial
activity
For the pH stability test, the pH of the cell-free supernatant
of the antagonistic strain was adjusted to pH 1–14, and
readjusted to neutral after 20 h before being applied to the
filter discs. The activity against S. scabiei was tested by agar
diffusion assay. To analyse the thermal stability, samples of
the cultured broth were exposed to 40, 60, 80 and 100
C for
30 min, and at 121
C for 15 min. The remaining activity
was tested against S. scabiei by disc diffusion assay.
Isolation of the antimicrobial compound by HPLC
and structural analysis
The Bacillus strain showing the strongest bioactivity against
S. scabiei was used to isolate the antimicrobial compound.
The inoculum for the production of antimicrobial compound
was prepared in 3 ml TSB broth at 37
C for 24 h in a rotary
shaker (150 rev min)1). GYM was used as production
medium with 0Æ1% inoculation size. A total of 15 l of culture
 BACILLUS SP. SUNHUA CONTROLS SCAB DISEASE
broth was obtained. After centrifugation at 8000 g for 30 min
at 4
C, the supernatant was extracted two times with the
same volume of ethyl acetate and was dried in vacuo. The
crude extract was separated into nine fractions by silica gel
RP-18 flash chromatography, with H2O–MeOH (50 : 50),
H2O–MeOH (40 : 60), H2O–MeOH (30 : 70), H2O–MeOH
(20 : 80), H2O–MeOH (10 : 90), MeOH (100%), acetone,
ethyl acetate and hexane for the collection of different
fractions. The two most active fractions were further purified
by preparative HPLC using a SymmetryprepTM C18 column
(Waters, Milford, MA, USA), and were monitored by a
refractive index detector. Elution was performed with 60%
aqueous acetonitrile, maintaining a flow rate of 1Æ5 ml min)1.
Individual detected fractions were collected and evaporated
under reduced pressure. Paper discs containing 0Æ5 mg of
each individual fraction were loaded onto the GYM plates
that had been spread with S. scabiei. Structural analysis of the
active compounds against S. scabiei was performed using
NMR and mass spectrometer. The purified compounds were
dissolved in D-methanol. NMR spectra were recorded on a
Bruker Avance DPX 600 MHz NMR spectrometer (for
1
H-NMR spectrum; Bruker, Karlsruhe, Germany) and then
by a Bruker Avance DPX 300 MHz NMR spectrometer (for
13
C-NMR spectrum). Chemical shifts are given in ppm,
using tetramethylsilane (TMS) as an internal standard.
FAB-MS spectra were measured on a JMS-AX 505 WA
spectrometer (JEOL, Tokyo, Japan).
Scanning electron microscope analysis
Paper discs containing 0Æ5 mg of major compound and
0Æ5 mg of minor compound were loaded on the GYM plates
that had been inoculated with S. scabiei. After incubation at
28
C for 7 d, the samples for scanning electron microscope
(SEM) analysis were isolated from the inhibition zone.
Primary fixation was performed by soaking the samples in
paraformaldehyde and 2% glutaraldehyde in a 0Æ05-mol l)1
sodium cacodylate buffer (pH 7Æ2) at 4
C. For postfixation,
the primarily fixed samples were soaked in 1% osmium
tetroxide in a 0Æ05-mol l)1 sodium cacodylate buffer
(pH 7Æ2) for 2 h at 4
C and were thoroughly washed with
distilled water at room temperature. The washed samples
