A ralstonia solanacearum, bacterial agent and application thereof

Abstract

The application provides a ralstonia insulae and a bacterial agent and application thereof, and relates to the field of microorganisms. Rouxiella aceris The ralstonia insulae is specifically ralstonia insulae (RA) with a preservation number of CGMCC No.35889. The strain has the ability to decompose inorganic phosphorus, and has a wide range of phosphorus decomposition, can decompose calcium phosphate, iron phosphate and aluminum phosphate, and has a strong ability to decompose inorganic phosphorus under low temperature conditions. The strain has the ability to produce siderophores and low-temperature IAA. The strain has inhibitory effect on a plurality of pathogenic bacteria causing field crop diseases, can inhibit corn root rot, sorghum leaf spot or wheat root rot and the like. The strain can colonize in the soil environment at 5-40 DEG C, and the bacterial agent prepared from the strain can promote crop growth and significantly improve crop yield, even under the condition of reducing the use amount of phosphorus fertilizer, the yield is still high.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a type of *Russula natans*, its inoculants, and their applications. Background Technology

[0002] In my country's farmland soils, over 95% of phosphorus exists in the form of ineffective phosphorus, such as tricalcium phosphate, ferric phosphate, and aluminum phosphate. This inorganic phosphorus is difficult for crops to directly absorb and utilize, leading to chronic phosphorus deficiency. Traditional agricultural production primarily relies on chemical phosphate fertilizers to alleviate this deficiency. However, chemical phosphate fertilizers have low utilization rates, with most phosphorus being fixed in the soil, failing to provide a continuous supply of phosphorus throughout the crop's growth cycle. Furthermore, excessive phosphate fertilizers can cause soil compaction. Phosphorus-solubilizing microorganisms can decompose insoluble inorganic phosphorus in the soil, sustainably providing absorbable phosphorus to crops and addressing phosphorus deficiency. However, in low-temperature environments, soil microbial activity significantly decreases, and existing phosphorus-solubilizing microbial strains often fail to function properly, unable to effectively activate insoluble inorganic phosphorus in the soil, further exacerbating the phosphorus shortage problem for crops or overwintering crops in low-temperature regions.

[0003] From the perspective of soil phosphorus forms, although tricalcium phosphate is a poorly soluble inorganic phosphorus, its chemical stability is relatively low compared to aluminum phosphate and iron phosphate, which also exist in the soil. Aluminum phosphate and iron phosphate are present in extremely high proportions in typical low-phosphorus soils in my country, such as acidic soils, red soils, and yellow soils. These two types of phosphorus compounds have extremely strong chemical stability and are the most stubborn and difficult-to-decompose inorganic phosphorus forms in the soil. Conventional phosphorus-solubilizing microorganisms cannot decompose iron phosphate and aluminum phosphate, severely limiting their application in improving low-yield fields such as acidic and infertile soils.

[0004] Indoleacetic acid (IAA) is an important plant growth regulator that effectively promotes root development and cell elongation in crops. Existing IAA-producing microorganisms typically rely on medium- to high-temperature environments; their IAA production efficiency decreases significantly under low-temperature conditions, making it difficult to meet the growth regulator needs of crops in cold regions. Furthermore, frequent crop diseases in field production are a major challenge in agricultural production. Major grain crops such as sorghum, corn, and wheat are frequently attacked by diseases such as root rot and sorghum leaf spot, leading to sharp yield reductions.

[0005] Therefore, in order to improve crop yield and quality, especially for crops grown in low-temperature regions such as high latitudes and high altitudes, or for overwintering crops, there is an urgent need for a multifunctional microorganism that can decompose inorganic phosphorus at low temperatures, produce IAA at low temperatures, and effectively inhibit various pathogens and promote crop growth. Summary of the Invention

[0006] The purpose of this invention is to provide a multifunctional microorganism capable of decomposing inorganic phosphorus at low temperatures, producing IAA at low temperatures, effectively inhibiting various pathogens, and promoting crop growth, thereby providing a *Russula natans* bacterium, its inoculant, and its applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a *Russella macrantha*, specifically *Russella macrantha* (…). Rouxiella aceris )RA, with accession number CGMCC No.35889.

[0009] The *Russula macrantha* provided by this invention ( Rouxiella aceris RA was deposited on September 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35889.

