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Natural seed coats provide protection against penetration by Callosobruchus maculatus (Coleoptera: Bruchidae) larvae
Crop Protection 30 (2011) 651e657
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Crop Protection journal homepage: www.elsevier.com/locate/cropro
Natural seed coats provide protection against penetration by Callosobruchus maculatus (Coleoptera: Bruchidae) larvae Amanda J. Souza a,1, Patrícia O. Santos a,1, Marcio S.T. Pinto a,1, Tierry T. Wermelinger a, Elane S. Ribeiro a, Simone C. Souza a, Mariana F. Deus a, Maria C. Souza b, José Xavier-Filho a, Kátia V.S. Fernandes a, Antonia Elenir A. Oliveira a, * a
Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, P5 sala 224, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil Colégio Estadual Nelson Pereira Rebel, Campos dos Goytacazes, RJ, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2010 Received in revised form 30 November 2010 Accepted 15 December 2010
The seed coat is the first host or non-host tissue contacted by bruchids, suggesting its participation in the evolutionary adaptation of bruchids to favor legume seeds. In the present work, we studied the influence of seed coat on the ability of Callosobruchus maculatus (Coleoptera: Bruchidae F.) larvae to penetrate, develop in and survive on seeds. Our results showed that the oviposition, larval eclosion and adult emergence of C. maculatus were drastically reduced in some seeds and that the time necessary for the surviving larvae to perforate the seed coat increased by up to 100% in these seeds. The surviving larvae that crossed the Phaseolus vulgaris (Leguminosae B.) seed coat reached only 55.6% of the mass of a normal larva. The seed coat of some seeds was very toxic to insect larval development. Despite individual variations, seed coats were generally highly restrictive to the development and survival of the bruchid. The study of the seed coat efficiency as a protection tissue against penetration of insects can provide an important tool for new strategies for crop protection. The strengthening of the seed coat defense mechanisms may represent an efficient strategy because the seed chemical defense barrier would be moved to the outer structures and damage to the embryo would be minimized or avoided. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Planteinsect interactions Seed defense mechanism Bruchid insect Seed coat
1. Introduction Several members of the seed-eating Bruchidae family are major pests afflicting cultivated legumes because they develop within and consume the seed tissues of crops that would otherwise be used for human consumption (Southgate, 1979). Among these insect species, Callosobruchus maculatus (F.) is of considerable importance as a cosmopolitan pest of stored cowpea Vigna unguiculata (L.) (Jackai and Daoust, 1986). The bruchid attacks seeds both in the field and during storage. On cowpea (V. unguiculata) host seeds, the oviposition and egg hatching phases of C. maculatus are completed in about 6 days, eclosion occurs within the seed, and adult beetles emerge some 25e30 days after oviposition. Larval development and pupation take place inside the seed, and adults emerge from the seed; both of these processes cause significant losses in seed weight, germination viability and marketability (Caswell, 1968; Southgate,
* Corresponding author. Tel.: þ55 (22) 2739 7132. E-mail address: [email protected] (A.E.A. Oliveira). 1 These authors contributed equally to the execution of this article. 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.12.014
1979; Oliveira et al., 2001). Before the C. maculatus larva reaches the cotyledons, it has to cross the seed coat, which may represent a critical event in the infestation of non-host seeds due to the physical characteristics and toxicity of this tissue (Oliveira et al., 1999, 2001). The seed coat consists of several layers of specialized maternal cell types that provide an important interface between the embryo and the external environment during embryogenesis, dormancy and germination (Haughn and Chaudhury, 2005). Among the major functions of the seed coat are preservation of the integrity of seed parts, regulation of aqueous and gaseous exchanges between the embryo and the external environment, dispersion of some seeds and protection of the embryo against mechanical damage and the attacks of pests and pathogens (Zeng et al., 2004). The seed coat is the first host or non-host species tissue contacted by bruchids (Oliveira et al., 1999), but the participation of the seed coat in seed resistance against insect penetration has not received enough attention, considering that the coat is the first natural barrier encountered by pests and pathogens. In this work, we studied the behavior of C. maculatus larvae during the perforation of the seed coat and investigated the toxicity of the seed coats of cultivated and native legume species to this insect.
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2. Materials and methods 2.1. Seeds Phaseolus vulgaris (Leguminosae L.) (butter, red and red butter cultivars) and V. unguiculata (Leguminosae L.) (cv. fradinho) seeds were commercially obtained from local markets (Campos dos Goytacazes, RJ, Brazil). Adenanthera pavonina (Leguminosae L.), Bauhinia variegate (Leguminosae L.), Peltogyne gracilipes (Leguminosae D.), Clitoria fairchildiana (Leguminosae H.), Galactia latisiliqua (Leguminosae D.), Macroptilium bracteatum (Leguminosae Nees & Mart), Sesbania virgata (Leguminosae C.), Tephrosia adunca (Leguminosae B.) and Vigna vexillata (Leguminosae L.) seeds were collected on the Campus of the Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil. Dioclea altissima (Leguminosae V.) and Caesalpinia ferrea (Leguminosae M.) seeds were obtained from the Empresa de Pesquisa Agropecuária do Rio de Janeiro, Campos dos Goytacazes, RJ, Brazil. Canavalia ensiformis (Leguminosae L.) and Vigna angularis (Leguminosae W.) seeds were obtained from the seed bank of the Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil. 2.2. Insects C. maculatus (Coleoptera: Bruchidae F.) was obtained from a colony maintained in the Laboratório de Química e Função de Proteínas e Peptídeos from the Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil. The insects were reared on cowpea seeds (cv. Fradinho) at 28 C, 60e80% r.h., with a photoperiod of L12:D12. 