& /#+&.o~‘~--- A+/+L.~ * J- F: ...
& /#+&.o~‘~---
A+/+L.~ * J- F:
Bd Rech. Agron. Gembloux [1993] 28 (4), 501-505
/3m z!‘- J+-- cd &LcclLL ”
~~~~~~~~
Tj&A;
NOTE
y58
* Y&
,‘-
LQcb
,Y
Identification of ‘elemental sulphur (Si)
in Bo&a senegalensis (PERS.) LAM ex POIR. leaves
bY
G. LOGNAY (‘), D. SECK (*‘), M. MAKLIER (‘), E. HAUBRUGE (“),
C. GASPAR (‘“), M. SEVERIN (‘)
Summary
Studies were undertaken to identify the molecules responsible of Boscia
senegalensis (PERS.) L.AM ex POIR. (Capparaceae) insecticide (fumigant) proper-
ties ; the volatiles were isolated from the leaves by steam distillation and
analyzed by Gas Chromatography - Mass Spectrometry. Only a few organic
molecules were detected and identified. The main constituent was elemental
sulphur (S,), a compound rarely found as such in the plant kingdom.
wwords : Boscia senegalensis, leaves, volatiles, elemental sulphur, GC-MS.
1. Introduction
&~@a senegaknsis (mKS.) LAM ex POIR. (Capparaceae) is a Little
shrub which grows throughout West African countries principally in the
Sahelian re’gion [B~KXIH, WICKENS, 19881. Several parts of the plant are
locally used for their medicinal properties [KERHARO, ADAMS, 19741 and
“) Depnrtmeqt of General and Organic Chemistry. Faculty of Agricultural
,..) :
‘cwnces. Passage des Déportés, 2 B-5030 GEMBWJX (Belgium).
Ueplrtment of General and Applied Zoology. Fzkulty of Agriculturnl
Siit,nces. Passage des Déportés, 2. B-5030 GEMRLOUX (BELCXJM).
502
G. LOGNAY et a1
sometimes the small cooked fruits are consumed by humans in case of
famine [SALIH et&., 19911. The 1 caves exhibit a strong biocide activity
against several stored grain insect species. A survey of the Senegalese
practices revealed that B. senegdezzsis fresh leaves are traditionally
added to stored grains in order to limit insect infestation and damages.
Chemical investigations proved that methylisothiocyanate (MITC)
enzymatically liberated from the methylglucosinolate (also called
glucocapparine) precursor was responsible for the insecticidal properties
[SECK et al. 19931. Since other volatile molecules could act as MITC
synergists, a characterization of 8. senegalensis steam distillate was
carried out. The results of this complementary study are reported herein.
2. Experimentals
B. senegalensis leaves were harvested in Thies (Senegal). They
were kept in the dark at subambiant temperature (-20°C) until use.
Hundred grams of freshly ground material were subjected to steam
distillation for 1 hour. The aqueous distillate was extracted three times
with 100 ml peroxide-free diethyl ether. The ethereal phases were
pooled, dried with anhydrous magnesium sulfate and the solvent was
distilled at 40°C to about 10 ml. The extract was further concentrated to
4 ml at room temperature under a stream of pure nitrogen and analyzed
by Cas Chromatography-Mass Spectrometry. A Nermag RlO-1OC Mass
Spectrometer coupled to a Delsi DI 700 Gas Chromatograph was used.
The operating conditions were the following :
- column CP-Sil-8 CI3 (Chrompack-The Netherlands) 25 m length,
0.32 mm I.D., 0.2 pm film thickness,
- temperature program : cold ‘on-column’ injection at 3O”C, T” rise at
5”C/min to 240°C then at lO”C/min to 29O”C,
- carrier gas : helium at 1 ml/min,
- ionisation voltage : 70 eV,
- source T” : 200°C.
The recorded fragmentation data were compared with those of the
EPA-NIH and WILEY-NBS mass spectra libraries (reference).
ELEMENTAL SULPHUR
503
3. Results and discussion
The total ion current (chromatographic profile) of 8. senegahsis
volatiles is presented in figure 1.
5 minutes
7
4
5
6
1
1
1
1
L
Stan number
Figure 1. - Total ion current of Bosch senegalensis steam-distillated volatiles.
As shown, only a few peaks were observed. MITC (compound 1)
liberated from methylglucosinolate during the distillation procedure was
detected as a shoulder in the tail of the solvent. In previous analyses
[SECK et&., 19931 using polar GC columns, this molecule was complete-
ly resolved and unambiguously identified. Compound 2 corresponds to
limonene whereas compound. 3 was identified as 2,6-di- tert-butyl-p-cre-
sol. The latter compound came from the diethyl ether to which it is
added as a stabilizer. Molecule 4 (M’ = 312, other main fragments at
m/z = 257, 256, 239, 227, 213, 129, 116, 71, 69, 57 and 56 [Base peak])
and its homologue 5 (M’ = 340, other main fragments at m/z = 285,284,
267, 129, 116, 71, 69, 57 and 56 [Base peak]) were tentatively attributed
to hexadecanoic and octadecanoic 2-methylpropyl esters. The phtalate
6 (Base peak at m/z = 149) eluted at 35.4 min is probably a contaminant
from the plastic bags used to store the leaves. The broad peak 7 at
-
--
-- ---if-
5 0 4
G.LQCNAY et al.
24.4 min exhibited a typical fragmentation pattem (Figure 2) with a
molecular ion at 256 (33 %) and two isotopic ions p + 1 (1 %) and p + 2
(10 %) rezecti‘vely at m/z = 257 and 258. This particular cluster (due to
33S and S natural isotopes) is indicative of a sulphur-containing
product. The nature of such a molecule is fully confirmed by a series of
similar clusters corresponding to a systematic loss of 32 atomic mass
units. Compound 7 was unambiguously identified as elemental sulphur
(S,). The comparison of the recorded S, mass spectrum with those of the
EPA-NIH and WILEY libraries supported our identification. The fact
that 8. senegahsis leaves samples came from trees not treated with
S-containing pesticides is an argument confirming that elemental
sulphur may not be considered as a contaminant.
2 5 6
128
160
192
@
'
9 6
l
2 2 4
Figure 2. - Mass spectrum of the elemental sulphur (S,).
A. from Boscih senegalensis ; B. reference.
TO the authors knowledge, it is only the second time that
elemental sulphur is detected in a plant material. Indeed, recently, the
same compound was identified in Capparis spinosa L. flower buds [BM-
VARD et&., 19921.
The biochemical pathway leading to the accumulation of elemental
sulphur remains unexplained.
ELEMENT.AL SULPHUR
505
Acknowledgements
The authors gratefully acknowledge Dr. E. DELCAKTE and Dr. R. PAUL
(Faculty of Agricultural Sciences of Gembloux) for their helpful cooperation.
References
BOOTII F., WICKENS G. (19881. Non-timber uses of selected arid zone trees and
shrubs in Africa. FAO Conservation Guide, n”19, 176 p.
BKEVAKD H., BRAMBILLA M., CFIAINTREAU A., MARION J.P., DISERENS H. [1992].
Occurrence of elemental sulphur in capers (Capparis spinosa L.) and first
investigation on the flavour profile. Havour Fragm. J, 7, 313-321.
KERFIARO J., ADAMS J. 119741. La pharmacopée sénégalaise traditionnelle. Plantes
médicinales et toxiques. Vigot, Paris, France, 314-315.
SALUI O., NOUR A., HARPER D. [1991]. Ch emical and nutritional
composition of
two famine food sources used in Sudan, mukheit (~OS& senega/ensis) and
maikah (Dobera roxburghr). J. Sci. Food Agric. 57, 367-377.
SECK D., LO(;NAY G., HAUBRUCX E., WATHELET J.P., MARLIER M., GASPAK C~I.,
SEVERIN M. [1993]. Biological activity of Boscia senegalensis (PERS.) L~M ex
POIR. (Capparaceae) on stored grain insects. 1. Chem. Ecol. 19 (2), 377-389.
--
---
-------
J. srored Pd Rex. Vol. 29, No. 4, pp, 311-318, 1993
0022-474X/93$6.00+0.00
Printed in Chat Britain. Al1 riglus mserved
Copyright 0 1993 Pergamon Press Ltd
F-
BIOLOGICAL ACTIVITY OF CAS%A OkCIDENTALIS L. 3 13 0
AGAINST CALLOSOBRUCHUS MACULATUS (F.)
5 ECB
(COLEOF’TERA: BRUCHIDAE)
V. LIENARD’, D. SECK’, G. LQGNAY’, C. GASPAR’ and M. SEVERIN~
‘Unité de Zoologie générale et appliquée, 2Unité de Chimie générale et organique, Faculté des Sciences
Agronomiqnes, 2 Passage des Déportés, B-5030 Gembloux, Belgium
(Receioed for publication 16 Jane 1993)
AIrstract-In developing countries, traditional control methods are commonly used against stored-product
insects and mites. In Senegal, the leaves of Cassis occidenralis L. (Caesalpiniaceae) are uscd to protect
cowpea seeds (Vigna ungutilata L. (Walpers)) against Caltosobruchus
nzacuiatus (Coleoptera: Bruchidae).
