Engineered bacterium Pseudomonas abietaniphila BHJ04-BLG4 controls pine wilt disease via nematocidal activity and induction of host systemic resistance

Abstract

Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus, is a destructive global forest disease for which microbial control offers promising potential. Pseudomonas abietaniphila BHJ04, an endophytic bacterium from Pinus massoniana, has been shown to induce host resistance. To enhance its biocontrol efficacy against PWD, we engineered this strain to improve nematicidal activity while maintaining resistance induction. An exogenous nematicidal gene encoding the extracellular alkaline serine protease BLG4 was introduced into BHJ04 by conjugation transfer, generating strain BHJ04-BLG4. Colonization in P. massoniana stems peaked 15 days post-inoculation (2.06 × 10⁴ copies/μL). In vitro assays demonstrated that purified BLG4 degraded nematode cuticles and killed nematodes (IC₅₀ = 35.98 μg/mL), while crude lysate supernatant from 48 h cultures achieved 100 % nematicidal activity. In greenhouse trials, BHJ04-BLG4 reduced PWD incidence to 40 % (control efficacy: 86.66 %). RT-qPCR showed significant induction of host defense genes: dehydration-responsive protein (315.06-fold at 48 h), and chitinase, PR-2 (β-1,3-glucanase), and PR-5 (thaumatin-like protein) genes (7.43–34.26-fold within 24–48 h). Preliminary ecological safety tests indicated that BHJ04-BLG4 caused no disease symptoms in tobacco, alfalfa, or P. thunbergii. These findings suggest that BHJ04-BLG4 confers enhanced resistance to PWD through synergistic mechanisms of direct nematicidal activity and host systemic resistance induction. This engineered strain represents a novel and sustainable biocontrol strategy for PWD management.