[0010] Secondly, this invention provides a *Russella asiatica* inoculum, derived from *Russella asiatica* with accession number CGMCC No. 35889. Rouxiella aceris RA was obtained by culturing in fermentation medium.

[0011] Further, based on the mass of the solvent distilled water, the fermentation medium comprises the following components: 0.3%~0.6% peptone, 0.2%~0.7% yeast extract, 0.04%~0.06% magnesium sulfate, 0.5%~2% brown sugar, 0.5%~1.5% sodium chloride, with the balance being water; the pH value of the fermentation medium is 7.0~7.2.

[0012] Furthermore, based on the mass of the solvent distilled water, the fermentation medium comprises the following components: 0.5% peptone, 0.5% yeast extract, 0.05% magnesium sulfate, 1% brown sugar, 1% sodium chloride, with the remainder being water; the pH value of the fermentation medium is 7.0~7.2.

[0013] Furthermore, the culture temperature is 20~30 ℃ and the rotation speed is 150~200 rpm.

[0014] Furthermore, the culture temperature was 28 ℃ and the rotation speed was 200 rpm.

[0015] Furthermore, the bacterial activity of the inoculant was 1.059 × 10⁻⁶. 9 ~2.035×10 9 CFU / mL.

[0016] Furthermore, the bacterial activity of the inoculant was 1.71 × 10⁻⁶. 9 ×10 9 CFU / mL.

[0017] Thirdly, the present invention provides the application of the aforementioned *Russula macrantha* or the aforementioned *Russula macrantha* inoculum in the decomposition of inorganic phosphorus in soil, the production of indoleacetic acid, or the production of ferrophosphate.

[0018] Furthermore, the decomposition of inorganic phosphorus in the soil is carried out at an ambient temperature of 5~30 ℃ (5, 10, 15, 20, 25, 30 ℃).

[0019] Furthermore, the indoleacetic acid production is carried out at an ambient temperature of 5~30℃ (5, 10, 15, 20, 25, 30℃).

[0020] Fourthly, the present invention provides the application of the aforementioned *Russula macrantha* or the aforementioned *Russula macrantha* inoculant in the suppression of crop diseases.

[0021] Furthermore, the types of crop diseases mentioned are maize root rot, sorghum leaf spot, or wheat root rot.

[0022] Furthermore, the aforementioned *Russula macrantha* or the aforementioned *Russula macrantha* inoculant is used to inhibit at least one of the following pathogens: *Fusarium oxysporum*, *Plasmodium sacchari*, *Fusarium solani*, *Alternaria sacchari*, *Rhizoctonia solani*, and *Cyclocarya granatum*.

[0023] Fifthly, the present invention provides the application of the aforementioned *Russula macrantha* or the aforementioned *Russula macrantha* inoculant in promoting crop growth.

[0024] Furthermore, the type of crop includes at least one of corn, sorghum, and wheat.

[0025] Furthermore, the crop is a crop grown in low-temperature regions (ambient temperature 5~15℃, or even 5~10℃) or an overwintering crop.

[0026] In a sixth aspect, the present invention provides a method for using the aforementioned *Russula natans* inoculant, wherein the *Russula natans* inoculant is applied with water before crop sowing.

[0027] Furthermore, the application rate of the *Russula natans* inoculant is 4-8 L / acre.

[0028] The technical solution provided by this invention has the following advantages:

[0029] 1. *Russula macrantha* RA possesses the ability to decompose inorganic phosphorus with a broad phosphorus solubility range, capable of decomposing tricalcium phosphate, ferric phosphate, and aluminum phosphate. At its optimum temperature of 15 ℃, it decomposes 363.30 mg / L of tricalcium phosphate; at 20 ℃, it decomposes 35.91 mg / L of ferric phosphate; and at 20 ℃, it decomposes 9.75 mg / L of aluminum phosphate. *Russula macrantha* RA also exhibits a strong ability to decompose inorganic phosphorus at low temperatures, demonstrating outstanding low-temperature phosphorus solubility: at 5 ℃, it decomposes 321.17 mg / L of tricalcium phosphate, 7.11 mg / L of ferric phosphate, and 5.75 mg / L of aluminum phosphate; at 10 ℃, it decomposes 346.52 mg / L of tricalcium phosphate, 16.75 mg / L of ferric phosphate, and 6.85 mg / L of aluminum phosphate.

[0030] 2. *Russula natans* RA has the ability to produce hematophiles, which facilitate the decomposition of insoluble iron phosphate.