‘fhe biological activity of the leaves, the seeds and oil of C. occidentalis was evaluated in controlled
conditions (28 f 2”C, 45 + 5% rh.) against C. maculatus. At the rate of 10% (w/w), both fresh and dry
leaves as well as whole and ground seeds had no contact toxicity on the cowpea beetle. In contras& seed
oil induced an increase in mortality of C. mandatas eggs and first larval instar at the concentration of
10 ml/kg cowpea. The basis of the ovicidal and larvicidal activities are discussed in this paper. Several triais
using pure compounds have highlighted that several fatty acids (linoleic, oleic and stearic) are responsible
for C. occidenza1i.s toxicity. C. occidentalis seed oil did not reduce the oviposition of C. maculatus at
10 ml/lcg seed.
Key worak-Callosobruchus maculatus (F.), Cassis occidentalis L., biological activity, stored cowpea seeds,
traditional method control, @na unguiczdata (L.) Walp.
INTRODZJCTION
In developing countries and especially on the African continent, the warm climate and vulnerable
storage conditions are favourable to the proliferation of numerous insect species and therefore
induce significant post-harvest losses.
At farm level where fmancial and technical means are limited, post-harvest losses in grain
legumes cari reach 100% in a few months (Labeyrie, 1981). In order to combat this major problem
inexpensively, people often use selected indigenous plants exhibiting insecticidal properties by
mixing them with the stored grains.
In Senegal, C. occhientalis L., a very widespread weed (Higgins et al., 1985) possessing medicinal
properties (Anon., 1987; Pandey, 1975) is used to preserve cowpea stocks. It is claimed that the
leaves mixed with cowpea seeds are protected a.gainst C. maculatus attacks. Like many other
traditional control methods, the mode of action of this plant is still unknown. The present study
was therefore directed to improve the understanding of the effects of C. occidentah on C.
maculatus. In a first series of trials, the effects of seeds and leaves were evaluated. Some authors
(Boughdad et al., 1987; Credland, 1992; Hi11 and Schoonhoven, 1981; Ivbijaro, 1990; Messina and
Renwick, 1983; Naik and Dumbre, 1984; Pereira, 1983; Schoonhoven, 1978; Singh et al., 1978; Su,
1991;, Su et al., 1972) reported on the potentialities of vegetable oils to protect cowpea seeds.
Therefore a second part of this work was focused on the biological properties of the oil of the seeds
of C. occidentah.
The effect of vegetable oil on stored product insects involves two different but complementary
mechanisms: a reduction of the respiratory activity and a direct toxicity of oil constituents on eggs
(Credland, 1992; Don-Pedro, 1989b; van Huis, 1991). In a study about insecticidal activity of
vegetable oils against C. maculatus, Don-Pedro (1990) demonstrated the toxic properties of several
fatty acids. The presence of other toxic minor lipids in the food of one or many developmental
instars of the insects could also enhance oil toxicity.
Miralles ana Gaydou (1986) have shown tha.t the unsaponifiable matter in C. occidentah
represents a relatively high proportion of the oil (8% w/w) in contrast to the other oils (for example,
Ca.ssia ahta: 5% w/w) and that this lipid fraction contained at least 40% sterol. Knowing their
31 II
-----
--
C_c_
--
-.-.
312
V: L~ENARD et ut.
role in insect growth regulation (Svoboda and Feldlaufer, 1991; Svodoba et af., 1991), we also
assesscd their potential effect on cowpea weevil development.
MATERIAL AND METHODS
Experimen ta1 condit ions
Phe strain of C. mactdatus came from the Department of Nioro du Rip (Senegal). It was reared
on cowpea seeds (Vigna unguiculata (L.) Walpers, variety Black-eyed no. 5) under controlled
conditions of temperature, 28 + 2°C and relative humidity, 45 f 5%. Seeds and dried leaves of C.
occidentalis came from the department of Nioro du Rip (Senegal) while fresh leaves were grown
in greenhouses at Gembloux (Belgium) using seeds from Senegal.
Four lots of experiments were performed in the same controlled conditions (T = 28 & 2°C;
r.h. = 45 + 5%).
1. Biological activity of leaves and see&
Four experiments were undertaken in 90 mm dia petri dishes containing 25 g of cowpea seeds.
Each was replicated 5 times. As a control treatment, 25 g of untreated cowpea seeds were similarly
infested with C. maculatus and placed in the same experimental conditions. Five replications were
i-
also made. The females from bath the “flightless” and “flight” forms were used for the experiments
1, 2 and 3. For the experiment 4, only females from the “flightless” form were used.
-Experiment 1. Whole seeds were mixed with cowpea seeds at 10% (w/w).
-Experiment 2. Seed powder was mixed with cowpea at 10% (w/w). TO obtain the Cassis
powder, seeds were ground for 2-3 min using a small laboratory mill. The powder was then passed
through a 500 dia sieve to separate fine particles from the pericarp which was not ground.
-Experiment 3. Dry leaves harvested in Senegal and dried outside in the shade were mixed with
cowpea seeds at 10% (w/w).
-Experiment 4. Fresh leaves were ground with a small laboratory mi11 and mixed with cowpea
seeds at 10% (w/w).
One day after the treatment of cowpea seeds with different forms of C. occident&, a11 petri
dishes were infested with 10 adults (5 males -t. 5 females) of C. maculatus less than 24 h old.
When the Fl population began to emerge 18 days after infestation (DAI), a daily Count of adults
emerged was made until 34 DAI, one day beiore F2 adults began to emerge.
2. Biological activity of C. occidentalis seed oil
I
2.1. Oil extraction. Seeds of C. occidentalis were ground and extracted with n-Hexane for 8 h
:
in a Soxhlet apparatus. The solvent was evaporated at 40°C under reduced pressure. The lipid
extract was kept at 4°C until use.
.t
2.2. Bioassay. Ten grams of c:owpea seeds contained in 55 mm dia petri dishes were treated with
C. occidentalis seed oil at 10 ml/kg. Cassis oil was uniformly distributed on cowpea seeds using
a “turbula” mixer at speed setting 2 for 10min. After the treatment, the seeds were put in an
incubator maintained at 28 f 2°C 45 ) 5% r.h. and infested one day later with four C. maculatus
adults (2 males + 2 females) of the “flightless” form (Taylor and Agbaje, 1974; Utida, 1972) that
were less than 24 h old. Untreated cowpea seeds were similarly infested and placed in the same
experimental conditions. 12 replicates of each procedure were conducted. 18 days later, dead adults
were taken out of the petri dishes and the eggs were counted. Thirty seeds were randomly sampled
from each replicate of treatment and the numbers of dead and hatched eggs as well as dead first
instar larvae were counted. The daily Count of Fl emergence was done from day 21 to day 40 after
infestation.
3. Biological activity of fatty aciak
Thisexperiment was undertaken to confirm or refute the hypothesis of Don-Pedro (1990) who
linked the toxicity of some vegetable oils to faitty acids contained in the oil.
3.1. Analysis of’ thefatty acids of C. occidentalis. The fatty acid composition of Cassis oil was
determined by gas chromatography after transesterification with methanol-BF3 according to the
Biological activity of Cawîa occidentaiis against Callosobruchus
maculatus
AOCS standard (1983). The chromatogiaphic conditions were as follows: column CPWAX 52 CB
(25 m long, 0.32 mm i.d., 0.2 m film thickness) from Chrompack; the carrier gas was helium at 0.7
bar; temperature program heating from 50 to 150°C at 30”C/min and then to 240°C at S”C/min;
gas chromatograph or Hewlett Packard 5880a fitted with an “on-column” injecter and an FID
detector maintained at 250°C. The fatty acids were identified by comparison of their retention times
with t:hat of pure acid.
3.2. Bioassay. Two sets of experiments were performed on fatty acids used either separately or
together. Trials were conducted in 55 mm dia petri dishes containing 10 g of cowpea seeds. One
kg of seeds was coated with the amount of fatty acid that would have been present in 10 ml of
the Cassis oil, volume which was used in the experiment with C. occidentalis seed oil. Untreated
seeds ‘were used for the control.
The experiment was conducted following the same procedure as describcd in Section 2.2.
4. Biological activity of C. occidentalis unsaponifable matter
4.1. Extraction. A sample of 2 g of oil was saponified for 1 h at 75°C with 100 ml of a 2N
methanolic solution of KOH. The unsaponifiable matter was extracted 3 times with 100ml of
diethyl ether. The pooled ethereal extracts were washed 3 times with 40 ml of water and finally
reduce:d to dryness at 35°C under reduced pressure.
4.2. Biomsay. The experiment was undertaken in 55 mm dia petri dishes containing 10 g of
cowpea seeds and was replicated 6 times. Two treatments were considered:
-control treatment: 1 kg of seeds was treated with x ml of solvent,
-experimental treatment: 1 kg of seeds was treated with x ml of unsaponifiable solution, where
x is the content of unsaponifiable matter contained in 10 ml of Cassia oil.
The Fl population began to emerge 18 days after the initial infestation. From that time to the
34th day after this infestation, newly emerged adults were counted daily.
Statistical analysis
Data were submitted to Student tests (Dagnelie, 1975) using the software package Minitab on
the Vax 8250 at the calculation and data processing Center of the Faculty of Agricultural Sciences
(Gembloux, Belgium).
