Interação entre esporos e proteínas Cry e Vip3 de Bacillus thuringiensis na mortalidade de larvas de Spodoptera frugiperda (Lepidoptera: Noctuidae

Abstract

Spodoptera frugiperda (J.E. Smith 1797) (Lepidoptera: Noctuidae) is a primary corn pest in Brazil. Biopesticides based on Bacillus thuringiensis (Berliner 1915) (Bt) are widely used to control this pest. However, the mechanism of action of Bt to kill its hosts is diverse. Therefore, the objective of this study was to identify the Bt mechanism type of action in larvae of S. frugiperda, evaluating the importance of the spores in its mortality. The study was conducted at the Biological Control Laboratory in Embrapa Maize and Sorghum. Were selected 2 Bt strains (HD1: express Vip3 protein; and 426: does not express Vip3) after mortality tests and molecular characterization. To separate the spores and crystals from the Bt strains, was adapted the Ludox purification method, and after that, another purification to achieve spores and crystals 100% purified. Was used Neubauer Chamber to determinate the spores concentration and the proteins (Cry – crystals and Vip3 – expressed in the supernatant) the software Quantity One (Biorad) using results from SDS Page gel. The bioassays were conducted adapting the droplet feeding method (0.08 µL/larva) with S. frugiperda lavae (1 day), 18 treatments with 4 replicates and 2 controls (PBSS and concentrated LB broth). Only spores from each strain (10 4 spores/larva), Cry proteins (HD1: 8 ng/larva and 426: 8 ng/larva), supernatant (with Vip3 HD1: 2 ng/larva and without Vip3 - 426: 0 ng/larva) were tested separately, and also its combination. During 7 days and each 24 hours, the dead larvae were counted, collected and stored at – 20 °C, also, at 14 th day after inoculation was made one last evaluation. To identify septicemia, the dead larvae were macerated and observed under the microscope to make the direct counting of vegetative cells, Neubauer Chamber counting and also determined CFU. The higher mortalities were the combination of HD1 spores and both Cry proteins (Crystals) (HD1 spore and crystal: 94,64% and HD1 spores and 426 crystal: 90,10%). However, the combination of spores, crystals and supernatant (Vip3) does not increased the mortality. The treatments with spores and crystals resulted in high number of cells counting, indicating septicemia. On the other hand, when combined spores and HD1 supernatant the cells counting were higher. In conclusion, the results suggest S. frugiperda larvae are Type III (Septcemia) of the Bt mechanism type of action against lepidopteran insects. However, only spores from Bt strains that express Vip3 proteins are important in S. frugiperda mortality, occurring synergism with Cry proteins.