Glucosinolate responses of oilseed rape mustard and kale to mechanical wounding and infestation by cabbage stem flea beetle (Psylliodes chrysocephala)


Koritsas, V.M.; Lewis, J.A.; Fenwick, G.R.

Annals of Applied Biology 118(1): 209-222

1991


Mechanical wounding for the petioles of six laboratory-grown rapeseed (Brassica napus) cultivars induced physiological changes in the plant, markedly affecting the levels of individual glucosinolates. Greatest increases were observed for the indole glucosinolates, glucobrassicin and neoglucobrassicin. Such changes were usually associated with large decreases in the levels of aliphatic glucosinolates. The total glucosinolate content of the wounded plant was thus a reflection of these two opposing trends and wounding produced a greater relative indole glucosinolate content in this total figure. Thus increasing wounding was associated with an increase in indole glucosinolates and a decrease in aliphatic compounds. Infestation of field- and laboratory-grown rapeseed with cabbage stem flea beetle (Psylliodes chrysocephala) produced similar effects, which were observed in various parts of the plant. Differences in response between field- and laboratory-grown infested plants are attributed to the different physiological ages of the harvested material. Laboratory-grown kale and mustards also showed wound-induced glucosinolate changes. The lake, cv. Fribor, produced elevated levels of both indoles and aliphatics after wounding. Total glucosinolate content in the mustards, which, unlike rape and kale, normally contain only traces of indole glucosinolates in the unstressed state, was increased following wounding. This was, however, not associated with elevated levels of indole glucosinolates, but with accumulation of aliphatic (Brassica nigra, B. juncea) and aromatic (Sinapis alba) glucosinolates. The significance of these findings is discussed.

Ann.
appl.
Biol.
(1991),
118,
209-221
Printed
in
Great
Britain
209
Glucosinolate
responses
of
oilseed
rape,
mustard
and
kale
to
mechanical
wounding
and
infestation
by
cabbage
stem
flea
beetle
(Psylliodes
chrysocephala)
By
V.
M.
KORITSAS",
J.
A.
LEWIS
2
and
G.
R.
FENWICK
2
'Wye
College,
University
of
London,
Ashford,
Kent
TN25
5AH,
UK
2
AFRC
Institute
of
Food
Research,
Norwich
Laboratory,
Colney
Lane,
Norwich,
Norfolk
NR4
7UA,
UK
(Accepted
16
November
1990)
Summary
Mechanical
wounding
of
the
petioles
of
six
laboratory-grown
rapeseed
(Brassica
napus)
cultivars
induced
physiological
changes
in
the
plant,
markedly
affecting
the
levels
of
individual
glucosinolates.
Greatest
increases
were
observed
for
the
indole
glucosinolates,
glucobrassicin
and
neoglucobrassicin.
Such
changes
were
usually
associated
with
large
decreases
in
the
levels
of
aliphatic
glucosinolates.
The
total
glucosinolate
content
of
the
wounded
plant
was
thus
a
reflection
of
these
two
opposing
trends
and
wounding
produced
a
greater
relative
indole
glucosinolate
content
in
this
total
figure.
Thus
increasing
wounding
was
associated
with
an
increase
in
indole
glucosinolates
and
a
decrease
in
aliphatic
compounds.
Infestation
of
field-
and
laboratory-grown
rapeseed
with
cabbage
stem
flea
beetle
(Psylliodes
chrysocephala)
produced
similar
effects,
which
were
observed
in
various
parts
of
the
plant.
Differences
in
response
between
field-
and
laboratory-
grown
infested
plants
are
attributed
to
the
different
physiological
ages
of
the
harvested
material.
Laboratory-grown
kale
and
mustards
also
showed
wound-induced
glucosinolate
changes.
The
kale,
cv.
Fribor,
produced
elevated
levels
of
both
indoles
and
aliphatics
after
wounding.
Total
glucosinolate
content
in
the
mustards,
which,
unlike
rape
and
kale,
normally
contain
only
traces
of
indole
glucosinolates
in
the
unstressed
state,
was
increased
following
wounding.
This
was,
however,
not
associated
with
elevated
levels
of
indole
glucosinolates,
but
with
accumulation
of
aliphatic
(Brassica
nigra,
B.
juncea)
and
aromatic
(Sinapis
alba)
glucosinolates.
The
significance
of
these
findings
is
discussed.
Key
words:
Glucosinolates,
oilseed
rape,
mustard,
kale,
cabbage
stem
flea
beetle,
Psylliodes
chrysocephala
Introduction
Glucosinolates,
formerly
referred
to
as
mustard
oil
glycosides,
are
found
mainly
throughout
the
order
Capparales
sensu
Cronquist
or
Taktajan,
constituting
the
Capparaceae,
Moringa-
ceae,
Resedaceae,
Tovariaceae
and
Cruciferae
(Kjaer,
1974).
The
Cruciferae
comprise
agriculturally
important
oilseeds,
vegetables,
condiments
and
salad
crops
and
the
significance
of
glucosinolates
in
this
family
has
been
discussed
by,
amongst
others,
Fenwick,
Heaney
&
Mullin
(1983),
Chew
(1988)
and
Fenwick,
Heaney
&
Mawson
(1989).
More
than
one
hundred
different
glucosinolates,
possessing
the
same
general
structure
(I,
Fig.
1),
have
now
been
Current
address:
Department
of
Pure
and
Applied
Biology,
University
of
Leeds,
Leeds
LS2
9JT,
UK
0
1991
Association
of
Applied
Biologists
210
V.
