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Effects of material and extracts of Trigonella foenum-graecum L. against the stored product pests Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae)
J. stored Prod. Ref. Vol. 33, No. 3. pp. 209-217. 1997 6 1997 Elsevier Science Ltd. All rights reserved
Pergamon
Printed in Great Britain
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0022-474X/97 $17.00 + 0.00
Effects of Material and Extracts of Trigonella foenum-graecum L. Against the Stored Product Pests Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) JEROME
PEMONGE,‘,’ MARIA JESUS PASCUAL-VILLALOBOS,‘* CATHERINE REGNAULT-ROGER’
and
‘Consejeria de Medio Ambiente, Agricultura y Agua, Centro de Investigacih y Desarrollo Agroalimentario, Estacih Sericicola, 30150 La Alberta, Murcia, Spain and ‘Sciences Biologiques, LEM-IBEAS, UniversitP de Pau et des Pays de I’Adour, 64000, Pau, France (Accepted 7 February 1997)
Abstract-Insecticidal activity of plant material (seeds and leaves) and extracts of Trigonellu foenum-graecum from two sources against the stored product pests Tribolium castaneum and Acanthoscelides ubtectus was investigated. Topical applications of extracts produced a high degree of mortality in both insects (at 6 and 30 pg/insect). Powdered fenugreek seeds or extracts applied to Phase&s udgaris beans produced mortality and inhibited oviposition and larval penetration by A. obtectus. The presence of the plant material decreased A. obfectus fecundity and reduced its longevity. Fenugreek seed appeared moderately toxic to young larvae of T. castaneum (LDB = 18% in diet), and surviving adults showed progressively decreased fecundity as the dose of seeds, especially ripe seed, was increased. Significant effects of dose, seed ripeness and their interaction were produced. The seed affected the fertility of both sexes. Some fertility was recovered on reversion to the standard diet. Possible active compounds and the implications of the results for pest control are discussed. 0 1997 Elsevier Science Ltd Key words-Trigonella foenum-graecum, Acanthoscelides obtectus
natural
insecticides,
reproduction,
Tribolium castaneum,
INTRODUCTION
Protection of agricultural stored products against insect pests is of utmost importance to secure a continuous and safe food supply all over the world. Conventional treatments have been used for this purpose, but nowadays, other ecologically sound methods based on the use of natural compounds are needed for an integrated approach to pest management. The interaction between plants and insects is mediated by chemical compounds as a result of joint evolution (Strebler, 1989). Therefore, the study of the effects of plant material and/or extracts upon pests will provide chances for finding alternative insecticides. *Author
to
whom all correspondence
should be addressed. 209
210
J. Pemonge et al.
In the south of France and in Mediterranean areas, one of the most damaging pests is Bruchidae) a pest of Phuseolus vulgaris L. (kidney bean), which produces losses (up to 30%) of stored beans. Its oviposition and growth are continuous, the larvae feeding on the seeds, and after emergence, the adults reproducing either in the field or in the stored seeds in a continuous cycle (Labeyrie, 1962). Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a cosmopolitan stored product pest with a preference for cereal grains and wheat flour. It is also a good model for animal breeding and entomological reseach (Orozco, 1967). The Leguminosae is a wide and chemically rich family (Pascual, 1978). The rotenoids, present in the genus Derris and Lonchocarpus (Ahmed et al., 1989), were one of the first insecticides discovered. The genus Trigonella belongs to the subfamily Lotoideae and, according to De Bolos and Vigo (1984), it includes 80 species widespread in the Mediterranean or subtropical arid regions. Trigonellu foenum-graecum L. (fenugreek) is a native herb to the Mediterranean area and Near East, which has been cultivated in India, Mediterranean regions and North Africa. Duke (1981) reported that it has been used for culinary and medicinal purposes and also for fodder. The plants and seeds have a strong distinctive odour. The legume has slender pointed curved pods over 10 cm long each with 10-20 seeds. The seeds have been used in folk medicine, having tonic, emollient and vermifugal properties (Duke, 1981). An hypoglycaemic effect was obtained when supplied to diabetic subjects (Sharma, 1986). Several allelochemicals are present in the plant. Fenugreekine is steroidal sapogenin with cardiotonic activity, and it is also a prophylactic against chicken pox (Ghosal et al., 1974). Trigonelline is an innocuous alkaloid present in the seed at 0.13% (Font Quer, 1981). The seed is a rich source of steroids useful in pharmacology (Provorov et al., 1993). Secoy and Smith (1983) reported that fenugreek leaves have been used to repel insects in stored grain. Grainge and Ahmed (1988) pointed out that the oil was repellent to stored grain pests. The objectives of this study were to test: (1) the effect of fenugreek seed intake on T. custaneum fecundity, larval growth and mortality, (2) adult mortality, antifecundity and reduced adult emergence of A. obtectus caused by the presence of fenugreek seeds or leaves or their extracts on beans, and (3) insecticidal activity by topical applications of extracts. Acanthoscefides obtectus (Say) (Coleoptera:
MATERIALS
AND METHODS
Plant materials and extracts
Seeds of T. foenum-graecum were obtained from the USDA germplasm bank. Two samples, corresponding to the following plant introduction numbers: PI515953 (from Iran) and PI532867 (from India) were sown at the Torreblanca Experimental Station belonging to CIDA (Centro de Investigation y Desarrollo Agroalimentario), Murcia in Spain, in 1995. Leaves were harvested at flowering time and unripe seeds before maturation, for the Iranian variety, and both were freeze dried prior to utilization. Ripe seeds were also harvested from both varieties and stored at room temperature. Extracts were obtained with organic solvents (of increasing polarity) either from leaves or from seeds previously ground to 1 mm particle size with a coffee mill. Hexane, acetone and 50% methanol were sequentially used to extract the plant material ( x 10) for a period of 48 h each, at room temperature. Seed cake was obtained by previous oil extraction with Soxhlet’s apparatus using petroleum ether as a solvent. Insects A. obtectus was reared in a growth cabinet at a constant temperature of 27°C and 70% r.h. under a photoperiod of 12 h light/l2 h dark. Adults oviposited on dry P. uulgaris beans and the larvae developed inside the beans until adult emergence. Newly emerged adults were used for the experiments, pairing off males and females when needed. T. custuneum cultures were kept on an artificial diet of wheat flour with 5% brewer’s yeast. Synchronized cultures were maintained in an incubator at 30°C and 70% r.h. in complete darkness. Larvae, pupae and adults of known age were collected for the experiments. When pairs were needed, sex had to be checked at the pupal stage.
