Antifungal activity of salaceyin A against Colletotrichum orbiculare and Phytophthora capsici

332

Journal of Basic Microbiology 2007, 47, 332 – 339

www.jbm-journal.com

Research Paper

Antifungal activity of salaceyin A against
Colletotrichum orbiculare and Phytophthora capsici

C. N. Park

, D. Lee

, W. Kim

, Y. Hong

, J. S. Ahn

and B. S. Kim

Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul, Korea

Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea

The antifungal activities of novel salicylic acid derivatives, salaceyin A, 6-(9-methyldecyl)
salicylic acid, and salaceyin B, 6-(9-methylundecyl) salicylic acid were evaluated against plant
pathogenic fungi. Salaceyin A showed antifungal activity against Cladosporium cucumerinum,
Colletotrichum orbiculare and Phytophthora capsici at 64

µg ml

–1

while salaceyin B was less effective.

In vitro antifungal activities of the compounds were influenced by the experimental pH value of
the MIC test medium wherein their antifungal activities were enhanced by increasingly acidic
conditions. Salaceyin A showed potent in vivo control efficacy against Phytophthora blight in
pepper plants. The disease was effectively suppressed at 500

µg ml

–1

, which was comparable to

the commercial fungicide, metalaxyl. Salaceyin A suppressed anthracnose development on
cucumber leaves in a concentration dependent manner. The control efficacy of salaceyin A
against C. orbiculare infection was similar to chlorothalonil when applied prior to pathogen
inoculation. Since the salaceyins are derivatives of salicylic acid, a known important signal
molecule critical to plant defenses against pathogen invasion, we investigated the possibility
that exogenous application of the salaceyin A would activate a systemic acquired resistance
against P. capsici infection and C. orbiculare development on pepper and cucumber plants
respectively. The addition of 500

µg ml

–1

of salaceyin A to the plant root systems did not

significantly decrease disease development in the hosts. We are led to conclude that the disease
control efficacy of salaceyin A against the Phytophthora blight and anthracnose diseases, mainly
originates from the direct interaction of the agent with the pathogens.

Keywords: Salaceyin A/Phytophthora blight/Antifungal activity/Colletotrichum orbiculare

Received: January 29, 2007; returned for modification: February 13, 2007; accepted: February 23, 2007

DOI 10.1002/jobm.200710325

Introduction

Modern plant disease management is in constant need
of new antifungal agents that differ from the fungi-
cides currently in use in terms of a mode of action and
their chemical properties (Kim and Hwang 2003). Con-
tinuous evolution of fungicide resistant plant patho-
gens and the mounting concerns of the public regard-
ing food safety underlie this demand (Knight et al.
1997). Fungicides in development should fulfill strin-
gent requirements beginning with biodegradability

Correspondence: Prof. Beom Seok Kim, Division of Biotechnology,
College of Life Sciences & Biotechnology, Korea University, Seoul 136 –
713, Republic of Korea
E-mail: biskim@korea.ac.kr

under natural conditions to minimize unexpected re-
sidual effects to the environment. Selectivity in fungi-
cidal activity is a second important characteristic
wherein the fungicide should be non-toxic to non-
fungal organisms yet capable of preventing or treating
pathogenic infections without harm to coexisting neu-
tral or benign fungal species e.g. saprotrophic soil fungi
and mycorrhizal associates. Microbial secondary me-
tabolites have merits as potential lead compounds for
new fungicides (Kim and Hwang 2003, Vining 1990)
since microorganisms are known to synthesize versatile
chemical structures with diverse biological activities
that are beyond the scope of synthetic organic chemis-
try (Vining 1990). Microbial metabolites are degraded
rapidly when exposed to the natural environment,
leading to low residual levels (Tanaka and Ōmura 1993).

