Biotransformation of the Brassica Phytoalexin Brassicanal A by the Blackleg Fungus

Article abstract of DOI:10.1021/jf960098u

The biotransformation of the brassica phytoalexin brassicanal A by the blackleg fungus [Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm] was investigated. Three main biotransformation products were detected and isolated; their chemical structures were determined by spectroscopic methods and concomitant synthesis. Additionally, the antifungal activities of brassicanal A and its biotransformation products were compared. Overall, the biotransformation pathway suggests that the blackleg fungus has enzymes to carry out this biotransformation different from those involved in the biotransformation of the brassica phytoalexin brassinin.

Full text of DOI:10.1021/jf960098u

J. Agric. Food Chem. 1996, 44, 3403
−3407
3403
Biotr a n sfor m a tion of th e Br a ssica P h ytoa lexin Br a ssica n a l A by th e
Bla ck leg F u n gu s
M. Soledade C. Pedras* and Abdul Q. Khan
Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon, Saskatchewan S7N 5C9, Canada
The biotransformation of the brassica phytoalexin brassicanal A by the blackleg fungus [Lep-
tosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm] was
investigated. Three main biotransformation products were detected and isolated; their chemical
structures were determined by spectroscopic methods and concomitant synthesis. Additionally, the
antifungal activities of brassicanal A and its biotransformation products were compared. Overall,
the biotransformation pathway suggests that the blackleg fungus has enzymes to carry out this
biotransformation different from those involved in the biotransformation of the brassica phytoalexin
brassinin.
Keyw or d s: Blackleg fungus; brassicanal A; brassinin; canola; Leptosphaeria maculans; Phoma
lingam; phytoalexin; rapeseed
INTRODUCTION
Brassica oilseeds are the third most important world
source of edible vegetable oils, after soybean and palm
(Luhs and Friedt, 1994). The oilseeds canola (Brassica
napus, Brassica rapa), rapeseed (Brassica napus, Bras-
sica rapa), and mustard (Brassica juncea) are among
the most economically valuable brassica crops (Labana
and Gupta, 1993). Canola, rapeseed, and other bras-
sicas (Cruciferae family, syn. Brassicaceae) have defense
mechanisms that can be associated with phytoalexin
biosynthesis (Pedras and Se´guin-Swartz, 1992; Pedras
et al., 1996; Rouxel et al., 1995). Phytoalexins are
secondary metabolites synthesized de novo by plants in
response to diverse forms of stress, including pathogen
attack (Brooks and Watson, 1985). The phytoalexins
from brassicas have an indole ring and at least one
sulfur atom as common structural features. Despite
their close biogenetic relationship (L-tryptophan is the
biogenetic precursor; Monde et al., 1994), brassica
phytoalexins possess significantly different structures
with various ring systems. These structural differences
suggest that these phytoalexins have different biological
activity; however, little is known about their antifungal
or other antimicrobial activity.
We are studying the transformation of brassica phy-
toalexins by phytopathogenic fungi as part of an inte-
grated research program aimed at understanding and
controlling fungal diseases of canola and other economi-
cally important brassicas. Pathogenic attack by the
blackleg fungus [Leptosphaeria maculans (Desm.) Ces.
et de Not., asexual stage Phoma lingam (Tode ex Fr.)
Desm.] on species of Brassica is a serious agricultural
problem worldwide (Gugel and Petrie, 1992). Initially,
we investigated the biotransformation of the phytoal-
exin brassinin (1) by the blackleg fungus and related
species (Pedras and Taylor, 1991, 1993; Pedras et al.,
1992). We determined that the virulence of the blackleg
fungus correlated with its ability to rapidly metabolize
and detoxify brassinin (1) (Pedras et al., 1992). Con-
sidering that brassinin (1) is an advanced precursor of
F igu r e 1. Structures of brassica phytoalexins.
spirobrassinin (2), cyclobrassinin (3) (Figure 1) (Monde
et al., 1994), and possibly other related phytoalexins,
the fast degradation of brassinin by the blackleg fungus
may deprive the plant of important chemical defenses.
Therefore, the overall result is a plant more susceptible
to further fungal attack.
A possible strategy for controlling the blackleg fungus
would be the inhibition of the enzymes involved in the
detoxification of brassinin (1) and/or related phytoal-
exins. However, before such inhibitors can be rationally
designed, it is important to determine whether the
blackleg fungus metabolizes and detoxifies other bras-
sica phytoalexins. Ultimately, a correlation between the
bioactivity of the phytoalexins and of their biotransfor-
mation products will allow an understanding of the
detoxification mechanisms utilized by the blackleg
fungus to overcome the plant’s defenses. It should then
be possible to biorationally design antifungal agents
selective against the blackleg fungus.
