Streptomyces californicus cd11 and uses thereof

Abstract

The present application relates to the field of microbial technology, in particular to streptomyces californicus CD11 and its application, the present application discloses streptomyces californicus CD11 and its application, the strain is isolated from the intestinal tract of bombyx mori, has good inhibitory effect on soil-borne fungal disease pathogen and soil-borne bacterial fusarium wilt, and has the abilities of secreting cellulase and beta-1, 3-glucanase, producing siderophore and auxin IAA.Pot experiment shows that the biocontrol efficiency of the bacterial agent on wheat stem base rot is 60.67%, the biocontrol efficiency on tomato fusarium wilt is 44.59%, and the plant height and fresh weight are significantly improved.The present application also provides isolation, purification and identification of the bacteriostatic metabolite of the strain, i.e.tenuazonic acid.The present application selects the streptomyces californicus CD11 with vigorous growth capacity, which can be applied in the preparation of biological bacterial fertilizer, biological pharmaceutical and other fields in large quantities.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Streptomyces calcareosa CD11 and its applications. Background Technology

[0002] Soil-borne diseases are a significant factor restricting healthy crop production. Among them, fungal wilt and bacterial bacterial wilt are two typical soil-borne vascular diseases that are serious, widespread, and difficult to control. Fungal wilt is mainly caused by soil-borne fungi such as Fusarium. The pathogen can infect the plant root system, leading to leaf yellowing, wilting, vascular bundle browning, stunted growth, and even death. Bacterial bacterial wilt is mainly caused by Ralstonia solanacearum. After invading the plant, the pathogen multiplies rapidly in the vascular bundles and blocks water transport, ultimately causing the plant to wilt and die quickly. Both of these diseases are characterized by a wide host range, long survival time in the soil, insidious transmission, and great difficulty in control, often causing serious losses in crop yield and quality.

[0003] Soil actinomycetes have simple nutritional requirements, and most groups can produce highly resistant conidia, which helps them adapt to complex and variable soil environments, making them a resource microorganism with broad application potential. Streptomyces is an important group of actinomycetes and also an important potential beneficial microorganism in soil. Existing studies have shown that Streptomyces often exhibits high relative abundance in disease-suppressing soils and can play an important role in the control of various soil-borne diseases by producing secondary metabolites related to antifungal, antibacterial, growth-promoting, and plant resistance-inducing effects. Therefore, exploring Streptomyces resources and their active metabolites with the potential to inhibit fungal wilt pathogens and bacterial bacterial wilt pathogens can provide new strain resources and theoretical basis for the green control of soil-borne diseases. Summary of the Invention

[0004] In view of the above, it is necessary to conduct microbial screening and verification for the prevention and control of soil-borne diseases, and to obtain strains with broad-spectrum antibacterial effects and the ability to promote plant growth, so as to provide new germplasm resources for the future development of biopesticides and biofertilizers.

[0005] To achieve the above objectives, this invention has screened out a new strain: *Streptomyces calacana* (… Streptomyces Californian Strain CD11, classified as: Streptomyces calacana Streptomyces CalifornianThe strain is classified and named as Streptomyces calica CD11 in Chinese, with accession number CGMCC No. 38498. It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 29, 2026.

[0006] The present invention also includes the aforementioned Streptomyces calacana ( Streptomyces Californian Application of strain CD11 in the preparation of bio-organic fertilizer, antibacterial agent and / or biopesticide.

[0007] Furthermore, the pathogen inhibited by the antibacterial agent is: *Fusarium oxysporum* var. *hypogon* (watermelon-specific *Fusarium oxysporum*). Fusarium oxysporum f. sp. snowy ), pepper anthracnose bacteria ( Colletotrichum capsici ), soybean-specific Fusarium oxysporum ( Fusarium oxysporum f. sp. wisteria ), Fusarium oxysporum ( Fusarium fujikuroi ), Fusarium graminearum ( Fusarium pseudogramineum Fusarium solani ( ) Fusarium solani Fusarium graminearum ( ), Fusarium gramineae ) and Ralstonia solanacearum ( Ralstonia solanacearum ).