2.3. Natural seed experiments Natural seeds were placed into glass flasks (size 12 6 cm) closed with perforated plastic lids for oxygenation. We used sixty seeds for flask of V. unguiculata (cv. fradinho), C. ensiformis, P. vulgaris (butter, red and red butter cultivars), V. angularis, V. vexilata and ten seeds for flask of D. altissima. Each flask was exposed to ten C. maculatus females (3 days old) over 24 h, inside a BOD incubator chamber at 28 C and 70% r.h. After this time, the females were removed, and the eggs laid on the seeds were counted for determination of oviposition ratio. After 8 days of incubation at 28 C, 70% r.h., larval eclosion was observed and registered, and the seeds were incubated for an additional 32 days, under the same conditions. Adult emergence was evaluated after the 40-day period. Larval development was tracked by daily observations with a stereoscopic microscope coupled to a digital CCD video camera. The necessary time (days) for the surviving larvae to perforate the seed coat completely was also recorded. Control experiments using cowpea host seeds were performed under the same conditions described above. The results were analyzed by Student’s t test, and significance was set at P < 0.05 (Bridge and Sawilowsky, 1999). 2.4. Artificial cowpea seeds covered with natural seed coats Seed coat and cotyledons from seeds are easily separated by manual testa peeling since there are no tissue adhesive connections among them. The effectiveness of seed coat pieces as barriers against the penetration of C. maculatus larvae was studied by using an artificial seed system (Macedo et al., 1993) modified to include a covering made of natural soybean seed coat. Artificial seeds (final mass 400 mg) were made using a finely ground decorticated cowpea seed meal from an insect-susceptible V. unguiculata genotype (cv. fradinho). Firstly 50 mg of the V. unguiculata flour was placed into a cylindrical brass mold, and
over that, powder was placed a piece of natural seed coat with the external part of the seed coat facing down. Next, 400 mg of V. unguiculata flour were added over the seed coat piece. The artificial seed was made by pressing the mixture together with the help of a hand press. After removal of the seed from the mold, the excess V. unguiculata flour (50 mg) was removed from the outer surface to expose the piece of seed coat. The parts of the artificial seed that were not covered by the seed coat were protected with a plastic film (parafilm M Laboratory filmePechiney plastic), and the seeds were presented to fertile females (3 days old) in glass flasks as previously described in Section 2.3. Excess eggs were removed so that each seed retained three eggs laid upon it. After 8 days of incubation at 28 C and 70% r.h., larval eclosion was observed and recorded. After the completion of 20 days of incubation, larvae that crossed the seed coat or died inside the eggs were counted. The seeds covered with P. vulgaris natural seed coats were opened, and the weights of all larvae were taken and compared with those of larvae grown in control artificial cowpea seeds. Experiments were run in triplicate (for a total of 9 eggs). These 9 eggs laid on seed coats were considered the standard of normalization when calculating the percentages of larval eclosion, larva that crossed the seed coat and larvae that died inside the eggs. Control seeds consisted of artificial cowpea seeds covered by a cowpea seed coat fragment, manufactured in a process identical to that described above. 2.5. Artificial cowpea seeds containing seed coat flour Seed coats were separated from cotyledons, ground to flour and added to the cowpea meal in concentrations of 1, 2, 4, 8 and 16%. These mixtures were used to create artificial seeds as described in Section 2.4 (Macedo et al., 1993). After 20 days, infested seeds were opened, and the weight and the number of larvae were recorded. Control artificial seeds were composed exclusively of V. unguiculata (cv. fradinho) seed meal. Experiments consisted of 3 seeds per assay and were run in triplicate (for a total of 9 seeds and 27 eggs per tested dose). Dose-response curves were drawn using the number and the average weight of the surviving larvae found for each tested dose, compared with larvae grown in the control artificial seeds. The results were analyzed with Student’s t test, and significance was set at P < 0.05 (Bridge and Sawilowsky, 1999). The doseresponse curve values were used to calculate the dose that reduced larval weight to 50% (the WD50), the dose that reduced the number of surviving larvae by 50% (the LD50) and the lethal dose (LD). 3. Results 3.1. Performance and survival of C. maculatus on natural seeds The survival of C. maculatus on natural non-host seeds was evaluated and compared with insect development in the control V. unguiculata (cv. fradinho) seeds (Fig. 1). The number of eggs laid in V. unguiculata seeds was 200, and this number (considered 100% oviposition) was used to calculate the percentage of reduction in oviposition for the other seeds. Female oviposition, larval eclosion and adult emergence were drastically reduced in some seeds when compared with the host seed. In V. unguiculata seeds (positive control), the females laid a total of 200 eggs; 6.5% of the eggs did not eclose, and 14% of the adult insects failed to emerge from the seed after 40 days. In other cultivars, the reduction varied from 52.5% in P. vulgaris (cv. butter) to 96.5% in P. vulgaris (cv. red) (Fig. 1A). The rates of larval eclosion and adult emergence were also affected. In C. ensiformis and D. altissima, larval eclosion was not observed (a 100% reduction). In other seeds, the reduction in larval eclosion varied from 16% in V. angularis to 68% in P. vulgaris (cv. red better). Twenty-five days after oviposition, adult emergence from
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Fig. 1. A: Performance and survival of Callosobruchus maculatus on natural seeds. a: Vigna unguiculata cv. fradinho (control experiments); b: Canavalia ensiformis; c: Phaseolus vulgaris (cv. butter); d: Phaseolus vulgaris (cv. red butter); e: Phaseolus vulgaris (cv. red); f: Vigna angularis, g: Dioclea altissima and h: Vigna vexilata. Data refer to the infestation of sixty seeds from each species by ten C. maculatus females. B: Time (days) requires by the surviving larvae to perforate the seed coats completely. The data represent the mean over twelve larvae per experiment. *Indicates those results statistically different from the control (V. unguiculata) (P < 0.05 by Student’s t test).
Fig. 2. Development of Callosobruchus maculatus larvae on natural seeds of Vigna unguiculata (EPACE 10 cv) from the first day of oviposition to the sixth day of development. A: Egg on the first day; B: Egg on the fifth day; C: Interior surface of the egg (fifth day); D: Egg on the sixth day; E: Perforation crossing the seed coat (sixth day); F: Perforation in the cotyledon (sixth day). G: Arrow showing the larva penetrating the cotyledon (sixth day); bars ¼ 0.5 mm. The bar in panel A is equal to those in panels BeD and G.
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Fig. 3. Development of Callosobruchus maculatus larvae on natural seeds of Phaseolus vulgaris (butter cv) from the second day of oviposition to the eleventh day of development. A: Egg on the second day; B: Egg on the fifth day; C: Eggs on the sixth day; D: Eggs on the seventh day; E: Eleventh day, arrow showing the perforation of the seed coat; F: Interior surface of the egg (eleventh day), arrow showing the perforation. Bars ¼ 0.5 mm. The bar in panel F is equal to those in panels AeE.
