Streptomyces AB_3 and solid fermentation microbial inoculant thereof and application

Abstract

This invention discloses a Streptomyces strain AB_3, classified as amylase-producing Streptomyces (…). Streptomyces diastatochromogenes The strain is preserved under the CCTCC No. M20252932. This invention also discloses a solid-state fermentation agent of *Streptomyces AB_3*, comprising the aforementioned *Streptomyces AB_3* strain. This invention further discloses the application of *Streptomyces AB_3* strain or the solid-state fermentation agent of *Streptomyces AB_3* in controlling soil-borne bacterial wilt and in promoting crop growth. *Streptomyces AB_3* possesses multiple functions, including disease resistance and growth promotion. This invention utilizes a novel solid-state fermentation method, significantly increasing the amount of *Streptomyces* spores and solving the problem of *Streptomyces* failing to produce spores during liquid culture.

Description

Technical Field

[0001] This invention relates to a Streptomyces AB_3 strain and its solid fermentation agent and applications, specifically to an amylase-producing Streptomyces diastatochromogenes AB_3 strain, the application of this Streptomyces in controlling soil-borne bacterial wilt disease and promoting crop growth, and the solid fermentation agent of Streptomyces AB_3 and its application in controlling soil-borne bacterial wilt disease and promoting crop growth. Background Technology

[0002] Streptomyces, as an important beneficial microbial resource, can produce a variety of secondary metabolites and plant hormones, significantly enhancing plant disease resistance and promoting plant growth, thus possessing great potential for development into bio-fertilizers. However, during liquid fermentation, Streptomyces mainly exists in mycelial form, making it difficult to form large quantities of spores, and it also suffers from problems such as high fermentation costs, insufficient utilization of metabolites, and the generation of large amounts of waste liquid. Therefore, its agricultural production mainly adopts solid-state fermentation technology. In recent years, solid-state fermentation technology has been continuously improved, demonstrating several advantages over liquid fermentation. However, current solid-state fermentation of Streptomyces generally uses grains such as rice and corn as fermentation substrates, which suffers from bottlenecks such as high costs, uneven fermentation processes, and susceptibility to contamination by other microorganisms, severely restricting the large-scale application and promotion of Streptomyces in agricultural production. Summary of the Invention

[0003] The inventors isolated a Streptomyces AB_3 strain from the rhizosphere soil of healthy tomato plants. This strain exhibits antagonistic activity against Ralstonia solanacearum and shows biocontrol potential against soil-borne bacterial wilt disease caused by Ralstonia solanacearum. Furthermore, the strain was found to produce siderophores and possess amylase, urease, and esterase activities, demonstrating strong growth-promoting potential. The inventors also developed a novel solid-state fermentation method suitable for Streptomyces AB_3, effectively solving the technical challenges of high cost, uneven material mixing, and high risk of contamination in traditional grain substrate fermentation. The solid-state fermentation preparation using this method not only significantly increases the biomass of Streptomyces AB_3 but also exhibits a significant plant growth-promoting effect.

[0004] The purpose of this invention is to provide a Streptomyces strain AB_3, classified as Streptomyces diastatochromogenes, which was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M20252932.

[0005] Another object of the present invention is to provide the application of the aforementioned Streptomyces strain AB_3 in the control of soil-borne bacterial wilt.

[0006] Preferably, the application is the use of Streptomyces strain AB_3 in the preparation of agents for controlling soil-borne bacterial wilt.

[0007] The soil-borne bacterial wilt mentioned above is a bacterial wilt disease of solanaceous crops caused by Ralstonia solanacearum, specifically, bacterial wilt of tomatoes caused by Ralstonia solanacearum.

[0008] Another object of the present invention is to provide the application of the aforementioned Streptomyces strain AB_3 in promoting crop growth.

[0009] The application described is the use of Streptomyces strain AB_3 in the preparation of fertilizers that promote crop growth or in seed coating agents that promote crop growth.

[0010] The crops mentioned are corn and ginger.

[0011] Another object of the present invention is to provide a Streptomyces AB_3 solid fermentation agent comprising the aforementioned Streptomyces strain AB_3.

[0012] Preferably, the number of spores in the Streptomyces AB_3 solid fermentation inoculum is 6.8 × 10⁻⁶. 9 ~8.5×10 9 5.5 × 10⁶ spores / g or spore concentration. 8 ~7.3×10 8 CFU / mL.

[0013] Preferably, the Streptomyces AB_3 solid fermentation agent uses a sterile mixture of perlite and wheat bran as a solid substrate. The Streptomyces AB_3 seed fermentation broth is added to the solid substrate to obtain a solid fermentation substrate. The solid fermentation substrate is spread evenly in an iron pan with a thickness of 3-5 cm, and solid fermentation is carried out at a temperature of 28-30℃ and an ambient relative humidity of 40-50% for 7-10 days to obtain a solid fermentation product. Alternatively, the solid fermentation product is mixed with sterile deionized water to obtain a Streptomyces AB_3 spore suspension.

