Jiajin Tan

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.

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.

Fusarium acuminatum is recognized as the causative agent of root rot in many forestry and agricultural plants. In recent years, root rot and foliage blight caused by F. acuminatum have become widespread and severe in China, particularly affecting Dianthus chinensis. The infection mechanism of F. acuminatum remains a pressing area for research. A crucial approach to elucidating its pathogenic mechanisms involves the genetic modification of candidate genes, which necessitates effective transformation systems. Currently, protoplast-mediated transformation (PMT) serves as a valuable tool for studying plant-pathogen interactions and offers several advantages over conventional transformation methods. In this study, we employed the PMT technique to establish a transformation system for the F. acuminatum strain FDCY-5 due to its benefits such as ease of operation, low cost, high conversion efficiency, and broad applicability. We successfully developed a transformation system capable of producing abundant high-quality protoplasts from F. acuminatum and generating green fluorescent protein (GFP) transformants. To verify whether GFP was constitutively expressed, we utilized fluorescence microscopy alongside PCR technology. The results demonstrated that GFP was effectively transformed into the protoplasts of F. acuminatum and expressed successfully. The established protoplast transformation system for F. acuminatum provides a foundational platform for analyzing functional genes within infected host plants as well as understanding the molecular mechanisms underlying host plant infections by F. acuminatum.

In order to provide a highly feasible research pathway for the control of larch shoot blight, healthy larch branches and leaves were collected from 13 sampling sites in 8 provinces in China. The antagonistic endophytic bacteria obtained from the screening were used to carry out disease control experiments in potted seedlings. The safety evaluation test was conducted on the antagonistic bacteria. Subsequently, the strains with better preventive effect and high safety were identified by morphological and molecular methods. A total of 391 strains of endophytic bacteria were isolated from healthy larch branches and leaves. Seventy-eight strains of larch endophytic bacteria with antagonistic effect were obtained by primary sieving. Ten strains of endophytic bacteria with obvious antagonism were further obtained by secondary sieving, and all of them had an inhibition rate of more than 57%. Among them, strains YN 2, JL 6, NMG 23, and JL 54 showed the highest inhibition rate of 63.16%–65.08%, which was significantly different from the other treatments. The results of the pot test showed that 14 days after inoculation with the pathogen, strains YN 2 and JL 54 were more effective in the control of larch shoot blight, with the preventive effects reaching 57.7% and 50.0%, respectively. Strains JL 6 and JL 54 were biologically safe in the safety evaluation test. Therefore, strain JL 54 was selected for identification. It was identified as Bacillus amyloliquefaciens through morphological observation, 16S rDNA sequence, gyrB gene sequence and 16S rDNA- gyrB tandem feature sequence analysis. IMPORTANCE: Larch shoot blight is a widely distributed, damaging, and rapidly spreading fungal disease of forest trees that poses a serious threat to larch plantations. Endophytic bacteria have biological effects on host plants against pests and diseases, and they have a growth-promoting effect on plants. In this paper, we investigated for the first time the biocontrol effect of endophytic bacteria on larch shoot blight by screening endophytic bacteria with the function of antagonizing dieback fungi. Bacillus amyloliquefaciens JL 54 has a better prospect of biocontrol against larch shoot blight, which lays the foundation for the application of this bacterium in the future.

