Effects of intact glucosinolates and products produced from glucosinolates in myrosinase-catalyzed hydrolysis on the potato cyst nematode (Globodera rostochiensis cv. Woll)

Article abstract of DOI:10.1021/jf010470s

The potato cyst nematode (Globodera rostochiensis cv. Woll) is responsible for large yield losses in the potato crop, and opportunities for reducing the attack of these plant nematode species are, therefore, important. This study has been devoted to the testing of the in vitro effects on the potato cyst nematode of eight glucosinolates [prop-2-enyl-, but-3-enyl-, (R)-4-methylsulfinylbut-3-enyl-, benzyl-, phenethyl-, 4-hydroxybenzyl-, (2S)-2-hydroxybut-3-enyl-, and (2R)-2-hydroxy-2-phenylethylglucosinolate] as well as the effects of the products of this myrosinase-catalyzed hydrolysis. The glucosinolates were used at three concentrations, 0.05, 0.3, and 1.0 mg/mL, in the presence or absence of the enzyme myrosinase. The effects of the compounds on the mortality were monitored every 8 h for a 72 h period. No effects were found for any of the intact glucosinolates. However, when active myrosinase was included with 1 mg/mL phenethylglucosinolate at pH 6.5, 100% mortality was observed within just 16 h. A similar effect was achieved at the same concentration of benzyl-and prop-2-enylglucosinolates in the myrosinase-containing solutions, although longer exposures were required (24 and 40 h, respectively). The main aglucone products released from the glucosinolates with pronounced effects on the nematodes were shown to be the corresponding isothiocyanates. The results suggest that mixtures of these specific glucosinolates and active myrosinase or autolysis of plant materials containing these enzymes and glucosinolates might be used to control the potato cyst nematode in the soil.

Full text of DOI:10.1021/jf010470s

690 J. Agric. Food Chem. 2002, 50, 690−695
Effects of Intact Glucosinolates and Products Produced from
Glucosinolates in Myrosinase-Catalyzed Hydrolysis on the
Potato Cyst Nematode (Globodera rostochiensis Cv. Woll)
S. BUSKOV, B. SERRA, E. ROSA,* H. SØRENSEN, AND J. C. SØRENSEN†
†
‡
,‡
†
Universidade de Tr a´ s-os-Montes e Alto Douro, Apartado 202, 5001-911 Vila Real Codex, Portugal,
and Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark
The potato cyst nematode (Globodera rostochiensis cv. Woll) is responsible for large yield losses in
the potato crop, and opportunities for reducing the attack of these plant nematode species are,
therefore, important. This study has been devoted to the testing of the in vitro effects on the potato
cyst nematode of eight glucosinolates [prop-2-enyl-, but-3-enyl-, (R)-4-methylsulfinylbut-3-enyl-,
benzyl-, phenethyl-, 4-hydroxybenzyl-, (2S)-2-hydroxybut-3-enyl-, and (2R)-2-hydroxy-2-phenyleth-
ylglucosinolate] as well as the effects of the products of this myrosinase-catalyzed hydrolysis. The
glucosinolates were used at three concentrations, 0.05, 0.3, and 1.0 mg/mL, in the presence or
absence of the enzyme myrosinase. The effects of the compounds on the mortality were monitored
every 8 h for a 72 h period. No effects were found for any of the intact glucosinolates. However,
when active myrosinase was included with 1 mg/mL phenethylglucosinolate at pH 6.5, 100% mortality
was observed within just 16 h. A similar effect was achieved at the same concentration of benzyl-
and prop-2-enylglucosinolates in the myrosinase-containing solutions, although longer exposures were
required (24 and 40 h, respectively). The main aglucone products released from the glucosinolates
with pronounced effects on the nematodes were shown to be the corresponding isothiocyanates.
The results suggest that mixtures of these specific glucosinolates and active myrosinase or autolysis
of plant materials containing these enzymes and glucosinolates might be used to control the potato
cyst nematode in the soil.
