Bacillus safensis HMQAU23223 and application thereof

Abstract

The invention relates to the field of biocontrol of plant diseases. The invention provides a bacillus safensis strain HMQAU23223, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 13, 2025, and the preservation registration number is CGMCC No.36623. The bacillus safensis strain HMQAU23223 has the advantages that the preservation number is CGMCC No.36623; the invention also provides a biocontrol inoculant. The invention also provides a preparation method of the biocontrol inoculant. The invention further provides the application of the bacillus safensis strain HMQAU23223 or the biocontrol microbial inoculum or the biocontrol microbial inoculum prepared by the preparation method. The invention further provides the application of the bacillus safensis strain HMQAU23223 or the biocontrol microbial inoculum prepared by the preparation method. The invention further provides a method for preventing and treating plant diseases. The invention also provides an application of the bacillus safensis strain HMQAU23223 in preparation of a compound for inhibiting phytopathogen or preventing and treating plant pathogenic nematodes. The fermentation filtrate of the bacillus safensis strain HMQAU23223 provided by the invention has the lethal effects of 89% or above on pine wood nematode, meloidogyne incognita, wheat cyst nematode, heterodera filipjevi and ditylenchus destructor, and has better biocontrol potential.

Description

A strain of Bacillus sarcodactylis HMQAU23223 and its application Technical Field

[0001] This invention belongs to the field of plant disease biocontrol, specifically involving a strain of Bacillus sabensis HMQAU23223 and its applications. Background Technology

[0002] Pine wilt disease, also known as pine blight, is a disease caused by the pine wood nematode (Bursaphelenchus xylophilus). This nematode is carried and spread by the pine sawyer beetle (Monochamus alternatus), and is a global forest disease (Pan Shaojie, 2023), causing enormous damage to my country's forest resources (Sun Yufeng, 2023).

[0003] Wheat cyst nematode disease is caused by 12 effective species, among which *Heterodera avenae* and *Heterodera filipjevi* are two highly damaging nematodes that seriously threaten wheat production (Xu Nianlong, 2021). Compared to *Heterodera avenae*, *Heterodera filipjevi* is more virulent, has a longer infection cycle, and can transmit various soil-borne fungal diseases (Han Kun, 2023). Currently, nematode control mainly relies on chemical pesticides combined with wheat field management to reduce cyst nematodes and a series of secondary soil-borne diseases induced by them (Lü Yan, 2022). While chemical pesticides can reduce nematode damage to some extent, they are costly, leave significant residues, and cause severe environmental damage. Therefore, finding safe, efficient, and environmentally friendly control measures for wheat cyst nematodes is of great significance for controlling nematode damage and promoting wheat production.

[0004] Pseudoperonospora cubensis and Meloidogyne incogita cause downy mildew and root-knot nematode disease in cucumbers, respectively. These are important leaf and root diseases in cucumber-producing areas worldwide (Thomas, 1986) and are also devastating diseases in protected cucumber cultivation, seriously affecting cucumber yield and quality (Fu Shuyun, 1983; Shi Yanxia, ​​2002).

[0005] Sweet potato stem nematode disease is caused by *Ditylenchus destructor*, a migratory endoparasitic nematode and an internationally recognized quarantine nematode (Hooper, 1973). This nematode primarily infests sweet potatoes, potatoes, and garlic. After infecting tubers, the skin wrinkles and cracks, the interior turns brown and gradually black, and secondary bacterial and fungal infections occur later, leading to the rotting and spoilage of the entire tuber. This nematode can cause a 30%-50% reduction in sweet potato and potato yields, and in severe cases, a reduction of over 80%, or even total crop failure (Zhou Zhong, 2003), seriously affecting yield and quality. Sweet potato stem nematode disease occurs in Beijing, Tianjin, Shandong, Hebei, Henan, Jiangsu, Xinjiang, and Gansu, with the most severe outbreaks in Shandong and Hebei provinces, becoming one of the most serious diseases affecting sweet potatoes in northern China (Yang Baojun, 1990).

[0006] However, existing technologies lack biocontrol agents with high efficacy against the above-mentioned agricultural and forestry plant diseases such as cucumber downy mildew, root-knot nematode disease, pine wilt disease, sweet potato stem nematode disease, and wheat cyst nematode disease. How to screen out a suitable biocontrol bacteria for the preparation of biocontrol agents is a technical problem that needs to be solved in the field of plant disease biocontrol. Summary of the Invention

[0007] This invention aims to address the shortcomings of existing technologies by providing a strain of Bacillus salsa HMQAU23223 and its applications.

[0008] The first objective of this invention is to provide a strain of biocontrol actinomycete Bacillus safensis HMQAU23223 isolated from the saline-alkali soil of the Yellow River Delta in Dongying City, Shandong Province, which enriches the strain resources of biocontrol bacteria for plant diseases and lays the foundation for the research and development of biocontrol bacteria.

[0009] The second objective of this invention is to provide a biocontrol agent.

[0010] A third objective of this invention is to provide a method for preparing the above-mentioned biocontrol agent.

[0011] A fourth object of the present invention is to provide the application of the above-described Bacillus sabolicus strain HMQAU23223 or the above-described biocontrol agent or the biocontrol agent prepared by the above-described preparation method in inhibiting plant pathogens, preparing products for inhibiting plant pathogens, preventing and controlling diseases caused by plant pathogens, and preparing products for preventing and controlling diseases caused by plant pathogens.

[0012] The fifth objective of this invention is to provide the application of the above-described Bacillus sabolicii strain HMQAU23223, or the above-described biocontrol agent, or the biocontrol agent prepared by the above-described preparation method, in the control of plant diseases.

[0013] The sixth objective of this invention is to provide a method for the prevention and control of plant diseases.

[0014] The seventh object of the present invention is to provide the application of the above-described Bacillus sabensis strain HMQAU23223 or the above-described biocontrol agent or the biocontrol agent prepared by the above-described preparation method in the control of plant pathogenic nematodes.

