Antimicrobial activities of novel mannosyl lipids isolated from the biocontrol fungus simplicillium lamellicola BCP against phytopathogenic bacteria

Article abstract of DOI:10.1021/jf500361e

The antagonistic fungus Simplicillium lamellicola BCP has been developed as a microbial biopesticide that effectively controls the development of various plant diseases caused by both pathogenic bacteria and pathogenic fungi. Antibacterial bioassay-directed fractionation was used to isolate mannosyl lipids from S. lamellicola BCP, and the structures of these compounds were elucidated using spectral analysis and chemical degradation. Three novel mannosyl lipids were characterized and identified as halymecins F and G and (3R,5R)-3-O-β-d-mannosyl-3,5-dihydrodecanoic acid. Massoia lactone and (3R, 5R)-3-hydroxydecan-5-olide were also isolated from S. lamellicola BCP. The three novel compounds inhibited the growth of the majority of phytopathogenic bacteria that were tested, and halymecin F displayed the strongest antibacterial activity. Agrobacterium tumefaciens was the most sensitive to the three novel compounds, with IC₅₀ values ranging from 1.58 to 24.8 μg/mL. The ethyl acetate extract of the fermentation broth from the antagonistic fungus effectively reduced the bacterial wilt caused by Ralstonia solanacearum on tomato seedlings. These results indicate that S. lamellicola BCP suppresses the development of plant bacterial diseases through the production of antibacterial metabolites.

Full text of DOI:10.1021/jf500361e

Article
pubs.acs.org/JAFC
Antimicrobial Activities of Novel Mannosyl Lipids Isolated from the
Biocontrol Fungus Simplicillium lamellicola BCP against
Phytopathogenic Bacteria
Quang Le Dang,†,‡ Teak Soo Shin,§ Myung Soo Park,† Yong Ho Choi,† Gyung Ja Choi,†
Kyoung Soo Jang,† In Seon Kim,# and Jin-Cheol Kim*,†
†Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology,
141 Gajeong-ro, Yuseong-Gu, Daejeon 305-600, Republic of Korea
‡Department of Phytochemistry, Vietnam Institute of Industrial Chemistry, 2 Pham Ngu Lao Street, Hanoi 10999, Vietnam
§Crop Protection Research Team, Dongbu Advanced Research Institute, Dongbu Farm Hannong Company, Ltd., 229 Munji-ro,
Yuseong-Gu, Daejeon 305-708, Republic of Korea
#Division of Applied Bioscience and Biotechnology, Institute of Environmentally Friendly Agriculture, College of Agriculture and Life
Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea
S
* Supporting Information
ABSTRACT: The antagonistic fungus Simplicillium lamellicola BCP has been developed as a microbial biopesticide that
effectively controls the development of various plant diseases caused by both pathogenic bacteria and pathogenic fungi.
Antibacterial bioassay-directed fractionation was used to isolate mannosyl lipids from S. lamellicola BCP, and the structures of
these compounds were elucidated using spectral analysis and chemical degradation. Three novel mannosyl lipids were
characterized and identified as halymecins F and G and (3R,5R)-3-O-β-D-mannosyl-3,5-dihydrodecanoic acid. Massoia lactone
and (3R, 5R)-3-hydroxydecan-5-olide were also isolated from S. lamellicola BCP. The three novel compounds inhibited the
growth of the majority of phytopathogenic bacteria that were tested, and halymecin F displayed the strongest antibacterial
activity. Agrobacterium tumefaciens was the most sensitive to the three novel compounds, with IC50 values ranging from 1.58 to
24.8 μg/mL. The ethyl acetate extract of the fermentation broth from the antagonistic fungus effectively reduced the bacterial
wilt caused by Ralstonia solanacearum on tomato seedlings. These results indicate that S. lamellicola BCP suppresses the
development of plant bacterial diseases through the production of antibacterial metabolites.
