Synergistic Activity Between Insecticidal Proteins Of Bacillus Thuringiensis

Every year, the damage from insects is one of the most important factors for loss of agricltual productions around the Globe. A large number of chemical pesticides were used to solve this problem. However, abusing of them had great harm to human health and the environment. At the same time, the pest resistance developed fast. Bacillus thuringiensis has been widely used as a microbial insecticide because of plenty advantages including specific insecticidal activity, safety to human and mammal, harmony to the environment.Single cry gene encountered problems in the applications, such as limited toxicity, narrow insecticidal spectrum, pest resistance, so-new methods to solve these problems is urgently needed.Firstly, inducing temperature of expressing Cry proteins were optimized.18℃、30℃and37℃were designed to test. The results revealed that expression levels of both the soluble and non-soluble fraction of Cry proteins reached highest at30℃.Bioassay against Plutella xylostella, important pests for vegetable were performed, and highly toxic proteins:Cry1Ai, Cry1Ea, Cry9Aa, Cry1Ie and Cry1Ib were identified respectively. The highest toxicity of them was from CrylAi with1.52(1.02-2.06)μg/g of LC50. Then, three combinations were mixed with different ratios respectively. CrylAi+CrylEa combination showed a weak synergism when the mixtures were1:2and1:4, synergistic factors were3.35and3.84respectively. An additive effect was revealed with toxin mixture (1:1). Cry1Ea+Cry9Aa and Cry1Ai+Cry9Aa combinations were mixed with1:2and1:4respectively, the result indicated that both of them had significant synergism. In addition, when they were combined in a1:1ratio respectively, LC50were0.60(0.37-0.85) and0.23(0.16-0.31)μg/g, the biggest synergistic factor were6.67and9.06respectively.1:1mixture of Cry9Aa+Cry1Ie、Cry9Aa+Cry1Ib、Cry1Ie+Cry1Ib combinations were made respectively, and the result showed that a combination of Cry9Aa+Cry1Ie had a weak synergism, synergistic factor was2.94, On the other hand, Cry9Aa+Cry1Ib and Cry1Ie+Cry1Ib toxin mixtures showed additive effect. Because the Cry9Aa+Cry1Ie combination showed synergistic effect, experiments were carried out with different ratios of these toxins (1:1,1:2,2:1,1:4,4:1,1:8, and8:1). that the results indicated that toxin mix (1:1) of Cry9Aa and Cry1Ie showed significant synergism, while, other toxin mixtures only showed additive effect at different levels。Bioassay against alien invasive pest Hyphantria cunea with Bt proteins was performed in the laboratory, and the results showed that high toxic proteins were Cry1Ac, Cry1Ah, Cry1Ai, Cry1Ea, Cry2Ab, and Cry9Aa. The high toxicity of them was from Cry2Ab, Cry1Ac and Cry1Ah with1.96(1.28-2.95),2.42(1.59-4.20),3.11(1.77-5.34) μg/g of LC50, respectively. When the mixture was1:1, nine combinations of Cry1Ai+Cry1Ea, Cry1Ea+Cry9Aa, Cry1Ai+Cry9Aa, Cry1Ac+Cry9Aa, Cry2Ab+Cry9Aa, Cry1Ah+Cry9Aa, Cry1Ah+Cry2Ab, Cry1Ac+Cry2Ab, Cry1Ea+Cry2Ab were assayed against the pests. The results revealed that two of these combinations, Cry1Ac+Cry9Aa and Cry1Ah+Cry2Ab had weak synergistic effect, synergistic factor was3.38and2.83respectively. The significant synergism combined by Cry1Ah+Cry9Aa was found with0.49(0.26-0.78)μg/g of LC50, synergistic factor was biggest with7.04. At the same time, other combinations had different degree of additive effect.Bioassay against Chilo suppressalis, very important insect pests for rice was finished. The results indicated that high toxic proteins were Cry1Ah, Cry1Be, Cry9Ee, Cry1Ie and Cry9Aa separately. The high toxicity of them was Cry1Be. Cry9Aa, Cry1Ie with0.10(0.05-0.16),0.14(0.09-0.20),0.40(0.25-0.59)μg/g of LC50, respectively. When the mixture was1:1, four combinations of Cry1Ai+Cry9Ee, Cry1Ie+Cry9Ee, Cry9Ee+Vip3Aa and Cry9Aa+Vip3Aa were assayed against the pests. The results indicated that a weak synergistic effect was revealed with a mixture of Cry1Ai+Cry9Ee, synergistic factor was2.09; Cry9Aa+Vip3Aa had strong synergistic effect with0.03(0.01-0.05) μg/g LC50, the synergistic factor reached to9.06; both Cry9Ee+Vip3Aa and Cry1Ie+Cry9Ee combinations showed additive effect at diffienent levels.To screen of highly toxic Bt strains and proteins against Ectropis oblique, a important pest of tea, we obtained sensitive, efficient and very effective strains, G033A powder and CrylBb protein. When treated with the three strains, the corrected mortality of Ectropis oblique was all above95%, about80%pests were dead by using G033A powder, and70%were killed when treated with CrylBb at the concentration of100μg/mL.