Bt Research 2024, Vol.15, No.5, 232-239 http://microbescipublisher.com/index.php/bt 236 sustainability by providing a diverse array of selection pressures that make it more difficult for pests to develop resistance (Gassmann and Reisig, 2022). Understanding the genetic and mechanistic basis of resistance can inform the design of more effective synergistic combinations and resistance management strategies (Huang et al., 2020). 6 Case Studies 6.1 Synergistic effects of Cry and Cyt proteins in Bt cotton The combination of Cry and Cyt proteins in Bt cotton has shown significant synergistic effects in controlling pest populations. For instance, Cry1Ac and Cry1F toxins have been used together in WideStrike cotton plants to manage resistant strains of the cabbage looper, Trichoplusia ni. The study demonstrated that the Cry1F resistance trait in T. ni was not associated with the Cry1Ac resistance mechanism, indicating that the combination of these toxins can effectively manage resistance (Figure 2) (Kain et al., 2022). the synergistic interaction between Cry and Cyt proteins, such as Cry10Aa and Cyt2Ba, has been shown to significantly enhance the mosquitocidal activity against Aedes aegypti larvae, with a synergistic potentiation of 68.6-fold. Figure 2 SDS-PAGE analysis of midgut BBMV proteins from individual T. ni 5th instar larvae from the Cornell strain (1 and 2), GLEN-Cry1Ac-R (3 and 4), GLEN-WideStrike-R (5 and 6), and GLEN-Cry1F-R (7 and 8) (Adopted from Kain et al., 2022) 6.2 Synergistic use of Bt toxins and non-Bt proteins in corn The use of Bt toxins in combination with non-Bt proteins has also been explored in corn. For example, the combination of Cry and Vip3Aa proteins in Bt corn has been effective in controlling Helicoverpa zea, a major pest. The study found that while H. zea populations have developed resistance to Cry1Ab, Cry2Ab2, and Cry1A.105 toxins, the Vip3Aa protein still provided high control efficacy, particularly in southern locations (Dively et al., 2023). This indicates that the combination of Cry and Vip3Aa proteins can delay resistance development and sustain the efficacy of Bt corn (Yang et al., 2022). 6.3 Lessons learned from the application of synergistic proteins The application of synergistic proteins in Bt crops has provided several valuable lessons. The combination of different Bt toxins, such as Cry and Vip proteins, can effectively manage resistance and extend the efficacy of Bt crops. For instance, the combination of Cry1Ac and Vip3Aa in Bt cotton has been shown to be effective against H. zea, even in populations resistant to Cry toxins. The use of sentinel plants, such as Bt sweet corn, can help monitor resistance development and inform resistance management strategies. The identification of novel synergistic interactions, such as those between Cry10Aa and Cyt2Ba, highlights the potential for developing new combinations of insecticidal proteins to enhance pest control (Valtierra-de-Luis et al., 2020). 7 Comparative Analysis of Synergistic vs. Standalone Proteins 7.1 Efficacy of synergistic combinations compared to single proteins The efficacy of synergistic combinations of Bacillus thuringiensis (Bt) toxins has been extensively studied, revealing that combinations often outperform single proteins in pest control. For instance, the combination of Cry1Ca and Vip3Aa was found to be the most effective against Spodoptera exigua, showing a high synergism factor (>4) at both 7 and 10 days after treatment (DAT). Similarly, the combination of Cry1Ac and Vip3Aa11
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