KEYWORDS: biocontrol, halymecin, mannosyl lipid, antimicrobial activity, Simplicillium lamellicola
morphological identification.4 However, re-examination of the
strain using sequence analyses of the 28S rRNA gene and ITS
regions identified it as S. lamellicola. The antagonistic fungus
effectively suppressed various plant diseases caused by
INTRODUCTION
■
Phytopathogenic fungi and bacteria cause massive damage and
reduce the yield of crops. The resistance of plant pathogens to
commercial synthetic agrochemicals, the desire for organic
food, and environmental concerns have led to intensive
pathogenic fungi, including B. cinerea, and S. lamellicola was
commercialized as the microbial fungicide Acre.5 Its antifungal
research efforts for integrated control using antagonistic
mechanism is both mycoparasitism and antibiosis.4,6 The
microorganisms and microorganism-derived natural prod-
ucts.1,2 As biodegradable compounds, microbial natural
antagonistic fungus overgrew the colonies of B. cinerea and
caused severe lysis of the host hyphae. Hyphal growth of BCP
products are attractive candidates for development as agro-
chemicals. A number of natural fungicides such as kasugamycin,
strain inside the mycelia of B. cinerea was observed.6 In
addition, it produces verlamelin as an antifungal metabolite,
polyoxins, validamycin, and blasticidin-S, and bactericides such
which is active in vitro to phytopathogenic fungi, but not to
as oxytetracyline and streptomycin have been isolated from
phytopathogenic bacteria.4 During evaluation of the disease
microbial resources. These compounds are well-known
control efficacy of this microbial fungicide in fields, we found
commercial agrochemicals derived from Streptomyces species
that it also highly suppressed the development of tomato
and have been effectively used to control phytopathogenic
bacterial wilt caused by Ralstonia solanacearum. Therefore, the
current study was performed to isolate and determine the
chemical structures of the antibacterial metabolites from the
diseases caused by the fungi Botrytis cinerea, Colletotrichum
coccodes, Magnaporthe oryzae, Phytophthora infestans, Puccinia
recondita, and Rhizoctonia solani and the bacteria Xanthomonas
oryzae, Xanthomonas citri, and Pseudomonas species.1,3
The fungal strain used to produce the mannosyl lipids in the
present study was Simplicillium lamellicola BCP, which was
isolated from the mycelia of Botrytis cinerea. This strain was
previously known as Acremonium strictum BCP on the basis of
Received: January 21, 2014
Revised: March 23, 2014
Accepted: March 24, 2014
Published: March 24, 2014
© 2014 American Chemical Society
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by preparative TLC using a mixture of hexane/EtOAc (1:3, v/v) to
yield 4.2 mg of compound 4 and 5 mg of compound 5.
fermentation broth of the biocontrol fungal strain S. lamellicola
BCP. In addition, the in vitro and in vivo activities of the ethyl
acetate layer and pure compounds were evaluated against
phytopathogenic bacteria.
The aqueous layer was concentrated to dryness and redissolved in
0.2 mL of 2 N HCl in a 7 mL screw-top vial. The vial was heated to
100 °C for 4 h to hydrolyze the sugar moiety. After hydrolysis, the
mixture of products was divided into two parts. One portion was
partitioned with DCM to remove the nonpolar impurities, and the
aqueous layer was evaporated off under an N2 stream. The residue was
dissolved in MeOH and analyzed by TLC using DCM/MeOH/W
(6:4:1, v/v/v) and was compared with authentic monosaccharides.
The second portion was neutralized with NaHCO3 and dried under an
N2 stream. The residue was acetylated with 0.2 mL of acetic
anhydride/pyridine (1:1, v/v) at 100 °C for 45 min. After the
acetylation reaction, the mixture of acetic anhydride/pyridine was
removed under an N2 stream. The residue containing the acetylated
monosaccharide was dissolved into 0.2 mL of 2% CuSO4 in water and
partitioned twice with 0.5 mL of EtOAc. The EtOAc layers were
pooled and dried under a nitrogen stream. The residue obtained after
drying was dissolved in 50 μL of chloroform for GC-MS analysis.
Authentic samples of glucose, rhamnose, mannose, galactose, and
sorbitol (0.5 mg each) were also acetylated. The acetylated
monosaccharides were analyzed by GC-MS on an SP2330 column
(30 m × 0.32 mm i.d.; 0.25 μm film thickness; Supelco, Inc.,
Bellefonte, PA, USA) using He as the carrier gas with a temperature
program of 120 °C (2 min) to 230 °C (5 °C min−1) maintained for 12
min.
Structure Determination of Antibacterial Substances. The
optical rotations of the purified compounds in MeOH were recorded
on a JASCO J-20 automatic recording spectropolarimeter using a 1 cm
path-length cell. The chemical structures were determined by mass and
nuclear magnetic resonance (NMR) spectroscopy. Fast atom
bombardment (FAB) analysis was performed with a glycerol matrix
and Ar as the bombarding gas using a high-resolution mass
spectrometer (JMS-AX505; JEOL Ltd., Tokyo, Japan). The electro-
spray ionization mass spectra (ESI-MS) of the purified compounds
were recorded on an MSD1100 single-quadrupole mass spectrometer
equipped with an ESI (Hewlett-Packard Co., Palo Alto, CA, USA). 1H
and 13C NMR, COSY, HMQC, and HMBC spectra were recorded in
CD3OD (E. Merck) with a Bruker AMX-500 spectrometer (Bruker
Analytische Messtechnik Gmbh, Rheinstetten, Germany).7 The
spectra were referenced to either tetramethylsilane (TMS) (1H) or
solvent signals (13C). The coupling constants are reported in hertz.
The chemical structures of the purified substances were analyzed on
the basis of the MS and NMR spectral data and comparisons to
reported values in the literature.