RESULTS
1. Biological activity of seeh and leaves
The mean numbei of adults which emerged varied from 154.2 for the dry leaves treatment to
293.2 for the fresh leaves. In the same conditions 157.7 to 276.8 adults emerged from untreated
cowpea seeds (Table 1).
2. Biological activity of seed oil
The mean numbers of eggs laid by C. maculatus females in the control treatment were 98.7 on
cowpea seeds and 7.9 on the petri dishes. In the same time, 98.7 and 15.9 eggs respectively were
laid in the presence of oil treated seeds. Statistical analysis of the data indicated that more eggs
Tabk 1. The biological activity of Cas.& occi&~falis kaves and
Tabk 2. The cffect of cowpea treatmcnt with Cmsia occidentafis seed
sœds o n Calkmbruchus maculatus
oil on the oviposition of CallosoLwuchur maculatus
Mean numba of adults emerged’
Mcan number of ergs laid on’
Treatment
0 (contr01)
10
Treatment
Cowpea sads Petri dishes
Total
Whok weds
166.2 f 61.8’
196.0 ri: 40.7’
C’ontrol
111.7f8.6’
7.9 f 8.@
119.5 * 15.T
Sd powder
m-
235.6 k 19.8’
229.0 f 71.4’
HI10
98.7 i 21.5*
15.9 f. 9.P
114.6 * 17.1’
m-y kavcr
157.7 f 43.8
154.2 + 28.2’
Fr& kwes2
HIO: se& treated with 10 ml of seed oil/kg of cowpea.
276.8 f 16.6’
293.2 f 17.3’
‘Within a column, means followed by the samc lettcn are not
‘Within a line, means followed by the samc ktter are not significantly
significantly different at the 5% k-4.
different at thc 5% kvel.
%fstation with femaks from the “Bightkss” form.
314
v. LIENAIW el a!.
Table 3. Thc biological effccts OF Caria occidtnfal~ sud oil on Colloso-
bruchus macula~us cggs, first instar iarva and progeay
Mean percentage rnortality’
Mem numbcr
Treatment
E8.P
Larvae
ofF1emergcd'
Control
1.8 f 1.8'
OI.~* 2.4*
100.1 f0.8'
HI0
28.4 + 6.ob
51.6 +l.4b
10.9 i: 10.8b
HIO: se& ttrated with 101111 oil/kg of cowpea.
‘Within a column, means followed by the same ktters are not sigaifi-
cantly ditTuent at the 5% level.
were deposited on cowpea seeds compared to pettii dishes even when seeds were treated with Cassis
oil (Table 2).
The mean percentage mortality of eggs and larvae were respectively 1.8 and 0.4 in the control
and 28.4 and 57.6 on oiled seeds. The mean number of C. maculatus which emerged was 100.1
insects in the control and was 10.9 on treated seeds (Table 3).
L- C l 6
,c18- C18:l
C18:2
Name
i
15.266
I
48.2
Cl&2 Linoleic acid
18.128
3.9
C18:3
1.2
c20
0.7
c20: 1
0.8
0.6
Fig. 1. Chromatogram of dosage of different Ifatty acids from Cawia occiaknralis
seed oil.
1
1,
*,
.^
..--. -_-_____ ___..._ .._ .
Biological activity of Cassis occidentah against Callosobruchus
maculatus
3 1 5
Tabk 4. The elfect of Ca.& occ~&n~alis oil fatty acida on the oviposition of Calloso-
brucbw >M~~I~S
Mean nmber of eggs laid’
Treatment
seeds
Petri dishes
T o t a l
COfltrOl
116 & 36.6’
20.2 * 16.8’
136.2 + 34.8’
linokic acid
138 k 21.7’
9.2 t 13.7’
147.3 + 29.1’
control
95.2 f 16.T
9.3 * 9.5’
104.5 f 21.7”
olcic acid
13.2 + 25.5’
35.8 f 19.6’
109 rt 27.9’
Conlrol
155.8 + 26.3’
8.8 f 14.r
164.7 f 30.48
palmitic acid
154.5 + 36.6’
3.8 f 7.8
158.3 & 42.4’
C o n t r o l
146 f 19.3’
6.7 f 9.4’
152.7 t 22.7
stearic acid
20.8 2 19.0b
1.2+ 1.31
22.0 f 19.4b
Control
129 f 23.1’
23.8 f 14.4’
152.8 + 28.4’
bknd of the 4 acids
128.5 + 31.Y
14.7 + 11.1’
143.2+41.4’
‘Within a column, means followed by the same ktters are not significantly different at
the 5% kvcl.
A decrease in the adherence of eggs on the seeds treated with the oil has been observed.
1
3. Biohgical activity of fatty acitis
The fatty acid profile of C. occidentah seed oil is shown in Fig. 1. The analysed samples
contained 48.2% of linoleic acid, 24.0% of oleic acid, 15.9% of palmitic acid and 4.7% of stearic
acid. T:hese results are in line with those of Miralles and Gaydou (1986).
The mean number of eggs laid by C. maculatus females in the control varied from 95.2 to 155.8
on cowpea seeds and from 6.7 to 23.8 on petri dishes. On treated seeds, their means varied
respectively from 20.8 to 154.5 and from 1.2 to 35.8. The total, mean number of eggs laid by females
varied from 104.5 to 158.3 eggs except to seeds treated with steak acid (only 22 eggs) (Table 4).
The average mortality of eggs and first Iarva instars were respectively less than 3.4% and 2.6%
on untreated seeds. On the other hand, mortality varied from 3 to 61.4% for the eggs and from
1.2 to Z!3.6% for the larvae on treated seeds (Table 5).
The mean numbers of C. maculatus emerged varied from 81.5 to 124.2 adults in the control and
from 5.2 to 82.8 adults on treated seeds (Table 6).
!
4. Biokgical activity of unsaponifiable
part of oil
i
The mean number of C. maculatus which emerged was 123.9 on untreated seeds and 141.2 on
treated seeds (Table 7).
Table 5. Thc cffect of Car~ia occidenrolis seed oil fatty acids on the mortality of Calloso-
bruchus macularus eggs and first instar larva
Mean percentage of eggû’
Mem percentage
of fint instar
Dead
Hatched
larva dead’
Control
2* 1.2’
91.4 + 1.6’
0.6 tr 1.V
linokic acid
25.4 i: 25.0b
73.6 & 24.4b
1.2 f 1.v
C o n t r o l
2.4 + 3.0’
97 f 3.8’
1.0 f 1.6’
OkiCWid
59.4 f 8.6C
36iJ.V
4.6 t 3.8’
Control
3.4 + 3.2’
94 f 3.8’
2.6 & 3.8’
palmitic acid
3 f 4.6’
93.6 f 5.V
3.4 + 3.8’
Control
1.5 f 1.v
98.5 F 1.0”
0.0 f 0.v
SRaric acid
6.1 -I: l.od
91.5 f 3.3”
2.4 f 2.8’
Control
4 k 4.2’
93.4 f 6.6’
2.6 k 2.8’
bknd of tbe 4 acids
61.4rt21.F
15.4 + 7.gd
23.6 2 17.0b
‘Within a cohmm, mean perccntagcs followed by the same letters arc not significantly
different et the 5% kvel.
““_.. .._. _, __
\\
IIFy*ll”?..i~
---
---
316
V. LIENARD et al.
Table 6. The effect of four fatty acids extractui
from Cassis
Tabk 7. The biological effects of Cmsia occidenfafis
occidenralis oil on the emcrgence of adults of Cakmbmchus macu-
unsaponifiable
matter on Callosobruchus macularus
latus
P’ogcnY
Mean n u m b e r o f F I cmcrged’
Mcan n u m b e r o f F I cmerged’
C o n t r o l
117.7 * 25.2’
C o n t r o l
123.9 f 20.7’
linokic acid
68.7 f Y9.5b
Tmatmeot
141.2 + 15.V
‘Within a cohunn, mean perccntages
followed by the
C o n t r o l
81.5 f 19.5’
samc ktters are not significantly different at the
okic acid
20.5 f 6.T
5% Ievel.
ChtrOl
124.2 + 22.0’
palmitic acid
82.8 + 4.@
C o n t r o l
117113.7’
steak acid
17.2 f 4.6’
C o n t r o l
83.4 + 23.3”
b k n d o f the 4 acids
5.2 f 3.P
‘Within a column, means followed by thc same ktter arc net
significantly different at the 5% kvel.
DISCUS,SION
In our experimental conditions, the whole seeds, the seed powder, the dry leaves and the fresh
leaves did not reduce the number of adult emerged and thus had no biological activity against C.
maculatus. It is noteworthy that fresh leaves used in our trials were harvested in greenhouses in
Belgium. Ermel et al. (1986), Levin and York (1978) and Singh (1986) reported the importance of
the origin of the Neem tree (Azadirachta
indica L.), a plant used as an alternative control method,
as a source of variability for chemical composition and probably for biological activity also.
Modification of growing conditions induced changes of azadirachtin content (the active principle
of Neem tree) and therefore led to significant changes in the insecticidal properties.
Moreover, it is important to recall that females from both the “Aightless” and “flight” forms
were used for the biological activity tests with whole seeds, the seed powder and the dry leaves.
Sano (1967) and Utida (1972) observed that females from the “flight” form had a low fecundity
and fertility compared to females from the “flightless” form. The use of these two forms explained
the great variability in the results obtained in trials with whole seeds, seed powder and dry leaves.