[0008] Furthermore, the diseases controlled by the biological pesticide are: wheat stem rot and / or tomato bacterial wilt.

[0009] The present invention also includes the aforementioned Streptomyces calacana ( Streptomyces Californian Application of strain CD11 in promoting wheat growth.

[0010] The present invention also includes the aforementioned Streptomyces calacana ( Streptomyces Californian Application of strain CD11 in improving wheat SPAD value and tiller number.

[0011] The present invention also includes the aforementioned Streptomyces calacana ( Streptomyces Californian Application of strain CD11 in the production of IAA, siderophore, β-1,3-glucanase, cellulase and / or purpuricin.

[0012] The present invention also includes the aforementioned Streptomyces calacana ( Streptomyces Californian A method for planting plants with strain CD11, wherein the method comprises adding a bacterial suspension containing strain CD11, conidia and / or bio-organic fertilizer during the seedling stage of the plant.

[0013] Furthermore, the plant is wheat or tomato.

[0014] The technical solution of this invention has the following beneficial effects: The *Streptomyces calcareae* CD11 strain obtained by this invention exhibits broad-spectrum antagonistic activity against soil-borne fungal diseases, mainly wheat stem rot, and good antibacterial activity against bacterial wilt of tomato, breaking through the limitations of existing biocontrol strains in controlling only a single pathogen. Studies have also found that the CD11 strain can produce cellulase, β-1,3-glucanase, and siderophores, while also producing IAA growth-promoting hormones to promote wheat growth. Therefore, the CD11 strain possesses dual functions of disease prevention and growth promotion. Pot experiments have demonstrated that the CD11 strain has significant resistance to wheat stem rot and good growth-promoting effects on wheat; it also shows good resistance to bacterial wilt of tomato. This application also uses wheat bran (45%), silkworm excrement (45%), and rice husks (10%) as a fermentation substrate, with a material-to-water ratio of 1:0.8, and obtains ≥10 spores through shallow tray fermentation (28–30℃, 7–10 days). 9 A stable bacterial agent at 1 / g provides a new, green, and environmentally friendly solution to the environmental pollution caused by silkworm excrement and other waste. The application also provides a method for the fermentation, isolation, purification, and quantification of purpuric acid produced by strain CD11. Furthermore, the yield of purpuric acid produced by strain CD11 can be further optimized by changing the culture medium, modifying the culture medium composition, inoculum size, fermentation conditions, and mutagenesis breeding, demonstrating the potential for high-yield purpuric acid production. Attached Figure Description

[0015] Figure 1 The colony morphology of strain CD11 on oat medium.

[0016] Figure 2 This is a phylogenetic tree diagram of strain CD11.

[0017] Figure 3 The figure shows antagonistic plates of strain CD11 inhibiting different plant pathogens. In the figure, A represents the effect of CD11 on the wilt pathogen of watermelon: Fusarium oxysporum, a specialized strain of watermelon. Fusarium oxysporum f. sp. snowy The antagonistic results of ) ; B is CD11 against the anthracnose pathogen of pepper: *Colletotrichum capsicis* ( Colletotrichum capsici The antagonistic results of CD11 against the soybean root rot pathogen: *Fusarium oxysporum* (a specific strain of *Fusarium oxysporum*). Fusarium oxysporum f. sp. wisteria The antagonistic results of CD11 against the rice seedling blight pathogen: Fusarium oxysporum (D) Fusarium fujikuroi The antagonistic results of CD11 against the wheat stem rot pathogen: *Fusarium graminearum* (E is the antagonistic effect of CD11 against Fusarium graminearum). Fusarium pseudograssesThe antagonistic results of CD11 against the peony root rot pathogen: Fusarium solanum (F11) Fusarium nightshade The antagonistic results of CD11 against the pathogen of maize stalk rot: Fusarium graminearum (G represents CD11). Fusarium grasses The antagonistic results of CD11 against the pathogen of bacterial wilt of tomato: Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia nightshade RS1115 antagonistic results.

[0018] Figure 4 The figures show the experimental results of cellulase and β-1,3-glucanase production by strain CD11. In the figure, A represents the experimental results of cellulase production, and B represents the experimental results of β-1,3-glucanase production.