Activity of fenugreek against stored product pests
Znfzuence ofT.
foenum-graecum
211
on A. obtectus
Mortality andfecundity of the beetles in the presence of plant material. Five pairs of adults were enclosed within a glass Petri dish (P, = 10 cm) in which 1 g of ground material (seeds or leaves), covered or uncovered with cloth, and six beans (not in direct contact with the plant) had been placed. When cloth was used, effects due to colour or direct contact of insects with the plant material were avoided. Each treatment was replicated five times. Mortality and fecundity (number of eggs) were recorded daily for a period of 8 days. Insecticidal e&t by topical application. Acetone extract from leaves and seeds was topically applied to A. obtectus adults using a Hamilton microsyringe. The carrier was acetone in all cases, including the control. Thirty micrograms of leaf and 6 pg of seed extracts were applied per insect, singly placed inside a glass test tube, using 10 replications (insects) per treatment. Mortality was recorded after 2 days. Powdered Trigonella seed or extract applied to beans. In one test, five pairs of adults were enclosed in a 15 ml vial with five beans, which were sprinkled with powdered seed at a dose of 10% (w/w). After 4 days, adult mortality was recorded and adults removed; after 45 days, adult emergence was assessed. Ripe and unripe seeds from Iran were tested against a control. Another test was similarly designed, but the treatments included the application of hexane, acetone and methanolic seed extracts (using acetone as a solvent and allowing evaporation before testing) at a dose of 130 pg/bean. Eggs laid were counted after 4 days. Each treatment was replicated three times in both experiments. Znfruence of T. foenum-graecum
on T. castaneum
Influence of fenugreek seed dose on T. castaneum fecundity. Firstly, an experiment testing the effect of ripe or unripe fenugreek seed, mixed with the diet in the proportion of 30%, on the fecundity of insect pairs, was set up. Adults were fed with the treated diet after emergence, and 5 days later, the number of eggs laid per female was counted. Each treatment was replicated 10 times. In a second experiment, newly-hatched larvae (n = 50) were fed with increasing doses of ripe or unripe fenugreek, mixed with the diet in proportions of 0, 5, 10, 15, 20, 25 and 30%. Those insects that successfully completed the cycle were paired off (n = 5-10) and fed with the same diet for a period of 5 days. Subsequently, the numbers of eggs laid per female were counted. This experiment enabled the regression of fecundity with fenugreek seed dose to be determined. The third experiment was based on the productivity of pairs in which neither (control), one, or both sexes were fed for a period of 10 days after adult emergence with fenugreek mixed to form 40% of the diet. After such preparation, the pairs were set up and maintained for another 10 days, feeding on a diet with or without fenugreek (dose of 40% w/w). The numbers of eggs laid were then counted for each treatment. Five replications were set up for each treatment. Larval growth inhibition and mortality of T. castaneum by mixing seed or cake extract with the diet. The material tested included ripe and unripe seed or seed cake from Iran and ripe seed or cake from India which were extracted with hexane and acetone. Extracts (dose of 5% w/w) were applied using acetone as a carrier, subsequently allowing solvent evaporation. One hundred milligrams of the corresponding diet plus a lo-day-old larva were placed in a 4 ml vial. After 10 days, the length (mm) of the larva was measured using a magnifying lens ( x 40), and mortality was recorded to compare with the control. Each treatment was replicated 10 times. Insecticidal e&ct by topical application. Acetone extract from leaves and seeds was topically applied at 30 and 6 pg, respectiveIy, per insect (25-day-old T. castaneum larvae) using a Hamilton microsyringe, as described above for adult A. obtectus. Statistics
Mean, range and coefficients of variation were computed for the variates studied. For comparisons between treatments and control, T or Mann-Whitney U-tests were used. Mortality was usually expressed as a percentage. Analyses of variance for bifactorial experiments were carried out to test the significance of the factors studied and their interaction, using F-tests. For mean comparisons, standard errors of the differences were computed.
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Table I. Average mortality and fecundity of A. obrecrus when exposed to leaves or seeds of fenugreek (covered (c) or uncovered (u) with a cloth) Treatments
Cumulative mortality (%)
Material Control Leaves Seeds Leaves Seeds ANOVA F F F
Cloth
2 days
Material Cloth Material x Cloth
4 days
0
0
0 0 0 0
6.1 IO 0 5
-
Cumulative fecundity (no. eggs)
6 days
8 days
2 days
3.3 6.7 13.3 0 5
3.3 IO 23.3 0 5
0 0
0.95 I .26 0.03
0.74 I .66 0.02
1.97 4.92* 0.43
: 0 -
4 days
6 days
8 days
4.6 I .Ol I.5 I.8 0.2
6.9 1.28 1.86 2 1.1
6.9 1.5 2.23 3.1 1.5
14.78** 0.13 0.22
4.74’ 0.28 0.79
15.10** 0.18 0.87
F = F-test: significant. *P < 0.05; **P < 0.01. n = 5 replications per treatment and 5 females plus 5 males per replication. Table 2. Adult mortality and emergence inhibition in A. obrecrus reared on 10% w/w ground fenugreek seed on P. vulgaris beans Seed
Adult mortality (no.) after 4 days Mean
Ripe Unripe Control
6.00 3.30 0
Adult emergence (no.) after 45 days
Range
Mean
Range
4-8 2-4
7.67 5.67 25.00
5-8 38 22-28
n = 3 replications per treatment and IO insects per replication.