were dehydrated in a series of ethanol concentrations (30,
50, 70, 80, 90, 100, 100 and 100%) and were mounted on
metal stubs for gold coating, followed by treatment with
100% hexamethyldisilazane for 15 min before observation.
The samples were observed under a JSM 5410 LV Scanning
Electron Microscope (5000·) (JEOL).
Pot assay
To test whether the isolated strain would have a suppressing
effect against scab disease in a potato field, a pot assay was
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
215
carried out. Experiments were performed in pots filled with
sterilized soil. S. scabiei was grown in oat meal broth (Loria
et al. 1995) at 28
C for 7 d (107 CFU ml)1). A 200-ml of
culture was centrifuged at 3000 g for 10 min. The mycelial
pellets were washed three times with distilled water and
were resuspended in 30 ml of sterile water in preparation for
mixing into the soil of each pot.
Solanum tuberosum tubers were washed with sterile
distilled water, planted in sterile soil, and grown in a
greenhouse at 28
C. Potato seedlings up to 12Æ5 cm in size
were selected and transferred to infested soil pots. The
seedlings were then placed in a growth chamber in which the
temperature was 15
C at night and 20
C during the day.
After 2 weeks, culture broth of the antagonistic strain
(108 CFU ml)1) in 200 ml GYM broth was mixed into the
S. scabiei-infested soil. At the same time, an identical
quantity of S. scabiei was also inoculated into the soil to
maximize the chances of infection. Again, after 2 weeks, the
same quantity of the antagonistic strain was inoculated into
the pot soil. Positive control treatments were also performed
by using S. scabiei as an inoculum, and noninfested soil pots
were prepared as untreated controls. Potatoes were harves-
ted after 3 months and the infection rate was calculated as
the percentage of infected potatoes with lesions over 0Æ5 cm
in diameter. This experiment was repeated twice. The
percentage was calculated according to the following
formula:
Infection rateð100%Þ
Number of potatoes with lesions over 0
5 cm
¼ :
Number of total potatoes (infected and noninfected)
Inhibitory spectrum of the antagonistic strain
All strains used as indicator organisms were provided by
the Korean Agricultural Culture Collection. Agar diffu-
sion assay was used to determine qualitatively the
suppressive ability of the metabolites against various
phytopathogens.
The phytopathogenic bacterial strains were previously
cultured in appropriate growth liquid mediums and
appropriate temperatures, with final OD600 c. 1Æ0. Then
100-ll of the inoculum was added to 100 ml of each
appropriate growth agar medium (Table 3) and, after being
mixed well, was poured into five plastic plates. The zone of
inhibition was measured after 24–48 h of incubation. The
phytopathogenic fungal strains were cultured in potato
dextrose agar (PDA) medium until they showed abundant
sporulation. The spores were harvested in sterile 0Æ8%
NaCl to yield a concentration of c. 106–107 spores ml)1.
Then, 2 ml of inoculum was added to 100 ml of PDA
medium. All the test plates were allowed to dry for 30 min
before sterilized paper discs (10 mm in diameter) contain-
216 J . S . H A N ET AL.

