Multilocus identification and genetic enhancement of Trichoderma spp. for entomopathogenic activity against Spodoptera littoralis.

Background: The Egyptian cotton leafworm (Spodoptera littoralis) is a highly destructive, pesticide-resistant pest affecting over 80 economically important crops across the Mediterranean and African regions. While chemical insecticides offer temporary relief, their long-term use poses environmental and health risks, and resistance development reduces their effectiveness. Biological control using entomopathogenic fungi, particularly Trichoderma spp., offers a sustainable alternative. Traditionally, it is used against plant pathogens, Trichoderma harzianum, T. viride, T. asperellum, and T. longibrachiatum have also shown insecticidal potential through the production of compounds like peptaibols, gliotoxins, and chitinases, and by inducing systemic resistance in plants. However, the entomopathogenic potential of native Trichoderma isolates in Egypt remains undiscovered, and field performance is often inconsistent. This study aims to identify and evaluate native Trichoderma strains against S. littoralis and enhance their biocontrol efficacy through interspecific protoplast fusion a promising parasexual technique for strain improvement.

Results: Multilocus sequence analysis targeting the tef1-α and rpb2 genes identified the isolates as T. harzianum, T. asperellum, and T. longibrachiatum. Phylogenetic analysis clustered the isolates into three well-distinctive clades corresponding to these species. Among the tested isolates, Tricho19 (T. longibrachiatum), Tricho5 (T. asperellum), and Tricho30 (T. harzianum) demonstrated the highest extracellular chitinase activity and larval mortality in oral bioassays against S. littoralis. Interspecific protoplast fusion led to the generation of fusants with significantly enhanced chitinase production and insecticidal activity relative to their parental strains. Greenhouse assays confirmed the superior performance of fusant Fus8, which exhibited the highest larval mortality and antifeedant activity, closely approaching the efficacy of a chemical insecticide.

Conclusion: Interspecific protoplast fusion significantly improved the entomopathogenic performance of Trichoderma strains against S. littoralis. The enhanced activity of fusant strains, particularly Fus8, highlights the potential of this cost-effective strategy to generate improved biocontrol agents. These findings contribute to the development of sustainable pest management alternatives that can reduce reliance on chemical pesticides in agriculture.