Previous Article | Next Article 
Applied and Environmental Microbiology, August 2001, p. 3739-3745, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3739-3745.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Isolation and In Vivo and In Vitro Antifungal
Activity of Phenylacetic Acid and Sodium Phenylacetate from
Streptomyces humidus
Byung Kook
Hwang,1,*
Song Won
Lim,1
Beom Seok
Kim,1
Jung Yeop
Lee,1 and
Surk Sik
Moon2
Laboratory of Molecular Plant Pathology,
College of Life and Environmental Sciences, Korea University, Seoul
136-701,1 and Department of Chemistry,
Kongju National University, Kongju 314-701,2
Korea
Received 7 February 2001/Accepted 30 May 2001
 |
ABSTRACT |
The antifungal substances SH-1 and SH-2 were isolated from
Streptomyces humidus strain S5-55 cultures by various
purification procedures and identified as phenylacetic acid and sodium
phenylacetate, respectively, based on the nuclear magnetic resonance,
electron ionization mass spectral, and inductively coupled plasma mass spectral data. SH-1 and SH-2 completely inhibited the growth of Pythium ultimum, Phytophthora capsici, Rhizoctonia solani,
Saccharomyces cerevisiae, and Pseudomonas syringae
pv. syringae at concentrations from 10 to 50 µg/ml. The two compounds
were as effective as the commercial fungicide metalaxyl in inhibiting
spore germination and hyphal growth of P. capsici. However,
the in vivo control efficacies of the two antifungal compounds against
P. capsici infection on pepper plants were similar to those
of H3PO3 and fosetyl-AI but less than that of metalaxyl.
| |
TEXT |
Streptomyces spp. are
capable of producing microbial antibiotics with a wide variety of
chemical structures. In particular, approximately 60% of antibiotics
developed for agricultural use were isolated from
Streptomyces spp. (32). It is interesting that
Streptomyces strains continue to provide a larger number and
wider variety of new antibiotics than any other actinomycete genus,
suggesting that substantial numbers of Streptomyces species or strains with novel antibiotic productivity exist in nature (27). In searches for bioactive antibiotics,
Streptomyces strains have been isolated from various types
of soils, including rice paddy, lake mud and water, deciduous forest,
tropical forest, wasteland, and cave soils (9, 16, 19, 30, 31,
34).
So far, various antifungal antibiotics active against the oomycete
plant pathogen Phytophthora have been isolated and
characterized from actinomycetes (2, 12, 13, 15, 20-23).
In our previous search program for microorganisms producing antifungal
antibiotics useful for the control of plant diseases,
Streptomyces humidus strain S5-55 was isolated from soils in
Korea, which showed substantial antagonistic activity against plant
pathogens (25). The antifungal substances active against
Phytophthora capsici and Magnaporthe grisea were
partially purified from the culture filtrates of S. humidus
strain S5-55. In the present study, the antifungal substances SH-1 and
SH-2, active against some plant-pathogenic fungi, were purified from
the culture broth of S. humidus strain S5-55 by various
purification procedures. By analyzing various spectral and other
physicochemical data, their chemical structures were elucidated and the
two compounds were identified as phenylacetic acid (SH-1) and sodium
phenylacetic acid (SH-2). In addition to an in vitro bioassay for
antifungal activity, we also evaluated the control efficacy of SH-1 and
SH-2 against phytophthora blight of pepper plants compared to those of
commercial fungicides.
Isolation of antifungal substances from S. humidus
cultures.
S. humidus strain S5-55 antagonistic to
various plant-pathogenic fungi was isolated from soil from Kwangwon
Province in Korea (25). The culture broth (100 liters) of
strain S5-55, which was incubated in soluble starch broth (5 g of
soluble starch, 10 g of glycerol, 4 g of yeast extract,
0.3 g of K2HPO4, 0.2 g of
KH2PO4, 0.5 g of MgSO4
· 7H2O [all in 1 liter of H2O]) at 28°C on a rotary shaker at 150 rpm for 14 days, was centrifuged at 1,250 × g for 30 min and filtered through Whatman no.
2 filter paper. The culture filtrate was extracted with
n-butanol (100 liters). The butanol phase was concentrated
in vacuo by using a rotary evaporator (Büchi, Switzerland). The
crude extracts were purified by C18 reversed-phase flash
column chromatography. The open glass column (150 by 200 mm) was packed
with C18 resin (Lichroprep RP-18, 40-63 µm; Merck,
Darmstadt, Germany). The column loaded with crude extracts was eluted
with stepwise gradients of methanol and water (0:100, 20:80, 40:60,
60:40, 80:20, and 100:0 [vol/vol]). Each fraction (2.5 liters) of the
eluate was concentrated in vacuo. The antifungal activity of each
fraction against P. capsici, M. grisea, and
Rhizoctonia solani was measured by a paper disk method
(21). The 40% methanol fraction (7.5 ml), which showed a
high antifungal activity, was further purified by preparative
thin-layer chromatography (TLC) (silica gel 60 F254 [0.2
mm thick]; Merck). TLC plates loaded with crude extracts were
developed with a chloroform-methanol (8:2 [vol/vol]) solvent system.
