Toxicity and larval growth inhibition of eleven insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 μg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect

Avilla Hernández, Carlos

Ferré, J.

González Cabrera, J.

González Zamora, José Enrique

Vargas Osuna, E.

idUS. Depósito de Investigación Universidad de Sevilla

  1 Title: Toxicity of several -endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain. Authors: C. Avilla(1), E. Vargas-Osuna(2), J. González-Cabrera(3), J. Ferré(3), J.E. González-Zamora(1) Affiliations: (1) Departamento de Ciencias Agroforestales, Universidad de Sevilla. Carretera de Utrera, km 1. E-41013 Seville (Spain)  (2) Departamento de Ciencias y Recursos Agrícolas y Forestales. Universidad de Córdoba. Apartado 3048. E-14080 Córdoba (Spain)  (3) Departament de Genètica, Facultat CC. Biològiques, Universitat de València. Av. Dr. Moliner, 50. E-46100 Burjassot (Valencia, Spain) Address for manuscript correspondence:  J.E. González-Zamora  Escuela Universitaria de Ingeniería Técnica Agrícola (Universidad de Sevilla)  Carretera de Utrera, km 1  E-41013 Sevilla (Spain)  E-mail: zamora@us.es  Phone number: +34-954 486 459  Fax number: +34-954 486 436 Available in: Journal of Invertebrate Pathology 90 (2005) 51–54 doi:10.1016/j.jip.2005.04.003   2 ABSTRACT Toxicity and larval growth inhibition of eleven insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 µg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect.  Keywords: Bacillus thuringiensis, Helicoverpa armigera, Microbial insect control, Cotton pests, Cry toxins, Growth inhibition, ICP.   3 INTRODUCTION Helicoverpa armigera (Hübner) is an important pest of different crops (cotton, tomato, among others) in Spain and other countries. To prevent the damage that larvae produce in these crops, a variety of methods are used for their control, including the use of chemical pesticides and also microorganisms. The most important of the latter is Bacillus thuringiensis (Berliner), a bacterium which produces different proteins (-endotoxins) toxic to larvae of different species of Lepidoptera and other insects (Schnepf et al., 1998).  B. thuringiensis  (Bt) has been used since the 50’s of the 20th century by spraying its spores and crystals on the plants. In the late 80’s and early 90’s different cultivars, such as tobacco, tomato, potato, cotton, and corn, among others, were genetically engineered to express -endotoxins, which allowed them to be tolerant to insect attack (Schnepf et al., 1998). The use of insect resistant transgenic crops has been adopted in many countries because of the higher protection they provide and the reduction in pesticide applications, but there is concern about the development of resistance in target pest populations. Different strategies, such as the use of refuges, gene rotation, and gene stacking, have been proposed to avoid (or at least delay) the development of resistance.  To date, the only transgenic crop with a Bt gene authorized in Spain is Bt-corn, which incorporates a gene that codes for the Cry1Ab toxin. However, it is expected that other transgenic cultivars be authorized in the near future, in particular Bt-cotton (which expresses the Cry1Ac toxin) because of the important losses that H.   4 armigera can produce in this crop. Several authors have studied the effect of Bt toxins on H. armigera populations from China, India and Australia (Chakrabarti et al., 1998; Gajendra Babu et al., 2002; Liao et al., 2002; Kumar et al., 2004; Fengxia et al., 2004; Jalali et al., 2004). However, to our knowledge there is no published study describing the activity of Bt toxins in European populations. Since susceptibility to a given Bt toxin may vary among populations (González-Cabrera et al., 2001; Wu et al, 1999; Jalali et al., 2004), it is of great interest to determine the effect of Bt toxins on local populations of H. armigera prior to the possible introduction of transgenic cultivars and also for the more rational use of