Pseudomonas putida ZF541 and application thereof in preventing and treating white mold of peanut

Abstract

The application provides a plurienibacter gilvus ZF541 and application thereof in prevention and treatment of peanut sclerotinia blight, and belongs to the technical field of microbial pesticides. Priestia megaterium The plurienibacter gilvus is plurienibacter gilvus (P. gilvus) ZF541, and the preservation number thereof in the China General Microbiological Culture Collection Center is CGMCC No. 34882. The fermentation liquor of ZF541 can inhibit the growth of sclerotium rolfsii. In a pot experiment, the prevention effect of ZF541 on peanut sclerotinia blight is 83.88%, and in a field experiment, the prevention effect reaches 72.93%, which is equivalent to that of a chemical fungicide thifluzamide and higher than that of trichoderma harzianum. The ZF541 fungicide has a good biocontrol prospect, and the prevention and treatment effect is stable.

Description

Technical Field

[0001] This invention relates to the field of microbial pesticide technology, specifically to a strain of Primus giantis ZF541 and its application in controlling peanut white mold disease. Background Technology

[0002] Peanut white mold disease is caused by *Sclerotium moniliforme* (… Sclerotium rolfsii Sclerotium affine (Sacc.) is a serious soil-borne disease of peanuts, affecting over 600 plant species, including important crops such as wheat, soybeans, peanuts, sesame, tomatoes, and onions. Peanuts can be infected at any growth stage by Sclerotium affine, primarily damaging the stem, pedicel, and pods. Affected plants exhibit stem rot at the base, cortex shedding, leaving fibrous tissue, and yellowing and scorched leaf edges, severely impacting commercial value and economic benefits.

[0003] Peanut white mold disease occurs in most peanut-producing areas and is showing a trend of increasing severity year by year. In recent years, due to factors such as high-density planting, continuous cropping, and large-scale planting of single varieties, white mold disease has become the main soil-borne disease of peanuts, generally causing yield losses of 10%-30%, and in severe cases, more than 80%, seriously affecting peanut production.

[0004] Given the limitations of conventional control measures in effectively managing peanut white mold, chemical pesticides remain the primary method of control. However, long-term use of chemical pesticides not only pollutes the environment, creates pesticide residues, and fosters drug resistance in pathogens, but also negatively impacts soil biodiversity. Biological control, as a green, sustainable, safe, and reliable method, has demonstrated enormous potential in controlling plant diseases and has attracted widespread attention.

[0005] Under artificial in vitro culture conditions, biocontrol strains have an inhibitory effect on mycelial growth, sclerotium germination, and the occurrence of white mold disease in peanuts, but there are few reports of successful large-scale application in production. The control efficacy of *Primulina megaterium* against peanut white mold disease has not yet been reported. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to antagonize *Sclerotium moniliforme* (…). Sclerotium rolfsii Sacc.) and / or control peanut white mold disease.

[0007] To solve the above-mentioned technical problems, the present invention first provides *Primatex megaterium*, wherein *Primatex megaterium* is *Primatex megaterium* (… Priestia megaterium ZF541, its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 34882.

[0008] The present invention also provides the application of the aforementioned Primate gigantea in the preparation of products.

[0009] The present invention also provides a microbial agent, which may contain the aforementioned *Primatex megalophylla*.

[0010] The bacterial agent may also be a culture, which is a substance obtained by culturing the giant Primus in a microbial culture medium.

[0011] The substance may be a fermentation product, such as a fermentation broth containing the aforementioned *Primatex megaterium* and substances secreted into a liquid culture medium, or a solid fermentation product containing the aforementioned *Primatex megaterium* and substances secreted into a solid culture medium.

[0012] The active ingredients of the above-mentioned microbial agents may also contain other biological or non-biological components. Other active ingredients of the above-mentioned compositions can be determined by those skilled in the art based on the effects of the compositions.

[0013] The active ingredient of the above-mentioned bacterial agent may be *Primatex megaterium* or / and the metabolites of *Primatex megaterium* or / and the culture of *Primatex megaterium*.

[0014] As stated above, the metabolites can be obtained from the shake culture broth of *Primatex megalophylla*. The metabolites can be bacterial metabolites of *Primatex megalophylla*. Specifically, the bacterial metabolites of *Primatex megalophylla* can be prepared by culturing *Primatex megalophylla* in LB liquid medium and collecting the bacterial solution, which is the bacterial metabolite of *Primatex megalophylla*.