M.
KORITSAS,
J.
A.
LEWIS
AND
G.
R.
FENWICK
S—fi—D—Glucose
R—C
NOS0,
--
Name
Ia
Progoitrin
Ib
Gluconapin
Ic
Gluconapoleiferin
Id
Glucobrassicanapin
Ie
Gluconasturtiin
If
Glucobrassicin
Ig
Neoglucobrassicin
Ih
4-Methoxyglucobrassicin
Ii
Sinigrin
Ij
Sinalbin
lk
Glucotropaeolin
II
Glucocapparin
Im
Glucoiberin
R
2-Hydroxy-3-butenyl
3-Butenyl
3-Hydroxy-4-pentenyl
4-Pentenyl
2-Phenethyl
3-Indolymethyl
1-Methoxy-3-indolylmethyl
4-Methoxy-3-indolylmethyl
2-Propenyl
p-Hydroxybenzyl
Benzyl
Methyl
3-Methylsulphinylpropyl
Fig.
1.
Glucosinolate
structure;
trivial
names
and
side
chain
(R)
structure
of
glucosinolates
discussed
in
this
paper.
reported.
Most
crucifers
contain
only
a
limited
number
of
major glucosinolates
(usually
less
than
six),
although
others
may
be
present
in
trace
amounts.
The
nature
and
amounts
of
the
individual
compounds
is
determined
by
genetic,
agronomic
and
environmental
factors
(Fenwick
et
al.,
1983).
Within
the
plant
itself,
physiological
age
and
plant
part
are
major
determining
factors.
The
glucosinolates
shown
(Fig.
1)
include
those
commonly
found
in
brassica
leaf,
stalk
and
root
tissue.
Glucosinolates,
or
the
products
of
their
hydrolysis
by
the
thioglucosidase,
myrosinase
(Fig.
2),
which
co-exists
in
these
plants,
have
been
shown
to
play
a
role
in
the
complex
interactions
between
crucifers
and
their
potential
herbivores,
pathogens,
competitors
and
symbionts
(VanEtten
&
Tookey,
1979;
Rosenthal
&
Janzen,
1979;
Chew,
1988;
Nielsen,
1988).
It
has
been
suggested
that
volatile
products
of
glucosinolate
hydrolysis
are
involved
in
the
attraction
of
insects
to
host
plants
(Finch,
1978,
1986)
and
traps
containing
allyl
isothiocyanate
(Fig.
2,
III)
have
been
demonstrated
to
capture
adult
flea
beetles,
Phyllotreta
cruciferae
(Goeze)
(Vincent
&
Stewart,
1984).
In
other
studies,
however,
intact
glucosinolates
rather
than
their
hydrolysis
products
were
found
to
be
important
in
host
location
and
acceptance
(Thorsteinson,
1953;
Nayer
&
Thorsteinson,
1963;
DeBoer
&
Hanson,
1984;
Larsen,
Nielsen,
Ploger
&
Sorensen,
1985).
Beetles
that
feed
on
cruciferous
plants
are
known
to
respond
differently
to
individual
glucosinolates,
that
is,
to
the
different
side
chain
groups
(R,I;
Fig.
1)
in
the
glucosinolate
molecule
(Nielsen,
1988;
Larsen
et
al.,
1985).
Whilst
sinigrin
(Ii)
elicited
feeding
in
adult
Phyllotreta
cruciferae,
it
failed
to
do
so
in
P.
striolata
F.
(Vincent
&
Stewart,
1984).
The
aromatic
glucosinolate,
glucotropaeolin
(Ik),
proved
highly
stimulating
to
several
flea
beetle
species,
including
P.
nemorum
L.,
P.
undulata
Kutsch.
and
Phaedon
cochleariae
F.,
but
glucocapparin
(II,
found
in
Capparis
species)
and
glucoiberin
(Im)
were
slightly
less
stimulating
to
P.
nemorum,
P.
undulata,
and
Phaedon
cochleariae
than
sinigrin
(Larsen
et
al.,
1985).
Relatively
little
is
known
about
the
effects
of
wounding
caused
by
insects,
pests
and
fungal
pathogens
on
the
content
of
total
(Lammerink,
MacGibbon
&
Wallace,
1984),
and
individual
Wounding,
cabbage
stem
flea
beetle
infestition
&
glucosinolates
211
S
Glucose
R
C
NOSO
--
I
H,0
Myrosinase
/
SH
+
Glucose
NOSO,
II
CH,=CHCH,NCS
III
C'H,
=CHCH,SCN
IV
CH
2
=CHCH
2
NC
V
CH
2
=
CH
—7--
\
0
NH
VII
CH,
=CH
CHCH
'
CN
VIII
I
I
N
OH
H
CH
CH,—
CHCHCH
2
CN
/
I
IX
N
N
S
OH
H
H
CH
2
CN
X
CH
2
OH
XI
1
XII
CH,_
—CHCH
2
CN
S
SC
N
-
VI
XIII
Fig.
2.
Schematic
representation
of
enzymic
breakdown
of
glucosinolates
(from
Watson,
D.H.
(Ed.),
Natural
Toxicants
in
Food,
Progress
and
Prospects,
Ellis
Harwood,
Chichester,
1987,
p
78,
with
permission).
(Butcher,
El-Tigani
&
Ingram,
1974;
Butcher,
Searle
&
Mousedale,
1976)
glucosinolates.