Herein we present results concerning the biotrans-
formation of the brassica phytoalexin, brassicanal A (4)
by the blackleg fungus. Additionally, the syntheses of
brassicanal A and the products of biotransformation are
described, the antifungal activities of these metabolites
are compared, and the implications of these results are
discussed.
MATERIALS AND METHODS
* Author to whom correspondence should be ad-
dressed [telephone (306) 966-4772; fax (306) 966-4730;
e-mail pedras@sask.usask.ca].
Gen er a l. All chemicals were purchased from Aldrich
Chemical Co., Inc., Madison, WI, or Sigma Chemical Co., St.
S0021-8561(96)00098-2 CCC: $12.00
© 1996 American Chemical Society

3404 J. Agric. Food Chem., Vol. 44, No. 10, 1996
Pedras and Khan
Ta ble 1. HP LC Reten tion Tim es of Br a ssin in ,
Louis, MO. All solvents were of HPLC grade and used as such,
unless indicated otherwise.
Br a ssica n a l A, a n d Biotr a n sfor m a tion P r od u cts
Preparative TLC: Merck, Kieselgel 60 F254, 20 × 20 cm ×
retention time
(min)
0.25 mm.
compound
Analytical TLC: Merck, Kieselgel 60 F254, aluminum sheets,
5 × 2 cm × 0.2 mm; compounds were visualized under UV
brassinin (1)
brassicanal A (4)
18.7
10.2
6.0
light and by dipping the plates in
a 5% aqueous (w/v)
brassicanal A sulfoxide (5)
phosphomolybdic acid solution containing a trace of ceric
sulfate and 4% (v/v) H2SO4, followed by heating at 200 °C.
Flash Column Chromatography (FCC): Silica gel Merck,
grade 60, mesh size 230-400, 60 Å.
3-(hydroxymethyl)indole 2-methylsulfoxide (6)
3-methylindole 2-methylsulfoxide (7)
indole-3-carboxaldehyde
3.4 (broad)
8.7
6.3
5.6
indole-3-carboxylic acid
HPLC analysis was carried out with a high-performance
Hewlett-Packard liquid chromatograph equipped with a qua-
ternary pump, automatic injector, and diode array detector
(wavelength range 190-600 nm), degasser, and Hypersil ODS
column (5 µm particle size silica, 4.6 i.d. × 200 mm), equipped
with a guard column filled with the same stationary phase.
Mobile phase: 75% H2O-25% CH3CN to 100% CH3CN, for 35
min, linear gradient, and a flow rate 1.0 mL/min. Organic
extracts were dried over MgSO4 and solvents removed under
reduced pressure by rotary evaporator.
followed by neutralization of the broth with NaOH and
extraction with CHCl3 [containing 1% NH4OH (v/v)]. The
extracts were analyzed by HPLC, as described below. Bras-
sicanal A (4) was stable in uninoculated medium for at least
6 days.
Meta bolism of Com p ou n d s 5-7. Solutions of 5-7 (final
concentration 2 × 10-4 M) in DMSO (final concentration of
DMSO in MM was 0.5% v/v) were administered separately to
35-h-old liquid cultures and to uninoculated media and
analyzed as described for brassicanal A. The compounds were
stable in uninoculated medium for at least 6 days.
An a lysis a n d Isola tion of Meta bolites. The analyses of
the organic extracts and biotransformation products were
performed with the HPLC system described above. The
retention times shown in Table 1 were obtained with the
mobile phase described under HPLC analysis. The samples
to be analyzed by HPLC were dissolved in CH3CN and filtered
through a tight cotton wool plug. Only the chromatograms of
the neutral organic extracts showed peaks not present in
chromatograms of extracts of control cultures; acidic and basic
extracts were similar to those of control cultures.
The isolation of products resulting from the fungal metabo-
lism of brassicanal A (4) was carried out by FCC over silica
gel (CH2Cl2-MeOH, 98:2 v/v), followed by preparative TLC.
After TLC development with CH2Cl2-MeOH (95:5 v/v; bras-
sicanal A, Rf 0.57; 5, Rf 0.40; 6, Rf 0.19; 7, Rf 0.38), the
fluorescent bands were scraped from the plate and eluted with
CH2Cl2-MeOH (90:10 v/v).