Kriechbaumerella dendrolimi (Hymenoptera, Chalcididae) is a dominant pupal parasitoid species of various significant pine caterpillars, including Dendrolimus houi Lajonquiere (Lepidoptera, Lasiocampidae), with great potential for utilization. So far, the mass rearing of K. dendrolimi has been successfully established using Antheraea pernyi (Lepidoptera, Saturniidae) pupae as alternative hosts and released in the forest to suppress D. houi populations. However, the outcome is still expected to be improved due to lower parasitism rates, which might be related to the autonomous immune function of A. pernyi pupae. In our study, we investigated the effects of K. dendrolimi parasitization on the immune responses of A. pernyi pupae by measuring the expression of key immune factors: superoxide dismutase (SOD), polyphenol oxidases (PPOs), Attacin, Lysozymes (LYSs), and serine proteases (PRSSs). Our results show that parasitization significantly upregulated these immune factors, with distinct temporal patterns observable between 4 and 48 h post-parasitization. This upregulation highlights a robust immune response, adapting over time to the parasitic challenge. These findings suggest that specific immune mechanisms in A. pernyi pupae are activated in response to K. dendrolimi, shedding light on potential targets for enhancing host resistance. Metabolomic analyses complemented these findings by illustrating the broader metabolic shifts associated with the immune response. Specifically, Attacin was significantly upregulated twice, hypothesizing that the parasitoid’s venom contains at least two parasitic factors. Metabolomics analysis revealed a significant metabolite difference within parasitized A. pernyi pupae. The highest number of differential expression metabolites (DEMs) was observed at 16 h post-parasitism (1184 metabolites), with fewer DEMs at 8 h (568 metabolites) and 32 h (693 metabolites), suggesting a close relationship between parasitism duration and the number of DEMs. These fluctuations reflected the fundamental process of immune interaction. KEGG enrichment results showed that the DEMs were mainly enriched in energy metabolism and immune-related pathways, indicating that parasitism is a process of continuous consumption and immune interaction in the host. These DEMs could also become future targets for regulating the immune functions of A. pernyi pupae and could provide reference data for optimizing mass-rearing techniques.

Bacillus pumilus plays a very important role in the biological control of plant diseases. In this study, we conducted indoor biocontrol experiments using the LYMC-3 strain of B. pumilus to assess its efficacy against poplar canker and tea gray blight. Simultaneously, we explored the optimal fermentation conditions for the production of inhibitory substances against Phomopsis macrospora and Pestalotiopsis vesicolor. Subsequently, the inhibitory substances were extracted crudely and subjected to a preliminary investigation of their properties. The results of the indoor biocontrol experiments showed that the LYMC-3 strain exhibited disease prevention rates of 44.12% and 48.59% against poplar canker and tea gray blight, respectively. The fermentation optimization experiment showed that a culture medium containing 10 g/L of glucose, 15 g/L of tryptone, and 5 g/L of sodium chloride favored the production of inhibitory substances via the LYMC-3 strain. Among the crude extracts obtained, the non-protein extracts demonstrated the most obvious inhibitory effects. Furthermore, the inhibitory substances exhibited high polarity, excellent thermal stability, and high sensitivity to strong acids and bases. This study establishes a theoretical foundation for the purification and identification of inhibitory substances using the LYMC-3 strain. Additionally, it contributes to elucidating biological control mechanisms and facilitates the application of the LYMC-3 strain in production.

