Paenibacillus elgii with broad-spectrum antagonistic activity and application thereof in eucalyptus disease prevention

Abstract

The application discloses a Paenibacillus elgii with broad-spectrum antagonistic activity and application thereof in eucalyptus disease prevention and treatment, and belongs to the field of forest disease biological prevention and agricultural microbial technology. The application provides a Paenibacillus elgii SHP-12 from eucalyptus rhizosphere soil, and the preservation number of the Paenibacillus elgii SHP-12 is CGMCC No. 39680. Experimental results show that the Paenibacillus elgii SHP-12 has obvious antagonistic effects on multiple eucalyptus pathogenic bacteria such as fusarium oxysporum, colletotrichum gloeosporioides, phytophthora palmivora, pseudoplectania nigrella and pseudomonas syringae, and is a biocontrol strain with broad-spectrum antagonistic activity, and can be used for preparing a biological control agent for preventing and treating eucalyptus root rot, eucalyptus dieback, eucalyptus scorch disease and eucalyptus bacterial wilt. The application provides a new strain resource for eucalyptus disease prevention and treatment, and has a good application prospect in the field of eucalyptus disease green prevention and control.

Description

Technical Field

[0001] This invention relates to the fields of biological control of forest tree diseases and agricultural microbial technology, and in particular to a Bacillus edilum with broad-spectrum antagonistic activity and its application in the control of eucalyptus diseases. Background Technology

[0002] Eucalyptus is an important fast-growing timber species, characterized by rapid growth, strong adaptability, and high economic value, playing a vital role in timber production, the pulp industry, and ecological construction. With the continuous expansion of eucalyptus plantations, diseases caused by various pathogenic microorganisms have gradually become a significant factor restricting the healthy growth of eucalyptus and the development of the industry. Currently, several pathogens have been reported to infect eucalyptus and cause different types of diseases. Among them, *Fusarium oxysporum* (…) Fusarium oxysporum ) and Phytophthora camphorata ( Phytophthora cinnamomi ) can cause eucalyptus root rot; Colletotrichum anthracnose ( Colletotrichum gloeosporioides ) can cause eucalyptus shoot blight, Pseudodendron calcarina ( Calonectria pseudoreteaudii ) can cause eucalyptus scorch disease; Solanaceae Raulella ( Ralstonia solanacearum These pathogens can cause diseases including bacterial wilt in eucalyptus trees; these pathogens not only seriously affect the growth of eucalyptus seedlings and the health of forest stands, but also threaten other trees or crops.

[0003] Currently, the control of eucalyptus diseases mainly relies on chemical agents, but long-term use can easily lead to drug resistance in pathogens and cause environmental pollution, pesticide residues, and soil microecological imbalance. Therefore, developing safe, efficient, and environmentally friendly microbial agents is of great value in promoting the green control of eucalyptus diseases. Currently, *Bacillus edilum* (a type of eucalyptus) derived from healthy eucalyptus tissue is a promising candidate. Paenibacillus elgii Research reports on the biological control of eucalyptus diseases are scarce, especially regarding biocontrol agents with broad-spectrum antagonistic activity. To address these shortcomings in existing technologies, there is an urgent need to provide a strain of *Bacillus edilum* derived from healthy eucalyptus tissue that exhibits broad-spectrum antagonistic activity against various important eucalyptus pathogens, and to utilize it for the green control of eucalyptus diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus edodes with broad-spectrum antagonistic activity and its application in the control of eucalyptus diseases, so as to solve the problems existing in the prior art. The Bacillus edodes SHP-12 provided by this invention has a strong antagonistic effect on five pathogens and can be used for green biological control of eucalyptus diseases.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of *Bacillus edodes* (…). Paenibacillus elgiiSHP-12, the Egeriazophora SHP-12, was deposited on July 13, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39680, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides the application of the above-mentioned Egeriazophora SHP-12 in the preparation of products for the prevention and control of eucalyptus diseases.

[0007] Furthermore, the eucalyptus diseases include eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt.

[0008] The present invention also provides a product for preventing and controlling eucalyptus diseases, with the above-mentioned Bacillus edilum SHP-12 as the active ingredient.