[0015] The microbial culture medium can be LB liquid medium, with the following specific formula: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, and distilled water to a final volume of 1000 mL.

[0016] The culture can be carried out at 28°C.

[0017] The culture conditions can be 180 r / min for 48 h.

[0018] The bacterial agent may be any one of the following: A1) is used to antagonize *Sclerotium sclerotiorum* ( ). Sclerotium rolfsii Sacc.) bacterial agent; A2) Inoculants used to control white mold disease in peanuts; A3) is used to prevent and control *Sclerotinia sclerotiorum* ( ). Sclerotium rolfsii Inoculants for diseases caused by Sacc.

[0019] This invention also provides the application of the aforementioned microbial agent in the preparation of products.

[0020] The present invention also provides a method for preparing a microbial agent, the method comprising the step of using the aforementioned Priestella megaterium or a culture thereof as a component of the microbial agent.

[0021] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.

[0022] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0023] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0024] The aforementioned carrier may specifically be at least one of LB medium, calcium phosphate, rice husk powder, wheat bran, rice bran, skim milk powder, maltodextrin, sucrose, glycerol, and starch.

[0025] In this invention, the product may have at least one of the following properties: B1) is used to antagonize *Sclerotium sclerotiorum* ( ). Sclerotium rolfsii Sacc.); B2) Used to prevent and control peanut white mold; B3) is used to prevent and control *Sclerotinia sclerotiorum* ( ). Sclerotium rolfsii Sacc . Inoculants for diseases caused by fungi.

[0026] The present invention also provides a method for controlling peanut white mold, the method comprising the step of contacting the plant with the aforementioned Pristella giantiflora or fungal agent to control peanut white mold.

[0027] In the above method, the contact with the plant can be inoculated by root irrigation.

[0028] In the above method, the plant can be any of the following: C1) angiosperms, C2) Magnoliopsida, C3) Fabales, C4) Leguminosae, C5) Arachis genus, C6) peanut.

[0029] The peanut mentioned can be a peanut seedling.

[0030] In one specific embodiment, healthy peanut seeds were disinfected by soaking in 1% sodium hypochlorite for 10 minutes, then rinsed three times with sterile water, germinated in a container moistened with sterile gauze for 24 hours, and then sown in pots (pot height 9.5 cm, inner diameter 11 cm, bottom diameter 7.5 cm), with 4 seeds planted in each pot, and 3 seedlings were retained after emergence.

[0031] Healthy peanut seedlings that had grown for 10 days were selected for pot experiments. Each pot was inoculated by root irrigation with 20 mL of ZF541 fermentation liquid (ZF541 treatment group).

[0032] Precipitella megacephala ( Priestia megaterium ZF541 is a biocontrol strain with high activity against peanut white mold. This study determined the preventive effect of its bacterial suspension against peanut white mold through in vivo experiments. In pot experiments, its control efficacy against peanut white mold was 83.88%, comparable to the chemical fungicide thifluzamide, and significantly higher than the control biocontrol agent *Trichoderma harzianum* water-dispersible granules (100 million CFU / g) (64.50%). In field experiments, the control efficacy of ZF541 bacterial suspension against peanut white mold reached 72.93%, comparable to the chemical fungicide thifluzamide (71.13%), and higher than *Trichoderma harzianum* (67.36%). This indicates that ZF541 has good biocontrol potential and its control effect remains stable.

[0033] Preservation Instructions Strain name: Primus megaterium Latin name: Priestia megaterium Strain number: ZF541 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: June 13, 2025 CGMCC Registration Number: CGMCC No. 34882 Attached Figure Description Figure 1 It is strain ZF541 based on 16S rDNA and rpoB , gyrB Phylogenetic tree of gene sequences.

[0034] Figure 2 This represents the inhibitory effect of strain ZF541 on *Sclerotium oliguriae*. A: Inhibitory effect of strain ZF541 on *Sclerotium oliguriae*; B: *Sclerotium oliguriae* colonies (control). Strain ZF541 was located around the periphery of plate A, while *Sclerotium oliguriae* was located in the center of plates A and B.

[0035] Figure 3 These are the results of biocontrol factor assays for *Primulina megaterium* ZF541. A represents the ability to solubilize inorganic phosphorus; B represents the ability to solubilize organic phosphorus; C represents the ability to fix nitrogen; D represents the ability to solubilize potassium; E represents the ability to produce protease; F represents the ability to produce cellulase; G represents the ability to produce siderophores; and H represents the ability to produce chitinase.