the host seed was observed (data not shown), and 40 days after oviposition, 86% of the insects developed on host seeds had emerged. In contrast to these figures, almost no adult insects emerged from tested seeds 40 days after oviposition, and most of the larvae were dead inside the eggs or on the surface of the cotyledons (data not shown). In C. ensiformis seeds, although a reduction in female oviposition was not observed, larval eclosion was reduced 100%. Consequently, even by 40 days there was no adult emergence from these seeds (Fig. 1A). 3.2. Influence of the seed coat on the survival of neonate larvae and their ability to perforate the seed Our results showed that in the host V. unguiculata seeds (positive control) the larvae had already completely crossed the seed coat and reached the cotyledons by the sixth day after oviposition. In other seeds the time taken to reach the cotyledon varied from 9 to 12 days (Fig. 1B), showing increases of up to 100%. Different behaviors of C. maculatus neonate larvae on the seed coat of their natural host, V. unguiculata, compared to that on a non-host seed coat were observed. At the first day after oviposition, the egg laid on the V. unguiculata seed coat is transparent (Fig. 2A). On the fifth day, the formation of the larva was apparent (Fig. 2B); on the interior surface of the egg on the fifth day, the perforation through which the larva bored into the surface of the seed toward the cotyledons becomes visible (Fig. 2C). On the sixth day, we observed flour inside the egg, giving it a completely white aspect (Fig. 2D), the seed coat was completely perforated (Fig. 2E), the larva was partially inside of the cotyledon (Fig. 2G), and it had also perforated the cotyledon (Fig. 2F). The behavior of neonate larvae on non-host seeds was altered for all studied seed coats. On natural P. vulgaris seed coats, we observed similar development in all studied cultivars (butter, red and red butter), and we illustrated the results for the butter cultivar
(Fig. 3). The eggs showed anomalies that were already visible after the second day of oviposition, consisting of alterations in the appearance of the egg content (Fig. 3A and B). Some surviving neonate larvae were apparently formed inside the egg at the sixth and seventh days (Fig. 3C and D). At the eleventh day, the complete perforation of the seed coat was observed (Fig. 3E), but the larva was still inside of the egg (Fig. 3F). Alterations were detected in the development of neonate larvae at the second and third days on V. vexilata (Fig. 4A and B); however, for these seeds, flour was observed inside some eggs at the fifth day after oviposition (Fig. 4C), indicating that the larva began to bore through into the seed cotyledon. Only at the tenth day after oviposition, the complete perforation of the seed coat was observed and the larva was partially inside the cotyledon (Fig. 4D). Similar alterations were detected in the development of neonate larvae on V. angularis seed coats (data not shown). At the first (Fig. 5A) and second days after oviposition on the P. lunatus seed coat, we observed spots of dark coloration inside some eggs (Fig. 5B). At the sixth day, some larvae appeared to have formed (Fig. 5C), and at the seventh day, flour was observed inside the egg, giving it a whitish appearance (Fig. 5D). The seed coat was completely perforated only on the twelfth day after oviposition (Fig. 5E), and the larva was observed to be partially inside the cotyledon (Fig. 5F). The eggs oviposited on the C. ensiformis seed coat were transparent (Fig. 6A), but by the second day, the majority of the eggs were withered (Fig. 6B). Some surviving neonate larvae were apparently formed by the seventh day (Fig. 6C). These larvae stayed alive until the sixteenth day after oviposition (Fig. 6D) but were unable to perforate the seed coat (Fig. 6E). Artificial seeds covered with natural seed coats (Fig. 7) confirmed the negative interference of seed coats in larval development. All seed coats reduced the number of eclosed larvae and the number of instances of complete perforation of the seed coat (Fig. 7A). The
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Fig. 4. Development of Callosobruchus maculatus larvae on natural seeds of Vigna vexilata from the second day of oviposition to the tenth day of development. A: Egg on the second day; B: Eggs on the third day; C: Egg on the fifth day; D: Tenth day, arrow showing the larva penetrating the cotyledon. Bar ¼ 0.5 mm. The bar in panel D is equal to those in panels AeC.
percentage of larvae that died inside the eggs varied from 25% for eggs oviposited onto P. vulgaris (cv. butter) seed coats to 100% for those oviposited onto D. altissima and C. ensiformis. About 62.5% of the larvae that survived in experiments with P. vulgaris (cv. red) seed coats succeeded in completely perforating the seed coat; however, after 20 days of development, these larvae had only about 55.6% (5.9 mg) of the mass of larvae developed in artificial seeds covered with V. unguiculata natural seed coats (Fig. 7B and B0 ).
3.3. Toxicity of the seed coat flour to larvae Toxicity experiments showed that flour from all non-host seeds interfered with normal development of the C. maculatus larvae, reducing the weight and survival of the 20 day old larvae considerably. Toxicity levels varied from cultivar to cultivar and were dosedependent. The values of the WD50 (the dose that reduced larval weight by 50%), the LD50 (the dose that reduced the number of
Fig. 5. Development of Callosobruchus maculatus larvae on natural seeds of Phaseolus lunatus from the first day of oviposition to the twelfth day of development. A: Egg on the first day; B: Egg on the fourth day; C: Egg on the sixth day; D: Egg on the seventh day; E: Twelfth day, arrow showing the perforation of the seed coat. F: twelfth day, arrow showing the larva penetrating the cotyledon Bar ¼ 0.5 mm. The bar in panel A is equal to those in panels BeD and F.
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Fig. 6. Development of Callosobruchus maculatus larvae on natural seeds of Canavalia ensiformis from the first day of oviposition to the sixteenth day of development. A: Egg on the first day; B: Egg on the second day; C: Egg on the seventh day; D: Interior surface of the egg (sixteenth day); E: Sixteenth day, surface of the seed coat. Bar ¼ 0.5 mm. The bar in panel E is equal to those in panels AeD.
surviving larvae by 50%) and the LD (lethal dose) were calculated, and the results are shown in Table 1. The smallest values of WD50 were found for the seed coats of T. adunca (0.12%), S. virgata (1.9%), C. ferrea (2.0%) and D. altissima (2.0%). Thus, these seed coats were the most efficient at reducing larval mass. The seed coat flours of
A. pavonina and P. gracilipes showed the highest WD50 values (9% and 8.4%, respectively), even though they were highly toxic to the bruchids when compared to control host seed coats. The lowest LD50 and LD values were found for T. adunca (0.15% and 0.4%, respectively) and S. virgata (2.3% and 4%, respectively) seed coats. Although great variation in the WD50, LD50 and LD values has been observed, all seed coats may be considered toxic to C. maculatus, as during insect development in natural seeds, the larvae that first enter into contact with seed coat tissue are continuously exposed to it and rely on a diet that is entirely composed of compounds from this tissue. 4. Discussion The seed coat is the first host or non-host tissue contacted by bruchids (Oliveira et al., 1999), suggesting its participation in the evolutionary adaptation of bruchids to favor legume seeds. Table 1 Toxicity of the seed coat flour on Callosobruchus maculatus larval development. The toxicity was expressed through the percentage values of the WD50 (the dose that reduced larval weight by 50%), the LD50 (the dose that reduced the number of surviving larvae by 50%) and the LD (lethal dose).
Cultivated seeds
Wild seeds
Fig. 7. A: Oviposition and performance of Callosobruchus maculatus larvae on artificial seeds covered with natural seed coats. B: Mean mass of Callosobruchus maculatus larva that survived on artificial seeds covered with the natural seed coats of Vigna unguiculata cv. fradinho (control larvae) or Phaseolus vulgaris (cv. red). Experiments were done in triplicate and the data shown are the mean of these results. B0 : Picture of artificial seeds covered with natural V. unguiculata seed coats. Bar ¼ 1 cm. *Indicates those results statistically different from the control (V. unguiculata) (P < 0.05 by Student’s t test).