Journal of Basic Microbiology 2007, 47, 332 – 339

Antifungal activity of salaceyin A

333

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Despite the apparent virtues of microbial metabolites as
a source of new fungicidal agents, studies dedicated to
screening for fungicidal compound has been minimal
in comparison to the efforts involving plant metabolites
useful for pharmaceutical industry. Several successful
antifungal agents originating from microbial secondary
metabolites, however, have proved their usefulness as
fungicides for plant protection. These agents include
Blasticidin S (Takeuchi et al. 1957), kasugamycin
(Umezawa et al. 1965), polyoxin (Isono et al. 1965), and
validamycin (Iwasa et al. 1970), that are widely used to
control economically important plant diseases.
Endophytic microorganisms can be expected to be
important sources of antifungal agents for the control
of plant diseases. The term endophyte refers to coloni-
zation of the interior of plants by bacterial or fungal
microorganisms (Coombs and Franco 2003, Quadt-
Hallmann and Kloepper 1996). Endophytes occupy an
ecological niche similar to that of colonization by plant
pathogens, but unlike the pathogens do not cause dam-
age to their host (Hallmann et al. 1997). Several bacte-
rial endophytes have been reported to support plant
growth and to improve the health of their plant host by
competing for space and for nutrients with plant
pathogens or by producing secondary metabolites that
protect the host plant from attack by the pathogens
(Hallmann et al. 2001, Reiter et al. 2002).
We recently launched a screening program to identify
endophytes that produce biologically active secondary
metabolites and have identified two novel salicylic acid
derivatives (salaceyin A and salaceyin B) from the cul-
ture broth of the endophytic Streptomyces laceyi MS53
that was isolated from a stem of Ricinus communis L (Kim
et al. 2006). The aim of the present study was to evaluate
the antifungal activity of the salaceyins against various
plant pathogenic fungi. In addition, we evaluated the in
vivo control efficacy of the agents on a Phytophthora cap-
sici infection on pepper plants and on the development
of Colletotrichum orbiculare on cucumber plants.

Materials and methods

Antifungal compounds
The salicylic acid derivatives, salaceyin A, 6-(9-methyl-
decyl)salicylic acid (

1) and salaceyin B, 6-(9-methyl-

undecyl) salicylic acid (

2) used in this study were iso-

lated from the culture broth of Streptomyces laceyi MS53
(Fig. 1.) as described previously (Kim et al. 2006). The
purity of each compound was confirmed by HPLC and

H-NMR spectroscopic analysis (Bruker, Billerica, MA,

USA). The systemic fungicide metalaxyl [methyl N-(me-

thoxyacetyl)-N-(2,6-xylyl)-DL-alaninate] was provided by
the Sungbo Chemical Co, Korea (technical grade mate-
rial at 95% a.i.) and a stock solution of metalaxyl was
prepared in acetone. The commercial fungicide chloro-
thalonil (tetrachloroisophthalonitrile; Daconil), was
obtained from the Kyungnong Co, Korea. Salicylic acid
was purchased from Sigma-Aldrich (St. Louis, MO,
USA).

In vitro bioassay for antifungal activity
In vitro antifungal activities of the salaceyins were ex-
amined in a 24-well microtiter dish (Cell Wells, Corning
Glass Works, Corning, NY, USA) using a method modi-
fied from Nair et al. (Nair et al. 1994). Zoospore suspen-
sions of Phytophthora capsici Leonian (10

zoospores ml

–1

)

and mycelia suspensions of Rhizoctonia solani Kuhn were
used as inocula in the experiment. The inocula of other
plant pathogenic fungi such as Alternaria mali Roberts,
Botrytis cinerea Pers : Pers, Cladosporium cucumerinum Ellis
& Arthur, Colletotrichum orbiculare (Berk & Mont) van
Arx, Cylindrocarpon destructans (Zinssm.) Scholten, Didy-
mella bryoniae (Fuckel) Rehm, Fusarium oxysporum f.sp.
lycopersici Snyder & H. N. Hansen, Magnaporthe grisea
(Herb) Barr were prepared as spore suspensions at con-
centrations of 10

spores ml

–1

Candida albicans (CP

Robin) Berkhout, Saccharomyces cerevisiae Meyer & Han-
sen, Bacillus subtilis (Ehrenberg) Cohn, Ralstonia solanacea-
rum (Smith) Yabuuchi et al., and Xanthomonas campestris
pv vesicatoria (Doidge) Dye were prepared at the concen-
tration of 10

cells ml

–1

. One milliliter of potato dex-

trose broth (PDB, Difco, Circle Sparks, MD, USA) con-
taining salaceyins A or B (0 – 256

µg ml

–1

) was dispensed

into each well. The inoculum suspension (10

µl) was

added to the PDB solution and the inoculated well
plates then incubated at 28(±1) °C on a rotary shaker at
100 rpm. Nutrient broth (PDB, Difco) was used for test-
ing the yeasts and bacteria. The inhibitory effects of the
salaceyins on the growth of the test microorganisms
were evaluated after incubation for 2 – 4 days. The low-
est concentration that inhibited the growth of the mi-
croorganism was considered as the minimum inhibi-
tory concentration (MIC) of the salaceyins.