[0014] The perlite and wheat bran are mixed in a volume ratio of 2:1.

[0015] The solid-state fermentation is carried out in a dedicated solid-state fermentation chamber for Streptomyces. The fermentation chamber is equipped with a support frame, each of which can hold multiple iron trays for shallow solid-state fermentation. The fermentation chamber is equipped with ultraviolet lamps, high-power air conditioners, and humidifiers, providing excellent temperature and humidity control.

[0016] The Streptomyces AB_3 seed fermentation broth was prepared by the following method: Streptomyces AB_3 spores were inoculated into NB liquid culture medium and cultured in a shaker at 28-30℃ for 48 hours to obtain the Streptomyces AB_3 seed fermentation broth.

[0017] Another objective of this invention is the application of the aforementioned Streptomyces AB_3 solid fermentation agent in the control of soil-borne bacterial wilt.

[0018] Preferably, the application is carried out 7 days after crop seedling transplantation, according to 10 9 CFU / strain will inoculate the crop roots with a suspension of AB_3 spores.

[0019] Another object of the present invention is to provide the application of the aforementioned Streptomyces AB_3 solid fermentation inoculant in promoting crop growth.

[0020] The application described is the use of Streptomyces strain AB_3 solid fermentation inoculant in the preparation of fertilizers that promote crop growth or in seed coating agents that promote crop growth.

[0021] Another object of the present invention is to provide a seed coating agent, which is prepared by mixing a coating powder premix with 4L to obtain a uniform suspension, then mixing the suspension with a seed coating pigment slurry, and finally adding a Streptomyces AB_3 spore suspension at a volume ratio of 100:15 between the total volume of the suspension and the seed coating pigment slurry and the volume of the Streptomyces AB_3 spore suspension, and mixing thoroughly.

[0022] The ratio of the coating powder premix to water is 1 kg: 4 L; the ratio of the coating powder premix to seed coating pigment paste is 1 kg: 1 L.

[0023] The spore concentration of the Streptomyces AB_3 spore suspension was 5.5 × 10⁻⁶. 8 ~7.3×10 8 CFU / mL.

[0024] The beneficial effects of this invention are:

[0025] Streptomyces AB_3 possesses multiple functions, including disease resistance and growth promotion. This invention utilizes a novel solid-state fermentation method to significantly increase the spore quantity of Streptomyces, solving the problem of Streptomyces' inability to produce spores during liquid culture. Attached Figure Description

[0026] Figure 1 The antagonistic effect of Streptomyces AB_3 on Ralstonia solanacearum Rs1115 is shown; where A: Streptomyces AB_3 (front), B: Streptomyces AB_3 (back), and C: Streptomyces W10.

[0027] Figure 2 The colony morphology of strain AB_3.

[0028] Figure 3 The image shows the morphology of AB_3 spore filaments and conidia under a scanning electron microscope.

[0029] Figure 4 The results are for enzyme activity detection of strain AB_3; where A: amylase activity detection result, B: urease activity detection result, and C: esterase activity detection result.

[0030] Figure 5 A phylogenetic tree for strain AB_3 constructed based on the 16S rRNA sequence.

[0031] Figure 6 The study aimed to assess the control effect of strain AB_3 on bacterial wilt of tomato; where A: Rs1115 treatment, B: AB_3 + Rs1115 treatment, and C: blank control (no inoculation).

[0032] Figure 7 For solid-state fermentation of Streptomyces AB_3; where A: solid-state fermentation chamber of Streptomyces, and B: solid-state shallow-tray fermentation product of Streptomyces AB_3.

[0033] Figure 8 The fermentation products of the solid-state fermentation control group are: A: corn stalks, B: perlite, C: wheat bran + perlite (2:1), and D: wheat bran.

[0034] Figure 9 Corn seeds coated with the solid fermentation product of Streptomyces AB_3.

[0035] Figure 10 The graph shows the growth-promoting effect of Streptomyces AB_3 solid fermentation coating agent on corn.

[0036] Figure 11 A bar graph showing the growth-promoting effect of Streptomyces AB_3 solid fermentation coating agent on corn.

[0037] Figure 12 This is a schematic diagram of ginger seedling cultivation in trays.

[0038] Figure 13 The graph shows the effect of Streptomyces AB_3 solid fermentation agent on promoting ginger growth; where A: plant height, B: chlorophyll content, C: leaf thickness, D: leaf width, E: leaf length, F: root length, G: number of leaves, H: number of roots, I: stem diameter, J: fresh root volume, and K: fresh root weight.

[0039] Figure 14 This is a typical ginger seedling that demonstrates the growth-promoting effect of Streptomyces AB_3 solid fermentation inoculant on ginger.