After the plate was air dried, a silica gel band which showed
antifungal activity against P. capsici and M. grisea at the position of Rf 0.7 was
collected by scraping off the band and then extracting it with
methanol. The inhibition zones produced on TLC plates were visualized
by the bioautographic technique (11).
The antifungal extract was concentrated to dryness and dissolved in 4 ml of methanol. The crude substances were purified on a Sephadex LH-20
column (26 by 950 mm column packed with Sephadex LH-20 resin;
Pharmacia, Uppsala, Sweden). Each fraction (2 ml) was collected using a
fraction collector (RediFrac; Pharmacia). The antifungal activity of
the fractions against P. capsici and M. grisea
was examined by the paper disk method. Fractions 71 to 79 (SH-1) and 89 to 97 (SH-2) showed antifungal activity against P. capsici.
The antifungal substances SH-1 and SH-2 were further purified by a
preparative high-performance liquid chromatographic system (Gilson,
Middleton, Wis.) with a C18 reversed-phase column (SymmetryPrep C18, 7 µm, 7.8 by 300 mm, Waters). The
antifungal substances SH-1 and SH-2 were eluted using a linear gradient
solvent system from 10% acetonitrile in water to 100% acetonitrile at a flow rate of 2 ml/min under the UV absorbance of 210 nm. The pure
antifungal substance SH-1 was obtained from a single peak with the
retention time of 22.06 min at 210 nm. The pure antifungal substance
SH-2 was also obtained from a peak at the retention time of 5.50 min at
210 nm. Finally, 150 and 100 mg of the antifungal substances SH-1 and
SH-2, respectively, were produced from 100 liters of the culture extracts.
Structure elucidation of SH-1 (phenylacetic acid) and SH-2 (sodium
phenylacetate) within S. humidus cultures.
The UV
absorption spectra of SH-1 and SH-2 were measured with a Beckman DU 650 spectrometer (Beckman Instruments Inc., Fullerton, Calif.). Nuclear
magnetic resonance (NMR) spectra of the purified antifungal substances
SH-1 and SH-2 were recorded on a Bruker AMX 500 NMR spectrometer
(Billerica, Mass.). 1H NMR and 13C NMR spectra
were measured in CD3OD. Low-resolution electron ionization
(EI) mass spectra were recorded with a VG70-VSEQ mass spectrometer (VG
ANALYTICAL, Manchester, United Kingdom) to elucidate the structures of
antifungal substances SH-1 and SH-2. Inductively coupled plasma (ICP)
mass spectra were recorded with an Elan 6100 mass spectrometer
(Perkin-Elmer, Norwalk, Conn.) to elucidate the structure of antifungal
substance SH-2.
The structure of antifungal substance SH-1 was elucidated by EI mass
spectral,
1H,
13C, and two-dimensional NMR
(COSY, HMQC, and HMBC) spectral analyses.
Based on the EI mass spectral
data, the molecular formula of SH-1
was deduced to be
C
8H
8O
2. The antifungal substance
SH-1 gave molecular
ion at
m/z 136 (M+): EI MS
m/z 65 (10%), 91 (97%), 92 (19%), and
136 (26%) (Fig.
1A). NMR data indicated a hydrogen count
of eight,
including one exchangeable proton, and a carbon count of
eight
in CD
3OD.
1H NMR (CD
3OD):
3.58 (2H, s) and 7.32-7.20 (5H, m) ppm;
13C NMR
175.56 (C-1), 136.07 (C-3), 130.34 (C-4, C-8), 129.45
(C-5, C-7), 127.89 (C-6), and 41.94 (C-2) ppm. The COSY and HMQC
spectral data revealed
that SH-1 has three partial structures
(Fig.
2A). With the HMBC spectral data, the
substructure aromatic
ring could be connected to methylene protons (
3.58). Methylene
protons were connected to the carbonyl carbon (
175.56) and C-3
(
139.41) in aromatic ring. With all the spectral
data, the structure
of antifungal substance SH-1 was determined to be
phenylacetic
acid (Fig.
2B). The structure of antifungal substance SH-2
was
elucidated by EI mass spectral, NMR, and ICP mass spectral
analyses.