KEYWORDS: Glucosinolates; isothiocyanates; myrosinase; potato cyst nematode
INTRODUCTION
glucosinolate derivative, prop-2-enylisothiocyanate, on the
potato cyst nematode was first reported by Ellenby (14) and,
later, methylisothiocyanate (Vapam) was found to be an effective
nematocide (15), which is still used today. The high costs of
this and other synthetic nematocides and their negative envi-
ronmental impact prompted several studies, which suggest
opportunities for new biotechnological processing to produce
environmentally friendly products (8, 16) and the use of nonhost
plants containing glucosinolates in crop rotation, particularly
rapeseed (17-23). From these studies it is clear that more
specific information is needed concerning the effects of each
individual glucosinolate and the concentrations at which they
exert their influence. In vitro studies (24) have revealed the
nematocidal effects of some glucosinolate breakdown products
against the cyst nematode Heterodera schachtii, whereas no
effects were achieved for the intact glucosinolates. Similar
findings were reported in experiments with prop-2-enylglucosi-
nolate by Donkin et al. (25) on the nematode Caenorhabditis
elegans and by Pinto et al. (26) on Globodera rostochiensis.
Consideration of opportunities for protection against pest
problems caused by the potato cyst nematode is important as
potato is one of the major vegetable crops grown worldwide,
Glucosinolates (Figure 1) are a group of allelochemicals that
occur in all plants of the order Capparales and in some few
other plants (1-4). More than 130 structurally different glu-
cosinolates have been identified (3, 4) and may, according to
the structure of the amino acid derived side chain (R), be divided
into three subclasses comprising aliphatic, phenyl, and indol-
3-ylmethylglucosinolates, respectively (4, 5). However, it is also
well-known that glucosinolates co-occur in the plant with the
group of â-thioglucoside glucohydrolase isoenzymes (EC
3.2.3.1), generally known as myrosinase (6-8). When the tissue
is damaged, glucosinolates are hydrolyzed to give a broad range
of compounds, such as isothiocyanates, thiocyanates, nitriles,
and oxazolidine-2-thiones, depending on the hydrolysis or
autolysis conditions (9-12). These hydrolysis products have
shown various bioactive effects, for instance, against some soil-
borne diseases and nematodes (5, 13). The effect of the
*
Corresponding author (telephone +351-259350446; fax +351-
2
59350327; e-mail erosa@utad.pt).
†
Royal Veterinary and Agricultural University.
Universidade de Tr a´ s-os-Montes e Alto Douro.
‡
1
0.1021/jf010470s CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/12/2002

Effect of Glucosinolates on the Potato Cyst Nematode
J. Agric. Food Chem., Vol. 50, No. 4, 2002 691
Figure 1. Structures of intact glucosinolates used for tests against the potato cyst nematode.
and severe yield losses have been reported in potato crops
produced in soils infested with this nematode (27), particularly
when no crop rotation is used. The suppressive effects of
incorporated Brassicaceae residues on nematodes has been
known since the beginning of the second half of the 20th century
Brassica crops have against the potato cyst nematode. The
objective has been the evaluation of in vitro effects on the
activity of the potato cyst nematode (G. rostochiensis cv. Woll
exerted by intact glucosinolates and the hydrolysis products
released from the glucosinolates in myrosinase-catalyzed reac-
tions, including isothiocyanates, oxazolidine-2-thiones, and
alcohols.
(28). Several of the commonly cultivated plants belonging to
the Brassicaceae are hosts to one or more species of the
nematode Meloidogyne (29). Results showed that collard
MATERIALS AND METHODS
(Brassica oleracea var. acephala cv. Georgia Southern) was
the least susceptible followed by broccoli (Brassica oleracea
var. botrytis cv. De Cicco). Isothiocyanates, apart from being
recognized as one of the major inhibitors of microbial activity,
have also shown insecticidal activity, especially the aromatic
isothiocyanates, as has been reported in several studies (5).