NMR spectra were recorded on a Bruker AM 300 or Bruker
1
AMX 500 spectrometer; for H (300 or 500 MHz), δ values were
referenced to CD3OD (3.31 ppm) and for 13C (75.5 or 125.8
MHz) to CD3OD (49.15 ppm).
Fourier transform infrared (FTIR) spectra were obtained on
a Bio-Rad FTS-40 spectrometer using diffuse reflectance cell.
Mass spectra (MS) were obtained on a VG 70 SE mass
spectrometer [high resolution (HR), electron impact (EI) or
chemical ionization (CI) with ammonia as carrier gas], em-
ploying a solids probe.
F u n ga l Cu ltu r es. The virulent blackleg isolates (P. lin-
gam) ENG-53 and BJ -125 and an avirulent isolate, Unity (now
considered a different species; Pedras et al., 1995), were
employed in this study. The fungal isolates were grown on
V8 agar [20% (v/v) V8 juice, 0.75 g/L CaCO3, 100 mg/L
streptomycin sulfate, 40 mg/L Rose Bengal, 15 g/L agar] plates
at 26 °C, under continuous light for 10 days. Spore suspen-
sions of each isolate were prepared by overlaying the V8 agar
plates with 10 mL of sterile distilled water, and the plate
surfaces were rubbed with a flamed glass rod to dislodge the
pycnidiospores. The suspension was filtered and transferred
to centrifuge tubes and the spores were separated by centrifu-
gation at 3000g for 10 min. After one washing with sterile
distilled water, the spores were stored at -20 °C. Liquid
cultures were grown in minimal medium supplemented with
thiamin (MM) (Pedras et al., 1992). The cultures were
incubated on a shaker at 150 rpm.
F u n ga l Sp or e Ger m in a tion Assa ys. The antifungal
activity (to isolate BJ -125) of brassicanal A (4), compounds
5-7, and indole-3-carboxaldehyde was investigated using the
following spore germination inhibition bioassay. Dimethyl
sulfoxide (DMSO, control) or a DMSO solution (final concen-
tration e1%) of the compound to be tested (final concentration
of each compound 1 × 10-3, 8 × 10-4, and 5 × 10-4 M) was
added to a spore suspension (2 × 106 spores/mL) in MM
containing 5% Tween 80 (1.0 mL in a microcentrifuge tube)
and incubated on a shaker at 150 rpm at room temperature.
Aliquots (withdrawn at different times after vortex mixing of
the microcentrifuge tubes containing the spore suspensions)
of each suspension (10 µL) were pipetted to a hemacytometer,
and the germinated and ungerminated spores were counted
under the microscope at 40 × 10 magnification. Each assay
was repeated at least twice.
The structures of the products were determined from
analyses of the spectroscopic data (NMR, MS, FTIR, UV) of
the purified metabolites and confirmed by synthesis, as
described below.
Br a ssica n a l A (4) Syn th esis. Brassicanal A was prepared
by formylation (Smith, 1954) of 2-(methylthio)indole (Hino et
al., 1969) as follows. Phosphorus pentasulfide (0.47 g, 2.0
mmol) was added to a mixture of oxindole (1.33 g, 10.0 mmol),
sea sand (1.5 g), and benzene (30 mL). The reaction mixture
was heated under reflux with stirring for 80 min and then
allowed to cool, and the benzene layer was decanted. The
insoluble residue was extracted first with benzene and then
with EtOAc. The benzene and EtOAc extracts were combined
and evaporated to give crude 2-indolinethione (1.1 g, 7.4
mmol). A mixture of 2-indolinethione (1.1 g, 7.4 mmol), MeI
(2.8 g, 20 mmol), Na2CO3 (1.2 g), and acetone (20 mL) was
stirred at room temperature for 4 h and filtered. The filtrate
was evaporated to leave a brown oily residue which was
extracted with benzene. The benzene extract was purified
(FCC, hexane-acetone, 9:1 v/v) to give 2-(methylthio)indole
(0.8 g, 5 mmol). Phosphorus oxychloride (1.66 g, 10.0 mmol,
freshly distilled) was added with stirring under argon atmo-
sphere to DMF (3 mL, freshly distilled). A solution of
2-(methylthio)indole (0.17 g, 1.0 mmol) in DMF (1 mL) was
added dropwise with continuous stirring. The reaction mix-
ture was kept at room temperature for 45 min and then poured
onto crushed ice. Aqueous NaOH (3.8 g in 20 mL) was added
to the reaction mixture (slow addition, exothermic reaction!),
and the product was filtered off, washed with water, and dried
to yield brassicanal A (0.11 g): Rf 0.57 (CH2Cl2-MeOH, 95:5
v/v); 1H NMR in Table 2; 13C NMR in Table 3; FTIR νmax 3151,
1626, 1581, 1448 cm-1; EIMS, mass (relative intensity) )
measured 191.0409 (M+ C10H9NOS, calculated 191.0408) (100),
158 (68), 148 (17), 130 (9), 115 (7); CIMS, mass (relative
intensity) ) 192 (M+ + 1) (100).