ing 100 ll of sterile solution of metabolites were placed on Table 1 Inhibition of Streptomyces scabiei by heat-treated metabolites
the agar surfaces. The diameter of inhibition zone around of Bacillus sp. sunhua
the discs was measured. Relative remaining
Temperature (
C) activity (%)

RESULTS Room temperature 100

40 94Æ1
Isolation of micro-organism antagonistic against 60 82Æ3
S. scabiei 80 58Æ8
100 45Æ0
A total of 206 micro-organisms were screened from the soil 121* 0
samples; 18 of the isolates were selected on the basis of their
activity against S. scabiei, among which the most active Supernatant of Bacillus sp. sunhua was exposed to various tempera-
strain was further characterized. This strain was named tures for 30 min and residual activities against S. scabiei were expressed
sunhua and was identified as a Bacillus species by 16S rRNA as a percentage of original activity.
sequence analysis. *Autoclaved at 121
C for 15 min.

activity against S. scabiei was found to be stable at 60
C with

pH and thermal stability
The antimicrobial activity of Bacillus sp. sunhua culture
filtrates was found to be the highest at neutral pH, but was
only moderately reduced when exposed to pH ranges from
5Æ0 to 13Æ0 (Fig. 1). Under acidic conditions in the pH range
of 1Æ0–4Æ0, biocontrol activity was reduced significantly or
completely eliminated. What is more important is that the
activity was found to be more stable in alkaline pH than in
acidic pH.
The supernatant of Bacillus sp. sunhua was exposed to
various temperatures for 30 min and residual activity was
measured (Table 1). Heat treatment of the broth showed the
antimicrobial activity was stable at a temperature of 100
C
for 30 min and antimicrobial activity remained above 50%
when broth was treated at 80
C for 30 min. Antimicrobial
82Æ3% of the original activity remaining.
Inhibition of S. scabiei sporulation by the
supernatant fluid of Bacillus sp. sunhua
When S. scabiei was cultured in the presence of Bacillus
sp. sunhua culture filtrate, inhibition of mycelium growth
was observed around the paper disc, and inhibition of
sporulation was observed around the mycelium inhibition
zone. The diameter of the sporulation inhibition zone was
much larger than that of the mycelium inhibition zone
(Fig. 2). This indicated that there were at least two

120
Relative remaining activity (%)

100

80

60

40

20

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH

Fig. 1 Effect of pH on the antagonism of Streptomyces scabiei growth Fig. 2 Antimicrobial activity of Bacillus sp. sunhua against Streptom-
by secreted metabolites of Bacillus sp. sunhua. Culture supernatant yces scabiei. A 100-ll of supernatant fluid of Bacillus sp. sunhua was
fluids were incubated at various pH values. Activity was expressed as applied to the disc. Mycelium inhibition zones were observed within
the percentage of relative residual activity the spore inhibition zones

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
 BACILLUS SP. SUNHUA CONTROLS SCAB DISEASE 217

Culture broth (15 l)

Centrifuged at 8000 rpm

Supernatant
Extracted with EtOAc

EtOAc extract Water layer discard

Silica gel RP – 18 chromatography

50% Aq MeOH

40% Aq MeOH

30% Aq MeOH

20% Aq MeOH

10% Aq MeOH

100% MeOH

100% Acetone

100% EtoAC

100% Hexane
Preparative HPLC
Fig. 3 Flow diagram of isolation procedures
of antibiotics rp14 rp5

antibiotic activities in the cultured filtrate of Bacillus sp. ture and all 13C-NMR assignments of the compound are
sunhua. identical to macrolactin A (Table 2).

Isolation and characterization of the active Observation of antimicrobial activity of the

compounds
Two active compounds against S. scabiei were isolated
from the ethyl acetate extraction by reversed phase flash
chromatography and preparative HPLC (Fig. 3). Of the
nine fractions obtained from the reversed phase flash
chromatography, the 30% aqueous MeOH fraction and the
20% aqueous MeOH fraction showed the strongest activity
against S. scabiei. The structures of these active com-
pounds were analysed by 1H-NMR, 13C-NMR, HMBC,
HMQC, NOESY and mass spectrometer. The mass
spectrum of the major compound in the 20% aqueous
MeOH fraction showed a molecular ion peak that
confirmed it to be iturin A at m/z 1043 (Peypoux et al.
1978). The minor compound in the 30% aqueous MeOH
fraction was identified as macrolactin A, with a formula of
C24H35O5 (Gustafson et al. 1989). This compound was
confirmed by the presence of 24 signals in 13C-NMR
spectrum composed of the following: a methyl carbon, six
methylene carbons, four oxygenated methine carbons, 12
olefinic methine carbons, and an ester carbonyl carbon.
1
H-1H, 13C-1H connectivity were elucidated by 1H-1H
COSY, HMQC, HMBC and DEPT spectra. The geom-
etry of the six olefins was determined by NOESY spectra
together with their coupling constants. The planar struc-
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
purified compounds by bioassay and SEM
Bioassay and SEM analysis also showed that the presence of
active compounds damaged both the mycelium and the
sporulation of S. scabiei. Normal mycelium and spore-
formation could be observed in the absence of any antibiotics
(Fig. 4a), while iturin A was observed to inhibit the
sporulation of S. scabiei; sporulation did not occur even
after a prolonged incubation for 2 weeks (Fig. 4b,d).
Treatment with macrolactin A led to inhibition of sporu-
lation as well as to the disruption of S. scabiei mycelium
(Fig. 4c,e).
Pot assay
In order to investigate the possibility of using Bacillus sp.
sunhua to suppress S. scabiei in soil, a pot assay
experiment was performed. The infection rate decreased
to 35% after treatment with Bacillus sp. sunhua compared
with a 75% infection rate for potatoes in the pot treated
alone with S. scabiei (positive control) (Fig. 5b). Further-
more, the lesions on potatoes treated with Bacillus sp.
sunhua were smaller and less serious than those of the
potatoes that had been treated with S. scabiei alone
(Fig. 4a).
218 J . S . H A N ET AL.

