Isolation and identification of antifungal fatty acids from the basidiomycete Gomphus floccosus

Article abstract of DOI:10.1021/jf8008662

Bioautography of extracts of the fruiting bodies of the basidiomycete Gomphus floccosus (Schw.) Singer indicated the presence of fungitoxic compounds in the ethyl acetate fraction against the plant pathogens Colletotrichum fragariae, Colletotrichum gloeosporioides, and Colletotrichum acutatum. Bioassay-guided fractionation of this fraction resulted in the isolation of the bioactive fatty acids (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (1), (9E,11Z)-13-oxo-9,11-octadecadienoic acid (2), and (10E,12E)-9-oxo-10,12- octadecadienoic acid (3). These three oxylipins were further evaluated for activity against a greater range of fungal plant pathogens (C. fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea, Fusarium oxysporum, Phomopsis obscurans, and Phomopsis viticola) in in vitro dose-response studies. Phomopis species were the most sensitive fungi to these compounds. At 120 h of treatment, the IC₅₀ values for compounds 1, 2, and 3 for P. obscurans were 1.0, 4.5, and 23 μM, respectively, as compared to 1.1 μM for the captan positive control.

Full text of DOI:10.1021/jf8008662

5062 J. Agric. Food Chem. 2008, 56, 5062–5068
Isolation and Identification of Antifungal Fatty Acids
from the Basidiomycete Gomphus floccosus
,
†
†
‡
CHARLES L. CANTRELL,* BETHANY P. CASE, E. EDWARD MENA,
‡
†
†
TOBY M. KNIFFIN, STEPHEN O. DUKE, AND DAVID E. WEDGE
Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of
Agriculture, University, Mississippi 38677, and LifePharms Inc., Groton, Connecticut 06340
Bioautography of extracts of the fruiting bodies of the basidiomycete Gomphus floccosus (Schw.)
Singer indicated the presence of fungitoxic compounds in the ethyl acetate fraction against the plant
pathogens Colletotrichum fragariae, Colletotrichum gloeosporioides, and Colletotrichum acutatum.
Bioassay-guided fractionation of this fraction resulted in the isolation of the bioactive fatty acids
(
(
9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (1), (9E,11Z)-13-oxo-9,11-octadecadienoic acid
2), and (10E,12E)-9-oxo-10,12-octadecadienoic acid (3). These three oxylipins were further evaluated
for activity against a greater range of fungal plant pathogens (C. fragariae, C. gloeosporioides, C.
acutatum, Botrytis cinerea, Fusarium oxysporum, Phomopsis obscurans, and Phomopsis viticola) in
in vitro dose-response studies. Phomopis species were the most sensitive fungi to these compounds.
At 120 h of treatment, the IC50 values for compounds 1, 2, and 3 for P. obscurans were 1.0, 4.5, and
23 µM, respectively, as compared to 1.1 µM for the captan positive control.
KEYWORDS: Gomphus floccosus; Botrytis cinerea; Fusarium oxysporum; Phomopsis obscurans;
Colletotrichum fragariae; Colletotrichum gloeosporioides; Colletotrichum acutatum; fatty acid; mushroom
INTRODUCTION
above-ground spore production and dispersal. They are
represented by almost all of the known 20000 species of
basidiomycetes. Furthermore, the majority of the 50000
species of ascomycetes are either lichen-associated fungi or
mushrooms. Thus, mushrooms represent two distinct branches
of the fungal kingdom and are virtually untapped sources of
novel, small molecules. Mushrooms, including shelf fungi
and other fungi that produce visible fruiting bodies, have been
largely uninvestigated by those searching for natural products
for pest management.
Evolved resistance to commercially available fungicides
and the desire for environmentally and toxicologically safer
agrochemicals are providing an impetus for fungicide dis-
covery (1). Natural product-based pesticides offer several
advantages, in that some are specific toward target species,
and they often have unique modes of action with little
mammalian toxicity. An additional benefit is that they
generally have a relatively short environmental half-life as
compared to synthetic pesticides.