Meta bolism of Br a ssica n a l A (4). Liquid shake cultures
(100 mL of media in 250-mL Erlenmeyer flasks) inoculated
with fungal spores (2 × 109) and incubated at 27 ( 2 °C were
used for the metabolism studies. Control cultures of each
isolate were grown separately. Solutions of brassicanal A
(final concentrations 5 × 10-4 and 2 × 10-4 M) in DMSO (final
concentration of DMSO in MM was 0.5% v/v) were adminis-
tered to 35-h-old liquid cultures and to uninoculated media.
Samples (10 mL) were withdrawn at 2-24-h intervals up to 6
days and were either immediately frozen or filtered; the broth
and the mycelium were extracted with Et2O. The extracted
broth was acidified to pH 2 with HCl and extracted with Et2O,

Biotransformation of Brassica Phytoalexin by the Blackleg Fungus
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3405
Ta ble 2. 1H NMR (300 MHz) Ch em ica l Sh ifts (p p m ) a n d Mu ltip licities (J in Hz) for Com p ou n d s 4-7 in CD3OD
H no.
brassicanal A (4)
5a
6a
7.76 dd (8.0, 1.0)
7.16 ddd (8.0, 7.0, 1.0)
7.33 ddd (8.0, 7.0, 1.0)
7.49 dd (8.0, 1.0)
7a
4
8.06 m
7.24 m
7.24 m
7.38 m
2.68 s, SCH3
10.0 s, CHO
8.10 dd (8.0, 1.5)
7.30 m
7.65 dd (8.0, 1.0)
7.00 ddd (8.0, 7.5, 1.0)
7.30 ddd (8.0, 7.0, 1.0)
7.42 dd (8.0, 1.0)
3.00 s, S(O)CH3
5/6
6/5
7
7.30 m
7.60 dd, (7.0, 1.0)
3.10 s, S(O)CH3
10.2 s, CHO
other
3.00 s, S(O)CH3
4.91 d (12.5), 4.89 d (12.5) CH2OH
2.47 s, CH3
a
Assignments for H-4 and H-7 may be interchanged.
Ta ble 3. 13C NMR (75.5 MHz) Ch em ica l Sh ifts (p p m ) a n d Mu ltip licities for Com p ou n d s 4-7 in CD3OD
C no.
brassicanal A (4)
5
6
7
2
3
3a
4
5
6
7
7a
148.76
117.63
127.45
121.19
123.86
124.77
112.33
138.91
147.70
116.98
127.94
121.40
124.65
126.25
114.08
138.60
134.47
120.68
127.83
121.16
121.34
126.09
113.29
139.30
132.52
118.43
128.81
121.13
121.19
126.37
113.19
139.49
other
185.4 CHO
16.95 SCH3
185.94 CHO
42.24 S(O)CH3
55.06 CH2OH
41.08 S(O)CH3
8.85 CH3
39.78 S(O)CH3
Br a ssica n a l A Su lfoxid e (5) Syn th esis. m-Chloroper-
benzoic acid (5 mg in 0.5 mL of MeOH) was added to a stirred
solution of brassicanal A (5 mg) in MeOH (1 mL) at 0 °C. After
30 min, the reaction mixture was treated with Me2S (200 µL),
concentrated, and separated by preparative TLC to yield
quantitatively brassicanal A sulfoxide (5): Rf 0.40 (CH2Cl2-
MeOH, 95:5 v/v); 1H NMR in Table 2; 13C NMR in Table 3;
FTIR νmax 3046, 1627, 1600, 1460 cm-1; EIMS, mass (relative
intensity) ) measured 207.0359 (M+, C10H9NO2S, calculated
207.0354) (26), 190 (100), 175 (12), 144 (3), 115 (3); CIMS, mass
(relative intensity) ) 208 (M+ + 1) (7), 192 (100).
of the HPLC chromatograms of extracts of fungal
cultures containing brassicanal A and control cultures
indicated that only the neutral extracts contained
possible biotransformation products of brassicanal A.