In vivo evaluation of antifungal activity
The control efficacy of salaceyin A against P. capsici
infection on pepper plants was evaluated in the con-
trolled environment of a growth room. Pepper seeds
(Capsicum annuum L cv Nokkwang) were sown in a plas-
tic tray (50

× 40 × 15 cm

) containing a steam-sterilized

soil mix (peat moss : perlite : vermiculite, 5 : 3 : 2, by vol-
ume), sand, and loam soil (1 : 1 : 1 by volume). At the
four-leaf stage, each pepper seedling was transplanted

334

C. N. Park et al.

Journal of Basic Microbiology 2007, 47, 332 – 339

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to a plastic pot (4

× 4 × 7 cm

) containing the identical

soil mix. The pepper plants were grown to the first-
branch stage in the growth room (28(±2) °C with 5,000
lux for 16

h per day). The commercial fungicide

metalaxyl was used for the comparison of in vivo control
efficacy of Phytophthora blight. Salaceyin A and metal-
axyl were dissolved in methanol and acetone, respec-
tively, and diluted with Tween 20 solution (0.5 g l

–1

) to

give concentrations of 10, 50, 100 and 500 mg l

–1

. The

salaceyin A solution was amended with lactic acid
(0.1%, v/v). To evaluate possible curative or protective
effects of the compounds, the pepper plants were
treated one day before and one day after inoculation
with P. capsici. The pepper plants treated or not yet
treated with salaceyin A and metalaxyl were purposely
wounded by making longitudinal slits (1 cm) on the
stems one cm from the soil surface. Sterile cotton was
soaked in zoospore suspensions (1

× 10

zoospores ml

–1

)

and placed on the wound sites. The inoculated sites
were covered with plastic tape to maintain a moist
condition. The severity of disease was rated on a daily
basis after inoculation using a scale of 0 to 5 as follows:
0, no visible disease symptoms; 1, leaves slightly wilted
with brownish lesions beginning to appear on the
stems; 2, 30 – 50% of the entire plant diseased; 3, 50 –
70% of the entire plant diseased; 4, 70 – 90% of the
entire plant diseased; 5, plant death. The data are pre-
sented as the statistical means of 10 plants per treat-
ment.
The ability of salaceyin A to suppress the infection of
C. orbiculare on cucumber plants was evaluated in the
growth room. Cucumber (Cucumis sativus L cv Baekrok-
dadaki) seeds were sown in a plastic pot containing the
previously described soil mix for the pepper plant
study. Cucumber plants were raised in the growth
room at 28(±2) °C with 5,000 lux for 16 h per day.
Salaceyin A and the commercial fungicide chlorothalo-
nil were diluted with Tween 20 solution (0.5 g l

–1

) to

give concentrations of 10, 50, 100, and 500 mg l

–1

. The

salaceyin A solution was amended with lactic acid
(0.1%, v/v). Each of the chemical solutions was sprayed
onto the primary leaves of the cucumber plants when
at their three-leaf stage one day before and one day
after inoculation with C. orbiculare. Conidial suspensions
(10

conidia ml

–1

) of C. orbiculare in Tween 20 solution

(0.5 g l

–1

) were sprayed onto the leaves of the cucumber

plants. The inoculated plants were placed in a dew
chamber at 28(±1) °C for 24 h and then transferred to
the growth room. Lesions on the primary leaves of the
cucumber plants were counted six days after inocula-
tion. Data are presented as the statistical means of le-
sion number per cm

leaf area of 10 cucumber plants.

