Application and methods of the bicolor long-beaked fungus Ob-BHS48 in enhancing plant resistance induction systems

Abstract

This invention discloses the application and method of the bicolor long-beaked fungus Ob-BHS48 in enhancing plant induced systemic resistance, belonging to the field of biocontrol technology. Through adjacent inoculation co-culture, no direct antagonistic or competitive effect was observed between the Ob-BHS48 strain and the Polish endothelial fungus strain CFCC53558. However, after inoculation of *Picea rubra* with the Ob-BHS48 strain, compared with uninoculated plants, a second inoculation with the Ep-53558 strain enhanced the disease resistance of *Picea rubra*, significantly reducing the area of ​​lesions on branches and trunks, similar to the effect of pretreatment with methyl jasmonate in increasing plant induced resistance. The accumulation of disease-resistant metabolites and the expression of disease-resistant genes were also upregulated. This indicates that the Ob-BHS48 strain can enhance the induced resistance of *Picea rubra*, achieving plant disease prevention. This invention broadens the application scope of dominant long-beaked fungi in forest disease control, opening up new avenues for developing biocontrol strategies for plant diseases. It has great application potential in forest protection and green agriculture, and can promote the green and sustainable development of these industries.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to the application and method of the two-colored long-beaked fungus Ob-BHS48 in enhancing plant resistance-inducing systems. Background Technology

[0002] Conifers have long been threatened globally by bark beetles and their associated long-beaked fungi. Conifers have evolved complex induced defense mechanisms to combat various biological stresses. When stressed, these mechanisms trigger a series of defense signal transduction processes, thereby activating the induced defense response. Non-pathogenic or weakly pathogenic microorganisms and their metabolites are typical inducers of this defense response, such as Bacillus, which induce systemic resistance. ) induces microorganisms and uses methyl jasmonate ( (represented by) Induced metabolites have shown great potential in green biological control in agriculture. Currently, the identification and research of fungi that activate induced defense responses in conifers are receiving increasing attention, which is crucial for developing green defense strategies against bark beetle damage.

[0003] Bark beetle-associated long-beaked fungi possess complex community structures and functions. Highly pathogenic endophytic sporophores (… As a pioneer species and the primary invasive host, it spreads through high-density bark beetle populations, and its spread is positively correlated with the severity of beetle infestation. During the colonization stage of low-density bark beetles, a close symbiotic relationship is formed with them. Some species become dominant and exhibit weaker pathogenicity to their host plants. It is well known that long-beaked fungi have many other potential roles and functions. Existing research indicates that the larch bark beetle is associated with pseudo-bicolor long-beaked fungi. It can improve the larch's resistance to Fuji long beak shell. Resistance to spruce resistance varies significantly among strains from different host sources, and not just any long-beaked fungus can enhance host resistance. Therefore, screening for long-beaked fungal strains that can induce resistance in spruce and clarifying their mechanisms of action and induction methods are crucial technical supports for constructing a green control system for spruce.

[0004] Spruce bark beetle ( (This is a species of beetle belonging to the order Coleoptera, subfamily Bark Boraeinae, and genus Bark Boraeus. It primarily damages the base to top of trees and can carry and transmit *Endothecia polioense*, a highly pathogenic fungus to spruce.) This fungus can cause symptoms such as canker, blue staining of sapwood, and dryness in coniferous hosts, and can lead to host death. Red spruce is affected by the bark beetle *Bark beetle* and its transmitted pathogens in Northeast my country. The main host tree species, The CFCC53558 strain (Ep-53558) was isolated by the inventors in the tunnels of the eight-toothed bark beetle in the mixed forest of red spruce and fir in Jingouling Forest Farm.