Table 2 Comparison of 1H- and 13C-NMR

Sunhua* Macrolactin A

data of a compound isolated from Bacillus sp.
Atom dc dH (J, Hz) dc dH (J, Hz) sunhua with those of macrolactin A

1 166Æ5 166Æ3
2 116Æ4 5Æ44 (m) 117Æ8 5Æ68 (d, 11Æ2)
3 143Æ7 6Æ54 (t, 11Æ5) 143Æ7 6Æ29 (dd, 11Æ2, 11Æ5)
4 129Æ0 7Æ12 (dd, 11Æ5, 14Æ8) 129Æ4 7Æ48 (dd, 11Æ5, 14Æ9)
5 141Æ0 6Æ07 (m) 142Æ4 5Æ85 (ddd, 7Æ2, 7Æ2, 14Æ9)
6 41Æ8 2Æ31 (m) 42Æ8 2Æ30 (m)
2Æ35 (m)
7 71Æ1 4Æ15 (m) 71Æ2 4Æ15 (m)
8 136Æ0 5Æ66 (dd, 5Æ8, 15Æ1) 138Æ3 5Æ58 (dd, 4Æ7, 15Æ0)
9 124Æ5 6Æ47 (dd, HÆl, 15Æ1) 124Æ9 6Æ73 (dd, 11Æ5, 15Æ0)
10 130Æ0 6Æ02 (m) 130Æ6 6Æ04 (dd, 11Æ2, 11Æ5)
11 127Æ0 5Æ44 (m) 128Æ6 5Æ42 (m)
12 35Æ0 2Æ22 (m) 36Æ7 2Æ43 (m)
2Æ38 (m) 2Æ70 (m)
13 67Æ3 3Æ75 (m) 68Æ8 4Æ03 (m)
14 42Æ7 1Æ50 (m) 43Æ9 1Æ74 (m)
1Æ83 (m)
15 68Æ0 4Æ20 (m) 69Æ2 4Æ62 (m)
16 133Æ6 5Æ46 (m) 136Æ6 5Æ70
17 129Æ9 6Æ07 (m) 131Æ2 6Æ35 (dd, 10Æ6, 15Æ3)
18 130Æ3 5Æ97 (dd, 10Æ5, 15Æ0) 129Æ6 6Æ08 (dd, 10Æ6, 14Æ8)
19 133Æ6 5Æ56 (m) 133Æ7 5Æ70
20 32Æ0 1Æ99 (m) 32Æ3 1Æ91 (m)
2Æ10 (m) 2Æ06 (m)
21 24Æ2 1Æ40 (m) 25Æ0 1Æ32 (m)
22 34Æ2 1Æ42 (m) 35Æ3 1Æ32 (m)
1Æ56 (m) 1Æ49 (m)
23 71Æ0 4Æ90 (m) 70Æ8 5Æ10 (m)
24 18Æ7 1Æ15 (d, 6Æ3) 19Æ9 1Æ09 (d, 6Æ5)

*CD3OD.

C6D6 for 1H-NMR and CD3OD for 13
C-NMR.