For a11 other tests, only females from the “flightless” form were used.
C. occidentalis
seed oil did not significantly affect the number and the distribution (on cowpea
seeds and on petri dishes) of eggs laid but did reduce the mean number of Fl adults which emerged.
The present results indicate that C. occidentah seed oil is effective in reducing damage to cowpea
seeds from C. maculatus. There is an increase in mortality of both eggs and first instar larvae which
results in a significant reduction in C. maculatus progeny and damage. Our results with
C. occidentah seed oil are in agreement with many other works on the use of oils against
.’ ’
stored-products insects (Boughdad et al., 1987; Credland, 1992; Don-Pedro, 1989a, b, 1990; Hi11
and Schoonhoven, 1981; Ivbijaro, 1990; Messina and Renwick, 1983; Naik and Dumbre, 1984;
Pereira, 1983; Schoonhoven, 1978; Singh er af., 1978; Su, 1991; Su er al., 1972; van Huis, 1991).
Four different hypotheses have been given by these authors to explain the toxicity of vegetable oils:
(1) The toxicity to the eggs and first instar larvae is the consequence of the occlusion of the
short funnel at the posterior end of the egg. This hypothesis cari explain the ovicidal and larvicidal
effects observed in our experiments;
(2) A reduction of egg adherence on the cowpe,a seed which prevents the first instar larva from
penetrating the seed testa. This decrease of adherence has been observed in the experiments
described and cari explain mortality of C. macularus first instar larvae;
(3) A direct toxic effect of some oil constitue@,;
(4) The toxicity of different fatty acids contained in the oil.
In our experimental conditions, the four fatty acids used separately or a11 htogether reduced
significantly the number of progeny. Oleic, stearic acids and the blend of the four fatty acids were
the most aective. Both linoleic ami oleic acids exhibit only ovicidal effects. This observation is
in line with Don-Pedro’s results (1990). Palmitic acid did not reduce oviposition and did not affect
either fecundity or first instar larva survival. Nevertlheless, stearic acid is the only lipid tested which
Biological activity of Cassis occiaéntalis against Callosobruchus maculatus
317
reduced the mean number of eggs laid by C. maculatus females. Moreover, stearic acid has also
an ovicidal effect, although the increase of mortality is less than with oleic and linoleic acids.
From the results (Table 7), it is clear that the unsaponifiable matter of Cassiu oil did not
significantly reduce the number of beetle progeny.
CONCLUSIONS
In the present experimental conditions, the fresh and dry leaves, whole and powdered seeds of
C. occidentah showed no contact toxicity. As modifications of growing conditions are known to
induce modifications of the chemical composition of plant material, it would be interesting to test
freshl harvested leaves from tropical areas to confirm or invalidate the aforementioned results.
The toxicity of Cassia occidentah oil cari be assigned to physical effects (suffocation of eggs and
decrease of eggs adherence) and to direct toxicity of fatty acids of the oil.
In tropical Africa, hachis oil is used by farmers to prote& stored cowpea seeds (Boughdad et al.,
1987; Don-Pedro, 1989a; Messina and Renwick, 1983; Pereira, 1983) but this oil could be valorized
for trade and export. It is also possible that other underexploited oils could have a potential role
in grain protection.
Acknowledgement-Sincere
thanks to Dr J.-L. Hemptinne for his helpful comments on the manuscript.
REFERENCES
Anonymous (1987) Fiche espèce: Cassia occidentalis L. (Caesalpiniaceae). Méd. Tradit. Pharmac. 1, (2), 143-170.
AOCS (1983) Officiai and Tentative Methods of the American Oil Chemists’ Society, Chicago, IL.
Boughdad A., Gillon Y. and Gagnepain C. (1987) Effect of Arachis hypogea seed fats on the larval development of
Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J. stored Prod. Res. 23, 99-103.
Credland P. F. (1992) The structure of bruchid eggs may explain the ovicidal effect of oils. J. srored Prod. Res. 28, 1 -9.
Dagnelie P. (1975) Théorie et Méthodes Statistiques, Vol. 2. Les presses agronomiques de Gembloux, Belgium.
Don-Pedro K. N. (1989a) Effects of fixed vegetable oils on oviposition and adult mortality of Callosobruchus maculatus
(F.) on cowpea. ht. Pest Control31, 34-37.
Don-Pedro K. N. (1989b) Mode of action of fixed oils against eggs of Callosobruchus maculatus (F.). Pestic. Sci. 26, 107-I 15.
Don-Pedro K. N. (1990) Insecticidal activity of fatty acid constituents of fixed vegetable oils against Callosobruchus
maculatus (F.) on cowpea. Pestic. Sci. 30, 209-211.
Ermel K., Pahlich E. and Schmutterer H. (1986) Azadirachtin content of necm kernels from different geographical locations,
and its dependence on temperature, relative humidity, and light. In Natural Pesticides from the Neem Tree and other
Tropical Plants. Proc. 3rd Int. Neem COI$ (Edited by Schmutterer H. and Ascher K. R. S.), pp. 171-184. Eschborn.
Higgins J. M., Walker R. H. and Whitwell T. (1985) Coffee serma (Cassis occidentalis) competition with cotton (Gossypium
hirsutum). Weed Sci. 34, 52-56.
Hi11 J. and Schoonhoven A. V. (1981) Effectiveness of vegetable oil fractions in controlling the mexican bean weevil on
stored beans. J. Econ. Entomol. 74, 478-479.
Huis van A. (1991) Biological methods of bruchid control in the tropics: a review. Insect Sci. Applic. 12, 87-102.
Ivbijaro M. F. (1990) The efficacy of seed oils Azadirachta indica A. Juss and Piper guineense Schum and Thonn on the
comtrol of Callobruchus
maculatus (F.). Insect Sci. Appl. 11, 149-152.
Labeyrie V. (1981) Vaincre la carence protéique par le développement des légumineuses alimentaires et la protection de
leurs récoltes contre les bruches. Food Nutr. Bull. 3, 24-38.
Levin D. A. aod York B. M. (1978) The toxicity of plant alkaloids: an ccogeographic perspective. Biochem. Syst. Ecol.
6, 61-76.
Messina F. J. and Renwick J. A. A. (1983) Effectiveness of oils in protecting stored cowpeas from the cowpea weevil
(Coleoptera: Bruchidae). J. Econ. Entomol. 76, 634-636.
Miralles J. and Gaydou E. M. (1986) Composition en acides gras des huiles extraites des graines de trois Cassis
(Caesalpinacées) d’origine sénégalaise. Reo. Fr. Corps gras 10, 381-384.
Naik IR. L. and Dumbre R. B. (1984) Effect of some vegetable oils used in protecting stored cowpea on biology of pulse
beetle, Callosobruchus maculatus (FABR.) (Coleoptera: Bruchidae). Bull. Grain Technol. 22, 2532.
Pande:y Y. N. (1975) Cassia seeds used as drug in the indigenous medical systems of India. Q. J. Crude Res. 13, 61-64.
Percim J. (1983) The effectiveness of six vegetable oils as protectants of cowpeas and bambara groundnuts against
infiestation by Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J. stored Prod. Res. 19, 5762.
Sano IL (1967) Density effèct and environmental temperature as the factors producing the active form of Callosobruchus
maculatus (F.) (Coleoptera, Bruchidae). J. stored Prod. Res. 2, 187-195.
Schoonhoven A. V. (1978) Use of vegetable oils to protect stored beans from bruchid attack. J. Econ. Entomol. 71,254-256.
Singh R. P. (198# C omparison of antifeeding efficacy and extract yields from different parts and ecotypes of neem
(Asadiruchta mdica A. Jus~) trees. In Natural Pesticides from the Neem tree and Other Tropical Plants. Proc. 3rd Int.
Neem Car$ (Edited by Schmutterer H. and Ascher K. R. S.), pp. 185-194. Eschborn.
Singh S. R., Luse R. A., Leuschner K. and Nangju D. (1978) Groundnut oil treatment for the control of Callosobruchus
maculatus
(F.) during cowpea storage. J. stored Prod. Res. 14, 77-80.
SU H. C. F. (1991) Laboratory evaluation of toxicity of Calamns oil against 4 specics of stored-product insects. J. Entomol.
sci’. 26, 76-80.
.
“.
.
I
Su H. C. F., Speirs R. D. and Mahamy P. (3. (1972) Citrus oil as protectnnts of black-eyed peas against cowpea weevils:
laboratory evaluation. J. Econ. Enromol,. 65, 1433-1436.
Svodoba J. A. and Feldlaufer M. F. (1991) Neutral sterol metabolism in insects. Lipiak 26, 614418.
1
Svodoba J. A., Weirich G. F. and Feldlaufer M. F. (1991) Recent advances in insect steroid biochemistry. In Physiology
and Biochemistry of S&ols (Fdited by Patterson G. W. and Nes W. D.), pp, 294326. American oii chemists’ Society,
‘T
Compaign, IL.
Taylor T. A. and Agbaje L. A. (1974) Flighrt activity in normal and active forms of Cullosobruchus
macula/us in a store
in Nigeria. J. stored Prod. Res. 10, 9-16.
Utida S. (1972) Density dependent polymorphism in the aduh of Calfosobruchus
maculafus (Coleoptera, Bruchidae).