[0015] The eighth object of the present invention is to provide the use of the aforementioned Bacillus salsa strain HMQAU23223 in the preparation of compounds for the control of plant pathogenic bacteria or plant pathogenic nematodes.

[0016] The above-mentioned objective of this invention is achieved through the following technical solution: a strain of Bacillus safensis HMQAU23223, which was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with the accession number CGMCC No. 36623.

[0017] Based on the same inventive concept, the present invention also provides a biocontrol agent, wherein the active ingredient of the biocontrol agent includes a bacterial suspension, fermentation broth or fermentation filtrate of Bacillus salsa strain HMQAU23223 as described above.

[0018] Based on the same inventive concept, the present invention also provides a method for preparing the biocontrol agent as described above, wherein the active ingredient of the biocontrol agent is fermentation filtrate, and the specific steps include: (1) preparing seed liquid; (2) taking 1-2% of the seed liquid and adding it to LB liquid culture medium, and culturing it in a constant temperature shaker at 200-400 rpm for 24-48h at 28-30℃ to prepare fermentation broth; (3) centrifuging the fermentation broth at 24-28℃ and 6000-8000 rpm for 15-20min, and filtering to obtain sterile fermentation filtrate, which is the biocontrol agent.

[0019] In one embodiment of the present invention, the specific steps for preparing the seed culture in step (1) include: activating strain HMQAU23223 on LB solid medium at 25-30℃ for 2-3 days, then inoculating the isolated and purified bacteria into an Erlenmeyer flask containing LB liquid medium, and culturing it in a constant temperature shaker at 200-300 rpm for 8-10 hours at 25-30℃ to obtain the seed culture. Most preferably, the activation conditions in step (1) are: activating strain HMQAU23223 on LB solid medium at 28℃ for 2 days; and the shaking culture conditions are: culturing in a constant temperature shaker at 28℃ at 200 rpm for 10 hours.

[0020] In one embodiment of the present invention, the shaking culture conditions in step (2) are: cultured at 200 rpm for 48 h in a constant temperature shaker at 28°C.

[0021] In one embodiment of the present invention, the centrifugation conditions in step (3) are to centrifuge the fermentation broth at 24°C and 8000 rpm for 15 min.

[0022] In some embodiments of the present invention, the composition and content of LB solid culture medium are as follows: 10g peptone, 5g yeast extract, 5g sodium chloride, 18g agar powder, and deionized water to a final volume of 1000mL, with a pH of 7.0-7.2. The composition and content of LB liquid culture medium are as follows: 10g peptone, 5g yeast extract, 5g sodium chloride, and deionized water to a final volume of 1000mL, with a pH of 7.0-7.2.

[0023] Based on the same inventive concept, this invention provides the application of the *Bacillus sabinatus* strain HMQAU23223 as described above, or the biocontrol agent as described above, or the biocontrol agent prepared by the preparation method as described above, in the following (a1) or (a2) or (a3) ​​or (a4): (a1) inhibiting plant pathogens; (a2) preparing a product for inhibiting plant pathogens; (a3) ​​preventing and controlling diseases caused by plant pathogens; (a4) preparing a product for preventing and controlling diseases caused by plant pathogens; wherein the plant pathogens are *Pseudoperonospora cubensis*, *Botrytiscinerea*, *Cladosporium tenuissimum*, *Corynesporacassiicola*, *Alternaria alternata*, *Nigrospora oryzae*, and *Stagonosporopsis*. *Cucurbitacearum*, *Phomopsis* sp., *Alternaria tenuissima*, *Fusarium* sp., *Trametes trogii*, *Colletotrichum plurivorum*, *Fusarium culmorum*, *Mycosphaerella* sp., *Lasiodiplodia pseudotheobromae*, *Botryosphaeria dothidea*, *Neopestalotiopsis clavispora*, *Colletotrichum* sp., *Curvularia* sp.The following fungi are listed: *Ulocladium chartarum*, *Stemphylium botryosum*, *Alternaria solani*, *Colletotrichum orbiculare*, *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, *Phytophthora capsici*, *Sclerotinia sclerotiorum*, *Pythium spinosum*, *Fusarium oxysporum*, and *Fusarium solani*.

[0024] Based on the same inventive concept, this invention also provides the application of the above-mentioned Bacillus spp. strain HMQAU23223, or the above-mentioned biocontrol agent, or the biocontrol agent prepared by the above-mentioned preparation method, in the control of plant diseases, wherein the plant diseases are any one of cucumber downy mildew, bean gray mold, blueberry leaf spot, blueberry twig blight, cucumber target spot, peanut leaf spot, lettuce leaf spot, eggplant early blight, cucumber anthracnose, strawberry anthracnose, pepper blight, celery sclerotinia stem rot, cucumber damping-off, cucumber wilt, garlic dry rot, and watermelon wilt.

[0025] Based on the same inventive concept, this invention also provides a method for preventing and controlling plant diseases. When preventing and controlling plant diseases, the biocontrol agent prepared by the above-mentioned preparation method or the strain of Bacillus spp. HMQAU23223 or the biocontrol agent prepared by the above-mentioned preparation method is applied to the disease-prone parts of the plant by coating, impregnation, spraying, atomizing, irrigating, dusting, sowing, foaming, coating or spraying.

[0026] Based on the same inventive concept, this invention also provides the application of the above-mentioned Bacillus sabensis strain HMQAU23223 or the above-mentioned biocontrol agent or the biocontrol agent prepared by the above-mentioned preparation method in the control of plant pathogenic nematodes, wherein the plant pathogenic nematodes are pine wood nematodes, southern root-knot nematodes, cereal cyst nematodes, Phillips cyst nematodes, and stem rot nematodes.

[0027] Based on the same inventive concept, this invention also provides the application of the above-mentioned Bacillus sabensis strain HMQAU23223 in the preparation of compounds for the control of plant pathogenic bacteria or plant pathogenic nematodes.