In Vitro Antibacterial Assay against Phytopathogenic
Bacteria. An in vitro antibacterial assay was performed in 96-well
microtiter plates based on a modification of the broth microdilution
method.8,9 The R. solanacearum strain was incubated in TSB medium,
and the other bacteria were incubated in NB medium. The stock
solutions of pure compounds dissolved in MeOH were diluted 100-
fold with the proper growth medium for each strain and tested at final
concentrations ranging from 0.39 to 100 μg/mL. Bacterial suspensions
(50 μL) of each strain with an inoculum of 2 × 106 cells/mL were
added to the wells containing 50 μL of 2-fold serially diluted pure
compounds in the proper growth medium for a final volume of 100 μL
in each well. The negative controls were treated with 1% MeOH,
which corresponds to the highest concentration. The blank wells were
prepared with culture medium containing pure compounds at the
same test concentrations. The dilutions were prepared with three
replicates, and the experiments were performed a minimum of three
times. The inoculated plates were incubated at 30 °C, except for X.
euvesicatoria at 25 °C, for 18−48 h after shaking at 300 rpm for 20 min
on a microplate shaker. The optical density (OD) of each well was
measured using a microplate reader (Bio-Rad, Benchmark Plus, USA)
at 600 nm. The growth inhibition of each dilution was determined
using the formula9
MATERIALS AND METHODS
■
Organisms and Culture Conditions. The following seven
phytopathogenic bacterial strains were used for the antibacterial
bioassay: Acidovorax konjaci, the causal agent of bacterial blight of
konjac; Agrobacterium tumefaciens, the causal agent of crown gall
disease; Burkholderia glumae, the causal agent of bacterial grain rot of
rice; Pectobacterium carotovorum subsp. carotovorum, the causal agent
of bacterial soft rot of potato; Pseudomonas syringae pv lachrymans, the
causal agent of cucumber angular leaf spot; R. solanacearum, the causal
agent of tomato bacterial wilt; and Xanthomonas euvesicatoria, the
causal agent of pepper bacterial spot. All strains were grown on
nutrient agar (NA) (Becton, Dickinson and Co., Sparks, MD, USA)
and nutrient broth (NB), except R. solanacearum, which was grown in
tryptone soy agar (TSA) (Becton, Dickinson and Co.) and tryptone
soy broth (TSB) (Becton, Dickinson and Co.). X. euvesicatoria was
cultured aerobically at 25 °C for 18−36 h, and the remaining strains
were cultured aerobically at 30 °C for 18−36 h.
Isolation of Antibacterial Substances. Strain BCP was cultured
on potato dextrose broth (PDB) medium (Becton, Dickinson and
Co.) on a rotary shaker at 150 rpm and at 25 °C for 10 days as
described previously by Kim et al.4 The broth culture (10 L) was
centrifuged at 10000g for 10 min. The obtained supernatant was mixed
with an equal volume of acetone and incubated in a fume hood for 24
h. The precipitates were filtered out using filter paper, and the filtrate
was concentrated under a vacuum to remove the acetone. The aqueous
layer was then partitioned twice with equal volumes of ethyl acetate
(EtOAc). The EtOAc layers were pooled and concentrated in a rotary
evaporator at 40 °C to remove the organic solvent. The EtOAc
concentrate (2.5 g) showed strong activity against R. solanacearum as
determined by a disk diffusion assay.4 The EtOAc concentrate was
subjected to chromatography on a silica gel column [100 g of Kiesel
gel 60 (230−400 mesh; E. Merck, Darmstadt, Germany), 3.4 cm × 40
cm, packed with EtOAc/methanol (MeOH) (9:1, v/v) ] with
successive elution with mixtures of EtOAc/MeOH/water (W)
(80:10:2, v/v/v, 200 mL), EtOAc/MeOH/W (80:10:5, v/v/v, 200
mL), EtOAc/MeOH/W/acetic acid (80/10/8/1, v/v/v/v, 500 mL),
and dichloromethane (DCM/MeOH/W (60:40:10, v/v/v, 500 mL),
yielding 15 fractions, ABE 1−15. The fractions were monitored using
thin-layer chromatography (TLC) (Kiesel gel 60GF 254, 0.25-mm
layer thickness; E. Merck) with the developing solvent EtOAc/
MeOH/W/acetic acid (80:10:8:1, v/v/v/v). The fractions were also
assayed in vitro for growth inhibition of R. solanacearum. The active
fractions ABE 5−8 displayed a similar pattern of primary components
based on the TLC analysis and were pooled and further purified
repeatedly using preparative HPLC (SCL-10A VP; Shimadzu Co.,
Kyoto, Japan) with a Capcell Pak C18 UG120 Å (5 μm, 20 mm × 250
mm; Shiseido Co., Tokyo, Japan). The combined fraction (460 mg)
was eluted with a linear gradient from 45% MeOH to 100% MeOH for
30 min and then maintained at 100% MeOH for 10 min. The eluent
(6.5 mL/min) was monitored at 204, 215, and 254 nm. Two
compounds, 1 (85 mg) and 2 (23 mg), were isolated to purity by
repeated preparative HPLC. Compound 3 (12 mg) was purified from
fraction ABE 13 by preparative TLC (Kiesel gel 60GF 254, 0.5 mm
layer thickness; E. Merck) with the developing solvent EtOAc/
MeOH/W/acetic acid (80:10:8:1, v/v/v/v).