J. srored Prod. Res. 8, 111-126.
A+/+L.~ * J- F:
Bd Rech. Agron. Gembloux [1993] 28 (4), 501-505
/3m z!‘- J+-- cd &LcclLL ”
~~~~~~~~
Tj&A;
NOTE
y58
* Y&
,‘-
LQcb
,Y
Identification of ‘elemental sulphur (Si)
in Bo&a senegalensis (PERS.) LAM ex POIR. leaves
bY
G. LOGNAY (‘), D. SECK (*‘), M. MAKLIER (‘), E. HAUBRUGE (“),
C. GASPAR (‘“), M. SEVERIN (‘)
Summary
Studies were undertaken to identify the molecules responsible of Boscia
senegalensis (PERS.) L.AM ex POIR. (Capparaceae) insecticide (fumigant) proper-
ties ; the volatiles were isolated from the leaves by steam distillation and
analyzed by Gas Chromatography - Mass Spectrometry. Only a few organic
molecules were detected and identified. The main constituent was elemental
sulphur (S,), a compound rarely found as such in the plant kingdom.
wwords : Boscia senegalensis, leaves, volatiles, elemental sulphur, GC-MS.
1. Introduction
&~@a senegaknsis (mKS.) LAM ex POIR. (Capparaceae) is a Little
shrub which grows throughout West African countries principally in the
Sahelian re’gion [B~KXIH, WICKENS, 19881. Several parts of the plant are
locally used for their medicinal properties [KERHARO, ADAMS, 19741 and
“) Depnrtmeqt of General and Organic Chemistry. Faculty of Agricultural
,..) :
‘cwnces. Passage des Déportés, 2 B-5030 GEMBWJX (Belgium).
Ueplrtment of General and Applied Zoology. Fzkulty of Agriculturnl
Siit,nces. Passage des Déportés, 2. B-5030 GEMRLOUX (BELCXJM).
502
G. LOGNAY et a1
sometimes the small cooked fruits are consumed by humans in case of
famine [SALIH et&., 19911. The 1 caves exhibit a strong biocide activity
against several stored grain insect species. A survey of the Senegalese
practices revealed that B. senegdezzsis fresh leaves are traditionally
added to stored grains in order to limit insect infestation and damages.
Chemical investigations proved that methylisothiocyanate (MITC)
enzymatically liberated from the methylglucosinolate (also called
glucocapparine) precursor was responsible for the insecticidal properties
[SECK et al. 19931. Since other volatile molecules could act as MITC
synergists, a characterization of 8. senegalensis steam distillate was
carried out. The results of this complementary study are reported herein.
2. Experimentals
B. senegalensis leaves were harvested in Thies (Senegal). They
were kept in the dark at subambiant temperature (-20°C) until use.
Hundred grams of freshly ground material were subjected to steam
distillation for 1 hour. The aqueous distillate was extracted three times
with 100 ml peroxide-free diethyl ether. The ethereal phases were
pooled, dried with anhydrous magnesium sulfate and the solvent was
distilled at 40°C to about 10 ml. The extract was further concentrated to
4 ml at room temperature under a stream of pure nitrogen and analyzed
by Cas Chromatography-Mass Spectrometry. A Nermag RlO-1OC Mass
Spectrometer coupled to a Delsi DI 700 Gas Chromatograph was used.
The operating conditions were the following :
- column CP-Sil-8 CI3 (Chrompack-The Netherlands) 25 m length,
0.32 mm I.D., 0.2 pm film thickness,
- temperature program : cold ‘on-column’ injection at 3O”C, T” rise at
5”C/min to 240°C then at lO”C/min to 29O”C,
- carrier gas : helium at 1 ml/min,
- ionisation voltage : 70 eV,
- source T” : 200°C.
The recorded fragmentation data were compared with those of the
EPA-NIH and WILEY-NBS mass spectra libraries (reference).
ELEMENTAL SULPHUR
503
3. Results and discussion
The total ion current (chromatographic profile) of 8. senegahsis
volatiles is presented in figure 1.
5 minutes
7
4
5
6
1
1
1
1
L
Stan number
Figure 1. - Total ion current of Bosch senegalensis steam-distillated volatiles.
As shown, only a few peaks were observed. MITC (compound 1)
liberated from methylglucosinolate during the distillation procedure was
detected as a shoulder in the tail of the solvent. In previous analyses
[SECK et&., 19931 using polar GC columns, this molecule was complete-
ly resolved and unambiguously identified. Compound 2 corresponds to
limonene whereas compound. 3 was identified as 2,6-di- tert-butyl-p-cre-
sol. The latter compound came from the diethyl ether to which it is
added as a stabilizer. Molecule 4 (M’ = 312, other main fragments at
m/z = 257, 256, 239, 227, 213, 129, 116, 71, 69, 57 and 56 [Base peak])
and its homologue 5 (M’ = 340, other main fragments at m/z = 285,284,
267, 129, 116, 71, 69, 57 and 56 [Base peak]) were tentatively attributed
to hexadecanoic and octadecanoic 2-methylpropyl esters. The phtalate
6 (Base peak at m/z = 149) eluted at 35.4 min is probably a contaminant
from the plastic bags used to store the leaves. The broad peak 7 at
-
--
-- ---if-
5 0 4
G.LQCNAY et al.
24.4 min exhibited a typical fragmentation pattem (Figure 2) with a
molecular ion at 256 (33 %) and two isotopic ions p + 1 (1 %) and p + 2
(10 %) rezecti‘vely at m/z = 257 and 258. This particular cluster (due to
33S and S natural isotopes) is indicative of a sulphur-containing
product. The nature of such a molecule is fully confirmed by a series of
similar clusters corresponding to a systematic loss of 32 atomic mass
units. Compound 7 was unambiguously identified as elemental sulphur
(S,). The comparison of the recorded S, mass spectrum with those of the
EPA-NIH and WILEY libraries supported our identification. The fact
that 8. senegahsis leaves samples came from trees not treated with
S-containing pesticides is an argument confirming that elemental
sulphur may not be considered as a contaminant.
2 5 6
128
160
192
@
'
9 6
l
2 2 4
Figure 2. - Mass spectrum of the elemental sulphur (S,).
A. from Boscih senegalensis ; B. reference.
TO the authors knowledge, it is only the second time that
elemental sulphur is detected in a plant material. Indeed, recently, the
same compound was identified in Capparis spinosa L. flower buds [BM-
VARD et&., 19921.
The biochemical pathway leading to the accumulation of elemental
sulphur remains unexplained.
ELEMENT.AL SULPHUR
505
Acknowledgements
The authors gratefully acknowledge Dr. E. DELCAKTE and Dr. R. PAUL
(Faculty of Agricultural Sciences of Gembloux) for their helpful cooperation.
References
BOOTII F., WICKENS G. (19881. Non-timber uses of selected arid zone trees and
shrubs in Africa. FAO Conservation Guide, n”19, 176 p.
BKEVAKD H., BRAMBILLA M., CFIAINTREAU A., MARION J.P., DISERENS H. [1992].
Occurrence of elemental sulphur in capers (Capparis spinosa L.) and first
investigation on the flavour profile. Havour Fragm. J, 7, 313-321.
KERFIARO J., ADAMS J. 119741. La pharmacopée sénégalaise traditionnelle. Plantes
médicinales et toxiques. Vigot, Paris, France, 314-315.
SALUI O., NOUR A., HARPER D. [1991]. Ch emical and nutritional
composition of
two famine food sources used in Sudan, mukheit (~OS& senega/ensis) and
maikah (Dobera roxburghr). J. Sci. Food Agric. 57, 367-377.
SECK D., LO(;NAY G., HAUBRUCX E., WATHELET J.P., MARLIER M., GASPAK C~I.,
SEVERIN M. [1993]. Biological activity of Boscia senegalensis (PERS.) L~M ex
POIR. (Capparaceae) on stored grain insects. 1. Chem. Ecol. 19 (2), 377-389.
--
---
-------
J. srored Pd Rex. Vol. 29, No. 4, pp, 311-318, 1993
0022-474X/93$6.00+0.00
Printed in Chat Britain. Al1 riglus mserved
Copyright 0 1993 Pergamon Press Ltd
F-
BIOLOGICAL ACTIVITY OF CAS%A OkCIDENTALIS L. 3 13 0
AGAINST CALLOSOBRUCHUS MACULATUS (F.)
5 ECB
(COLEOF’TERA: BRUCHIDAE)
V. LIENARD’, D. SECK’, G. LQGNAY’, C. GASPAR’ and M. SEVERIN~
‘Unité de Zoologie générale et appliquée, 2Unité de Chimie générale et organique, Faculté des Sciences
Agronomiqnes, 2 Passage des Déportés, B-5030 Gembloux, Belgium
(Receioed for publication 16 Jane 1993)
AIrstract-In developing countries, traditional control methods are commonly used against stored-product
insects and mites. In Senegal, the leaves of Cassis occidenralis L. (Caesalpiniaceae) are uscd to protect
cowpea seeds (Vigna ungutilata L. (Walpers)) against Caltosobruchus
nzacuiatus (Coleoptera: Bruchidae).