Evaluation of the systemic protection ability
of salaceyin A
Pepper and cucumber plants were grown as described
in the previous section. Pepper plants at the four-leaf
stage and cucumber plants at the three-leaf stage were
removed from their pots and the root part placed into
an aerated hydroponics vessel constructed from a Ma-
genta tissue culture box (10

× 6 × 6 cm) (Sigma Chemi-

cal Co., St. Louis, MO, USA). The hydroponics vessels
were supplied with 250 ml of 0.5

× Murashige and Skoog

(MS) basal salts medium (Murashige and Skoog 1962)
amended with 250 mg l

–1

of salicylic acid or 500 mg l

–1

of salaceyin A. The pepper and cucumber plants were
maintained in this environment for 24 h. Before inocu-
lation with a pathogen, the plants were transplanted
into soil pots containing the soil mixture described
previously. The pepper plants to be treated were
wounded by making longitudinal slits (1 cm) on the
stems 4.5 cm from the soil surface. Sterile cotton was
soaked in zoospore suspensions (1

× 10

zoospores ml

–1

)

and placed on the wound sites. The inoculated sites
were then covered with plastic tape to maintain a moist
condition. The severity of disease was rated as described
previously.
Conidial suspensions (10

conidia ml

–1

) of C. orbiculare

in Tween 20 solution (0.5 g l

–1

) were sprayed onto the

primary leaves of the cucumber plants treated with
salicylic acid or salaceyin A by way of root feeding. The
inoculated plants were placed in a dew chamber at
28(±1) °C for 24 h and then transferred to the growth
room. Lesions on the primary leaves of the cucumber
plants were counted six days after inoculation. Data are
presented as the statistical means of lesion number per
cm

leaf area of 10 cucumber plants.

Determination of endogenous plant salicylic acid
levels
Leaf samples were harvested at 24 h after root feeding
of the chemicals to determine the endogenous level of
total salicylic acid (free salicylic acid and

β-gluco-

sylsalicylic acid) in the pepper and cucumber plants
that had been treated with 250 mg l

–1

salicylic acid or

500 mg l

–1

salaceyin A. Leaves were obtained from all

portions of the respective plant, dissected into 5 mm
wide pieces and combined to ensure heterogeneity of
plant tissue age. Leaf samples (500 mg) were flash-
frozen in liquid nitrogen and stored at – 80

°C until

analyzed for their salicylic acid content. The salicylic
acid content was quantified by spectrofluorescence
high-performance liquid chromatography using a
method described by Spletzer and Enyedi (Spletzer and
Enyedi 1999).

Journal of Basic Microbiology 2007, 47, 332 – 339

Antifungal activity of salaceyin A

335

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Results and discussion

Recently we identified the novel salicylic acid deriva-
tives, salaceyin A, 6-(9-methyldecyl)salicylic acid (

1) and

salaceyin B, 6-(9-methylundecyl)salicylic acid (

2) from

the culture broth of Streptomyces laceyi MS53 (Fig. 1).
These salicylic derivatives showed potent in vitro anti-
fungal activity against several plant pathogenic fungi
(Table 1), which led us to evaluate their disease control
efficacy against plant diseases. The growth of C. cucu-
merinum, C. orbiculare P. capsici was completely inhibited
in vitro at 64

µg ml

–1

whereas the minimum inhibitory

concentrations (MICs) on A. mali, M. grisea, F. oxysporum
and R. solani were 256

µg ml

–1

. The growth of other

plant pathogenic fungi tested in this study, however,
were not inhibited, even at the higher concentration of
256

µg ml

–1

. The antifungal activity of salaceyin B was

consistently less effective than that of salaceyin A.
Salicylic acid, the derivative of benzoic acid hydroxy-
lated at the ortho-position, is known to play a central
role in plant disease resistance (Mauch-Mani and
Metraux 1998). Numerous citations in the literature
indicate that increases of salicylic acid in plants are
correlated with a resistance against pathogen infections
(Malamy et al. 1990, Mauch-Mani and Metraux 1998,
Mills and Wood 1984). Despite the ability to induce
resistance against plant pathogens, it has been sug-
gested that salicylic acid has no direct antifungal activ-
ity against several specific plant pathogens (Mills and
Wood 1984). Other data have shown, however, that
salicylic acid does alter various aspects of fungal devel-
opment such as spore viability in S. cerevisiae (Romano

COOH

1 R = CH

2 R = CH

Figure 1. Structure of salaceyin A (1) and salaceyin B (2).