Based on the EI mass spectral data, the antifungal substance
SH-2
gave molecular ion at
m/z 136 (M+): 136 (70%), 92 (60%), 91 (99%),
65 (35%), and 63 (15%) (Fig.
1B). NMR data
indicated a hydrogen
count of seven and a carbon count of eight in
CD
3OD.
1H NMR:
3.46 (2H, s) and 7.14 (1H,
tt,
J = 7.3, 1.9 Hz), 7.24
(2H, brt,
J = 7.4 Hz), 7.31 (2H, brd,
J = 7.5 Hz) ppm;
13C NMR:
180.55 (C-1), 139.41 (C-3), 130.28 (C-4, C-8),
129.15
(C-5, C-7), 126.92 (C-6), and 46.43 (C-2) ppm. Analyses of
two-dimensional
NMR spectrum indicated that the organic portion of the
structure
of SH-2 was identical to that of SH-1. However, the ICP mass
spectral
data confirmed that Na ion exists in the structure of SH-2
(Fig.
1C). Based on all the spectral data, the antifungal substance
SH-2 was determined to be sodium phenylacetate (Fig.
2B). When
the SH-2
powder (1 mg) was hydrolyzed with a small amount of 0.01
N HCl in
methanol, the active peak appeared at the same retention
time of SH-1
as the original SH-2 peak by high-performance liquid
chromatography.
The melting point of SH-1 was dramatically higher
than that of SH-2
(data not shown). The only difference in the
NMR spectral data was that
there was no proton at carbonyl residue
in SH-2. ICP mass spectral data
were further examined to confirm
whether SH-2 is a salt form of SH-1.
The intensity level (in counts
per second) of Na was higher than that
of standard Na level in
ICP mass spectral data.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 1.
EI mass spectrum (A) of the antifungal substances SH-1
(phenylacetic acid), and EI (B) and ICP (C) mass spectra of SH-2
(sodium phenylacetate).
|
|
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 2.
(A) Correlations of partial structures of the antifungal
substance SH-1 from HMBC spectra and (B) structures of the antifungal
substances SH-1 (R = H) and SH-2 (R = Na) isolated from
S. humidus strain S5-55.
|
|
In vitro antimicrobial activity of SH-1 (phenylacetate acid) and
SH-2 (sodium phenylacetate).
Microorganisms such as
Alternaria mali, Colletotrichum orbiculare, Cylindrocarpon
destructans, Fusarium moniliforme, Fusarium oxysporum f. sp.
cucumerinum, M. grisea, Didymella bryoniae, R. solani, P. capsici, Pythium ultimum, Bacillus subtilis, Pseudomonas syringae
pv. syringae, Saccharomyces cerevisiae, and Candida
albicans were used to determine the MICs of SH-1 (phenylacetic
acid) and SH-2 (sodium phenylacetate) using a modified version of the
antimicrobial bioassay method of Nair et al. (26). Potato
dextrose broth (1 ml) supplemented with SH-1 and SH-2 at concentrations
from 0 to 1,000 µg/ml was pipetted into each well of a 24-well
microtiter dish (Cell Wells; Corning Glass Works, Corning, N.Y.) to
ascertain the MICs against fungi. Nutrient broth was also used for to
ascertain the MICs against bacteria and yeasts. Germ suspension (10 µl) was added to each well. The concentration of fungal spores or zoospores tested was 104 spores/ml. Bacteria and yeasts
were adjusted to 104 CFU/ml. The inoculated plates were
incubated at 28°C on a rotary shaker at 120 rpm. The inhibition of
microbial growth was evaluated after incubation for 3 or 4 days. The
lowest concentrations of SH-1 and SH-2 that completely inhibited
microbial growth were considered to be MICs. SH-1 and SH-2 completely
inhibited the growth of P. capsici, R. solani, S. cerevisiae, and P. syringae pv. syringae at the
concentration of 50 µg/ml (Table 1).
The growth of P. ultimum was also completely inhibited at 10 µg/ml, whereas A. mali, C. destructans, F. moniliforme,
and F. oxysporum f. sp. cucumerinum showed little
inhibition even at 500 or 1,000 µg/ml. The antifungal substances SH-1
and SH-2 exhibited an intermediate level of inhibitory activity against
C. orbiculare, C. albicans, and B. subtilis, with
MICs ranging from 50 to 100 µg/ml.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
MICs of antifungal substances SH-1 (phenylacetic acid)
and SH-2 (sodium phenylacetate) from S. humidus strain S5-55
against various microorganisms
|
|
Zoospore suspension of
P. capsici was prepared by the method
of Kim et al. (
24) using the culture plates grown on
oatmeal
agar for 10 days at 28°C. The zoospore suspensions were mixed
with SH-1, SH-2, metalaxyl, fosetyl-AI, and
H
3PO
3 to give the
concentrations of 0, 1, 10, 50, 100, and 500 µg/ml. After incubation
for 4 h at 28°C,
zoospore germination was microscopically examined
in two experiments
with five replicates. SH-1 and SH-2 completely
inhibited zoospore
germination of
P. capsici at 50 µg/ml, whereas
the
commercial fungicide metalaxyl was not effective against zoospore
germination at concentrations up to 100 µg/ml (data not shown).
