Synergistic Insecticidal Activity Against Hyphantria cunea by Cry9Aa3 Mutants and Cry1Ah Combinations

The larvae of Hyphantria cunea feed on plant leaves, causing significant losses to forestry and agricultural production. At present, cry1 genes such as cry1Ac and cry1Ah are mainly used to control H. cunea. To delay the problem of pest resistance induced by a single insecticidal gene, it is crucial to discover and develop new insecticidal genes or gene combinations. This study found cry9Aa3 and cry9Aa4 showed insecticidal activity against H. cunea. The toxicity of 14 mutants of Cry9Aa3 was analyzed and the LC₅₀ of the triple-amino-acid substitution mutant 316LRG318AAA was 3.69 μg/g, which represents a 1.49-fold increase in insecticidal activity compared to Cry9Aa3. Additionally, enhanced stability of this mutant was detected in the midgut juice of H. cunea. Cry9Aa3 and 316LRG318AAA, in combination with Cry1Ah, demonstrated synergistic effects against H. cunea, with synergistic factors of 4.76 and 8.33, respectively. This study has identified the mutant 316LRG318AAA and its combination with Cry1Ah as exhibiting high toxicity against H. cunea, providing valuable genetic resources for the development of transgenic poplars and holding significant importance for delaying resistance in this pest.