[0040] Information on the preservation of biological materials

[0041] Streptomyces strain AB_3, classified as Streptomyces diastatochromogenes, was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M20252932. Detailed Implementation

[0042] Gao's No. 1 medium containing 0.1% cycloheximide and 0.1% naphthylpyrone acid: Dissolve 50 mg of cycloheximide in 1 mL of anhydrous ethanol to prepare an cycloheximide solution; dissolve 10 mg of naphthylpyrone acid in 1 mL of sterile water to prepare a naphthylpyrone acid solution; add both the cycloheximide solution and the naphthylpyrone acid solution to the Gao's No. 1 medium at a volume ratio of 0.1%.

[0043] SMSA selective medium: Crystal violet 50 mg / L, 1% TTC 50 mg / L, polymyxin 100 mg / L, bacitracin 20 mg / L, chloramphenicol 5 mg / L, actinomycin 50 mg / L, penicillin 0.5 mg / L, pH 7.2.

[0044] NB liquid medium: peptone 5 g / L, yeast extract 0.5 g / L, beef extract 3 g / L, glucose 10 g / L, pH 7.2.

[0045] ISP3 medium (International Streptomyces Project medium 3): Oat flakes 3 g / L, potassium nitrate 0.2 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L, agar 10 g / L, pH 7.0. Preparation method: Before sterilization, weigh an appropriate amount of oat flakes, cook them in deionized water, filter, add the filtrate to the medium, bring the volume to 1L with deionized water, add the other ingredients listed above, adjust the pH, sterilize, and set aside.

[0046] LB solid medium: 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 30 g agar, bring the volume to 1000 mL with deionized water, set to natural pH, and autoclave at 115°C for 30 min.

[0047] LB liquid medium: 10 g peptone, 5 g yeast extract, 10 g sodium chloride, deionized water to a final volume of 1000 mL, pH at rest, autoclave at 115°C for 30 min.

[0048] Physiological saline: Dissolve 0.9 g NaCl in 100 mL of ultrapure water and autoclave at 115℃ for 30 min.

[0049] MKB iron-limiting medium: 15 mL glycerol, 5 g acid-hydrolyzed casein, 2.5 g K2HPO4, 1.0 g MgSO4, bring the volume to 1000 mL with deionized water, set to natural pH, and sterilize at 115°C for 30 min.

[0050] CAS detection solutions: Prepare the following four solutions separately: 1) 1 mM ferric chloride stock solution: Dissolve 0.2703 g of ferric chloride hexahydrate in 1 L of 10 mM hydrochloric acid, store at room temperature protected from light for later use; 2) CAS stock solution: Dissolve 0.2421 g of chromazine in 200 mL of deionized water, store at 4 ℃ protected from light for later use; 3) Hexadecyltrimethylammonium bromide solution: Dissolve 0.0219 g of hexadecyltrimethylammonium bromide (HTDMA) in 50 mL of deionized water, prepare fresh before use; 4) Piperazine buffer: Dissolve 4.3079 g of anhydrous piperazine in 30 mL of water, adjust the pH to 5.6 with concentrated hydrochloric acid, prepare fresh before use. Mixed formulation: Take 7.5 mL of CAS stock solution, add 1.5 mL of 1 mM ferric chloride stock solution, add 50 mL of HTDMA solution while stirring, then add 30 mL of piperazine buffer solution, and finally add deionized water to make up to 100 mL to obtain 100 mL of CAS detection solution.

[0051] Example 1

[0052] Screening, identification and biological characteristics of Streptomyces AB3

[0053] 1. Screening of Rhizosphere Streptomyces

[0054] Healthy tomato plants were collected from a tomato plantation in Houcun Village, Qilin Town, eastern suburbs of Nanjing (118°57'E, 32°03'N). The plants were dug up with their roots intact, and loose soil attached to the roots was gently shaken to remove them. The plants were immediately placed in sterile self-sealing bags and stored at 4°C. Before the experiment, the samples were removed, and the remaining rhizosphere soil was scraped off using a sterile scraper. The rhizosphere soil sample was transferred to a sterile mortar and ground into powder. Approximately 5 g of the soil sample was weighed and placed in a 250 mL Erlenmeyer flask containing 45 mL of sterile water and an appropriate amount of sterile glass beads. The flask was placed on a shaker (28°C, 280 rpm) and shaken for 12-20 min to fully disperse the soil sample. At this point, the soil sample was diluted 10 times. -1 Then, it was serially diluted with sterile water, and 10 was taken. -3 10 -4 10 -5Soil suspensions of varying dilutions were used for bacterial screening. 200 μL of each dilution of soil suspension was evenly spread onto the surface of Gao's No. 1 agar medium containing 0.1% actinone and 0.1% nalidixic acid. After the spreads dried, the plates were incubated at 28°C for 5-7 days, during which time colony growth was observed regularly. Once a single Streptomyces colony matured, it was promptly picked and purified by streaking on ISP3 medium to obtain Streptomyces AB_3, which was then transferred to an agar slant for storage.