The optimum incubation time (in MM) for isolation of
the putative products was determined by TLC and by
HPLC analysis of each extract. Subsequently, the
components of the neutral Et2O extracts obtained from
larger scale cultures were separated by chromatogra-
1
phy, and each constituent was analyzed by H and 13C
3-(Hyd r oxym eth yl)in d ole 2-Meth ylsu lfoxid e (6) Syn -
th esis. NaBH4 (5 mg) was added to stirred solution of
brassicanal A sulfoxide (10 mg) in MeOH (2 mL) at room
temperature. After 30 min, the mixture was quenched with
water and the solvent was evaporated under reduced pressure.
The crude product was purified by preparative TLC (CH2Cl2-
MeOH, 93:7 v/v, developed twice) to yield 3-(hydroxymethyl)-
indole 2-methylsulfoxide (6) (7.6 mg): Rf 0.19 (CH2Cl2-MeOH,
95:5 v/v); 1H NMR in Table 2; 13C NMR in Table 3; FTIR νmax
3326, 2920, 1623, 1452, 1027 cm-1; EIMS, mass (relative
intensity) ) measured 209.0499 (C10H11NO2S, calculated
209.0510) (68), 192 (99), 176 (100), 117 (63); CIMS, mass
(relative intensity) ) 210 (M+ + 1) (13), 192 (100).
NMR, FTIR, and MS. Three main products were
isolated after incubation of P. lingam with brassicanal
A (4): brassicanal A sulfoxide (5), 3-(hydroxymethyl)-
indole 2-methylsulfoxide (6), and 3-methylindole 2-
methylsulfoxide (7). The structure of each compound
was deduced from comparison of their spectroscopic data
and those of brassicanal A as described below and
confirmed by synthesis.
1
The H NMR spectrum of each compound (CD3OD)
showed the four hydrogens characteristic of a 2,3-
disubstituted indole nucleus. In addition, the sulfoxide
5 showed the aldehyde hydrogen, as well as the signal
for the Me group, which was shifted downfield in both
3-Meth ylin d ole 2-Meth ylsu lfoxid e (7) Syn th esis. Bras-
sicanal A (10 mg), 10% palladium-charcoal (5 mg) and NaBH4
(20 mg) in 2-propanol (3 mL) was refluxed with stirring; 60
min later a further quantity (10 mg) of NaBH4 was added to
the reaction mixture (Heacock and Hutzinger, 1964). After 2
h, the reaction mixture was filtered and treated as described
for 6. The residue was dissolved in MeOH and treated with
m-chloroperbenzoic acid as described for 5. The crude product
was purified by preparative TLC (CH2Cl2-MeOH, 96:4) to
yield 3-methylindole 2-methylsulfoxide (7.0 mg): Rf 0.38 (CH2-
1
the H (2.68 ppm in 4 vs 3.1 ppm in 5) and the 13C (16.9
ppm in 4 vs 42.2 ppm in 5) NMR spectra. These
changes in the chemical shifts suggested that the SsMe
group present in brassicanal A (4) had been oxidized to
the corresponding MesSdO by the fungus. EIMS of 5
(molecular ion mass at 207 and base peak at 190)
confirmed that oxygen addition had occurred. Further
corroboration of the structure was provided by oxidation
of brassicanal A (4) with m-chloroperbenzoic acid, as
described under Materials and Methods.
Both compounds 6 and 7 showed, in addition to the
indolyl hydrogens, a Mes(SdO) group, a methylene
group in 6 (doublets at 4.89 and 4.91 ppm), and a methyl
group in 7 (2.47 ppm); the aldehyde hydrogens were
absent in both 6 and 7. EIMS of 6 (molecular ion mass
at 209 and base peak at 176) and of 7 (molecular ion
mass at 193 and base peak at 178) confirmed that the
aldehyde group of brassicanal A (4) had been reduced
to the corresponding alcohol in 6 or to the corresponding
methyl group in 7. The structures of both 6 and 7 were
also confirmed by synthesis. Metabolites 5-7 were not
detected in uninoculated media containing brassicanal
A (4) or in control fungal cultures.
1
Cl2-MeOH, 95:5 v/v); H NMR in Table 2; 13C NMR in Table
3; FTIR νmax 3160, 1617, 1449, 1020 cm-1; EIMS, mass (relative
intensity) ) measured 193.0562 (M+, C10H11NOS, calculated
193.0561) (63), 178 (100), 160 (57), 117 (35); CIMS, mass
(relative intensity) ) 194 (M+ + 1) (100).