‘fhe biological activity of the leaves, the seeds and oil of C. occidentalis was evaluated in controlled
conditions (28 f 2”C, 45 + 5% rh.) against C. maculatus. At the rate of 10% (w/w), both fresh and dry
leaves as well as whole and ground seeds had no contact toxicity on the cowpea beetle. In contras& seed
oil induced an increase in mortality of C. mandatas eggs and first larval instar at the concentration of
10 ml/kg cowpea. The basis of the ovicidal and larvicidal activities are discussed in this paper. Several triais
using pure compounds have highlighted that several fatty acids (linoleic, oleic and stearic) are responsible
for C. occidenza1i.s toxicity. C. occidentalis seed oil did not reduce the oviposition of C. maculatus at
10 ml/lcg seed.
Key worak-Callosobruchus maculatus (F.), Cassis occidentalis L., biological activity, stored cowpea seeds,
traditional method control, @na unguiczdata (L.) Walp.
INTRODZJCTION
In developing countries and especially on the African continent, the warm climate and vulnerable
storage conditions are favourable to the proliferation of numerous insect species and therefore
induce significant post-harvest losses.
At farm level where fmancial and technical means are limited, post-harvest losses in grain
legumes cari reach 100% in a few months (Labeyrie, 1981). In order to combat this major problem
inexpensively, people often use selected indigenous plants exhibiting insecticidal properties by
mixing them with the stored grains.
In Senegal, C. occhientalis L., a very widespread weed (Higgins et al., 1985) possessing medicinal
properties (Anon., 1987; Pandey, 1975) is used to preserve cowpea stocks. It is claimed that the
leaves mixed with cowpea seeds are protected a.gainst C. maculatus attacks. Like many other
traditional control methods, the mode of action of this plant is still unknown. The present study
was therefore directed to improve the understanding of the effects of C. occidentah on C.
maculatus. In a first series of trials, the effects of seeds and leaves were evaluated. Some authors
(Boughdad et al., 1987; Credland, 1992; Hi11 and Schoonhoven, 1981; Ivbijaro, 1990; Messina and
Renwick, 1983; Naik and Dumbre, 1984; Pereira, 1983; Schoonhoven, 1978; Singh et al., 1978; Su,
1991;, Su et al., 1972) reported on the potentialities of vegetable oils to protect cowpea seeds.
Therefore a second part of this work was focused on the biological properties of the oil of the seeds
of C. occidentah.
The effect of vegetable oil on stored product insects involves two different but complementary
mechanisms: a reduction of the respiratory activity and a direct toxicity of oil constituents on eggs
(Credland, 1992; Don-Pedro, 1989b; van Huis, 1991). In a study about insecticidal activity of
vegetable oils against C. maculatus, Don-Pedro (1990) demonstrated the toxic properties of several
fatty acids. The presence of other toxic minor lipids in the food of one or many developmental
instars of the insects could also enhance oil toxicity.
Miralles ana Gaydou (1986) have shown tha.t the unsaponifiable matter in C. occidentah
represents a relatively high proportion of the oil (8% w/w) in contrast to the other oils (for example,
Ca.ssia ahta: 5% w/w) and that this lipid fraction contained at least 40% sterol. Knowing their
31 II
-----
--
C_c_
--
-.-.
312
V: L~ENARD et ut.
role in insect growth regulation (Svoboda and Feldlaufer, 1991; Svodoba et af., 1991), we also
assesscd their potential effect on cowpea weevil development.
MATERIAL AND METHODS
Experimen ta1 condit ions
Phe strain of C. mactdatus came from the Department of Nioro du Rip (Senegal). It was reared
on cowpea seeds (Vigna unguiculata (L.) Walpers, variety Black-eyed no. 5) under controlled
conditions of temperature, 28 + 2°C and relative humidity, 45 f 5%. Seeds and dried leaves of C.
occidentalis came from the department of Nioro du Rip (Senegal) while fresh leaves were grown
in greenhouses at Gembloux (Belgium) using seeds from Senegal.
Four lots of experiments were performed in the same controlled conditions (T = 28 & 2°C;
r.h. = 45 + 5%).
1. Biological activity of leaves and see&
Four experiments were undertaken in 90 mm dia petri dishes containing 25 g of cowpea seeds.
Each was replicated 5 times. As a control treatment, 25 g of untreated cowpea seeds were similarly
infested with C. maculatus and placed in the same experimental conditions. Five replications were
i-
also made. The females from bath the “flightless” and “flight” forms were used for the experiments
1, 2 and 3. For the experiment 4, only females from the “flightless” form were used.
-Experiment 1. Whole seeds were mixed with cowpea seeds at 10% (w/w).
-Experiment 2. Seed powder was mixed with cowpea at 10% (w/w). TO obtain the Cassis
powder, seeds were ground for 2-3 min using a small laboratory mill. The powder was then passed
through a 500 dia sieve to separate fine particles from the pericarp which was not ground.
-Experiment 3. Dry leaves harvested in Senegal and dried outside in the shade were mixed with
cowpea seeds at 10% (w/w).
-Experiment 4. Fresh leaves were ground with a small laboratory mi11 and mixed with cowpea
seeds at 10% (w/w).
One day after the treatment of cowpea seeds with different forms of C. occident&, a11 petri
dishes were infested with 10 adults (5 males -t. 5 females) of C. maculatus less than 24 h old.
When the Fl population began to emerge 18 days after infestation (DAI), a daily Count of adults
emerged was made until 34 DAI, one day beiore F2 adults began to emerge.
2. Biological activity of C. occidentalis seed oil
I
2.1. Oil extraction. Seeds of C. occidentalis were ground and extracted with n-Hexane for 8 h
:
in a Soxhlet apparatus. The solvent was evaporated at 40°C under reduced pressure. The lipid
extract was kept at 4°C until use.
.t
2.2. Bioassay. Ten grams of c:owpea seeds contained in 55 mm dia petri dishes were treated with
C. occidentalis seed oil at 10 ml/kg. Cassis oil was uniformly distributed on cowpea seeds using
a “turbula” mixer at speed setting 2 for 10min. After the treatment, the seeds were put in an
incubator maintained at 28 f 2°C 45 ) 5% r.h. and infested one day later with four C. maculatus
adults (2 males + 2 females) of the “flightless” form (Taylor and Agbaje, 1974; Utida, 1972) that
were less than 24 h old. Untreated cowpea seeds were similarly infested and placed in the same
experimental conditions. 12 replicates of each procedure were conducted. 18 days later, dead adults
were taken out of the petri dishes and the eggs were counted. Thirty seeds were randomly sampled
from each replicate of treatment and the numbers of dead and hatched eggs as well as dead first
instar larvae were counted. The daily Count of Fl emergence was done from day 21 to day 40 after
infestation.
3. Biological activity of fatty aciak
Thisexperiment was undertaken to confirm or refute the hypothesis of Don-Pedro (1990) who
linked the toxicity of some vegetable oils to faitty acids contained in the oil.
3.1. Analysis of’ thefatty acids of C. occidentalis. The fatty acid composition of Cassis oil was
determined by gas chromatography after transesterification with methanol-BF3 according to the
Biological activity of Cawîa occidentaiis against Callosobruchus
maculatus
AOCS standard (1983). The chromatogiaphic conditions were as follows: column CPWAX 52 CB
(25 m long, 0.32 mm i.d., 0.2 m film thickness) from Chrompack; the carrier gas was helium at 0.7
bar; temperature program heating from 50 to 150°C at 30”C/min and then to 240°C at S”C/min;
gas chromatograph or Hewlett Packard 5880a fitted with an “on-column” injecter and an FID
detector maintained at 250°C. The fatty acids were identified by comparison of their retention times
with t:hat of pure acid.
3.2. Bioassay. Two sets of experiments were performed on fatty acids used either separately or
together. Trials were conducted in 55 mm dia petri dishes containing 10 g of cowpea seeds. One
kg of seeds was coated with the amount of fatty acid that would have been present in 10 ml of
the Cassis oil, volume which was used in the experiment with C. occidentalis seed oil. Untreated
seeds ‘were used for the control.
The experiment was conducted following the same procedure as describcd in Section 2.2.
4. Biological activity of C. occidentalis unsaponifable matter
4.1. Extraction. A sample of 2 g of oil was saponified for 1 h at 75°C with 100 ml of a 2N
methanolic solution of KOH. The unsaponifiable matter was extracted 3 times with 100ml of
diethyl ether. The pooled ethereal extracts were washed 3 times with 40 ml of water and finally
reduce:d to dryness at 35°C under reduced pressure.
4.2. Biomsay. The experiment was undertaken in 55 mm dia petri dishes containing 10 g of
cowpea seeds and was replicated 6 times. Two treatments were considered:
-control treatment: 1 kg of seeds was treated with x ml of solvent,
-experimental treatment: 1 kg of seeds was treated with x ml of unsaponifiable solution, where
x is the content of unsaponifiable matter contained in 10 ml of Cassia oil.
The Fl population began to emerge 18 days after the initial infestation. From that time to the
34th day after this infestation, newly emerged adults were counted daily.
Statistical analysis
Data were submitted to Student tests (Dagnelie, 1975) using the software package Minitab on
the Vax 8250 at the calculation and data processing Center of the Faculty of Agricultural Sciences
(Gembloux, Belgium).
RESULTS
1. Biological activity of seeh and leaves
The mean numbei of adults which emerged varied from 154.2 for the dry leaves treatment to
293.2 for the fresh leaves. In the same conditions 157.7 to 276.8 adults emerged from untreated
cowpea seeds (Table 1).