There
was no difference between SH-1 and SH-2 in inhibiting zoospore
germination. Treatment with H
3PO
3 began to
inhibit zoospore germination
at 1 µg/ml and the inhibitory effect was
maximum at 100 µg/ml.
To examine the inhibitory effects of the chemicals on hyphal growth of
P. capsici, SH-1, SH-2, metalaxyl, fosetyl-AI, and
H
3PO
3 were added to a suspension of germinated
zoospores with
an average length of 30 µm. After further incubation
of the mixtures
for 3 h at 28°C, hyphal growth of
P. capsici was measured under
a light microscope, as previously
described (
15). The effects
of these chemicals on the
hyphal growth of
P. capsici were determined
by comparing the
hyphal length of the oomycete pathogen in each
of the chemicals with
that of a control preparation. The experiments
were repeated twice with
three replicates. Hyphal growth of
P. capsici was strongly
inhibited by treatment with SH-1, metalaxyl,
and
H
3PO
3. However, the inhibitory effect of SH-2
against hyphal
growth was not as great as those of other compounds
tested (data
not
shown).
Mycelial disks (7-mm diameter) of
P. capsici were placed in
the center of the V8 agar plates supplemented with phenylacetic
acid (Sigma), metalaxyl, fosetyl-AI, or H
3PO
3.
The mycelial growth
of
P. capsici was rated after incubation
for 7 days at 28°C. The
percentage inhibition of mycelial growth by
the chemical was calculated
by the following formula: [1
(diameter of mycelial growth in
the chemical-treated plate/diameter of
mycelial growth in the
untreated control)] × 100. Treatment with
phenylacetic acid strongly
inhibited mycelial growth of
P. capsici in potato dextrose agar
(PDA) plates supplemented with the
compound at various concentrations
(see Fig.
4A). Compared to
phenylacetic acid, metalaxyl was highly
active against
P. capsici. However, H
3PO
3 was less effective
than
phenylacetic acid in inhibiting the growth of the oomycete
pathogen.
The commercial fungicide fosetyl-AI did not show any
antifungal
activity against
P. capsici, even at 500 µg/ml.
Taken together, the in vitro data obtained by the microtiter broth
dilution, zoospore germination, and mycelial growth inhibition
tests
strongly suggested that phenylacetic acid (SH-1) and sodium
phenylacetate (SH-2) have antifungal activity against the
plant-pathogenic
oomycete
P. capsici. In zoospore
germination tests, both compounds
inhibited zoospore germination of
P. capsici. The phosphonate
fungicide fosetyl-AI, which is
being used for control of oomycetes,
was highly inhibitory to some
Phytophthora spp. (
6). Phenylacetic
acid
strongly inhibited the hyphal growth of
P. capsici at 100
µg/ml, whereas sodium phenylacetate did not show inhibitory activity
against hyphal growth at the same concentration without lysis
of the
zoospores. However, both compounds were shown to be inhibitory
to
zoospore germination of
P. capsici compared to fosetyl-AI
(or
H
3PO
3) and phenylacetic acid (or sodium
phenylacetate) (data not
shown). Some compounds which showed in vitro
antifungal activity
were often found to have negligible in vivo control
efficacy against
plant diseases (
7). Because
phenylacetic acid and sodium phenylacetate
exhibited a high
antifungal activity against
P. capsici in vitro,
their in
vivo control efficacy of plant diseases should be further
examined.
In vivo antifungal activity of SH-1 (phenylacetic acid) and SH-2
(sodium phenylacetate).
The antifungal substance SH-1 was
evaluated for the ability to suppress phytophthora blight on pepper
plants in a growth room. Seeds of pepper (Capsicum annuum
L.) cv. Hanbyul were sown in a plastic tray (55 by 15 by 10 cm)
containing steam-sterilized soil mix (peat moss, perlite, and
vermiculite [5:3:2, vol/vol/vol]), sand, and loam soil (1:1:1,
vol/vol/vol). Six seedlings at the four-leaf stage were transplanted
into a plastic pot (5 by 15 by 10 cm). Pepper plants were raised to the
first-branch stage in a growth chamber at 28°C (±2°C) for 16 h a day. Antifungal substances SH-1 and SH-2 and the commercial
fungicide metalaxyl dissolved in methanol and acetone, respectively,
were diluted to give concentrations of 0, 10, 100, 500, and 1,000 µg/ml. The 30-ml chemical solution was soil drenched into each pot 1 day before inoculation of P. capsici on pepper plants. A
zoospore suspension was prepared by the method of Kim et al.