[0036] Figure 4 These are images of peanuts after treatment with the experimental group (ZF541) and the pathogen control group (disease control). In the image, A shows the control effect of ZF541 on peanut white mold, and B shows the control treatment directly targeting the pathogen. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] The following examples use SPSS statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The Duncan's new multiple range test was used for the test. Different lowercase letters indicate significant differences at the 0.05 level.

[0040] Example 1: Isolation, Identification and Application of Strain ZF541 I. Materials and Methods 1.1 Test Materials 1.1.1 The tested pathogens were uniformly shaped as *Sclerotium sclerotiorum*. Sclerotium rolfsii The Sacc. was purchased from the China Agricultural Microbial Culture Collection Center, strain number: ACCC 39139. The public may obtain this biological material from the applicant in accordance with relevant national biosafety regulations. This biological material is solely for the purpose of replicating the relevant experiments of this invention and may not be used for any other purpose.

[0041] 1.1.2 Test culture media: LB medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, distilled water to 1000 mL; PDA medium: 200 g potato, 20 g glucose, 15 g Agar, distilled water to 1000 mL.

[0042] 1.1.3 The peanut variety used for testing, “Four-Grain Red Peanut,” was purchased from Shouguang Xinxinran Horticulture Co., Ltd.

[0043] 1.2 Isolation and purification of strains from soil Soil samples from Wushan Village, Lingshui, Hainan Province, were used to isolate antagonistic bacteria using a gradient dilution method. Following the method described by Wei Xinchen et al. (Wei Xinchen, Liu Yi, Yang Ruwei, Duan Guangrong, Xie Xuewen, Shi Yanxia, ​​Chai Ali, Li Baoju, Li Lei, Isolation, Identification and Preliminary Study on Biocontrol Effect of Antagonistic Bacterium ZF517 for Potato Scab, Chinese Journal of Biological Control 41(02) (2025) 411-422.), 10 g of the soil sample was weighed after mixing and dissolved in 90 mL of sterile water. The solution was shaken on a shaker at 28℃ and 180 r / min for 0.5 h to ensure complete suspension. Suspensions diluted 10⁻³ to 10⁻⁷ were evenly spread on LB agar and incubated at 28℃ for 48 h. Subsequently, single colonies with different morphologies were picked using a sterile inoculation loop and purified a second time. After obtaining stable single colonies, they were purified and stored in a -80°C freezer with glycerol.

[0044] 1.3 Screening of antagonistic strains of *Sclerotium sclerotiorum* According to the method of Huang Yishuo et al. (Huang Yishuo, Xie Xuewen, Shi Yanxia, ​​Chai Ali, Li Lei, Li Baoju, Control effect of Bacillus polymyxa ZF197 on stem base rot of Chinese cabbage, Journal of Horticulture 47(06) (2020) 1059-1071.), the inhibition rate of the above colonies against Sclerotium truncatum was determined by the plate confrontation method. A 5 mm diameter Sclerotium truncatum mycelium was inoculated in the center of a PDA plate. After incubation at 28℃ for 1 day, 5 µL OD was added at four opposite points 3 cm from the center of the plate using the cross-hatching method. 600 The target bacterial strain was cultured at 0.8 μg / mL, with a control strain not inoculated, at 28°C for 3 days. Each treatment was repeated three times. The diameters of the control and treated colonies of the target bacteria were measured. Inhibition rate (%) = [(control colony diameter – treated colony diameter) / control colony diameter] × 100%.

[0045] 1.4 Identification of antagonistic bacteria ZF541 1.4.1 Morphological Identification of Strain ZF541 Strain ZF541 was inoculated onto LB medium using the streak plate method, and colony morphology was observed after 36 h. Electron microscopy samples were fixed with 2.5% glutaraldehyde and dehydrated seven times (10 min each time) with 20%, 30%, 40%, 50%, 60%, 80%, and 100% ethanol, followed by transmission electron microscopy (TEM) observation.

[0046] 1.4.2 Physiological and biochemical determination of strain ZF541: The physiological and biochemical characteristics of strain ZF541 were determined according to Bergey's Manual of Bacterial Identification, 8th Edition and the Manual of Systematic Identification of Common Bacteria. The unique carbon source utilization of strain ZF541 was determined by the China Agricultural Microbial Culture Collection Center using the BIOLOG GEN III kit.

