(1) Background: The cabbage flea beetle (CFB; Phyllotreta striolata) seriously damages the production of Chinese flowering cabbage (CFC; Brassica campestris L. ssp. chinensis var. utilis), which is a key leafy vegetable in South China. A large number of chemical insecticides have been sprayed to control this pest; as a result, residues and resistances are becoming an issue. It is necessary to develop biocontrol technologies to address this issue. (2) Methods: Fungal strains were selected based on bioactivity against CFB, and CFC seed pelletization with fungal conidia was subject to evaluation of control efficacy against CFB. The effective mixture of fungus and chemical insecticide was determined based on safety and joint toxicology tests. (3) Results: The screening of 103 strains from 14 genera identified the Metarhizium anisopliae strain MaGX19S02 (Ma) as the one with the highest virulence. The LC₅₀s of Ma to CFB adult and second instar larvae on day 9 post-treatment were 3.04 × 10⁶ and 27.2 × 10⁶ spores/mL, respectively. In the pot test, the pelletization of CFC seeds with Ma conidia (50/25/12.5 mg in 1 g seed with 4 g fillers) demonstrated significant CFB mortalities (45–82%) 20 days after the larvae were introduced. In the field test, the seed pelletization achieved 57–81% control efficacy 14 days after sowing. Furthermore, the combination of Ma with chlorfenapyr (Chl) demonstrated a synergistic effect against CFB; based on this result, we prepared the mixture formulation of 20% Ma-Chl wettable powder (WP). The assessment of the effects of 20% Ma-Chl WP (500× diluent) against CFB revealed 93.33% mortality in the pot test and 61.3% control efficacy in the field test on day 7 post-treatment. (4) Conclusions: The findings demonstrate the potential of Ma to control CFB in the field. Seed pelletization with Ma conidia effectively controlled CFB larvae and protected CFC seedlings, wherein a mixture formulation of 20% Ma-Chl WP had substantial efficacy in controlling CFB adults. Our research provides new methods for CFB biocontrol.

The striped flea beetle, Phyllotreta striolata, is a worldwide pest that causes severe damage to cruciferous crops. Pyridaben, an inhibitor of mitochondrial complex I, has been widely used for the control of P. striolata. Identifying key genes involved in pyridaben tolerance is vital for establishing effective resistance management strategies. A total of 94 full-length cytochrome P450 (CYP) genes were identified in P. striolata. The identified genes were classified into four clans (mitochondrial, CYP2, CYP3, and CYP4), 23 families, and 57 subfamilies. Transcriptomic analysis across developmental stages and tissues revealed distinct expression patterns were seen in these P450 genes. Pyridaben exposure significantly upregulated the expression of CYP6TH1 and CYP6TH2. Tissue-specific expression profiling indicated that CYP6TH1 and CYP6TH2 were highly expressed in the head and fat body. RNA interference-mediated knockdown of these genes significantly increased the susceptibility of P. striolata to pyridaben. Molecular docking confirmed stable binding between pyridaben and both CYP proteins, with binding free energies of −7.73 and −7.57 kcal/mol for CYP6TH1 and CYP6TH2, respectively. This study demonstrates that CYP6TH1 and CYP6TH2 play critical roles in pyridaben tolerance in P. striolata, which highlights promising genetic targets for future resistance management strategies.