2. Biological activity of seed oil
The mean numbers of eggs laid by C. maculatus females in the control treatment were 98.7 on
cowpea seeds and 7.9 on the petri dishes. In the same time, 98.7 and 15.9 eggs respectively were
laid in the presence of oil treated seeds. Statistical analysis of the data indicated that more eggs
Tabk 1. The biological activity of Cas.& occi&~falis kaves and
Tabk 2. The cffect of cowpea treatmcnt with Cmsia occidentafis seed
sœds o n Calkmbruchus maculatus
oil on the oviposition of CallosoLwuchur maculatus
Mean numba of adults emerged’
Mcan number of ergs laid on’
Treatment
0 (contr01)
10
Treatment
Cowpea sads Petri dishes
Total
Whok weds
166.2 f 61.8’
196.0 ri: 40.7’
C’ontrol
111.7f8.6’
7.9 f 8.@
119.5 * 15.T
Sd powder
m-
235.6 k 19.8’
229.0 f 71.4’
HI10
98.7 i 21.5*
15.9 f. 9.P
114.6 * 17.1’
m-y kavcr
157.7 f 43.8
154.2 + 28.2’
Fr& kwes2
HIO: se& treated with 10 ml of seed oil/kg of cowpea.
276.8 f 16.6’
293.2 f 17.3’
‘Within a column, means followed by the samc lettcn are not
‘Within a line, means followed by the samc ktter are not significantly
significantly different at the 5% k-4.
different at thc 5% kvel.
%fstation with femaks from the “Bightkss” form.
314
v. LIENAIW el a!.
Table 3. Thc biological effccts OF Caria occidtnfal~ sud oil on Colloso-
bruchus macula~us cggs, first instar iarva and progeay
Mean percentage rnortality’
Mem numbcr
Treatment
E8.P
Larvae
ofF1emergcd'
Control
1.8 f 1.8'
OI.~* 2.4*
100.1 f0.8'
HI0
28.4 + 6.ob
51.6 +l.4b
10.9 i: 10.8b
HIO: se& ttrated with 101111 oil/kg of cowpea.
‘Within a column, means followed by the same ktters are not sigaifi-
cantly ditTuent at the 5% level.
were deposited on cowpea seeds compared to pettii dishes even when seeds were treated with Cassis
oil (Table 2).
The mean percentage mortality of eggs and larvae were respectively 1.8 and 0.4 in the control
and 28.4 and 57.6 on oiled seeds. The mean number of C. maculatus which emerged was 100.1
insects in the control and was 10.9 on treated seeds (Table 3).
L- C l 6
,c18- C18:l
C18:2
Name
i
15.266
I
48.2
Cl&2 Linoleic acid
18.128
3.9
C18:3
1.2
c20
0.7
c20: 1
0.8
0.6
Fig. 1. Chromatogram of dosage of different Ifatty acids from Cawia occiaknralis
seed oil.
1
1,
*,
.^
..--. -_-_____ ___..._ .._ .
Biological activity of Cassis occidentah against Callosobruchus
maculatus
3 1 5
Tabk 4. The elfect of Ca.& occ~&n~alis oil fatty acida on the oviposition of Calloso-
brucbw >M~~I~S
Mean nmber of eggs laid’
Treatment
seeds
Petri dishes
T o t a l
COfltrOl
116 & 36.6’
20.2 * 16.8’
136.2 + 34.8’
linokic acid
138 k 21.7’
9.2 t 13.7’
147.3 + 29.1’
control
95.2 f 16.T
9.3 * 9.5’
104.5 f 21.7”
olcic acid
13.2 + 25.5’
35.8 f 19.6’
109 rt 27.9’
Conlrol
155.8 + 26.3’
8.8 f 14.r
164.7 f 30.48
palmitic acid
154.5 + 36.6’
3.8 f 7.8
158.3 & 42.4’
C o n t r o l
146 f 19.3’
6.7 f 9.4’
152.7 t 22.7
stearic acid
20.8 2 19.0b
1.2+ 1.31
22.0 f 19.4b
Control
129 f 23.1’
23.8 f 14.4’
152.8 + 28.4’
bknd of the 4 acids
128.5 + 31.Y
14.7 + 11.1’
143.2+41.4’
‘Within a column, means followed by the same ktters are not significantly different at
the 5% kvcl.
A decrease in the adherence of eggs on the seeds treated with the oil has been observed.
1
3. Biohgical activity of fatty acitis
The fatty acid profile of C. occidentah seed oil is shown in Fig. 1. The analysed samples
contained 48.2% of linoleic acid, 24.0% of oleic acid, 15.9% of palmitic acid and 4.7% of stearic
acid. T:hese results are in line with those of Miralles and Gaydou (1986).
The mean number of eggs laid by C. maculatus females in the control varied from 95.2 to 155.8
on cowpea seeds and from 6.7 to 23.8 on petri dishes. On treated seeds, their means varied
respectively from 20.8 to 154.5 and from 1.2 to 35.8. The total, mean number of eggs laid by females
varied from 104.5 to 158.3 eggs except to seeds treated with steak acid (only 22 eggs) (Table 4).
The average mortality of eggs and first Iarva instars were respectively less than 3.4% and 2.6%
on untreated seeds. On the other hand, mortality varied from 3 to 61.4% for the eggs and from
1.2 to Z!3.6% for the larvae on treated seeds (Table 5).
The mean numbers of C. maculatus emerged varied from 81.5 to 124.2 adults in the control and
from 5.2 to 82.8 adults on treated seeds (Table 6).
!
4. Biokgical activity of unsaponifiable
part of oil
i
The mean number of C. maculatus which emerged was 123.9 on untreated seeds and 141.2 on
treated seeds (Table 7).
Table 5. Thc cffect of Car~ia occidenrolis seed oil fatty acids on the mortality of Calloso-
bruchus macularus eggs and first instar larva
Mean percentage of eggû’
Mem percentage
of fint instar
Dead
Hatched
larva dead’
Control
2* 1.2’
91.4 + 1.6’
0.6 tr 1.V
linokic acid
25.4 i: 25.0b
73.6 & 24.4b
1.2 f 1.v
C o n t r o l
2.4 + 3.0’
97 f 3.8’
1.0 f 1.6’
OkiCWid
59.4 f 8.6C
36iJ.V
4.6 t 3.8’
Control
3.4 + 3.2’
94 f 3.8’
2.6 & 3.8’
palmitic acid
3 f 4.6’
93.6 f 5.V
3.4 + 3.8’
Control
1.5 f 1.v
98.5 F 1.0”
0.0 f 0.v
SRaric acid
6.1 -I: l.od
91.5 f 3.3”
2.4 f 2.8’
Control
4 k 4.2’
93.4 f 6.6’
2.6 k 2.8’
bknd of tbe 4 acids
61.4rt21.F
15.4 + 7.gd
23.6 2 17.0b
‘Within a cohmm, mean perccntagcs followed by the same letters arc not significantly
different et the 5% kvel.
““_.. .._. _, __
\\
IIFy*ll”?..i~
---
---
316
V. LIENARD et al.
Table 6. The effect of four fatty acids extractui
from Cassis
Tabk 7. The biological effects of Cmsia occidenfafis
occidenralis oil on the emcrgence of adults of Cakmbmchus macu-
unsaponifiable
matter on Callosobruchus macularus
latus
P’ogcnY
Mean n u m b e r o f F I cmcrged’
Mcan n u m b e r o f F I cmerged’
C o n t r o l
117.7 * 25.2’
C o n t r o l
123.9 f 20.7’
linokic acid
68.7 f Y9.5b
Tmatmeot
141.2 + 15.V
‘Within a cohunn, mean perccntages
followed by the
C o n t r o l
81.5 f 19.5’
samc ktters are not significantly different at the
okic acid
20.5 f 6.T
5% Ievel.
ChtrOl
124.2 + 22.0’
palmitic acid
82.8 + 4.@
C o n t r o l
117113.7’
steak acid
17.2 f 4.6’
C o n t r o l
83.4 + 23.3”
b k n d o f the 4 acids
5.2 f 3.P
‘Within a column, means followed by thc same ktter arc net
significantly different at the 5% kvel.
DISCUS,SION
In our experimental conditions, the whole seeds, the seed powder, the dry leaves and the fresh
leaves did not reduce the number of adult emerged and thus had no biological activity against C.
maculatus. It is noteworthy that fresh leaves used in our trials were harvested in greenhouses in
Belgium. Ermel et al. (1986), Levin and York (1978) and Singh (1986) reported the importance of
the origin of the Neem tree (Azadirachta
indica L.), a plant used as an alternative control method,
as a source of variability for chemical composition and probably for biological activity also.
Modification of growing conditions induced changes of azadirachtin content (the active principle
of Neem tree) and therefore led to significant changes in the insecticidal properties.
Moreover, it is important to recall that females from both the “Aightless” and “flight” forms
were used for the biological activity tests with whole seeds, the seed powder and the dry leaves.
Sano (1967) and Utida (1972) observed that females from the “flight” form had a low fecundity
and fertility compared to females from the “flightless” form. The use of these two forms explained
the great variability in the results obtained in trials with whole seeds, seed powder and dry leaves.