Seed coat
Values (%) WD50
LD50
LD
Phaseolus vulgaris (butter cv.) Phaseolus vulgaris (red cv.) Phaseolus vulgaris (red butter cv.) Caesalpinia ferrea Dioclea altissima Vigna vexillata Tephrosia adunca Sesbania virgata Macroptilium bracteatum Galactia latisiliqua Clitoria fairchildiana Peltogyne gracilipes Bauhinia variegata Adenanthera pavonina
4 3 4 2 2 4.5 0.12 1.9 4.3 4.1 3.3 8.4 3 9
4 2 4 6 4 7.1 0.15 2.3 8.9 6.6 6.8 9.5 9 9
15 12 8 12 10 8 0.4 4 10 8 8 14 10 12
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Artificial seed coats have been used for crop protection. The use of chitosan (a derivative of chitin) in formulations that include TMTD (a fungicide) as an artificial seed coating has been documented as an efficient strategy to prevent fungal colonization (Goosen Mattheus, 1997; Ziani et al., 2010). The results obtained in this work showed that the C. maculatus female oviposition, larval eclosion and adult emergence were drastically reduced in some non-host seeds. The time necessary for the surviving larvae to perforate the seed coat completely was also affected, showing increases of up to 100%. Janzen (1977) analyzed the perforation capacity of C. maculatus larvae in seeds of 73 species and showed that the seed coat prevented the penetration of larvae in 69.5% of the studied seeds: There was no direct relationship between hardness and/or thickness of the seed coat and the ability of the larva to cross this tissue. The study showed high larval mortality rates during the perforation of the seed coats of Erythrina berteroana (Leguminosae D.) and Ormosia venezolana (Leguminosae R) seeds (Janzen, 1977). Results obtained with P. lunatus seeds showed that Acanthoscelides obtectus (Coleoptera: Bruchidae D.) larvae were unable to penetrate the seed coat. The reasons for this are probably not physical, as the seed coat of P. lunatus is not as hard as those of the other species of Phaseolus (Thiéry, 1984). Thiéry et al. (1994) showed that the survival of A. obtectus larvae depends on the ability of the first instar to bore through the seed coat of P. vulgaris and that only 57% of the larvae successfully penetrated the seeds. Previous work has shown the efficiency of seed coats in preventing insect penetration. The seed coats of the fourteen V. faba genotypes acted as barriers to the penetration of two bruchid species, C. chinensis (L.) and C. maculatus. Only 45e58% of the neonate larvae perforated the seed coat and reached the cotyledons, whereas the other larvae began to perforate the seed coat and died while trying to penetrate it (Boughdad et al., 1986; Desroches et al., 1995). Our results showed that the surviving larvae that crossed the P. vulgaris seed coat reached only 55.6% of the mass of a larva that crossed the seed coat of the host seed (V. unguiculata). When the seed coat flours of non-host seeds were added to the insect artificial diet we observed that some were very toxic to C. maculatus larval development. These results indicate the presence of toxic compounds in seed coat tissues of non-host seeds. We have shown previously that vicilin-like 7S storage proteins are present in the seed coats of C. ensiformis (Oliveira et al., 1999), P. lunatus (Moraes et al., 2000) and P. vulgaris (Silva et al., 2004). These proteins are toxic to C. maculatus larvae. Oliveira et al. (2001) isolated a water-soluble polysaccharide from the C. ensiformis seed coat that was shown to be highly detrimental to C. maculatus larval development. Understanding the natural defense mechanisms used by plants to adversely affect the beetle could be useful to the development of strategies for protecting other C. maculatus-susceptible legumes. Although an enormous number of toxic proteins and peptides found in seed cotyledons have been related to the resistance of some seeds against insects, almost no work has considered the toxicity of the seed coat as an important factor for such resistance, despite the knowledge that this tissue is the first natural barrier encountered by pests of stored seeds. We suggest that the components of the seed coat may be important to the avoidance of these seeds by C. maculatus. We believe that the expression of these detrimental compounds in the seed coats of non-host seeds may
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have been important for the evolutionary development of discrimination among various legume seeds by this bruchid and could represent an efficient defense mechanism that prevents the arrival of insects to seed cotyledons and favors the integrity and viability of the embryo. Thus, manipulation of seed coat toxicity through genetic engineering might be a promising component of crop protection strategies, preventing insect penetration in seed cotyledons and consequently minimizing losses of seed production and food quality problems.
Acknowledgments This work was supported by grants from the Brazilian agencies FAPERJ, CNPq, and the Universidade Estadual do Norte Fluminense Darcy Ribeiro.
