- Email: [email protected]
Laboratory evaluation of transgenic Bt rice resistance against rice leaf roller, Cnaphalocrocis medinalis
Journal of Asia-Pacific Entomology 20 (2017) 221–224
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Short Communication
Laboratory evaluation of transgenic Bt rice resistance against rice leaf roller, Cnaphalocrocis medinalis Jong Hoon Kim a, Sue Yeon Lee b, Jae Young Choi b, Seok Hee Lee a, Ying Fang a, Kyu Baik Ha a, Dong Hwan Park a, Min Gu Park a, Ra Mi Woo a, Woo Jin Kim a, Ju-Kon Kim c, Yeon Ho Je a,b,⁎ a b c
Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 08826, Republic of Korea Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Republic of Korea
a r t i c l e
i n f o
Article history: Received 4 October 2016 Revised 16 December 2016 Accepted 20 December 2016 Available online 22 December 2016 Keywords: Bacillus thuringiensis cry1Ac Transgenic rice Cnaphalocrocis medinalis Pest resistance
a b s t r a c t Pest resistance of 8 transgenic Bt rice events with a synthetic cry1Ac gene linked to rice rbcS-tp sequence were assessed under laboratory conditions. Bioassays were conducted with neonates and third instar larvae of Cnaphalocrocis medinalis, which is a significant pest of rice in Asia. C. medinalis larvae were shown to be susceptible to all eight events, even though there were differences between the causes of death. The results differed between developmental stages of the larvae, despite the fact that all 8 events led to high mortalities. Neonates displayed feeding avoidance and death by starvation on six Bt rice events. In the case of third instar larvae however, only two events resulted in feeding avoidance. Nevertheless, all Bt rice events were shown to be highly effective against C. medinalis larvae in laboratory conditions. These results may be a significant foundation for the evaluation of improved transgenic Bt rice. © 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
Introduction Bacillus thuringiensis (Bt) is a gram-positive, sporulating bacterium that produces a variety of insecticidal crystal (Cry) proteins, and widely used as one of the most successful biological control agents (Roh et al., 2007). Several Bt cry genes have been introduced into a variety of crops for protection against pests, starting first with tobacco (Vaeck et al., 1987) and now with major crops such as cotton, maize, potato, rice, broccoli, lettuce, walnut, apple, alfalfa and soybean (Shelton et al., 2002). Recently, studies that introduce cry genes into crops to create pest resistance have made much progress, and the total area of land planted with Bt crops has increased substantially (James, 2014; Tabashnik et al., 2003). Several Bt genes were cloned and successfully transferred into higher plants in the 1980s. However, the insecticidal activities of these transgenic plants were relatively low, mostly due to low protein expression caused by mRNA degradation. Deletion of sequences that are irrelevant to insecticidal activity lead to the creation of synthetic truncated Bt genes, which significantly increased their transgenic expression within plants (Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). To further enhance Bt gene expression in plants, the genes were either inserted into the chloroplast genome (Kota et al., 1999; Liu et al., 2008; McBride et al., 1995), or had their bacterial coding
sequences modified to plant-preferred coding sequences (Perlak et al., 1993; Sharma et al., 2000). The first reported transformation of rice with a Bt gene was in 1993 (Fujimoto et al., 1993), and many other transgenic rice lines have been developed since then. To confer resistance to rice against significant lepidopteran pests such as Chilo suppressalis, Scirpophaga incertulas and Cnaphalocrocis medinalis, Bt genes such as cry1Ac, cry1Ab and cry1Ab/ Ac fusion genes were introduced into rice cultivars (Chen et al., 2008; Khanna and Raina, 2002; Ye et al., 2001). It has been reported that the targeting of foreign gene to the chloroplasts using the small subunit gene of the ribulose biphosphate carboxylase/oxygenase (rbcS) promotor and its transit peptide sequence (tp) drastically increased the foreign gene product levels in transgenic rice (Jang et al., 2002; Jang et al., 1999). Previously, chloroplast-targeted expression has been achieved through the linking of a synthetic truncated cry1Ac gene to the rice rbcS-tp sequence (Kim et al., 2009). In this study, we assessed the pest resistance of these transgenic Bt rice events (RbcS3:Cry1Ac) against C. medinalis, which is a prominent rice pest that is widely distributed over the rice fields of Asia. Materials and methods Insects
⁎ Corresponding author at: Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea. E-mail address: [email protected] (Y.H. Je).