[0028] In the application described above, the plant pathogen is cucumber downy mildew fungus; the plant pathogenic nematodes are pine wood nematode, southern root-knot nematode, cereal cyst nematode, Phillips cyst nematode, and stem rot nematode.

[0029] As described above, the compound is shogaol, [8]-shogaol, trans-cinnamic acid, 3-methylthiopropanol, methyl trans-cinnamate, pimecrolimus, and doxorubicin hydrochloride. Preferably, the compound is any one or at least two of [8]-shogaol, trans-cinnamic acid, 3-methylthiopropanol, methyl trans-cinnamate, and doxorubicin hydrochloride. Optionally, the compound is any one or at least two of [8]-shogaol, trans-cinnamic acid, and methyl trans-cinnamate. These three compounds or their combination have a good lethal effect on pine wood nematode and southern root-knot nematode and a good inhibitory effect on cucumber downy mildew.

[0030] In the application described above, the most preferred embodiment is a combination of trans-cinnamic acid and methyl trans-cinnamate, wherein the mass ratio of trans-cinnamic acid to methyl trans-cinnamate is 5:5. Under these conditions, the co-toxicity coefficient of the compound is 445.72, exhibiting a synergistic effect in killing pine wood nematodes.

[0031] Compared with existing technologies, the effects and advantages of the present invention are: (1) The fermentation filtrate of Bacillus salsa strain HMQAU23223 provided by the present invention has a lethal effect of more than 89% on pine wood nematode, southern root-knot nematode, cereal cyst nematode, Philippine cyst nematode and stem rot nematode, and has good biocontrol potential.

[0032] (2) The fermentation filtrate of Bacillus sabinatus strain HMQAU23223 provided by this invention exhibits varying degrees of inhibitory effects on a variety of pathogens. It showed varying degrees of inhibitory effects on 32 tested pathogens. The best inhibitory effect was observed against Botrytis cinerea (96.67%). It also showed good inhibitory effects against Cladosporium fasciatus, Diplostomum simonii, and Botrytis cinerea, with inhibition rates between 72.50% and 77.78%. The next best inhibitory effects were against Cladosporium spp., Alternaria alternata, Bacillus anthracis, Cladosporium pachyphyllum, Scutellaria baicalensis, Polysporus cucurbita, Pseudomonas spp., and Alternaria alternata, with inhibition rates between 51.56% and 69.44%. The inhibitory effect on Fusarium oxysporum was relatively poor. This indicates that strain HMQAU23223 has a broad antibacterial spectrum and good biocontrol potential.

[0033] (3) The metabolic compounds of the Bacillus sabinatus strain HMQAU23223 provided by the present invention, especially [8]-shogaol, trans-cinnamic acid and trans-cinnamic acid methyl ester, have a good lethal effect on pine wood nematode and southern root-knot nematode, and have a high inhibition rate on cucumber downy mildew. Attached Figure Description

[0034] Figure 1 shows the lethal effect of a 5-fold dilution of bacterial fermentation filtrate on pine wood nematodes after 24 hours of treatment; where A: water control; B: HMQAU23039; C: HMQAU23217; D: HMQAU23233; E: HMQAU23239; F: HMQAU23259; G: HMQAU23258; H: HMQAU23246; I: HMQAU23256; J: HMQAU23005; Bar: AJ=200μm.

[0035] Figure 2 shows the colony morphology of bacteria HMQAU23223.

[0036] Figure 3 shows the phylogenetic tree of bacteria HMQAU23223 constructed based on the 16S rDNA sequence.

[0037] Figure 4 shows the inhibitory effect of the tested bacterial strain HMQAU23223 on different pathogenic fungi; 1-32 (top): pathogenic fungi control; 1-32 (bottom): inhibitory effect of different pathogenic fungi on plate confrontation; 1: HMQAU140073 (Hypericum pseudococcoccus); 2: HMQAU170042 (Botrytis cinerea); 3: HMQAU200087 (Cyclocarya paliurus); 4: HMQAU210027 (Fusarium solani); 5: HMQAU210069 (Pseudomonas aeruginosa); 6: HMQAU240336 (Phytophthora capsici); 7: HMQAU240283 (Anthrax siamensis); 8: HMQAU230414 (Fusarium oxysporum); 9: HMQAU230413 (Fusarium oxysporum); 10: HMQAU230410 (Pythium spp.); 11: HMQAU230064 (Alternaria alternata); 12: HMQAU210122 (Alternaria pumilus); 13: HMQAU240135 (Colletotrichum gloeosporioides); 14: HMQAU240368 (Colletotrichum gloeosporioides); 15: HMQAU230334 (Cladosporium spp.); 16: HMQAU240005 (Fusarium oxysporum); 17: HMQAU 140073 (Diplosporium spp.); 18: HMQAU150053 (Staphylococcus aureus); 19: HMQAU160060 (Rhizopus oryzae); 20: HMQAU160061 (Fusarium oxysporum); 21: HMQAU160066 (Polysporium cucumeritum); 22: HMQAU160067 (Polysporium clavatum); 23: HMQAU170147 (Alternaria alternata); 24: HMQAU210070 (Fusarium oxysporum); 25: HMQAU170142 (Cyclophorus spp.); 26: HMQAU2 30337 (Multi-hosted Anthracnose); 27: HMQAU250033 (Alternaria spp.); 28: HMQAU250037 (Trichophyton spp.); 29: HMQAU250039 (Neoprothiolane spp.); 30: HMQAU250050 (Clonorhynchus spp.); 31: HMQAU250103 (Anthracnose); 32: HMQAU250104 (Curvularia spp.).

[0038] Figure 5 shows the 24-hour lethal effect of different concentrations of [8]-shogaol on pine wood nematodes; A: control; B: 80 μg / mL [8]-shogaol; C: 100 μg / mL [8]-shogaol.