Degradative products of 1 were prepared by directed lactonization
of fatty acids in the structure of 1 (12 mg), with the catalysis of 0.1 N
HCl in CH3CN (2 mL of a mixture of 1 mL of 0.1 HCl aq + 1 mL of
CH3CN). The reaction was performed at 80 °C for 2 h. The product
was monitored by TLC with hexane/EtOAc (1:3, v/v) as the
developing solvent. After working up the reaction, the nonpolar
product was extracted twice with 1 mL of a mixture of EtOAc and
hexane (3:1, v/v). The organic layers were pooled and concentrated
under an N2 stream at 40 °C until dryness. The residue was separated
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Figure 1. Chemical structures of the compounds isolated from the ethyl acetate layer of the Simplicillium lamellicola BCP broth. 1, halymecin F; 2,
halymecin G; 3, (3R,5R)-3-O-β-D-mannosyl-3,5-dihydroxydecanoic acid; 4, (+)-(3R,5R)-3-hydroxydecan-5-olide; 5, (−)-massoia lactone; DDA, 3, 5-
dihydroxydecanoic acid.
using the following formula: control value (%) = 100 × (disease
% inhibition = 100 × [1 − OD of treated well
severity of control − disease severity of treatment)/disease severity of
control.
/OD of negative control well]
Statistical Analysis. The data obtained in this study were
where OD values of the negative control well and the OD of the
treated well were corrected with the OD of the blank wells
corresponding to each concentration. The IC50 values were derived
from Probit analysis of the concentration−response data, with serially
diluted concentrations of the pure compounds.
evaluated by one-way ANOVA, and the significance of the treatments
was determined by Tukey’s HSD for multiple comparisons (P ≤ 0.05).
The IC50 values were calculated on the basis of Probit analysis using
WINPEPI software version 11.4.13
Disease Control Efficacy of the Ethyl Acetate Layer of S.
lamellicola BCP against Tomato Bacterial Wilt. To evaluate the
disease control efficacy of the EtOAc extract of S. lamellicola BCP
culture broth against tomato bacterial wilt caused by R. solanacearum
SL1944 (race 1, biovar 4),10,11 3-week-old tomato plants at the 4−5-
true-leaf stage of ‘Seokwang’ were used. The plants were grown in
vinyl pots with a volume of 170 mL in a greenhouse and were then
transplanted into vinyl pots with a diameter of 7 cm (one plant per
pot). Three different amounts of the EtOAc layer, 50, 100, and 200
mg, were dissolved in 2 mL of MeOH and then poured into 98 mL of
distilled water containing 250 μg/mL Tween-20 to obtain test
concentrations of 500, 1000, and 2000 μg/mL. R. solanacearum was
cultured on TSA Petri dishes at 30 °C for 48 h and were subsequently
washed twice from the dishes with sterile distilled water. The inoculum
suspension was adjusted to an optical density OD600 of 0.1
(approximately 1.5 × 108 cells/mL) using sterile distilled water. The
20 mL aliquots of three EtOAc extract solutions were evenly applied to
the soil of each pot. After 3 h, R. solanacearum was inoculated by
applying 20 mL of the inoculum suspensions into the soil. The second
treatment with the EtOAc layer was performed 5 days after the first
treatment. Streptomycin sulfate (SM), a bactericidal antibiotic with a
broad activity against both Gram-negative and Gram-positive bacteria,
was used as a positive control at a concentration of 200 μg/mL. Pots
treated with water containing Tween-20 (250 μg/mL) and MeOH
(2%) served as the negative controls (CK). The plants were
maintained in a controlled climate at 30 °C with a relative humidity
of 70−80%. The disease severity was recorded on a scale of 0−5 as
described by Winstead and Kelman.12 The following scales were used:
0 = no symptoms, 1 = one leaf partially wilted, 2 = one to two leaves
wilted, 3 = two to three leaves wilted, 4 = four or more leaves wilted,
and 5 = death of the entire plant. The pots were arranged as a
randomized complete block with five replicates per treatment. The
experiment was repeated three times. The control value was calculated
RESULTS AND DISCUSSION
■
This study focused on the identification of antibacterial
metabolites produced by the biocontrol agent S. lamellicola
BCP and the in vitro and in vivo activities of these compounds
against phytopathogenic bacteria to identify either alternatives
to currently used crop protection agents or lead compounds to
synthesize derivatives with enhanced activities or environmental
friendliness. The S. lamellicola BCP strain, previously identified
as A. strictum BCP on the basis of morphological characteristics,
was developed as a microbial fungicide for the control of
various plant diseases, such as tomato gray mold and rice
blast.5,6 However, in the present study, we identified the strain
as S. lamellicola BCP on the basis of sequence analysis of the
28S rRNA gene and ITS regions (Figures S1 and S2 in the
Supporting Information). In addition, we found that the
microbial fungicide also effectively suppressed the development
of tomato bacterial wilt in fields. We previously reported that
the antagonistic fungus produces verlamelin as an antifungal
metabolite.4 However, the metabolite did not show any
antibacterial activity. Therefore, in this study, we characterized
the antibacterial metabolites produced by S. lamellicola BCP.
Characterization of Substances from the Culture
Broth of S. lamellicola BCP. When investigating the
bioactivities of the EtOAc extract from the S. lamellicola BCP
culture broth, we found that the EtOAc layer significantly
inhibited the growth of R. solanacearum and also contained
trimers and tetramers of (3R,5R)-3,5-dihydroxydecanoic acids
(DDA) as the primary metabolites. Using bioassay-guided
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fractionation, the antibacterial metabolites were isolated from
the EtOAc layer by various chromatographic methods.
of halymecin B,14 except for an additional acetyl group attached
to the C-5 of DDA′. Compound 1 was named halymecin F, and
its chemical structure is presented in Figure 1.