and Suzzi 1985), reduction of conidial germination in
Sphaerotheca fuliginea (Conti et al. 1996), inhibition of
sclerotial differentiation and growth in Sclerotium rolfsii
and Sclerotina minor (Georgiou et al. 2000), and the inhibi-
tion of Eutypa lata mycelia growth (Amborabe et al. 2002).
In the referenced experiment with E. lata, the causal
agent of eutypa dieback, salicylic acid and other chemi-
cally synthesized derivatives were tested for their anti-
fungal properties. Salicylic acid delayed and hindered
mycelia growth when applied at 1 mM concentration.
Few derivative compounds were found to match the
antifungal activity of salicylic acid, however, since even
a minor modification to the molecule e.g. substitution
on the benzene ring with hydroxyl, sulfurhydryl, chlo-
ride, aldehyde, or nitrate, dramatically reduced the
antifungal effect. Some chlorinated derivatives such as
4-chlorosalicylic acid, 3-chlorobenzoic acid and 3,5-
dichlorobenzoic acid did show antifungal activities simi-
lar to salicylic acid. The novel salicylic acid derivatives
that we have identified, salaceyin A (

1) and B (2), are

natural salicylic acid derivatives which have a long
branched acyl chain at the C2 position. Salaceyin A was
shown to have superior antifungal activity compared
to that of salicylic acid. In our experiment, salicylic
acid inhibited P. capsici mycelia growth at 128

µg ml

–1

Table 1. Minimum inhibitory concentrations (MIC) of salaceyins A and B against various microorganisms

MIC (

µg ml

–1

)

Microorganism

Salaceyin A

Salaceyin B

Alternaria mali

256 >256

Botrytis cinerea

>256

Cladosporium cucumerinum

256

Colletotrichum orbiculare

256

Cylindrocarpon destructans

>256 >256

Didymella bryoniae >256

>256

Fusarium oxysporum f.sp. lycopersici

256 >256

Magnaporthe grisea

256

Phytophthora capsici

128

Rhizoctonia solani

256 >256

Candida albicans >256

>256

Saccharomyces cerevisiae

256 >256

Bacillus subtilis >256

>256

Ralstonia solanacearum >256

>256

Xanthomonas campestris pv. vesicatoria >256

>256

Growth of the test microorganism was not inhibited at the concentration of 256

µg ml

–1

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C. N. Park et al.

Journal of Basic Microbiology 2007, 47, 332 – 339

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Table 2. Effects of the test medium pH on antifungal activities of salaceyins A and B

MIC (

µg ml

–1

)

Compound pH

Phytophthora capsici

Colletotrichum orbiculare

Salaceyin A

128

6 128

256

Salaceyin B

8 128

256

whereas the MIC of salaceyin A was an even lower
64

µg ml

–1

(Table 2). When we observed the mycelical

shape under light microscopy after the treatments of
salaceyin A and salicylic acid at the MIC, any morpho-
logical difference between the treatments was not ob-
served.
Salaceyins A and B had enhanced antifungal activities
in the MIC test when a medium with lower pH was used
(Table 2). The complete inhibition of mycelial growth of
P. capsici was obtained with 16

µg ml

–1

of salaceyin A

when the pH of the test medium was close to 4.0,
while 32

µg ml

–1

was required at pH 6.0 and

µg ml

–1

at pH 8.0. Salaceyin B showed a similar

enhanced antifungal activity against C. orbicular and
P. capsici in acidic media (Table 2). The growth of the
fungi was not influenced by the experimental pH values
used in this study. The enhanced antifungal activity of
salaceyins in acidic media is possibly due to an increased
uptake rate of the salicylic acid derivatives into the fun-
gal cell. Salicylic acid has a pKa of 2.98 which would
indicate that the amount in the charged state increases
toward a neutral pH while the cell membranes provide a
reasonable barrier to the diffusion of charged molecules
(Amborabe et al. 2002). Once salicylic acid derivatives

enter the fungal cell, however, they seem to exert their
harmful effects on fungal cell functions.
The in vivo control efficacy of salaceyin A on Phyto-
phthora blight in pepper plants was evaluated under a
growth room condition (Fig. 2). As the concentration of
the test compound increased, the blight disease was
markedly suppressed. The disease was effectively con-
trolled at 500 mg l

–1

of salaceyin A. The treatments one

day before or just prior to inoculation were more effec-
tive than those applied one day after inoculation in
terms of controlling for the disease and suggests that
salaceyin A has more of a preventive than curative
property. When the test treatment was applied just
before inoculation, the control efficacy of salaceyin A
was similar to that of metalaxyl.
The

development

C. orbiculare on the leaves of the

cucumber plants treated with salaceyin A and the
commercial fungicide chlorothalonil were evaluated six
days after inoculation with C. orbiculare (Fig. 3). The
infection C. orbiculare generates severe chlorosis and
cell collapse, thereby facilitating penetration and colo-
zization of plant tissues. The anthracnose development
on cucumber leaves was observed to be gradually inhib-
ited as the concentration of salaceyin A increased.