[0039] Figure 6 shows the sporangium morphology of cucumber downy mildew under different treatments. A: control; B: original fermentation filtrate of HMQAU23223; C: 80 μg / mL[8]-shogaol; D: 100 μg / mL[8]-shogaol. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0041] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] In this invention, Bacillus sarcodactylis HMQAU23223 may be simply referred to as "strain HMQAU23223" or "HMQAU23223".

[0045] Example 1 Isolation and screening of biocontrol bacterial strains 1.1 Isolation of bacterial strains In 2023, bacterial strains were isolated and purified in the Yellow River Delta saline-alkali land of Dongying City, Shandong Province by dilution plating method and streak plate method and preserved in 30% glycerol.

[0046] The preparation method of the bacterial cell preservation solution is as follows: dilute glycerol with 0.9% physiological saline to a concentration of 30% to obtain the bacterial cell preservation solution.

[0047] 1.2 Screening of biocontrol bacterial strains 1.2.1 Test materials Pine wood nematode and Botrytis cinerea were provided by the Nematode Laboratory and Fungal Laboratory of Qingdao Agricultural University, respectively.

[0048] 1.2.2 Preparation of Bacterial Fermentation Filtrate and Seed Culture: Sixty-eight bacterial strains, including strain HMQAU23223, isolated from saline-alkali land, were activated on LB solid medium at 28℃ for 2 days. The purified bacteria (one loop) were inoculated into Erlenmeyer flasks containing 50 mL of LB liquid medium and cultured at 200 rpm for 10 hours at 28℃ to prepare the seed culture. The composition and concentration of the LB solid medium were as follows: 10 g peptone, 5 g yeast extract, 5 g sodium chloride, 18 g agar powder, and deionized water to a final volume of 1000 mL, pH 7.0–7.2. The composition and concentration of the LB liquid culture medium were as follows: 10 g peptone, 5 g yeast extract, 5 g sodium chloride, and deionized water to a final volume of 1000 mL, pH 7.0–7.2.

[0049] Preparation of fermentation broth: 1% (v / v) seed culture was added to 50 mL of fresh LB liquid medium and cultured at 200 rpm for 48 h in a constant temperature shaker at 28℃ to prepare the fermentation broth. The composition and content of the LB liquid culture medium were as follows: 10 g peptone, 5 g yeast extract, 5 g sodium chloride, and deionized water to a final volume of 1000 mL, pH 7.0–7.2.

[0050] Preparation of fermentation filtrate: After centrifuging the fermentation broth at 24℃ and 8000rpm for 15min, the filtrate was filtered through a 0.22μm microporous filter to obtain sterile fermentation filtrate of each bacterial strain, and stored in a refrigerator at 4℃ for later use.

[0051] 1.2.3 Screening of Bacterial Strains Initial screening was performed using the immersion method to determine the toxicity of the fermentation filtrate of different bacterial strains to *Pinus wiltii*. 1 mL of fermentation filtrate from different tested strains and 50 μL of *Pinus wiltii* suspension (approximately 200 nematodes) were added to 24-well cell culture plates and mixed. The plates were incubated at 25°C. After 24 h and 48 h of treatment, the mixtures were observed under a microscope, and the number of dead nematodes and the total number of nematodes were recorded. Uninoculated liquid culture medium served as a control. Each treatment was performed in triplicate, and the experiment was repeated twice.

[0052] The toxicity of the fermentation filtrate of seven selected strains to pine wood nematodes was determined using the immersion method. The original fermentation filtrate of the selected strains was diluted 5-, 10-, 25-, and 50-fold, respectively. 1 mL of the fermentation filtrate and 50 μL of pine wood nematode suspension (approximately 200 nematodes) were then added to 24-well cell culture plates and mixed. The plates were incubated at 25°C. After 24 and 48 hours of treatment, the mixture was observed under a microscope, and the number of dead nematodes and the total number of nematodes were recorded. Uninoculated liquid LB medium served as a control. Each treatment was repeated in triplicate, with two replicates. Observation was performed by pipetting 50 μL of the nematode-containing mixture onto a slide and observing it under a microscope. Each treatment had three replicates, with at least 30 nematodes observed each time. Due to the phenomenon of feigned death in nematodes, the total number of nematodes was first observed and recorded under a microscope. During this period, 50 μL of water was repeatedly used to stimulate the nematodes; if they remained stiff and immobile after 4 hours, they were considered dead. The criteria for judging the activity level are based on the mortality rate of pine wilt nematodes after 24 hours. A nematode mortality rate ≥90% is grade "++++"; 50% ≤ nematode mortality rate <90% is grade "++++"; 30% ≤ nematode mortality rate <50% is grade "++"; 10% ≤ nematode mortality rate <30% is grade "+"; and nematode mortality rate <10% is grade "-" (Zhu Limei, 2007).

[0053]

[0054] 1.3 Initial Screening Results of Biocontrol Bacterial Strains The 66 bacterial strains previously isolated in the laboratory were initially screened using the immersion method, with uninoculated liquid culture medium serving as a blank control. The results showed that among the original fermentation filtrate of the 66 bacterial strains, 54 strains exhibited a lethality of over 80% against pine wood nematodes after 24 hours, and 47 strains exhibited a corrected mortality rate of over 90%. Among these, 13 strains achieved a corrected mortality rate of 100% after 24 hours: HMQAU23239, HMQAU23056, HMQAU23214, HMQAU23215, HMQAU23225, HMQAU23157, HMQAU23226, HMQAU23229, HMQAU23231, HMQAU23233, HMQAU23237, HMQAU23238, and HMQAU23265, as shown in Table 1. The lethality of the bacterial fermentation filtrate diluted 5 times after treatment with pine wood nematodes for 24 hours is shown in Table 2 and Figure 1. In the determination of the lethality of the bacterial fermentation filtrate diluted 5 times and 10 times against pine wood nematodes, it was found that HMQAU23223 had a high and stable lethality against pine wood nematodes; therefore, this strain was selected for further analysis.

[0055] Table 1. Lethal effect of bacterial fermentation filtrate on pine wood nematode.