[0005] Currently, the main control techniques for the bark beetle of spruce and its associated fungi include creating mixed forests, removing weak trees, using tree traps, pheromone trapping, and chemical pesticide spraying or fumigation. These methods have drawbacks such as long control cycles, complex operations, strong environmental dependence, and the risk of pollution. my country has not yet implemented a systematic approach to controlling diseases transmitted by bark beetles, and there is an urgent need to develop a simple, effective, and environmentally friendly control method. The BHS48 strain (Ob-BHS48) is a bicolor long-beaked fungal strain isolated by the inventors in the tunnels of *Picea rubra* var. *rubra* in Wangqing County, Yanbian Korean Autonomous Prefecture, Jilin Province. Therefore, investigating whether the Ob-BHS48 strain can induce systemic resistance in plants, especially *Picea rubra*, and its effects on host resistance and induction methods, is of great significance for broadening the application of long-beaked fungi in forest disease control. Summary of the Invention

[0006] To achieve the above-mentioned technical objectives and effects, the present invention provides the following technical solution:

[0007] Application of the bicolor long-beaked fungus Ob-BHS48 in enhancing plant resistance-inducing systems. The Ob-BHS48 strain has the accession number CGMCC No.42479, is classified as Ophiostoma bicolor, and is deposited at the China General Microbiological Culture Collection Center on December 19, 2025.

[0008] Furthermore, the enhanced plant-induced resistance system is manifested in: increasing the expression levels of disease resistance R genes, disease resistance enzyme genes, and genes related to the sucrose metabolism pathway, increasing the accumulation of disease resistance-related metabolites, and / or reducing the area of ​​lesions on plant branches and trunks.

[0009] Furthermore, the disease-resistant R gene is MA_907632g0010 (PR1), and the disease-resistant enzyme genes include MA_10427891g0010 (CAT) and MA_96757g0010 (POX).

[0010] Furthermore, the gene related to the sucrose metabolism pathway is MA_113423g0010 (SUS).

[0011] Furthermore, the disease-resistant metabolites include amino acid metabolites and inositol phosphate metabolites; wherein the amino acid metabolites are selected from at least one of L-aspartic acid, L-threonine, D-aspartic acid, and D-phenylalanine; and the inositol phosphate metabolites are selected from at least one of 1D-inositol-1,4-bisphosphate and D-glucose-6-phosphate.

[0012] On the other hand, the present invention also provides a method for enhancing plant-induced resistance by inoculating plants with the above-mentioned Ob-BHS48 strain mycelium to enhance plant-induced resistance, thereby achieving disease control.

[0013] Furthermore, the culture conditions for the Ob-BHS48 strain were as follows: cultured in 2% MEA medium at 25°C in the dark for 7 days.

[0014] Furthermore, the specific inoculation operation includes: using a sterilized 6mm diameter cork punch to extract mycelial moss from the edge of the Ob-BHS48 colony; drilling a hole to the xylem at a height of 1.5m on one side of the plant trunk; inserting the mycelial moss into the inoculation hole; covering the bark; and sealing the inoculation site by wrapping with sealing film and fixing with tape.

[0015] Furthermore, the layout of the inoculation site is as follows: three rows of inoculation points are set at 5cm intervals in the vertical direction, with four inoculation points in each row and a 3cm interval between adjacent inoculation points.

[0016] Furthermore, the plant is red spruce, and the plant disease is spruce blue stain canker.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention discloses the application and method of the bicolor long-beaked fungus Ob-BHS48 in enhancing plant resistance induction systems. Using a co-culture method with adjacent inoculation, this invention found no antagonistic or competitive interaction between the Ob-BHS48 strain and the Polish endophora ep-53558. In further experiments, the Ob-BHS48 strain was first inoculated into the xylem of *Picea rubra*, followed by inoculation with *Picea rubra* ep-53558. The results showed a significant increase in the disease resistance of *Picea rubra*, and a marked reduction in lesion area. Metabolomics analysis was performed on the fourth day after secondary inoculation of *Picea rubra* with the Ob-BHS48 strain, MeJA pretreated, and untreated *Picea rubra* with the ep-53558 strain. The results showed that, similar to MeJA pretreatment, pretreatment with the Ob-BHS48 strain increased the accumulation of various disease-resistant compounds in the phloem of *Picea rubra*. Simultaneous RNA-seq analysis showed that the two pretreatments had essentially the same effect on gene expression in *Picea rubra*, but some differences also existed. We performed GO and KEGG enrichment analyses on common DEGs from the two comparison groups, revealing significant overlap in the genetic changes of the two pretreatments in spruce. Combined differential gene and metabolite KEGG co-enrichment revealed significant enrichment of genes involved in pathways such as phenylpropane, flavonoids, glycolysis / gluconeogenesis, and carotenoid biosynthesis in both pretreatments. These genes / metabolites are involved in plant defense responses. Subsequent real-time quantitative PCR (RT-qPCR) detection of disease resistance-related genes confirmed that pretreatment of the Ob-BHS48 strain enhanced the expression of disease resistance-related genes in red spruce, including disease resistance R genes, enzyme genes, and genes involved in disease resistance-related metabolite pathways. Attached Figure Description