Inhibitory spectrum of Bacillus sp. sunhua against DISCUSSION

other phytopathogens
The cell-free culture supernatant of Bacillus sp. sunhua
was tested against other phytopathogens, as shown in
Table 3. S. scabiei was found to be the most sensitive. The
supernatant showed moderate activity against Fusarium
oxysporum, the phytopathogen causing potato dry rot
disease, and slight antagonistic activity toward Pseudomonas
syringae pv. syringae, Alternaria mali, and Penicillium
digitatum, which are responsible for angular leaf spot
disease, Alternaria leaf spot, and blue and green citrus
mould respectively. It should be pointed out that the
supernatant of Bacillus sp. sunhua showed no inhibitory
activity towards beneficial bacteria, such as Rhizobium
meliloti, a symbiotic nitrogen-fixing micro-organism, or
Pseudomonas fluorescens, which can produce a toxic insec-
ticidal compound.
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
For plant disease control, chemical agents are now widely
applied. However, the use of chemicals leads to environ-
mental pollution and ecological destruction. In order to
overcome these problems, soil micro-organisms are being
extensively studied as potential biocontrol agents (Hoitink
and Boehm 1999; Raaijmakers et al. 2002). Many antibiotics
produced by antagonistic strains have the advantage of being
easily decomposed and leave no harmful residues (Cook
1993). Until recently, common scab disease has been
controlled by the chemical treatment of potato seeds,
irrigation, and soil amendment. Eckwall et al. controlled
scab disease by using S. diastatochromogenes PonSSII, which
demonstrated two biocontrol mechanisms, antibiosis and
competition, but the structure of the antibiotics was not
characterized (Eckwall and Schottel 1997; Neeno-Eckwall
et al. 2001). Until now, there has been no report of using a
 BACILLUS SP. SUNHUA CONTROLS SCAB DISEASE 219

Table 3 Antagonistic activity of Bacillus sp. sunhua against phyto-

(a)
pathogens*

Inhibition zone
Organism diameter (mm)

Streptomyces scabiei. 22
Rhizobium meliloti –
Pseudomonas fluorescens –
Pseudomonas syringae pv. syringae 14
(d)
(b) Fusarium oxysporum 20
Alternaria mali 16
Penicillium digitatum ±

–, Absence of inhibition; ±, activity zone was not clear.

*A 100-ll minor supernatant compound eluted in 30% aqueous
MeOH were applied to discs.

Diameter of the inhibition zone (mm) around the disc.

(c) (e) Bacillus species as a suppressive strain to control common

Fig. 4 Antimicrobial activity of Bacillus sp. sunhua and scanning
electron microscopy of Streptomyces scabiei grown on GYM agar plate.
(a) Normal mycelium and spores of S. scabiei taken from the untreated
area, (b) Sporulation was inhibited by iturin A and (c) Growth of
S. scabiei mycelia was disrupted by macrolactin A. (d) Only sporulation
inhibition was observed around the paper disc when S. scabiei was
cultured in the presence of iturin A. (e) Cells of S. scabiei were dead
when treated with macrolactin A and a clear inhibition zone was
observed
scab disease; here we report the use of Bacillus sp. sunhua as
a biocontrol agent and analyse the structure of the antibiotics
produced by Bacillus sp. sunhua for the first time.
Bacillus species have been used as biocontrol agents
against pathogenic fungi, by producing lipopeptide antibi-
otics (Fravel 1988; Zuber et al. 1993; Kim and Kim 1994;
Potera 1994; Leifert et al. 1995). Iturin A is one of them
(Eshita and Roberto 1995; Moyne et al. 2001; Hiradate et al.
2002; Cho et al. 2003). In our study, we found that the
major compound iturin A produced by Bacillus sp. sunhua
can inhibit the sporulation of S. scabiei. A minor compound
produced by Bacillus sp. sunhua was macrolactin A, which
was first isolated from a marine bacterium (Gustafson et al.
1989). It is known to possess antibacterial, cytotoxic and
antiviral activity (Gustafson et al. 1989) and can be used as a
neuronal cell-protecting substance (Kim et al. 1997). How-
ever, until now, there had been no report that macrolactin A