[0056] Table 2. Lethal effect of 5-fold dilution of bacterial fermentation filtrate on pine wood nematode.

[0057] Table 3. Lethal effect of 10-fold dilution of bacterial fermentation filtrate on pine wood nematode.

[0058] 1.4 Results of rescreening of biocontrol bacterial strains Table 4: Lethality of 7 bacterial strains after 24 hours of treatment with different dilutions on pine wood nematodes

[0059] As shown in Table 4, through the rescreening test of 7 bacterial strains at different dilutions, and considering the corrected mortality rate after 24 hours of treatment with pine wood nematodes, 4 bacterial strains, HMQAU23039G1, HMQAU23190, HMQAU23223, and HMQAU23239, showed higher mortality rates against pine wood nematodes. Among them, HMQAU23223 had the best lethality, laying the foundation for the next step of the experiment.

[0060] Example 2 Identification of biocontrol bacteria 2.1 Materials and methods 2.1.1 Test strain: Strain HMQAU23223 was provided by the Fungal Laboratory of the College of Plant Medicine, Qingdao Agricultural University.

[0061] 2.1.2 Identification of biocontrol bacteria for pine wilt disease: Morphological identification: The method was based on the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu et al., 2001).

[0062] Physiological and biochemical identification: The methods were based on the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu et al., 2001).

[0063] Molecular identification: After extracting the genome of HMQAU23223 using the OMEGA bacterial DNA extraction kit, 16S rDNA fragments were amplified using the universal bacterial primers 27F: 5'-AGA GTT TGA TCC TGG CTC AG-3' and 1492R: 5'-TAC GGC TAC CTT GTTACG ACT T-3'. Sequencing was then performed for alignment and evolutionary analysis.

[0064] 2.2 Results and Analysis 2.2.1 Morphological and Physiological Biochemical Identification Strain HMQAU23223 was cultured on LB solid medium at 28℃ for 2 days. As shown in Figure 2, the colonies were raised, orange-red, opaque, round, with irregular edges and wrinkled surfaces.

[0065] Physiological and biochemical tests showed that strain HMQAU23223 was negative for methyl red, starch hydrolysis, and oxidase tests, but positive for citrate and catalase tests. Based on the morphological and physiological and biochemical characteristics of HMQAU23223, the strain was preliminarily identified as Bacillus.

[0066] 2.2.2 Molecular Identification: After PCR amplification of the 16S rDNA sequence of strain HMQAU23223, it was cloned and sequenced by Beijing Qingke Biotechnology Co., Ltd. The strain sequence was submitted to the NCBI gene database, and a phylogenetic tree was constructed. The results are shown in Figure 3. Strain HMQAU23223 clustered together with strain CP192519, indicating the closest phylogenetic relationship. Based on the morphological characteristics, physiological and biochemical characteristics, and phylogenetic relationship of this strain, strain HMQAU23223 was finally identified as Bacillus safensis.

[0067] The Bacillus safensis strain HMQAU23223 was deposited at the China General Microbiological Culture Collection Center on November 13, 2025, with accession number CGMCC No. 36623.

[0068] Example 3 Inhibitory effect of strain HMQAU23223 on different plant pathogens and pathogenic nematodes 3.1 Plant diseases and their pathogens and pathogenic nematodes tested Bean gray mold: Botrytis cinerea HMQAU170042; Blueberry leaf spot: Cladosporium tenuissimum HMQAU230334, Corynesporacassiicola HMQAU250050, Alternaria alternata HMQAU170147, Nigrosporaoryzae HMQAU160060, Stagonosporopsis cucurbitacearum HMQAU160066, Phopsis sp. HMQAU210069, Alternaria tenuissima HMQAU250033, Fusarium sp. HMQAU 210070, *Trametes trogii* HMQAU250037, *Colletotrichum plurivorum* HMQAU230337, *Fusarium culmorum* HMQAU160061, *Mycosphaerella sp.* HMQAU170142, Blueberry twig blight: *Lasiodiplodia pseudotheobromae* HMQAU140073, *Botryosphaeria dothidea* HMQAU150053, *Neopestalotiopsis clavispora* HMQAU250039, Cucumber target spot: *Corynespora* Peanut anthracnose: Colletotrichum sp. HMQAU200087; Peanut leaf spot: Curvularia sp.HMQAU250104, Lettuce leaf spot: *Ulocladium chartarum* HMQAU210122, *Stemphylium botryosum* HMQAU210121; Eggplant early blight: *Alternaria solani* HMQAU230064; Cucumber anthracnose: *Colletotrichum orbiculare* HMQAU240368; Strawberry anthracnose: *Colletotrichum siamense* HMQAU240283, *Colletotrichum gloeosporioides* HMQAU240135; Pepper blight: *Phytophthora capsici* HMQAU240336; Celery sclerotinia rot: *Sclerotinia* * *Sclerotiorum* HMQAU170216, *Pythium spinosum* HMQAU230410, *Fusarium wilt* HMQAU230414 and HMQAU230413, *Fusarium oxysporum* HMQAU210027, *Fusarium oxysporum* HMQAU240005, *Pine wilt nematode*, *Southern root-knot nematode*, *Stem rot nematode*, *Corydalis gracilis nematode*, and *Philippine cyst nematode* were all provided by the Mycology and Nematology Laboratory of Qingdao Agricultural University.

[0069] 3.2 Experimental Methods Broad-spectrum antibacterial activity: The antibacterial spectrum of strain HMQAU23223 was determined using the plate confrontation method (Wang Qing et al., 2013). Bacteria were inoculated in the center of a 9 cm diameter PDA plate, and fungal colonies were inoculated at a distance of 2.25 cm from the bacteria. Plates without bacterial colonies served as the control group. The plates were incubated at 28 °C until the control colony area reached 3 / 4 of the plate. The radius of the fungal colony was measured, and the inhibition rate was calculated. Each treatment was repeated in triplicate.

[0070] Inhibition rate = (colon radius of control group - colony radius of treatment group) / colony radius of control group × 100%.