[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 In this embodiment, the two-color long-beaked fungus Ob-BHS48 strain and the Polish endophytic fungus Ep-53558 strain were co-cultured adjacently; where green * represents the inoculation site of Ob-BHS48 strain or blank medium; red * represents the inoculation site of Ep-53558.

[0021] Figure 2 This example shows the lesion area diagram and lesion area statistics of the two-color long-beaked fungus Ob-BHS48 strain, MeJA pretreated and untreated red spruce after inoculation with Ep-53558;

[0022] Figure 3This example shows the differential metabolite changes four days after inoculation of Ep-53558 with the pretreated and untreated red spruce strain Ob-BHS48 (Ep4 vs ObEp4).

[0023] Figure 4 This example illustrates the KEGG enrichment pathway for the differential metabolites of Ep4 vs ObEp4.

[0024] Figure 5 This example shows the differential metabolite changes four days after MeJA-pretreated and untreated red spruce inoculated with Ep-53558 (Ep4 vs. MeJAEp4).

[0025] Figure 6 This example illustrates the KEGG enrichment pathway for differential metabolites of Ep4 vs MeJAEp4.

[0026] Figure 7 This is a Veen plot showing the differences in metabolites between Ep4 vs ObEp4 and Ep4 vs MeJAEp4 in this embodiment;

[0027] Figure 8 This example shows the KEGG enrichment pathway, a common differential metabolite of Ep4 vs ObEp4 and Ep4 vs MeJAEp4.

[0028] Figure 9 This example shows the differential gene changes between Ep4 and ObEp4 and between Ep4 and MeJAEp4.

[0029] Figure 10 This example shows the differential gene changes in Ep4 vs ObEp4 and Ep4 vs MeJAEp4.

[0030] Figure 11 In this embodiment, Ep4 vs ObEp4 and Ep4 vs MeJAEp4 share the GO enrichment pathway of DEGs.

[0031] Figure 12 In this embodiment, Ep4 vs ObEp4 and Ep4 vs MeJAEp4 share the KEGG enrichment pathway for DEGs.

[0032] Figure 13 This example shows the KEGG co-enrichment maps of differentially expressed genes and metabolites in Ep4 vs ObEp4 and Ep4 vs MeJAEp4.