[0047] 1.4.3 Multigene Amplification and Sequence Analysis of Strains ZF541 After extracting a small amount of genomic DNA from strain ZF541 using the TIANGEN bacterial genomic DNA extraction kit, 16S rDNA was selected... rpoB and gyrB Gene-specific primers were used to amplify the gene of strain ZF541, and the amplification product was sent to Beijing Bomeide Biotechnology Co., Ltd. for sequencing. The 16S rDNA gene amplification primers were: 27F: 5'-AGAGTTTGATCCTGGCTAG-3'; 1492R: 5'-AAGGAGGTGATCCAGCCGCA-3'. rpoB The gene amplification primers were BF: 5'-ATCGAAACGCCTGAAGGTCCAAACAT-3'; BR: 5'-ACACCCTTGTTACCGTGACGACC-3'. groE The gene amplification primers were EF: 5'-GTGCGAACCCAATGGGTCTTC-3'; ER: 5'-CCTTGTTGTACCACTTGCTC-3'. The amplification system was 50µL, including 1µL each of upstream and downstream primers, 1µL template DNA, 25µL T-Taq Mix, and 22µL ddH2O. The PCR reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 34 cycles; 72℃ extension for 10 min. The obtained PCR products were sequenced by Bomeide Biotechnology Co., Ltd. The 16S rDNA of strain ZF541 was obtained using the above method. rpoB and gyrB Gene sequences were obtained, and all other sequences were downloaded from the NCBI website. MEGA 7.0, Sequence Matrix, and Seaview 4 were used to sequentially ligate and compare the 16S rDNA of different strains. rpoB and gyrB Gene sequences were used to construct phylogenetic trees using the maximum likelihood method to analyze their taxonomic positions.

[0048] 1.5 Biocontrol Factor Detection of Strain ZF541 1.5.1 Phosphate-solubilizing ability test: A single colony of ZF541 was picked using a sterile inoculation needle and inoculated into the center of PVK selective medium and organophosphate bacteria medium, with three replicates. The plates were incubated upside down in a 28°C incubator for 24 h. If a transparent digestion zone appeared in the inoculated area, it indicated that the strain had phosphate-solubilizing ability.

[0049] PVK selective medium formulation: Calcium phosphate (Ca3(PO4)2): 5 g; yeast extract: 0.5 g; potassium chloride (KCl): 0.2 g; magnesium sulfate (MgSO4·7H2O): 0.1 g; agar: 15 g (for solid media); distilled or deionized water: bring to 1 L. Adjust pH to 7.0 ± 0.2, which can be adjusted with sodium hydroxide or hydrochloric acid.

[0050] Organophosphate bacteria culture medium formula: Glucose 10.0g; Ammonium sulfate ((NH4)2SO4) 0.5g; Yeast extract 0.5g; Sodium chloride (NaCl) 0.3g; Potassium chloride (KCl) 0.3g; Magnesium sulfate (MgSO4·7H2O) 0.3g; Ferrous sulfate (FeSO4·7H2O) 0.03g; Manganese sulfate (MnSO4·H2O) 0.03g; Lecithin 0.2g; Calcium carbonate (CaCO3) 1.0g; Agar (for solid culture medium) 15.0g. Make up to 1 L, and adjust the pH to 7.0-7.5.

[0051] 1.5.2 Nitrogen fixation capacity test: A single colony of ZF541 was picked up using a sterile inoculation needle and inoculated into the center of a nitrogen-fixing solid medium, with three replicates. The plates were incubated upside down in a 28°C incubator for 24 h. If colonies grew in the inoculated area, it indicated that the strain had nitrogen-fixing capacity.

[0052] Nitrogen-fixing solid culture medium formula: glucose 10.0 g, potassium dihydrogen phosphate (KH2PO4) 0.2 g, magnesium sulfate (MgSO4·7H2O) 0.2 g, sodium chloride (NaCl) 0.2 g, potassium sulfate (K2SO4) 0.1 g, calcium carbonate (CaCO3) 5.0 g, distilled water to 1 L, pH adjusted to 7.0, and agar added at 15-18 g / L.

[0053] 1.5.3 Potassium-Solubilizing Ability Test: A single colony of ZF541 was picked up using a sterile inoculation needle and inoculated into the center of a potassium-solubilizing solid medium, with three replicates. The plates were incubated upside down in a 28°C incubator for 24 h. If oil droplet-like colonies grew in the inoculated area, it indicated that the strain had potassium-solubilizing ability.