[0019] Figure 5 This is a schematic diagram of the qualitative detection of siderophore produced by strain CD11 on a plate.

[0020] Figure 6 This is a comparison of the siderophore production capacity of strain CD11 with other strains of the same genus.

[0021] Figure 7 This is a comparison of the IAA production capacity of strain CD11 with other strains of the same genus.

[0022] Figure 8 The effect of strain CD11 on SPAD value and tiller number.

[0023] Figure 9 The effects of applying strain CD11 to the aboveground and underground growth of wheat stem rot in pot experiments were investigated.

[0024] Figure 10 The effect of applying strain CD11 to tomato bacterial wilt pot experiment and its control effect.

[0025] Figure 11 The liquid chromatography and mass spectrometry results for the isolation, purification, and identification of purpuricin, the antibacterial metabolite of strain CD11, and the comparative verification results of the biosynthetic gene cluster of purpuricin from strain CD11 are shown. Among them, a is the HPLC chromatogram of the CD11 metabolite, b is the inhibition zone experiment diagram, c is the HPLC of the purified CD11 antibacterial component, d is the antibacterial experiment of the purified CD11 antibacterial component, e is the HR-MS mass spectrometry of the purified CD11 antibacterial component, f is the MS / MS secondary mass spectrometry, g is the chemical structural formula of purpuricin, and h is the gene cluster comparison diagram.

[0026] Figure 12 Chromatographic analysis of purpuric acid produced by strain CD11 and other strains of the same genus.

[0027] Information on the preservation of biological materials.

[0028] The strain information deposited in this application is: *Streptomyces calacana* ( Streptomyces Californian Strain CD11, classified as: Streptomyces calacana Streptomyces Californian The strain is classified and named as Streptomyces calica CD11 in Chinese, with accession number CGMCC No. 38498. It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 29, 2026. Detailed Implementation

[0029] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0030] Unless otherwise stated, each feature disclosed in this specification is merely one example of a series of equivalent or similar features.

[0031] Example 1.

[0032] This example illustrates the screening, identification, and biological characteristics of Streptomyces calica CD11 strain.

[0033] 1. Strain screening: Sixty 3rd-4th instar larvae were collected from the silkworm breeding area in Wujiang District, Suzhou City, Jiangsu Province. The larvae were surface-sterilized with 75% ethanol, and then homogenized with a small amount of sterile water in a sterile mortar. 100 μL of the homogenate was added to 900 μL of sterile water and serially diluted to 10⁻⁶. -6 Select 10 -4 10 -5 10 -6 Three dilutions were plated on Gao's No. 1 medium plates and incubated upside down at 30°C for 5-7 days. Single colonies of actinomycetes were picked, streaked on Gao's No. 1 medium plates for purification, and the obtained single colonies were transferred to slant culture and stored at 4°C. Gao's No. 1 medium contained the following components: 20g soluble starch, 1.0g KNO3, 0.5g K2HPO4·7H2O, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 20g agar, 1000mL deionized water, pH 7.2.

[0034] 2. Identification of strain CD11:

[0035] ① Morphological identification: When strain CD11 was inoculated onto oat agar plates, it formed colonies. The colony color gradually changed from white to light gray and continued to deepen, while releasing the characteristic "earthy" odor of actinomycetes. The colony surface was powdery, the aerial hyphae and spore hyphae were light gray, the mycelium within the substrate was purple, and conidia were abundant (see details). Figure 1 ).

[0036] ② Molecular identification: 16S rRNA sequencing was performed on strain CD11. The sequencing results were then subjected to BLAST alignment analysis using the EzBioCloud platform. The sequences of the top 9 strains with high homology to CD11 were selected. A phylogenetic tree was constructed using neighbor-joining (NJ) with MEGA 11.0 software. The results are as follows: Figure 2 As shown in the figure, strain CD11 is similar to Streptomyces calacana (…). Streptomyces Californian The homology reached 99.98%; and based on the strain's morphology and physiological and biochemical indicators, Streptomyces CD11 was identified as Streptomyces calcareosa, and this strain has been patented.