RESULTS AND DISCUSSION
Virulent isolates of P. lingam were grown as described
under Materials and Methods. After 35 h, brassicanal
A (4), synthesized as described above, in DMSO was
administered to fungal cultures and to uninoculated
media (final concentrations 5 × 10-4 and 2 × 10-4 M).
Control cultures of the fungus were grown separately.
Cultures were incubated and samples were withdrawn
at 2-24-h intervals (up to 6 days), extracted first with
Et2O, and then acidified and reextracted. Comparison

3406 J. Agric. Food Chem., Vol. 44, No. 10, 1996
Pedras and Khan
Ta ble 4. P r od u cts of Meta bolism of Com p ou n d s 4-6 by P . lin ga m Isola tes BJ -125, ENG-53, a n d Un ity
fungal isolate
(virulence on rapeseed)
compound added to
fungal culturesa
products (%) of metabolism
BJ -125 (virulent)
ENG 53 (virulent)
BJ -125 (virulent)
BJ -125 (virulent)
BJ -125 (virulent)
Unity (avirulent)
4
4
5
6
7
4
5 (10%), 6 (30%), 7 (40%) after 60 h; 5 (e5%), 6 (30%), 7 (50%) after 6 days
5 (10%), 6 (30%), 7 (40%) after 60 h; 5 (e5%), 6 (30%), 7 (50%) after 6 days
6 (>80%), 7 (e5%) after 60 h
7 (<10%) after 60 h; 7 (50%) after 6 days
no products of metabolism detected for 6 days
5 (10%), 6 (e5%), 7 (e5%) after 6 daysb
Compounds at 5 × 10-4 M were dissolved in DMSO and added to germinated spores incubated in MM. 80% of brassicanl A remaining
a
b
in cultures.
Ta ble 5. Resu lts of Bioa ssa ys w ith Sp or es of P . lin ga m , Isola te BJ -125, In cu ba ted w ith Com p ou n d s 4-7 a n d
In d ole-3-ca r boxa ld eh yd e
compound added to ungerminated fungal spores
concentrationa (µg/mL)
% of ungerminated spores
control
brassicanal A (4)
50% after 38 h; <1% after 72 h
92% after 38 h; 25% after 72 h
75% after 38 h; <1% after 72 h
55% after 38 h; <1% after 72 h
75% after 38 h; <1% after 72 h
65% after 38 h; <1% after 72 h
50% after 38 h; <1% after 72 h
similar to control
1 × 10-3 M (200)
8 × 10-4 M (150)
5 × 10-4 M (100)
5
1 × 10-3
8 × 10-4
5 × 10-4
1 × 10-3
1 × 10-3
1 × 10-3
5 × 10-4
M
M
M
M
M
M
M
6
7
similar to control
65% after 38 h; <1% after 72 h
similar to control
indole-3-carboxaldehyde
a
Compounds were dissolved in DMSO and added to ungerminated spores incubated in MM.
to the 3-methylindole 7. The final product of the
biotransformation of brassicanal A, metabolite 7, was
detected in the cultures 2 h after incubation and
remained in the cultures for at least 6 days. Perhaps
significantly, the biotransformation of brassicanal A (4)
was different from that of brassinin (1) (Pedras and
Taylor, 1993) and occurred much more slowly. While
the biotransformation of brassinin (1) by virulent black-
leg isolates rapidly yielded the corresponding oxidation
products indole-3-carboxaldehyde and indole-3-carbox-
ylic acid, the biotransformation of brassicanal A pro-
ceeded with reduction of the aldehyde. No traces of
indole-3-carboxaldehyde or indole-3-carboxylic acid, the
final metabolites of brassinin (1), were detected at any
time during the biotransformation of 4. It is also worthy
to note that, in contrast with the inhibitory effect of
brassinin (1) on the biosynthesis of the nonspecific
blackleg phytotoxins, brassicanal A (4) did not have any
similar effect.
F igu r e 2. Biotransformation of brassicanal A by the blackleg
fungus: (a) Phoma lingam or m-chloroperbenzoic acid; (b) P.
lingam or NaBH4; (c) P. lingam; (d) 1, NaBH4/Pd-C; 2,
m-chloroperbenzoic acid.