[0054] Potassium-solubilizing solid culture medium formula: 5.0 g sucrose, 5.0 g glucose, 0.5 g ammonium sulfate ((NH4)2SO4), 0.5 g yeast extract, 0.3 g magnesium sulfate (MgSO4·7H2O), 2.0 g disodium hydrogen phosphate (Na2HPO4), 0.03 g ferric sulfate (FeSO4·7H2O), 0.03 g manganese sulfate (MnSO4·H2O), 2.0 g potassium feldspar powder (passed through a 200-mesh sieve; potassium feldspar does not dissolve; continuous stirring throughout preparation; shake well after sterilization before pouring into plates to prevent mineral sedimentation and aggregation), 15.0 g agar, distilled water to a final volume of 1 L, pH adjusted to 7.2–7.4.

[0055] 1.5.4 Protease Synthesis Capacity Assay: A single colony of ZF541 was picked using a sterile inoculating needle and inoculated into the center of a protease-selective medium, with three replicates. The plates were incubated upside down in a 28°C incubator for 24 h. If a clear digestion zone appeared in the inoculated area, it indicated that the strain had protease synthesis capacity.

[0056] Protease-selective culture medium formulation: 10.0 g casein, 3.0 g beef extract, 2.0 g disodium hydrogen phosphate (Na2HPO4), 5.0 g sodium chloride (NaCl), 15.0 g agar, distilled water to a final volume of 1 L, pH adjusted to 7.2.

[0057] 1.5.5 Cellulase Synthesis Capacity Assay: A single colony of ZF541 was picked using a sterile inoculating needle and inoculated into the center of the cellulase assay medium and the cellulase activity assay medium, with three replicates. The plates were incubated upside down in a 28°C incubator for 7 days. After incubation, 2.5 mL of 0.2 mg / mL Congo red staining solution was added to the surface of the plate. After staining for 30 min, the staining solution was discarded, and the plate surface was slowly rinsed with 1 mL of NaCl solution. If a clear zone appears in the inoculated area, it indicates that the strain has cellulase synthesis capacity.

[0058] The culture medium formula for cellulase detection is as follows: sodium carboxymethyl cellulose (CMC-Na) 10.0 g, ammonium sulfate ((NH4)2SO4) 2 g, dipotassium hydrogen phosphate (K2HPO4) 1.0 g, potassium dihydrogen phosphate (KH2PO4) 0.5 g, magnesium sulfate (MgSO4·7H2O) 0.2 g, sodium chloride (NaCl) 1.0 g, agar 15 g~18 g, and distilled water to a final volume of 1 L.

[0059] 1.5.6 Siderophore Synthesis Capacity Detection: A single colony of ZF541 was picked using a sterile inoculation needle and inoculated into the center of CAS medium, with three replicates. The plates were incubated upside down in a 28℃ incubator for 48 h. If a pale yellow digestion zone appeared in the inoculated area, it indicated that the strain had siderophore synthesis capacity.

[0060] CAS detection culture medium: purchased from Beijing Solarbio Science & Technology Co., Ltd., product number LB0680.

[0061] 1.5.7 Chitin Synthesis Capacity Detection: A single colony of ZF541 was picked using a sterile inoculation needle and inoculated into the center of the chitin detection medium, with three replicates. The plates were incubated upside down in a 28℃ incubator for 48 h. If a clear zone appeared in the inoculated area, it indicated that the strain had chitin synthesis capacity.

[0062] Chitin assay medium formulation: Colloidal chitin (wet weight) 4.0 g, ammonium sulfate ((NH4)2SO4) 2 g, potassium dihydrogen phosphate (KH2PO4) 0.3 g, dipotassium hydrogen phosphate (K2HPO4) 0.7 g, magnesium sulfate (MgSO4·7H2O) 0.5 g, ferric sulfate (FeSO4·7H2O) 0.01 g, agar 18.0 g, distilled water to 1 L.

[0063] 1.6 Effect of fermentation broth of strain ZF541 on the growth of *Sclerotium oliguriae* Single colonies of strain ZF541 were picked and inoculated into Erlenmeyer flasks containing 200 mL of LB liquid medium. The broth was incubated at 28°C and 180 r / min for 48 h to obtain the ZF541 fermentation broth. The broth was diluted 10-fold with PDA medium and poured onto plates. A neatly arranged *Sclerotium sclerotium* mycelial cake was placed in the center of each plate. An equal volume of LB medium was added as a control. The plates were incubated at 25°C. When the hyphae of the control group reached the edge of the PDA medium, the colony diameter was measured, and the inhibition rate was calculated.