Mushrooms have extremely complex nutritional require-
ments as compared with the more easily cultured fungi. Many
of them form mycorrhizal associations with plants and grow
poorly without their host, while others are obligate mycor-
rhizal species that perish without their micorrhizal associa-
tions (3). In their natural habitats, mushrooms are challenged
by a wide variety of pests, such as bacteria, nematodes,
insects, browsing animals, and other fungi. As a result, these
fungi produce a wide and virtually unexplored variety of
defensive chemicals. Investigating field-collected fungal
fruiting bodies has the potential to yield new secondary
metabolites, which may be used as novel chemical plant
protectants against pathogens or other pests.
Fungi are a source of novel compounds (1, 2). Most discovery
efforts have relied on culturing the fungus to supply natural
products; however, less than 1% of the estimated 1.6 billion
species of fungi have been cultured. Most culture collections
and screening practices have been carried out on easily cultured
zygomycetes and primitive ascomycete species (3). Hence,
discovery efforts that limit themselves to those species that can
be cultured are sampling an extremely narrow part of the total
fungal spectrum.
Mushrooms represent a fungal group that are composed
of diploid mycelial filaments that unite with other individuals
of the same species to form a highly specialized reproductive
organ (the fruiting body or sporocarp) that is dedicated to
We initially evaluated approximately 6000 sporocarp extracts
for their antifungal activity against the plant pathogens Colle-
totrichum acutatum, Colletotrichum fragariae, and Colletotri-
chum gloeosporioides. Among the lead candidates was the
basidiomycete woolly chanterelle, Gomphus floccosus (Schw.)
*
To whom correspondence should be addressed. Tel: +1-662-915-
5
898. Fax: +1-662-915-1035. E-mail: charles.cantrell@ars.usda.gov.
†
U.S. Department of Agriculture.
LifePharms Inc.
‡
1
0.1021/jf8008662 CCC: $40.75  2008 American Chemical Society
Published on Web 06/17/2008

Antifungal Fatty Acids from the Basidiomycete G. floccosus
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5063
Table 1. Fungal Growth Inhibition for G. floccosus Crude Extract and
Isolated Oxylipins Using Direct Bioautography
Bioassay-Guided Fractionation. Initially, 1.4 g of crude ethyl
acetate extract was dissolved in 100 mL of CH
placed into a 500 mL separatory funnel. The phase was extracted thrice
using 100 mL portions of hexane, dried over MgSO , and evaporated
to dryness providing 872 mg of material. A final percent composition
of 70/30 (CH OH/H O) was obtained by adding 28.5 mL of H O to
the aqueous phase. This aqueous phase was again extracted thrice using
00 mL portions of CHCl , dried over MgSO , and evaporated to
dryness providing 371 mg of material. CH OH was removed from the
3 2
OH/H O (90/10) and
a
diameter of zone of inhibition (mm)
4
concentration
test material
crude extract
(µg)
C. acutatum C. fragariae C. gloeosporioides
3
2
2
80
diffuse zone
diffuse zone
19 ( 0.6
7.7 ( 0.6 diffuse zone
4.3 ( 0.6 n/a
10
1
3
4
compound 1
compound 2
compound 3
80
10.0 ( 0.6 7.0 ( 0.6
6.0 ( 0.6 3.0 ( 0.6
8.0 ( 0.6 7.0 ( 0.6
6.0 ( 0.6 6.0 ( 0.6
6.0 ( 0.6 7.0 ( 0.6
4.0 ( 0.6 6.0 ( 0.6
20
5.0 ( 0.6
19 ( 0.6
3
80
aqueous phase by rotary evaportation, returned to the separatory funnel,
and extracted thrice using 50 mL portions of ethyl acetate. Drying with
20
4.0 ( 0.6
17.0 ( 0.6
8.0 ( 0.6
80
MgSO
O was removed from the aqueous phase by freeze drying providing
1 mg.
A portion of the CHCl
a reversed-phase C-18 HPLC column (Zorbax, 9.4 mm × 250 mm, 5
µm) running a linear gradient from 40/60 (H O with 0.1% trifloroacetic
acid/acetonitrile) to 0/100 (H O with 0.1% trifloroacetic acid/acetoni-
4
and evaporation of the ethyl acetate provided 7 mg of material.