References Boughdad, A., Gillon, Y., Gagnepain, C., 1986. Influence du tegument des graines mures de Vicia faba sur le developpement larvaire de Callosobruchus maculatus. Entomol. Exp. Appl. 42, 219e223. Bridge, P.D., Sawilowsky, S.S., 1999. Increasing physicians’ awareness of the impact of statistics on research outcomes: comparative power of the t-test and Wilcoxon rank-sum test in small samples applied research. J. Clin. Epidemiol. 52, 229e235. Caswell, G.H., 1968. The storage of cowpeas in the northern states of Nigeria. Proc. Agric. Soc. Nigeria 5, 4e6. Desroches, P., El Shazly, E., Mandon, N., Duc, G., Huignard, J., 1995. Development of Callosobruchus chinensis (L.) and Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in seeds of Vicia faba L. differing in their tannin, vicine and convicine contents. J. Stored Prod. Res. 31 (1), 83e89. Goosen Mattheus, F.A., 1997. Applications of Chitin and Chitosan. CRC Press LLC, Boca Raton, Florida. Haughn, G., Chaudhury, A., 2005. Genetic analysis of seed coat development in Arabidopsis. Trends Plant Sci. 10, 472e477. Jackai, L.E.N., Daoust, R.A., 1986. Insect pests of cowpea. Annu. Rev. Entomol. 31, 95e119. Janzen, D.H., 1977. How southern cowpea weevil larvae (Bruchidae: Callosobruchus maculatus) die on nonhost seeds. Ecology 58, 921e927. Macedo, M.L.R., Andrade, L.B.S., Moraes, R.A., Xavier-Filho, J., 1993. Vicilin variants and the resistance of cowpea (Vigna unguiculata) seeds to the cowpea weevil (Callosobruchus maculatus). Comp. Biochem. Physiol. 105, 89e94. Moraes, R., Sales, M.P., Pinto, M.S.P., Silva, L.B., Oliveira, A.E.A., Machado, O.L.T., Fernandes, K.V.S., Xavier-Filho, J., 2000. Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus). Braz. J. Med. Biol. Res. 33, 191e198. Oliveira, A.E.A., Sales, M.P., Machado, O.L.T., Fernandes, K.V.S., Xavier-Filho, J., 1999. The toxicity of Jack bean (Canavalia ensiformis) cotyledon and seed coat proteins to the cowpea weevil (Callosobruchus maculatus). Entomol. Exp. Appl. 92, 249e255. Oliveira, A.E.A., Sassaki, G.L., Iacomini, M., Cunha, M., Fernandes, K.V.S., Xavier-Filho, J., 2001. Isolation and characterization of a galactorhamnan polysaccharide from the seed coat of Canavalia ensiformis that is toxic to the cowpea weevil Callosobruchus maculatus. Entomol. Exp. Appl. 101, 225e231. Silva, L.B., Sales, M.P., Oliveira, A.E.A., Machado, O.L.T., Fernandes, K.V.S., XavierFilho, J., 2004. The seed coat of Phaseolus vulgaris interferes with the development of the cowpea weevil [Callosobruchus maculatus (F) (Coleoptera: Bruchidea)]. An. Acad. Bras. Cienc. 76, 57e65. Southgate, B.J., 1979. Biology of the Bruchidae. Annu. Rev. Entomol. 24, 449e473. Thiéry, D., 1984. Hardness of some Fabaceous seed coats in relation to larval penetration by Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). J. Stored Prod. Res. 20, 177e181. Thiéry, D., Jarry, M., Pouzat, J., 1994. To penetrate or not penetrate? A behavioral choice by bean beetle first-instar larvae in response to Phaseolus vulgaris seed surface quality. J. Chem. Ecol. 20, 1867e1875. Zeng, C.L., Wang, J.B., Liu, A.H., Wu, X.M., 2004. Seed coat microsculpturing changes during seed development in diploid and amphidiploid Brassica species. Ann. Bot. 93, 555e566. Ziani, K., Ursúa, B., Maté, J., 2010. Application of bioactive coatings based on chitosan for artichoke seed protection. Crop Prot. 29, 853e859.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Natural seed coats provide protection against penetration by Callosobruchus maculatus (Coleoptera: Bruchidae) larvae Amanda J. Souza a,1, Patrícia O. Santos a,1, Marcio S.T. Pinto a,1, Tierry T. Wermelinger a, Elane S. Ribeiro a, Simone C. Souza a, Mariana F. Deus a, Maria C. Souza b, José Xavier-Filho a, Kátia V.S. Fernandes a, Antonia Elenir A. Oliveira a, * a
Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, P5 sala 224, Parque Califórnia, 28013-602 Campos dos Goytacazes, RJ, Brazil Colégio Estadual Nelson Pereira Rebel, Campos dos Goytacazes, RJ, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2010 Received in revised form 30 November 2010 Accepted 15 December 2010
The seed coat is the first host or non-host tissue contacted by bruchids, suggesting its participation in the evolutionary adaptation of bruchids to favor legume seeds. In the present work, we studied the influence of seed coat on the ability of Callosobruchus maculatus (Coleoptera: Bruchidae F.) larvae to penetrate, develop in and survive on seeds. Our results showed that the oviposition, larval eclosion and adult emergence of C. maculatus were drastically reduced in some seeds and that the time necessary for the surviving larvae to perforate the seed coat increased by up to 100% in these seeds. The surviving larvae that crossed the Phaseolus vulgaris (Leguminosae B.) seed coat reached only 55.6% of the mass of a normal larva. The seed coat of some seeds was very toxic to insect larval development. Despite individual variations, seed coats were generally highly restrictive to the development and survival of the bruchid. The study of the seed coat efficiency as a protection tissue against penetration of insects can provide an important tool for new strategies for crop protection. The strengthening of the seed coat defense mechanisms may represent an efficient strategy because the seed chemical defense barrier would be moved to the outer structures and damage to the embryo would be minimized or avoided. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Planteinsect interactions Seed defense mechanism Bruchid insect Seed coat
1. Introduction Several members of the seed-eating Bruchidae family are major pests afflicting cultivated legumes because they develop within and consume the seed tissues of crops that would otherwise be used for human consumption (Southgate, 1979). Among these insect species, Callosobruchus maculatus (F.) is of considerable importance as a cosmopolitan pest of stored cowpea Vigna unguiculata (L.) (Jackai and Daoust, 1986). The bruchid attacks seeds both in the field and during storage. On cowpea (V. unguiculata) host seeds, the oviposition and egg hatching phases of C. maculatus are completed in about 6 days, eclosion occurs within the seed, and adult beetles emerge some 25e30 days after oviposition. Larval development and pupation take place inside the seed, and adults emerge from the seed; both of these processes cause significant losses in seed weight, germination viability and marketability (Caswell, 1968; Southgate,
* Corresponding author. Tel.: þ55 (22) 2739 7132. E-mail address: [email protected] (A.E.A. Oliveira). 1 These authors contributed equally to the execution of this article. 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.12.014
1979; Oliveira et al., 2001). Before the C. maculatus larva reaches the cotyledons, it has to cross the seed coat, which may represent a critical event in the infestation of non-host seeds due to the physical characteristics and toxicity of this tissue (Oliveira et al., 1999, 2001). The seed coat consists of several layers of specialized maternal cell types that provide an important interface between the embryo and the external environment during embryogenesis, dormancy and germination (Haughn and Chaudhury, 2005). Among the major functions of the seed coat are preservation of the integrity of seed parts, regulation of aqueous and gaseous exchanges between the embryo and the external environment, dispersion of some seeds and protection of the embryo against mechanical damage and the attacks of pests and pathogens (Zeng et al., 2004). The seed coat is the first host or non-host species tissue contacted by bruchids (Oliveira et al., 1999), but the participation of the seed coat in seed resistance against insect penetration has not received enough attention, considering that the coat is the first natural barrier encountered by pests and pathogens. In this work, we studied the behavior of C. maculatus larvae during the perforation of the seed coat and investigated the toxicity of the seed coats of cultivated and native legume species to this insect.