100
90
80
70
Infection rate (%)

60
50
40
30
20
10
a b c
0
a b c
Treatment

Fig. 5 Suppressive effect of Bacillus sp. sunhua on common scab disease caused by Streptomyces scabiei. (a) Potato from uninfested soil. (b) Potato
treated with S. scabiei. (c) Potato treated with S. scabiei and Bacillus sp. sunhua

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
220 J . S . H A N ET AL.
could be used as a biocontrol agent. Here we have shown
that macrolactin A can inhibit the sporulation and mycelium
formation of S. scabiei, and we observed the disruption of
mycelia. Polyene macrolides combines with ergosterol and
damages the cell membrane which then results in loss of cell
integrity (Strohl 1997). Our results imply that the mechan-
ism of macrolactin A is identical to the action of macrolide
antibiotics. This antibiotic mechanism is different from that
of lysozyme produced by the Trichoderma and Pseudomonas
species (Wells 1988; Lim et al. 1991).
Metabolites produced by Bacillus sp. sunhua also showed
antifungal activity against Fusarium oxysporum, the causal
organism of dry rot disease. We suppose that this effect was
caused by the antifungal compound iturin A. Therefore,
Bacillus sp. sunhua is effective not only against the growth of
S. scabiei, but also against other potato phytopathogens. We
suggest that it has potential for controlling potato diseases.
In the pot assay, the rate of scab disease was reduced by 40%
after treatment with Bacillus sp. sunhua. Most importantly,
the antibiotics produced by Bacillus sp. sunhua are more stable
at alkaline pH, making it suitable for the treatment of scab
disease which is more serious in alkaline soil. In addition,
compared with other bacterial species, Bacillus species produce
various biologically active metabolites, and offer the further
advantage of forming endospores that are resistant to heat,
desiccation, organic solvents and UV irradiation. Therefore,
Bacillus sp. sunhua has great potential as a biological control
agent for the control of common scab disease.
In summary, we present the first report of the compound
macrolactin A having inhibitory action towards potato scab
disease. To our knowledge, this is the first report of a
Bacillus species biocontrol strain that may function effect-
ively to control scab disease. Additionally, our studies
suggest that Bacillus sp. sunhua is a good candidate for
controlling potato disease in the field. To test this possibil-
ity, we are now in the process of using this strain as a
biocontrol agent in a large potato planting area, in collabor-
ation with Korea’s Rural Development Administration.
ACKNOWLEDGEMENTS
This study was supported by the Technology Development
Program for Agriculture and Forestry, Ministry of Agricul-
ture and Forestry, Republic of Korea. This study was also
partially supported by the Korean National Research
Resource Center (KNRRC) Project to ECUM (Extract
Collection of Useful Microorganism) from the Korean
Ministry of Science and Technology.
REFERENCES
Agrios, G.N. (1997) Plant Pathology, 4th edn. San Diego, CA:
Academic Press.
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
Bouchek-Mechiche, K., Pasco, C., Andrivon, D. and Jouan, B. (2000)
Differences in host range, pathogenicity to potato cultivars and
response to soil temperature among Streptomyces species causing
common and netted scab in France. Plant Pathol 49, 3–10.