To ascertain the sequence of the biotransformation
steps, compounds 5-7 (final concentration 2 × 10-4 M)
were separately administered to cultures of P. lingam
isolate BJ-125. The cultures were incubated and samples
withdrawn at 2-24-h intervals, extracted, and analyzed
by TLC and HPLC. The results of these experiments
are summarized in Table 4. As predicted, 6 and 7 were
detected in the cultures incubated with 5, whereas only
7 was detected in the cultures incubated with either 6
or 7. After incubation with 5 for 2 h, more than 50% of
5 had been converted to 6 (determined by HPLC
analysis of the ethereal extract; traces of 7 present);
after 60 h, 6 was the major metabolite (determined by
HPLC analysis of the ethereal extract; traces of 5 and
7 present). On the other hand, the biotransformation
of 6 to 7 was slower; after 6 days only 50% of 6 had
been converted to 7; compound 7 was stable in the
cultures for at least 6 days.
The suggested sequence of reactions that brassicanal
A (4) undergoes when incubated with virulent isolates
of P. lingam is given in Figure 2. In the first biotrans-
formation step, the SMe group of brassicanal A was
oxidized to the corresponding sulfoxide 5; this product
was detected in the cultures 2 h after incubation with
brassicanal A (4). Unexpectedly, however, the aldehyde
group of 4 was reduced to the alcohol 6 and then further
The avirulent isolate Unity was also incubated with
brassicanal A (4); in contrast with the results obtained
for the two virulent isolates, the metabolism of 4
occurred much more slowly. As shown in Table 4, only
10% of 4 was metabolized to 5 (traces of 6 and 7 were
detected) after 6 days.
The antifungal activities (to virulent blackleg isolates)
of brassicanal A (4), its biotransformation products 5,
6, and 7, and indole-3-carboxaldehyde were compared
using a spore germination inhibition assay (Table 5).
After 24 h of incubation, 20% of the fungal spores
started to germinate in controls and in assays contain-
ing 6 or 7 (all three concentrations), whereas no
germination was observed in spores incubated with
either brassicanal A (4) or 5 (all three concentrations).
After 38 h of incubation, 50% of the spores were found
ungerminated in control assays and in assays with
compounds 6 and 7, whereas in assays with compounds
4, 5, and indole-3-carboxaldehyde (higher concentration,
1 × 10-3 M) most of the spores were ungerminated (92%,
75%, and 65%, respectively). After 72 h of incubation,
ungerminated spores (ca. 25%) were only found in
assays containing brassicanal A (1 × 10-3 M), whereas

Biotransformation of Brassica Phytoalexin by the Blackleg Fungus
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3407
Heacock, R. A.; Hutzinger, O. The preparation of hydroxyska-
toles and 5,6-dihydroxyskatoles. Can. J . Chem. 1964, 42,
514-521.
mycelium appeared in the rest of the assays. These
results (Table 5) indicate that brassicanal A (4) is
significantly more inhibitory to blackleg spore germina-
tion than its biotransformation products or indole-3-
carboxaldehyde.
Hino, T.; Tsuneoka, K.; Nakagawa, M.; Akaboshi, S. Thiation
of oxindoles. Chem. Pharm. Bull. 1969, 17, 550-558.
Holland, H. L. Chiral sulfoxidation by biotransformation of
organic sulfides. Chem. Rev. 1988, 88, 473-485.
Labana, K. S.; Gupta, M. L. Importance and origin. In Breeding
Oilseed Brassicas; Labana, K. S., Banga, S. S., Banga, S.
K., Eds.; Springer-Verlag: Berlin, Germany, 1993; pp 1-7.
Luhs, W.; Friedt, W. Major oil crops. In Designer Oil Crops;
Murphy, D. J ., Ed.; VCH: Weinheim, Germany, 1994; pp
1-71.
Monde, K.; Takasugi, M.; Ohnishi, T. Biosynthesis of crucif-
erous phytoalexins. J . Am. Chem. Soc. 1994, 116, 6650-
6657.
Pedras, M. S. C.; Se´guin-Swartz, G. The blackleg fungus:
phytotoxins and phytoalexins. Can. J . Plant Pathol. 1992,
14, 67-75.
Pedras, M. S. C.; Taylor, J . L. Metabolic transformation of the
phytoalexin brassinin by the blackleg fungus. J . Org. Chem.
1991, 56, 2619-2621.
Pedras, M. S. C.; Taylor, J . L. Metabolism of the phytoalexin
brassinin by the blackleg fungus. J . Nat. Prod. 1993, 56,
731-738.