Bt spray formulations. The results obtained will also be useful to search for alternatives in the case resistance evolves in the target populations to some of the currently used Bt toxins. MATERIALS AND METHODS The H. armigera colony was established from larvae and egg masses collected in cotton fields in June-July 2001-02 in Andalucia, southern Spain. Bioassays were carried out by incorporating the Bt toxins into the artificial diet (Poitout and Bues, 1974). Cry1Aa3, Cry1Ab3, Cry1Ac4, Cry1Ca2, Cry1Da, Cry1Ea, Cry1Fa1, Cry1Ja1, Cry2Aa1, and Cry2Ab2 were purified from recombinant Bt strains EG1273, EG7077, EG11070, EG1081, EG7300, EG11901, EG11069, EG7279 (obtained from Ecogen Inc., Langhorne, Pa.), EG7543, and EG7699 (from Monsanto Co., Chesterfield, Mo.), respectively. Bt strains were grown at 29ºC for 48 h in CCY medium (Stewart et al., 1981) supplemented with the suitable antibiotic. Spores and crystals were collected by centrifugation at 22000 ×g for 10 min at 4ºC. The pellet was washed four times with 1 M NaCl/10 mM EDTA and suspended in 10 mM KCl. Protoxin inclusions were   5 solubilized in 50 mM sodium carbonate buffer, pH 10.5, containing 10 mM dithiothreitol. Protoxins were trypsin-activated (trypsin type XI: from bovine pancreas, Sigma Chemical, St. Louis, MO) at 37ºC for two hours (1 mg trypsin per 10 mg protoxin). Purified and trypsin activated Cry9Ca was obtained from Jeroen Van Rie (Bayer BioScience, Ghent, Belgium). Protein concentration was measured by the method of Bradford (Bradford, 1976).  The diet was poured into 100 ml beakers and the toxin was added to final concentrations of 1, 2, 4, 8, and 16 µg of toxin per ml of diet. A control was prepared with distilled water. The solidified diet was cut into small cylinders and placed into boxes of 30 mm diameter and 15 mm height, with no ventilation hole in the lid. One neonate larva was added to each box. Three groups of ten boxes were used with each concentration and the control. Mortality was determined after 7 days and surviving larvae were also weighed. Boxes were kept in a rearing room at 26±1ºC, 60±10% relative humidity, and a photoperiod of 16:8 (L:D). The results of bioassays were evaluated by probit analysis and the 50% lethal concentration (LC50) values were estimated using the POLO-PC program (LeOra Software, Berkeley, Calif.). The ratio between the weight of treated and untreated larvae at day seven was used to calculate larval growth inhibition. The effective concentrations that produce a reduction in larval growth of 50 % (EC50) and 99 % (EC99) were calculated by adjusting the curve as proposed by Sims et al. (1996).   6 RESULTS AND DISCUSSION Among the eleven Bt toxins tested, Cry1Ac4 and Cry2Aa1 were the most toxic and produced a considerable mortality on H. armigera larvae (Table 1), with Cry1Ac4 being almost twice as much active as Cry2Aa1. These results are in agreement with those of other authors, who found Cry1Ac more or equally active than Cry2Aa with similar bioassay protocols (but using protoxins), although the LC50 ratio between toxins differs. Thus, the Cry1Ac/Cry2Aa LC50 ratio was 6.5-fold (Gajendra Babu et al., 2002) and 35-fold (Chakrabarti et al., 1998) in two independent populations from India, but it was non-significantly different in a population from Australia (Liao et al., 2002). In the range of concentrations tested, the rest of toxins did not give enough mortality as to allow estimation of LC50 values. However, most of the toxins with no detectable lethal effect showed a clear effect on larval growth (Fig. 1), with several of them with an EC50 below or near 1 μg/ml or included in the range of concentrations tested (Table 1), like Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1. Toxins Cry1Aa3, Cry1Ca2, and Cry1Ea did not have any effect on either mortality or larval growth.  