2.3. Extraction of DNA from the Guts of the IS and IR M. usitatus

A 15 mm × 75 mm flat-bottomed test tube was soaked in 1 × 10⁷ conidia.mL⁻¹ spore suspension for 2 h, dried naturally, and prepared for use. Meanwhile, fresh cowpeas were cut into 1 cm sections without holes at both ends, soaked in spore suspension for 15 s, removed, dried, and placed in the above flat-bottomed test tube, and M. usitatus adults were released into the flat-bottomed test; the tubes were then sealed with cotton, and placed at 26 ± 1 °C and 12 L:12 D in a lighted incubator. Sterile water treatment with 0.05% Tween-80 was used as a blank control. Three replicates of each treatment were set up, and the surviving test worms were collected following 1 day, 2 days, and 3 days. Their abdomens were cut under aseptic conditions and sent to Guangzhou Kidio Biotechnology for bacterial 16S High-throughput sequencing. The sensitive control was recorded as S-CK, and the resistance control was recorded as R-CK. The 1 × 10⁷ conidia.mL⁻¹-treated sensitive populations were recorded as B1, B2, and B3 at 1 day, 2 days, and 3 days, respectively, 1 × 10⁷ conidia.mL⁻¹-treated resistance populations were recorded as D1, D2, and D3 at 1 day, 2 days, and 3 days, respectively. The DNA was individually extracted from the adult guts of each population using the HiPure Stool DNA Kits (Magen, Guangzhou, China) following the manufacturer’s instructions. The extracted DNA was assessed for quality using electrophoresis on a 1% agarose gel and stained with ethidium bromide, following the manufacturer’s protocol. The amplification products from the second round were purified using AMPure XP Beads. The extracted DNA was stored at −80 °C. DNA from adults of each population was pooled, resulting in eight samples selected for amplicon library construction.