[0064] 1.7 Determination of the control effect of strain ZF541 on peanut white mold disease The pot experiment was conducted in a greenhouse with a cultivation temperature of (25±1)℃ and a photoperiod of 16 h light to 8 h darkness. Healthy peanut seeds were disinfected by soaking in 0.1% NaCl for 10 min, then rinsed three times with sterile water, and germinated for 24 h in a container moistened with sterile gauze. After germination, the seeds were sown in pots (9.5 cm high, 11 cm inner diameter, and 7.5 cm bottom diameter), with 4 seeds planted per pot. After emergence, 3 seedlings were retained.

[0065] Soak dried oat grains in water overnight, drain, and divide into sterile bags, filling 1 / 5 of the bag's volume. Autoclave at 121℃ for 20 minutes. After cooling, inoculate with 10 neatly arranged mycelial cakes (5mm in diameter) of *Sclerotium sclerotiorum* and incubate in the dark at 25℃. Shake the oat grains once daily until all grains are fully covered with mycelium. Prepare the ZF541 fermentation broth according to method 1.6.

[0066] In the pot experiment, healthy peanut seedlings that had grown for 10 days were selected. Each pot was inoculated with 20 mL of ZF541 fermentation broth (ZF541 treatment group) by root drenching. A control was prepared by inoculating each pot with 20 mL of a 1500-fold dilution of 240 g / L thifluzamide suspension (purchased from Sichuan Lier Crop Science Co., Ltd.). A biocontrol control was prepared by inoculating each pot with 20 mL of a 500-fold dilution of 100 million CFU / g Trichoderma harzianum water-dispersible granules (purchased from Chengdu Tepu Biotechnology Co., Ltd.). A pathogen control was prepared by inoculating an equal volume of LB liquid medium (disease control). The 500-fold dilution of 100 million CFU / g Trichoderma harzianum water-dispersible granules was prepared by diluting each g of Trichoderma harzianum with 500 mL of water.

[0067] Twenty-four hours after root drenching, 10 oat grains wrapped with neatly arranged Sclerotium truncatum were inoculated at the base of each peanut stem. After culturing for another 3 weeks, the control effect was calculated according to the method of Dong Weibao et al. (Dong Weibao, Shi Yanmao, Sun Aixiang, Geng Yingxiu, Liu Jiayu, Greenhouse inoculation technology and resistance identification of peanut white mold, Peanut Journal (03) (2001) 17-20.). Three pots each of control and treatment were used, and the results were repeated three times.

[0068] Field Experiment: Field Efficacy Determination of Strain ZF541 against Peanut White Rot. Plump, disease-free peanut seeds were selected and sown in an area of ​​20m². 2In each plot, 50 peanut seeds were sown. Ten to 15 days after emergence, artificial inoculation with *Sclerotium affine* was performed using the soil-inoculation method: cultured sclerotia / mycelium of *Sclerotium affine* were mixed with fine soil and evenly spread at the base of the peanut rhizomes. The mixture was then covered with soil to retain moisture, inducing uniform disease development in the field. Twenty-four hours after inoculation, the first root drenching treatment was applied, with 20 mL of ZF541 bacterial solution per plant. A second treatment was applied after 7 days, for a total of two applications. A control group was sprayed / drenched at the same time, while a blank control group was irrigated with the same amount of water. The timing, water volume, and method of operation were completely identical for all treatments. A 1500-fold dilution of 240 g / L thifluzamide suspension (purchased from Sichuan Lier Crop Science Co., Ltd.) was used as a chemical fungicide control, and a 100 million CFU / g Trichoderma harzianum water-dispersible granule (purchased from Chengdu Tepu Biotechnology Co., Ltd.) was used as a biocontrol control. The 100 million CFU / g Trichoderma harzianum water-dispersible granule was prepared by diluting each g of Trichoderma harzianum with 500 mL of water to a 500-fold dilution.

[0069] Disease grading standards: Grade 0, no symptoms in the plant; Grade 1, lesions only appear at the base of the stem; Grade 2, constriction symptoms appear at the base of the stem, and less than one-third of the whole plant shows systemic symptoms (wilt, death, withering, etc.); Grade 3, less than two-thirds of the whole plant shows systemic symptoms; Grade 4, more than two-thirds of the whole plant shows systemic symptoms.

[0070] Disease index = ∑ (number of diseased plants at each level × disease level) / (total number of plants surveyed × highest disease level) × 100%; Prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0071] 1.8 Data Statistics and Analysis The experimental data in this study were statistically analyzed using Excel and SPSS software.