For a11 other tests, only females from the “flightless” form were used.
C. occidentalis
seed oil did not significantly affect the number and the distribution (on cowpea
seeds and on petri dishes) of eggs laid but did reduce the mean number of Fl adults which emerged.
The present results indicate that C. occidentah seed oil is effective in reducing damage to cowpea
seeds from C. maculatus. There is an increase in mortality of both eggs and first instar larvae which
results in a significant reduction in C. maculatus progeny and damage. Our results with
C. occidentah seed oil are in agreement with many other works on the use of oils against
.’ ’
stored-products insects (Boughdad et al., 1987; Credland, 1992; Don-Pedro, 1989a, b, 1990; Hi11
and Schoonhoven, 1981; Ivbijaro, 1990; Messina and Renwick, 1983; Naik and Dumbre, 1984;
Pereira, 1983; Schoonhoven, 1978; Singh er af., 1978; Su, 1991; Su er al., 1972; van Huis, 1991).
Four different hypotheses have been given by these authors to explain the toxicity of vegetable oils:
(1) The toxicity to the eggs and first instar larvae is the consequence of the occlusion of the
short funnel at the posterior end of the egg. This hypothesis cari explain the ovicidal and larvicidal
effects observed in our experiments;
(2) A reduction of egg adherence on the cowpe,a seed which prevents the first instar larva from
penetrating the seed testa. This decrease of adherence has been observed in the experiments
described and cari explain mortality of C. macularus first instar larvae;
(3) A direct toxic effect of some oil constitue@,;
(4) The toxicity of different fatty acids contained in the oil.
In our experimental conditions, the four fatty acids used separately or a11 htogether reduced
significantly the number of progeny. Oleic, stearic acids and the blend of the four fatty acids were
the most aective. Both linoleic ami oleic acids exhibit only ovicidal effects. This observation is
in line with Don-Pedro’s results (1990). Palmitic acid did not reduce oviposition and did not affect
either fecundity or first instar larva survival. Nevertlheless, stearic acid is the only lipid tested which
Biological activity of Cassis occiaéntalis against Callosobruchus maculatus
317
reduced the mean number of eggs laid by C. maculatus females. Moreover, stearic acid has also
an ovicidal effect, although the increase of mortality is less than with oleic and linoleic acids.
From the results (Table 7), it is clear that the unsaponifiable matter of Cassiu oil did not
significantly reduce the number of beetle progeny.
CONCLUSIONS
In the present experimental conditions, the fresh and dry leaves, whole and powdered seeds of
C. occidentah showed no contact toxicity. As modifications of growing conditions are known to
induce modifications of the chemical composition of plant material, it would be interesting to test
freshl harvested leaves from tropical areas to confirm or invalidate the aforementioned results.
The toxicity of Cassia occidentah oil cari be assigned to physical effects (suffocation of eggs and
decrease of eggs adherence) and to direct toxicity of fatty acids of the oil.
In tropical Africa, hachis oil is used by farmers to prote& stored cowpea seeds (Boughdad et al.,
1987; Don-Pedro, 1989a; Messina and Renwick, 1983; Pereira, 1983) but this oil could be valorized
for trade and export. It is also possible that other underexploited oils could have a potential role
in grain protection.
Acknowledgement-Sincere
thanks to Dr J.-L. Hemptinne for his helpful comments on the manuscript.
REFERENCES
Anonymous (1987) Fiche espèce: Cassia occidentalis L. (Caesalpiniaceae). Méd. Tradit. Pharmac. 1, (2), 143-170.
AOCS (1983) Officiai and Tentative Methods of the American Oil Chemists’ Society, Chicago, IL.
Boughdad A., Gillon Y. and Gagnepain C. (1987) Effect of Arachis hypogea seed fats on the larval development of
Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J. stored Prod. Res. 23, 99-103.
Credland P. F. (1992) The structure of bruchid eggs may explain the ovicidal effect of oils. J. srored Prod. Res. 28, 1 -9.
Dagnelie P. (1975) Théorie et Méthodes Statistiques, Vol. 2. Les presses agronomiques de Gembloux, Belgium.
Don-Pedro K. N. (1989a) Effects of fixed vegetable oils on oviposition and adult mortality of Callosobruchus maculatus
(F.) on cowpea. ht. Pest Control31, 34-37.
Don-Pedro K. N. (1989b) Mode of action of fixed oils against eggs of Callosobruchus maculatus (F.). Pestic. Sci. 26, 107-I 15.
Don-Pedro K. N. (1990) Insecticidal activity of fatty acid constituents of fixed vegetable oils against Callosobruchus
maculatus (F.) on cowpea. Pestic. Sci. 30, 209-211.
Ermel K., Pahlich E. and Schmutterer H. (1986) Azadirachtin content of necm kernels from different geographical locations,
and its dependence on temperature, relative humidity, and light. In Natural Pesticides from the Neem Tree and other
Tropical Plants. Proc. 3rd Int. Neem COI$ (Edited by Schmutterer H. and Ascher K. R. S.), pp. 171-184. Eschborn.
Higgins J. M., Walker R. H. and Whitwell T. (1985) Coffee serma (Cassis occidentalis) competition with cotton (Gossypium
hirsutum). Weed Sci. 34, 52-56.
Hi11 J. and Schoonhoven A. V. (1981) Effectiveness of vegetable oil fractions in controlling the mexican bean weevil on
stored beans. J. Econ. Entomol. 74, 478-479.
Huis van A. (1991) Biological methods of bruchid control in the tropics: a review. Insect Sci. Applic. 12, 87-102.
Ivbijaro M. F. (1990) The efficacy of seed oils Azadirachta indica A. Juss and Piper guineense Schum and Thonn on the
comtrol of Callobruchus
maculatus (F.). Insect Sci. Appl. 11, 149-152.
Labeyrie V. (1981) Vaincre la carence protéique par le développement des légumineuses alimentaires et la protection de
leurs récoltes contre les bruches. Food Nutr. Bull. 3, 24-38.
Levin D. A. aod York B. M. (1978) The toxicity of plant alkaloids: an ccogeographic perspective. Biochem. Syst. Ecol.
6, 61-76.
Messina F. J. and Renwick J. A. A. (1983) Effectiveness of oils in protecting stored cowpeas from the cowpea weevil
(Coleoptera: Bruchidae). J. Econ. Entomol. 76, 634-636.
Miralles J. and Gaydou E. M. (1986) Composition en acides gras des huiles extraites des graines de trois Cassis
(Caesalpinacées) d’origine sénégalaise. Reo. Fr. Corps gras 10, 381-384.
Naik IR. L. and Dumbre R. B. (1984) Effect of some vegetable oils used in protecting stored cowpea on biology of pulse
beetle, Callosobruchus maculatus (FABR.) (Coleoptera: Bruchidae). Bull. Grain Technol. 22, 2532.
Pande:y Y. N. (1975) Cassia seeds used as drug in the indigenous medical systems of India. Q. J. Crude Res. 13, 61-64.
Percim J. (1983) The effectiveness of six vegetable oils as protectants of cowpeas and bambara groundnuts against
infiestation by Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J. stored Prod. Res. 19, 5762.
Sano IL (1967) Density effèct and environmental temperature as the factors producing the active form of Callosobruchus
maculatus (F.) (Coleoptera, Bruchidae). J. stored Prod. Res. 2, 187-195.
Schoonhoven A. V. (1978) Use of vegetable oils to protect stored beans from bruchid attack. J. Econ. Entomol. 71,254-256.
Singh R. P. (198# C omparison of antifeeding efficacy and extract yields from different parts and ecotypes of neem
(Asadiruchta mdica A. Jus~) trees. In Natural Pesticides from the Neem tree and Other Tropical Plants. Proc. 3rd Int.
Neem Car$ (Edited by Schmutterer H. and Ascher K. R. S.), pp. 185-194. Eschborn.
Singh S. R., Luse R. A., Leuschner K. and Nangju D. (1978) Groundnut oil treatment for the control of Callosobruchus
maculatus
(F.) during cowpea storage. J. stored Prod. Res. 14, 77-80.
SU H. C. F. (1991) Laboratory evaluation of toxicity of Calamns oil against 4 specics of stored-product insects. J. Entomol.
sci’. 26, 76-80.
.
“.
.
I
Su H. C. F., Speirs R. D. and Mahamy P. (3. (1972) Citrus oil as protectnnts of black-eyed peas against cowpea weevils:
laboratory evaluation. J. Econ. Enromol,. 65, 1433-1436.
Svodoba J. A. and Feldlaufer M. F. (1991) Neutral sterol metabolism in insects. Lipiak 26, 614418.
1
Svodoba J. A., Weirich G. F. and Feldlaufer M. F. (1991) Recent advances in insect steroid biochemistry. In Physiology
and Biochemistry of S&ols (Fdited by Patterson G. W. and Nes W. D.), pp, 294326. American oii chemists’ Society,
‘T
Compaign, IL.
Taylor T. A. and Agbaje L. A. (1974) Flighrt activity in normal and active forms of Cullosobruchus
macula/us in a store
in Nigeria. J. stored Prod. Res. 10, 9-16.
Utida S. (1972) Density dependent polymorphism in the aduh of Calfosobruchus
maculafus (Coleoptera, Bruchidae).
J. srored Prod. Res. 8, 111-126.