20
H
4
2
a
Mean dimensions of zones (mm) of fungal inhibition ( SD. Benomyl, captan,
cyprodinil, and azoxystrobin served as positive controls; data not shown.
3
phase from above was further purified using
Singer (Cantharellaceae) (syn. Cantharellus floccosus). In
previous phytochemical investigations of G. floccosus, the
constituents responsible for the delayed gastrointestinal distur-
bances in individuals consuming the fruiting bodies were
isolated (4, 5). Norcaperatic acid was isolated and found to be
the cause of this effect. An additional paper reported the
presence of norcaperatic acid and mannitol from G. floccosus
2
2
trile). Three compounds were collected providing 14 mg of 1, 13 mg
of 2, and 8 mg of 3.
(
9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic Acid (1). High-
-
resolution ESI-MS m/z 295.2296 [M - H] , calculated for C18
95.2273; m/z 591.4624; [2M - H] , calculated for C36
91.4625. H NMR (400 MHz in CDCl ): δ 6.44 (dd, 1H, J ) 11.2,
H
31
O
O
3
,
,
-
2
5
1
6
1
1
H
63
6
1
3
(
6). Mannitol was also reported in this species by Dominguez
5.2 Hz, H-11), 5.93 (t, 1H, J ) 10.8 Hz, H-12), 5.62 (dd, 1H, J )
.8, 15.2 Hz, H-10), 5.42 (dt, 1H, J ) 7.6, 10.8 Hz, H-13), 4.11 (m,
H, H-9), 2.30 (t, 2H, J ) 7.2 Hz), 2.15 (m, 2H), 2.05 (m, 2H, H-8),
.6 (m, 2H, H-3), 1.2-1.4 (overlapping), 0.80 (t, 3H, J ) 6.8 Hz, H-18)
et al. (7). Min et al. (8) reported that an ethanol extract of G.
floccosus had antifungal activity against Microsporum gypseum,
with an MIC of 1 mg/mL; however, the active constituents were
not identified. In this paper, we report the identity and antifungal
activity of several fatty acids isolated by bioassay-guided
fractionation of G. floccosus.
1
3
(9). C NMR (100 MHz in CDCl ): δ 179.6 (s, C-1), 135.8 (d, C-10),
3
133.2 (d, C-13), 127.9 (d, C-12), 126.1 (d, C-11), 73.1 (d, C-9), 37.5
(t), 34.2 (t, C-2), 31.7 (t, C-17 or 16), 24.5 (t), 29.3 (t), 29.2 (t), 29.1
(
t), 27.9 (t, C-14?), 25.5 (t), 24.9 (t, C-3), 22.7 (t, C-17 or 16), 14.2 (q,
C-18).
9E,11Z)-13-Oxo-9,11-octadecadienoic Acid (2). High-resolution
MATERIALS AND METHODS
(
1
13
Instrumentation. H and C NMR spectra were recorded in CDCl
and/or C on a Varian 400 MHz spectrometer model number
EUR0020 (Palo Alto, CA). All C multiplicities were deduced from
0 and 135° DEPT experiments. Gas chromatography-mass spec-
3
-
ESI-MS m/z 293.2128 [M - H] , calculated for C18
87.4323 [2M - H] , calculated for C36
29 3
H O , 293.2117;
H O , 587.4312. H NMR
59 6
6
D
6
-
1
5
(
1
7
1
3
400 MHz in CDCl ): 7.49 (dd, 1H, J ) 11.4, 15.4 Hz, H-11), 6.16 (d,
3
9
H, J ) 15.2, H-12), 6.11 (d, 1H, J ) 11.2, H-10), 5.90 (dt, 1H, J )
.6, 10.8 Hz, H-9), 2.54 (t, 2H, J ) 7.4), 2.32 (m, 2H), 1.62 (m, 2H,
trometry (GC-MS) analysis was performed on a Varian CP-3800 GC
coupled to a Varian Saturn 2000 MS/MS. High-resolution mass spectra
were obtained using an Agilent 1100 HPLC coupled to a JEOL
AccuTOF (JMS-T100LC) (Peabody, MA). High-performance liquid
chromatography (HPLC) method development work was performed
using an Agilent 1100 system equipped with a quaternary pump,
autosampler, diode array detector, and vacuum degasser. Semiprepara-
tive HPLC purifications were performed using a Waters Delta-Prep
system (Milford, MA) equipped with a diode array detector and a binary
pump while monitoring at 254 nm.