[0031] 3. *Russula natans* RA has the ability to produce IAA at low temperatures. The IAA production capacity is 57.38 mg / L at the optimum temperature of 15 ℃ and 40.03 mg / L at a low temperature of 5 ℃.

[0032] 4. Rhus oryzae RA has an inhibitory effect on a variety of pathogens that cause diseases in field crops (Fusarium oxysporum, Fusarium oxysporum, Fusarium solani, Alternaria alternata, Rhizoctonia solani, and Coelomyces gracilis), and can inhibit diseases such as corn root rot, sorghum leaf spot, or wheat root rot.

[0033] 5. *Russula macrantha* RA can colonize in soil environments ranging from 5 to 40 °C, with the strongest colonization ability at 20 °C. Inoculants (fermentation broth) prepared from this bacterium can promote crop growth and significantly increase crop yield, achieving high yields even with reduced phosphate fertilizer use. Therefore, *Russula macrantha* RA and its inoculants can promote crop growth, increase crop yield, and reduce phosphate fertilizer use, showing promising application prospects. Attached Figure Description

[0034] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is the phylogenetic tree of Rhus erythromycosis RA provided by the present invention;

[0036] Figure 2 This is a colony morphology diagram of *Russula natans* RA provided by the present invention;

[0037] Figure 3 This is a microscopic image of the cell morphology of *Russula natans* RA provided by the present invention.

[0038] Figure 4 This is a graph showing the ability of *Rhus aceae* RA to produce ferophiles, as provided by this invention. Detailed Implementation

[0039] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0040] The culture media and their composition or preparation methods involved in the examples are as follows:

[0041] Buffered nutrient medium: 2.5 g yeast extract, 2.5 g bacteriological peptone (Oxoid brand LP0037), 2.5 g glucose, 7.5 g piperazine-1,4-diethanesulfonic acid (PIPES), 1 L distilled water, pH 7.0~7.2.

[0042] Buffered nutrient agar plate: 2.5 g yeast extract, 2.5 g bacteriological peptone (Oxoid brand LP0037), 2.5 g glucose, 7.5 g piperazine-1,4-diethanesulfonic acid (PIPES), 20 g agar, 1 L distilled water, pH 7.0~7.2.

[0043] NB liquid medium: 10 g peptone, 3 g beef meal, 5 g sodium chloride, 1 L water, pH 7.0~7.2.

[0044] Meng Jinna's Inorganic Phosphorus Plate Culture Medium: 10 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 5 g Ca3(PO4)2, 20 g agar, 1 L water, pH 7.0~7.2, sterilized at 115 ℃ for 20 min.

[0045] NB medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L distilled water, pH 7.0~7.2.

[0046] Meng Jinna's Inorganic Phosphorus Liquid Culture Medium: 10 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 5 g Ca3(PO4)2 / AlPO4 / FePO4, 1 L water, pH 7.0~7.2, sterilized at 115 ℃ for 20 min.

[0047] Detection medium for ferrophosphate production: (1) 60.5 mg of CAS chromaine dissolved in 50 mL of water; (2) 10 mL of 10 mmol / L FeCl3 solution (containing 10 mmol / L HCl); (3) 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA) dissolved in 40 mL of water; (4) 750 mL of water + 100 mL of salt solution (3 g KH2PO4 + 5 g NaCl + 10 g NH4Cl to 100 mL of water) + 20 g agar + 30.24 g PIPES, with pH adjusted to 6.8 using NaOH; (5) 30 mL of 10% acid hydrolyzed casein solution; (6) 10 mL of 20% glucose solution. Mix (1) and (2) thoroughly and add them to (3) while stirring. Sterilize (3), (4), (5), and (6) separately at 115℃ for 20 min. After sterilization, cool to 50℃ and add (5) and (6) to (4) first, then slowly add (3) to the plate.

[0048] PDA medium: 200 g potato, 20 g glucose, 15 g agar, 1 L distilled water, boiled, filtered, dissolved and dispensed, then autoclaved at 121 °C for 30 min.

[0049] Fermentation medium (using distilled water as solvent, the following percentages are the mass ratio of components to distilled water): peptone 0.5%, yeast extract 0.5%, magnesium sulfate 0.05%, brown sugar 1%, sodium chloride 1%, pH 7.0~7.2.