[0072] 2 Results and Analysis 2.1 Isolation and screening of strain ZF541 and observation of mycelial morphology of the pathogen A total of 186 bacterial strains were isolated and purified from soil samples from Wushan Village, Lingshui, Hainan. Preliminary screening of these 186 strains was conducted using the plate confrontation method. Ten antagonistic bacteria showed good inhibitory effects on *Sclerotium sclerotium*, inhibiting mycelial growth with inhibition rates exceeding 50%. Strain ZF541 showed the most significant effect, with an inhibition rate of 67.25%. (Table 1) Table 1. Inhibition rates of the 10 screened antagonistic strains against *Sclerotium riberi* on agar plates.

[0073] Note: Data are mean ± standard error. Different lowercase letters indicate significant differences at the 0.05 level.

[0074] 2.2 Identification of strain ZF541 Physiological and biochemical characteristics of strain ZF541 were determined, indicating that it is a Gram-negative bacterium with an optimal growth environment of pH 6 and can grow in NaCl concentrations ranging from 1% to 8%. Biolog results showed that strain ZF541 can utilize dextran, D-maltose, sucrose, etc., and the bacterial cells exhibit motility (Table 2).

[0075] The 16S rDNA of strain ZF541 is SEQ ID NO: 1 (1395bp), as follows:

[0076] The PCR amplification primers for 16S rDNA were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3').

[0077] strain ZF541 rpoB For SEQ ID NO: 2 (1134bp), the details are as follows:

[0078] strain ZF541 gyrB It is SEQ ID NO: 2 (1074bp), as follows:

[0079] With 16S rDNA and rpoB , gyrB A multi-gene phylogenetic tree constructed from gene sequences shows that strain ZF541 is related to Bacillus subtilis. Priestia megaterium SR7(CP022674.1) clusters in the same branch ( Figure 1 Based on the above morphological characteristics analysis, physiological and biochemical indicators, BIOLOG unique carbon source utilization characteristics, and multi-gene sequence analysis results, strain ZF541 was identified as *Primatex megaterium*. Priestia megaterium .

[0080] Table 2 Physiological and biochemical reactions of strain ZF541

[0081] Note: + indicates that the experimental result is positive; - indicates that the experimental result is negative.

[0082] 2.3 Effects of strain ZF541 bacterial suspension on the growth of *Sclerotium truncatum* mycelia After being cultured on LB agar plates, strain ZF541 produced pale yellow, round or oval, slightly raised, opaque colonies with smooth surfaces and neat edges. Plate contrast experiments showed that, compared to the control (… Figure 2 Compared to (B), treatment with ZF541 bacterial suspension (B) Figure 2 After treatment with A), the growth rate of *Sclerotium moniliforme* slowed down significantly, and the inhibition rate reached 67.25%. Figure 2 The inhibitory effect of strain ZF541 on *Sclerotium sclerotium* is shown in Figure A, where strain ZF541 inhibits *Sclerotium sclerotium*, with *Sclerotium sclerotium* located in the center of the plate and strain ZF541 located around the periphery of the plate; Figure B is the control *Sclerotium sclerotium* colony, where *Sclerotium sclerotium* was inoculated only in the center of the plate.

[0083] 2.4 Identification of biocontrol factors in strain ZF541 2.4.1 Phosphorus solubility test Positive result criteria: This is a common method to distinguish between high and low phosphate solubility: Measure the diameter of the clear zone (D) and the colony diameter (d), and calculate the D / d value. The larger the ratio, the stronger the phosphate solubility.

[0084] 2.4.2 Nitrogen fixation capacity test Positive criteria: If colonies can grow in the inoculated area and form visible colonies on a nitrogen-fixing solid culture medium plate, it is considered positive.

[0085] 2.4.3 Potassium solubilization capacity test Positive result criteria: On potassium-solubilizing solid medium, colonies grow as viscous, translucent, glossy oil droplets. This describes the typical characteristics of this strain.

[0086] 2.4.4 Detection of protease synthesis capacity Positive result criteria: A clear disappearance zone appears around the colony on a protease-selective medium.

[0087] 2.4.5 Detection of cellulase synthesis capacity Positive result criteria: After staining, a clear transparent hydrolysis zone is observed around the colony.

[0088] 2.4.6 Detection of siderophore synthesis capacity Positive result criteria: An orange (or yellow) halo appears around the colony on a CAS agar plate. This is because the siderophore removes iron ions from the blue CAS complex, causing the medium to turn from blue to yellow.