[0009] This invention also provides the application of the above-mentioned *Bacillus erythropoietinus* SHP-12 or the above-mentioned product in inhibiting eucalyptus pathogens, said eucalyptus pathogens including *Fusarium oxysporum* (…). Fusarium oxysporum ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), Phytophthora camphorata ( Phytophthora cinnamomi ), Pseudomonas calceolus ( Calonectria pseudoreteaudii ) and Solanaceae Raulella ( Ralstonia solanacearum ).

[0010] The present invention also provides a method for inhibiting eucalyptus pathogens, comprising the step of applying the above-mentioned Bacillus edilum SHP-12 or the above-mentioned product; The eucalyptus pathogens include Fusarium oxysporum, Colletotrichum gloeosporioides, Phytophthora camphorata, Pseudomonas calceolaria, and Solanaceae Raulella.

[0011] The present invention also provides the application of the above-mentioned Bacillus edilum SHP-12 or the above-mentioned product in the prevention and control of eucalyptus diseases, including eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt.

[0012] The present invention also provides a method for preventing and controlling eucalyptus diseases, including the step of applying the above-mentioned Bacillus edodes SHP-12 or the above-mentioned product; The eucalyptus diseases mentioned include eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt.

[0013] The present invention also provides the application of the above-mentioned Bacillus edilum SHP-12 or the above-mentioned product in promoting eucalyptus growth.

[0014] The present invention also provides a method for promoting eucalyptus growth, comprising the step of applying the above-mentioned Bacillus edodes SHP-12 or the above-mentioned product.

[0015] The present invention discloses the following technical effects: This invention provides a strain of *Bacillus edodes* SHP-12 from the rhizosphere soil of eucalyptus trees, with the preservation number CGMCC No. 39680. Experimental results show that *Bacillus edodes* SHP-12 exhibits significant antagonistic activity against various eucalyptus pathogens, including *Fusarium oxysporum*, *Colletotrichum gloeosporioides*, *Phytophthora camphorata*, *Pseudomonas aeruginosa*, and *Raulella solanaceae*. It is a biocontrol strain with broad-spectrum antagonistic activity and can be used to prepare biological control agents for eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt. This invention provides a new strain resource for the control of eucalyptus diseases and has promising application prospects in the field of green control of eucalyptus diseases. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the 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.

[0017] Figure 1 Bacillus edilum ( Paenibacillus elgii The images show the morphological characteristics of SHP-12 cultured on LB medium; where A is a macroscopic colony morphology image; B is a single colony morphology image; and C is a Gram staining result image. Figure 2 Phylogenetic tree diagram of Bacillus edilum SHP-12; Figure 3 The active fermentation broth of Bacillus erythropoietin SHP-12 is used to target Fusarium oxysporum ( Fusarium oxysporum The results of the influence of the growth curve on the figure; Figure 4 The image shows the inhibition bands of *Bacillus erythropoietinus* SHP-12 against *Fusarium oxysporum*; where A represents the treatment group and B represents the control group. Figure 5 Bacillus erythropoietinus SHP-12 is a counterpart to Colloidal anthrax ( Colletotrichum gloeosporioides The inhibition band diagram of (A) is shown; where A is the treatment group and B is the control group. Figure 6 For Egeriazolidinedioides SHP-12 against Phytophthora camphorata ( Phytophthora cinnamomi Inhibition band diagram; where A is the treatment group and B is the control group; Figure 7 For Egeriazolidinedioides SHP-12 against Pseudomonas calceolaria ( Calonectria pseudoreteaudii The inhibition band diagram of (A) is shown; where A is the treatment group and B is the control group. Figure 8Bacillus erythropoietinus SHP-12 against Solanaceae Raulella ( Ralstonia solanacearum The inhibitory effect of ) on the transparent circle diagram; where A is the treatment group and B is the control group. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The experimental data in the specific embodiments of the present invention are all derived from at least three independent repeated trials. The results are expressed as mean ± standard deviation (Mean ± SD), and P < 0.05 indicates that the difference is statistically significant.

[0024] Example 1: Isolation, screening, and molecular identification of antagonistic strains 1. Collection of rhizosphere soil samples Soil samples were collected from eucalyptus plantations in Qingyuan City, Guangdong Province. Before sampling, surface debris, fallen leaves, and weeds were removed. Rhizosphere soil (including fine roots) was excavated from a depth of 30 cm within a 50-100 cm radius of the eucalyptus trunk. The soil samples were placed in 50 mL sterile centrifuge tubes and quickly stored in a foam incubator containing dry ice for low-temperature preservation before being transported back to the laboratory for subsequent separation operations.