[0055] 2. Streptomyces AB3 antagonistic test against Ralstonia solanacearum

[0056] Using an inoculation needle, colonies of *Ralstonia solanacearum* QL-Rs1115 (denoted as Rs1115, GenBank accession: GU390462) preserved in the laboratory were picked and streaked onto SMSA selective medium plates. The plates were incubated at 30°C for 36 h. Free-floating red cells were picked and inoculated into NB liquid medium, then incubated overnight at 30°C and 170 rpm on a shaker to obtain a *Ralstonia solanacearum* bacterial suspension. The suspension was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. The suspension was resuspended in 0.9% physiological saline and the concentration was adjusted to 10. 7 CFU / mL (OD) 600 = 0.5), to obtain a suspension of Ralstonia solanacearum Rs1115.

[0057] The antagonistic ability of Streptomyces AB_3 against Ralstonia solanacearum Rs1115 was detected using a single-point spray method: Streptomyces AB_3 treatment group: spores of Streptomyces AB_3 were picked up with a sterile toothpick and inoculated at a single point in the center of an NA plate, and incubated at 28℃ for 3-5 days until single colonies of Streptomyces formed; subsequently, a suspension of Ralstonia solanacearum Rs1115 (10 g / L) was used. 7 A spray bottle containing CFU / mL of Rs1115 bacterial suspension was evenly sprayed once (approximately 0.2 mL) onto an NA plate containing a single colony of Streptomyces AB_3. The plate was incubated at 30°C for 16 h, and the formation of inhibition zones was observed. The diameter of the inhibition zone (D) and the diameter of the Streptomyces colony (d) were measured. Streptomyces pratensis W10, which has no inhibitory effect, was used as a control instead of Streptomyces AB_3. Each treatment was performed in triplicate. Comparison with Streptomyces W10, which has no inhibitory effect, clearly shows that Streptomyces AB_3 has an antagonistic effect against Ralstonia solanacearum. The average diameter of the inhibition zone of Streptomyces AB_3 was 1.93 cm (see [link to article]). Figure 1 ).

[0058] 3. Identification of antagonistic Streptomyces AB3

[0059] (1) Morphological observation of strain AB_3

[0060] Strawberry strain AB_3 was streaked onto ISP3 medium and incubated at 28°C for 5 days. The colony color was compared using "The ISCC-NBS COLORCHARTS Standard samples NO.2106," and the results showed that its aerial hyphae were white. Figure 1 , Figure 2 The mycelium within its substrate is pale yellow. Figure 1 Using a sterile scalpel, cut the culture medium containing freshly cultured AB_3 colonies for 5 days into sections with an observation surface ≤ 6 × 6 mm. 2 Samples with a thickness ≤ 2 mm were fixed with 2.5% glutaraldehyde for at least 8 hours, rinsed three times with phosphate buffer for 10 min each time, dehydrated in a gradient of 50%, 70%, 80%, and 90% ethanol for 15 min each, dehydrated three times with 100% ethanol for 30 min each time, replaced three times with tert-butanol for 30 min each time, and then freeze-dried. The samples were then attached to the sample stage with double-sided tape, observation side facing upwards, and coated with a 10 nm gold film using an ion sputtering apparatus. The morphology of the antagonistic strain AB_3 conidia and spores was observed using a scanning electron microscope. The conidia of AB_3 were short rod-shaped with a rough surface, and the spore chains were linear. Figure 3 ).

[0061] (2) Physiological and biochemical characteristics of strain AB_3

[0062] Physiological and biochemical indicators were determined according to the "Streptomyces Identification Manual". The results are shown in Table 1 and... Figure 4 .

[0063] Table 1. Physiological and biochemical characteristics of strain AB_3

[0064] Physicochemical characteristics result Physicochemical characteristics result Growth temperature range 10-37℃ lactose - Growth pH range 5-11 Mannitol - Salt tolerance 5% Mannose - Starch hydrolysis + Raffinose - Cellulose hydrolysis + Rhamnose - Twain 20 - Ribose + Twain 40 + Sorbitol - Twain 80 - sucrose - <![CDATA[Producing H2S]]> - Xylose - Produced by IAA - alanine - Urease + Arginine + Nitrate reduction + Asparagine + Gelatin liquefaction - Aspartic acid - Arabic sugar + glutamic acid + fructose - glycine + Galactose - proline + Galactitol - Serine + glucose + Threonine + Inositol - Tyrosine +

[0065] Note: + indicates positive; − indicates negative.

[0066] (3) Molecular biological identification of strain AB_3

[0067] Strains AB_3 were sent to Shanghai Lingen Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The resulting gene sequence was 1454 bp. BLAST alignment analysis was performed using the EzBioCloud platform, and the sequences of the top 8 valid strains with high homology to strain AB_3 were selected as reference sequences from the database. A phylogenetic tree was constructed using MEGA 7.0 software and the maximum likelihood (ML) method.