1
3
J ) 10.4), 1.31 (d, 2H, J) 4.4), 0.88 (t, 3H, J ) 6.6, H-18) (10).
C
NMR (100 MHz in CDCl
3
): 201.3 (s, C-13), 179.2 (s, C-1), 142.9 (d,
C-9), 137.3 (d, C-11), 129.6 (d, C-12), 127.1 (d, C-10), 41.2 (t, C-14),
4.1 (t, C-2), 31.6 (t), 29.3 (t), 29.4 (t), 29.3 (t), 29.1 (t), 28.6 (t, C-8),
4.8 (t, C-3), 24.5 (t, C-15), 22.7 (t, C-17), 14.2 (q, C-18).
3
2
(
10E,12E)-9-Oxo-10,12-octadecadienoic Acid (3). High-resolution
-
ESI-MS m/z 293.2130 [M - H] , calculated for C18
H
30
O
3
, 293.2117.
): 7.20 (dd, J ) 10.5, 15.2, H-11), 6.04 (d,
J ) 16, H-10), 5.96 (dd, J ) 10.4, H-12), 5.8 (dt, J ) 6.8, 15.0, H-13),
1
6 6
H NMR (400 MHz in C D
GC-MS Analysis. Reaction products were analyzed by GC-MS
using a DB-5 column (30 m × 0.25 mm fused silica capillary column,
film thickness of 0.25 µm) operated using the following conditions:
injector temperature, 240 °C; column temperature, 60-240 °C at 3
2
1
(
(
.73 (t, J ) 6.8, H-2), 2.08 (d, J ) 3.6, H-14), 1.88 (d, J ) 6.4, H-8),
.59 (t, J ) 6.8, H-15), 1.48 (s, H-3), 1.27-1.10 (4-7, 16, 17), 0.92
13
t, J ) 7.2, H-18). C NMR (100 MHz in C
6 6
D ): 199.6 (s, C-9), 180.1
s, C-1), 145.2 (d, C-13), 142.7 (d, C-11), 129.7 (d, C-12), 128.8 (d,
°
C/min then held at 240 °C for 5 min; carrier gas, He; and injection
C-10), 41.2 (t, C-8), 33.6 (t, C-2), 32.0 (t, C-14), 30.9 (t), 30.0 (t),
9.7 (t), 29.5 (t), 29.0 (d,C-4), 25.3 (t, C-5), 23.2 (t, C-17), 14.6 (q,
C-18).
Diazomethane Generation. An Aldrich (St. Louis, MO) Mini
volume, 5 µL (splitless).
2
High-Resolution LC-MS Analysis. All isolated compounds were
dissolved in MeOH and injected directly into a 0.3 mL/min stream of
a MeOH. Twenty microliters of sample (approximately 0.1 mg/mL)
was injected manually at 0.5 min, while mass drift compensation
standards (L-tryptophan) were injected at 1.5 min over the course of a
Diazald Apparatus was used for the production of diazomethane in
ether. Briefly, 2.5 g of KOH was dissolved in 4 mL of deionized water
and placed in the reaction vessel followed by the addition of 5 mL of
ethanol. A separatory funnel containing 2.5 g of diazald dissolved in
22.5 mL of ether was placed above the reaction vessel. The reaction
vessel was warmed to 65 °C using a water bath followed by the
dropwise addition of the diazald solution over a period of 50 min. The
receiving flask and condenser coldfinger were cooled using a dry ice/
acetone bath. The codistilled diazomethane in ether solution was stored
in sealed vials at -20 °C until needed.
2
min run. Mass drift compensations were performed relative to
-
-
-
L-tryptophan [M - H] , [2M - H] , and [3M - H] ions for negative
ion analysis.