C. medinalis larvae were collected from paddy fields in Taean-gun, Chungcheongnam-do, Korea and reared on acryl cages containing
http://dx.doi.org/10.1016/j.aspen.2016.12.010 1226-8615/© 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
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Fig. 1. Mortalities of neonates of C. medinalis fed on T3 generation Bt rice. Bars with diagonal lines represent mortality caused by starvation due to larval feeding avoidance.
tillering stage of rice plants. The rearing conditions were 25 °C, 45 ± 100% RH, and a photoperiod of 16 h light:8 h dark. Transgenic Bt rices A total of 8 Bt rice events of T3 generations (608102, 608103, 608104, 608106, 608107, 608108, 608110 and 608111), which are linked of a synthetic truncated cry1Ac gene to the rice rbcS-tp sequence for chloroplast-targeted expression, were used in this study (Kim et al., 2009). Also, non-Bt rice (Oryza sativa L. cv. Ilmi) was used as control. These Bt and non-Bt rice seeds were germinated in the greenhouse and grown for 3 weeks, and then seedlings were transplanted to a paddy field in isolated GMO field, Suwon, Korea. Two months after transplantation, Bt rice and non-Bt rice were examined in the laboratory.
608107, 608110 and 608111 showed cessation of feeding (Fig. 2) and 100% mortality due to starvation (Fig. 1). These results were consistent with the previous reports that C. medinalis and Chilo suppressalis larvae avoided feeding on Bt rice (Chen et al., 2008; Zheng et al., 2011). Many studies have shown that larval feeding avoidance occurs against various Bt toxins (Berdegué et al., 1996; Chen et al., 2008; Gore et al., 2005; Gould and Anderson, 1991; Ramachandran et al., 1993). These reports
Bioassay The pest resistance of Bt rice events were determined against the neonates and 3rd instar larvae of C. medinalis. In the case of neonates, leaves of booting stage rice (1.0–1.5 cm wide and 10 cm long) were fixed to pieces of filter paper. The basal sections of filter paper with rice leaves were placed in 60 mm petri-dishes along with moistened cotton. One neonate was placed per petri-dish, and a total of 30 neonates were used for each sample. For 3rd instar larvae, leaves of booting stage rice (1.0–1.5 cm wide and 10 cm long) were wrapped with moistened cotton to prevent withering of the leaves. Each leaf was placed in a plastic vial along with one larva and the vial was covered with a lid to prevent larval escape. A total of 30 larvae were used for each sample. Treated larvae were maintained under rearing conditions and the larval mortality was scored after five days. All assays were performed in triplicates. To investigate feeding avoidance disorder, feeding area was checked quantitatively via microscopic observation. Results and discussion To evaluate the pest resistance of eight Bt rice events under laboratory conditions, bioassays were performed against neonates and 3rd instar. In case of neonates, all Bt rice events showed high level of pest resistance compared to control non-Bt rice (Fig. 1). While cumulative feeding areas of two Bt rice events (608103 and 608108) were similar to that of control non-Bt rice (Fig. 2), C. medinalis larvae fed on these two events showed high level of mortality (Fig. 1). Particularly, C. medinalis larvae fed on Bt rice events 608102, 608104, 608106,
Fig. 2. Feeding behavior of neonates of C. medinalis fed on control non-Bt rice (A) and T3 generation Bt rice 608103 (B). Arrow heads indicate feeding area of C. medinalis larvae.