[0033] Figure 14This example illustrates the effect of the two-colored long-beaked fungus Ob-BHS48 strain and MeJA pretreatment on the expression of resistance-related genes induced by red spruce. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] This invention employs a proximity inoculation co-culture method, using the long-beaked fungus Ob-BHS48 as the research material. It was found that Ob-BHS48 showed no antagonistic or competitive effect against Polish endophora Ep-53558. Pretreatment of *Picea rubra* with Ob-BHS48 followed by inoculation with *Picea rubra* Ep-53558 resulted in significant disease resistance in the *Picea rubra*, with a marked reduction in lesion area. Metabolomics and RNA sequencing (RNA-seq) analyses were performed on the fourth day after secondary inoculation of *Picea rubra* with Ob-BHS48, MeJA pretreated, and untreated *Picea rubra* with Ep-53558. The results showed that, compared to the control group and similarly to MeJA pretreatment, Ob-BHS48 pretreatment increased the accumulation of relevant disease-resistant compounds in the phloem of *Picea rubra*. Furthermore, while the effects of Ob-BHS48 and MeJA pretreatment on gene expression in *Picea rubra* were similar, differences also existed. We performed GO and KEGG enrichment analyses on common DEGs and found significant overlap in the genetic changes of spruce under the two pretreatments. DEGs from both groups were enriched in five biological processes, including environmental information processes. Combined differential gene and metabolite KEGG co-enrichment revealed significant enrichment of genes involved in pathways such as phenylpropane, flavonoids, glycolysis / gluconeogenesis, and carotenoid biosynthesis in both pretreatments. These genes / metabolites are involved in plant defense responses. Real-time quantitative PCR (RT-qPCR) detection of disease resistance-related genes in *Picea rubra* leaves showed that pretreatment of *Picea rubra* with the Ob-BHS48 strain enhanced the expression of disease resistance-related genes, such as disease resistance R genes, enzyme genes, and disease resistance-related metabolite pathway genes.

[0036] This invention provides the application of the long-beaked fungus Ob-BHS48 strain in enhancing plant resistance-inducing systems.

[0037] The Ob-BHS48 strain exhibits advantages in inducing systemic resistance, primarily in two aspects: First, this strain does not need to directly inhibit pathogen growth to exert its effects, fundamentally avoiding the selective pressure that leads to drug resistance in pathogens due to long-term exposure to antimicrobial substances, thus providing a more sustainable path for long-term disease control. Second, the plant defense response activated by it is not a localized, short-term stress response, but a systemic defense mechanism covering the entire plant, with a longer duration of defense. It can not only cope with the initial infection of pathogens but also enhance the plant's resistance to subsequent potential infections. At the same time, this ISR characteristic can also form a synergistic effect with other control methods, further improving control efficiency.

[0038] Furthermore, the enhancement of plant-induced system resistance involves increasing the expression levels of disease-resistant R genes and enzyme genes, increasing the expression levels of sucrose metabolism pathways, increasing the accumulation of disease-resistant metabolites, and / or reducing the area of ​​lesions on plant branches and trunks.

[0039] Furthermore, the disease-resistant genes include MA_907632g0010 (PR1), MA_10427891g0010 (CAT), and MA_96757g0010 (POX).

[0040] Furthermore, the disease-related metabolite pathway-related gene includes MA_113423g0010 (SUS).

[0041] The present invention also provides a method for enhancing the resistance of plant-induced systems to control plant diseases, wherein the method involves inoculating plants with Ob-BHS48 strain mycelium.

[0042] Furthermore, the application method involves using a sterilized cork punch with a diameter of 6 mm to extract mycelial moss from the edge of the Ob-BHS48 colony and inoculating it onto the bark of a red spruce tree at a height of 1.5 m on one side of the trunk.

[0043] Furthermore, the Ob-BHS48 strain of the long-beaked fungus was cultured for 7 days in the dark at 25°C on 2% MEA medium.

[0044] The specific method is as follows: The strain was cultured on 2% MEA medium in the dark at 25°C for 7 days. On a 25-year-old red spruce, at a height of approximately 1.5m on one side of the trunk, three rows were inoculated vertically at 5cm intervals, with four inoculation points per row, spaced 3cm apart. A sterile 6mm diameter cork punch was used to drill holes down to the xylem. The mycelium was then inserted into the inoculation holes using a sterile toothpick. The holes were then covered with bark, sealed with sealing film, and secured with tape to ensure good sealing of the inoculation site.

[0045] Furthermore, the plant in question is red spruce.

[0046] The invention will now be further described with reference to the accompanying drawings.

[0047] In the following embodiments and comparative examples of the present invention, the test spruce was obtained from 24-year-old red spruce with a diameter at breast height of 13.30±1.91cm. Seedlings were provided by Jiangshanjiao Experimental Forest Farm, Mudanjiang City, Heilongjiang Province (43°52′50″N, 129°2′55″E, 700 m above sea level).