A linear regression analysis was performed to relate T. castaneum fecundity with fenugreek seed intake. RESULTS Leaving the material uncovered significantly increased insect mortality (P < 0.05), especially in the presence of Trigonella seeds (Table 1). Female fecundity decreased in the presence of plant material (either leaves or seeds), and this effect was more evident on the third day (P < 0.01) because no females (control or treatments) oviposited during the first 2 days. The solvent acetone did not affect A. obtectus but showed some toxicity to T. castaneum larvae. However, the insecticidal effect of topical application of Trigonella leaf and seed extracts was noticeable on both insects (Fig. 1). Seed extracts were more active than those from leaves against A. obtectus, in spite of being applied at a lower dose (6 pg/insect for seed and 30 pg/insect for leaf extracts), but the reverse applied to T. castaneum. Powdered fenugreek seed or extract on beans produced mortality in the adult of A. obtectus and inhibited larval development, which resulted in decreased adult emergence compared to the controls (Tables 2 and 3). Ripe seed was more active (Table 2), but differences among extracts were unclear (Table 3). Fenugreek seed mixed with the diet in the proportion of 30% after adult emergence produced a decrease of T. castaneum fecundity (Table 4) from 79.9 eggs/female for a period of 5 days on the standard diet to half or a third of this value on the treated diets. When each treatment (ripe and unripe seed) was compared with the control, a very highly significant difference was obtained Table 3. Oviposition and adult emergence inhibition in A. obrecrus by application of fenugreek seed extracts on P. vulgaris beans Extract
H A M Control
Laid eggs (no.) after 4 days
Adult emergence (no.) after 45 days
Mean
Range
Mean
Range
63.3 46.7 44.0 80.0
55-80 20-85 9-80 65-90
4.3 4.7 8.0 22.3
2-8 l-11 l-14 18-25
n = 3 replications per treatment and 5 females plus 5 males per replication. Extracts were obtained sequentially with hexane (H), acetone (A) and 50% methanol (M).
213
Activity of fenugreek against stored product pests
,oo Mortality 4%) I
I
00 -
= Control I A.obtectus IC-T = Control I T.castaneum
=C-A
60.
aL-A IL-T=
40-
= Leaves I A.obtectus
Leaves I
T.castaneum
EX-A
= Seeds / A.obtectus
IS-T
= Seeds / Txastaneum
zo-
0
1
C-A
C-T
L-A
L-T
S-A
S-T
Extracts Fig. 1. Mortality of T. cosraneum larvae (25 days) and A. obrectus adults by topical application of fenugreek leaf (30 fig/insect) and seed (6 pg/insect) acetone extracts (n = IO).
(P < O.OOl), but the same was true for the comparison between ripe and unripe seed. A reduction in fecundity was more pronounced if insects were fed with ripe seed. When young larvae were fed with a diet in which fenugreek seed was mixed in the range O--30%, some mortality was produced during the first 10 days: the lethal dose (LD) calculated by probit method was LD,, = 18% and LD,, = 33.5% for ripe seeds, LD,, = 30% and LD,, = 45.5% For unripe seeds. However, there were always insects completing the cycle, and therefore couples could be paired to test for effects in fecundity. The analysis of variance indicated significant effects of the dose (P < 0.001) but also of seed ripeness and their interaction (P < 0.05) (Table 5). Linear regression analysis of number of eggs laid (per T. cusraneum female over 5 days) with dose of seed was very highly significant (P < 0.001) for both unripe and ripe seed. Figure 2a and b show the fitted regression lines for unripe and ripe seeds, respectively, indicating a negative relation in both cases. Increasing the amount of seed in the diet from egg eclosion produced increasing sterility in adults. Complete sterility was predicted with 26.5% of ripe seed in the diet (Fig. 2b). When adults were always fed with the standard diet (control), the number of eggs laid in 10 days was 75.6 per pair. With 40% fenugreek in the diet, almost complete egg laying inhibition was produced (1.4 eggs/pair). However, this seems to be a reversible effect since the pair recovered some
Table 4. Decrease of T. castaneum fecundity by intake of fenugreek seed mixed in diet (30% w/w) after adult emergence Number of eggs/couple (during 5 days)
Treatments
Standard diet Unripe fenugreek seed Ripe fenugreek seed
Mean zt SE
Variance
Range
CV(%) . ,
U
79.9 f 5.21 44.1 * 3.97 24.7 k 2.52
271.9 157.2 63.6
45-96 29-68 12-38
22.62 28.43 32.28
g*** 0***
U = Mann-Whitney U-test for comparisons (n = 10) with the standard diet: ***significant at P < 0.001 (comparison between ripe and unripe seed U = 6.5***).
Table 5. Mean fecundity (number of eggs/pair over 5 days) in T. casfaneum fed on a diet mixed with fenugreek seed at different doses Dose of fenugreek seed in diet (%)
Seed
Unripe Rioe
F SED
0
5
62.3 62.3
32.0 45.0
IO 28.4 27.3
I5
20
25
30
26.8 18.4
20.8 6.0
11.8 2.8
9.4 I .2
Seed ripeness
Dose
Ripeness x dose
6.97* I .906
52.97*** 3.876
3.10* 5.48 1
F = F-test: significant at *O.OI c P < 0.05, ***P -C0.001. SED = standard error of difference for mean comparison by t-test. n = 5-10 pairs per treatment.
214
J. Pemonge ef al.
0
5
10
15
20
25
J 30
Dose of unripe seed in diet (%)
-20 ’ o
5
10
15
20
25
I 30
Dose of ripe seed in diet (%) Fig. 2. Linear regression of number of eggs laid per T. custaneum female (during 5 days) on the dose of T. foenum-graecum seed in the diet since egg eclosion. (a) unripe seed y = (- 1.37 k 0.186)x + (47.3 f 3.52); (b) ripe seed y = ( - 1.98 f 0.158)x + (52.54 f 3.0).
fecundity on reversion to the standard diet (24.2 eggs). The inhibition of oviposition was a consequence of seed intake in either or both males or females (Table 6). Extracts of low- to mid-polarity incorporated into the diet (5%) generally did not produce a high mortality, but did inhibit growth (Table 7).
Table 6. Mean fertility (number of eggs in a IO-day period) in T. custaneum pairs in which one or both Sexes were fed with fenugreek seed (40% w/w) 10 days after adult emergence and before pairing OK, with reversion or not to standard diet Pair: 0 x 6 Diet after oairinn off With 40% w/w reversion F SED
No x No
No x Yes
Yes x No
Yes x Yes
56.8 75.6
18.0 38.8
14.4 38.4
1.4 24.2
Pair
Diet after pairing off
263.69*** 1.999
233.57*** 1.413
F = F-test: significant at **+P < 0.001, non-significant at P > 0.05. SED = standard error of difference for mean comparison by r-test. n = 5 replications per treatment.