An
investigation
was
therefore
conducted
to
examine
the
effect
on
glucosinolate
content
of
rapeseed
of
pest
infestation
and
mechanical
damage.
Preliminary
results
of
the
effects
of
cabbage
stem
flea
beetle
(Psylliodes
chrysocephala
L.)
infestation
and
tissue
puncturing
on
laboratory-
and
field-grown
B.
napus
L.,
cv.
Rafal
have
recently
been
reported
(Koritsas,
Lewis
&
Fenwick,
1989).
In
the
present
paper
we
describe
the
effects
of
similar
infestation
and
damage
for
B.
napus
L.
cv.
Ariana,
discuss
the
effects
of
mechanical
damage
on
four
additional
rapeseed
cultivars
and
indicate
the
effect
of
such
damage
on
glucosinolate
profiles
of
individual
plant
parts.
The
consequences
of
mechanical
damage
to
kale
and
mustards
are
discussed.
Materials
and
Methods
Plant
raising
and
insect
material
Plants
of
oilseed
rape
(Brassica
napus
L.,
cvs
Ariana,
Bienvenu,
Jet
Neuf,
Korina,
Mikado
and
Rafal),
brown
mustard
(B.
juncea
L.,
cv.
Trowse),
black
mustard
(B.
nigra
L.,
cv.
Sutton),
white
mustard
(Sinapis
alba
L.
Sutton
Seeds),
and
kale
(B.
oleracea
L.
var.
acephala,
cv.
Fribor,
212
V.
M.
KORITSAS,
J.
A.
LEWIS
AND
G.
R.
FENWICK
`F
1
hybrid')
were
raised
in
a
controlled
environment
chamber
at
a
photon-flux
density
of
120
it
E
m
-2
s
-1
,
and
a
light
regime
of
12
h
light
(15
°
C),
and
12
h
dark
(10
°
C).
The
soil
was
a
3:1
peat/sand
mixture
containing
potassium
nitrate
(0.15
g),
potassium
sulphate
(0.15
g),
magnesium
limestone
(2.95
g),
superphosphate
(1.20
g),
CaCO
3
(2.35
g),
and
Fortone
G
(fritted
trace
element,
containing
B,
Cu,
Fe,
Mn,
Mo
and
Zn
in
unknown
quantities,
0.37
g)
per
litre
of
soil
(Matkin
&
Chandler,
1957).
Individual
plants
were
physically
damaged
by
puncturing
the
petiole
with
a
hyperdermic
syringe
or
infested
with
seven
first-instar
P.
chrysocephala
larvae
when
their
fifth
leaf
was
emerging,
watered
every
two
days
with
tap
water
and
given
Hoaglands'
nutrient
solution
(25%
strength)
on
the
eighth
day
of
the
experiment.
Damaged
and
infested
plants
of
B.
napus,
cvs
Ariana
and
Rafal
were
collected
after
10
days.
B.
napus
cv.
Rafal
was
sown
in
experimental
plots
on
1
Sept
1987
on
a
calcareous
loam
soil
(pH
7.8)
which
had
received
50
kg
ha
-1
each
of
N,
K
and
P,
following
a
previous
crop
of
winter
wheat.
Two
experiments
were
carried
out
with
Ariana.
The
appropriate
parts
(petioles,
laminae,
stems
and
roots)
of
the
laboratory-
and
field-
grown
plants
were
excised
from
groups
of
ten
plants,
combined,
freeze-dried,
and
stored
at
20
°C.
Extract
preparation
The
ground,
freeze-dried
samples
(typically
0.3
-
0.5
g)
were
extracted
with
boiling
70%
(v/v)
methanol,
and
re-extracted
twice
more,
with
boiling
70%
methanol.
This
procedure
inactivated
myrosinase.
Following
removal
of
the
volatile
solvent
in
vacuo
at
40
°C,
the
samples
were
made
up
to
volume
(10
-
25
ml,
depending
on
size
of
initial
plant
material)
with
distilled
water,
and
stored
at
—20
°C
until
required
for
analysis.
Samples
were
subsequently
thawed
and
an
aliquot
added
to
100
/21
of
a
1
:1
(v/v)
mixture
of
barium
acetate
(0.5
m)
and
lead
acetate
(0.5
M)
solution
and
the
internal
standard
glucotropaeolin
(Peterka
&
Fenwick,
1988).
The
mixture
was
spun
at
2000
g
for
5
min
and
the
supernatant
applied
to
a
microcolumn
of
DEAE
-
Sephadex
A25
(40
mg)
in
a
Pasteur
pipette
(Heaney
&
Fenwick,
1980).
The
column
was
washed
with
2.0
ml
of
distilled
water,
allowed
to
drain
and
2
x
0.5
ml
of
0.02
M
pyridine-
acetate,
pH
7,
loaded
and
again
allowed
to
drain.
The
column
containing
the
glucosinolates
was
treated
with
75
/./1
of
a
sulphatase
preparation
(from
Helix
pomatia,
Sigma)
and
left
overnight
(16
-
18
h)
before
eluting
the
desulphoglucosinolates
with
3
x
0.5
ml
H
2
0,
combining
the
eluates.
Chromatographic
analysis
High
performance
liquid
chromatographic
analyses
were
conducted
in
duplicate
according
to
the
method
of
Spinks,
Sones
&
Fenwick
(1984),
using
a
Perkin
Elmer
Sigma
3B
system.
The
desulphated
glucosinolate
eluate
(10
ill)
was
injected
onto
a
250
x
4.6
mm
Spherisorb
ODS2
(5
pm)
column,
oven
temperature
30
°
and
flow
rate
1.5
ml/min.