(24) using the culture plates grown on oatmeal agar for 11 days at 28°C. The pepper plants were inoculated with a zoospore
suspension (105 zoospores/ml) by the stem wound inoculation
method. Antifungal substance SH-1, SH-2, phenylacetic acid, metalaxyl,
fosetyl-AI, and H3PO3 dissolved in water
were diluted to give concentrations of 0, 10, 100, 500, and 1,000 µg/ml. The chemical solutions were sprayed to the pepper plants until
they ran off 1 day before inoculation of P. capsici. The
pepper plants were also inoculated with a zoospore suspension
(105 zoospores/ml) by the soil drench method. Disease
severity on pepper plants was rated daily after inoculation based on a
scale from 0 to 5 as follows: 0 for no visible disease symptoms, 1 for slightly wilted leaves, with brownish lesions beginning to appear on
the stems, 2 for 30 to 50% of the entire plant diseased, 3 for 50 to
70% of the entire plant diseased, 4 for 70 to 90% of the entire plant
diseased, and 5 for a dead plant. Data are the means of 10 plants per
treatment. Statistical analyses were conducted with the Statistical
Analysis System for personal computers (SAS Institute, Cary, N.C.).
Percent data were subjected to an angular transformation (arcsine
square root) to normalize the variance prior to analysis. Fisher's
protected least significant difference with a P of 0.05 was
used to separate the means. In vivo efficacy of SH-1 and SH-2 for the
control of phytophthora blight in pepper plants was examined after
inoculation of P. capsici using stem wound and soil drench
methods under controlled environmental conditions (Fig.
3). The symptoms of phytophthora blight
began to appear on pepper plants 4 days after inoculation. When
inoculated with P. capsici, brownish lesions occurred on the
pepper stem and extended rapidly to the upper part of plants,
accompanied by wilting of the entire plant, leaf defoliation, and
damping-off. Treatment with the antifungal substances SH-1 and SH-2
greatly inhibited the phytophthora disease in pepper plants. The
suppressing effect of both compounds against phytophthora blight was
pronounced at 1,000 µg/ml (Fig. 3A). In contrast, the commercial
fungicide metalaxyl completely inhibited the development of
phytophthora blight in pepper plants at the concentration of 10 µg/ml, irrespective of stem wound or soil drench inoculation methods.
When soil drenched 1 day before inoculation of P. capsici,
the control efficacy of SH-1 was less than that of
H3PO3 or fosetyl-AI (Fig. 3B).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 3.
In vivo control efficacy of SH-1, SH-2,
H3PO3, fosetyl-AI, and metalaxyl against
P. capsici infection on pepper plants at the first-branch
stage. (A) Foliar spray treatment on pepper plants just before stem
wound inoculation. (B) Soil drench treatment 1 day before soil drench
inoculation. The disease severity rating is based on a scale of 0 to 5 scale, with a score of 0 for no visible symptoms and a score of 5 for a
dead plant. Means at each concentration followed by the same letter are
not significantly different (P = 0.05) according to the
least significant difference test.
|
|
In vivo efficacy of phenylacetic acid, metalaxyl, fosetyl-AI, and
H
3PO
3 for the control of phytophthora blight in
pepper plants
was evaluated under greenhouse conditions (Fig.
4B). As the concentration
of phenylacetic
acid and other compounds increased, the phytophthora
disease was
gradually inhibited on the pepper plants at the first-branch
stage.
Treatments with 500 or 1,000 µg of phenylacetic acid, fosetyl-AI,
and
H
3PO
3 per ml showed a relatively high level of
protective
activity against
P. capsici infection. The
control efficacy of
phenylacetic acid against phytophthora blight was
in general similar
to those of fosetyl-AI and
H
3PO
3 but less than that of metalaxyl,
which
showed complete control at 500 and 1,000 µg/ml.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 4.
In vitro and in vivo efficacy of the authentic
phenylacetic acid, H3PO3, fosetyl-AI, and
metalaxyl against mycelial growth of P. capsici (A) and the
disease development in pepper plants (B). Mycelial growth was measured
on potato dextrose agar containing different concentrations of the
chemicals when the control plates (9 cm in diameter) were completely
covered by the fungus. Each chemical was sprayed on the foliage of
plants 1 day before inoculation. Disease severity was rated 7 days
after inoculation on pepper plants at the first-branch stage. Means at
each concentration followed by the same letter are not significantly
different (P = 0.05) according to the least significant
difference test.
|
|
Concluding remarks.