[0089] 2.4.7 Chitin Ability Test Positive result criteria: A clear zone appears around the colony on the chitin detection medium.

[0090] Strain ZF541 formed distinct clear digestion zones on protease-selective medium; formed oil droplet-like colonies on potassium-solubilizing medium; and showed inhibited colony growth on nitrogen-fixing medium. No clear zones were produced on PVK medium and organophosphorus-selective medium, and no pale yellow digestion zones were observed on CAS detection medium. No digestion zones were observed on cellulase detection medium. These results indicate that strain ZF541 possesses potassium-solubilizing ability and protease activity, but lacks siderophore synthesis ability, biofilm formation ability, cellulase activity, and nitrogen-fixing ability.

[0091] 2.5 Pot control efficacy of strain ZF541 against peanut white mold disease The disease index of potted peanuts treated with strain ZF541 showed no significant difference compared to that of 24% thifluzamide suspension at a dilution of 1500 times, but both were significantly lower than the water control (P<0.05). In terms of control efficacy, strain ZF541 achieved 83.88% control of peanut white mold, comparable to the chemical fungicide 240 g / L thifluzamide suspension, and significantly higher than the control biocontrol agent 100 million CFU / g Trichoderma harzianum water-dispersible granules (64.50%). ZF541 shows promising potential for biocontrol.

[0092] Table 3. Potted control efficacy of strain ZF541 against peanut white mold disease.

[0093] Note: Data are mean ± standard error. Different lowercase letters indicate significant differences at the 0.05 level.

[0094] Figure 4 In Figure A, ZF541 is shown as the control effect on peanut white mold, and in Figure B, it is shown as the control treatment against the direct pathogen.

[0095] 2.6 Field control efficacy of strain ZF541 against peanut white mold disease In April 2025, an evaluation of the biocontrol efficacy of ZF541 against peanut white mold was conducted in Shunyi District, Beijing. The results showed that ZF541 exhibited significant biocontrol efficacy against peanut white mold, achieving a control efficacy of 72.93%, comparable to the chemical fungicide 24% thifluzamide suspension (71.13%), and higher than the 100 million CFU / g Trichoderma harzianum water-dispersible granules treatment (67.36%). This trend is consistent with the pot experiment results, and the control efficacy remained stable.

[0096] Table 4. Field control efficacy of strain ZF541 against peanut white mold.

[0097] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Primate macrocephala, characterized by: The giant praseodymium is giant praseodymium ( Priestia megaterium ZF541, its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 34882.

2. The use of the giant Priestella as described in claim 1 in the preparation of the product.

3. A microbial agent, characterized in that: The bacterial agent contains *Primatex megalophylla* as described in claim 1.

4. The microbial agent according to claim 3, characterized in that: The bacterial agent is a culture, which is a substance obtained by culturing the giant Primus in a microbial culture medium.

5. The microbial agent according to claim 3 or 4, characterized in that: The bacterial agent is any one of the following: A1) is used to antagonize *Sclerotium sclerotiorum* ( ). Sclerotium rolfsii Sacc.) bacterial agent; A2) Inoculants used to control white mold disease in peanuts; A3) is used to prevent and control *Sclerotinia sclerotiorum* ( ). Sclerotium rolfsii Inoculants for diseases caused by Sacc.

6. The use of the microbial agent according to claims 3-5 in the preparation of products.

7. A method for preparing a microbial agent, characterized in that, The bacterial agent is the bacterial agent according to any one of claims 3-5, and the method includes the step of using the giant Priestella or its culture according to claim 1 as a component of the bacterial agent.

8. The product according to claim 2 or 6, characterized in that, The product has at least one of the following properties: B1) is used to antagonize *Sclerotium sclerotiorum* ( ). Sclerotium rolfsii Sacc.); B2) Used to prevent and control peanut white mold; B3) is used to prevent and control *Sclerotinia sclerotiorum* ( ). Sclerotium rolfsii Inoculants for diseases caused by Sacc.

9. A method for controlling peanut white mold, characterized by: The method includes the step of contacting plants with the *Pristina macrocarpa* of claim 1 or the fungal agent of claims 3-5 to control peanut white mold disease.

10. The method according to claim 9, characterized in that: The plant is any one of the following: C1) Angiosperms, C2) Magnolia class plants, C3) Leguminosae (family C3) C4) Leguminosae (family legumes) C5) Arachis plants, C6) Peanuts.

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