The
solvent
systems
employed
were
:-
A)
Water,
distilled
and
passed
through
a
Norganic
cartridge
(Millipore,
Harrow).
B)
20%
acetonitrile
(Koch
Light,
HPLC
grade)
in
water
(as
A,
above).
The
programme
consisted
of
99%
A
+
1%
B
for
1
min,
followed
by
a
linear
gradient
over
20
min
to
1%
A
+
99%
B
and
held
at
99%
B
for
3
min.
The
programme
returned
to
99%
A
+
1%
B
by
a
linear
gradient
over
5
min,
followed
by
10
min
equilibrium.
The
eluted
desulphoglucosinolates
were
monitored
with
a
Perkin
Elmer
LC75
spectrophotometer
detector
(8
1
cm
path
length
flow
cell)
at
229
nm
and
0.04
a.u.f.s.
Response
factors
were
as
recommended
by
an
Expert
Group
of
the
Commission
of
the European
Community
(Buchner,
1987)
with
benzyl
glucosinolate
(glucotropaeolin)
being
employed
as
internal
standard,
except
Wounding,
cabbage
stem
flea
beetle
infestation
&
glucosinolates
213
in
the
case
of
S.
alba
where
sinigrin
was
used.
Detection
limits
were
<
20
itg
g
-1
for
Id,
Ie
and
<
10
pg
g
-1
for
all
other
glucosinolates.
The
area
of
desulphoglucosinolates
was
compared
with
the
area
of
the
internal
standard,
and
the
concentration
of
the
glucosinolate
calculated
according
to:-
Area
of
glucosinolate
x
response
factor
x
innol
standard/g
material
Area
of
standard
Results
The
glucosinolate
contents
and
composition
of
the
undamaged,
control
oilseed
rape,
mustard
and
kale
plants
were
consistent
with
earlier
investigations
of
these
species
(Fenwick
et
al.,
1983,
1989).
Differences
in
the
absolute
glucosinolate
levels
of
the
field-
and
laboratory-
grown
plants
(Figs
3
and
5)
are
considered
to
be
due
primarily
to
the
differing
physiological
ages
of
the
material
selected
for
analysis.
Field
studies
The
changes
in
glucosinolate
levels
of
various
parts
of
rapeseed
cv.
Rafal
collected
from
the
field
in
April
1988
following
infestation
by
cabbage
stem
flea
beetle
larvae
are
presented
in
Figs
3
and
4.
In
parts
directly
attacked
by
insects
(Figs
3A
and
3B),
the
predominant
indole
glucosinolates,
glucobrassicin
(If),
and
neoglucobrassicin
(Ig)
increased,
but
levels
of
all
7
0
A
B
C
D
6.0
5.0
7
4.0
ou
ro
6
0
30
0
2
0
2
J l
1.0
7
5
1
g
7
7
0
-J71
.
/41
fgh
abcdefgh
abcdet
gh
abcde fgh
labcde
Individual
Glucosinolate
Fig.
3.
Levels
of
individual
glucosinolates
in
leaves
of
field
collected
oilseed
rape
(cv.
Rafal)
infected
(0
)
or
non-infected
(
)
by
cabbage
stem
flea
beetle
larvae.
(A)
proximal
end
of
petiole,
(B)
distal
end
of
petiole
adjacent
to
lamina,
(C)
lamina
adjacent
to
(B),
(C)
petiole
between
(A)
and
(B)
but
not
directly
damaged
by
the
insect,
Glucosinolates
Ia-h
labelled
as
in
Fig.
1.
7
0
10-
0
G
lu
co
s
ino
la
te
(mg.
g
FD
W)
6.0
5
0
40-1
3.0
7
0
214
V.
M.
KORITSAS,
J.
A.
LEWIS
AND
G.
R.
FEN
WICK
8.9
16.5
A
z
b
cde
f
g
h
a
b
c
de fgh
a
b
cde
f
g
h
a
b
c
de
fgh
Individual
Glucosinolate
Fig.
4.
Levels
of
individual
glucosinolates
in
the
stem
base
(A),
stem
apex
(B),
secondary
branch
(C)
and
roots
(D)
of
field-collected
oilseed
rape
(cv.
Rafal)
infested
(0)
or
non-infested
(
)
by
the
cabbage
stem
flea
beetle
larvae.
Glucosinolates
Ia-h
labelled
as
in
Fig.
1.
aliphatic
glucosinolates
decreased.
The
indole
compounds
present
in
the
infested
parts
increased
more
at
the
distal
(Fig.
3B)
than
at
the
proximal
end
(Fig.
3A)
of
the
petiole.
Five-fold
and
approximately
2,5-fold
increase
in
neoglucobrassicin
and
glucobrassicin
contents,
respectively
were
seen
in
tissue
not
directly
attacked
by
the
flea
beetle
(Figs
3C
and
3D).
In
laminae
adjacent
to
infested
sites
(Fig.
3C),
the
indole,
aromatic
and
most
aliphatic
compounds
increased,
contributing
to
the
elevated
total
glucosinolate
levels
observed
in
this
tissue
(infested
plant
13.5
mg
g
-1
freeze
dried
weight,
control
11.5
mg
g
-
').
Whilst
a
general
reduction
in
aliphatic
glucosinolates
was
seen
in
apparently
healthy
regions
of
infested
petioles,
the
accumulation
of
the
glucobrassicin
and
neoglucobrassicin
was
clear
(Fig.