100

1000

100

1000

100

500

100

500

100

500

Concentration of antifungal compounds (mg L

-1

)

iseas

erit

Salaceyin A

Metalaxyl

Figure 2. Effects of salaceyin A and the commercial fungicide metalaxyl on Phytophthora capsici infection in pepper plants. (a) Treatment
with salaceyin A and metalaxyl one day before inoculation, (b) Treatment with salaceyin A and metalaxyl just before inoculation,
(c) Treatment of salaceyin A and metalaxyl one day after inoculation. Vertical bars represent the standard deviation.

Journal of Basic Microbiology 2007, 47, 332 – 339

Antifungal activity of salaceyin A

337

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100

1000

ion

100

1000

100

500

100

500

100

500

Concentration of antifungal compounds (mg L

-1

)

Salaceyin A

Chlorothalonil

Figure 3. Effects of salaceyin A and the commercial fungicide chlorothalonil on Colletotrichum orbiculare infection in cucumber plants.
(a) Treatment of salaceyin A and chlorothalonil one day before inoculation, (b) Treatment of salaceyin A and chlorothalonil just before
inoculation, (c) Treatment of salaceyin A and chlorothalonil one day after inoculation. Vertical bars represent the standard deviation.

Starting with a 100 mg l

–1

concentration of salaceyin A,

the number of typical symptomatic lesions decreased
on the treated cucumber leaves. A few lesions were
observed on the leaves treated with 500 mg l

–1

. The

control efficacy of salaceyin A was similar between the
treatments one day and just prior to the inoculation of
C. orbiculare. Treatment one day after the inoculation,
however, could not effectively suppress the formation
of symptomatic lesions. In general, salaceyin A was
somewhat less effective than chlorothalonil in inhibit-
ing anthracnose development.
We performed additional preventive activity test of
salaceyin A to examine the longevity of the disease
control efficacy on the host plants. When the spore
suspension of C. orbiculare and zoospore suspension of
P. capsici were inoculated onto the host plants 10 days
after the salaceyin treatment, the disease control effi-
cacies of salaceyin A were greatly reduced as compared
to those on the host plants infected one day after the
salaceyin A treatment. The disease severity of Phy-
tophthora blight was 3.8 when 500 mg l

–1

of salaceyin A

treated. And the lesion number generated by C. orbicu-
lare infection was 3.1 lesions per cm

of leaf area.

Therefore, we suspected that the salaceyin A may be
continuously degraded after the application onto the
hosts. To identify the reason of the reduced disease
control activity of salaceyin. After the prolonged expo-
sure on the host plants, detailed studies on the residual
effect of salaceyin A will be necessary.
Since the salaceyins are derivatives of salicylic acid,
an important signal molecule that plays a critical role
in plant defenses against pathogen invasion (Malamy
et al. 1990, Mauch-Mani and Metraux 1998, Spletzer and
Enyedi 1999), we investigated the exogenous applica-

tion of the salaceyins as potential activators of a sys-
temically acquired resistance against P. capsici infection
and C. orbiculare development on pepper and cucumber
plants respectively. We treated the plants with salicylic
acid or salaceyin A by way of root feeding as described
previously (Spletzer and Enyedi 1999). Thus, after soak-
ing the roots of pepper and cucumber plants for 24 h in
MS basal salt medium amended with 250 mg l

–1

of sali-

cylic acid or 500 mg l

–1

of salaceyin A, the pepper and

cucumber plants were transplanted to soil pots. The
pepper and cucumber plants were inoculated with
P. capsici and C. orbiculare, respectively. The pepper
plants treated with salicylic acid showed reduced dis-
ease severity as compared to non-treated control plants
(Fig. 4a). In cucumber plants treated with salicylic acid,
systemic protection against C. orbiculare infection could
be observed (Fig. 4b). In both plant types treated with
salaceyin A, however, no significant disease control
effects were observed (Fig. 4a and 4b). The endogenous
foliar levels of total salicylic acid (free salicylic acid
+