[0050] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0051] The following sections will provide a detailed description of the isolation and identification of this strain, its physiological and biochemical properties, growth characteristics, ability to decompose inorganic phosphorus, ability to produce heparin, ability to produce indoleacetic acid (IAA), ability to inhibit pathogens, ability to colonize in soil, and application in field crop cultivation.

[0052] Example 1: Screening of phosphate-solubilizing bacteria

[0053] Soil samples were collected from Changli County, Qinhuangdao City, with corn as the previous crop. 10 g of soil sample was added to an Erlenmeyer flask containing 100 mL of sterile water and glass beads, shaken at 200 rpm for 30 min, and then serially diluted. 10 g samples were then selected. -5 10 -6 10 -7 10 -8 The diluted solution was spread onto buffered nutrient medium plates and incubated at 28 °C for 3 days. Single colonies of different morphologies were picked and incubated on buffered nutrient medium plates for another 3 days at 28 °C. A total of 15 bacterial strains were isolated. The 16S rDNA of these 15 bacterial strains was amplified, and the amplified fragments were mailed to BGI Genomics for sequencing. The obtained 16S rDNA sequences were compared with BLAST on the NCBI website, and the identification results are shown in Table 1.

[0054] Fifteen isolated bacterial strains were inoculated into NA liquid medium and cultured at 28 ℃ and 200 rpm for 2 days. Wells were then punched on Munkina inorganic phosphate plates using a φ7 mm punch, and 30 μL of the fermentation broth from each of the 15 bacterial strains was inoculated into the wells of the Munkina inorganic phosphate plates. The plates were cultured at 28 ℃ for 7 days, and the width of the zona pellucida was measured. A wider zona pellucida indicates a better phosphate-solubilizing effect of the strain. The phosphate-solubilizing abilities of the 15 bacterial strains are shown in Table 1. It is evident that strain RA exhibits the most outstanding phosphate-solubilizing ability, with a zona pellucida width of 6.0 mm.

[0055] Table 1. Identification results and phosphorus solubilization capacity of 15 bacteria isolated from soil.

[0056]

[0057] Example 2 Identification of strain RA

[0058] Bacterial cells of strain RA were collected, and genomic DNA was extracted using a Trans Genomic DNA Extraction Kit. 16S rDNA was amplified using a PCR system consisting of: 5 μl 10× buffer, 1 μl dNTPs, 1 μl 27F, 1 μl 1492R, and 0.5 μl Taq enzyme. Primers for 27F were: 5'-AGA GTT TGA TCC TGG CTCA-3'; primers for 1492R were: 5'-GGT TACCTT GTT ACG ACTT-3'. PCR conditions were: 94 ℃ for 4 min, 94 ℃ for 30 s, 60 ℃ for 30 s, 72 ℃ for 30 s, with a final extension at 72 ℃ for 10 min. The PCR products were ligated into the Trans pEASY-T3 vector, transformed into *E. coli* T1 competent cells, and positive clones were screened and sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing.

[0059] The 16S rDNA sequence of strain RA was compared with BLAST on the NCBI website, and the result was *Russula maculata*. Rouxiella aceris The homology was 99.53%.

[0060] The 16S rDNA sequence is shown in SEQ ID No. 1 of the sequence listing:

[0061]

[0062] Phylogenetic trees were constructed using MEGA software, based on 16S rDNA sequences, using the Neighbour-Joining method with a Btoostrap value of 1000. The phylogenetic trees are shown below. Figure 1 As shown, strain RA and Rouxiella aceris The strain was named *Russula maculatus* because it is most closely related to the bacteria. Rouxiella aceris RA.

[0063] Acer rumeniae Rouxiella aceris RA has been deposited at the China General Microbiological Culture Collection Center (CGMCC), a depository designated by the State Intellectual Property Office. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 11, 2025, and the deposit number is CGMCC No. 35889.

[0064] The colony morphology of *Russula maculata* RA grown on buffered nutrient agar plates is as follows: Figure 2 As shown, the colonies are white and translucent. The morphology of the bacteria is observed under an optical microscope (10×100x). Figure 3 As shown, the bacterial cells are short rod-shaped.

[0065] Example 3: Physiological and biochemical characteristics of Rhus erythromycosis RA

[0066] Physiological and biochemical identification of *Russula natans* RA was performed with reference to the relevant contents of *Manual of Systematic Identification of Common Bacteria* and *Bergey's Manual of Bacterial Identification (8th Edition)*. The results are shown in Table 2.