Recent in vitro research has revealed the susceptibility of the
potato nematode (G. rostochiensis) to prop-2-enylisothiocyanate
Glucosinolates and Other Chemicals. All glucosinolates have been
isolated as potassium salts according to standard procedures described
elsewhere (2). This procedure comprises an initial isolation of the total
glucosinolate fraction in a mixture with other anions, using the described
(2, 4) anion exchange techniques. Separation and purification of the
individual glucosinolates were subsequently performed by using the
described (2) Sephadex G-10/G-25 (Fine), Polyclar AT, and SPE
column chromatographic techniques and, finally, crystallization and
recrystallization. The glucosinolates thus obtained and used in this
project comprise prop-2-enylglucosinolate (sinigrin), but-3-enylglu-
cosinolate (gluconapin), (R)-4-methylsulfinylbut-3-enylglucosinolate
(glucoraphenin), benzylglucosinolate (glucotropaeolin), phenethylglu-
cosinolate (gluconasturtiin), 4-hydroxybenzylglucosinolate (sinalbin),
(26), and Heterodera schachtii has shown to be suppressed by
glucosinolate hydrolysis products using an initial glucosinolate
concentration of 0.5-5.0 mg/mL (24). Furthermore, green
manure of Brassica napus, probably containing glucosinolates
or glucosinolate hydrolysis products, was reported to reduce
the number of nematodes (Pratylenchus neglectus) to 50% of
the initial number (30). These results show the potential
antinematocidal effects of some of the glucosinolate-derived
hydrolysis products, but because of the limited number of
glucosinolates studied, further research is required.
(
2S)-2-hydroxybut-3-enylglucosinolate (epiprogoitrin), and (2R)-2-
hydroxy-2-phenylethyl glucosinolate (epiglucobarbarin) (Figure 1). The
identity and purity of the glucosinolates were determined by UV and
NMR spectroscopy (4) and by micellar electrokinetic capillary chro-
matography (MECC) according to the procedures described in Sørensen
et al. (31). All other chemicals were of analytical reagent grade.
Nematodes. Cysts of the potato nematode G. rostochiensis cv. Woll
were collected in a naturally infested field near Vila Real, Portugal,
In this study we have performed a systematic investigation
of the effects a number of common glucosinolates found in

692 J. Agric. Food Chem., Vol. 50, No. 4, 2002
Buskov et al.
Figure 2. Structures of glucosinolate-derived hydrolysis products obtained.
and kept in a refrigerator at 4 °C. Second-stage juveniles were induced
to hatch from the eggs within the cysts by placing the cysts in a small
Petri dish for 3 days at room temperature (∼20 °C), followed by 6
days in water and an extra 3 days in contact with potato root exudate
by placing the cysts on sieves made of muslin and supported on glass
blocks. During the initiation and release of juveniles from the cysts,
these were kept in the darkness at room temperature (∼20 °C).
Experiments. Tests were performed in Petri dishes (diameter ) 5
cm) containing 2 mL of a solution of each of the glucosinolates (2
mL) either with or without co-addition of myrosinase. All experiments
were carried out in 100 mM phosphate buffer, pH 6.5 (31). Phosphate
buffer alone was used as a control. Each test was replicated five times
using 15 second-stage juveniles per replicate. The Petri dishes were
kept in darkness at room temperature (20 °C) throughout the experiment.