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2. Materials and methods 2.1. Seeds Phaseolus vulgaris (Leguminosae L.) (butter, red and red butter cultivars) and V. unguiculata (Leguminosae L.) (cv. fradinho) seeds were commercially obtained from local markets (Campos dos Goytacazes, RJ, Brazil). Adenanthera pavonina (Leguminosae L.), Bauhinia variegate (Leguminosae L.), Peltogyne gracilipes (Leguminosae D.), Clitoria fairchildiana (Leguminosae H.), Galactia latisiliqua (Leguminosae D.), Macroptilium bracteatum (Leguminosae Nees & Mart), Sesbania virgata (Leguminosae C.), Tephrosia adunca (Leguminosae B.) and Vigna vexillata (Leguminosae L.) seeds were collected on the Campus of the Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil. Dioclea altissima (Leguminosae V.) and Caesalpinia ferrea (Leguminosae M.) seeds were obtained from the Empresa de Pesquisa Agropecuária do Rio de Janeiro, Campos dos Goytacazes, RJ, Brazil. Canavalia ensiformis (Leguminosae L.) and Vigna angularis (Leguminosae W.) seeds were obtained from the seed bank of the Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil. 2.2. Insects C. maculatus (Coleoptera: Bruchidae F.) was obtained from a colony maintained in the Laboratório de Química e Função de Proteínas e Peptídeos from the Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil. The insects were reared on cowpea seeds (cv. Fradinho) at 28 C, 60e80% r.h., with a photoperiod of L12:D12. 2.3. Natural seed experiments Natural seeds were placed into glass flasks (size 12 6 cm) closed with perforated plastic lids for oxygenation. We used sixty seeds for flask of V. unguiculata (cv. fradinho), C. ensiformis, P. vulgaris (butter, red and red butter cultivars), V. angularis, V. vexilata and ten seeds for flask of D. altissima. Each flask was exposed to ten C. maculatus females (3 days old) over 24 h, inside a BOD incubator chamber at 28 C and 70% r.h. After this time, the females were removed, and the eggs laid on the seeds were counted for determination of oviposition ratio. After 8 days of incubation at 28 C, 70% r.h., larval eclosion was observed and registered, and the seeds were incubated for an additional 32 days, under the same conditions. Adult emergence was evaluated after the 40-day period. Larval development was tracked by daily observations with a stereoscopic microscope coupled to a digital CCD video camera. The necessary time (days) for the surviving larvae to perforate the seed coat completely was also recorded. Control experiments using cowpea host seeds were performed under the same conditions described above. The results were analyzed by Student’s t test, and significance was set at P < 0.05 (Bridge and Sawilowsky, 1999). 2.4. Artificial cowpea seeds covered with natural seed coats Seed coat and cotyledons from seeds are easily separated by manual testa peeling since there are no tissue adhesive connections among them. The effectiveness of seed coat pieces as barriers against the penetration of C. maculatus larvae was studied by using an artificial seed system (Macedo et al., 1993) modified to include a covering made of natural soybean seed coat. Artificial seeds (final mass 400 mg) were made using a finely ground decorticated cowpea seed meal from an insect-susceptible V. unguiculata genotype (cv. fradinho). Firstly 50 mg of the V. unguiculata flour was placed into a cylindrical brass mold, and
over that, powder was placed a piece of natural seed coat with the external part of the seed coat facing down. Next, 400 mg of V. unguiculata flour were added over the seed coat piece. The artificial seed was made by pressing the mixture together with the help of a hand press. After removal of the seed from the mold, the excess V. unguiculata flour (50 mg) was removed from the outer surface to expose the piece of seed coat. The parts of the artificial seed that were not covered by the seed coat were protected with a plastic film (parafilm M Laboratory filmePechiney plastic), and the seeds were presented to fertile females (3 days old) in glass flasks as previously described in Section 2.3. Excess eggs were removed so that each seed retained three eggs laid upon it. After 8 days of incubation at 28 C and 70% r.h., larval eclosion was observed and recorded. After the completion of 20 days of incubation, larvae that crossed the seed coat or died inside the eggs were counted. The seeds covered with P. vulgaris natural seed coats were opened, and the weights of all larvae were taken and compared with those of larvae grown in control artificial cowpea seeds. Experiments were run in triplicate (for a total of 9 eggs). These 9 eggs laid on seed coats were considered the standard of normalization when calculating the percentages of larval eclosion, larva that crossed the seed coat and larvae that died inside the eggs. Control seeds consisted of artificial cowpea seeds covered by a cowpea seed coat fragment, manufactured in a process identical to that described above. 2.5. Artificial cowpea seeds containing seed coat flour Seed coats were separated from cotyledons, ground to flour and added to the cowpea meal in concentrations of 1, 2, 4, 8 and 16%. These mixtures were used to create artificial seeds as described in Section 2.4 (Macedo et al., 1993). After 20 days, infested seeds were opened, and the weight and the number of larvae were recorded. Control artificial seeds were composed exclusively of V. unguiculata (cv. fradinho) seed meal. Experiments consisted of 3 seeds per assay and were run in triplicate (for a total of 9 seeds and 27 eggs per tested dose). Dose-response curves were drawn using the number and the average weight of the surviving larvae found for each tested dose, compared with larvae grown in the control artificial seeds. The results were analyzed with Student’s t test, and significance was set at P < 0.05 (Bridge and Sawilowsky, 1999). The doseresponse curve values were used to calculate the dose that reduced larval weight to 50% (the WD50), the dose that reduced the number of surviving larvae by 50% (the LD50) and the lethal dose (LD). 3. Results 3.1. Performance and survival of C. maculatus on natural seeds The survival of C. maculatus on natural non-host seeds was evaluated and compared with insect development in the control V. unguiculata (cv. fradinho) seeds (Fig. 1). The number of eggs laid in V. unguiculata seeds was 200, and this number (considered 100% oviposition) was used to calculate the percentage of reduction in oviposition for the other seeds. Female oviposition, larval eclosion and adult emergence were drastically reduced in some seeds when compared with the host seed. In V. unguiculata seeds (positive control), the females laid a total of 200 eggs; 6.5% of the eggs did not eclose, and 14% of the adult insects failed to emerge from the seed after 40 days. In other cultivars, the reduction varied from 52.5% in P. vulgaris (cv. butter) to 96.5% in P. vulgaris (cv. red) (Fig. 1A). The rates of larval eclosion and adult emergence were also affected. In C. ensiformis and D. altissima, larval eclosion was not observed (a 100% reduction). In other seeds, the reduction in larval eclosion varied from 16% in V. angularis to 68% in P. vulgaris (cv. red better). Twenty-five days after oviposition, adult emergence from
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Fig. 1. A: Performance and survival of Callosobruchus maculatus on natural seeds. a: Vigna unguiculata cv. fradinho (control experiments); b: Canavalia ensiformis; c: Phaseolus vulgaris (cv. butter); d: Phaseolus vulgaris (cv. red butter); e: Phaseolus vulgaris (cv. red); f: Vigna angularis, g: Dioclea altissima and h: Vigna vexilata. Data refer to the infestation of sixty seeds from each species by ten C. maculatus females. B: Time (days) requires by the surviving larvae to perforate the seed coats completely. The data represent the mean over twelve larvae per experiment. *Indicates those results statistically different from the control (V. unguiculata) (P < 0.05 by Student’s t test).