[0067] Table 2. Physiological and biochemical characteristics of Rhus erythromycosis RA

[0068]

[0069] Example 4: Growth characteristics of Rhus rubrum RA (a type of bacterium)

[0070] I. Experimental Methods

[0071] 1. Growth temperature

[0072] A suspension of *Russella maculata* RA was inoculated at 1‰ onto buffered nutrient medium and incubated for 1 day at 5 ℃, 10 ℃, 20 ℃, 25 ℃, 28 ℃, 30 ℃, 40 ℃, and 45 ℃ on a shaker at 200 rpm. Each treatment was repeated in triplicate, and OD values ​​were measured. 600nm Absorbance value.

[0073] 2. Salt tolerance

[0074] A suspension of *Russella maculata* RA was inoculated at 1‰ into buffered nutrient media with different salt concentrations. The sodium chloride concentrations were adjusted to 0%, 1%, 2%, 3%, 5%, and 6%, respectively. Each treatment was repeated in triplicate. The cultures were incubated at 28 °C and 200 rpm for 2 days, and the OD was measured. 600nm Absorbance value.

[0075] 3. Acid and alkali resistance

[0076] A suspension of *Russella maculata* RA was inoculated at 1‰ into NB medium at different pH values. The pH was adjusted to 4, 5, 6, 7, 8, 9, 10, 11, and 12, with three replicates for each treatment. The cultures were incubated at 28 ℃ and 200 rpm for 2 days, and the OD values ​​were measured. 600nm Absorbance value.

[0077] Preparation method of *Russella maculata* RA bacterial suspension: *Russella maculata* RA was inoculated into buffered nutrient medium and cultured at 28℃ and 200 rpm for 1 day. The bacterial cells were collected by centrifugation at 6000 rpm, washed three times with physiological saline, and then added to prepare a bacterial suspension with a viability of 1×10⁻⁶. 8 CFU / mL.

[0078] II. Experimental Results

[0079] The experimental results of the growth characteristics of Rhus erythromycosis RA are shown in Tables 3-5. The data are expressed as mean ± standard deviation.

[0080] Table 3. Growth temperature range of *Rhus erythrophagus* RA

[0081]

[0082] Table 4. Salt tolerance range of *Russula natans* RA

[0083]

[0084] Table 5. pH range of acid and alkali tolerance for *Russula natans* RA

[0085]

[0086] As shown in Table 3, the growth temperature range of Rhus erythromycosis RA is 5~40 ℃, and the optimal growth temperature is 28 ℃.

[0087] As shown in Table 4, the salt tolerance range of *Russula natans* RA is 0% to 5%, with an optimal salt concentration of 1%.

[0088] As shown in Table 5, the pH range of acid and alkali tolerance of *Russula natans* RA is 5-11, with an optimal pH of 7.0.

[0089] Example 5: Ability of *Russula natans* RA to decompose inorganic phosphorus

[0090] I. Experimental Methods

[0091] Preparation of molybdenum antimony sulfate stock solution:

[0092] Prepare solution A: Weigh 0.5 g of potassium antimony tartrate and dissolve it in 100 mL of water; Prepare solution B: Weigh 10 g of ammonium molybdate and dissolve it in 450 mL of water, slowly add 153 mL of concentrated H2SO4 while stirring; Add solution A to solution B, and finally add water to 1 L, shake well, and store in a brown bottle.

[0093] Preparation of molybdenum-antimony anti-colorimetric reagent: Add 1.50 g of levozyscic acid to 100 mL of molybdenum-antimony sulfate stock solution. This reagent is effective for 24 hours and should be prepared before use.

[0094] Preparation of phosphorus standard stock solution: Accurately weigh 0.2195 g of potassium dihydrogen phosphate dried at 105 ℃ for 2 h, dissolve it in distilled water, add 5 mL of sulfuric acid solution (ρ=1.84 g / mL), cool and standardize to 1000 mL to obtain a phosphorus standard stock solution with a phosphorus (P) concentration of 50 mg / L.

[0095] Determination of phosphorus standard curve: Pipette 0, 200, 400, 600, 800, and 1000 μL of 50 mg / L phosphorus standard stock solution into 50 mL volumetric flasks, corresponding to concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg / L, respectively. Add 5 mL of molybdenum-antimony anti-chromic reagent, and dilute to 50 mL with ddH₂O. Shake well, let stand for 30 min, and measure the absorbance at 660 nm. Plot the standard curve with concentration on the x-axis and the corresponding measured absorbance on the y-axis.