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Fig. 3. Mortalities of 3rd instar larvae of C. medinalis fed on T3 generation Bt rice. Bars with diagonal lines represent mortality caused by starvation due to larval feeding avoidance.
suggested that the feeding avoidance of C. medinalis larvae observed in this study might be due to the Cry1Ac protein produced in Bt rice events. Third instar larvae of C. medinalis fed on Bt rice events also showed high level of mortalities (Fig. 3). However, feeding avoidance and death by starvation were observed only at larvae fed on two Bt rice events, 608106 and 608111 (Fig. 3). C. medinalis larvae fed on six Bt rice events (608102, 608103, 608104, 608107, 608108 and 608111)
showed death by Cry1Ac protein (Fig. 4). These results suggested that the feeding behavior of C. medinalis larvae could be affected by developmental stage of the insects. Furthermore, growth stage of the Bt rice and differences in Cry1Ac expression level between Bt rice events also could affect the feeding behavior of C. medinalis (Han et al., 2007; Zheng et al., 2011). In this study, we assessed pest resistance of eight Bt rice events against C. medinalis larvae. Among them, two events caused larval feeding avoidance and death by starvation regardless of developmental stage of C. medinalis. These results could be useful in the establishment of more effective Bt rice along generation.
Acknowledgements This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01113502)” Rural Development Administration, Republic of Korea. J. H. Kim, S. H. Lee, Y. Fang, K. B. Ha, D. H. Park, M. G. Park and R. M. Woo were supported by the Brain Korea 21 plus project, Seoul National University, Republic of Korea. References
Fig. 4. Feeding behavior of 3rd instar larvae of C. medinalis fed on control non-Bt rice (A) and T3 generation Bt rice 608103 (B). Arrow heads indicate feeding area of C. medinalis larvae.
Barton, K.A., Whiteley, H., Yang, N.-S., 1987. Bacillus thuringiensis §-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects. Plant Physiol. 85, 1103–1109. Berdegué, M., Trumble, J.T., Moar, W.J., 1996. Effect of CryIC toxin from Bacillus thuringiensis on larval feeding behavior of Spodoptera exigua. Entomol. Exp. Appl. 80, 389–401. Chen, H., Zhang, G., Zhang, Q., Lin, Y., 2008. Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae). J. Econ. Entomol. 101, 182–189. Fischhoff, D.A., Bowdishi, K.S., Perlak, F.J., Marrone, P.G., McCormick, S.M., Niedermeyer, J.G., Dean, D.A., Kusano-Kretzmer, K., Mayer, E.J., Rochester, D.E., 1987. Insect Tolerant Transgenic Tomato Plants. Fujimoto, H., Itoh, K., Yamamoto, M., Kyozuka, J., Shimamoto, K., 1993. Insect resistant rice generated by introduction of a modified δ-endotoxin gene of Bacillus thuringiensis. Nat. Biotechnol. 11, 1151–1155. Gore, J., Adamczyk, J., Blanco, C., 2005. Selective feeding of tobacco budworm and bollworm (Lepidoptera: Noctuidae) on meridic diet with different concentrations of Bacillus thuringiensis proteins. J. Econ. Entomol. 98, 88–94. Gould, F., Anderson, A., 1991. Effects of Bacillus thuringiensis and HD-73 delta-endotoxin on growth, behavior, and fitness of susceptible and toxin-adapted strains of Heliothis virescens (Lepidoptera: Noctuidae). Environ. Entomol. 20, 30–38. Han, L., Wu, K., Peng, Y., Wang, F., Guo, Y., 2007. Efficacy of transgenic rice expressing Cry1Ac and CpTI against the rice leaffolder, Cnaphalocrocis medinalis (Guenee). J. Invertebr. Pathol. 96, 71–79. James, C., 2014. Global Status of Commercialized Biotech/GM Crops: 2014. International Service for the Acquisition of Agri-biotech Applications (ISAAA) Ithaca, NY, USA.
