Abstract: 【Aim】This study aims to explore the role of autophagy in the resistance of Bursaphelenchus xylophilus to environmentally friendly pesticide emamectin benzoate stress, so as to address the issue of drug resistance caused by long-term single-agent application.【Methods】The dip method was employed to determine the toxicity of emamectin benzoate to B. xylophilus adults in 24 h. Autophagy was induced in B. xylophilus using 0.5 and 50 mmol/L rapamycin followed by treatment with emamectin benzoate at the concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 mg/L for 24 h, and the mortality rate of B. xylophilus adults was calculated. The reproduction rates of B. xylophilus adults were measured at 7 d after exposure to emamectin benzoate at LC₂₀ (0.30 mg/L) and median lethal concentration (LC₅₀)(1.14 mg/L) following the treatment with 50 mmol/L rapamycin for 1 h. Autophagy-related genes BxATG2, BxATG11 and BxATG18 of B. xylophilus were cloned by PCR and bioinformatically analyzed. qRT-PCR was employed to detect the expression levels of the key autophagy-related genes (BxATG2, BxATG6, BxATG10, BxATG11, BxATG12 and BxATG18) in B. xylophilus adults after exposure to 50 mmol/L rapamycin followed by emamectin benzoate at LC₅₀ (1.14 mg/L) and a high concentration (3.20 mg/L) for 24 h.【Results】 The mortality rates of B. xylophilus adults increased with the concentrations of emamectin benzoate rising. The LC₂₀ and LC₅₀ values of emamectin benzoate against B. xylophilus adults in 24 h were 0.30 and 1.14 mg/L, respectively. After rapamycin-induced autophagy, the lethality of emamectin benzoate to B. xylophilus adults decreased. The reproduction rates of B. xylophilus adults declined with the concentrations of emamectin benzoate increasing, and showed a slight but non-significant increase following rapamycin induction. The proteins encoded by the successfully cloned autophagy-related genes BxATG2, BxATG11 and BxATG18 exhibited high amino acid sequence identities with the homologs from the closely related nematodes. BxATG2, BxATG11 and BxATG18 were acidic and hydrophobic proteins, lacking signal peptides and transmembrane domains. BxATG2, BxATG11 and BxATG18 were most closely related to their counterparts in nematodes of the genus Bursaphelenchus. Rapamycin pretreatment (50 mmol/L) alone significantly altered the expression of BxATG11 and BxATG12, both 1.14 and 3.20 mg/L emamectin benzoate upregulated the expression of BxATG2, BxATG6, BxATG10, BxATG11 and BxATG18, and after induction by 50 mmol/L rapamycin, the upregulated effects of emamectin benzoate (particularly at the high concentration of 3.20 mg/L) on BxATG2, BxATG6, BxATG11, BxATG12 and BxATG18 were further enhanced.【Conclusion】 Cellular autophagy plays a critical role in the defense of B. xylophilus adults against emamectin benzoate stress. Resistance to emamectin benzoate in B. xylophilus adults can be enhanced by the expression induction of autophagy-related genes (such as BxATG2, BxATG6, BxATG10, BxATG11 and BxATG18) through rapamycin. This study deepens the understanding of biological adaptive evolution and provides a theoretical basis and reference for developing novel prevention and control strategies against pine wilt disease based on regulating the autophagy pathway.

Key words: Bursaphelenchus xylophilus, autophagy-related genes, emamectin benzoate, rapamycin, survival, reproduction