[0025] 2. Preparation of the test culture medium The culture medium formulation used in this embodiment is as follows: NA medium (beef extract peptone agar): beef extract powder 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, agar powder 15.0 g / L.

[0026] LB medium (Luria-Bertani agar): tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 5.0 g / L, agar powder 15.0 g / L.

[0027] PDA medium (potato glucose agar): 200.0 g / L peeled potatoes (boiled and juiced), 20.0 g / L glucose, 20.0 g / L agar powder.

[0028] V8 medium: V8 vegetable juice 100.0 mL / L, calcium carbonate 3.0 g / L, agar powder 20.0 g / L.

[0029] CMA medium (corn flour agar): 10.0 g / L corn flour, 15.0 g / L agar powder.

[0030] After the above culture media are prepared, distilled water is added to bring the volume to 300ml, and the media is sterilized in a high-pressure steam sterilizer at 121℃. After cooling, the media is ready for use.

[0031] 3. Isolation and purification of antagonistic strains The collected rhizosphere soil samples were added to a sterile conical flask, along with an appropriate amount of sterile water and sterile steel balls. The mixture was shaken at 150 r / min for 20 min to prepare a soil suspension. The suspension was then subjected to 10... -1 -10 -4 Serial dilutions were performed, with 80 μL each spread onto NA, LB, PDA, V8, and CMA solid agar plates, and incubated at 28°C for 1–2 days. After colony growth, single colonies of different morphologies were picked and purified by streak plating to obtain pure cultures. The purified strains were stored in 50% glycerol at -20°C for later use.

[0032] 4. Activation of Eucalyptus Root Rot Pathogen The tested pathogens were *Fusarium oxysporum* and *Phytophthora camphorata*. The pathogens were inoculated onto PDA medium and incubated at 28°C until vigorous colony growth was observed. Mycelial cakes were then cut from the edge of the colonies using a 4 mm diameter punch for subsequent antagonistic experiments.

[0033] 5. Primary and secondary screening of antagonistic strains Antagonistic strains were screened using the plate confrontation method. Pathogen mycelial discs (4 mm in diameter) were inoculated in the center of PDA plates, and test strains were symmetrically inoculated on both sides of the discs. A control group inoculated only with pathogens served as the blank control. The plates were incubated at 28°C. When the control colonies nearly covered the entire plate, the inhibition of mycelial growth was observed, and strains with antagonistic activity were screened. The candidate strains obtained from the initial screening were then re-screened using the same method, with three replicates for each treatment. After incubation, the diameter of the pathogen colonies was measured, and the mycelial growth inhibition rate was calculated using the following formula: Relative inhibition rate = (control colony diameter - treatment colony diameter) / control colony diameter × 100%; Colony diameter = hyphae diameter - colony block diameter.

[0034] The results showed that strain SHP-12 inhibited Fusarium oxysporum and Phytophthora camphorata by 73.53% and 78.89% respectively, exhibiting strong antagonistic activity and significantly inhibiting the mycelial growth of the pathogens.

[0035] 6. Morphological and physiological-biochemical identification of strain SHP-12 Morphological observation and physiological and biochemical characteristics analysis of strain SHP-12 were performed according to the *Handbook of Systematic Identification of Common Bacteria*. Gram staining was performed according to standard methods, and the results are as follows: Figure 1 As shown, strain SHP-12 is a Gram-positive bacterium with short rod-shaped cells. This strain can grow well on both LB and TSA media. On LB solid medium, it forms milky white, round colonies with neat edges, smooth and moist surfaces, and a glossy appearance. Other indicators are shown in Table 1.

[0036] Table 1. Biological characteristics of strain SHP-12 7. Molecular biological identification of strain SHP-12 Genomic DNA of strain SHP-12 was extracted using a boiling and heating method. The specific procedures are as follows: Take 1.5 mL of overnight cultured bacterial solution, centrifuge at 13000 r / min for 15 min, discard the supernatant, resuspend the precipitate in sterile water, heat and lyse it in a boiling water bath, centrifuge it, and take the supernatant as a PCR amplification template.

[0037] PCR amplification was performed using universal primers for bacterial 16S rDNA: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2).