Only two studies with H. armigera have tested the toxicity of a considerable number of Bt toxins (Chakrabarti et al., 1998; Liao et al., 2002). For the Australian population Cry1Ac and Cry2Aa were the most active toxins, whereas for the Indian population these two toxins were the most active ones together with Cry1Aa, which gave no toxicity in our study. In agreement with our results, a severe effect on larval growth   7 has been observed for Cry1Ab, Cry1D and Cry1F in the Indian population (Chakrabarti et al., 1998), and Cry1Ab and Cry2Ab in the Australian population (Liao et al., 2002), and no effect (or negligible) for Cry1Ca and Cry1Ea in both populations.  The absolute values of LC50 estimated in our study for Cry1Ac4 (3.5 µg/ml) and Cry2Aa1 (6.3 µg/ml) are generally higher than those reported in previous papers using the similar bioassay conditions (diet incorporation): 0.02 µg/ml for Cry1Ac and 0.71 µg/ml for Cry2Aa (Chakrabarti et al., 1998), 0.14-0.18 µg/ml for Cry1Ac (Kranthi et al., 2000), 0.24 µg/ml for Cry1Ac and 1.57 µg/ml for Cry2Aa (Gajendra Babu et al., 2002), 0.17-0.54 µg/ml for Cry1Ac (Fengxia et al., 2004), 0.09-91 µg/ml (with a mean of 1.4 µg/ml for the 24 populations studied) for Cry1Ac (Wu et al., 1999), and 0.11-0.71 μg/ml for Cry1Ac (Jalali et al., 2004). These discrepancies might be due to differences in the nature and mode of application of the toxins, the method employed to estimate protein concentration, the temperature at which the bioassay was performed (this varies from 26 to 30ºC), and unquestionably, the origin of the insects. Regarding the nature of the toxin, our tests were carried out with purified and activated Cry proteins, whereas the rest of studies used protoxins or a mixture of crystals and spores. We have chosen activated toxins because we tried to simulate the form of toxin found in transgenic crops and to avoid the synergistic effect of spores. Transgenic crops seem to be the most widespread way of using Bt toxins in the future. The area dedicated to these cultivars is steadily growing since 1996 and it is expected to keep this tendency in the future (James, 2004). In conclusion, the reported results represent the first study on the toxicity of Bt toxins to an European population of H. armigera . The Spanish population was very   8 susceptible to Cry1Ac4 and Cry2Aa1, as it has been found for populations of this species from other parts of the globe. Furthermore, growth inhibition was severe with Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1. Either in sprayable formulations or in Bt-crops, the combination/rotation of toxins with a different binding site is desirable from a resistance management point of view. The information on the toxicity of the different Bt toxins, along with that from binding studies of Bt toxins to binding sites in the midgut membrane (Estela et al., 2004), should allow to choose those toxins that, not binding to the same sites, are the most active against this pest.  ACKNOWLEDGEMENTS  This work was funded by the Spanish Ministry of Science and Technology (Project No. AGL2000-0840-C03 and AGL2003-09282-C03). The authors would like to thank Sofia Camúñez and Silvia Pérez for their technical support.  REFERENCES Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Chakrabarti, S.K., Mandaokar, A., Ananda Kumar, P.,  Sharma, R.P., 1998. Efficacy of lepidopteran specific delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera.  J. Invertebr. Pathol. 72, 336-337.   9 Estela, A., Escriche, B., Ferré, J., 2004. Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae). Appl. Environ. Microbiol. 70, 1378-1384. Fengxia, M., Shen, J., Zhou, W., Cen, H., 2004. Long-term selection for resistance to transgenic cotton expressing Bacillus thuringiensis toxin in Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae). Pest. Manag. Sci. 60, 167-172. Gajendra Babu, B., Udayasuriyan, V., Asia Mariam, M., Sivakumar, N.C., Bharathi, M.,  Balasubramanian, G., 2002. Comparative toxicity of Cry1Ac and Cry2Aa delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (H.). Crop Prot. 21, 817-822. González-Cabrera, J., Herrero, S., Sayyed, A.H., Escriche, B., Liu, Y.B., Meyer, S.K., Wright, D.J., Tabashnik, B.E., and Ferré, J., 2001. Variation in susceptibility to Bacillus thuringiensis toxins among unselected strains of Plutella xylostella. Appl. Environ. Microbiol. 