Pair x Diet 0.65 ns 2.827
Activity of fenugreek against stored product pests
215
Table 7. Larval growth inhibition and mortality, after 10 days, of T. custaneum by intake of ripe (r) or unripe (u) fenugreek seed (s) or cake (c) extracts at 5% in diet Treatments
Larval length
Origin
ripeness
material
extract
Mortality (%)
(mm)
Iran Iran Iran Iran Iran Iran Iran Iran India India India India
r r u
S
H A H A H A H A H A H A Control
10 0 0 0 60 20 60 0 0 0 40 0 0
2.7 + 0.71 3.1 * 0.21 3.2 + 0.25 3.5 & 0.16 3.0 + 0.44 2.7 + 0.22 3.0 f. 0.35 2.8 f 0.14 2.7 f 0.23 2.7 f 0.11 3.7 * 0.47 2.9 + 0.15 4.2 + 0.16
S S
U
S
r r u
c
U
C
r r r r
S
C C
S C
c
T *** *** ** ** *** *** *** *** ***
*** ***
Extracts were obtained sequentially with hexane (H) and acetone (A). T = t-test: significant **PC 0.01 and ***P
DISCUSSION
T. foenum-graecum leaves, seeds and extracts showed insecticidal effects on several targets. The presence of leaves or seeds in a closed space and volume arena induced A. obtectus adult mortality with a 2-day delayed effect and also a reduction in oviposition, which may have been due to the odour or colour of the plant. Topical application of plant extracts also produced mortality in the adult and decreased female fecundity. Fenugreek seed extracts are known to have contact activity. Afifi et al. (1988) reported that acetone seed extracts of T. foenum-graecum seeds were toxic to the stored grain pests Sitophilus oryzae (L.) and Rhyzopertha dominica (F.) at 500 ppm. Also, Afifi and Hafez (1988) obtained 100% mortality of the mite Tyrophagus putrescentiae (Schrank) at 100 ppm on flour. A. obtectus is an insect very well adapted to its host-plant, P. vulgaris, but it can also grow in other non-host Leguminosae plants (Hamraoui and Regnault-Roger, 1995). In a current experiment to see whether the beetle could adapt to fenugreek seeds, no larval development was observed. Powdering ground fenugreek seed or applying plant extracts to P. uulgaris seeds produced more rapid adult mortality, and larvae could not penetrate the seeds. Roche and Simanca (1987) reduced the emergence of bruchids by treating stored grains with sunflower oil. Plant extracts can form a film covering the surface of the pulse. This causes a reduction in respiration and gas exchange between the seeds and their environment. The toxicity of compounds penetrating the seed or the accumulation of the toxic metabolites (Don Pedro, 1989) also kills insects. The structure of bruchid eggs could also contribute to the ovicidal effect of the extracts (Credland, 1992). A. obtectus could be controlled by such extracts if the final use of the beans is not for food. Fenugreek was also toxic to T. casfaneum. A contact effect occurred and also antifeedancy, which interrupted larval development. The suppression of reproductive activity caused by T. foenum-graecum seed seems to affect both males and females. However, we have not tested whether a full recovery of fecundity is possible if insects are fed with untreated seed soon after egg eclosion or if an alteration in the reproductive system may cause irreversible sterility. T. foenum-graecum contains some antinutritional factors such as saponins and phytic acid (Singh et al., 1994). Bohannon et al. (1974) reported the presence of steroidal sapogenins, mainly diosgenin, in T. foenum-graecum, at a maximum concentration of 0.2-0.8% in one variety. Trigonella is not the only genus containing diosgenin. Mangla and Kamal (1989) studied the concentration of steroidal sapogenins in berries of Solanum pseudocapsicum L. at different stages of growth and stated that in ripe fruits (in a comparison of unripe or overripe), diosgenin was present in the highest amount. According to Dawson (1991), Dioscorea deltoidea was planted to produce rhizomes rich in the compound, but plantations were soon abandoned because of the need
216
J. Pemonge et al.
for an abundant water supply and the decrease of diosgenin if the rhizome yield was increased. Kamal et al. (1987) considered that cultivation of fenugreek was more feasible than Dioscorea for diosgenin production, and there is variation in diosgenin content among genotypes giving good prospects for breeding more productive strains (Jain and Agrawal, 1987). Saponin extracts have shown a range of activities, among them haemolytic activity (Khalid and El-Adawy, 1994). With diosgenin, Aradhava and Kale (1992) observed oestrogenic action in rats. Diosgenin is a precursor for the manufacture of corticosteroids and steroid hormones and has been used to produce oral contraceptives (Djerassi, 1992). In addition to these applications, saponins play a role in plant-insect relationships, e.g. some saponin glycosides exert a deterrent activity on insects and disturb their feeding (Jain and Tripathi, 1991), providing protection against pests. Our results indicate that they also may produce insecticidal effects, but further experiments would be necessary to establish the activity of each component of the extract. CONCLUSION The use of plant allelochemicals is now recognized as a promising alternative method for an integrated approach to pest management. T. foenum-gruecum has shown considerable insecticidal activity: extracts act directly on adults by contact, and powdered seeds inhibit reproduction by decreasing fecundity and exerting a larvicidal effect in A. obtectus and T. castaneum. Consequently, the plant could be considered as a new source of stored product pest control. Acknowledgemenrs-This research has been part of a Spanish project funded by INIA (SC94039). We also thank the Leonardo da Vinci (E.U.) program, which has financially supported a 6-month scholarship.
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Kamal R., Yadav R. and Sharma G. L. (1987) Diosgenin content in fenugreek collected from different geographical regions of south India. Indian Journal of Agricultural Sciences 57, 674-676. Khalid A. H. and El-Adawy T. A. (1994) Isolation, identification and toxicity of saponin from different legumes. Food Chemistry 50, 197-201. Labeyrie V. (1962) Les Acanthoscelides. In Entomologie Appliqub a l’dgriculture, ed. Balachowsky A. S., Vol. 1, pp. 469-490. Masson, Paris. Mangla M. and Kamal R. (1989) Steroidal sapogenins and glycoalkaloids from berries of the Solanum pseudocapsicum L. at different stages of growth. Indian Journal of Experimental Biology 27, 370-371. Oroxco F. (1967) El Tribolium castaneum en mejora animal. Separata de Avictdtura Tecnica, Vol. XVIII, no. 4, 10 pp. Pascual H. (1978) Leguminosas de la Peninsula lberica y Baleares. Instituto National de Investigacir_GtAgraria. MAPA, Madrid, 207 pp.