[0048] All raw materials used in the following embodiments and comparative examples of the present invention are commercially available products.

[0049] Example 1: Co-culture of a bicolor long-beaked fungal strain with a Polish endophora strain:

[0050] The co-culture method of dichromatic long-beaked fungi and Polish endophora strain by adjacent inoculation: A 0.5 cm diameter perforator was used to punch holes in the culture plate of Polish endophora strain Ep-53558, and agar blocks from the edge of the colonies were inoculated into the center of a PDA plate. A 0.5 cm diameter perforator was also used to punch holes in the culture plate of dichromatic long-beaked fungi Ob-BHS48, and these blocks were inoculated into PDA plates approximately 2 cm away from Ep-53558 strains for co-culture. A blank culture medium was used as a control to observe the interaction between the dichromatic long-beaked fungi and Polish endophora strains.

[0051] The results of the co-culture experiment between the bicolor long-beaked fungal strain and the Polish endophora strain are as follows: Figure 1 As shown, compared with the control group, the bicolor long-beaked fungal strain had no effect on the growth of the Polish endophora strain. The hyphae of Polish endophora Ep-53558 could cover the surface of the Ob-BHS48 colony and grow, indicating that the bicolor long-beaked fungal Ob-BHS48 strain does not have a direct inhibitory or competitive effect on the growth of Polish endophora Ep-53558.

[0052] Example 2: Effects of the two-colored long-beaked fungus Ob-BHS48 strain and MeJA pretreatment on the disease resistance of red spruce:

[0053] To determine whether the bicolor long-beaked fungus strain Ob-BHS48 could enhance the disease resistance of red spruce, Ob-BHS48 and... Trees were pretreated with MEA (control) and cultured as a whole. After 35 days post-treatment (dpi), all pretreated trees were inoculated with the pathogenic fungus Ep-53558. Pathogenicity was assessed both after pretreatment and inoculation. The lesion area (length × width) was calculated by measuring the length and width of the lesion at each inoculation site. Each tree was considered an independent entity; therefore, for each 25-year-old tree, the sum of the lesion areas at all inoculation sites was calculated. Based on literature and preliminary experimental results, lesions at adjacent inoculation sites may merge into one large lesion over time. Therefore, if lesions around multiple inoculation sites were connected, the area of ​​the large lesion was measured. Subsequently, a one-way ANOVA was performed on the lesions produced by different strains using IBM SPSS Statistics 19, followed by post-hoc multiple comparisons using the LSD test, with the significance level set at [value missing]. =0.05.

[0054] 35 days after vaccination, compared to the control group (Ep), and... Pretreated red spruce ( Similarly, red spruce pretreated with Ob-BHS48 strain ( It also showed enhanced resistance, characterized by a decrease in pathogenicity induced by Ep-53558 strain 35 days after inoculation. Figure 2 In summary, these results indicate that the bicolor long-beaked fungus strain Ob-BHS48 can enhance the resistance of spruce.

[0055] Example 3 Metabolomics analysis of the two-color long-beaked fungus Ob-BHS48 strain and Differences in the accumulation of resistance-related metabolites in early pretreatment of red spruce:

[0056] Weigh 50 mg of vacuum-frozen Ob-BHS48 strain and Pretreated and untreated samples of red spruce phloem inoculated with Ep-53558 strain four days prior were placed in centrifuge tubes, and 1000 μL of a methanol:acetonitrile:water solution in a 2:2:1 ratio was added. The samples were vortexed for 30 s and then homogenized at 45 Hz for 10 min. They were then sonicated in an ice-water bath for 10 min and allowed to stand at -20℃ for 1 h. After centrifugation at 12000g for 15 min at 4℃, 500 μL of the supernatant was transferred to a new centrifuge tube. The extract was dried using a vacuum concentrator, and 160 μL of an acetonitrile:water 1:1 mixture was added to reconstitute the dried metabolite. The mixture was vortexed for 30 s, then sonicated in an ice-water bath for 10 min, and centrifuged at 12000g for 15 min at 4℃. 120 μL of the supernatant was transferred to a sample vial for analysis.