The activity of the nematode juveniles was monitored every 8 h during
a 72 h period. It was assumed that a nematode was dead when it was
immobile in the solution and did not react to stimuli caused by pricking
the body with a needle or if the larvae did not regain mobility when
placed in water slightly heated by an incandescent light bulb. The
mortality ratio was determined as defined by the Abbot (32) formula:
Prior to HPLC analysis of degradation products, all samples were
treated with NH
To 1 mL of each sample was added 1 mL of 2 M NH
3
to transform isothiocyanates into thiourea derivatives.
in ethanol, and
3
samples were mixed thoroughly. After 1 h at room temperature, samples
were freeze-dried and analyzed by HPLC. All samples were analyzed
using a YMC Basic HPLC column [250 × 4.6 mm (i.d.), 5 µm particles]
2 4 2 4
and gradient elution. Eluent A was 25 mM NaH PO /Na HPO , pH
6.5, with 10% acetonitrile (ACN). Buffer B was 100% ACN. Buffers
were degassed and subsequently filtered through a 0.40 µm filter prior
to use. Initially, the eluent was 100% A/0% B. After 3 min, the eluent
was linearly changed to 55% A/45% B in 12 min (3.75% B/min) and
-1
then returned to initial conditions. Flow rate was 1.5 mL min , column
temperature was held at 30 °C, and detections were performed at 230
and 240 nm.
RESULTS
Analysis of Glucosinolates and Degradation Products of
Glucosinolates. The myrosinase-catalyzed hydrolysis of glu-
cosinolates used in the tests of nematocidal activity toward the
potato cyst nematode yielded a broad range of different products
comprising both isothiocyanates, alcohols, and oxazolidine-2-
thiones (Figure 2). No remaining intact glucosinolates, for
example, sinigrin, gluconapin, glucoraphenin, glucotropaeolin,
gluconasturtiin, epiprogoitrin, and epiglucobarbarin, could be
detected following myrosinase reaction, thus indicating that the
myrosinase-catalyzed hydrolysis reactions had proceeded to
completion. Small amounts of nondegraded intact glucosinolate
could be detected in the experiments utilizing sinalbin and its
hydrolysis products. Generally, three different groups of deg-
radation products from the selected glucosinolates could be
detected. From sinigrin, gluconapin, glucoraphenin, glucotro-
paeolin, and gluconasturtiin, the respective isothiocyanates were
identified by HPLC after transformation into their thiourea
derivatives, which were formed upon addition of NH3 in ethanol
to the solutions. 4-Hydroxybenzyl alcohol was detected as the
main degradation product from sinalbin, in agreement with the
limited stability of the initially formed 4-hydroxybenzylisothio-
cyanate. Myrosinase-catalyzed hydrolysis of both epiprogoitrin
mortality corrected )
%
mortality induced - % mortality control
×
100
1
00 - % mortality control
Analysis of Glucosinolates. Analyses of individual glucosinolates
were performed by high-performance capillary electrophoresis (HPCE)
using the described procedures based on MECC; cetyltrimethylammo-
nium bromide was used as surfactant (31, 33).
HPLC Analysis of Glucosinolate Degradation Products. Samples
containing products produced during myrosinase-catalyzed hydrolysis
of the tested glucosinolates were analyzed by reversed phase HPLC.
The procedure is a modification of the previously described reversed
phase HPLC and MECC methods, adapted to determinations of the
glucosinolate degradation products (4, 9, 11, 31, 34). The Gilson HPLC
system (Middleton, WI) consisted of two Gilson 306 pumps, a Gilson
8
11C mixer (1.5 mL), a Gilson 233 XL autosampler (20 µL sample
loop), a Gilson 402 syringe pump, a Gilson 831 column oven, and a
Gilson 119 UV-vis detector. Column back-pressure was measured by
a Gilson 821 pressure regulator. The system was controlled by Unipoint
system software v. 1.91 (Gilson).