Fig. 2. Development of Callosobruchus maculatus larvae on natural seeds of Vigna unguiculata (EPACE 10 cv) from the first day of oviposition to the sixth day of development. A: Egg on the first day; B: Egg on the fifth day; C: Interior surface of the egg (fifth day); D: Egg on the sixth day; E: Perforation crossing the seed coat (sixth day); F: Perforation in the cotyledon (sixth day). G: Arrow showing the larva penetrating the cotyledon (sixth day); bars ¼ 0.5 mm. The bar in panel A is equal to those in panels BeD and G.
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Fig. 3. Development of Callosobruchus maculatus larvae on natural seeds of Phaseolus vulgaris (butter cv) from the second day of oviposition to the eleventh day of development. A: Egg on the second day; B: Egg on the fifth day; C: Eggs on the sixth day; D: Eggs on the seventh day; E: Eleventh day, arrow showing the perforation of the seed coat; F: Interior surface of the egg (eleventh day), arrow showing the perforation. Bars ¼ 0.5 mm. The bar in panel F is equal to those in panels AeE.
the host seed was observed (data not shown), and 40 days after oviposition, 86% of the insects developed on host seeds had emerged. In contrast to these figures, almost no adult insects emerged from tested seeds 40 days after oviposition, and most of the larvae were dead inside the eggs or on the surface of the cotyledons (data not shown). In C. ensiformis seeds, although a reduction in female oviposition was not observed, larval eclosion was reduced 100%. Consequently, even by 40 days there was no adult emergence from these seeds (Fig. 1A). 3.2. Influence of the seed coat on the survival of neonate larvae and their ability to perforate the seed Our results showed that in the host V. unguiculata seeds (positive control) the larvae had already completely crossed the seed coat and reached the cotyledons by the sixth day after oviposition. In other seeds the time taken to reach the cotyledon varied from 9 to 12 days (Fig. 1B), showing increases of up to 100%. Different behaviors of C. maculatus neonate larvae on the seed coat of their natural host, V. unguiculata, compared to that on a non-host seed coat were observed. At the first day after oviposition, the egg laid on the V. unguiculata seed coat is transparent (Fig. 2A). On the fifth day, the formation of the larva was apparent (Fig. 2B); on the interior surface of the egg on the fifth day, the perforation through which the larva bored into the surface of the seed toward the cotyledons becomes visible (Fig. 2C). On the sixth day, we observed flour inside the egg, giving it a completely white aspect (Fig. 2D), the seed coat was completely perforated (Fig. 2E), the larva was partially inside of the cotyledon (Fig. 2G), and it had also perforated the cotyledon (Fig. 2F). The behavior of neonate larvae on non-host seeds was altered for all studied seed coats. On natural P. vulgaris seed coats, we observed similar development in all studied cultivars (butter, red and red butter), and we illustrated the results for the butter cultivar
(Fig. 3). The eggs showed anomalies that were already visible after the second day of oviposition, consisting of alterations in the appearance of the egg content (Fig. 3A and B). Some surviving neonate larvae were apparently formed inside the egg at the sixth and seventh days (Fig. 3C and D). At the eleventh day, the complete perforation of the seed coat was observed (Fig. 3E), but the larva was still inside of the egg (Fig. 3F). Alterations were detected in the development of neonate larvae at the second and third days on V. vexilata (Fig. 4A and B); however, for these seeds, flour was observed inside some eggs at the fifth day after oviposition (Fig. 4C), indicating that the larva began to bore through into the seed cotyledon. Only at the tenth day after oviposition, the complete perforation of the seed coat was observed and the larva was partially inside the cotyledon (Fig. 4D). Similar alterations were detected in the development of neonate larvae on V. angularis seed coats (data not shown). At the first (Fig. 5A) and second days after oviposition on the P. lunatus seed coat, we observed spots of dark coloration inside some eggs (Fig. 5B). At the sixth day, some larvae appeared to have formed (Fig. 5C), and at the seventh day, flour was observed inside the egg, giving it a whitish appearance (Fig. 5D). The seed coat was completely perforated only on the twelfth day after oviposition (Fig. 5E), and the larva was observed to be partially inside the cotyledon (Fig. 5F). The eggs oviposited on the C. ensiformis seed coat were transparent (Fig. 6A), but by the second day, the majority of the eggs were withered (Fig. 6B). Some surviving neonate larvae were apparently formed by the seventh day (Fig. 6C). These larvae stayed alive until the sixteenth day after oviposition (Fig. 6D) but were unable to perforate the seed coat (Fig. 6E). Artificial seeds covered with natural seed coats (Fig. 7) confirmed the negative interference of seed coats in larval development. All seed coats reduced the number of eclosed larvae and the number of instances of complete perforation of the seed coat (Fig. 7A). The
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Fig. 4. Development of Callosobruchus maculatus larvae on natural seeds of Vigna vexilata from the second day of oviposition to the tenth day of development. A: Egg on the second day; B: Eggs on the third day; C: Egg on the fifth day; D: Tenth day, arrow showing the larva penetrating the cotyledon. Bar ¼ 0.5 mm. The bar in panel D is equal to those in panels AeC.
percentage of larvae that died inside the eggs varied from 25% for eggs oviposited onto P. vulgaris (cv. butter) seed coats to 100% for those oviposited onto D. altissima and C. ensiformis. About 62.5% of the larvae that survived in experiments with P. vulgaris (cv. red) seed coats succeeded in completely perforating the seed coat; however, after 20 days of development, these larvae had only about 55.6% (5.9 mg) of the mass of larvae developed in artificial seeds covered with V. unguiculata natural seed coats (Fig. 7B and B0 ).
3.3. Toxicity of the seed coat flour to larvae Toxicity experiments showed that flour from all non-host seeds interfered with normal development of the C. maculatus larvae, reducing the weight and survival of the 20 day old larvae considerably. Toxicity levels varied from cultivar to cultivar and were dosedependent. The values of the WD50 (the dose that reduced larval weight by 50%), the LD50 (the dose that reduced the number of
Fig. 5. Development of Callosobruchus maculatus larvae on natural seeds of Phaseolus lunatus from the first day of oviposition to the twelfth day of development. A: Egg on the first day; B: Egg on the fourth day; C: Egg on the sixth day; D: Egg on the seventh day; E: Twelfth day, arrow showing the perforation of the seed coat. F: twelfth day, arrow showing the larva penetrating the cotyledon Bar ¼ 0.5 mm. The bar in panel A is equal to those in panels BeD and F.