[0057] exist The system was used to analyze the sample extracts. An HSS-T3 column was used, with solvent A (0.1% formic acid and 5 mM ammonium acetate aqueous solution) and solvent B (acetonitrile containing 0.1% formic acid) as the mobile phase. A gradient program was employed: initial conditions were 98% A and 2% B, held for 1.5 min; within 5.0 min, the linear gradient was adjusted to 50% A and 50% B; within 9.0 min, the linear gradient was adjusted to 2% A and 98% B, held for 1 min. Within 1 min, the composition was adjusted to 98% A and 2% B, held for 3 min. The flow rate was set to 0.35 mL / min, the column temperature to 50 °C, and the injection volume to 2 μL. The electrospray ionization (ESI) source temperature was 550℃, and the ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode). Ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 55, and 35 psi, respectively, and the collision-induced ionization (CAD) parameter was set to a moderate level. Instrument tuning and mass calibration were performed using 10 and 100 μmol / L polypropylene glycol solutions in triple quadrupole (QQQ) and LIT modes, respectively. Multiple reaction monitoring (MRM) mode was used for QQQ scans, with nitrogen set to a moderate level. The declustering voltage (DP) and collision energy (CE) of the MRM ion pairs were further optimized. Specific MRM ion pairs were monitored for each eluent phase based on the metabolites eluted in each phase. Qualitative analysis was based on secondary spectroscopy, and quantitative analysis was performed using MRM technology. Differential metabolite screening criteria were FC>1, VIP>1, and P<0.05. Composite classification and path retrieval were performed using the KEGG, LipidMaps, and HMDB databases.

[0058] In Ob-BHS48 pretreated red spruce, 205 DAMs were observed 4 days after inoculation with the pathogen (Ep4 vs ObEp4), of which 114 were upregulated and 91 were downregulated (e.g., ...). Figure 3 (As shown). When 205 DAMs were mapped to the KEGG database, the top 5 enriched metabolic pathways were: biosynthesis of various alkaloids (ko00996), D-amino acid metabolism, arginine biosynthesis (ko00220), various plant secondary metabolite synthesis pathways, and plant hormone signal transduction (ko04075). Figure 4 (As shown).

[0059] In MeJA-pretreated red spruce, 234 DAMs were observed 4 days post-inoculation (Ep4 vs. MeJAEp4), of which 118 were upregulated and 116 were downregulated (e.g., ...). Figure 5 (As shown). 234 DAMs were mapped to the KEGG database, and the top 5 enriched metabolic pathways were C5-branched dibasic acid metabolism (ko00660), D-amino acid metabolism, flavonone and flavonol biosynthesis (ko00944), phenylalanine metabolism (ko00360), and alanine, aspartate, and glutamate metabolism (ko00250). Figure 6 (As shown).

[0060] To compare the differences in the accumulation of resistance-related metabolites induced by Ob-BHS48 strain and MeJA pretreatment in *Picea purpurea*, Wien analysis was performed on the differential metabolites between the two pretreatments and untreated *Picea purpurea*. The results showed that the two pretreatments of *Picea purpurea* had 114 identical DAMs (e.g., ...) 4 days after inoculation with *E. polonica* (Ep4 vs ObEp4 and Ep4 vs MeJAEp4). Figure 7 As shown), 114 DAMs were aligned to the KEGG database, and the top 5 enriched metabolic pathways were D-amino acid metabolism, carbon fixation in photosynthesis, biosynthesis of flavonoids and flavonols, aminoacyl biosynthesis, purine metabolism (ko00230) (as shown). Figure 8 (As shown). In summary, similar to pretreatment of MeJA, pretreatment of the Ob-BHS48 strain increased the accumulation of relevant resistance metabolites in spruce.

