Establishment of Laboratory Bioassay System for Phyllotreta striolata Larvae and Screening of Novel Bt Cry Proteins

Phyllotreta striolata is a global pest of cruciferous vegetables, and controlling its soil-dwelling larvae is challenging. The lack of standardized larval bioassay methods hinders the screening of effective biocontrol agents. In this study, we established a stable and standardized laboratory-efficacy trial system for P. striolata larvae. Indoor rearing techniques were optimized for Brassica juncea var. foliosa and Brassica juncea var. megarrhiza were identified as the optimal host plants, with ideal oviposition conditions at 26-28 °C using black flannel substrate, and soil-cultured Brassica rapa var. pekinensis as the host plant. Based on these findings, a larval bioactivity assay was established using B. juncea var. megarrhiza slices on water-agar. This system maintained a natural larval mortality rate below 5% within 48 h, meeting the bioassay requirements. The reliability of the system was validated by evaluating the activity of the engineered Bacillus thuringiensis (Bt) strain G033A against larvae, where the LC₅₀ value decreased from 23.013 mg/mL to 7.295 mg/mL with an extended treatment time (12-48 h). Using this standardized method, novel Cry proteins with high activity against P. striolata larvae were screened. Cry8Ca and Cry8Ga proteins exhibited LC₅₀ values of 2.243 mg/mL and 1.649 mg/mL, respectively.