[0038] The PCR reaction system (25 µL) includes: 4 µL DNA template, 12.5 µL 2×EasyTaqMasterMix, 1 µL forward primer 27F, 1 µL reverse primer 1492R, and ddH2O to bring the total volume to 25 µL.

[0039] The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 30 cycles; 72℃ final extension for 10 min.

[0040] After verification by agarose gel electrophoresis, the amplified products were sent to Beijing Nuosai Genome Research Center Co., Ltd. for Sanger bidirectional sequencing.

[0041] The 16S rDNA sequence of strain SHP-12 is shown in SEQ ID NO.3.

[0042] SEQ ID NO.3: GGCTCCTTGCGGTTACCCCACCGACTTCGGGTGTTGTAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCAATTCCGACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGACCGGCTTTCTTAGGATTGGCTCCATCTCGCGACTTCGCTTCCCGTTGTACCGGCCATTGTAGTACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCAACTTAGAGTGCCCAACTTAATGCTGGCAACTAAGTTCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCTCTCTGTCCCGAAGCTCCTATCTCTAGGAATTCAGAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATACTCCACTGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGAGCGTACTCCCCAGGCGGAGTGCTTATTGTGTTTACTTCGGCACCAAGGGTATCGAAACCCCTAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGGCCAGAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCGCTTTCCTCTCCTGCACTCAAGCCACCCAGTTTCCAGTGCGAACCGAGGTTGAGCCTCGGGCTTAAACACCAGACTTAAGCGGCCGCCTGCGCGCGCTTT.

[0043] After the sequencing results are assembled using SEQMAN software, and subjected to multiple alignment and trimming using MEGA6.0 software, a phylogenetic tree is constructed using the Neighbor-Joining method, as shown in Figure 2 the figure.

[0044] Based on the comprehensive morphological characteristics, physiological and biochemical properties, and 16S rDNA phylogenetic analysis, strain SHP-12 was identified as a type of *Bacillus edilum* (…). Paenibacillus elgii It was named Egeriazolinone SHP-12.

[0045] 8. Strain preservation The present invention is a type of Bacillus edilum ( Paenibacillus elgii SHP-12 was deposited on July 13, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39680, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Example 2: Inhibitory effect of Bacillus erythropoietin SHP-12 sterile fermentation broth on the growth of Fusarium oxysporum. After activating Bacillus edilum SHP-12, a single colony was picked and inoculated into 200 mL of LB liquid medium. The colony was then cultured at 30℃ and 180 r / min for 5 days to obtain the SHP-12 fermentation broth.

[0047] The SHP-12 fermentation broth was filtered twice through a 0.22 μm microporous membrane to obtain sterile SHP-12 fermentation broth. Fusarium oxysporum was inoculated onto PDA solid medium and cultured. After colony growth, a spore suspension was prepared.

[0048] Treatment and control groups were set up. In the treatment group, 16 mL of SHP-12 sterile fermentation broth was added to 40 mL of PDB liquid medium and inoculated with 80 μL of Fusarium oxysporum spore suspension. In the control group, PDB liquid medium was used instead of SHP-12 sterile fermentation broth, while other conditions remained the same. Each treatment was performed in triplicate and cultured at 30℃ and 180 r / min with shaking. Samples were taken every 4 h during the culture process to measure the absorbance (OD) of the culture medium. 600 ), plot the growth curve of Fusarium oxysporum.

[0049] like Figure 3 As shown, the results indicate that, compared with the control group, the addition of SHP-12 sterile fermentation broth significantly inhibited the growth of Fusarium oxysporum, suggesting that SHP-12 sterile fermentation broth has a potential biocontrol effect on eucalyptus root rot caused by Fusarium oxysporum.

[0050] Example 3: Determination of the broad-spectrum antibacterial activity of Bacillus edodes SHP-12 against major pathogens of eucalyptus diseases. This example evaluates the inhibitory effect of *Egeriazolinium* SHP-12 on various eucalyptus disease-related pathogens. The tested strain was *Egeriazolinium* SHP-12, and the five eucalyptus disease-related pathogens were all obtained from the China Forestry Microbial Culture Collection Center, including *Fusarium oxysporum* (…). Fusarium oxysporum ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), Phytophthora camphorata ( Phytophthora cinnamomi ), Pseudomonas calceolus ( Calonectria pseudoreteaudii ) and Solanaceae Raulella ( Ralstonia solanacearum ).