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Jang, I.-C., Nahm, B.H., Kim, J.-K., 1999. Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system. Mol. Breed. 5, 453–461. Jang, I.-C., Lee, K.-H., Nahm, B.H., Kim, J.-K., 2002. Chloroplast targeting signal of a rice rbcS gene enhances transgene expression. Mol. Breed. 9, 81–91. Khanna, H.K., Raina, S.K., 2002. Elite indica transgenic rice plants expressing modified Cry1Ac endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borer (Scirpophaga incertulas). Transgenic Res. 11, 411–423. Kim, E.H., Suh, S.C., Park, B.S., Shin, K.S., Kweon, S.J., Han, E.J., Park, S.-H., Kim, Y.S., Kim, J.K., 2009. Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection. Planta 230, 397–405. Kota, M., Daniell, H., Varma, S., Garczynski, S.F., Gould, F., Moar, W.J., 1999. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc. Natl. Acad. Sci. 96, 1840–1845. Liu, C.-W., Lin, C.-C., Yiu, J.-C., Chen, J.J., Tseng, M.-J., 2008. Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theor. Appl. Genet. 117, 75–88. McBride, K.E., Svab, Z., Schaaf, D.J., Hogan, P.S., Stalker, D.M., Maliga, P., 1995. Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Nat. Biotechnol. 13, 362–365. Perlak, F.J., Stone, T.B., Muskopf, Y.M., Petersen, L.J., Parker, G.B., McPherson, S.A., Wyman, J., Love, S., Reed, G., Biever, D., 1993. Genetically improved potatoes: protection from damage by Colorado potato beetles. Plant Mol. Biol. 22, 313–321.
Ramachandran, R., Raffa, K., Miller, M., Ellis, D., McCown, B., 1993. Behavioral responses and sublethal effects of spruce budworm (Lepidoptera: Tortricidae) and fall webworm (Lepidoptera: Arctiidae) larvae to Bacillus thuringiensis Cry1A (a) toxin in diet. Environ. Entomol. 22, 197–211. Roh, J.Y., Choi, J.Y., Li, M.S., Jin, B.R., Je, Y.H., 2007. Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control. J. Microbiol. Biotechnol. 17, 547. Sharma, H.C., Sharma, K.K., Seetharama, N., Ortiz, R., 2000. Prospects for using transgenic resistance to insects in crop improvement. Electron. J. Biotechnol. 3, 21–22. Shelton, A.M., Zhao, J.-Z., Roush, R.T., 2002. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annu. Rev. Entomol. 47, 845–881. Tabashnik, B.E., Carrière, Y., Dennehy, T.J., Morin, S., Sisterson, M.S., Roush, R.T., Shelton, A.M., Zhao, J.-Z., 2003. Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J. Econ. Entomol. 96, 1031–1038. Vaeck, M., Reynaerts, A., Höfte, H., Jansens, S., De Beuckeleer, M., Dean, C., Zabeau, M., Montagu, M.V., Leemans, J., 1987. Transgenic plants protected from insect attack. Nature 328, 33–37. Ye, G.-Y., Shu, Q.-Y., Yao, H.-W., Cui, H.-R., Cheng, X.-Y., Hu, C., Xia, Y.-W., Gao, M.-W., Altosaar, I., 2001. Field evaluation of resistance of transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis Berliner to two stem borers. J. Econ. Entomol. 94, 271–276. Zheng, X., Yang, Y., Xu, H., Chen, H., Wang, B., Lin, Y., Lu, Z., 2011. Resistance performances of transgenic Bt rice lines T2A-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). 104, 1730–1735.