β-glucosylsalicylic acid) were significantly increased

in both the pepper and cucumber plants treated by
exogenous root feeding with salicylic acid (250 mg l

–1

Twenty four hours after treatment with salicylic acid,
the endogenous level of total salicylic acid in the pep-
per plants increased significantly to 43.3

± 6.7 µg g

–1

fresh weight (FW) from a basal level of 1.8

± 0.2 µg g

–1

FW. In the cucumber plants treated with salicylic acid,
the endogenous level of total salicylic acid was also
increased to 76.1

± 7.9 µg g

–1

fresh weight (FW) from a

basal level of 1.3

± 5 µg g

–1

FW. In the pepper and cu-

cumber plants treated with salaceyin A, however, no
significant increase of total salicylic acid was observed,
which indicates that salaceyin A is neither converted to

338

C. N. Park et al.

Journal of Basic Microbiology 2007, 47, 332 – 339

www.jbm-journal.com

control

salicylic acid

salaceyin A

treatment

rity

control

salicylic acid

salaceyin A

treatment

ion

num

leaf

Figure 4. Systemic protection of pepper plants against
Phytophthora capsici infection (a) and cucumber plants against
Colletotrichum orbiculare infection (b) by way of root feeding with
salicylic acid and salaceyin A. Bars represent the means

(± standard deviation) of 10 replicates. *indicates a significant
difference between control and treated plants (P < 0.05).

salicylic acid nor is salicylic acid induced to accumu-
late. These results revealed that salaceyin A does not
systemically protect the pepper and cucumber plants
from their infection with P. capsici and C. orbiculare nor
does salaceyin A have the ability to induce resistance at
the concentration used in this study. Therefore, we are
led to conclude that the disease control efficacy of
salaceyin A against Phytophthora blight and anthracnose
diseases mainly originates from the direct interaction
of the agent with the pathogens.
This study suggests the possibility that the salaceyins
may be useful as fungicides for the control of Phy-
tophthora blight and anthracnose diseases in host plants
or as lead compounds for further fungicide develop-
ment. Detailed field studies and persistence tests, how-
ever, must be completed in exploring the compounds as
potential fungal disease control agents.

Acknowledgements

This work was supported by the BioGreen21 Program,
Rural Development Administration, Korea. J.S.A. and

H.Y. are supported in part by the 21C Frontier Micro-
bial Genomics and Application Center Program.

References

Amborabe, B., Fleurat-Lessard, P., Chollet, J. and Roblin G.,

2002. Antifungal activity of salicylic acid and other benzoic
acid derivatives towards Eutypa lata: structure-activity rela-
tionship. Plant Physiol. Biochem., 40, 1051 – 1060.

Conti, G.G., Pianezzola, A., Violini, G., Maffi, D. and Arnoldi,

A., 1996. Possible involvement of salicylic acid in systemic
acquired resistance of Cucumis sativus against Sphaerotheca
fuliginea Eur. J. Plant Pathol., 102, 537 – 544.

Coombs,

J.T. and Franco,

C.M.M., 2003. Visualisation of an

endophytic Streptomyces sp. in wheat seed using green fluo-
rescent protein. Appl. Environ. Microbiol., 69, 4260 – 4262.

Georgiou,

C.D., Tairis,

N. and Sotiropoulou,

A., 2000. Hydroxyl

radical scavengers inhibit lateral-type sclerotial differentia-
tion and growth in phytopathogenic fungi. Mycologia, 92,
825 – 834.

Hallmann,

J., Quadt-Hallmann,

A., Mahaffee,

W.F. and Kloep-

per,

J.W., 1997. Endophytic Bacteria in Agricultural Crops.

Can. J. Microbiol., 43, 895 – 914.

Hallmann,

J., Quadt-Hallmann,

A., Miller,

W.G., Sikora,

R.A.

and Lindow,

S.E., 2001. Endophytic colonization of plants

by the biocontrol agent Rhizobium etli G12 in relation to Me-
loidogyne incognita infection. Phytopathology, 91, 415 – 422.