[0068] The results showed that the 16S rRNA gene sequence similarity between strain AB_3 and Streptomyces diastatochromogenes (LIQL01000147) was 97.33%. Figure 5 Based on the analysis of bacterial morphology, physiological and biochemical indicators, and 16S rRNA gene sequence, strain AB_3 was preliminarily identified as *Streptomyces diastatochromogenes*.

[0069] Streptomyces strain AB_3, classified as Streptomyces diastatochromogenes, was deposited on December 17, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No. M20252932.

[0070] (4) Detection of siderophore production capacity of strain AB_3

[0071] The production of siderophores is determined using a CAS detection solution. Substances in the detection solution bind with the siderophores and produce a colorimetric reaction.

[0072] First, the glycerol-preserved AB_3 bacterial strain was streaked and purified on LB solid medium, and incubated upside down at 30°C for 24 hours. Single colonies obtained after activation were then cultured in liquid medium: single colonies were picked and transferred to LB liquid medium (1.5 mL per well in a 24-well plate), and incubated at 30°C and 170 rpm for 12 hours. OD was adjusted using physiological saline. 600 To 0.5. Pipette 2 μL of each different bacterial culture into a 96-well plate containing 198 μL of MKB iron-limiting medium and incubate at 170 rpm for 48 h in a shaker at 30 ℃. Centrifuge using a microplate centrifuge and a 96-well plate filter membrane (centrifugation speed 3300 rpm). -1 Centrifuge for 10 min, collect the supernatant, mix the supernatant with CAS detection solution at a volume ratio of 1:1, let stand for 2 h, and measure OD using an ELISA reader. 630 (Referred to as A); Control treatment: Sterile MKB iron-limiting medium and CAS detection solution were mixed at a volume ratio of 1:1 and allowed to stand for 2 hours. OD was also measured using an enzyme-linked immunosorbent assay (ELISA) reader. 630 (denoted as Ar).

[0073] The formula for calculating the relative content of ferrocarriers (SU) is: SU = 1-A / Ar.

[0074] The concentration of siderophores produced by strain AB_3 was 32.87±1.45μmol / L, indicating that strain AB_3 has the ability to produce siderophores.

[0075] Example 2

[0076] The control effect of strain AB_3 on bacterial wilt.

[0077] (1) Preparation of conidia of Streptomyces AB3

[0078] Spread the AB_3 spore suspension onto ISP3 solid plates and incubate at 28°C for 5-7 days. Spores are scraped off using a sterile spatula, mixed with an appropriate amount of sterile water, and counted using a hemocytometer. The spore concentration is then adjusted to 102. 8 Quantity / mL, for later use.

[0079] (2) Pot experiment on the control of bacterial wilt of tomato by AB_3 inoculant

[0080] The tomato variety "Red Dwarf" was selected.

[0081] Before the experiment, the seeds underwent surface sterilization: seeds were soaked in sterile water for 16 hours, disinfected with 75% alcohol for 30 seconds, rinsed four times with sterile water, then soaked in 10% NaOCl for 20 minutes, and finally rinsed at least four times with sterile water and blotted dry with sterile filter paper. The surface-sterilized seeds were transferred to 1 / 2 MS medium and incubated in the dark at 28°C for 3 days. After germination, the seeds were sown in seedling trays containing 50 g of sterile seedling substrate per well. Seedling conditions were: temperature 22°C-32°C, relative humidity 70%, photoperiod 16 / 8h (light / dark), 200 µmol·m⁻¹ -2 ·s -1 PPFD (Photosynthetic Photon Flux Density) was measured, and the plants were watered daily with sterile water. Around 17 days after sowing (three-leaf stage), uniformly growing tomato seedlings were transplanted into containers of 200g of yellow-brown soil (organic carbon content 8.1 g·kg⁻¹). -1 The total nitrogen content was 0.79 g·kg⁻¹. -1 The seedlings were cultured in pots (pH 6.5) under the same conditions as the seedling stage. Three treatments were set up: a blank control (no microorganisms inoculated), an AB3 treatment (AB3 + Rs1115), and an Rs1115 treatment (inoculated only with Ralstonia solanacearum Rs1115). Seven days after transplanting the tomato seedlings, the AB3 spore suspension was... 9 The final concentration of CFU / plant tomato was inoculated into the roots of AB_3-treated tomato seedlings, while an equal volume of water was added to the roots of the blank control tomato seedlings. Seven days after inoculation with Streptomyces spore suspension, Ralstonia solanacearum Rs1115 bacterial suspension (Example 1) was inoculated at 10... 8The final concentration of CFU / tomato seedling was applied to the roots of tomato seedlings in the AB_3 and Rs1115 treatments, while the roots of the control tomato seedlings were treated with an equal volume of water. Each treatment consisted of 3 biological replicates, with each replicate containing 6 tomato plants (18 plants in total per treatment). The tomato plants were grown under greenhouse conditions identical to those used for seedling cultivation, and were regularly watered with sterile water. During the treatment period, the plant arrangement within the same treatment was randomly changed every 2 days. The treatment ended 50 days after inoculation with Rs1115 (small flags indicate diseased plants). Figure 6 ).