[0096] *Russula maculata* RA was inoculated into buffered nutrient medium and cultured at 28 ℃ and 200 rpm for 1 day. The bacterial cells were collected by centrifugation at 6000 rpm, washed three times with physiological saline, and then a bacterial suspension was prepared by adding physiological saline (1×10⁻⁶ viable cells). 8 CFU / mL was inoculated into Mengjina inorganic phosphorus liquid medium at 1% (v / v) and cultured at 28 ℃ with shaking at 200 rpm. The bacterial culture was collected at 7 days and 14 days, centrifuged at 10000 rpm for 10 min, and the supernatant was retained. The supernatant was diluted 50-500 times, 5 mL of molybdenum anti-chromic reagent was added, and ddH2O was added to bring the volume to 50 mL. The mixture was shaken well, allowed to stand for 30 min, and the absorbance was measured at 660 nm. The phosphorus concentration was determined according to the phosphorus standard curve. Uninoculated Mengjina inorganic phosphorus liquid medium was used as a blank control.

[0097] II. Experimental Results

[0098] The ability of *Rhus aceae* RA to decompose inorganic phosphorus is shown in Table 6.

[0099] Table 6. Ability of *Rhus erythrozoae* RA to decompose inorganic phosphorus (unit: mg / L)

[0100]

[0101] As shown in Table 6, *Russella maculata* RA has the ability to decompose tricalcium phosphate, ferric phosphate, and aluminum phosphate. At the optimum temperature of 15 ℃, it decomposes 363.30 mg / L of tricalcium phosphate, 35.91 mg / L of ferric phosphate, and 9.75 mg / L of aluminum phosphate. *Russella maculata* RA also exhibits a strong ability to decompose inorganic phosphorus at low temperatures. At 5 ℃, it decomposes 321.17 mg / L of tricalcium phosphate, 7.11 mg / L of ferric phosphate, and 5.75 mg / L of aluminum phosphate. At 10 ℃, it decomposes 346.52 mg / L of tricalcium phosphate, 16.75 mg / L of ferric phosphate, and 6.85 mg / L of aluminum phosphate.

[0102] *Russula maculata* has the strongest ability to decompose tricalcium phosphate, followed by ferric phosphate, and then aluminum phosphate. Generally, the ease with which microorganisms decompose different inorganic phosphorus compounds, from most difficult to easiest, is tricalcium phosphate, aluminum phosphate, and then ferric phosphate. *Russula maculata*'s ability to decompose ferric phosphate is stronger than that of aluminum phosphate, which may be related to the fact that this bacterium can produce heparin, which facilitates the decomposition of insoluble ferric phosphate.

[0103] Example 6: Ability of *Rhus erythromycosis* RA to produce heptaphilin

[0104] I. Experimental Methods

[0105] Streak *Rhus aceae* RA onto a buffer nutrient medium plate and grow at 28 °C for 1 day. Single colonies from the plate are picked and transferred to a ferophile production test medium plate and incubated at 28 °C for 3 days. The colony diameter (d) and the diameter of the ferophile zone (D) are measured, and the D / d value represents the ferophile production capacity of the strain.

[0106] II. Experimental Results

[0107] like Figure 4 As shown, *Russula natans* RA has the ability to produce hematophiles, with a D / d value of 2.5.

[0108] Example 7: IAA production capacity of *Rutella maculata* RA

[0109] I. Experimental Methods

[0110] Rhus erythromycosis RA was administered at a dose of 1×10 6CFU / mL of bacteria were inoculated into buffered nutrient medium containing 500 mg / mL L-tryptophan and cultured at 200 rpm for 2 days at 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, and 30 ℃, respectively. The IAA content was determined by the Salkowski method.

[0111] II. Experimental Results

[0112] The ability of *Rhus aceae* RA to produce IAA is shown in Table 7.

[0113] Table 7. IAA production capacity of *Russula natans* RA (unit: mg / L)

[0114]

[0115] As shown in Table 7, the ability to synthesize IAA at the optimal temperature of 15 ℃ is 57.38 mg / L, and the ability to synthesize IAA at a low temperature of 5 ℃ is 40.03 mg / L, which means that *Russula natans* RA has the ability to produce IAA at low temperatures.