Compound 1, a main component (Figure 1) ([α]20D −11.9°;
c 0.27, MeOH), was obtained as a colorless oil, and its
molecular formula is C50H88O20 as determined by FAB-MS
([M + Na]+ 1032) and ESI-MS ([M + Na]+ 1031.6) data. The
FAB-MS and positive ESI-MS data (Figure 2) of 1 exhibited
Compound 2 ([α]20 −8.99°; c 0.2, MeOH) was named
D
halymecin G. Its molecular formula is C38H68O16 as determined
from the quasi-molecular ion peaks at m/z [M + Na]+ 803.2
and m/z [M − H]− 779.7 in positive and negative ESI-MS
spectra, respectively. These data suggest that 2 lacks one acetyl
group and a DDA compared to 1. The positive-FAB-MS
spectrum of 2 showed significant fragment peaks at m/z 601
[M + H − 180]+, m/z 373 [C20H37O6]+, and m/z 187.1
[C10H19O3]+. The fragmentation of 373 indicated a dimer of
two DDA units without an acetyl group, and these data suggest
that compound 2 is a partial structure of 1 and has an acetyl
1
group located at DDA′. The H and 13C NMR spectra of 2
showed the presence of one acetyl group, one mannosyl
moiety, four carbonyl carbons, and three terminal methyl
groups. The location of the mannose moiety is at the C-3 of
DDA′ via glycosidic linkage as determined by the HMBC
correlation peaks at H-1 (4.61, mannose) with a carbon signal
at 74.94. The stereochemical analysis for the DDAs of 2 was
also performed as described for 1, and the chemically degraded
product was determined to be (3R,5R)-3-hydroxydecan-5-olide.
Analysis of the 1D-NMR spectroscopic data and interpretation
of the HMBC and HSQC allowed us to assign all signals of 2
and identified the compound as a monoacetate trimer of DDA
units (Table 1 and Figure 1). Compound 2 was named
halymecin G.
Figure 2. Fragmentation of halymecin F (1) (C50H88O20, quasi-
molecular ion peak m/z 1031.6 [M + Na]+) generated by ESI-MS.
significant fragment peaks at m/z 829.2 [M + H − 180]+, m/z
601 [M + H − 180 − C12H20O4]+, m/z 583 [601 −H2O]+, m/z
523 [583 − 60]+, m/z 415.3 [C22H39O7]+, m/z 355.3
[C22H39O7 − 60]+, m/z 229.1 [C12H21O4]+, and m/z 187.1
[C10H19O3]+. Hydrolysis of 1 after lactonization yielded a
hydrolysate containing a monosaccharide moiety. TLC analysis
revealed that the sugar moiety is D-mannose on the basis of
comparison to the authentic compound. GC-MS analysis of the
acetylated product of the hydrolysate also confirmed the
presence of pentaacetyl-β-D-mannose. These data suggest that 1
contains β-D-mannose, two acetyl groups (two losses of 60 in
the MS spectrum), and C10H19O3 units. The 1H NMR
spectrum of 1 (Table 1) displayed signals corresponding to
four terminal methyl groups at δ 0.89 (12H, t, J = 7.01), two
acetyl groups at δ 2.01 (3H, s) and 2.02 (3H, s), an anomeric
proton at δ 4.61 (1H, brs), and a region of protons of
oxygenated carbons. The 13C NMR spectrum of 1 revealed the
presence of 1 anomeric carbon at δ 101.18, 13 oxygenated
carbons from δ 62.97 to δ 78.28, 6 carbonyl groups in the range
of δ 172.47−173.63, and the methyl carbons of 2 acetyl groups
at δ 21.24 (Table 1). On the basis of analysis of the COSY and
HMQC spectra, assignment of the β-D-mannose moiety was
verified. The proton spin system of the H1−H6 of each fatty
acid was determined by COSY, and the position of the carbonyl
group was determined by a cross peak between the methylene
protons of the C-2 and the carbon of the carbonyl group. The
long-range correlations of H-3 (δ 4.21, DDA′) with an
anomeric carbon (101.18) on the HMBC spectrum demon-
strated that the β-D-mannose moiety was situated at the C-3 of
DDA′. The linkage of the C-5 with an acetyl group was
deduced from cross peaks between the protons of the acetyl
group and the C-4 (δ 39.84), C-5 (δ 72.78), and C-6 (δ 35.41)
in the HMBC spectrum and the fragment peak at m/z 602 on
the FAB-MS spectrum. The ester linkages between the carbonyl
carbons with the C-5 in the remaining fatty acid chains were
also deduced from the HMBC data. Furthermore, the HMBC
data led to the assignment of the other acetyl group at the C-3
of DDA‴. The lactonization of 1 produced a known δ-lactone
(4), which was identified as 3-hydroxydecan-5-olide. Its carbon
chiral centers were determined as (3R, 5R) on the basis of the
1H and 13C NMR spectra and optical rotary power. A review of
the literature and a comparison of the spectral data of 1 with
mannosyl lipids suggested that the structure of 1 was a tetramer
of 3,5-dihydrodecanoyl and that its structure was similar to that
Compound 3 ([α]20 −2.9°; c 0.176, MeOH) has the
D
molecular formula C16H30O9 as determined by ESI-MS ([M +
Na]+ 389.2 and [M − H]− 365.4). The negative-ESI-MS
spectrum of 3 showed a fragment peak at m/z 179.2 [C10H19O3
− H2O]+, indicating a dehydrated DDA. The 1H NMR
spectrum of 3 exhibited a singlet of the anomeric proton H-1
(mannose) at δ 4.66 and a terminal methyl group at δ 0.91. The
13C NMR spectrum had 16 carbon signals, including 6 carbons
belonging to a β-D-mannose moiety and 10 carbons for one
DDA. The position of the glycosidic linkage of the mannosyl
and DDA was also determined by the HMBC analysis. Finally,
compound 3 was identified as (3R,5R)-3-O-β-D-mannosyl-3,5-
dihydroxydecanoic acid, and its NMR data and structure are
presented in Table 1 and Figure 1.