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Short Communication
Laboratory evaluation of transgenic Bt rice resistance against rice leaf roller, Cnaphalocrocis medinalis Jong Hoon Kim a, Sue Yeon Lee b, Jae Young Choi b, Seok Hee Lee a, Ying Fang a, Kyu Baik Ha a, Dong Hwan Park a, Min Gu Park a, Ra Mi Woo a, Woo Jin Kim a, Ju-Kon Kim c, Yeon Ho Je a,b,⁎ a b c
Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 08826, Republic of Korea Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Republic of Korea
a r t i c l e
i n f o
Article history: Received 4 October 2016 Revised 16 December 2016 Accepted 20 December 2016 Available online 22 December 2016 Keywords: Bacillus thuringiensis cry1Ac Transgenic rice Cnaphalocrocis medinalis Pest resistance
a b s t r a c t Pest resistance of 8 transgenic Bt rice events with a synthetic cry1Ac gene linked to rice rbcS-tp sequence were assessed under laboratory conditions. Bioassays were conducted with neonates and third instar larvae of Cnaphalocrocis medinalis, which is a significant pest of rice in Asia. C. medinalis larvae were shown to be susceptible to all eight events, even though there were differences between the causes of death. The results differed between developmental stages of the larvae, despite the fact that all 8 events led to high mortalities. Neonates displayed feeding avoidance and death by starvation on six Bt rice events. In the case of third instar larvae however, only two events resulted in feeding avoidance. Nevertheless, all Bt rice events were shown to be highly effective against C. medinalis larvae in laboratory conditions. These results may be a significant foundation for the evaluation of improved transgenic Bt rice. © 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
Introduction Bacillus thuringiensis (Bt) is a gram-positive, sporulating bacterium that produces a variety of insecticidal crystal (Cry) proteins, and widely used as one of the most successful biological control agents (Roh et al., 2007). Several Bt cry genes have been introduced into a variety of crops for protection against pests, starting first with tobacco (Vaeck et al., 1987) and now with major crops such as cotton, maize, potato, rice, broccoli, lettuce, walnut, apple, alfalfa and soybean (Shelton et al., 2002). Recently, studies that introduce cry genes into crops to create pest resistance have made much progress, and the total area of land planted with Bt crops has increased substantially (James, 2014; Tabashnik et al., 2003). Several Bt genes were cloned and successfully transferred into higher plants in the 1980s. However, the insecticidal activities of these transgenic plants were relatively low, mostly due to low protein expression caused by mRNA degradation. Deletion of sequences that are irrelevant to insecticidal activity lead to the creation of synthetic truncated Bt genes, which significantly increased their transgenic expression within plants (Barton et al., 1987; Fischhoff et al., 1987; Vaeck et al., 1987). To further enhance Bt gene expression in plants, the genes were either inserted into the chloroplast genome (Kota et al., 1999; Liu et al., 2008; McBride et al., 1995), or had their bacterial coding
sequences modified to plant-preferred coding sequences (Perlak et al., 1993; Sharma et al., 2000). The first reported transformation of rice with a Bt gene was in 1993 (Fujimoto et al., 1993), and many other transgenic rice lines have been developed since then. To confer resistance to rice against significant lepidopteran pests such as Chilo suppressalis, Scirpophaga incertulas and Cnaphalocrocis medinalis, Bt genes such as cry1Ac, cry1Ab and cry1Ab/ Ac fusion genes were introduced into rice cultivars (Chen et al., 2008; Khanna and Raina, 2002; Ye et al., 2001). It has been reported that the targeting of foreign gene to the chloroplasts using the small subunit gene of the ribulose biphosphate carboxylase/oxygenase (rbcS) promotor and its transit peptide sequence (tp) drastically increased the foreign gene product levels in transgenic rice (Jang et al., 2002; Jang et al., 1999). Previously, chloroplast-targeted expression has been achieved through the linking of a synthetic truncated cry1Ac gene to the rice rbcS-tp sequence (Kim et al., 2009). In this study, we assessed the pest resistance of these transgenic Bt rice events (RbcS3:Cry1Ac) against C. medinalis, which is a prominent rice pest that is widely distributed over the rice fields of Asia. Materials and methods Insects
⁎ Corresponding author at: Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul 151-742, Republic of Korea. E-mail address: [email protected] (Y.H. Je).