3D).
Differences
in
total
glucosinolates
of
infested
and
non-infested
aerial
tissues
of
oilseed
rape
cv.
Rafal
were
not
evident,
as
infestation
was
associated
with
increased
indole,
but
decreased
aliphatic
and
aromatic,
compounds.
Thus
total
glucosinolate
contents
of
stem
base,
stem
apex
and
secondary
branches
from
infested
plants
were
7.5,
8.4,
14.3
mg
g
-1
freeze
dried
weight,
respectively
compared
with
7.9,
8.8
and
14.2
mg
g
-
',
respectively,
from
controls.
The
largest
glucosinolate
levels
prior
to
infestation
were
found
in
the
expanding
secondary
branch
and
roots
of
the
plant.
In
tissue
from
infested
plants,
glucobrassicin
(If)
increased
gradually
at
ascending
height
along
the
plant
stem
(Figs
4A-C),
with
progoitrin
(Ia)
and
glucobrassicana-
pin
(Id)
being
the
dominant
aliphatic
glucosinolates.
Aerial
infestation
increased
all
root
glucosinolates
(Fig.
4D)
resulting
in
total
glucosinolate
content
of
32.3
mg
g
- '
freeze
dried
root
weight,
compared
with
18.2
mg
g
-1
from
non-infested,
control,
plants.
4-Methoxyglucobras-
sicin
(Ih)
was
not
detected
in
the
roots.
Wounding,
cabbage
stem
flea
beetle
infestation
&
glucosinolates
215
Table
1.
Glucosinolate
contents
(mg
g
-1
freeze-dried
material)
of
petioles
of
laboratory-grown
oilseed
rape
(cv.
Ariana)
plants
infested
(+)
or
non-infested
(-)
with
P.
chrysocephala
larvae
Glucosinolates*
Infestation
Aliphatic
Indole
Category
r
.k
Total
A
romatic
r----L---,
Total
(+/-)
la
Ib
Ic
Id
aliph.
le
If
Ig
Ih
indoles
Total
February
1989
-
2.19
1.00
0.82
3.70
(7.71)
0.16
0.03
0.05
0.08
(0.16)
8.05
+
1.26
0.37
0.73
1.40
(3.76)
0.14
0.31
1.03
0.11
(1.45)
5.34
December
1988
0.51
0.34
0.05
N.D.
(0.90)
0.25
0.23
0.36
0.18
(0.77)
2.85
0.42
0.15
0.06
(0.63)
0.32
1.11
2.71
0.24
(4.06)
5.63
*
Glucosinolates
as
listed
Fig.
1.
N.D
not
detected
(see
'Materials
and
Methods'
section
for
detection
limits).
Laboratory
studies
-
effect
of
infestation
The
effect
of
infestation
by
P.
chrysocephala
on
the
glucosinolate
content
of
petioles
of
B.
napus
L.
cv.
Ariana
is
shown
in
Table
1.
A
consistent
finding
was
an
increase
in
the
levels
of
the
indole
glucosinolates,
glucobrassicin
(If)
and
neoglucobrassicin
(Ig),
although
the
first
study
showed
a
decline
in
total
glucosinolate
content
from
8.1
to
5.3
mg
g
-
freeze
dried
weight
with
infestation,
whilst
the
second
showed
an
increase
from
2.9
to
5.6
mg
g
-1
following
infestation.
Whilst
the
former
is
due
mainly
to
a
reduction
in
aliphatic
glucosinolates
(7.7
,
3.8
mg
g
-1
),
the
latter
corresponds
almost
exactly
to
the
increase
in
indoles
(0.84.1
mg
g
-
')
following
infestation.
Marked
reductions
in
aliphatic
and
aromatic,
as
well
as
large
increases
in
indole
glucosinolates,
were
seen
in
infested
leaf
tissues
and
stems
of
laboratory-grown
B.
napus
cv.
Rafal
(Figs
5A-F).
Very
large
increases
in
indole
glucosinolate
levels
were
evident
in
the
young
petiole
and
lamina
tissues
(Figs
5A
and
5C,
respectively),
whereas
in
older
tissues
only
neoglucobrassicin
increased,
in
petioles
(Fig.
5B).
Aerial
infestation
resulted
in
undetectable
levels
of
aliphatic
and
aromatic
glucosinolates
in
stem
tissues
(Fig.
5E),
but
stimulated
the
production
of
glucobrassicin
(six-fold),
neogluco-
brassicin
(23-fold),
and
4-methoxy-glucobrassicin
(two-fold),
which
contributed
almost
wholly
to
the
total
gain
in
glucosinolate
levels
(infested
7.7
mg
g
-1
freeze
dried
weight;
control
2.4
mg
g
-
').
Similarly,
in
the
roots
of
infested
plants
(Fig.
5F)
the
indole
glucosinolate,
Ig,
increased
substantially
whilst
the
aliphatic
and
aromatic
compounds
declined,
it
was
observed
that
there
were
less
roots
in
the
infested,
as
compared
to
control,
material.
The
major
compound
in
root
tissue
from
non-infested
plants
was
the
aromatic
glucosinolate,
gluconasturtiin
(Ie).
Effect
of
wounding
In
the
five
laboratory-grown
cultivars
of
rapeseed
tested,
indole
glucosinolate
contents
increased
in
response
to
wounding
whilst,
with
the
exception
of
cv.
Jet
Neuf,
aliphatic
glucosinolate
content
declined
(Table
2).