Raw Material and Extract Preperation. Fruiting bodies of G.
floccosus were collected at Sleeping Giant State Park in Hamden,
Connecticut. Their identity was verified by Ed Bosman, mycology
advisor and founder of the Connecticut Valley Mycology Society. (A
voucher specimen was deposited at the New York Botanical Garden
Herbarium). The collection (LifePharms assession #3641) totaled 555 g
wet weight. The sample was lyophilized (62 g), pulverized, and
extracted with ethyl acetate (20 wt %/vol). The ethyl acetate was
evaporated to give a residue (1.4 g).
Methylation of Linoleic Acids. A 1.0 mg solution of fatty acid 1,
2, or 3 in 2.0 mL of MeOH was treated at room temperature with
a solution of CH
2 2
N in diethyl ether (2 mL). The solution was placed
in a laboratory fume hood overnight to complete the reaction and

5064 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Cantrell et al.
Figure 1. Representative HPLC chromatogram (254 nm) used during the purification of the chloroform partition. (9S,10E,12Z)-9-Hydroxy-10,12-
octadecadienoic acid (1), (9E,11Z)-13-oxo-9,11-octadecadienoic acid (2), and (10E,12E)-9-oxo-10,12-octadecadienoic acid (3) identified bioactive compounds
were purified.
Figure 2. Growth inhibition/stimulation of C. acutatum after 48 and 72 h using 96 well microtiter format in a dose response to 1-3 and the commercial
fungicide standards azoxystrobin, benomyl, and captan. Means from percent growth inhibition/stimulation were pooled from two experiments replicated
in time.
allow for evaporation of solvent and CH
2
N
2
. Methyl ester products
Preparation of the Trimethylsilylated Linoleic Acid Methyl
Esters. Following methylation of compounds 1, 2, or 3, solvent was
evaporated, 0.3 mL of BSTFA containing 1% trimethylchlorosilane
1
were dissolved in CH
NMR.
2
Cl
2
and analyzed by GC-MS and
H

Antifungal Fatty Acids from the Basidiomycete G. floccosus
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5065
Figure 3. Growth inhibition of P. obscurans after 144 h using 96 well microdilution broth in a dose response to 1-3 and the commercial fungicide
standard captan. Percent growth inhibition means were pooled from two experiments replicated twice in time.
Figure 4. Growth inhibition of P. viticola after 144 h using 96 well microdilution broth assay in a dose response to 1-3 and the commercial fungicide
standard captan. Percent growth inhibition means were pooled from two experiments replicated twice in time.
was added, and the mixture was carefully heated in an oven for 60
Table 2. Concentrations of Test Compounds Causing 50% Growth
Reduction (IC50) Determined from Dose-Response Studies Using a 96
Well, Liquid Bioassay
min at 60 °C. Secondary containment was used as a precaution. The
solvents were evaporated under N
and analyzed by GC-MS.
2
, dissolved in methylene chloride,
Fungal Isolates and Media. Isolates of C. acutatum Simmonds, C.
fragariae Brooks, and C. gloeosporioides (Penz.) Penz. & Sacc. were
obtained from Barbara J. Smith (Agricultural Research Service, U.S.
Department of Agriculture, Popularville, MS). Cultures of Phomopsis
Viticola and Phomopsis obscurans were obtained from Mike Ellis (Ohio
State University, OH), and Botrytis cinerea Pers. and Fusarium
oxysporum Schlechtend were isolated in our laboratory at Oxford,
Mississippi. F. oxysporum identification was confirmed by Wade H.
Elmer (Connecticut Agricultural Experiment Station, New Haven, CT),
and the identity of Botrytis cinerea was confirmed by Kenneth Curry
IC50 (µM)
time
compound compound compound
organism
B. cinerea
point (h)
1
2
3
captan
48
72
48
>333
>333
>333
>333
>333
>333
>333
>333
>333
>333
10
120
>333
>333
>333
160
90
1.4
2.8
2.5
5.2
1.6
3.0
2.1
4.7
2.8
16
>333
>333
>333
91
>333
>333
>333
>333
>333
23
C. acutatum
C. fragariae
72
48
72
270
C. gloeosporioides
F. oxysporum
P. obscurans
P. viticola
48
>333
>333
>333
>333
(
University of Southern Mississippi, Hattiesburg, MS). The three
72
Colletotrichum species and P. obscurans were isolated from strawberry
48
(
Fragaria × ananassa Duchesne), while P. Viticola and Botrytis cinerea
72
were isolated from commercial grape (Vitis Vinifera L.) and F.