In order to explore the changes in the fatty acid metabolism of Bursaphelenchus xylophilus under the treatment of Bacillus cereus NJSZ-13, the surface changes in lipid droplets were observed under a Zeiss microscope after the B. xylophilus was stained with modified Oil Red O. The triglyceride (TG) content in B. xylophilus was determined according to the TG kit instructions. The type and content of fatty acids in B. xylophilus were detected by gas chromatography–mass spectrometry (GC-MS); the stearyl coenzyme A of B. xylophilus was analyzed by real-time fluorescence quantitative PCR. The change in Bx-SCD (gene regulating stearyl CoA desaturase in B. xylophilus) expression was observed. The results showed that the lipid droplets of B. xylophilus treated with NJSZ-13 were broken to varying degrees, and the TGs in B. xylophilus decreased continuously. The total fatty acid content in the bodies of treated B. xylophilus decreased: the difference between the fermentation broth treatment and the control was extremely significant (p < 0.01); that between the fermentation filtrate and the control was significant (p < 0.05); and that between the bacterial suspension and the control was not significant (p > 0.05). Saturated fatty acids decreased in all treatments, but not significantly. Compared with the control group, the unsaturated fatty acid content in fermentation broth and fermentation filtrate treatments was extremely significantly reduced, and the unsaturated fatty acid content of the bacterial suspension group was significantly decreased, which indicated that NJSZ-13 mainly caused a decrease in the unsaturated fatty acids in B. xylophilus. The trend in changes in monounsaturated fatty acids and unsaturated fatty acids was the same, but for polyunsaturated fatty acids, the fermentation broth and fermentation filtrate treatments caused a significant decrease in content, but the bacterial suspension resulted in no significant change. The results showed that NJSZ-13 mainly caused a decrease in monounsaturated fatty acid content in B. xylophilus. In addition, the contents of C16:1, C18:1, and C18:2 fatty acids were significantly decreased after treatment with strain NJSZ-13 for 48 h, and the contents of C16:1, C18:1, C18:2, and C20:4 were extremely significantly decreased after the fermentation broth and filtrate treatments. The expression of Bx-SCD in B. xylophilus was significantly lower than that of the control (p < 0.0001). This study analyzed the changes in the content of related substances and relative gene expression in fatty acid metabolism of B. xylophilus treated with strain NJSZ-13, and preliminarily reveals the nematicidal mechanism of strain NJSZ-13 against B. xylophilus. This provides a theoretical basis for further exploration of the key cause of death induced by this strain in B. xylophilus.

Pine wood nematode disease is a destructive disease to the ecological environment and forest resources. The disease is produced by Bursaphelenchus xylophilus, and the onset rate is extremely fast and the pathogenesis of the disease is not clear. Prevention of this disease is still important in production, so it is of great significance to explore its early diagnosis. In this study, the changes of chlorophyll fluorescence dynamic induction parameters, non-photochemical quenching parameters and organic acid content in needles of 7-year-old-infected Pinus hwangshanensis were studied by chlorophyll fluorescence technology and organic acid mass combined technology. The results showed that in the chlorophyll fluorescence dynamic induction group, Fm/Fo, Fv/Fo, Fm, Psi_o and Pi_Abs showed a significant downward trend, while Phi_Do, ABS/RC, TRO/RC and DIO/RC showed a significant uptrend. In non-photochemical quenching, Fm showed a downward trend, while Qp_L1, Qp_L2, QY_L1, QY_L2 and QY_Lss showed a significant upward trend. In the change of organic acid content, ferulic acid, syringic acid, gallic acid and jasmonic acid showed a significant increasing trend, while benzoic acid and salicylic acid showed a first increasing trend and then decreasing trend. Experimental results show that pine wood nematode had significant effects on photosynthesis and the organic acid content of pine before it showed symptoms of the disease. After comprehensive analysis, chlorophyll fluorescence dynamic induction parameters Pi_Abs, Fm, Phi_Do, TRO/RC, Psi_o, non-photochemical quenching parameters QY_L2, Qp_L1, QY_Lss, QY_L1 and organic acids ferulic acid, syringic acid, benzoic acid and gallic acid contents are ideal indicators for early diagnosis of pine wood nematodiasis.

In previous research, the pine wood nematode Bxadh2 gene significantly increased its expression in pine seedlings inoculated with endophytic Bacillus cereus GD2 and pine wood nematode. To explore pine wood nematode Bxadh2 gene function, we cloned and analyzed its biological information, and we preliminarily studied its physiological function through RNA interference. We found that the Bxadh2 gene’s full CDS length is 1269 bp, which encodes 422 amino acids, and presents a relatively stable hydrophobic protein. The protein encoded by the Bxadh2 gene has no signal peptide or transmembrane structure, and it is an intracellular protein that does not participate in transmembrane movement. The RNAi interference results showed that when the pine wood nematode’s Bxadh2 gene was suppressed, its survival rate and fecundity significantly decreased, indicating that the expression of the Bxadh2 gene was related to the growth and development of pine wood nematodes.

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.