δ-Lactones 4 and 5 were identified as (+)-(3R,5R)-3-
hydroxy-decan-5-olide (EI-MS m/z 168 [M − H2O]+,
C10H18O3; [α]20 +39.9°; c 0.05, CH2Cl2), and (−)-massoia
D
lactone (EI-MS m/z 168 [M]+, C10H16O2; [α]20D −65.9°; c 0.2,
CH2Cl2),15,16 respectively, on the basis of the analysis of the EI-
MS, GC-MS, and 1D-NMR data (Figure 1; Table S1 and
Figure S3 in the Supporting Information). In a previous study,
compounds 4 and 5 were found as biologically active δ-lactones
and were chemically synthesized from streptenol A. Optical
rotary powers (4, [α]20 +36.9°; c 0.92, CH2Cl2; 5, [α]20
D
D
−112.5°; c 1 CHCl3) and MS and 1D-NMR data of these
synthesized compounds were reported by Romeyke et al.15
Although compounds 4 and 5 are chemically degraded
products of compounds 1−3, their presence in the EtOAc
layer of the S. lamellicola BCP culture broth was also confirmed
with TLC and GC-MS.
Halymecins were previously reported to be tetramers and
trimers of (3R,5R)-3,5-dihydroxydecanoic acid isolated from
Fusarium spp. and Exophiala pisciphila. All of the micro-
organism resources were collected from marine algae and
sponges,14,17 whereas S. lamellicola BCP was isolated as a
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a
1
Table 1. H and 13C NMR (500 MHz) Data of Compounds 1−3 in Methanol-d4
1
2
3
b
b
position
mannose
1′
13C
1H
4.61 brs
13C
1H
13C
1H
4.66 brs
101.18
72.76
75.27
68.56
78.27
62.97
101.23
72.70
75.25
68.60
78.29
62.99
4.61 brs
100.7
72.71
75.21
68.55
78.44
62.93
2′
3′
4′
5′
6′
3.83 m
3.83 m
3.87 m
3.44 m
3.53 m
3.22 m
3.44 m
3.43 m
3.53 m
3.52 m
3.22 m
3.2 m
3.71 m; 3.82 m
3.69 m; 3.83 m
3.69 m; 3.84 m
c
DDA′
1
173.06
41.83
173.14
42.10
74.94
39.82
72.80
35.39b
25.94c
32.79d
23.62
14.42
21.29
172.65
173.06
37.76
78.01
36.63
70.83
33.75
25.65
32.77
23.61
14.41
2
2.57 m; 2.85 m
4.21 m
2.36 m; 2.48 m
4.17 m
2.76 m; 2.84 m
4.67 m
3
74.93
4
39.84
1.74−1.92 m
5.03 m
1.82−1.91 m
5.03 m
1.64−1.73 m
4.40 m
5
72.84
6
35.41b
25.97c
32.83d
23.71e
14.44f
21.24
1.60 brs
1.59 brs
1.62 brs
7
1.31 brs
1.29 brs
1.34 brs
8
1.31 brs
1.29 brs
1.34 brs
9
1.31 brs
1.29 brs
1.34 brs
10
0.89 t (7.01)
2.02 s
0.89 t (7.06)
2.02 s
0.91 t (6.9)
d
CH3CO
CH3CO
172.61
DDA″
1
2
172.47
40.76
173.06
43.19
2.43−2.49 m
4.09 m
2.38−2.48 m
3.54 m
3
66.77a
42.11
66.76a
42.22
4
1.72−1.78 m
5.05 m
1.72−1.79 m
5.07 m
5
73.71
73.13
6
35.10b
25.91c
32.78d
23.60e
14.37
1.61 brs
35.11b
25.94c
32.79d
23.62
1.59 brs
7
1.31 brs
1.29 brs
8
1.31 brs
1.29 brs
9
1.31 brs
1.29 brs
10
0.89 t (7.01)
14.42
0.89 t (7.06)
DDA‴
1
172.63
43.57
175.69
43.55
2
2.48−2.51 m
5.28 m
2.38−2.48 m
3.54 m
3
70.02
66.85a
42.36
4
39.30
1.81 m
1.72−1.79 m
5.04 m
5
72.61
4.93 m
72.86
6
35.14b
25.91c
32.82d
23.66e
14.40f
21.24
1.61 brs
1.31 brs
1.31 brs
1.31 brs
0.89 t (7.01)
2.01 s
35.11b
25.94c
32.79d
23.62
1.59 brs
7
1.29 brs
8
1.29 brs
9
1.29 brs
10
14.42
0.89 t (7.06)
CH3CO
CH3CO
172.47
DDA″″
1
173.16
43.55
2
2.43−2.48 m
4.09 m
3
66.82a
42.37
4
1.72−1.78 m
5.03 m
5
72.84
6
35.06b
25.91c
32.72d
23.59e
14.36f
1.61 brs
7
1.31 brs
8
1.31 brs
9
1.31 brs
10
0.89 t (7.01)
a
The chemical shifts (δ) are in ppm from TSM. The coupling constant J values (in hertzz) are given in parentheses. All assignments are based on the
b
c
COSY, HMQC, and HMBC experiments. Assignments followed by the same letters (a−f) in the same column may be interchangeable. DDA, 3,5-
dihydroxydecanoic acid. CH3CO, acetyl group