[0082] The severity of bacterial wilt was quantified using the disease index (DI) and biocontrol efficiency (BE). Disease severity ranged from 0 to 4, where 0 indicated healthy plants; 1 indicated ≤25% wilted leaves; 2 indicated 26%-50% wilted leaves; 3 indicated 51%-75% wilted leaves; and 4 indicated 76%-100% wilted leaves.

[0083] The formula for the Disease Index (DI) is as follows:

[0084]

[0085] Wherein, Nd: number of diseased plants for a given disease level, Cd: corresponding disease level, Ts: total number of plants surveyed, and Ad: actual highest disease level.

[0086] The formula for biocontrol efficiency (BE) is as follows:

[0087]

[0088] Among them, DI CK Rs1115 treatment of the disease index, DI T : AB_3 treatment disease index.

[0089] The results are shown in Table 2. The disease index of the Rs1115 treatment was 66.67, and the incidence rate was 72.22%. The disease index of the AB_3 treatment was 11.11, the incidence rate was 11.11%, and the biocontrol rate was 84.62%. This indicates that S. diastatochromogenes AB_3 has excellent control effects against Ralstonia solanacearum.

[0090] Table 2. Control effects of different treatments on bacterial wilt in tomato (pot experiment)

[0091] deal with Disease index Incidence rate / % Prevention and control effectiveness / % Rs1115 processing 66.67±29.07 72.22±28.33 / AB_3 processing 11.11±9.07 11.11±9.07 84.62

[0092] Example 3

[0093] Preparation of Streptomyces AB3 solid inoculum

[0094] (1) Preparation of Streptomyces AB3 seed fermentation broth

[0095] Streptomyces AB3 was streaked on ISP3 medium and cultured at 28℃ for 5 days until the spores were fully mature. Under aseptic conditions, a loop of spores was scraped out and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of NB liquid medium. The flask was then placed in a shaker at 28℃ and 170 rpm for 48 h to obtain the Streptomyces AB3 seed fermentation broth.

[0096] (2) Preparation of solid fermentation agent of Streptomyces AB3

[0097] Solid base material: Perlite (particle size 2-7 mm) and wheat bran are mixed evenly at a volume ratio of 2:1, sterilized, and dried in an oven to obtain solid base material (pH=7.0).

[0098] Streptomyces-specific solid fermentation chamber ( Figure 7 A): The fermentation chamber area is 15 m². 2 It is equipped with ultraviolet lamps, high-power air conditioners and humidifiers, and has good temperature and humidity control functions. There are racks in the fermentation chamber, and each rack can hold multiple iron trays for solid shallow tray fermentation.

[0099] Solid-state fermentation treatment group: Following an inoculation ratio of 1L:1kg for *Streptomyces AB3* seed fermentation broth, the *Streptomyces AB3* seed fermentation broth was added to the solid substrate and mixed thoroughly to obtain the solid fermentation substrate. The solid fermentation substrate was then spread evenly in an iron pan to a thickness of 3-5 cm and fermented in a dedicated *Streptomyces* solid-state fermentation chamber. The fermentation temperature was controlled at 28-30℃, the relative humidity at 40%, and the fermentation time was 10 days. After solid-state fermentation, 5g of the solid fermentation product (i.e., the *Streptomyces AB3* solid fermentation inoculum) was collected. Figure 7 B) The product was mixed with 45 mL of sterile water to obtain a diluted solution. The diluted solution was streaked onto ISP3 medium and incubated at 28°C for 5 days, showing no contamination. The diluted solution was filtered through sterile absorbent cotton, and the filtrate was serially diluted with sterile water. Spores were counted using a hemocytometer. The results showed that the spore count in the solid-state fermentation product of *Streptomyces AB_3* was 8.5 × 10⁻⁶. 9 spores / g.

[0100] The fermentation substrates for the four solid-state fermentation control groups were: sterilized and dried corn stalks, sterilized and dried wheat bran, sterilized and dried perlite, and a solid substrate with a volume ratio of sterilized and dried perlite and wheat bran of 1:2. The treatment methods and fermentation conditions were consistent with those of the "solid-state fermentation treatment group".

[0101] See results Figure 8 The results indicate that solid-state fermentation using corn stalks and perlite as substrates only produced a small number of Streptomyces spores and could not be effectively fermented. Furthermore, solid substrates consisting of wheat bran, perlite, and wheat bran in a 1:2 volume ratio all resulted in contamination by other microorganisms due to poor substrate permeability.