oxysporum from orchid (Cynoches sp.). Fungi were grown on potato
dextrose agar (PDA, Difco, Detroit, MI) in 9 cm Petri dishes and
incubated in a growth chamber at 24 ( 2 °C under cool-white
120
144
120
144
4.5
1.1
2.0
<1.0
<1.0
6.0
>333
100
10
115
38
26
110
65
2
fluorescent lights (55 ( 5 µmol/m /s) with a 12 h photoperiod.

5066 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Cantrell et al.
Conidia Preparation. Conidia were harvested from 7-10 day old
fungal growth inhibition using bioautography against C. fragar-
iae and diffuse zones against both C. acutatum and C.
gloeosporioides (Table 1). Diffuse inhibitory zones are regions
on the bioautography plate where fungal growth is visually
reduced and interspersed with few mycelia and reproductive
structures and are often characteristic of fungistatic compounds.
These results indicated potential “selective” activity of G.
floccosus extract to C. fragariae.
cultures by flooding plates with 5 mL of sterile distilled water and
dislodging conidia by softly brushing the colonies with an L-shaped
plastic rod. Aqueous conidial suspensions were filtered through sterile
Miracloth (Calbiochem-Novabiochem Corp., La Jolla, CA) to remove
mycelia. Conidia concentrations were determined photometrically (11, 12)
from a standard curve based on absorbance at 625 nm, and suspensions
were adjusted with sterile distilled water to a concentration of 1.0 ×
6
1
0 conidia/mL.
Standard conidial concentrations were determined from a standard
The ethyl acetate extract was partially dissolved in methanol/
water (90/10) and extracted thrice using hexane followed by
three extractions using chloroform and three with ethyl acetate.
Biological evaluation of the partitions using bioautography
against C. acutatum, C. fragariae, and C. gloeosporioides was
conducted. The chloroform extract had the highest activity
against all three species. This chloroform extract was further
purified using semipreparative reversed phase HPLC utilizing
an acetonitrile/water gradient providing three compounds 1, 2,
and 3 (Figure 1).
curve for each fungal species. Standard turbidity curves were periodi-
cally validated using both a Bechman/Coulter Z1 particle counter and
hemocytometer counts. Conidial and mycelial growth for microdilution
broth experiments were evaluated using a Packard model SpectraCount
microplate photometer (Packard Instrument Co., Meriden, CT).
Direct Bioautography. A number of bioautography techniques were
used as primary screening systems to detect antifungal activity. Matrix,
one-dimensional (1D) protocols on silica gel thin-layer chromatography
(
TLC) plates along with Colletotrichum spp. as the test organisms were
used to identify the antifungal activity according to published
methods (13, 14). Matrix bioautography was used to screen large
numbers of crude extract at 80 µg/spot. One-dimensional TLC was
subsequently used to purify and identify the number of antifungal agents
Negative ion high-resolution LC-MS indicated that compound
1
gave a parent molecular ion of m/z 295.2296 corresponding
-
to [M - H] and suggesting a possible molecular formula for
1 of C18H32O3 and three sites of unsaturation. H NMR analysis
in extracts. Each plate was subsequently sprayed with a spore suspension
1
5
(
10 spores/mL) of the fungus of interest and incubated in a moisture
indicated the presence of four olefinic protons, one oxygenated
chamber for 4 days at 26 °C with a 12 h photoperiod. Clear zones of
fungal growth inhibition on the TLC plate indicated the presence of
antifungal constituents in each extract. Fungal growth inhibition was
evaluated 4-5 days after treatment by measuring zone diameters.
Antifungal metabolites were readily located on the plates by visually
observing clear zones where the active compounds inhibited fungal
growth (15).
Microdilution Broth Assay. A reference method [M27-A from the
National Committee for Clinical Laboratory Standards (NCCLS)] for
broth dilution antifungal susceptibility testing of yeast was adapted for
evaluation of antifungal compounds against sporulating filamentous
fungi (12). This 96 well microdilution assay was used to determine
and compare the sensitivity of fungal plant pathogens to natural and
synthetic compounds with known fungicidal standards (16).