d
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Table 2. Antibacterial Activity of the Isolated Compounds against the Growth of Seven Phytopathogenic Bacteria
a
IC50 (95% CI)
name
1
2
3
A. konjaci
22.5(8.5−59.9)
1.58 (0.91−2.74)
33.2 (18.9−58.4)
>100
>100
>100
A. tumefaciens
1.49 (0.18−2.95)
>100
24.8 (16.9−36.4)
b
B. glumae
NI
P. carotovora subsp. carotovora
P. syringae pv lachrymans
R. solanacearum
>100
NI
>100
>100
>100
>100
>100
73.8 (41.4−106.1)
72.1 (40.6−103.6)
75.8 (44.3−107.4)
>100
X. euvesicatoria
a
The antibacterial activity is expressed as the IC50 (concentration causing 50% growth inhibition) as determined by the broth microdilution method.
b
Growth inhibition was observed 2 days after treatment. 95% CI, 95% confidence interval. No inhibition.
mycoparasite of B. cinerea. The present study identifies
compounds 1−3 for the first time.
Antibacterial Activity. The in vitro antibacterial activity of
compounds 1−3 was tested in 96-well microtiter plates, and the
results are presented in Table 2. Compound 1 displayed a
broad spectrum and strong antibacterial activity compared to
compounds 2 and 3. Compound 1 caused growth inhibition of
all seven phytopathogenic bacteria and was highly active against
A. konjaci, A. tumefaciens, and B. glumae, with IC50 values of
25.5, 1.58, and 33.2 μg/mL, respectively. R. solanacearum (IC50
73.8 μg/mL) and X. euvesicatoria (IC50 72.1 μg/mL) were
moderately sensitive to compound 1. However, P. carotovora
subsp. carotovora and P. syringae pv lachrymans were highly
tolerant to this compound. Compound 2 also affected the cell
growth of all bacteria tested. It effectively inhibited the growth
of A. tumefaciens with an IC50 value of 1.49 μg/mL. R.
solanacearum growth was inhibited by compound 2, with an
IC50 value of 75.8 μg/mL. Compound 3 caused good growth
inhibition of A. tumefaciens, but had no effect against B. glumae
and P. carotovora subsp. carotovora. Of the three compounds,
Figure 3. Control efficacy of the ethyl acetate layer of the Simplicillium
compound 1 showed the strongest antibacterial activity against
the phytopathogenic bacteria tested.
lamellicola BCP culture broth in reducing wilting disease severity
caused by Ralstonia solanacearum on tomato seedlings (A) and the
treated plants 9 days after inoculation (B). BCP500, 500 μg/mL;
BCP1000, 1000 μg/mL; and BCP2000, 2000 μg/mL, ethyl acetate
extracts from the S. lamellicola BCP culture broth; ST200, 200 μg/mL
of streptomycin sulfate; CK, untreated control. Each value represents
The EtOAc extract from the S. lamellicola BCP broth was
evaluated for in vivo antibacterial activity against R.
solanacearum on 3-week-old tomato seedlings (Figure 3). The
extract suppressed the development of bacterial wilt in a dose-
dependent manner. R. solanacearum caused severe wilting
damage on seedlings treated with distilled water and 500 μg/
mL EtOAc extract 9 days after inoculation (Figure 3B).
However, the EtOAc extract at concentrations of 1000 and
2000 μg/mL displayed strong control efficacies of 85 and 100%,
respectively, against R. solanacearum 9 days after inoculation.
These disease control efficacies were higher than that of
streptomycin sulfate (200 μg/mL). These results suggest that
the EtOAc extract and the fermentation broth of S. lamellicola
BCP may be used as a natural bactericide to control tomato
bacterial wilt.