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Provorov N. A., Soskov Y. D., Lutova Ludmila A., Sokolova 0. and Simarov B. V. (1993) Initial material for breeding fenugreek (T.,foenum-graecum) for increased seed productivity, symbiotic activity and synthesis of steroids in seeds and cell culture. Second European Symposium on Industrial Crops and Products, 22-24 November 1993, Pisa, Italy. Roche R. and Simanca M. E. (1987) Control of Acanthoscelides obrectus and Zabrotes subfusciatus with mineral and vegetable oils. Ciencias de la Agricultura 31, 34-39. Secoy D. M. and Smith A. E. (1983) Use of plants in control of agricultural and domestic pests. Economic Botany 37,28-57. Sharma R. D. (1986) Effect of fenugreek seeds and leaves on blood glucose and serum insulin responses in human subjects. Nutrition Research 6, 1353-l 364. Singh J., Gupta K. and Arora S. K. (1994) Changes in the antinutritional factors of developing seeds and pod wells of fenugreek (Trigonella ,foenum-graecum L.). Plant Foods for Human Nutrition 46, 77-84. Strebler G. (1989) Les Mediateurs Chimiques. Technique et Documentation. Lavoisier, Paris, 246 pp.
SPR 33/3-B
Pergamon
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0022-474X/97 $17.00 + 0.00
Effects of Material and Extracts of Trigonella foenum-graecum L. Against the Stored Product Pests Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) JEROME
PEMONGE,‘,’ MARIA JESUS PASCUAL-VILLALOBOS,‘* CATHERINE REGNAULT-ROGER’
and
‘Consejeria de Medio Ambiente, Agricultura y Agua, Centro de Investigacih y Desarrollo Agroalimentario, Estacih Sericicola, 30150 La Alberta, Murcia, Spain and ‘Sciences Biologiques, LEM-IBEAS, UniversitP de Pau et des Pays de I’Adour, 64000, Pau, France (Accepted 7 February 1997)
Abstract-Insecticidal activity of plant material (seeds and leaves) and extracts of Trigonellu foenum-graecum from two sources against the stored product pests Tribolium castaneum and Acanthoscelides ubtectus was investigated. Topical applications of extracts produced a high degree of mortality in both insects (at 6 and 30 pg/insect). Powdered fenugreek seeds or extracts applied to Phase&s udgaris beans produced mortality and inhibited oviposition and larval penetration by A. obtectus. The presence of the plant material decreased A. obfectus fecundity and reduced its longevity. Fenugreek seed appeared moderately toxic to young larvae of T. castaneum (LDB = 18% in diet), and surviving adults showed progressively decreased fecundity as the dose of seeds, especially ripe seed, was increased. Significant effects of dose, seed ripeness and their interaction were produced. The seed affected the fertility of both sexes. Some fertility was recovered on reversion to the standard diet. Possible active compounds and the implications of the results for pest control are discussed. 0 1997 Elsevier Science Ltd Key words-Trigonella foenum-graecum, Acanthoscelides obtectus
natural
insecticides,
reproduction,
Tribolium castaneum,
INTRODUCTION
Protection of agricultural stored products against insect pests is of utmost importance to secure a continuous and safe food supply all over the world. Conventional treatments have been used for this purpose, but nowadays, other ecologically sound methods based on the use of natural compounds are needed for an integrated approach to pest management. The interaction between plants and insects is mediated by chemical compounds as a result of joint evolution (Strebler, 1989). Therefore, the study of the effects of plant material and/or extracts upon pests will provide chances for finding alternative insecticides. *Author
to
whom all correspondence
should be addressed. 209
210
J. Pemonge et al.
In the south of France and in Mediterranean areas, one of the most damaging pests is Bruchidae) a pest of Phuseolus vulgaris L. (kidney bean), which produces losses (up to 30%) of stored beans. Its oviposition and growth are continuous, the larvae feeding on the seeds, and after emergence, the adults reproducing either in the field or in the stored seeds in a continuous cycle (Labeyrie, 1962). Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a cosmopolitan stored product pest with a preference for cereal grains and wheat flour. It is also a good model for animal breeding and entomological reseach (Orozco, 1967). The Leguminosae is a wide and chemically rich family (Pascual, 1978). The rotenoids, present in the genus Derris and Lonchocarpus (Ahmed et al., 1989), were one of the first insecticides discovered. The genus Trigonella belongs to the subfamily Lotoideae and, according to De Bolos and Vigo (1984), it includes 80 species widespread in the Mediterranean or subtropical arid regions. Trigonellu foenum-graecum L. (fenugreek) is a native herb to the Mediterranean area and Near East, which has been cultivated in India, Mediterranean regions and North Africa. Duke (1981) reported that it has been used for culinary and medicinal purposes and also for fodder. The plants and seeds have a strong distinctive odour. The legume has slender pointed curved pods over 10 cm long each with 10-20 seeds. The seeds have been used in folk medicine, having tonic, emollient and vermifugal properties (Duke, 1981). An hypoglycaemic effect was obtained when supplied to diabetic subjects (Sharma, 1986). Several allelochemicals are present in the plant. Fenugreekine is steroidal sapogenin with cardiotonic activity, and it is also a prophylactic against chicken pox (Ghosal et al., 1974). Trigonelline is an innocuous alkaloid present in the seed at 0.13% (Font Quer, 1981). The seed is a rich source of steroids useful in pharmacology (Provorov et al., 1993). Secoy and Smith (1983) reported that fenugreek leaves have been used to repel insects in stored grain. Grainge and Ahmed (1988) pointed out that the oil was repellent to stored grain pests. The objectives of this study were to test: (1) the effect of fenugreek seed intake on T. custaneum fecundity, larval growth and mortality, (2) adult mortality, antifecundity and reduced adult emergence of A. obtectus caused by the presence of fenugreek seeds or leaves or their extracts on beans, and (3) insecticidal activity by topical applications of extracts. Acanthoscefides obtectus (Say) (Coleoptera:
MATERIALS
AND METHODS
Plant materials and extracts
Seeds of T. foenum-graecum were obtained from the USDA germplasm bank. Two samples, corresponding to the following plant introduction numbers: PI515953 (from Iran) and PI532867 (from India) were sown at the Torreblanca Experimental Station belonging to CIDA (Centro de Investigation y Desarrollo Agroalimentario), Murcia in Spain, in 1995. Leaves were harvested at flowering time and unripe seeds before maturation, for the Iranian variety, and both were freeze dried prior to utilization. Ripe seeds were also harvested from both varieties and stored at room temperature. Extracts were obtained with organic solvents (of increasing polarity) either from leaves or from seeds previously ground to 1 mm particle size with a coffee mill. Hexane, acetone and 50% methanol were sequentially used to extract the plant material ( x 10) for a period of 48 h each, at room temperature. Seed cake was obtained by previous oil extraction with Soxhlet’s apparatus using petroleum ether as a solvent. Insects A. obtectus was reared in a growth cabinet at a constant temperature of 27°C and 70% r.h. under a photoperiod of 12 h light/l2 h dark. Adults oviposited on dry P. uulgaris beans and the larvae developed inside the beans until adult emergence. Newly emerged adults were used for the experiments, pairing off males and females when needed. T. custuneum cultures were kept on an artificial diet of wheat flour with 5% brewer’s yeast. Synchronized cultures were maintained in an incubator at 30°C and 70% r.h. in complete darkness. Larvae, pupae and adults of known age were collected for the experiments. When pairs were needed, sex had to be checked at the pupal stage.