To
examine
the
response
of
oilseed
rape
petiole
and
lamina
to
increasing
mechanical
damage,
petiole
tissues
of
cv.
Rafal
were
punctured
1,
4,
8
and
16
times.
Although
damage
increased
the
total
glucosinolates
of
young
petioles
(control
4.2
mg
g
-1
freeze
dried
weight;
6.7
mg
g
-
'
for
tissue
subjected
to
16
punctures),
the
levels
of
individual
glucosinolates
varied
markedly
(Fig.
6A).
There
was,
however,
an
apparent
association
between
glucobrassicin
216
V.
M.
KORITSAS,
J.
A.
LEWIS
AND
G.
R.
FENWICK
4.0
-
A
3.0
-
2.0
-
1.0
-
n
a
bcde
fgh
0
Ia
b
c
d
e
f
g
h
6
0
-
5.0
-
4.0
-
3.0
-
2.0
-
C
D
1
0
-
0
1
F1
abcde
f
gh abcde
f
gh
5.0
-
4
0
-
3.0
-
2.0
-
1.0
-
0
abcde
f
gh
abcde
f
g
h
Individual
Glucosinolate
Fig.
5.
Individual
glucosinolate
response
of
infested
(Z
)
and
non-infested
(
)
laboratory-grown
rape
(cv.
Rafal)
plants.
Young
petiole
(A),
mature
petiole
(B),
young
lamina
(C),
mature
lamina
(D),
stem
(E)
and
root
(F).
Glucosinolates
Ia-h
labelled
as
in
Fig.
I.
content
and
degree
of
damage,
and
an
inverse
relationship
between
the
latter
and
the
levels
of
progoitrin
and
gluconapin
(Ia,b).
Analysis
of
old
tissue
showed
similar
trends
(not
illustrated)
but
the
levels
of
glucosinolates
were
much
reduced.
The
kale
plant
(cv.
Fribor)
included
in
this
study
showed
an
increase
in
indole
glucosinolate
content
after
damage
(total
figures
0.12-496
mg
g
- '
freeze-dried
weight).
Increases
in
progoitrin
(Ia)
and
glucobrassicanapin
(Id)
contents
were
also
observed,
such
that
total
aliphatic
glucosinolates
rose
from
3.9-6.4
mg
g
- '
freeze-dried
weight
(Table
3).
Gluco
s
ino
la
te
(tng.
g
-
'
FD
W)
Wounding,
cabbage
stem
flea
beetle
infestation
&
glucosinolates
217
Table
2.
Glucosinolate
contents
(mg
g
-1
freeze
dried
material)
of
petioles
of
laboratory-grown
rapeseed
plants
with
(+)
or
without
(-)
mechanical
damage
Glucosinolates
Aliphatic
indole
Damage
(
A
Total
Aromatic
(
1
)
Total
Cultivar
(+1-)
Ia
Ib
Ic
Id
aliph.
Ie
If
Ig
lh
indoles
Total
Ariana
-
0.55
0.40
0.04
0.42
(1.41)
0.14
0.11
0.20
0.12
(0.43)
2.15
+
0.15
0.17
N.D.
0.09
(0.41)
0.07
0.22
0.22
0.14
(0.56)
1.13
Bienvenu
-
0.44
0.17
0.06
0.11
(0.78)
0.27
0.19
0.07
0.07
(0.33)
1.48
+
0.37
0.10
0.05
N.D.
(0.52)
0.22
1.00
0.28
0.16
(1.44)
2.27
Jet
Neuf
-
0.31
0.13
0.07
0.19
(0.60)
0.11
0.18
0.11
0.04
(0.33)
1.13
+
0.36
0.18
0.04
0.17
(0.75)
0.25
1.00
0.53
0.11
(1.64)
2.70
Korina
-
1.67
0.78
0.07
1.08
(3.60)
0.39
0.13
0.23
0.15
(0.90)
4.71
+
1.20
0.40
0.08
0.90
(2.58)
0.27 0.87
0.80
0.30
(2.24)
5.05
Mikado
-
0.61
0.39
0.14
0.51
(1.65)
0.59
0.28
0.21
0.14
(1.12)
3.02
+
0.48
0.41
0.09
0.34
(1.32)
0.64
0.49
0.33
0.15
(1.61)
3.06
Glucosinolates
as
listed
in
Fig.
1.
A
2.0
-
10
-
Gluc
o
s
ino
la
te
(mg.
g
FD
W)
3
0
-
2.0
-
1
0
-
0
0
0
16
0
16
d
B
r
TF
0
16
0
16
b
c
1
014816
0
16
Ia
r
0
16
e
=0=to
0
16
0
16
0
16
f
g
h
0
16
0
16
0
16
com.
0
16
0
16
0
16
la
Number
of
stabs
Fig.
6.
The
effect
on
the
individual
glucosinolate
levels
of
puncturing
laboratory-grown
oilseed
rape
(cr.
Rafal)
tissue
1,
4,
8
or
16
times
with
a
syringe.
Young
petiole
(A),
young
lamina
(B).
Glucosinolates
Ia-h
labelled
as
in
Fig.
1.
The
mustard
plants
examined
showed
a
rather
different
response
to
mechanical
damage
(Table
3)
from
those
of
the
oilseed
rapes
and
kale.
Large
increases
in
the
amounts
of
total
glucosinolates
following
such
damage
resulted
from
the
increase
of
sinigrin
(Ti)
in
B.
nigra
and
B.
juncea,
and
of
sinalbin
(Ij)
in
the
S.
alba
plants.