[0051] Single colonies of SHP-12 were picked and inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 48 h to prepare a concentration of 1×10⁻⁶. 8 CFU / mL bacterial suspension. Four pathogenic fungi, excluding *Raulella solanaceae*, were inoculated onto PDA medium. After the hyphae had fully colonized the plate, 4 mm diameter mycelial discs were collected from the edge of the colony and inoculated into the center of the PDA plate. Sterile filter paper discs soaked in SHP-12 bacterial suspension were symmetrically placed 2.5 cm to each side of the mycelial discs, with three replicates. Filter paper discs soaked in LB liquid medium served as a control.

[0052] *Raouli* of the Solanaceae family was inoculated onto LB liquid medium and cultured for 24 h to prepare a pathogen suspension. The pathogen suspension was then mixed with melted LB solid medium to prepare bacterial plates (volume ratio 1:10). Filter paper discs soaked in SHP-12 bacterial suspension were placed in the center of the plates, with filter paper discs containing LB liquid medium serving as a control. All treated plates were incubated at 28℃ for 7 days. Pathogen growth was observed, and the colony diameter in each treatment group was measured using the cross-crossing method. The expansion diameter of the colonies in two perpendicular directions was measured, and the average value (cm) was taken. The inhibition rate was calculated using the following formula: ; like Figure 4-Figure 8 As shown in Table 2, the results indicated that the growth of all five pathogens was significantly inhibited after SHP-12 treatment compared to the control group. Specifically, the inhibition rates for *Fusarium oxysporum*, *Colletotrichum gloeosporioides*, *Phytophthora camphorata*, *Pseudomonas aeruginosa*, and *Raulella solanaceae* reached 79.53%, 71.26%, 78.89%, 77.14%, and 73.33%, respectively.

[0053] This indicates that Bacillus edilum SHP-12 has a strong antagonistic effect on a variety of eucalyptus disease-related pathogens, exhibiting broad-spectrum antibacterial properties, and has good application prospects in the biological control of eucalyptus diseases.

[0054] Table 2 Pathogen Inhibition Rate Example 4: Effect of SHP-12 on chlorophyll content in eucalyptus seedlings under pathogen stress The tested pathogens were Fusarium oxysporum and Phytophthora camphorata, abbreviated as 40 and 79, respectively.

[0055] The tested strains included growth-promoting bacteria SHP-0-1-118 ( Pseudomonas eucalyptoterra The paper, Liu, T., Jiang, Y., Zhang, W. et al., has been published. It discusses a sustainable solution for eucalyptus cultivation under environmental stresses. Pseudomonas species with plant growth-promoting traits. Plant Soil (2025). https: / / doi.org / 10.1007 / s11104-025-07505-4) and antagonistic bacteria SHP-12 (i.e., the Egeriaceae Bacillus SHP-12 of the present invention) were treated individually or in combination.

[0056] Take fresh functional leaves from eucalyptus seedlings, wipe them clean with a damp gauze, remove the midrib, and cut them into pieces about 1 mm thick. 2 Weigh the fragments accurately and place them in a 2 mL centrifuge tube. Add approximately 10 mg of calcium carbonate powder and two 3 mm diameter stainless steel beads, and tighten the cap. Fix the centrifuge tube on a vortex mixer and grind at 2800-3000 rpm for 5 min, until the leaf residue turns white and the liquid turns dark green. Remove the steel beads and rinse their surface with 0.5 mL of 95% ethanol, returning the washings to the tube. Balance the mixture and centrifuge at 4000 rpm for 5 min. Carefully transfer the supernatant to a 10 mL brown volumetric flask. Resuspend the precipitate in 2.0 mL of 95% ethanol by vortexing, centrifuge under the same conditions, and combine the supernatants; repeat the washing once. Finally, bring the volume to 10 mL with 95% ethanol and invert to mix. Using 95% ethanol as a blank, measure the absorbance at 665 nm, 645 nm, and 652 nm.

[0057] The experiment was divided into two main groups based on the pathogen: 40-group and 79-group. Each main group was further divided into three subgroups: CK group, 12-group, and M group. The CK group was inoculated with the pathogen, the 12-group was inoculated with the pathogen and the antagonistic bacterium SHP-12, and the M group was inoculated with the pathogen, the antagonistic bacterium SHP-12, and the growth-promoting bacterium SHP-0-1-118. There were a total of 6 treatment groups: 40-CK, 40-12, 40-M, 79-CK, 79-12, and 79-M.