C. medinalis larvae were collected from paddy fields in Taean-gun, Chungcheongnam-do, Korea and reared on acryl cages containing
http://dx.doi.org/10.1016/j.aspen.2016.12.010 1226-8615/© 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
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Fig. 1. Mortalities of neonates of C. medinalis fed on T3 generation Bt rice. Bars with diagonal lines represent mortality caused by starvation due to larval feeding avoidance.
tillering stage of rice plants. The rearing conditions were 25 °C, 45 ± 100% RH, and a photoperiod of 16 h light:8 h dark. Transgenic Bt rices A total of 8 Bt rice events of T3 generations (608102, 608103, 608104, 608106, 608107, 608108, 608110 and 608111), which are linked of a synthetic truncated cry1Ac gene to the rice rbcS-tp sequence for chloroplast-targeted expression, were used in this study (Kim et al., 2009). Also, non-Bt rice (Oryza sativa L. cv. Ilmi) was used as control. These Bt and non-Bt rice seeds were germinated in the greenhouse and grown for 3 weeks, and then seedlings were transplanted to a paddy field in isolated GMO field, Suwon, Korea. Two months after transplantation, Bt rice and non-Bt rice were examined in the laboratory.
608107, 608110 and 608111 showed cessation of feeding (Fig. 2) and 100% mortality due to starvation (Fig. 1). These results were consistent with the previous reports that C. medinalis and Chilo suppressalis larvae avoided feeding on Bt rice (Chen et al., 2008; Zheng et al., 2011). Many studies have shown that larval feeding avoidance occurs against various Bt toxins (Berdegué et al., 1996; Chen et al., 2008; Gore et al., 2005; Gould and Anderson, 1991; Ramachandran et al., 1993). These reports
Bioassay The pest resistance of Bt rice events were determined against the neonates and 3rd instar larvae of C. medinalis. In the case of neonates, leaves of booting stage rice (1.0–1.5 cm wide and 10 cm long) were fixed to pieces of filter paper. The basal sections of filter paper with rice leaves were placed in 60 mm petri-dishes along with moistened cotton. One neonate was placed per petri-dish, and a total of 30 neonates were used for each sample. For 3rd instar larvae, leaves of booting stage rice (1.0–1.5 cm wide and 10 cm long) were wrapped with moistened cotton to prevent withering of the leaves. Each leaf was placed in a plastic vial along with one larva and the vial was covered with a lid to prevent larval escape. A total of 30 larvae were used for each sample. Treated larvae were maintained under rearing conditions and the larval mortality was scored after five days. All assays were performed in triplicates. To investigate feeding avoidance disorder, feeding area was checked quantitatively via microscopic observation. Results and discussion To evaluate the pest resistance of eight Bt rice events under laboratory conditions, bioassays were performed against neonates and 3rd instar. In case of neonates, all Bt rice events showed high level of pest resistance compared to control non-Bt rice (Fig. 1). While cumulative feeding areas of two Bt rice events (608103 and 608108) were similar to that of control non-Bt rice (Fig. 2), C. medinalis larvae fed on these two events showed high level of mortality (Fig. 1). Particularly, C. medinalis larvae fed on Bt rice events 608102, 608104, 608106,
Fig. 2. Feeding behavior of neonates of C. medinalis fed on control non-Bt rice (A) and T3 generation Bt rice 608103 (B). Arrow heads indicate feeding area of C. medinalis larvae.