Activity of fenugreek against stored product pests
Znfzuence ofT.
foenum-graecum
211
on A. obtectus
Mortality andfecundity of the beetles in the presence of plant material. Five pairs of adults were enclosed within a glass Petri dish (P, = 10 cm) in which 1 g of ground material (seeds or leaves), covered or uncovered with cloth, and six beans (not in direct contact with the plant) had been placed. When cloth was used, effects due to colour or direct contact of insects with the plant material were avoided. Each treatment was replicated five times. Mortality and fecundity (number of eggs) were recorded daily for a period of 8 days. Insecticidal e&t by topical application. Acetone extract from leaves and seeds was topically applied to A. obtectus adults using a Hamilton microsyringe. The carrier was acetone in all cases, including the control. Thirty micrograms of leaf and 6 pg of seed extracts were applied per insect, singly placed inside a glass test tube, using 10 replications (insects) per treatment. Mortality was recorded after 2 days. Powdered Trigonella seed or extract applied to beans. In one test, five pairs of adults were enclosed in a 15 ml vial with five beans, which were sprinkled with powdered seed at a dose of 10% (w/w). After 4 days, adult mortality was recorded and adults removed; after 45 days, adult emergence was assessed. Ripe and unripe seeds from Iran were tested against a control. Another test was similarly designed, but the treatments included the application of hexane, acetone and methanolic seed extracts (using acetone as a solvent and allowing evaporation before testing) at a dose of 130 pg/bean. Eggs laid were counted after 4 days. Each treatment was replicated three times in both experiments. Znfruence of T. foenum-graecum
on T. castaneum
Influence of fenugreek seed dose on T. castaneum fecundity. Firstly, an experiment testing the effect of ripe or unripe fenugreek seed, mixed with the diet in the proportion of 30%, on the fecundity of insect pairs, was set up. Adults were fed with the treated diet after emergence, and 5 days later, the number of eggs laid per female was counted. Each treatment was replicated 10 times. In a second experiment, newly-hatched larvae (n = 50) were fed with increasing doses of ripe or unripe fenugreek, mixed with the diet in proportions of 0, 5, 10, 15, 20, 25 and 30%. Those insects that successfully completed the cycle were paired off (n = 5-10) and fed with the same diet for a period of 5 days. Subsequently, the numbers of eggs laid per female were counted. This experiment enabled the regression of fecundity with fenugreek seed dose to be determined. The third experiment was based on the productivity of pairs in which neither (control), one, or both sexes were fed for a period of 10 days after adult emergence with fenugreek mixed to form 40% of the diet. After such preparation, the pairs were set up and maintained for another 10 days, feeding on a diet with or without fenugreek (dose of 40% w/w). The numbers of eggs laid were then counted for each treatment. Five replications were set up for each treatment. Larval growth inhibition and mortality of T. castaneum by mixing seed or cake extract with the diet. The material tested included ripe and unripe seed or seed cake from Iran and ripe seed or cake from India which were extracted with hexane and acetone. Extracts (dose of 5% w/w) were applied using acetone as a carrier, subsequently allowing solvent evaporation. One hundred milligrams of the corresponding diet plus a lo-day-old larva were placed in a 4 ml vial. After 10 days, the length (mm) of the larva was measured using a magnifying lens ( x 40), and mortality was recorded to compare with the control. Each treatment was replicated 10 times. Insecticidal e&ct by topical application. Acetone extract from leaves and seeds was topically applied at 30 and 6 pg, respectiveIy, per insect (25-day-old T. castaneum larvae) using a Hamilton microsyringe, as described above for adult A. obtectus. Statistics
Mean, range and coefficients of variation were computed for the variates studied. For comparisons between treatments and control, T or Mann-Whitney U-tests were used. Mortality was usually expressed as a percentage. Analyses of variance for bifactorial experiments were carried out to test the significance of the factors studied and their interaction, using F-tests. For mean comparisons, standard errors of the differences were computed.
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Table I. Average mortality and fecundity of A. obrecrus when exposed to leaves or seeds of fenugreek (covered (c) or uncovered (u) with a cloth) Treatments
Cumulative mortality (%)
Material Control Leaves Seeds Leaves Seeds ANOVA F F F
Cloth
2 days
Material Cloth Material x Cloth
4 days
0
0
0 0 0 0
6.1 IO 0 5
-
Cumulative fecundity (no. eggs)
6 days
8 days
2 days
3.3 6.7 13.3 0 5
3.3 IO 23.3 0 5
0 0
0.95 I .26 0.03
0.74 I .66 0.02
1.97 4.92* 0.43
: 0 -
4 days
6 days
8 days
4.6 I .Ol I.5 I.8 0.2
6.9 1.28 1.86 2 1.1
6.9 1.5 2.23 3.1 1.5
14.78** 0.13 0.22
4.74’ 0.28 0.79
15.10** 0.18 0.87
F = F-test: significant. *P < 0.05; **P < 0.01. n = 5 replications per treatment and 5 females plus 5 males per replication. Table 2. Adult mortality and emergence inhibition in A. obrecrus reared on 10% w/w ground fenugreek seed on P. vulgaris beans Seed
Adult mortality (no.) after 4 days Mean
Ripe Unripe Control
6.00 3.30 0
Adult emergence (no.) after 45 days
Range
Mean
Range
4-8 2-4
7.67 5.67 25.00
5-8 38 22-28
n = 3 replications per treatment and IO insects per replication.