[0061] Example 4: RNA-seq analysis of the similarities and differences in the expression of genes related to resistance to red spruce induced by the two-colored long-beaked fungus Ob-BHS48 and MeJA pretreatment:

[0062] The selected samples included phloem samples of *Picea abies* inoculated with *Ep-53558* strain four days after pretreatment with *Ob-BHS48* and *MeJA*, and samples without pretreatment. Three biological replicates were randomly selected for each treatment. Transcriptome sequencing was performed at BMI (Beijing, China). Clean data were aligned to the *P. abies* reference genome for analysis and annotation using HISAT2 software, and aligned reads were assembled and quantified using StringTie software. Differentially expressed genes (DEGs) were analyzed using DESeq2 software, with a standard of |log2(fold change)| ≥ 1 and p-value ≤ 0.01. Differentially expressed genes (DEGs) were compared with the GO database (http: / / www.blast2go.com / b2ghome). KEGG enrichment analysis was performed using the KOBAS database and clusterProfiler software.

[0063] The number of differentially expressed genes (DEGs) between the Ep group and the ObEp group was 4493 (2001 upregulated and 2492 downregulated), and the number of DEGs between the Ep group and the MeJAEp group was 5665 (2821 upregulated and 2844 downregulated). Figure 9 (As shown). The two comparison groups shared 2609 DEGs, indicating that the two pretreatments had similar effects on spruce gene expression, but differences also existed (e.g., ...). Figure 10 (As shown). We performed GO and KEGG enrichment analyses on the common DEGs of the two groups (as shown). Figure 11 and Figure 12 (As shown). The results showed that the two pretreatments significantly overlapped in their effects on the genetic changes in spruce. Furthermore, the DEGs from both groups were co-enriched, covering pathways such as terpene and polyketide metabolism and the synthesis of secondary metabolites. These results indicate that terpene metabolism plays a central role in the enhanced defense initiation process of Ob-BHS48 pretreatment in spruce. In the plant-pathogen interaction pathway, 113 genes are involved in regulating 26 key substances, including calcium-dependent protein kinases, LRR receptor-like serine / threonine protein kinase FLS2, and resistance proteins (RPM1, RPS2, RPS4), which are involved in the perception of resistance signals and the defense response. In summary, similar to the increase in induced resistance in plants after MeJA pretreatment, pretreatment of the dichroic long-beaked fungus Ob-BHS48 also increased the expression of spruce-related resistance genes. These changes in gene expression profiles may be core factors regulating the formation of spruce defense initiation.

[0064] Example 5: KEGG co-enrichment analysis of differentially expressed genes and metabolites to differentiate the resistance of the two-color long-beaked fungus Ob-BHS48 strain and MeJA pretreatment-induced red spruce:

[0065] like Figure 13As shown, samples of *Picea rubra* pretreated with Ob-BHS48, 4 days after re-inoculation with Ep-53558, were compared with untreated healthy *Picea rubra* samples 4 days after inoculation with Ep-53558. KEGG co-enrichment analysis of differentially expressed genes and metabolites identified co-enriched metabolic pathways. Similarly, samples of *Picea rubra* pretreated with MeJA, 4 days after re-inoculation with Ep-53558, were compared with untreated healthy *Picea rubra* samples 4 days after inoculation with Ep-53558. KEGG co-enrichment analysis of differentially expressed genes and metabolites identified co-enriched metabolic pathways. Genes related to phenylpropane, flavonoids, glycolysis / gluconeogenesis, and carotenoid biosynthesis were significantly enriched in both pretreatments. These genes / metabolites are involved in plant defense responses. In Ep vs ObEp, genes related to terpene synthesis were significantly enriched, but this pathway was not included in the significantly enriched genes in Ep vs MeJAEp. This suggests that terpene synthesis-related genes may be key genes in inducing plant defense and alertness in attenuated virulent fungi.