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Fig. 6. Development of Callosobruchus maculatus larvae on natural seeds of Canavalia ensiformis from the first day of oviposition to the sixteenth day of development. A: Egg on the first day; B: Egg on the second day; C: Egg on the seventh day; D: Interior surface of the egg (sixteenth day); E: Sixteenth day, surface of the seed coat. Bar ¼ 0.5 mm. The bar in panel E is equal to those in panels AeD.
surviving larvae by 50%) and the LD (lethal dose) were calculated, and the results are shown in Table 1. The smallest values of WD50 were found for the seed coats of T. adunca (0.12%), S. virgata (1.9%), C. ferrea (2.0%) and D. altissima (2.0%). Thus, these seed coats were the most efficient at reducing larval mass. The seed coat flours of
A. pavonina and P. gracilipes showed the highest WD50 values (9% and 8.4%, respectively), even though they were highly toxic to the bruchids when compared to control host seed coats. The lowest LD50 and LD values were found for T. adunca (0.15% and 0.4%, respectively) and S. virgata (2.3% and 4%, respectively) seed coats. Although great variation in the WD50, LD50 and LD values has been observed, all seed coats may be considered toxic to C. maculatus, as during insect development in natural seeds, the larvae that first enter into contact with seed coat tissue are continuously exposed to it and rely on a diet that is entirely composed of compounds from this tissue. 4. Discussion The seed coat is the first host or non-host tissue contacted by bruchids (Oliveira et al., 1999), suggesting its participation in the evolutionary adaptation of bruchids to favor legume seeds. Table 1 Toxicity of the seed coat flour on Callosobruchus maculatus larval development. The toxicity was expressed through the percentage values of the WD50 (the dose that reduced larval weight by 50%), the LD50 (the dose that reduced the number of surviving larvae by 50%) and the LD (lethal dose).
Cultivated seeds
Wild seeds
Fig. 7. A: Oviposition and performance of Callosobruchus maculatus larvae on artificial seeds covered with natural seed coats. B: Mean mass of Callosobruchus maculatus larva that survived on artificial seeds covered with the natural seed coats of Vigna unguiculata cv. fradinho (control larvae) or Phaseolus vulgaris (cv. red). Experiments were done in triplicate and the data shown are the mean of these results. B0 : Picture of artificial seeds covered with natural V. unguiculata seed coats. Bar ¼ 1 cm. *Indicates those results statistically different from the control (V. unguiculata) (P < 0.05 by Student’s t test).
Seed coat
Values (%) WD50
LD50
LD
Phaseolus vulgaris (butter cv.) Phaseolus vulgaris (red cv.) Phaseolus vulgaris (red butter cv.) Caesalpinia ferrea Dioclea altissima Vigna vexillata Tephrosia adunca Sesbania virgata Macroptilium bracteatum Galactia latisiliqua Clitoria fairchildiana Peltogyne gracilipes Bauhinia variegata Adenanthera pavonina
4 3 4 2 2 4.5 0.12 1.9 4.3 4.1 3.3 8.4 3 9
4 2 4 6 4 7.1 0.15 2.3 8.9 6.6 6.8 9.5 9 9
15 12 8 12 10 8 0.4 4 10 8 8 14 10 12
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Artificial seed coats have been used for crop protection. The use of chitosan (a derivative of chitin) in formulations that include TMTD (a fungicide) as an artificial seed coating has been documented as an efficient strategy to prevent fungal colonization (Goosen Mattheus, 1997; Ziani et al., 2010). The results obtained in this work showed that the C. maculatus female oviposition, larval eclosion and adult emergence were drastically reduced in some non-host seeds. The time necessary for the surviving larvae to perforate the seed coat completely was also affected, showing increases of up to 100%. Janzen (1977) analyzed the perforation capacity of C. maculatus larvae in seeds of 73 species and showed that the seed coat prevented the penetration of larvae in 69.5% of the studied seeds: There was no direct relationship between hardness and/or thickness of the seed coat and the ability of the larva to cross this tissue. The study showed high larval mortality rates during the perforation of the seed coats of Erythrina berteroana (Leguminosae D.) and Ormosia venezolana (Leguminosae R) seeds (Janzen, 1977). Results obtained with P. lunatus seeds showed that Acanthoscelides obtectus (Coleoptera: Bruchidae D.) larvae were unable to penetrate the seed coat. The reasons for this are probably not physical, as the seed coat of P. lunatus is not as hard as those of the other species of Phaseolus (Thiéry, 1984). Thiéry et al. (1994) showed that the survival of A. obtectus larvae depends on the ability of the first instar to bore through the seed coat of P. vulgaris and that only 57% of the larvae successfully penetrated the seeds. Previous work has shown the efficiency of seed coats in preventing insect penetration. The seed coats of the fourteen V. faba genotypes acted as barriers to the penetration of two bruchid species, C. chinensis (L.) and C. maculatus. Only 45e58% of the neonate larvae perforated the seed coat and reached the cotyledons, whereas the other larvae began to perforate the seed coat and died while trying to penetrate it (Boughdad et al., 1986; Desroches et al., 1995). Our results showed that the surviving larvae that crossed the P. vulgaris seed coat reached only 55.6% of the mass of a larva that crossed the seed coat of the host seed (V. unguiculata). When the seed coat flours of non-host seeds were added to the insect artificial diet we observed that some were very toxic to C. maculatus larval development. These results indicate the presence of toxic compounds in seed coat tissues of non-host seeds. We have shown previously that vicilin-like 7S storage proteins are present in the seed coats of C. ensiformis (Oliveira et al., 1999), P. lunatus (Moraes et al., 2000) and P. vulgaris (Silva et al., 2004). These proteins are toxic to C. maculatus larvae. Oliveira et al. (2001) isolated a water-soluble polysaccharide from the C. ensiformis seed coat that was shown to be highly detrimental to C. maculatus larval development. Understanding the natural defense mechanisms used by plants to adversely affect the beetle could be useful to the development of strategies for protecting other C. maculatus-susceptible legumes. Although an enormous number of toxic proteins and peptides found in seed cotyledons have been related to the resistance of some seeds against insects, almost no work has considered the toxicity of the seed coat as an important factor for such resistance, despite the knowledge that this tissue is the first natural barrier encountered by pests of stored seeds. We suggest that the components of the seed coat may be important to the avoidance of these seeds by C. maculatus. We believe that the expression of these detrimental compounds in the seed coats of non-host seeds may
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have been important for the evolutionary development of discrimination among various legume seeds by this bruchid and could represent an efficient defense mechanism that prevents the arrival of insects to seed cotyledons and favors the integrity and viability of the embryo. Thus, manipulation of seed coat toxicity through genetic engineering might be a promising component of crop protection strategies, preventing insect penetration in seed cotyledons and consequently minimizing losses of seed production and food quality problems.
Acknowledgments This work was supported by grants from the Brazilian agencies FAPERJ, CNPq, and the Universidade Estadual do Norte Fluminense Darcy Ribeiro.
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