A linear regression analysis was performed to relate T. castaneum fecundity with fenugreek seed intake. RESULTS Leaving the material uncovered significantly increased insect mortality (P < 0.05), especially in the presence of Trigonella seeds (Table 1). Female fecundity decreased in the presence of plant material (either leaves or seeds), and this effect was more evident on the third day (P < 0.01) because no females (control or treatments) oviposited during the first 2 days. The solvent acetone did not affect A. obtectus but showed some toxicity to T. castaneum larvae. However, the insecticidal effect of topical application of Trigonella leaf and seed extracts was noticeable on both insects (Fig. 1). Seed extracts were more active than those from leaves against A. obtectus, in spite of being applied at a lower dose (6 pg/insect for seed and 30 pg/insect for leaf extracts), but the reverse applied to T. castaneum. Powdered fenugreek seed or extract on beans produced mortality in the adult of A. obtectus and inhibited larval development, which resulted in decreased adult emergence compared to the controls (Tables 2 and 3). Ripe seed was more active (Table 2), but differences among extracts were unclear (Table 3). Fenugreek seed mixed with the diet in the proportion of 30% after adult emergence produced a decrease of T. castaneum fecundity (Table 4) from 79.9 eggs/female for a period of 5 days on the standard diet to half or a third of this value on the treated diets. When each treatment (ripe and unripe seed) was compared with the control, a very highly significant difference was obtained Table 3. Oviposition and adult emergence inhibition in A. obrecrus by application of fenugreek seed extracts on P. vulgaris beans Extract
H A M Control
Laid eggs (no.) after 4 days
Adult emergence (no.) after 45 days
Mean
Range
Mean
Range
63.3 46.7 44.0 80.0
55-80 20-85 9-80 65-90
4.3 4.7 8.0 22.3
2-8 l-11 l-14 18-25
n = 3 replications per treatment and 5 females plus 5 males per replication. Extracts were obtained sequentially with hexane (H), acetone (A) and 50% methanol (M).
213
Activity of fenugreek against stored product pests
,oo Mortality 4%) I
I
00 -
= Control I A.obtectus IC-T = Control I T.castaneum
=C-A
60.
aL-A IL-T=
40-
= Leaves I A.obtectus
Leaves I
T.castaneum
EX-A
= Seeds / A.obtectus
IS-T
= Seeds / Txastaneum
zo-
0
1
C-A
C-T
L-A
L-T
S-A
S-T
Extracts Fig. 1. Mortality of T. cosraneum larvae (25 days) and A. obrectus adults by topical application of fenugreek leaf (30 fig/insect) and seed (6 pg/insect) acetone extracts (n = IO).
(P < O.OOl), but the same was true for the comparison between ripe and unripe seed. A reduction in fecundity was more pronounced if insects were fed with ripe seed. When young larvae were fed with a diet in which fenugreek seed was mixed in the range O--30%, some mortality was produced during the first 10 days: the lethal dose (LD) calculated by probit method was LD,, = 18% and LD,, = 33.5% for ripe seeds, LD,, = 30% and LD,, = 45.5% For unripe seeds. However, there were always insects completing the cycle, and therefore couples could be paired to test for effects in fecundity. The analysis of variance indicated significant effects of the dose (P < 0.001) but also of seed ripeness and their interaction (P < 0.05) (Table 5). Linear regression analysis of number of eggs laid (per T. cusraneum female over 5 days) with dose of seed was very highly significant (P < 0.001) for both unripe and ripe seed. Figure 2a and b show the fitted regression lines for unripe and ripe seeds, respectively, indicating a negative relation in both cases. Increasing the amount of seed in the diet from egg eclosion produced increasing sterility in adults. Complete sterility was predicted with 26.5% of ripe seed in the diet (Fig. 2b). When adults were always fed with the standard diet (control), the number of eggs laid in 10 days was 75.6 per pair. With 40% fenugreek in the diet, almost complete egg laying inhibition was produced (1.4 eggs/pair). However, this seems to be a reversible effect since the pair recovered some
Table 4. Decrease of T. castaneum fecundity by intake of fenugreek seed mixed in diet (30% w/w) after adult emergence Number of eggs/couple (during 5 days)
Treatments
Standard diet Unripe fenugreek seed Ripe fenugreek seed
Mean zt SE
Variance
Range
CV(%) . ,
U
79.9 f 5.21 44.1 * 3.97 24.7 k 2.52
271.9 157.2 63.6
45-96 29-68 12-38
22.62 28.43 32.28
g*** 0***
U = Mann-Whitney U-test for comparisons (n = 10) with the standard diet: ***significant at P < 0.001 (comparison between ripe and unripe seed U = 6.5***).
Table 5. Mean fecundity (number of eggs/pair over 5 days) in T. casfaneum fed on a diet mixed with fenugreek seed at different doses Dose of fenugreek seed in diet (%)
Seed
Unripe Rioe
F SED
0
5
62.3 62.3
32.0 45.0
IO 28.4 27.3
I5
20
25
30
26.8 18.4
20.8 6.0
11.8 2.8
9.4 I .2
Seed ripeness
Dose
Ripeness x dose
6.97* I .906
52.97*** 3.876
3.10* 5.48 1
F = F-test: significant at *O.OI c P < 0.05, ***P -C0.001. SED = standard error of difference for mean comparison by t-test. n = 5-10 pairs per treatment.
214
J. Pemonge ef al.
0
5
10
15
20
25
J 30
Dose of unripe seed in diet (%)
-20 ’ o
5
10
15
20
25
I 30
Dose of ripe seed in diet (%) Fig. 2. Linear regression of number of eggs laid per T. custaneum female (during 5 days) on the dose of T. foenum-graecum seed in the diet since egg eclosion. (a) unripe seed y = (- 1.37 k 0.186)x + (47.3 f 3.52); (b) ripe seed y = ( - 1.98 f 0.158)x + (52.54 f 3.0).
fecundity on reversion to the standard diet (24.2 eggs). The inhibition of oviposition was a consequence of seed intake in either or both males or females (Table 6). Extracts of low- to mid-polarity incorporated into the diet (5%) generally did not produce a high mortality, but did inhibit growth (Table 7).