Effect of Glucosinolates on the Potato Cyst Nematode
J. Agric. Food Chem., Vol. 50, No. 4, 2002 693
Table 1. Effect of the Eight Glucosinolates after Enzymatic Hydrolysis by Myrosinase To Release the Corresponding Isothiocyanate at Three
a
Concentrations during Nine Sampling Times (Every 8 h)
concn
glucosinolate
prop-2-enyl-
(mg/mL)
8 h
16 h
24 h
32 h
40 h
48 h
56 h
64 h
72 h
0.05
0
1.3
6.7
0
0
4
0
0
0
0
0
1.3
0
0
0
0
0
0
0
0
1.3
20
0
2.7
6.7
0
0
1.3
0
0
1.3
0
0
2.7
0
0
1.3
8
18.7
100
0
54.7
93.3
0
6.6
29.3
2.7
2.7
13.3
1.3
0
4
0
0
2.7
0
0
5.3
0
1.3
9.3
14.7
38.7
2.7
15.9
56
2.7
4
28
2.7
0
16
34.6
100
2.7
18.7
30.7
2.7
2.7
8
0
0
2.7
2.7
4
12
4
6.7
13.3
30.7
54.7
28
50.6
29.4
50.6
38.7
58.6
42.7
65.3
0.3
1
0.05
13
21.3
36
10
13.2
21.4
41.4
5.3
8
12
1.3
0
4
5.3
8
13.3
9.3
17.3
22.7
50.7
65.3
14.2
25.7
44.3
9.3
12
13.3
1.3
1.3
4
8
9.3
13.3
10.7
17.3
22.7
57.3
70.7
but-3-enyl-
0.3
1
17.1
37.1
4
5.3
10.7
1.3
0
2.7
5.3
8
0.05
4
4-hydroxybenzyl-
0.3
1
2.7
9.3
0
6.7
0
0
0.05
4-methylsulfinylbut-3-enyl-
0.3
1
0
2.7
2.7
1.3
9.3
2.7
5.3
10.7
25.3
46.7
2.7
5.3
8
0.05
2-hydroxybut-3-enyl-
0.3
1
12
12
0.05
6.7
14.7
16
38.7
57.3
9.3
17.3
18.7
40
2-hydroxy-2-phenylethyl-
0.3
1
0.05
phenethyl-
0.3
1
1.3
82.7
0
21.3
82.7
64
0.05
5
70.7
100
12
73.3
15
80
21
84
21
86.7
24
86.7
25
90.7
benzyl-
0.3
1
a
Values are expressed in percentage of the cumulative mortality corrected according to Abbott’s formula.
and epiglucobarbarin initially released the corresponding isothio-
cyanates, which were rapidly transformed into the oxazolidine-
depended on the type of compound released during the
myrosinase-catalyzed hydrolysis of the glucosinolates, of which
the strongest effects were observed with phenethyl- and ben-
zylglucosinolates, both yielding isothiocyanates as the initial
quantitatively dominating product (11, 34). Similar results have
been reported by Lazzeri et al. (24), who found benzylisothio-
cyanate to have a nematocidal effect within 48 h of treatment
of Heterodera schachtii. Now, it is, however, also shown that
the homologous isothiocyanate, phenethylisothiocyanate, also
demonstrates good antinematocidal effects.
2-thiones (5R)-5-vinyl-1,3-oxazolidine-2-thione and (5S)-5-
phenyl-1,3-oxazolidine-2-thione.
Effect of Intact and Enzyme-Hydrolyzed Glucosinolates.
Intact glucosinolates induced no mortality on the second-stage
juveniles of the potato cyst nematode G. rostochiensis. When
myrosinase was co-added with each of the tested glucosinolates,
a highly significant difference (P < 0.001) between glucosi-
nolates and between concentrations was observed. The effect
of each concentration was dependent on the glucosinolate as
shown by the significant (P < 0.001) interaction glucosinolate
At a concentration of 6 mΜ, phenethyl- and benzylisothio-
cyanate showed nearly identical antinematocidal effects during
the first 8 h, but after 16 h, phenethylisothiocyanate was slightly
more effective, whereas 0.3 mM phenethylisothiocyanate was
more effective than benzylisothiocyanate; an opposite tendency
at 2 mΜ was noted with 21.3% mortality within the first 8 h of
benzylisothiocyanate treatment compared to 1.3% for pheneth-
ylisothiocyanate. After 16 h, >50% of the larvae were dead
after being exposed to the benzylisothiocyanate, whereas only
×
concentration. The cumulative mortality effects of each
glucosinolate at the three tested concentrations (0.05, 0.3, and
.0 mg/mL) are shown in Table 1. A 100% mortality was
1
observed for phenethylisothiocyanate at a concentration of 1.0
mg/mL corresponding to 6 mΜ within just 16 h. Only this
concentration resulted in a 100% mortality when the benzyl-
and prop-2-enylglucosinolates were used and after larvae
exposure of 16 and 40 h, respectively (Table 1). It is interesting
to note that both phenethyl- and benzylisothiocyanate induced
the same mortality within the first 8 h, but phenethylisothio-
cyanate was slightly more effective in the following period.
