Bacillus pumilus is widely used as a biocontrol agent. To further develop the biological control potential of B. pumilus LYMC-3 against pine blight, a statistical experimental design was used to optimize a liquid medium using low-cost substrates to improve its antagonistic activity against Sphaeropsis sapinea. Through the plate antagonism test and greenhouse control effect test, this study determines the antifungal effect of strain LYMC-3 against S. sapinea and pine blight. Moreover, response surface optimization methodologies were used to systematically optimize the medium composition and culture conditions of the LYMC-3 strain. The plate antagonism test showed that the inhibition rate of LYMC-3 fermentation filtrate (diluted 5-fold) was 66.09%. The greenhouse control effect test showed that the control effect of its fermentation filtrate on shoot blight reached 89.99%. The response surface optimization test ultimately determined that a higher inhibition rate can be achieved under these conditions: the optimal medium components were 7.2 g/L glucose, 15 g/L peptone, and 7.1 g/L magnesium sulfate; the optimal culture conditions were 52% liquid volume, 28 °C culture temperature, an initial pH of 7, and 1% bacterial inoculation volume. Under the optimized system, the five-fold diluted LYMC-3 fermentation filtrate inhibition rate against S. sapinea was 81.23%, which was 15.84% higher than that before optimization. Meanwhile, optimize the selection of lower-cost and more commonly used glucose instead of beef paste as the carbon source for the culture medium, and choose cheaper magnesium sulfate instead of sodium chloride as the nitrogen source.

Larch dieback disease, caused by Neofusicoccum laricinum, severely affects forest health and productivity. To effectively curb the occurrence of this disease, a powder formulation of Bacillus amyloliquefaciens JL54 was developed through spray drying. The research commenced with the optimization of fermentation medium and culture conditions through statistical design, aiming to maximize both bacterial viability and antagonistic activity. The optimal medium included 12 g/L yeast extract, 11.8 g/L yeast powder, and 7.2 g/L magnesium sulfate. Optimal culture conditions included 30% loading volume, 1% inoculum, 37 °C incubation temperature, 31.8 h shaking time, and initial pH 6.4. Under these conditions, the viable count of strain JL54 reached 4.45 × 10⁹ cfu/mL, a 296.67-fold increase compared with the unoptimized system. To evaluate its practical applicability, field trials were conducted, showing a 54% control efficiency against larch dieback disease, significantly suppressing disease progression. Subsequently, the spray drying process was optimized with a 1:10 protective agent-to-water ratio, 100 °C inlet temperature, and 630 mL/h feed flow rate, achieving a 78.41% powder recovery rate. Collectively, this study demonstrates the potential of B. amyloliquefaciens JL54 as an effective biocontrol agent for managing larch dieback and supports its application in spray-dried formulations for forest disease control.

Larix olgensis, a keystone timber species in Northeast China, is increasingly threatened by Neofusicoccum laricinum-induced shoot blight, a devastating disease that compromises forest health and necessitates sustainable management strategies. Here, we demonstrate that the endophytic bacterium Bacillus amyloliquefaciens JL54 elicits multifaceted defense responses in L. olgensis, enhancing resistance to pathogen infection. Greenhouse assays revealed that JL54 pretreatment reduced disease incidence by 12.5% and achieved 43.75% control efficacy while maintaining host vigor. Histochemical analyses identified JL54-induced rapid hydrogen peroxide (H₂O₂) accumulation, extensive lignin deposition, and localized programmed cell death (PCD), indicative of a primed immune response. Transcriptomic analyses uncovered distinct temporal defense patterns: early-stage responses (0 h post-inoculation) were characterized by upregulation of cutin, suberin, and wax biosynthesis pathways, reinforcing physical barriers, whereas late-stage responses (12 h post-inoculation) were dominated by ribosome- and proteostasis-related pathways (e.g., heat shock proteins [HSPs], glutathione S-transferases [GSTs]) to mitigate cellular damage. Biochemical assays corroborated these findings, with JL54 colonization reducing membrane lipid peroxidation (27.2% decrease in malondialdehyde content) and significantly elevating the activity of key defense enzymes, including peroxidase (POD), phenylalanine ammonia-lyase (PAL), and GST. Phytohormone profiling implicated jasmonic acid (JA) as the central mediator of induced systemic resistance (ISR), with JL54-potentiated JA signaling preceding pathogen containment. Collectively, these results demonstrate that JL54 contributes to a coordinated defense strategy in L. olgensis, integrating structural reinforcement (cuticle/lignin), oxidative stress management, and JA-mediated immune priming. These insights advance the understanding of endophyte-conferred resistance in conifers and highlight JL54’s potential as a biocontrol agent for sustainable forestry.