Table 6. Mean fertility (number of eggs in a IO-day period) in T. custaneum pairs in which one or both Sexes were fed with fenugreek seed (40% w/w) 10 days after adult emergence and before pairing OK, with reversion or not to standard diet Pair: 0 x 6 Diet after oairinn off With 40% w/w reversion F SED
No x No
No x Yes
Yes x No
Yes x Yes
56.8 75.6
18.0 38.8
14.4 38.4
1.4 24.2
Pair
Diet after pairing off
263.69*** 1.999
233.57*** 1.413
F = F-test: significant at **+P < 0.001, non-significant at P > 0.05. SED = standard error of difference for mean comparison by r-test. n = 5 replications per treatment.
Pair x Diet 0.65 ns 2.827
Activity of fenugreek against stored product pests
215
Table 7. Larval growth inhibition and mortality, after 10 days, of T. custaneum by intake of ripe (r) or unripe (u) fenugreek seed (s) or cake (c) extracts at 5% in diet Treatments
Larval length
Origin
ripeness
material
extract
Mortality (%)
(mm)
Iran Iran Iran Iran Iran Iran Iran Iran India India India India
r r u
S
H A H A H A H A H A H A Control
10 0 0 0 60 20 60 0 0 0 40 0 0
2.7 + 0.71 3.1 * 0.21 3.2 + 0.25 3.5 & 0.16 3.0 + 0.44 2.7 + 0.22 3.0 f. 0.35 2.8 f 0.14 2.7 f 0.23 2.7 f 0.11 3.7 * 0.47 2.9 + 0.15 4.2 + 0.16
S S
U
S
r r u
c
U
C
r r r r
S
C C
S C
c
T *** *** ** ** *** *** *** *** ***
*** ***
Extracts were obtained sequentially with hexane (H) and acetone (A). T = t-test: significant **PC 0.01 and ***P
DISCUSSION
T. foenum-graecum leaves, seeds and extracts showed insecticidal effects on several targets. The presence of leaves or seeds in a closed space and volume arena induced A. obtectus adult mortality with a 2-day delayed effect and also a reduction in oviposition, which may have been due to the odour or colour of the plant. Topical application of plant extracts also produced mortality in the adult and decreased female fecundity. Fenugreek seed extracts are known to have contact activity. Afifi et al. (1988) reported that acetone seed extracts of T. foenum-graecum seeds were toxic to the stored grain pests Sitophilus oryzae (L.) and Rhyzopertha dominica (F.) at 500 ppm. Also, Afifi and Hafez (1988) obtained 100% mortality of the mite Tyrophagus putrescentiae (Schrank) at 100 ppm on flour. A. obtectus is an insect very well adapted to its host-plant, P. vulgaris, but it can also grow in other non-host Leguminosae plants (Hamraoui and Regnault-Roger, 1995). In a current experiment to see whether the beetle could adapt to fenugreek seeds, no larval development was observed. Powdering ground fenugreek seed or applying plant extracts to P. uulgaris seeds produced more rapid adult mortality, and larvae could not penetrate the seeds. Roche and Simanca (1987) reduced the emergence of bruchids by treating stored grains with sunflower oil. Plant extracts can form a film covering the surface of the pulse. This causes a reduction in respiration and gas exchange between the seeds and their environment. The toxicity of compounds penetrating the seed or the accumulation of the toxic metabolites (Don Pedro, 1989) also kills insects. The structure of bruchid eggs could also contribute to the ovicidal effect of the extracts (Credland, 1992). A. obtectus could be controlled by such extracts if the final use of the beans is not for food. Fenugreek was also toxic to T. casfaneum. A contact effect occurred and also antifeedancy, which interrupted larval development. The suppression of reproductive activity caused by T. foenum-graecum seed seems to affect both males and females. However, we have not tested whether a full recovery of fecundity is possible if insects are fed with untreated seed soon after egg eclosion or if an alteration in the reproductive system may cause irreversible sterility. T. foenum-graecum contains some antinutritional factors such as saponins and phytic acid (Singh et al., 1994). Bohannon et al. (1974) reported the presence of steroidal sapogenins, mainly diosgenin, in T. foenum-graecum, at a maximum concentration of 0.2-0.8% in one variety. Trigonella is not the only genus containing diosgenin. Mangla and Kamal (1989) studied the concentration of steroidal sapogenins in berries of Solanum pseudocapsicum L. at different stages of growth and stated that in ripe fruits (in a comparison of unripe or overripe), diosgenin was present in the highest amount. According to Dawson (1991), Dioscorea deltoidea was planted to produce rhizomes rich in the compound, but plantations were soon abandoned because of the need
216
J. Pemonge et al.
for an abundant water supply and the decrease of diosgenin if the rhizome yield was increased. Kamal et al. (1987) considered that cultivation of fenugreek was more feasible than Dioscorea for diosgenin production, and there is variation in diosgenin content among genotypes giving good prospects for breeding more productive strains (Jain and Agrawal, 1987). Saponin extracts have shown a range of activities, among them haemolytic activity (Khalid and El-Adawy, 1994). With diosgenin, Aradhava and Kale (1992) observed oestrogenic action in rats. Diosgenin is a precursor for the manufacture of corticosteroids and steroid hormones and has been used to produce oral contraceptives (Djerassi, 1992). In addition to these applications, saponins play a role in plant-insect relationships, e.g. some saponin glycosides exert a deterrent activity on insects and disturb their feeding (Jain and Tripathi, 1991), providing protection against pests. Our results indicate that they also may produce insecticidal effects, but further experiments would be necessary to establish the activity of each component of the extract. CONCLUSION The use of plant allelochemicals is now recognized as a promising alternative method for an integrated approach to pest management. T. foenum-gruecum has shown considerable insecticidal activity: extracts act directly on adults by contact, and powdered seeds inhibit reproduction by decreasing fecundity and exerting a larvicidal effect in A. obtectus and T. castaneum. Consequently, the plant could be considered as a new source of stored product pest control. Acknowledgemenrs-This research has been part of a Spanish project funded by INIA (SC94039). We also thank the Leonardo da Vinci (E.U.) program, which has financially supported a 6-month scholarship.
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