Cho, S.J., Lee, S.K., Cha, B.J., Kim, Y.H. and Shin, K.S. (2003)
Detection and characterization of the Gloeosporium gloeosporioides
growth inhibitory compound iturin A from Bacillus subtilis strain
KS03. FEMS Microbiol Lett 223, 47–51.
Chun, J. and Goodfellow, M. (1995) A phylogenetic analysis of the
genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol
45, 240–245.
Cook, R.J. (1993) Making greater use of introduced microorganisms for
biological control of plant pathogens. Annu Rev Phytopathol 31, 53–80.
Doyle, J.J. and Maclean, A.A. (1960) Relationships between Ca:K ratio,
pH, and prevalence of potato scab. Can J Plant Sci 40, 616–619.
Eckwall, E.C. and Schottel, J.L. (1997) Isolation and characterization
of an antibiotic produced by the scab disease-suppressive Streptom-
yces diastatochromogenes strain PonSSII. J Ind Microbiol Biotechnol
19, 220–225.
Eshita, S.M. and Roberto, N.H. (1995) Bacillomycin Lc, a new
antibiotic of the iturin group: isolations, structures, and antifungal
activities of the congeners. J Antibiot 48, 1240–1247.
Ferreira, J.H.S., Matthee, F.N. and Thomas, A.C. (1991) Biological
control of Eutypa lata on grapevine by an antagonistic strain of
Bacillus subtilis. Phytopathology 81, 283–287.
Fravel, D.R. (1988) Role of antibiosis in the biocontrol of plant
diseases. Ann Rev Phytopathol 26, 75–91.
Gustafson, K., Roman, M. and Fenical, W. (1989) The macrolactins, a
novel class of antiviral and cytotoxic macrolides from a deep-sea
marine bacterium. J Am Chem Soc 111, 7519–7524.
Healy, F.G., Wach, M., Krasnoff, S.B., Gibson, D.M. and Loria, R.
(2000) The txtAB genes of the plant pathogen Streptomyces
acidiscabies encode a peptide synthetase required for phytotoxin
thaxtomin A production and pathogenicity. Mol Microbiol 38, 794–
804.
Hiradate, S., Yoshida, S., Sugie, H., Yada, H. and Fujii, Y. (2002)
Mulberry anthracnose antagonists (iturins) produced by Bacillus
amyloliquefaciens RC-2. Phytochemistry 61, 693–698.
Hoitink, H and Boehm, M (1999) Biocontrol within the context of soil
microbial communities: a substrate-dependent phenomenon. Annu
Rev Phytopathol 37, 427–446.
Hooker, W.J. (1981) Common scab. In Compendium of Potato Disease
ed. Hooker, W.J.J. pp. 33–34. St Paul, MN: American Phytopath-
ological Society.
Iwasa, T., Higashide, E., Yamamoto, H. and Shibata, M. (1971)
Studies on validamycins, new antibiotics. II. Production and biolo-
gical properties of validamycins A and B. J Antibiot 24, 107–113.
Keinath, A.P. and Loria, R. (1989) Population dynamics of Streptom-
yces scabies and other actinomycetes as related to common scab of
potato. Phytopathology 79, 681–687.
Kim, Y.S. and Kim, S.D. (1994) Antifungal mechanism and properties
of antibiotic substances produced by Bacillus subtilis YB-70 as a
biological control agent. J Microbiol Biotechnol 4, 296–304.
Kim, H.H., Kim, W.G., Ryoo, I.J., Kim, C.J., Suk, J.E., Han, K.H.,
Hwang, S.Y. and Yoo, I.D. (1997) Neuronal cell protection activity
of macrolactin A produced by Actinomadura sp. J Microbiol
Biotechnol 7, 429–434.
 BACILLUS SP. SUNHUA CONTROLS SCAB DISEASE
Kimura, H., Sashihara, T., Matsusaki, H., Sonomoto, K. and Ishizaki,
A. (1998) Novel bacteriocin of Pediococcus sp. ISK-1 isolated from
well-aged bed of fermented rice bran. Ann NY Acad Sci 864, 345–348.
Lawrence, C.H., Clark, M.C. and King, R.R. (1990) Induction of
common scab symptoms in aseptically cultured potato tubers by the