[0071] Broad-spectrum nematicidal activity: The broad-spectrum activity of strain HMQAU23223 was determined using the immersion method. 1 mL of the fermentation filtrate of the strain was mixed with 50 μL of suspensions of pine wood nematodes (approximately 200 nematodes), southern root-knot nematodes (approximately 200 nematodes), stem rot nematodes (approximately 200 nematodes), cereal cyst nematodes (approximately 200 nematodes), and Phillips cyst nematodes (approximately 100 nematodes), respectively, and added to 24-well cell culture plates. After incubation at 25°C for 24 h, the mixtures were observed under a microscope, and the number of dead nematodes and the total number of nematodes were recorded. Liquid culture medium without inoculation served as a control. Each treatment was replicated in triplicate.

[0072] 3.3 Inhibitory Effects of HMQAU23223 on Different Plant Pathogens and Pathogenic Nematodes Table 5 shows that strain HMQAU23223 exhibited varying degrees of inhibitory effects against the 33 tested pathogens. The best inhibitory effect was observed against *Botrytis cinerea* (96.67%). It also showed good inhibitory effects against *Cladosporium fasciatus*, *Diplostomum simonii*, and *Botrytis cinerea*, with inhibition rates ranging from 72.50% to 77.78%. Following closely were *Cladosporium spp.*, *Alternaria alternata*, *Amycosis anthracnose*, *Cladosporium pachyphyllum*, *Nitrophus oryzae*, *Polystomium cirrhifolium*, *Pseudomonas spp.*, and *Alternaria alternata*, with inhibition rates ranging from 51.56% to 69.44%. The inhibitory effect against *Fusarium oxysporum* was relatively poor. This indicates that strain HMQAU23223 has a broad antibacterial spectrum and good biocontrol potential. The inhibitory effects of the tested bacterial strain HMQAU23223 on different pathogenic fungi are shown in Figure 4.

[0073] Table 5. Inhibitory effects of bacterium HMQAU23223 against different pathogenic fungi

[0074] Table 6 shows that bacteria HMQAU23223 has good lethal effects against various nematodes. Among them, the 5-fold dilution of the fermentation filtrate has a lethal effect of over 89% against pine wood nematode, southern root-knot nematode, cereal cyst nematode, Phillips cyst nematode, and stem rot nematode, indicating good biocontrol potential.

[0075] Table 6. Lethal effects of different dilutions of HMQAU23223 fermentation filtrate on different nematodes.

[0076] Example 4. Lethal Effect of Metabolites from Strain HMQAU23223 on Pine Wood Nematode 4.1 Experimental Methods: The fermentation filtrate of strain HMQAU23223 was mixed with pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v / v), vortexed, sonicated at low temperature for 30 min, allowed to stand at -20℃ for 10 min, centrifuged at 14000 g at 4℃ for 20 min, and the supernatant was collected. The supernatant was vacuum dried, and for mass spectrometry analysis, 100 μL of acetonitrile / water solution (acetonitrile:water = 1:1, v / v) was added to reconstitute the mixture, vortexed, centrifuged at 14000 g at 4℃ for 15 min, and the supernatant was injected. Untargeted metabolomics analysis was performed using UHPLC-Q-TOF MS. Seven compounds (Table 7) were selected from the upregulated metabolites and used to treat pine wood nematodes for 24 h before determining their lethal effects. The drugs and their concentration dilution ratios are as follows: 12.13, 15.17, 20.22, 30.34, 40.45, 60.67 μg / mL of tsedge; 20, 30, 50, 80, 100, 120, 140, 160, 180, 200 μg / mL of [8]-shogaol; 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL of pimecrolimus; 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μg / mL of trans-cinnamic acid, 3-methylthiopropanol, methyl trans-cinnamate, and doxorubicin hydrochloride. Except for doxorubicin hydrochloride, which was prepared using sterile water, the other six drugs were prepared as stock solutions using 1 mL of pure dimethyl sulfoxide (DMSO), containing 6.0672 mg / mL of styraxetine, 1 mg / mL of shogaol, 100 mg / mL of trans-cinnamic acid, 100 mg / mL of methyl trans-cinnamate, 100 mg / mL of 3-methylthiopropanol, 25 mg / mL of doxorubicin hydrochloride, and 10 mg / mL of pimecrolimus. Subsequently, except for shogaol, which was diluted with 1% DMSO aqueous solution and doxorubicin hydrochloride, which was diluted with sterile water, the other five stock solutions were serially diluted with 0.1% Tween 80 aqueous solution. The control groups consisted of 1% DMSO aqueous solution, sterile water, 1% DMSO, and 0.1% Tween 80 aqueous solution.

[0077] Table 7 Screening of differentially expressed metabolites

[0078] 4.2 Results and Analysis: A total of 2755 metabolites (ESI+1472, ESI-1283) were detected in all samples. Based on the chemical classification information of the metabolites, these metabolites can be classified into 12 major categories. Among these categories, organic acids and their derivatives accounted for the largest proportion, accounting for 24.75% of the total identified metabolites. This was followed by lipids and lipid molecules, accounting for 22.9% of the total metabolites. Then came organic heterocyclic compounds (19.09%), benzene series compounds (10.64%), organic oxygen compounds (6.75%), etc.

[0079] Based on the t-test (P<0.05), and combined with the VIP≥1 obtained from the OPLS-DA model (VIP value represents the influence of the inter-group difference of the corresponding metabolite on the classification of samples in the model, and metabolites with VIP≥1 are generally considered to be significantly different), differential metabolites were screened and identified, and 1869 significantly differential metabolites were screened and identified, of which 973 metabolites were upregulated and 896 metabolites were downregulated.