[0066] Example 6: Quantitative real-time PCR analysis of the effects of two-color long-beaked fungus Ob-BHS48 strain and MeJA pretreatment on the expression of resistance-related genes in red spruce:

[0067] This example selected four DEGs involved in fungal stress response and used the CFX96™ real-time system (Bio-Rad Laboratories, USA) and SYBR Green FP205 kit (Tiangen, China) to quantitatively analyze the expression levels of these four genes in phlox subsp. erythrorhizon phlox spp. ... Relative expression levels were calculated using a method that normalizes the expression using the actin gene (NCBI accession number: AAF03692) as an internal reference. Primer sequences were designed using Primer 5.0 software and synthesized by a company called Sangon Biotech.

[0068] Quantitative results such as Figure 14 As shown, due to the large FPKM value, it was transformed using the lg method, which may result in negative values. However, this does not affect the observation of the relative gene expression in different treatments. Although the expression levels of FPKM and RT-qPCR values ​​differed in different treatments according to transcriptome data, the expression patterns of these four DEGs in RT-qPCR were consistent with the transcriptome results, demonstrating that the expression patterns of the four genes have high reliability. Figure 14(As shown). Compared with the control, in Ob-BHS48 strain and MeJA pretreatment, the expression of MA_10427891g0010(CAT), MA_113423g0010(SUS), MA_907632g0010(PR1), and MA_96757g0010(POX) was upregulated. Among them, MA_907632g0010(PR1) is a disease resistance R gene, MA_10427891g0010(CAT) and MA_96757g0010(POX) are disease resistance enzyme genes, and MA_113423g0010(SUS) is a gene related to the disease resistance-related metabolite pathway.

[0069] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0070] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The application of the bicolor long-beaked fungus strain Ob-BHS48 in enhancing plant resistance-inducing systems, characterized in that, The preservation number of the Ob-BHS48 strain is CGMCC No. 42479.

2. The application according to claim 1, characterized in that, The enhanced plant-induced resistance system is characterized by: increasing the expression levels of disease resistance R genes, disease resistance enzyme genes, and genes related to the sucrose metabolism pathway, increasing the accumulation of disease resistance-related metabolites, and / or reducing the area of ​​lesions on plant branches and trunks.

3. The application according to claim 2, characterized in that, The disease-resistant R gene is MA_907632g0010 (PR1), and the disease-resistant enzyme genes include MA_10427891g0010 (CAT) and MA_96757g0010 (POX).

4. The application according to claim 2, characterized in that, The gene associated with the sucrose metabolism pathway is MA_113423g0010 (SUS).

5. The application according to claim 2, characterized in that, The disease-resistant metabolites include amino acid metabolites and inositol phosphate metabolites; wherein the amino acid metabolites are selected from at least one of L-aspartic acid, L-threonine, D-aspartic acid and D-phenylalanine; and the inositol phosphate metabolites are selected from at least one of 1D-inositol-1,4-bisphosphate and D-glucose-6-phosphate.

6. A method for enhancing plant-induced resistance systems, characterized in that, Disease control can be achieved by pretreating plants with the Ob-BHS48 strain mycelium as described in claims 1-5 to enhance the plant's induced system resistance.

7. The method according to claim 6, characterized in that, The culture conditions for the Ob-BHS48 strain were as follows: cultured in 2% MEA medium at 25°C in the dark for 7 days.

8. The method according to claim 6, characterized in that, The specific inoculation procedure includes: using a sterilized 6mm diameter cork punch to extract mycelial moss from the edge of the Ob-BHS48 colony; drilling a hole to the xylem at a height of 1.5m on one side of the plant trunk; inserting the mycelial moss into the inoculation hole; covering the bark; and sealing the inoculation site by wrapping with sealing film and fixing with tape.

9. The method according to claim 8, characterized in that, The layout of the inoculation sites is as follows: three rows of inoculation points are set at 5cm intervals in the vertical direction, with four inoculation points in each row and a 3cm interval between adjacent inoculation points.

10. The method according to any one of claims 6-9, characterized in that, The plant in question is red spruce, and the plant disease is spruce blue stain canker.

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