[0058] Each treatment group was cultured under the same conditions for 21 days, and leaves were collected at the same time for chlorophyll content determination.

[0059] Table 3. Absorbance values ​​and chlorophyll content (mg / L) of each treatment group Table 3 shows that the effect of *Bacillus edodes* SHP-12 on increasing chlorophyll content in eucalyptus leaves under pathogen stress varied among different pathogen lines. In the pathogen 79 system, SHP-12 alone (79-12) showed the most significant effect, with a total chlorophyll content of 5.496 mg / L, far exceeding the control's 2.938 mg / L, directly alleviating the pathogen's inhibition of chlorophyll synthesis. In the pathogen 40 system, the combined treatment of SHP-12 and the growth-promoting bacterium SHP-0-1-118 (40-M) resulted in a total chlorophyll content of 11.623 mg / L, significantly exceeding the control (7.093 mg / L) and the antagonistic bacterium treatment alone, demonstrating a synergistic effect. This antagonistic bacterium can be used in combination with the growth-promoting bacterium. All treatments showed significant differences from the control, with the 40-M and 79-12 groups exhibiting the most pronounced promoting effects. In some systems, combining SHP-12 with growth-promoting bacteria can further enhance its growth-promoting effect. Overall, Bacillus edodes SHP-12 can effectively improve the chlorophyll content of eucalyptus leaves under pathogen stress, exhibiting both good biocontrol and growth-promoting potential.

[0060] Example 5: Potted Plant Live Control Experiment This embodiment evaluates the control effect of Bacillus erygii SHP-12 alone and in combination with growth-promoting bacteria SHP-0-1-118 on eucalyptus root rot caused by Fusarium oxysporum and Phytophthora indica through a pot inoculation experiment.

[0061] The soil was sterilized at 121℃ under high pressure and then used for later use. 100 g of sterilized soil was added to each flowerpot. Eucalyptus seedlings of uniform growth were selected, their roots were washed, and they were transplanted into flowerpots. They were allowed to acclimate for 7 days under the same environmental conditions.

[0062] The tested pathogens were Fusarium oxysporum and Phytophthora camphorata, and the tested microorganisms were the antagonistic bacterium Egeriazolinium SHP-12 and the growth-promoting bacterium SHP-0-1-118.

[0063] After each strain has been cultured to a suitable inoculation state, it is ready for use. The bacterial solution is diluted or mixed with PBS buffer at pH 5.5.

[0064] The experiment was divided into two main groups based on the pathogens: 40-group and 79-group. Each main group was further divided into three subgroups: CK group, 12-group, and M group. The CK group was the pathogen treatment group, inoculated only with the pathogen; the 12-group was the antagonistic bacteria treatment group, inoculated with the pathogen and SHP-12 in a ratio of 1:1:2; and the M group was the combined treatment group, inoculated simultaneously with the pathogen, SHP-12, and growth-promoting bacteria SHP-0-1-118 in a ratio of 1:0.5:0.5:2. A total of six treatment groups were established: 40-CK, 40-12, 40-M, 79-CK, 79-12, and 79-M. Each treatment group had 10 biological replicates.

[0065] Inoculation was performed via root drenching, with each plant initially inoculated with 10 mL of bacterial solution. A second inoculation of the same amount was performed 7 days post-inoculation, using the same method. During the cultivation period, environmental conditions were maintained consistently for all treatments. Plant growth was observed regularly, and disease symptoms were assessed. Disease incidence was assessed 21 days post-inoculation and graded according to the following criteria.

[0066] Grade 0: The plant is growing normally, the leaves have no obvious symptoms, the root system is intact, and there is no rot.

[0067] Grade 1: Mild disease, with some roots showing browning or slight rot, plant growth is affected to some extent, and leaves slightly wilting or yellowing.

[0068] Level 2: Moderate to severe disease, with obvious root rot, partial root necrosis, obvious wilting and yellowing of the plant, and stunted growth.

[0069] Level 3: Severe disease, with extensive root rot or necrosis, severe wilting of the above-ground parts, and the plant near death or dying.

[0070] The formula for calculating the Disease Index (DI) is as follows: ; In the formula, n i v represents the number of plants at each disease level. i The values ​​represent the corresponding disease severity levels, where N is the total number of plants surveyed and V is the highest disease severity level.