To our knowledge, this is the first study
to demonstrate the in vivo efficacy of phenylacetic acid and sodium
phenylacetate for the control of phytophthora blight in pepper plants,
although it should be noted that Burkhead et al. (3)
previously provided preliminary evidence for antifungal activity
against Gibberella pulicaris. In the present study,
phenylacetic acid and sodium phenylacetate were found to be very
effective not only in inhibiting zoospore germination and mycelial
growth of P. capsici but also in controlling phytophthora
blight in pepper plants. The synthetic fungicides such as prothiocarb,
propamocarb, phosphate, and acyanilide including metalaxyl have
practically been used to control the plant diseases caused by oomycetes
(6). Among the oomycetes, P. capsici, which
causes root and crown rot and blight of pepper (Capsicum
annum L.) plants, is one of the limiting factors in production of
pepper in pepper-growing fields worldwide (14).
Phenylacetic acid, a deamination product of phenylalanine, has been
known to possess a positive effect on the growth and development
of
maize (
29). The plants and microorganisms which produce
phenylalanine
ammonia lyase can derive phenylacetic acid from
phenylalanine
in nature (
29). Wightman and Lighty
(
33) found that phenylacetic
acid acts as a natural auxin
in the shoots of higher plants, such
as barley, corn, tobacco, and
tomato. Some microorganisms can
utilize phenylacetic acid during
their metabolic process.
Penicillium chrysogenum takes up
phenylacetic acid as a precursor of penicillin
G (
10). The
transport system of phenylacetic acid in
P. crysogenum is
well understood (
8).
Ralstonia solanacearum was
shown to
utilize phenylalanine and phenylacetic acid as the sole carbon
and nitrogen source (
1). Kawazu et al. (
17,
18) have also
demonstrated that phenylacetic acid produced by
Bacillus subtilis strain HY-16,
Bacillus cereus
strain HY-3, and
Bacillus megaterium strain HY-17 has in
vitro toxic effect against the pine wood nematode
Barsaphelenchus
xylophilusi.
Phenylacetic acid suppressed phytophthora blight at the concentration
of 1,000 µg/ml, but sodium phenylacetate showed less
efficacy at
1,000 µg/ml. Fosetyl-AI, which breaks down rapidly
in soil and plant
tissues to phosphorous acid (H
3PO
3) and
CO
2 (
5), was known to have low activity
against
Phytophthora and
Pythium spp. in vitro
(
6). Phosphorous acid was highly inhibitory
to mycelial
growth, sporangium development, and zoospore release
of several
Phytophthora spp. even at low concentrations (
4,
5). Because it seems likely that
P. capsici could not
utilize
phenylacetic acid, phenylacetic acid and sodium phenylacetate
may successfully inhibit the growth of
P. capsici in vitro.
Papavizas
and Bowers (
28) demonstrated that metalaxyl was
effective in
inhibiting zoospore germination of
P. capsici
at 100 µg/ml. Phenylacetic
acid and sodium phenylacetate were more
effective than metalaxyl
in reducing zoospore germination of
P. capsici. However, in vivo
modes of actions of phenylacetic acid
against phytophthora blight
in pepper plants remain to be elucidated in
detail. In an earlier
study, phenylacetic acid was found to act as a
natural auxin in
some higher plants (
33), which suggests
that treatment with
phenylacetic acid may enhance the growth rate of
plants. Phenylacetic
acid may induce some resistance in pepper plants
against infection
by
P. capsici. However, it will be
difficult to determine whether
phenylacetic acid can trigger systematic
acquired resistance in
pepper plants, because the chemical has direct
antifungal activity
in vitro against
P. capsici. Application
of phenylacetic acid
may also result in the reduction of the primary
inoculum density
of
P. capsici in soils of pepper-growing
fields.
| |
ACKNOWLEDGMENTS |
This research was financially supported from 1999 to 2002 by the
special research fund of the Ministry of Agriculture and Forestry of Korea.
We thank E. J. Bang and J. J. Seo (Korea Basic Science
Institute, Seoul, Korea) for NMR, El mass spectroscopy, and ICP mass spectroscopy. We also thank D. A. Holte critically for reading our manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Molecular Plant Pathology, College of Life and Environmental Sciences, Korea University, Seoul 136-701, Korea. Phone: (82) 2 3290 3061. Fax:
(82) 2 925 1970. E-mail: bkhwang{at}korea.ac.kr.
| |
REFERENCES |
| 1.
|
Agrawal, P.,
S. Latha, and A. Mahadevan.
1996.