[0102] Example 4

[0103] Seed coating of Streptomyces AB3 solid fermentation inoculum and its application

[0104] Streptomyces AB_3 spore suspension: The solid fermentation product of Streptomyces AB_3 (Example 3) was mixed with sterile deionized water to obtain a spore concentration of 10. 8 Streptomyces AB3 spore suspension at CFU / mL (by hemocytometer).

[0105] Coating agent: Add 1 kg of coating powder premix (manufacturer: Shenzhen Xingkaiyue Biotechnology Co., Ltd.) to 4 L of water and stir to obtain a uniform coating powder premix suspension; mix the above coating powder premix suspension with 1 L of seed coating pigment slurry (manufacturer: Shenzhen Yicai New Materials Co., Ltd.), and then mix the total volume of the coating powder premix suspension and the seed coating pigment slurry with the Streptomyces AB_3 spore suspension (10... 8 A coating agent was prepared by adding Streptomyces AB_3 spore suspension at a volume ratio of 100:15 (CFU / mL) and mixing thoroughly.

[0106] Seed coating of 100 kg of corn seeds was carried out using the above coating agent: First, select healthy corn seeds that are plump, uniform in size, and undamaged. Disinfect with 70% ethanol for 1-2 minutes, rinse three times with sterile water, then disinfect with 5% sodium hypochlorite solution for 10-15 minutes, and rinse 5-8 times with sterile water to ensure that any residual sodium hypochlorite is completely removed. After the final rinse, drain as much water as possible and air dry until the moisture content is 13%. Pour the corn seeds into a large basin or plastic bucket, filling it no more than 2 / 3 full. Slowly and evenly pour in the coating agent while quickly stirring by hand (wearing gloves) for 5-10 minutes until all seeds are evenly coated. Remove the seeds and place them in a cool, ventilated place to air dry. The coated seeds will have a uniform color, a smooth, non-sticky film, and will not stick together. The film should not easily peel off when gently rubbed. Figure 9 This indicates that the solid-state fermentation products of Streptomyces AB_3 can be used for seed coating.

[0107] To further investigate the growth-promoting effect of the Streptomyces coating agent, the inventors conducted a pot experiment on corn.

[0108] Two groups were set up: a control group (uncoated) and an AB3 treatment group (treated with Streptomyces AB3). Each treatment had three pots, with three seedlings per pot. 10 kg of seedling substrate was evenly distributed into the pots. For the AB3 treatment group, three coated seeds were sown per pot at a depth of 2 cm; for the control group, three uncoated seeds were sown per pot at a depth of 2 cm. The seedlings were cultivated in a constant temperature greenhouse at 28 ℃ with a photoperiod of 14 h light / 10 h dark and a light intensity of 300 µmol·m⁻¹. 2 • s, relative humidity 60-70%, and timely irrigation with an equal amount of sterile water. After 30 days of cultivation, the aboveground and underground growth indicators of maize seedlings were recorded (underground growth indicators: root length; aboveground growth indicators: stem diameter, fresh weight, leaf width, number of leaves, leaf length, and plant height, etc.) to evaluate the growth-promoting effect of Streptomyces solid fermentation coating agent on maize. The results showed that seed coating of strain AB_3 had a significant growth-promoting effect on maize. Figure 10 , Figure 11 (Table 3).

[0109] Table 3. Growth-promoting effects of Streptomyces AB_3 solid fermentation coating agent on maize.

[0110] index CK group AB_3 processing group Plant height (cm) 39.17±1.23 57.13±1.89 Stem diameter (mm) 5.78±0.19 7.83±0.72 Number of leaves 4 5 Leaf length (cm) 28.53±0.19 42.83±2.55 Leaf width (cm) 2.13±0.21 3.23±0.047 Root length (cm) 4.57±0.21 7±1.18 Fresh weight (g) 4.85±0.29 11.35±1.28

[0111] Example 4

[0112] Application of Streptomyces AB3 solid-state fermentation products in ginger seedling cultivation

[0113] Preparation of ginger seedling substrate: Crush and sieve (0.5mm sieve) the solid fermentation product obtained from the solid fermentation of the solid fermentation treatment group in Example 3. Mix the solid fermentation product and the seedling substrate thoroughly at a mass ratio of 1:100 to prepare the ginger seedling substrate for later use.

[0114] Ginger Seed Preparation: Select ginger rhizomes that are plump, have bright buds, are uniform in size, have a glossy skin, and are free from pests, diseases, and external damage. Sun-drying: On a sunny, warm day, from 10:00 AM to 3:00 PM, sun-dry the ginger on a sunny surface at 10℃ for 2-3 days, taking it in and out daily. Avoid direct or intense sunlight. Cutting: Cut the ginger rhizomes into pieces, each weighing 25-35g. Ensure each piece contains 1-2 rhizome nodes, and crucially, retain intact, plump buds at the nodes. Keep the cut surface of each piece as small as possible. Disinfection: Soak the ginger rhizomes in a 0.5% potassium permanganate solution for 30 minutes. Sprouting: Place the disinfected ginger rhizomes in a temperature range of 22℃-28℃ (around 23℃ in the early stage, around 26℃ in the middle stage, and around 24℃ in the later stage) at a relative humidity of 80%-85%. When the sprouts reach a length of 2-5 mm, they are ready for planting.