A 96 well microdilution assay was used to determine the sensitivity
of C. acutatum, C. fragariae, C. gloeosporioides, F. oxysporum, B.
cinerea, P. obscurans, and P. Viticola to the various antifungal agents
isolated. The fungicide captan was used as a positive standard in all
assays. Azoxystrobin and benomyl were used in some in addition to
captan in some studies. Each fungal species was initially challenged
in microdilution broth assay using three-point dose-response format
so that the final test concentrations of 45, 90, and 135 µM were achieved
methine, a methyl triplet, and several aliphatic methylene
1
3
protons. C and DEPT NMR analysis indicated the presence
of one carbonyl, four olefinic carbons, one hydroxylated carbon,
one methyl, and several methylene carbons.
The above data indicated that 1 was possibly a hydroxylated
octodecadienoic acid isomer, and reaction with diazomethane
and conversion to its corresponding trimethylsilyl ether provided
a compound that gave a parent molecular ion of m/z 382 when
analyzed by GC-MS. The above reactions confirmed the
presence of both carboxylic acid and hydroxyl functional groups
1
and inspection of the literature, and comparison of H NMR
data with that previously reported (9) indicated complete
agreement providing structural confirmation of 1 as (9S,10E,12Z)-
9
-hydroxy-10,12-octadecadienoic acid. Stereochemistry at C-9
was established by comparison of optical rotation data with that
which had been previously reported for 1 (23). We report here
13
for the first time C NMR data in CDCl3 for 1 along with
1
1
carbon assignment data established by inspection of H- H
correlation spectroscopy (COSY), heteronuclear single quantum
coherence (HSQC), and heteronuclear multiple-bond correlation
(HMBC) spectral data.
(
in duplicate) in the different columns of the 96 well plate. Subse-
quently, six-point formats with the final test compound concentrations
of 1, 3.3, 10, 33, 100, and 333 µM were used to establish IC50 values.
Experiments were performed twice, and the mean values were pooled
for all experiments. Linear growth phases were utilized for computing
activities of both the standard positive control, captan, and the test
compounds; all solvents were tested for their effects on fungal growth
previously when the linear phase of growth for each isolate was
determined (12).
Fungal growth was evaluated by measuring the absorbance of each
well at 620 nm at 0, 24, 48, and 72 h, except for P. obscurans and P.
Viticola, where the data were also recorded at 120 and 144 h. Mean
absorbance values and standard errors were determined and represented
graphically, and the graphs were used to evaluate fungal growth
inhibition. Differences in spore germination and mycelial growth in
each of the wells in the 96 well plate demonstrated sensitivity to
particular concentrations of pure compounds and indicated fungistatic
or fungicidal effects.
Negative ion high-resolution LC-MS analysis of compound
2
gave a parent molecular ion of m/z 293.2128 corresponding
-
to [M - H] and suggesting a possible molecular formula for
2
1
of C18H30O3 and four sites of unsaturation. H NMR analysis
indicated the presence of four olefinic protons, a methyl triplet,
1
3
and several aliphatic methylene protons. C and DEPT NMR
analysis indicated the presence of two carbonyls one being a
ketone, four olefinic carbons, one methyl, and several methylene
carbons. The above data indicated 2 was likely an oxo-
octodecadienoic acid isomer, and inspection of the literature
1
13
and comparison of H and C NMR data with that previously
reported (11, 17) indicated complete agreement providing
structural confirmation of 2 as (9E,11Z)-13-oxo-9,11-octadeca-
dienoic acid.