Utilization of phenylalanine and phenylacetic acid by Pseudomonas solanacearum.
Appl. Biochem. Biotechnol.
61:379-391.
|
| 2.
|
Aizawa, S.,
H. Akutsu,
H. Seto, and N. Otaka.
1982.
Capsimycin, an antibiotic active against phytopathogenic fungi.
The Fifth International Congress of Pesticide Chemistry (IUPAC)., Kyoto, Japan.
|
| 3.
|
Burkhead, K. D.,
P. J. Slininger, and D. A. Schisler.
1998.
Biological control bacterium Enterobacter cloacae S11:T:07 (NRRL B-21050) produces the antifungal compound phenylacetic acid.
Soil Biol. Biochem.
30:665-667[CrossRef].
|
| 4.
|
Coffey, M. D., and L. A. Bower.
1984.
In vitro variability among isolates of eight Phytophthora species in response to phosphorous acid.
Phytopathology
74:738-742.
|
| 5.
|
Coffey, M. D., and M. C. Joseph.
1985.
Effects of phosphorous acid and fosetyl-AI on the life cycle of Phytophthora cinnamomi and P. citricola.
Phytopathology
75:1042-1046.
|
| 6.
|
Cohen, Y., and M. D. Coffey.
1986.
Systemic fungicides and the control of oomycetes.
Annu. Rev. Phytopathol.
24:311-338.
|
| 7.
|
Fawcett, C. H., and D. M. Spencer.
1970.
Plant chemotherapy with natural products.
Annu. Rev. Phytopathol.
8:403-418.
|
| 8.
|
Fernandez-Canon, J. M.,
A. Reglero,
H. Martinez-Blanco, and J. M. Luengo.
1989.
Uptake of phenylacetic acid by Penicillium chrysogenum Wis 54-1255: a critical regulatory point in benzylpenicillin biosynthesis.
J. Antibiot.
42:1398-1409[Medline].
|
| 9.
|
Hayakawa, M.,
K. Ishizawa, and H. Nonomura.
1988.
Distribution of rare actinomycetes in Japanese soils.
J. Ferment. Technol.
66:367-373[CrossRef].
|
| 10.
|
Hillenga, D. J.,
J. M. Hanneke,
S. Versantvoort,
A. van der Molen,
J. M. Driessen, and W. N. Konings.
1995.
Penicillium chrysogenum takes up the penicillin G precursor phenylacetic acid by passive diffusion.
Appl. Environ. Microbiol.
61:2589-2595[Abstract].
|
| 11.
|
Homans, A. L., and A. Fuchs.
1970.
Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances.
J. Chromatogr.
51:327-329[Medline].
|
| 12.
|
Hwang, B. K.,
S. J. Ahn, and S. S. Moon.
1994.
Production, purification, and antifungal activity of the antibiotic nucleoside, tubercidin, produced by Streptomyces violaceoniger.
Can. J. Bot.
72:480-485.
|
| 13.
|
Hwang, B. K., and B. S. Kim.
1995.
In-vivo efficacy and in-vitro activity of tubercidin, an antibiotic nucleoside, for control of Phytophthora capsici blight in Capsicum annuum.
Pestic. Sci.
44:255-260.
|
| 14.
|
Hwang, B. K., and C. H. Kim.
1995.
Phytophthora blight of pepper and its control in Korea.
Plant Dis.
79:221-227.
|
| 15.
|
Hwang, B. K.,
J. Y. Lee,
B. S. Kim, and S. S. Moon.
1996.
Isolation, structure elucidation, and antifungal activity of a manumycin-type antibiotic from Streptomyces flaveus.
J. Agric. Food Chem.
44:3653-3657[CrossRef].
|
| 16.
|
Jiang, C. L., and L. H. Xu.
1996.
Diversity of aquatic actinomycetes in lakes of the middle plateau, Yunnan, China.
Appl. Environ. Microbiol.
62:249-253[Abstract].
|
| 17.
|
Kawazu, K.,
H. Zhang, and H. Kanzaki.
1996.
Accumulation of benzoic acid in suspensions of cultured cells of Pinus thunbergii Parl. in response to phenylacetic acid administration.
Biosci. Biotechnol. Biochem.
60:1410-1412[Medline].
|
| 18.
|
Kawazu, K.,
H. Zhang,
H. Yamashita, and H. Kanzaki.
1996.
Relationship between the pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus, and phenylacetic acid.
Biosci. Biotechnol. Biochem.
60:1413-1415[Medline].
|
| 19.
|
Kim, B. S.,
J. Y. Lee, and B. K. Hwang.
1998.