[0080] 4.3 Lethal Effects of Different Metabolites on Pine Wood Nematode Table 8 shows that different concentrations of shogaol had no lethal effect on pine wood nematode. Table 9 shows that the lethal effect of [8]-shogaol on pine wood nematode first increased and then decreased with increasing concentration. When the concentration was 80 μg / mL, the corrected mortality rate was 93.18%; when the concentration was 100 μg / mL, the corrected mortality rate was 97.75%; and as the concentration increased to 200 μg / mL, the corrected mortality rate was 53.77%. The 24-hour lethal effect of different concentrations of [8]-shogaol on pine wood nematode is shown in Figure 5. Pimecrolimus had a poor lethal effect on pine wood nematode, and the corrected mortality rate at 100 μg / mL was still 0. Table 10 shows that the lethal effects of different concentration gradients of trans-cinnamic acid, 3-methylthiopropanol, methyl trans-cinnamate, and doxorubicin hydrochloride on pine wood nematodes varied significantly. The corrected mortality rates of 300 μg / mL methyl trans-cinnamate and 500 μg / mL trans-cinnamic acid were both 100%. However, the lethal effects of 3-methylthiopropanol and doxorubicin hydrochloride on pine wood nematodes were poor, with corrected mortality rates of 1.22% and 7.14% for 900 μg / mL 3-methylthiopropanol and 500 μg / mL doxorubicin hydrochloride, respectively.

[0081] Table 8. Lethal effect of different concentrations of styrax extract on pine wood nematode.

[0082] Table 9. Lethal effects of different concentrations [8]-shogaol and pimecrolimus on pine wood nematode.

[0083] Table 10. Lethality of different concentrations of compounds on pine wood nematode

[0084] Example 5 Inhibitory effect of HMQAU23223 fermentation broth and its metabolites on cucumber downy mildew and southern root-knot nematode 5.1 Materials and methods The inhibitory effect of HMQAU23223 fermentation filtrate stock solution, 80 μg / mL, 100 μg / mL [8]-shogaol, 300 μg / mL, 400 μg / mL, 500 μg / mL trans-cinnamic acid, 300 μg / mL, 400 μg / mL, 500 μg / mL trans-cinnamic acid methyl ester on cucumber downy mildew was determined by concave slide method. 1 mL of HMQAU23223 fermentation filtrate stock solution and the corresponding agents were respectively mixed with cucumber downy mildew sporangia (2×10 5 The suspension of *Cucumber downy mildew* sporangia was mixed at a volume ratio of 1:1, with sterile water and a suspension of *Cucumber downy mildew* sporangia mixed at a volume ratio of 1:1 serving as a control. Subsequent experimental procedures followed Yan Lei's method (Yan Lei, 2013). Each treatment was replicated in triplicate.

[0085] The lethality of the above-mentioned agents against *Symplocos spp.* was determined using the immersion method. 1 mL of the HMQAU23223 fermentation filtrate stock solution and the corresponding agent were added to 50 μL of *Symplocos spp.* (containing approximately 200 nematodes) and mixed in a 24-well cell culture plate. After incubation at 25°C for 24 h, the mixture was observed under a microscope, and the number of dead nematodes and the total number of nematodes were recorded. Uninoculated liquid culture medium served as a control. Each treatment was repeated in triplicate.

[0086] 5.2 Effects of HMQAU23223 and its metabolites on cucumber downy mildew and southern root-knot nematode Table 11 Effects of HMQAU23223 fermentation broth and its metabolites on cucumber downy mildew and southern root-knot nematode

[0087] As shown in Table 11, the original fermentation filtrate of HMQAU23223 showed a 100% inhibition rate against cucumber downy mildew. The differential metabolites of bacteria HMQAU23223[8]-shogaol and methyl trans-cinnamate showed good inhibitory effects against cucumber downy mildew. The inhibition rates of 80 μg / mL[8]-shogaol and 300 μg / mL methyl trans-cinnamate were 98% and 96.37%, respectively, while the inhibition rates of 100 μg / mL[8]-shogaol, 400 μg / mL and 500 μg / mL methyl trans-cinnamate were all 100%, as shown in Figure 6.

[0088] Meanwhile, the corrected mortality rates of HMQAU23223 fermentation filtrate stock solution, 80 μg / mL and 100 μg / mL [8]-shogaol, 300 μg / mL, 400 μg / mL and 500 μg / mL trans-cinnamic acid and trans-cinnamic acid methyl ester were all 100%.

[0089] Example 6 Inhibitory Effects of Single and Compound Agents of HMQAU23223 Metabolites on Pine Wood Nematode 6.1 Materials and Methods The lethality of single and compound agents of the HMQAU23223 metabolites trans-cinnamic acid and trans-cinnamic acid methyl ester against pine wood nematode was determined using the immersion method. The corresponding concentrations of trans-cinnamic acid and trans-cinnamic acid methyl ester, and the calculated EC50 of the single agent were respectively measured. 50 A 5:5 mixture of the drug and its dilution was added to 50 μL of pine wood nematodes (approximately 200 nematodes) and placed in a 24-well cell culture plate. After 24 hours incubation at 25°C, the mixture was aspirated and observed under a microscope. The number of dead nematodes and the total number of nematodes were recorded, and the corrected mortality rate was calculated. Each treatment was repeated in triplicate. The corresponding solvent without the drug served as a control. Based on linear regression analysis between the logarithm of the drug concentration and the corrected mortality probability, the toxicity regression equation was derived, and the inhibitory median concentration (EC50) was calculated. 50 Value. The co-toxicity coefficient of the 5:5 compound preparation was determined using the method of Sun Yunpei (1950).

[0090] 6.2 Results and Analysis: Corresponding toxicity equations, correlation coefficients, and EC50 of single-agent and combined formulations of trans-cinnamic acid and trans-methyl cinnamate. 50 The equations are: y = -20.65 + 9.13x, r = 0.983, EC = 0.983. 50 =182.666μg / mL; y=-9.5+4.76x, r=0.965, EC 50 =103.525μg / mL; y=-34.01+23.21x, r=0.944, EC 50 =29.649 μg / mL. The co-toxicity coefficient of the 5:5 compound is 445.72, indicating a synergistic effect, as shown in Table 12.