Negative ion high-resolution LC-MS of compound 3 gave a
parent molecular ion of m/z 293.2130 corresponding to [M -
RESULTS AND DISCUSSION
-
H] and suggesting a possible molecular formula for 3 of
1
An ethyl acetate extract of the fruiting bodies of G. floccosus
contained sufficient quantities of crude extract for chemical
investigations. Spots of 10 and 80 µg generated clear zones of
C18H30O3 and four sites of unsaturation. H NMR analysis
indicated the presence of four olefinic protons, a methyl triplet,
1
3
and several aliphatic methylene protons. C and DEPT NMR

Antifungal Fatty Acids from the Basidiomycete G. floccosus
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5067
analysis indicated the presence of two carbonyls one being a
ketone, four olefinic carbons, one methyl, and several methylene
carbons. The above data indicated that 3 was also an oxo-
octodecadienoic acid isomer. Inspection of the literature and
or no activity against seven bacterial plant pathogens and 3 to
be mildly active to three of the test species. In their study, 1
and 3 had good activity against Phytophthora parasitica and
Cladosprium herbarum, with little or no activity against B.
cinerea, Phytophthora infestans, F. oxysprorum, Rhizopus spp.,
or Alternaria brassicicola. Although these workers used only
one dose, making it difficult to compare results, their findings
do not conflict with ours. Their conclusions were that this family
of compounds can contribute to plant protection not only by
the induction of defensive responses to plant pathogens but also
by direct antimicrobial activity in some cases.
In the present study, we used a bioassay-guided approach to
isolate active antifungal metabolites from the basidiomycete G.
floccosus, confirming the presence of some oxylipins in the
sporocarp of this basidiomycete. Oxylipins are known to be
important in fungal life cycle processes, including basidi-
omycetes (e.g., 22). Our study demonstrated that Phomopsis
species are most sensitive to compounds 1-3, with other fungi
showing considerably less sensitivity. Our results suggest that
the oxylipins might deserve further testing for activity as plant
protectants to control Phomopsis species, which cause small
fruit blight, leaf spot, and rot. Because oxylipins are quite
common as phytochemicals in the food supply, naturally
occurring ones might have reduced regulatory requirements for
approval.
1
comparison of mass spectrometry fragmentation data and H
1
3
and C NMR data with that previously reported (18) indicated
complete agreement providing structural confirmation of 3 as
(
10E,12E)-9-oxo-10,12-octadecadienoic acid.
The activities of the three compounds were evaluated
individually in a 96 well microdilution format against the three
Colletotrichum species used in the bioautography trials, as well
as F. oxysporum, B. cinerea, P. obscurans, and P. Viticola.
Bioautographical data were collected after 4 days of growth,
and microtiter data were collected at 24, 48, 72, 120, and 144 h,
depending on the growth curves for each organism. The most
sensitive species were those in the genus Phomopsis, with P.
obscurans being most susceptible.
Bioautography of compound 1 demonstrated clear zones of
antifungal activity against all three Colletotrichum species
(
Table 1). In the microtiter plate bioassays, compound 1
inhibited growth of C. acutatum 79% at a dose of 90 µM after
8 h (Figure 2). This activity dropped to 37% after 72 h. Doses
4
lower and higher than this had significant activity, which also
decreased over time. Stimulation of growth at a subtoxic
concentration (hormesis) is common with fungicides (e.g., 19).
In fact, the term hormesis was coined in a study with a natural
product fungicide (20). Against the other two species of
Colletotrichum in the microtiter format, compound 1 stimulated
growth at doses up to 100 µM and had no effect at 333 µM
ACKNOWLEDGMENT
We thank Solomon Green III, Amber Callahan, Dewayne
Harries, and Linda Robertson for technical assistance.
(
see the Supporting Information). Compound 1 also showed
hormetic activity against F. oxysporum, although stimulation
was highest at the highest dose (333 µM) tested in this case
Supporting Information Available: Figures of growth inhibi-
tion/stimulation and six-point inhibition curves. This material
is available free of charge via the Internet at http://pubs.acs.org.
(
see the Supporting Information). Concentrations higher than
this were not tested, since they would be unrealistic for potential
practical use. Against P. obscurans, compound 1 caused
increasing inhibition with increasing dose at 144 h (Figure 3).
Against P. Viticola at 144 h of treatment, doses of 1-10 µM
were hormetic, and those of 33-333 µM were increasingly
inhibitory with concentation (Figure 4).
Compounds 2 and 3 appeared slightly more active in the
microtiter environment than compound 1 (Figures 2–4). How-
ever, they had similar activity to 1 in the bioautography assay
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Received for review March 19, 2008. Revised manuscript received April
3, 2008. Accepted April 28, 2008.
2
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