[0115] Ginger seedling tray cultivation: 21-cell trays are recommended, with a single cell depth of 6-7 cm. AB_3 treatment group: First, add a 2 cm thick layer of ginger seedling substrate to the bottom of the tray, then place the ginger piece inside, ensuring the bud faces upwards (e.g., ...). Figure 12 After covering the seedlings with substrate level with the top of the planting hole, water thoroughly and move them to a greenhouse for routine ginger seedling management. Simultaneously, a seedling substrate without solid fermentation products was used as a control (CK) group. Fifty days after ginger sowing, aboveground growth indicators such as plant height, stem diameter, number of leaves, leaf length, leaf width, chlorophyll content, and leaf thickness, as well as underground growth indicators such as number of roots, root length, root fresh weight, and fresh root volume, were recorded to evaluate the growth-promoting effect of Streptomyces solid fermentation products on ginger. The results showed that the solid fermentation products of strain AB_3 had a significant growth-promoting effect on ginger (Table 4). Figure 13 , Figure 14 ).

[0116] Table 4. Effects of Streptomyces AB_3 solid fermentation inoculum on ginger growth promotion in treatment groups

[0117] index CK group AB_3 processing group Plant height (cm) 31.52±2.38 43.31±2.93 Chlorophyll content 38.25±2.28 44.32±0.87 Blade thickness (mm) 0.16±0.005 0.18 Leaf width (cm) 2.28±0.093 2.54±0.019 Leaf length (cm) 12.13±0.94 15.93±0.5 Root length (cm) 10.38±1.25 14.9±0.57 Number of leaves 4.33±0.29 5.34±0.12 Number of roots 6.43±0.82 14.14±0.82 Stem diameter (mm) 5.49±0.14 7.35±0.28 Fresh root volume (mL) 2.16±0.08 5.71±0.22 Fresh root weight (g) 1.65±0.14 5.46±0.45 .

Claims

1. A Streptomyces strain AB_3, classified as Streptomyces diastatochromogenes, with the strain preservation number CCTCC No. M20252932.

2. A Streptomyces AB_3 solid fermentation inoculum, characterized in that: The Streptomyces AB_3 solid fermentation agent comprises the Streptomyces strain AB_3 as described in claim 1.

3. The Streptomyces AB_3 solid fermentation agent according to claim 2, characterized in that: The number of spores in the solid-state fermentation product of Streptomyces AB_3 was 6.8 × 10⁻⁶. 9 ~8.5×10 9 5.5 × 10⁶ spores / g or spore concentration. 8 ~7.3×10 8 CFU / mL.

4. The Streptomyces AB_3 solid fermentation agent according to claim 2, characterized in that: It is a solid fermentation product obtained by adding Streptomyces AB_3 seed fermentation broth to a sterile mixture of perlite and wheat bran as a solid substrate, spreading the solid fermentation substrate evenly in an iron pan with a thickness of 3-5 cm, and fermenting it for 7-10 days at a temperature of 28-30℃ and an ambient relative humidity of 40-50%. Alternatively, it can be obtained by mixing the solid fermentation product with sterile deionized water to obtain a Streptomyces AB_3 spore suspension. The Streptomyces AB_3 seed fermentation broth is prepared by the following method: Streptomyces AB_3 spores are inoculated into NB liquid culture medium and cultured in a shaker at 28-30℃ for 48 hours to obtain the Streptomyces AB_3 seed fermentation broth.

5. The Streptomyces AB_3 solid fermentation agent according to claim 4, characterized in that: The perlite and wheat bran are mixed in a volume ratio of 2:

1.

6. The application of the Streptomyces strain AB_3 according to claim 1 or the Streptomyces AB_3 solid fermentation agent according to claim 2 in the control of soil-borne bacterial wilt.

7. The application according to claim 6, characterized in that: The application described is the use of Streptomyces strain AB_3 or Streptomyces AB_3 solid fermentation agent in the preparation of formulations for controlling soil-borne bacterial wilt.

8. The application according to claim 6, characterized in that: The soil-borne bacterial wilt mentioned above is a bacterial wilt disease of solanaceous crops caused by Ralstonia solanacearum.

9. The application of the Streptomyces strain AB_3 according to claim 1 or the Streptomyces AB_3 solid fermentation agent according to claim 2 in promoting crop growth.

10. The application according to claim 9, characterized in that: The application refers to the use of Streptomyces strain AB_3 or Streptomyces AB_3 solid fermentation agent in the preparation of fertilizers that promote crop growth or in seed coating agents that promote crop growth.

Images