[0091] Table 12. Lethality of single and compound agents of cinnamic acid and trans-cinnamic acid against pine wood nematode over 24 hours.

[0092] In summary, this invention has successfully screened three metabolites that have good lethal effects on pine wood nematode and southern root-knot nematode and good inhibitory effects on cucumber downy mildew.

[0093] [8]-Shogaol, English name: [8]-Shogaol, CAS No.: 36700-45-5, Molecular formula: C 19 H28 O3, molecular weight: 304.42, molecular structure formula: .

[0094] Trans-cinnamic acid, molecular formula: C9H8O2, molecular weight: 148.16, molecular structure:

[0095] Methyl trans-cinnamate, molecular formula: C 10 H 10 O2, molecular weight: 162.19, molecular structure formula:

[0096] Currently, there are no reports at home and abroad on the lethal effects of [8]-shogaol, trans-cinnamic acid and trans-cinnamic acid methyl ester on pine wood nematode and southern root-knot nematode and on the inhibition of Cuban pseudoperennial fungus, which lays the foundation for further in-depth research on the compounds.

[0097] It should be noted that the specific embodiments are merely representative examples of the present invention, and the technical solution of the present invention is obviously not limited to the above embodiments, and there can be many variations. Those skilled in the art who obtain the present invention based on its explicit disclosure or without objection from the written description should consider it to be within the scope of protection of this patent.

Claims

1. A strain of Bacillus safensis, HMQAU23223, characterized in that, This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 13, 2025, with accession number CGMCC No. 36623.

2. A biocontrol agent, characterized in that, The active ingredient of the biocontrol agent includes a bacterial suspension, fermentation broth, or fermentation filtrate of Bacillus salsa strain HMQAU23223 as described in claim 1.

3. The method for preparing the biocontrol agent according to claim 2, characterized in that, The active ingredient of the biocontrol agent is fermentation filtrate. The specific steps include: (1) preparing seed liquid; (2) adding 1-2% of the seed liquid to LB liquid culture medium and culturing it in a constant temperature shaker at 200-400 rpm for 24-48h at 28-30℃ to prepare fermentation broth; (3) centrifuging the fermentation broth at 24-28℃ and 6000-8000 rpm for 15-20min and filtering to obtain sterile fermentation filtrate, which is the biocontrol agent.

4. The application of the *Bacillus sabolicii* strain HMQAU23223 as described in claim 1, or the biocontrol agent as described in claim 2, or the biocontrol agent prepared by the preparation method as described in claim 3, in the following (a1) or (a2) or (a3) ​​or (a4), characterized in that, (a1) Inhibiting plant pathogens; (a2) Preparing products for inhibiting plant pathogens; (a3) ​​Preventing and treating diseases caused by plant pathogens; (a4) Preparing products for preventing and treating diseases caused by plant pathogens; wherein the plant pathogens are *Pseudoperonospora cubensis*, *Botrytiscinerea*, *Cladosporium tenuissimum*, *Corynesporacassiicola*, *Alternaria alternata*, *Nigrospora oryzae*, *Stagonosporopsis cucurbitacearum*, *Phomopsis* sp., *Alternaria tenuissima*, *Fusarium* sp., *Trametes trogii*, *Colletotrichum plurivorum*, and *Fusarium* sp. *Culmorum*, *Mycosphaerella* sp., *Lasiodiplodia pseudotheobromae*, *Botryosphaeria dothidea*, *Neopestalotiopsis clavispora*, *Colletotrichum* sp., *Curvularia* sp., *Ulocladium chartarum*, *Stemphylium botryosum*, *Alternaria solani*, *Colletotrichum orbiculare*, *Colletotrichum siamense*, *Colletotrichum gloeosporioides*, *Phytophthora capsici*, *Sclerotinia sclerotiorum*, *Pythium* Fusarium spinosum, Fusarium oxysporum, and Fusarium solani.

5. The application of the *Bacillus sabolicii* strain HMQAU23223 as described in claim 1, or the biocontrol agent as described in claim 2, or the biocontrol agent prepared by the preparation method described in claim 3, in the control of plant diseases, characterized in that... The plant diseases mentioned are any one of the following: cucumber downy mildew, bean gray mold, blueberry leaf spot, blueberry twig blight, cucumber target spot, peanut leaf spot, lettuce leaf spot, eggplant early blight, cucumber anthracnose, strawberry anthracnose, pepper blight, celery sclerotinia stem rot, cucumber damping-off, cucumber wilt, garlic dry rot, and watermelon wilt.

6. A method for controlling plant diseases, characterized in that, When controlling plant diseases, the *Bacillus sabinatus* strain HMQAU23223 as described in claim 1, or the biocontrol agent as described in claim 2, or the biocontrol agent prepared by the preparation method described in claim 3, can be applied to the disease-prone parts of the plant by coating, impregnation, spraying, atomizing, irrigating, dusting, sowing, foaming, coating, or spraying.

7. The application of the *Bacillus sabolicus* strain HMQAU23223 as described in claim 1, or the biocontrol agent as described in claim 2, or the biocontrol agent prepared by the preparation method described in claim 3, in the control of plant pathogenic nematodes, characterized in that, The plant pathogenic nematodes mentioned are pine wood nematode, southern root-knot nematode, cereal cyst nematode, Philippine cyst nematode, and stem rot nematode.

8. The use of the Bacillus sabinatus strain HMQAU23223 as described in claim 1 in the preparation of compounds for the control of plant pathogenic bacteria or plant pathogenic nematodes.

9. The application according to claim 8, characterized in that, The compound is any one or at least two of the following: shogaol, [8]-shogaol, trans-cinnamic acid, 3-methylthiopropanol, methyl trans-cinnamic acid, pimecrolimus, and doxorubicin hydrochloride.

10. The application according to claim 9, characterized in that, The compound is a combination of trans-cinnamic acid and trans-cinnamic acid methyl ester, with a mass ratio of 5:5.

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