Similarly, phenethylisothiocyanate was more effective than
benzylisothiocyanate at a concentration of 0.05 mg/mL (0.3
mΜ), whereas the opposite occurred at 0.3 mg/mL. The lowest
mortality effect was observed for 4-methylsulfinylbut-3-enyl-
isothiocyanate at any of the tested concentrations. With the other
glucosinolate hydrolysis products, the larvae mortality was
1
8.7% mortality occurred in the presence of phenethylisothio-
cyanate. Benzylisothiocyanate was less effective in controlling
the second-stage juveniles of G. rostochiensis than it was for
juveniles of H. schachtii because a mortality of 100% was
achieved for this nematode after 48 h at 3.3 mM (24). In our
study the (2S)-2-hydroxybut-3-enylglucosinolate-derived com-
pound, (5R)-5-vinyl-1,3-oxazolidine-2-thione, resulted in a
nematode mortality as high as 13.3%, but greater effects are
likely to occur when concentration increases, as has been shown
in experiments with other nematodes (25).
<
50% for the 72 h period even when the highest concentration
was used.
In previous studies with Brassica crops (5) we found the
highest phenethylglucosinolate concentration in the roots, thus
leading to the suggestion of using these plants in association
with potatoes, at least during the first growth stages to control
the potato cyst nematode. Roots seem to be a poor host for
some nematode species as reported by Johnson et al. (21),
probably due to the high phenethylglucosinolate content.
DISCUSSION
To achieve mortality effect for any of the eight tested
glucosinolates, it was necessary to co-add active myrosinase,
as has been reported elsewhere (24-26). The mortality effect

694 J. Agric. Food Chem., Vol. 50, No. 4, 2002
Buskov et al.
Kirkegaard (35) and Potter (30) also suggested that root-type
glucosinolates are more potent than leaf-type glucosinolates in
soil biofumigation, mainly due to the effect from phenethyl-
isothiocyanate. However, the plant root biomass represents only
a small proportion of the total plant biomass, which depends
on the Brassica species. Thus, if these plants are used in soil
amendments, higher root biomass is required to increase the
beneficial effects, unless the concentration in the roots can
compensate for their lower biomass. Aerial plant parts containing
glucosinolates and particularly prop-2-enyl- and/or benzylglu-
cosinolates will induce, according to the results of this study, a
synergetic effect on the control of this nematode depending on
the glucosinolate concentration and biomass production. Envi-
ronmental conditions during hydrolysis (soil pH, temperature,
and humidity) might also affect the release of the desired
isothiocyantes and other glucosinolate-derived compounds.
Method of Analysis for the Determination of 4-Hydroxybenzyl-
glucosinolate Degradation Products. J. Biochem. Biophys. Meth-
ods 2000, 43, 157-174.
(
12) Buskov, S.; Hansen, L. B.; Olsen, C. E.; Sørensen, J. C.;
Sørensen, H.; Sørensen, S. Determination of Ascorbigens in
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(
13) Rosa, E. Daily variation in glucosinolate concentrations in the
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(
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(
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(
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sinase-catalyzed hydrolysis products are released. The magnitude
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isothiocyanate.
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(
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