Biocontrol provides an alternative to the use of synthetic
agrochemicals and is used as part of an overall integrated plant
disease management program. Several filamentous fungal
species are known mycoparasites against various phytopatho-
genic fungi.18−20 The biocontrol mechanism is most likely due
to the action of cell wall degradative enzymes and secondary
metabolites.21−23 In addition, the microorganisms that produce
microbial polyesters (polyhydroxyalkanoates) have long been
considered important in various sectors, such as the food,
pharmaceutical, cosmetic, fragrance, flavor, and biofuel
industries.17
the mean
standard deviation of three experiments with five
replicates each. Means ( SD) followed by the same letters above the
bars are not significantly different (P = 0.05) in a Tukey’s HSD test.
S. lamellicola BCP was reported to be a biocontrol fungus
with good efficacy against B. cinerea and M. grisea.5,6 S.
lamellicola was previously known as Cephalosporium lamellicola
and Verticillium lamellicola, which exhibited mycoparasitic
interactions with phytopathogenic fungi causing rust dis-
eases.20,24 The strain BCP was also reported to produce
verlamelin that significantly suppressed tomato late blight
caused by P. infestans, wheat leaf rust caused by P. recondita, and
barley powdery mildew caused by B. graminis f. sp. hordei.4,25 In
the present study, we demonstrated that S. lamellicola BCP
produces different antimicrobial metabolites and may be
considered a producer of polyesters of (3R,5R)-3,5-dihydrox-
ydecanoic acid.
For the antimicrobial activities of substances containing
(3R,5R)-3,5-dihydroxydecanoic acid, verbalactone isolated from
the plant roots of Verbascum undulatum exhibited moderate
antibacterial activity against Salmonella enteritidis, Staphylococcus
aureus, and S. epidermidis.26 Only a few tetramers and trimers of
(3R,5R)-3,5-dihydroxydecanoic acid from three fungal strains
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Journal of Agricultural and Food Chemistry
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have previously been reported. Halymecins A, B, and C were
isolated from Fusarium spp. FE-71-1, and halymecins D and E
were isolated from Acremonium spp. FK-N30. Of these
compounds, halymecin A is a tetramer of DDA and causes
growth inhibition of bacteria, such as Enterococcus faecium,
Klebsiella pneumoniae, and Proteus vulgaris. Halymecin A also
showed strong antimicroalgal and cytotoxic activities.14
Exophilin A was reported to be a trimer of (3R,5R)-3,5-
dihydroxydecanoic acid isolated from Exophiala pisciphila
NI10102. The trimer displayed antimicrobial activity against
some Gram-positive bacterial strains, such as Enterococcus
faecium and Staphylococcus aureus.17 In our study, the mannosyl
lipids halymecin F (1), halymecin G (2), and (3R,5R)-3-O-β-D-
mannosyl-3,5-dihydroxydecanoic acid (3) were novel mannosyl
lipids isolated from S. lamellicola BCP with antibacterial
activities against phytopathogenic bacteria.
In addition, we successfully isolated and confirmed the
presence of massoia lactone (5), an active metabolite member
of the Trichoderma family,23 in the culture broth of S. lamellicola
BCP. Massoia lactone represents a minor component of the
crude extracts of various Trichoderma spp., including
Trichoderma viride. This compound was patented because it
controls B. cinerea and Phytopthora species.22 The role of
Trichoderma antimicrobial metabolites in biocontrol remains
unclear. Some of these compounds may be the major
contributor to biocontrol activity for a certain strain but not
for other strains.21 The presence of massoia lactone (5) in S.
lamellicola BCP suggests a relationship between the biocontrol
mechanism of the strain and Trichoderma species. Our findings
confirmed the production of mannosyl lipids 1−3 and massoia
lactone from S. lamellicola BCP and the antibacterial activities
of the novel mannosyl lipids against phytopathogenic bacteria.
This strain may be considered a producer of polyesters of
(3R,5R)-3,5-dihydroxydecanoic acid. The presence of the three
novel mannosyl lipids in the fermentation broth cultures of S.
lamellicola BCP was demonstrated in this study. Furthermore,
the in vivo activities of the EtOAc extract against tomato
bacterial wilt in a climate-controlled room and disease control
of the microbial biopesticide Acre against tomato bacterial wilt
in fields were also elucidated. These results suggest that S.
lamellicola BCP and its mannosyl lipids could be used to
control plant diseases caused by phytopathogenic bacteria.
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ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectral data and EI-MS spectrum of compound 4, in
vivo antifungal activity of ABE, and neighbor-joining trees
based on sequence analysis of the 28S rRNA gene and ITS
regions. This material is available free of charge via the Internet
at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
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*(J.-C.K.) Phone: +82-42-860-7436. Fax: +82-42-861-4913. E-
mail: kjinc@krict.re.kr.
Funding
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Garcia, A. Effect of mycoparasitic fungi on the development of
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Effects of Simplicillium lanosonivieum on Phakopsora pachyrhizi, the
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This study was performed with support from the Cooperative
Research Program for Agricultural Science and Technology
Development (Project PJ010207022014), Rural Development
Administration, Republic of Korea.
Notes
The authors declare no competing financial interest.
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