[0116] Example 8: Antibacterial activity of Rhus erythromycosis RA

[0117] I. Experimental Methods

[0118] A cross was drawn on the back of a 90 mm diameter agar plate, with the intersection of the cross as the center of the plate. 7 mm mycelial discs of pathogens (Fusarium oxysporum, Epicorioides sacchariformis, Fusarium solani, Alternaria sacchariformis, Rhizoctonia solani, and Coelophyte gracilis) were inoculated into the center of the PDA plate. A RA cell of *Russella asiatica* was picked and inoculated 20 mm from the center as the treatment group. The control group consisted of no *Russella asiatica* RA cells. The plates were incubated at 28 ℃ for 7 days. The pathogen radii in the control and treatment groups were measured, and the inhibition rate was calculated.

[0119] Inhibition rate % = (radius of pathogens in control group - radius of pathogens in treatment group) / radius of pathogens in control group × 100%.

[0120] II. Experimental Results

[0121] The inhibition rates of *Russula natans* RA against the above six pathogens are shown in Table 8.

[0122] Table 8. Inhibition rate of *Rhus aceae* RA against 6 pathogens.

[0123]

[0124] As shown in Table 8, *Russula natans* RA has an inhibitory effect on a variety of pathogens that cause diseases in field crops (corn root rot, sorghum leaf spot, and wheat root rot).

[0125] Example 9: Preparation of *Russula natans* RA inoculant

[0126] Single colonies of *Russella macrantha* RA were picked and inoculated into fermentation medium. The culture was carried out at 28 ℃ and 200 rpm for 1 day. The culture was then transferred at 10% (v / v) to the fermentation medium, with a sample volume of 100 mL / 500 mL. The culture was carried out at 28 ℃ and 200 rpm for 1 day to obtain *Russella macrantha* RA inoculum (fermentation broth), with a bacterial activity of 1.71 × 10⁻⁶. 9 CFU / mL.

[0127] Example 10 Colonization ability of Rhus erythromycosis RA in soil

[0128] I. Experimental Methods

[0129] Add 1% (mL / g) of *Russula natans* RA inoculant (prepared in Example 9) to sterilized field soil (sampling location: Changli County, Qinhuangdao City), add sterile water to adjust the soil moisture to 35%, and determine the initial bacterial activity as 1.53 × 10⁻⁶. 7 CFU / g; Field soil was placed in incubators at 5 ℃, 10 ℃, 15 ℃, 20 ℃, 30 ℃ and 40 ℃ respectively, and samples were taken at 7 d, 20 d and 30 d to determine the bacterial activity of Amygdalina RA in the field soil.

[0130] II. Experimental Results

[0131] The colonization capacity of *Rhus aceae* RA in soil is shown in Table 9.

[0132] Table 9. Colonization capacity of *Russula maculata* RA in soil at different temperatures (unit: CFU / g)

[0133]

[0134] As shown in Table 9, *Russula natans* RA can colonize in soil environments ranging from 5 to 40 °C, with the strongest colonization ability at a soil temperature of 20 °C.

[0135] Example 11: Application of Rhus erythrophagus RA inoculant in field crop cultivation

[0136] I. Experimental Methods

[0137] The *Russella asiatica* RA inoculum prepared in Example 9 (1.71 × 10⁻⁶ live bacteria) 9 A field trial was conducted to promote wheat growth using CFU / mL (experiment location: Baizhuang Village, Changli County, Qinhuangdao City; trial period: October 8, 2023 to June 14, 2024). Four treatments were included, with four replicates, using a completely randomized block design. Each plot area was 10.00 m × 4.50 m = 45.00 m². 2The row spacing is 15 cm, with approximately 94 effective plants per meter. Field management follows conventional methods. During the growing season, abamectin and flufenoxuron are used to control pests such as spider mites and aphids, while flutriafol and pyraclostrobin are used to control diseases such as powdery mildew and sheath blight.

[0138] Treatment 1: Acer rubrum RA inoculant + conventional fertilization

[0139] Treatment 2: Acer rubrum RA inoculant + phosphorus-reduced fertilizer

[0140] Treatment 3: Conventional fertilization

[0141] Treatment 4: Reduced phosphorus fertilization

[0142] The conventional fertilization for treatments 1 and 3 was as follows: 1000 kg / mu of farmyard manure, 18 kg / mu of urea, and 30 kg / mu of blended fertilizer (25-8-10) were applied as basal fertilizer before wheat sowing. On March 20, 2024, 10 kg / mu of high-nitrogen compound fertilizer (27-15-8) was applied as top dressing, and on April 25, 10 kg / mu of superphosphate was applied as top dressing.