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Fig. 3. Mortalities of 3rd instar larvae of C. medinalis fed on T3 generation Bt rice. Bars with diagonal lines represent mortality caused by starvation due to larval feeding avoidance.
suggested that the feeding avoidance of C. medinalis larvae observed in this study might be due to the Cry1Ac protein produced in Bt rice events. Third instar larvae of C. medinalis fed on Bt rice events also showed high level of mortalities (Fig. 3). However, feeding avoidance and death by starvation were observed only at larvae fed on two Bt rice events, 608106 and 608111 (Fig. 3). C. medinalis larvae fed on six Bt rice events (608102, 608103, 608104, 608107, 608108 and 608111)
showed death by Cry1Ac protein (Fig. 4). These results suggested that the feeding behavior of C. medinalis larvae could be affected by developmental stage of the insects. Furthermore, growth stage of the Bt rice and differences in Cry1Ac expression level between Bt rice events also could affect the feeding behavior of C. medinalis (Han et al., 2007; Zheng et al., 2011). In this study, we assessed pest resistance of eight Bt rice events against C. medinalis larvae. Among them, two events caused larval feeding avoidance and death by starvation regardless of developmental stage of C. medinalis. These results could be useful in the establishment of more effective Bt rice along generation.
Acknowledgements This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01113502)” Rural Development Administration, Republic of Korea. J. H. Kim, S. H. Lee, Y. Fang, K. B. Ha, D. H. Park, M. G. Park and R. M. Woo were supported by the Brain Korea 21 plus project, Seoul National University, Republic of Korea. References
Fig. 4. Feeding behavior of 3rd instar larvae of C. medinalis fed on control non-Bt rice (A) and T3 generation Bt rice 608103 (B). Arrow heads indicate feeding area of C. medinalis larvae.
Barton, K.A., Whiteley, H., Yang, N.-S., 1987. Bacillus thuringiensis §-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidopteran insects. Plant Physiol. 85, 1103–1109. Berdegué, M., Trumble, J.T., Moar, W.J., 1996. Effect of CryIC toxin from Bacillus thuringiensis on larval feeding behavior of Spodoptera exigua. Entomol. Exp. Appl. 80, 389–401. Chen, H., Zhang, G., Zhang, Q., Lin, Y., 2008. Effect of transgenic Bacillus thuringiensis rice lines on mortality and feeding behavior of rice stem borers (Lepidoptera: Crambidae). J. Econ. Entomol. 101, 182–189. Fischhoff, D.A., Bowdishi, K.S., Perlak, F.J., Marrone, P.G., McCormick, S.M., Niedermeyer, J.G., Dean, D.A., Kusano-Kretzmer, K., Mayer, E.J., Rochester, D.E., 1987. Insect Tolerant Transgenic Tomato Plants. Fujimoto, H., Itoh, K., Yamamoto, M., Kyozuka, J., Shimamoto, K., 1993. Insect resistant rice generated by introduction of a modified δ-endotoxin gene of Bacillus thuringiensis. Nat. Biotechnol. 11, 1151–1155. Gore, J., Adamczyk, J., Blanco, C., 2005. Selective feeding of tobacco budworm and bollworm (Lepidoptera: Noctuidae) on meridic diet with different concentrations of Bacillus thuringiensis proteins. J. Econ. Entomol. 98, 88–94. Gould, F., Anderson, A., 1991. Effects of Bacillus thuringiensis and HD-73 delta-endotoxin on growth, behavior, and fitness of susceptible and toxin-adapted strains of Heliothis virescens (Lepidoptera: Noctuidae). Environ. Entomol. 20, 30–38. Han, L., Wu, K., Peng, Y., Wang, F., Guo, Y., 2007. Efficacy of transgenic rice expressing Cry1Ac and CpTI against the rice leaffolder, Cnaphalocrocis medinalis (Guenee). J. Invertebr. Pathol. 96, 71–79. James, C., 2014. Global Status of Commercialized Biotech/GM Crops: 2014. International Service for the Acquisition of Agri-biotech Applications (ISAAA) Ithaca, NY, USA.
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