Diversity of actinomycetes antagonistic to plant pathogenic fungi in cave and sea-mud soils of Korea.
J. Microbiol.
36:86-92.
|
| 20.
|
Kim, B. S.,
S. S. Moon, and B. K. Hwang.
1999.
Isolation, identification, and antifungal activity of a macrolide antibiotic, oligomycin A, produced by Streptomyces libani.
Can. J. Bot.
77:850-858[CrossRef].
|
| 21.
|
Kim, B. S.,
S. S. Moon, and B. K. Hwang.
1999.
Isolation, antifungal activity, and structure elucidation of the glutarimide antibiotic, streptimidone, produced by Micromonospora coerulea.
J. Agric. Food Chem.
47:3372-3380[CrossRef][Medline].
|
| 22.
|
Kim, B. S.,
S. S. Moon, and B. K. Hwang.
2000.
Structure elucidation and antifungal activity of an anthracycline antibiotic, daunomycin, isolated from Actinomadura roseola.
J. Agric. Food Chem.
48:1875-1881[CrossRef][Medline].
|
| 23.
|
Kim, C. J.,
I. K. Lee,
B. S. Yun, and I. D. Yoo.
1993.
Concanamycin B, active substance against Phytophthora capsici produced by Streptomyces neyagawaensis 38D10 strain.
Korean J. Microbiol. Biotechnol.
21:322-328.
|
| 24.
|
Kim, Y. J.,
B. K. Hwang, and K. W. Park.
1989.
Expression of age-related resistance in pepper plants infected with Phytophthora capsici.
Plant Dis.
73:745-747.
|
| 25.
|
Lim, S. W.,
J. D. Kim,
B. S. Kim, and B. K. Hwang.
2000.
Isolation and numerical identification of Streptomyces humidus strain S5-55 antagonistic to plant pathogenic fungi.
Plant Pathol. J.
16:189-199.
|
| 26.
|
Nair, M. G.,
S. K. Mishra, and A. R. Putnam.
1992.
Antifungal anthracycline antibiotics, spartanamicins A and B from Micromonospora spp.
J. Antibiot.
45:1738-1745[Medline].
|
| 27.
|
Okami, Y., and K. Hotta.
1988.
Search and discovery of new antibiotics, p. 33-67.
In
M. Goodfellow, S. T. Williams, and M. Mordaski (ed.), Actinomycetes in biotechnology. Academic Press, London, United Kingdom.
|
| 28.
|
Papavizas, G. C., and J. H. Bowers.
1981.
Comparative fungitoxicity of captafol and metalaxyl to Phytophthora capsici.
Phytopathology
71:123-128.
|
| 29.
|
Sarwar, M., and W. T. Frankenberger, Jr.
1995.
Fate of L-phenylalanine in soil and its effect on plant growth.
Soil Sci.
59:1625-1630[Abstract/Free Full Text].
|
| 30.
|
Shomura, T.
1993.
Screening for new products of new species of Dactylosporangium and other actinomycetes.
Actinomycetology
7:88-98.
|
| 31.
|
Suzuki, K.,
K. Nagai,
Y. Shimizu, and Y. Suzuki.
1994.
Search for actinomycetes in screening for new bioactive compounds.
Actinomycetology
8:122-127.
|
| 32.
|
Tanaka, Y. T., and S.  mura.
1993.
Agroactive compounds of microbial origin.
Annu. Rev. Microbiol.
47:57-87[CrossRef][Medline].
|
| 33.
|
Wightman, F., and D. L. Lighty.
1982.
Identification of phenylacetic acid as a natural auxin in the shoot of higher plants.
Physiol. Plant.
55:17-24.
|
| 34.
|
Xu, L. H.,
Q. R. Li, and C. L. Jiang.
1996.
Diversity of soil actinomycetes in Yunnan, China.
Appl. Environ. Microbiol.
62:244-248[Abstract].
|
Applied and Environmental Microbiology, August 2001, p. 3739-3745, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3739-3745.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Somers, E., Ptacek, D., Gysegom, P., Srinivasan, M., Vanderleyden, J.
(2005). Azospirillum brasilense Produces the Auxin-Like Phenylacetic Acid by Using the Key Enzyme for Indole-3-Acetic Acid Biosynthesis. Appl. Environ. Microbiol.
71: 1803-1810
[Abstract]
[Full Text]
-
Lee, J. Y., Moon, S. S., Hwang, B. K.
(2003). Isolation and Antifungal and Antioomycete Activities of Aerugine Produced by Pseudomonas fluorescens Strain MM-B16. Appl. Environ. Microbiol.
69: 2023-2031
[Abstract]
[Full Text]
Top
Abstract
Text