218
V.
M.
KORITSAS,
J.
A.
LEWIS
AND
G.
R.
FEN
WICK
Table
3.
Glucosinolate
contents
(mg
g
-
'
freeze
dried
material)
of
laboratory-grown
mustard
and
kale
plants
with
(+)
or
without
(-)
mechanical
damage
Species
Damage
(+/-)
Aliphatic
A
Glucosinolates
Aromatic
1
If
l
Indole
Ig
Ih
Total
(
Ia
lb
Id
Ii
le
Ij
lk
Sinapis
alba
-
2.08
1.26
<0.01
<0.01 <0.01
3.37
+
7.07
2.21
<0.01
0.10
0.04
8.09
Brassica
nigra
<0.01
6.71
0.77
<0.01 <0.01
7.50
+
0.07
10.64
0.46
0.10
0.01
11.30
Brassica
juncea
3.96
0.32
0.01
0.03
4.30
+
10.46
0.48
0.15
0.10
11.46
Brassica
napus
-
1.96
0.98
0.98
0.40
0.08
0.04
<0.01
4.45
var.
acephala
+
2.78
0.97
1.62
0.53
0.57
0.24
0.15
6.85
Glucosinolates
as
listed
in
Fig.
1.
Discussion
In
oilseed
rape
plants
the
content
of
aliphatic
glucosinolates
initially
greatly
exceeded
that
of
the
indoles,
primarily
as
a
result
of
the
presence
of
high
levels
of
progoitrin
and
glucobrassicanapin.
The
effect
of
laboratory
infestation
of
cv.
Ariana
was
to
greatly
increase
the
levels
of
indole
glucosinolates,
especially
those
of
glucobrassicin
and
neoglucobrassicin,
a
finding
in
agreement
with
the
earlier
field
and
laboratory
studies
on
cv.
Rafal
(Koritsas
et
al.,
1989).
Infestation
also
caused
a
reduction
in
the
levels
of
aliphatic
glucosinolates.
Previous
reports
of
tissue
damage
affecting
glucosinolate
content
were
described
by
Butcher
and
his
colleagues
(Butcher
et
al.,
1974,
1976;
Searle,
Chamberlain,
Rausch
&
Butcher,
1982;
Rausch,
Butcher
&
Hilgenberg,
1983)
and
Lammerink
et
al.
(1984).
The
former
workers
also
found
that
infection
of
swede
(B.
napus
L.)
root
tissue
with
clubroot
organism
(Plasmodiphora
brassicae
Wor.)
led
to
increased
amounts
of
glucobrassicin
but
that
other
indole
glucosinolates
were
unaffected.
The
present study
showed
large
increases
in
amounts
of
gluconasturtiin,
together
with
rises
in
other
glucosinolates,
both
aliphatic
and
indole,
in
apparently
healthy
roots
of
infested
oilseed
rape
plants.
The
analytical
procedures
employed
by
Butcher
et
al.
(1974),
and
Searle
et
al.
(1982)
were
specific
for
the
indole
glucosinolates,
however,
and
it
cannot
be
ruled
out
that
similar
changes
in
other
glucosinolates
as
found
here
were
not
also
occurring.
Studies
to
examine
the
effect
of
the
clubroot
organism
on
the
glucosinolate
content
of
susceptible
and
non-susceptible
brassicas
are
now
in
progress.
The
differing
response
of
aliphatic,
aromatic
and
indole
glucosinolates
is
consistent
with
the
probable
differing
biosynthetic
pathways
of
the
indole
compounds
(Fenwick
et
al.,
1983),
although
knowledge
of
this
area
is
fragmentary.
Given
the
wide-ranging
biological
properties
of
the
hydrolysis
products
from
aliphatic
and
aromatic
glucosinolates
(as
exemplified
by
the
isothiocyanates),
it
is
perhaps
at
first
sight
unexpected
that,
in
oilseed
rape
and
other
plants,
these
compounds
should
so
greatly
accumulate.
However,
whilst
they
have
been
much
less
studied
(due
primarily
to
the
lack
of
purified
material),
indole
glucosinolates
have
been
shown
to
be
highly
stimulating
to
several
adult
flea
beetle
species
(Larsen
et
al.,
1985),
and
their
products
were
fungitoxic
toward
the
stem
canker
organism
Leptosphaeria
maculans
(Mithen,
Lewis
&
Fenwick,
1986).
In
the
present
work,
an
increase
in
indole
glucosinolates
was
also
consistently
observed
in
mechanically
wounded
oilseed
rape
tissues
of
cvs
Ariana,
Bienvenu,
Jet
Neuf,
Korina
and
Mikado.
This
finding
again
supports
the
finding
in
cv.
Rafal
(Koritsas
et
al.,
1989)
and
suggests
the
physiological
effects
occurring
after
mechanical
damage
are
general
and
not
Wounding,
cabbage
stem
flea
beetle
infestation
&
glucosinolates
219
cultivar-specific.
These
damaged
tissues
contained
2
-p
125
times
more
indolic
compounds
than
those
found
in
non-damaged
tissues.
The
role
of
indole
glucosinolates,
however,
in
normal
plant
metabolism
remains
uncertain.
Kutacek
&
Kefeli
(1968)
suggested
that
glucobrassicin
may
be
able
to
act
as
a
precursor
of
indole
auxins
at
certain
stages
of
the
life
cycle
of
the
crucifers;
for
example,
during
the
'bolting'
stage
of
the
flowering
shoot.
