The invasive tomato leafminer, Tuta absoluta, poses a severe global threat to solanaceous crops, necessitating sustainable biocontrol solutions. Through systematic bioprospecting across several Moroccan soils, we constructed a novel library of indigenous fungal isolates using complementary Tenebrio molitor baiting and selective media methods. High-throughput phenotyping identified 49 highly pathogenic isolates, which were characterized for conidial production, thermotolerance, and virulence against T. absoluta. We discovered two lead isolates, Beauveria bassiana UCA-350 and Metarhizium robertsii UCA-329, that demonstrated superior virulence, reducing median survival time and achieving lower LC₅₀ values than most commercial reference strains. Notably, virulence was positively correlated with in vitro conidial yield, revealing a key trait linkage for strain selection. Furthermore, genus-level divergence in thermotolerance was observed, with Beauveria isolates exhibiting significantly higher heat resilience. Our integrated multi-trait screening pipeline not only delivers two potent, regionally sourced biocontrol candidates but also establishes a phenotypic selection framework that prioritizes both efficacy and production scalability, advancing the rational development of next-generation mycoinsecticides.

Fusarium oxysporum f. sp. Albedinis (Foa) is the causal agent of Bayoud disease, responsible for the loss of 75% of date palm trees in Morocco and posing a threat to its cultivation across North Africa. This study examined ten Foa isolated from various Moroccan locations for the presence of the transposable element Fot1 and the distribution of “Secreted in Xylem” (SIX) genes. Pathogenicity assays on date palm seedlings revealed varying levels of aggressiveness among isolates, with a positive correlation between aggressiveness and SIX gene count. Highly aggressive isolates harbored 9–12 SIX genes, while hypo-aggressive and moderately aggressive isolates carried 0–6. SIX2, SIX6, SIX7, SIX11, SIX12, and SIX13 were differently dispersed among aggressive isolates, whereas SIX12 and SIX13 were present in all aggressive isolates, suggesting their potential role in virulence. This study is the first to highlight a correlation between Foa aggressiveness and SIX gene distribution, providing a foundation for future functional analyses to elucidate their role in pathogenicity.