vivotoxin, thaxtomin. Phytopathology 80, 606–608.
Leifert, C., Li, H., Chidburee, S., Hampson, S., Workman, S., Sigee,
D., Epton, H.A. and Harbour, A. (1995) Antibiotic production and
biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus
CL45. J Appl Bacteriol 78, 97–108.
Lim, H.S., Kim, Y.S. and Kim, S.D. (1991) Pseudomonas stutzeri YPL-
1 genetic transformation and antifungal mechanism against Fusarium
solani, an agent of plant root rot. Appl Environ Microbiol 57, 510–516.
Liu, Daqun., Anderson, N.A. and Kinkel, L.L. (1995) Biological
control of potato scab in the field with antagonistic Streptomyces
scabies. Phytopathology 85, 827–831.
Liu, Daqun., Anderson, N.A. and Kinkel, L.L. (1996) Selection and
characterization of strains of Streptomyces suppressive to the potato
scab pathogen. Can J Microbiol 42, 487–502.
Loria, R., Bukhalid, R.A., Creath, R.A., Leiner, R.H., Olivier, M. and
Steffens, J.C. (1995) Differential production of thaxtomins by
pathogenic Streptomyces species in vitro. Phytopathology 85, 537–541.
Loria, R., Bukhalid, R.A. and Fry, B.A. (1997) Plant pathogenicity in
the genus Streptomyces. Plant Dis 81, 836–846.
Moyne, A.-L., Shelby, R., Cleveland, T.E. and Tuzun, S. (2001)
Bacillomycin D: an iturin with antifungal activity against Aspergillus
flavus. J Appl Microbiol 90, 622–629.
Munimbazi, C. and Bullerman, L.B. (1998) Isolation and partial
characterization of antifungal metabolites of Bacillus pumilus. J Appl
Microbiol 84, 959–968.
Neeno-Eckwall, E.C., Kinkel, L.L. and Schottel, J.L. (2001) Compe-
tition and antibiosis in the biological control of potato scab. Can J
Microbiol 47, 332–340.
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 99, 213–221, doi:10.1111/j.1365-2672.2005.02614.x
221
Peypoux, F., Guinand, M., Michel, G., Delcambe, L., Das, B.C. and
Lederer, E. (1978) Structure of iturine A, a peptidolipid antibiotic
from Bacillus subtilis. Biochemistry 17, 3992–3996.
Pietro, A.D., Gut-Rella, M., Pachlatko, J.P. and Schwinn, F.J. (1992)
Role of antibiotics produced by Chaetomium globosum in biocontrol of
Pythium ultimum, a causal agent of damping-off. Phytopathology 82,
131–135.
Potera, C. (1994) From bacteria: a new weapon against fungal infection.
Science 265, 605.
Potter, H.S., Hooker, W.J., Cargo, W. and Stachwick, G.T. (1958)
Pentachloronitrobenzene and urea-formaldehyde for potato scab
control in Michigan. Plant Dis Rep 43, 633–637.
Raaijmakers, J.M., Vlami, M. and de Souza, J.T. (2002) Antibiotic
production by bacterial biocontrol agents. Antonie Van Leeuwenhoek
81, 537–547.
Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–
425.
Strohl, W.R. (1997) Biotechnology of Antibiotics, 2nd edn. New York:
Marcel Dekker, Inc.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL
W: improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap penal-
ties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Tominaga, A. and Kobayashi, Y. (1978) Kasugamycin-resistant
mutants of Bacillus sutilis. J Bacteriol 135, 1149–1150.
Wells, H.D. (1988) Trichoderma as a biocontrol agent. In Biocontrol of
Plant Diseases ed. Mukerji, Y. and Garg, K.L. pp. 71–82. Boca
Raton, FL: CRC Press, Inc.
Zuber, P., Nakano, M.M. and Marahiel, M.A. (1993) Peptide
antibiotics. In Bacillus subtilis and Other Gram-positive Bacteria ed.
Sonenshein, A.L., Hoch, J.A. and Losick, R. pp. 897–916.
Washington, DC: American Society for Microbiology.