[0143] Phosphorus-reducing fertilization for treatments 2 and 4 was as follows: 1000 kg / mu of farmyard manure, 18 kg / mu of urea, and 30 kg / mu of blended fertilizer (25-8-10) were applied as basal fertilizer before wheat sowing. On March 20, 2024, 10 kg / mu of high-nitrogen compound fertilizer (27-15-8) was applied as top dressing [A1].

[0144] For treatments 1 and 2, apply 4 L / acre of Amygdalinia RA inoculant and irrigate with water before sowing.

[0145] The yield of each plot was statistically analyzed, and the data were analyzed using Duncan's new multiple range test in DPS software.

[0146] II. Experimental Results

[0147] The results of the field trials are shown in Table 10.

[0148] Table 10 Wheat Plot Yield Statistics

[0149]

[0150] As shown in Table 10, the yield of treatment 1 (Russula azedarach RA inoculant + conventional fertilization) was significantly better than other treatments, increasing by 7.60% compared to treatment 3 (conventional fertilization). The yield of treatment 2 (Russula azedarach RA inoculant + reduced phosphorus fertilization) was significantly better than treatment 4 (reduced phosphorus fertilization), indicating that Russula azedarach RA inoculant can significantly improve yield. There was no significant difference between treatment 2 (Russula azedarach RA inoculant + reduced phosphorus fertilization) and treatment 3 (conventional fertilization), indicating that reducing phosphorus fertilizer application after using Russula azedarach RA inoculant does not affect yield. Therefore, Russula azedarach RA inoculant can promote crop growth, increase crop yield, and reduce phosphorus fertilizer application, showing good application prospects.

[0151] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A Ralstonia solanacearum strain, characterized in that, Rouxiella aceris The specific species mentioned is *Russula natans* RA, with the accession number CGMCC No. 35889. ​ 2. A bacterial inoculum of Ralstonia solanacearum, characterized in that, The maple rumeniaceae fungus with accession number CGMCC No. 35889 ( Rouxiella aceris RA was obtained by culturing in fermentation medium.

3. The Erwinia amylovora inoculant of claim 2, wherein, The fermentation medium comprises the following components by mass of distilled water: 0.3%–0.6% peptone, 0.2%–0.7% yeast extract, 0.04%–0.06% magnesium sulfate, 0.5%–2% brown sugar, and 0.5%–1.5% sodium chloride; the pH of the fermentation medium is 7.0–7.

2.

4. The Erwinia amylovora inoculant of claim 2, wherein, The fermentation medium comprises the following components by mass of distilled water: 0.5% peptone, 0.5% yeast extract, 0.05% magnesium sulfate, 1% brown sugar, and 1% sodium chloride; the pH of the fermentation medium is 7.0-7.

2.

5. The Ralstonia solanacearum inoculant of claim 2, wherein, The culture temperature is 20~30 ℃, the rotation speed is 150~200 rpm; the bacterial activity is 1.059×10 9 ~2.035×10 9 CFU / mL.

6. The Erwinia amylovora inoculant of claim 2, wherein, The culture temperature was 28 ℃, the rotation speed was 200 rpm; the bacterial activity was 1.71 x 10 9 CFU / mL.

7. The application of *Russula macrantha* as described in claim 1 or the *Russula macrantha* inoculant as described in any one of claims 2 to 6 in the decomposition of inorganic phosphorus in soil, production of indoleacetic acid, production of ferrophosphate, inhibition of pathogens, inhibition of crop diseases, or promotion of wheat growth, wherein... The crop disease is corn root rot, sorghum leaf spot, or wheat root rot; the pathogen is at least one of the following pathogens: Fusarium oxysporum, Epistylis sorghum, Fusarium solani, Alternaria sorghum, Rhizoctonia solani, and Cyclospora gracilis.

8. The use of an Erwinia amylovora bacterial agent according to any one of claims 2 to 6, characterized in that, The *Russula natans* inoculant is applied with water before crop sowing.

9. The method of using an Erwinia amylovora bacterial agent of claim 8, wherein, The application rate of the *Russula natans* inoculant is 4-8 L / acre.

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