Pedras, M. S. C.; Borgmann, I.; Taylor, J . L. Biotransformation
of brassinin is a detoxification mechanism in the blackleg
fungus. Phytochem. (Life Sci. Adv.) 1992, 11, 1-7.
Pedras, M. S. C.; Taylor, J . L.; Morales, V. M. Phomaligin A
and other yellow pigments in Phoma lingam and P. wasa-
biae. Phytochemistry 1995, 38, 1215-1222.
Pedras, M. S. C.; Khan, A. Q.; Taylor, J . L. Phytoalexins from
brassicas: overcoming plant’s defenses. In Phytochemical
Pest Control Agents; Hedin, P., Ed.; ACS Symposium Series;
American Chemical Society: Washington, DC, 1996; in
press.
It is now well documented that phytopathogens use
phytoalexin metabolism as a detoxification mechanism
to overcome plant’s chemical defenses (Van Etten et al.,
1989). We have previously determined that the blackleg
fungus metabolizes (oxidative transformations) and
detoxifies brassinin (1) effectively (Pedras et al., 1992;
Pedras and Taylor, 1993). However, results of this
study indicate that the virulent blackleg fungus cannot
metabolize brassicanal A (4) as effectively. In fact, while
brassinin (1) was completely metabolized in less than
24 h by virulent isolates, brassicanal A (4) was detected
in mycelial extracts even after 60-h incubations with
the same virulent isolates. On the other hand, but most
importantly, we also determined in this study that the
so-called “avirulent” isolates of P. lingam (Unity group,
now considered a species related to the virulent group;
Pedras et al., 1995) metabolized brassicanal A (4) at a
much slower rate than the virulent isolates; after 6-day
incubations, less than 20% of 4 was biotransformed to
5 (traces of 6 and 7 present). This result indicates that
virulent blackleg isolates are significantly more effective
in detoxifying brassicanal A than the related “avirulent”
isolates.
Overall, our results suggest that the virulent blackleg
fungus may utilize different enzymes to transform
diverse phytoalexins; the enzyme(s) involved in the
biotransformation of brassicanal A could be nonselective
enzymes used in general detoxification processes. In
any case, the process appears to be a detoxification
beneficial to the virulent blackleg pathogen. Addition-
ally, considering that preparation of chiral sulfoxides
is of interest in synthetic methodology (Holland, 1988),
the pathogenic blackleg fungus may be a useful bioagent
for the oxidation of sulfides. Further work to under-
stand mechanisms that allow the blackleg fungus to
overcome the plant’s chemical defenses is in progress.
Rouxel, T.; Kolman, A.; Ballesdent, M. H. Phytoalexins from
the crucifers. In Handbook of Phytoalexin Metabolism and
Action; Daniel, M., Purkayastha, R. P., Eds.; Dekker: New
York, 1995; pp 229-261.
Smith, G. F. Indoles. Part I. Formylation of indole and some
reactions of 3-formylindole. J . Chem. Soc. 1954, 3842-3846.
Van Etten, H. D.; Mathews, D. E.; Mathews, P. S. Phytoalexin
detoxification: importance for pathogenicity and practical
implications. Annu. Rev. Phytopathol. 1989, 27, 143-164.
ACKNOWLEDGMENT
We thank G. Se´guin-Swartz and R. K. Gugel, Agri-
culture and Agri-Food Canada Research Station, Saska-
toon, SK, for kindly providing isolates of P. lingam
(virulent isolates ENG-53 and BJ -125 and avirulent
isolate Unity) and B. Chatson, Plant Biotechnology
Institute, Saskatoon, SK, for NMR spectral determina-
tions.
Received for review February 13, 1996. Revised manuscript
received J uly 25, 1996. Accepted J uly 30, 1996.X Part of this
work was presented at the Phytochemical Pest Control Agents
Symposium, Pacifichem 95, Honolulu, HI. Support for the
authors’ work was obtained in part from the University of
Saskatchewan and the Natural Sciences and Engineering
Research Council of Canada (research and equipment grants
to M.S.C.P.).
LITERATURE CITED
Brooks, C. J .; Watson, D. G. Phytoalexins. Nat. Prod. Rep.
1985, 427-459.
J F960098U
Gugel, R. K.; Petrie, G. A. History, occurrence, impact, and
control of blackleg of rapeseed. Can. J . Plant Pathol. 1992,
14, 36-45.
X Abstract published in Advance ACS Abstracts, Sep-
tember 1, 1996.