67, 4610-4613.  Jalali, S.K., Mohan, K.S., Singh, S.P., Manjunath, T.M.,  Lalitha, Y., 2004. Baseline-susceptibility of the old-world bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) populations from India to Bacillus thuringiensis Cry1Ac insecticidal protein.  Crop Prot. 23, 53-59. James, C., 2004. Preview: Global status of commercialized biotech/GM crops: 2004. International Service for the Acquisition of Agri-biotech Applications, ISAAA Briefs No. 32. Ithaca, N. Y. Kranthi, K.R., Kranthi, S., Ali, S., Banerjee, S.K., 2000. Resistance of Cry1Ac δ-endotoxin of Bacillus thuringiensis in a laboratory selected strain of Helicoverpa armigera (Hübner). Curr. Sci. 78 (8), 1001-1004.   10 Kumar, S., Udayasuriyan, V., Sangeetha, P., Bharathi, M., 2004. Analysis of Cry2A proteins encoded by genes cloned from indigenous isolates of Bacillus thuringiensis for toxicity against Helicoverpa armigera. Curr. Sci. 86 (4), 566-570. Liao, Ch., Heckel, D.G., Akhurst, R., 2002. Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton. J. Invertebr. Pathol. 80, 55-63. Poitout, S., Bues, R., 1974. Élevage des chenilles de vingthuit especes de lépidoptères Noctuidae et de deux especes d’Arctiidae sur milieu artificiel simple. Particularités de l’élevage selon les especes. Ann. Zool. Écol. Anim. 6, 431-441. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R., Dean, D.H., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775-806. Sims, S.R., Greenplate, J.T., Stone, T.B., Caprio, M.A., Gould, F.L., 1996. Monitoring strategies for early detection of Lepidoptera resistance to Bacillus thuringiensis insecticidal proteins. ACS Symposium series, no. 645, pp. 229-242. In Brown T.M. [ed] Molecular genetics and evolution of pesticide resistance. American Chemical Society, Whasington D.C. Stewart, G.S.A.B., Johnstone, K., Hagelberg, E.,  Ellar, D. J., 1981. Commitment of bacterial spores to germinate. Biochem. J. 198, 101-106. Wu, K., Guo, Y.,  LV, N., 1999. Geographical variation in susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis insecticidal protein in China.  J. Econom. Entomol. 92 (2), 273-278.   11 Table 1: Effect of activated B. thuringiensis toxins on mortality (LC50) and larval growth rate (EC50 and EC90) of neonate larvae of H. armigera exposed for 7 days.    a EC50 was much lower than the lowest concentration tested (1 μg/ml). b The EC99 was higher than the highest concentration tested (16 μg/ml). c Not detectable because the mortality found at the highest concentration (16 μg/ml) was lower or near 50 % of the individuals and POLO PC (LeOra Software, Berkeley, Calif.) could not adjust to a regression line. d EC50 was lower than the lowest concentration tested (1 μg/ml). e The EC99 was much higher than the highest concentration tested (16 μg/ml). f No effect on growth inhibition. Crystal protein LC50 (μg/ml) 95% FL EC50±s.e. (μg/ml) EC99±s.e. (μg/ml) Cry1Ac4 3.5 2.7-4.5 <<1.0a 3.7±0.5 Cry2Aa1 6.3 4.9-8.5 <<1.0 >16b Cry9Ca NDc  <<1.0 >16 Cry1Fa1 ND  <1.0d >16 Cry1Ab3 ND  <1.0 >16 Cry2Ab2 ND  2.3±0.5 >16 Cry1Da ND  3.8±1.6 >>16e Cry1Ja1 ND  9.4±4.5 >>16 Cry1Aa3 ND  NEf NE Cry1Ca2 ND  NE NE Cry1Ea ND  NE NE   12 FIGURE LEGENDS  Fig. 1. Percent larval growth inhibition of H. armigera by different B. thuringiensis proteins at the concentrations: 1 μg/ml ( ), 4 μg/ml ( ), and 16 μg/ml ( ).   13 020406080100Cry1Ab3 Cry1Ac4 Cry1Da Cry1Fa1 Cry1Ja1 Cry2Aa1 Cry2Ab2 Cry9CaPercent  inhibition (%) (%)  

Toxicity of several d-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

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Toxicity of several d-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

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idUS. Depósito de Investigación Universidad de Sevilla