Isono,

K., Nagatsu,

J., Kobinata,

K., Sasaki,

K. and Suzuki,

S.,

1965. Studies on polyoxins, antifungal antibiotics. Part I.
Isolation and characterization of polyoxins A and B. Agric.
Biol. Chem., 29, 848 – 854.

Iwasa,

T., Higashide,

E., Yamamoto,

H. and Shibata,

M., 1970.

Studies on validamycins, new antibiotics. ll. Production and
biological properties of validamycins A and B. J. Antibiot.,
23, 595 – 602.

Kim,

B.S. and Hwang,

B.K.,

2003. Biofungicides. In: Handbook

of Fungal Biotechnology (D.K. Arora, P.D. Bridge, D. Bhat-
nager, Ed.), pp. Dekker, New York.

Kim,

N., Shin,

J.C., Kim,

W., Hwang,

B.Y., Kim,

B.S., Hong,

and Lee,

D., 2006. Cytotoxic 6-alkylsalicylic acids from the

endophytic Streptomyces laceyi. J. Antibiot., 59, 797 – 800.

Knight,

S.C., Anthony,

V.M., Brady,

A.M., Greenland,

A.J.,

Heaney,

S.P., Murray,

D.C., Powell,

K.A., Schulz,

M.A., Sinks,

C.A., Worthington,

P.A. and Youle,

D., 1997. Rationale and

perspectives on the development of fungicides. Annu. Rev.
Phytopathol., 35, 349 – 372.

Malamy,

J., Carr,

J.P., Klessig,

D.F. and Raskin,

I., 1990. Salicylic

acid: a likely endogenous signal in the resistance response of
tobacco to viral infection Science, 250, 1002 – 1004

Mauch-Mani,

B. and Metraux,

J., 1998. Salicylic acid and

systemic acquired resistance to pathogen attack. Ann. Bot.,
82, 535 – 540.

Mills,

P.R. and Wood,

R.K.S., 1984. The effects of polyacrylic

acid, acetylsalicylic acid and salicylic acid on resistance of
cucumber to Colletotrichum lagenarium. Phytopathol. Z., 111,
201 – 216.

Murashige,

T. and Skoog,

F., 1962. A revised medium for rapid

growth and bioassays with tobacco tissue cultures. Physiol.
Plant., 15, 473 – 497.

Journal of Basic Microbiology 2007, 47, 332 – 339

Antifungal activity of salaceyin A

339

www.jbm-journal.com

Nair,

M.G., Chandra,

A. and Thorogood,

D.L., 1994. Gopalami-

cin, an antifungal macrodiolide produced by soil actinomy-
cetes. J. Agric. Food Chem., 42, 2308 – 2310.

Quadt-Hallmann,

A. and Kloepper,

J.W., 1996. Immunological

detection and localization of a cotton endophyte, Enterobac-
ter asburiae, strain JM22 in different plant species. Can. J.
Microbiol., 42, 1144 – 1154.

Reiter,

B., Pfeifer,

U.S.H. and Sessitsch,

A., 2002. Response of

endophytic bacterial communities in potato plants to infec-
tion with Erwinia carotovora subsp. atroseptica. Appl. Environ.
Microbiol., 68, 2261 – 2268.

Romano,

P. and Suzzi,

G.., 1985. Sensitivity of Saccharomyces

cerevisiae vegetative cells and spores to antimicrobial com-
pounds. J. Appl. Bacteriol., 59, 299 – 302.

Spletzer,

M.E. and Enyedi,

A.J., 1999. Salicylic acid induces

resistance to Alternaria solani in hydrophonically grown to-
mato. Phytopathology, 89, 722–727.

Takeuchi,

S., Hirayama,

K., Ueda,

K., Sakai,

H. and Yonehara,

H., 1957. Blasticidin S, a new antibiotic. J. Antibiot. Ser. A,
11, 1 – 5.

Tanaka,

Y.T. and Ōmura,

S., 1993. Agroactive compounds of

microbial origin. Annu. Rev. Microbiol., 47, 57 – 87.

Umezawa,

H., Okami,

Y., Hashimoto,

T., Suhara,

Y., Hamada,

M. and Takeuchi,

T., 1965. A new antibiotic, Kasugamycin.

J. Antibiot. Ser. A, 18, 101 – 103.

Vining,

L.C., 1990. Function of secondary metabolites. Annu.

Rev. Microbiol., 44, 395 – 427.