In
Brassica
root
tissue
infected
with
P.
brassicae,
Bausch
et
al.
(1983)
demonstrated
the
involvement
of
glucobrassicin
in
indoleacetic
acid
biosynthesis,
even
though
its
conversion
to
indoleacetonitrile
(Fig.
2,X)
has
been
questioned
for
healthy
crucifer
tissues
(Schraudolf
&
Weber,
1969).
Evidence
suggests
that
potential
indoleacetic
acid
precursors,
glucobrassicin
and
indoleacetonitrile,
as
well
as
indoleacetic
acid
itself,
and
cytokinins,
are
present
at
increased
levels
in
clubbed
roots
(Raa,
1971
;
Tamura,
Nomoto
&
Nagao,
1972;
Butcher
et
al.,
1976;
Dekhuijzen,
1976,
1980).
From
other
studies,
auxins
and
cytokinins
appear
to
regulate
the
development
of
crown
gall
tumor
induced
by
the
bacterium
Agrobacterium
tumefaciens
(Drummond,
1983),
and
olive
knot
disease
(Pseudomonas
savastranoi)
in
Oleaceae
(Misaghi,
1982).
These
pathogens
evoke
at
least
a
part
of
their
symptoms
by
altering
the
hormonal
balance
of
the
host,
and
in
so
doing,
may
disturb
the
translocation
flow
of
the
plant.
The
weevils
Ceutorrhynchus
pleurostigma
and
C.
chalybaeus
are
known
to
induce
gall
and
gall-like
structures
in
crucifers
(Meyer,
1987)
and
may
also
disturb
the
hormonal
balance
of
the
plants,
creating
assimilate
sinks
that
provide
nutrients
to
the
insects.
Similarly,
infested
or
wounded
rape
petioles
having
increased
indoles
and
perhaps
cytokinins,
may
also
act
as
metabolic
sinks
diverting
assimilates
such
as
carbohydrates
and
N-
compounds.
In
the
case
of
P.
chrysocephala
this
would
represent
a
classic
example
of
a
pest
benefiting
from
exploiting
its
host
by
altering
its
metabolism.
The
mustards
which
have
been
examined
in
this
preliminary
investigation
show
markedly
different
behaviour,
in
that
the
levels
of
sinigrin
and
sinalbin
are
increased
in
B.
juncea,
B.
nigra
and
S.
alba,
respectively.
Fenwick
et
al.
(1983),
and
more
recently
Chew
(1988)
have
discussed
the
manifest
biological
properties
of
sinigrin
and
allyl
isothiocyanate,
its
major
hydrolysis
product.
Information
on
sinalbin
is
more
sparse,
but
the
compound
stimulated
feeding
by
the
seed
weevil,
Ceutorhynchus
assimilis
(Chew,
1988),
and
produced
response
characteristics
in
the
moth
Mamestra
brassicae
(Wieczorek,
1986).
It
has
been
suggested
that
glucosinolates,
rather
than
hydrolysis
products,
are
the
active
content
stimuli
in
the
feeding
specificity
of
cruciferous
insects
(David
&
Gardiner,
1966;
Blau,
Feeny
&
Contado,
1978;
Chew,
1988).
Indeed,
glucosinolates
in
different
proportions
could
provide
a
basis
for
insect
preference
of
different
crucifers.
In
the
present
study,
larvae
preferred
young
to
old
tissues.
Young
petioles
appeared
to
be
more
responsive
to
infestation
in
terms
of
glucosinolate
changes
than
old
tissue.
The
ability
of
the
young
tissue
to
develop
high
levels
of
indole
glucosinolates
may
be
one
reason
why
larvae
favoured
these
tissues.
Biochemical
and
physiological
changes
resulting
from
altered
indole
levels
in
such
tissue
may
make
it
nutritionally
acceptable
to
the
insect.
The
full
significance
of
the
findings
reported
here,
and
their
extrapolation
to
other
pathogen
infections
and
insect
damage
has
yet
to
be
assessed.
However,
the
present
findings
do
perhaps
call
into
question
the
reliability
of
using
glucosinolate
'profiles'
from
unchallenged
(healthy)
plants
as
a
means
for
predicting
host
status.
Whilst
chromatographic
methods
are
available
for
such
analyses,
and
can
be
of
use
to
plant
pathologists
it
is
likely
that
special
benefit
will
accrue
from
the
introduction
of
rapid,
simple
and
specific
immunochemical
procedures,
the
development
of
which
are
currently
in
progress
(R.
Mithen,
M.
R.
A.
Morgan
&
G.
R.
Fenwick,
unpublished
observation).
The
results
presented
here
also
suggest
that
current
ideas
regarding
the
role
and
relative
importance
of
indole
glucosinolates
in
plants
(McDanell
et
al.,
1988)
need
to
be
re-evaluated.
220
V.
M.
KORITSAS,
J.
A.
LEWIS
AND
G.
R.
FENWICK
Acknowledgements
The
authors
are
grateful
to
Science
and
Engineering
Research
Council
(V.
M
Koritsas)
and
Ministry
of
Agriculture
Fisheries
and
Food
(J.
A.
Lewis
&
G.
R.
Fenwick)
for
funding
the
work
described
here
and
thanks
Ms
Sue
Stickels
for
maintaining
the
insect
cultures.
Brassica
nigra
and
B.
juncea
seed
was
kindly
provided
by
Richard
Foss,
Crops
Department,
Colmans
of
Norwich.
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