[0071] The formula for calculating the efficacy (CE) is as follows: .

[0072] Table 4. Effects of different treatments on the incidence and control of eucalyptus root rot. As shown in Table 4, the results indicate that *Bacillus edodes* SHP-12 has a significant in vivo control effect against bacterial wilt of eucalyptus. In 79 groups, the control effect of SHP-12 treatment was 56.25%, which increased to 62.50% after combined application with growth-promoting bacteria; in 40 groups, the control effect of SHP-12 treatment was 73.68%. This demonstrates that *Bacillus edodes* SHP-12 provided by this invention can effectively inhibit the occurrence of eucalyptus root rot in living plants and has the potential to be developed into a biological agent for eucalyptus root rot.

[0073] Table 5. Differences in average growth indicators of eucalyptus seedlings under different pathogenic stresses After the pot experiment, the growth indicators of eucalyptus seedlings in each treatment group were measured. The results showed that compared with the control group treated with pathogen alone, the seedling height, root length, and biomass of eucalyptus seedlings were all increased after inoculation with SHP-12. Under the stress of pathogen No. 40, the seedling height, root length, fresh weight, and dry weight of group 40-12 reached 17.33 cm, 9.23 cm, 0.237 g, and 0.0434 g, respectively; under the stress of pathogen No. 79, the seedling height, root length, fresh weight, and dry weight of group 79-12 reached 20.33 cm, 12.10 cm, 0.181 g, and 0.0325 g, respectively, all of which were higher than the corresponding control groups, indicating that SHP-12 can alleviate the inhibitory effect of pathogens on the growth of eucalyptus seedlings. After being treated in combination with the growth-promoting bacterium SHP-0-1-118, some growth indicators were further improved, indicating that SHP-12 has a good disease prevention and growth-promoting effect and shows a certain synergistic effect with the growth-promoting bacterium.

[0074] In summary, Bacillus edilum SHP-12 can effectively reduce eucalyptus root rot caused by Fusarium oxysporum and Phytophthora camphorata, and has the potential to be developed into a biological control agent for eucalyptus diseases.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Bacillus erythropoietinus* ( Paenibacillus elgii SHP-12, characterized in that, The *Bacillus edilum* SHP-12 was deposited on July 13, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39680, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of the Bacillus sp. SHP-12 of claim 1 in the preparation of products for the prevention and control of eucalyptus diseases.

3. The application as described in claim 2, characterized in that, The eucalyptus diseases mentioned include eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt.

4. A product for preventing and controlling eucalyptus diseases, characterized in that, The active ingredient is the *Bacillus edodes* SHP-12 as described in claim 1.

5. The application of the *Bacillus edodes* SHP-12 of claim 1 or the product of claim 4 in inhibiting eucalyptus pathogens, characterized in that... The eucalyptus pathogens include Fusarium oxysporum (… Fusarium oxysporum ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), Phytophthora camphorata ( Phytophthora cinnamomi ), Pseudomonas calceolus ( Calonectria pseudoreteaudii ) and Solanaceae Raulella ( Ralstonia solanacearum ).

6. A method for inhibiting eucalyptus pathogens, characterized in that, Includes the step of applying the *Bacillus edodes* SHP-12 of claim 1 or the product of claim 4; The eucalyptus pathogens include Fusarium oxysporum, Colletotrichum gloeosporioides, Phytophthora camphorata, Pseudomonas calceolaria, and Solanaceae Raulella.

7. The application of the *Bacillus erythropoietinus* SHP-12 as described in claim 1 or the product as described in claim 4 in the control of eucalyptus diseases, characterized in that... The eucalyptus diseases mentioned include eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt.

8. A method for controlling eucalyptus diseases, characterized in that, Includes the step of applying the *Bacillus edodes* SHP-12 of claim 1 or the product of claim 4; The eucalyptus diseases mentioned include eucalyptus root rot, eucalyptus shoot blight, eucalyptus scorch, and eucalyptus bacterial wilt.

9. The use of the Bacillus edodes SHP-12 of claim 1 or the product of claim 4 in promoting eucalyptus growth.

10. A method for promoting eucalyptus growth, characterized in that, The step includes applying the Egeriaceae SHP-12 of claim 1 or the product of claim 4.

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