[0037] 3. The physiological and biochemical characteristics of Streptomyces CD11 are shown in Table 1.

[0038]

[0039] Table 1 shows the physicochemical properties of strain CD11 after 4-30 days of growth on gelatin liquefaction, milk peptone, starch hydrolysis, cellulose decomposition, Chesna agar, and carbon-nitrogen source utilization media. These results indicate that strain CD11 can hydrolyze starch, liquefy gelatin, produce hydrogen sulfide, decompose cellulose, and utilize D-arabinose, D-xylose, D-raffinose, rhamnose, and glucose as carbon sources, and serine, glutamic acid, and aspartic acid as nitrogen sources.

[0040] Example 2.

[0041] This example demonstrates the antibacterial activity of Streptomyces calendula CD11 strain.

[0042] Take fresh slant agar of cultured actinomycetes, add a small amount of sterile water to prepare a spore suspension. Spread 0.1 mL of the spore suspension onto a Gao Shi No. 1 plate and incubate at 30℃ for 5-7 days. After the spores are fully developed, use a 5 mm punch to collect the actinomycete mycelial cake. The pathogen of watermelon wilt is *Fusarium oxysporum* (specifically *Fusarium oxysporum*). Fusarium oxysporum f. sp. snowy Anthracnose pathogen of pepper: *Colletotrichum capsicis* ( Colletotrichum capsici Soybean root rot pathogen: Soybean-specific Fusarium oxysporum ( Fusarium oxysporum f. sp. wisteria), the pathogen of rice seedling blight: Fusarium oxysporum (Fusarium oxysporum) Fusarium fujikuroi ), against the pathogen of wheat stem rot: Fusarium graminearum ( Fusarium pseudograsses ), the pathogen of peony root rot: Fusarium solanum ( Fusarium solani ), the pathogen of corn stalk rot: Fusarium graminearum ( Fusarium gramineae The bacteria were simultaneously inoculated onto PDA plates along with actinomycete mycelium, and cultured in opposition at 30°C for 5-7 days. The plates were then observed, and the antagonistic activity was compared by measuring the size of the antagonistic zone.

[0043] Ralstonia solanacearum ( Ralstonia solanacearum RS1115 was inoculated into NB liquid medium (5 g / L peptone, 0.5 g / L yeast extract, 3 g / L beef extract, 10 g / L glucose, pH 7.2) and incubated overnight at 30°C and 170 rpm in a shaker to obtain a Ralstonia solanacearum RS1115 suspension. 0.1 mL of the RS1115 suspension was spread on NA plates, and CD11 bacterial blocks were placed in the center of the plates. Three replicates were set up and incubated at 30°C for 3-5 days. The plates were observed, and the inhibition level was determined by the size of the inhibition zone.

[0044] The antibacterial effects of strain CD11 against various pathogens are as follows: Figure 3 As shown, Figure 3 In the middle: A represents CD11 against the pathogen of watermelon wilt: *Fusarium oxysporum* var. *hypophyte* (watermelon-specific type). Fusarium oxysporum f. sp. snowy The antagonistic results of ) ; B is CD11 against the anthracnose pathogen of pepper: *Colletotrichum capsicis* ( Colletotrichum capsici The antagonistic results of CD11 against the soybean root rot pathogen: *Fusarium oxysporum* (a specific strain of *Fusarium oxysporum*). Fusarium oxysporum f. sp. wisteria The antagonistic results of CD11 against the rice seedling blight pathogen: Fusarium oxysporum (D) Fusarium fujikuroi The antagonistic results of CD11 against the wheat stem rot pathogen: *Fusarium graminearum* (E is the antagonistic effect of CD11 against Fusarium graminearum). Fusarium pseudogramineum The antagonistic results of CD11 against the peony root rot pathogen: Fusarium solanum (F11) Fusarium solani The antagonistic results of CD11 against the pathogen of maize stalk rot: Fusarium graminearum (G represents CD11). Fusarium gramineae The antagonistic results of CD11 against the pathogen of bacterial wilt of tomato: Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum The RS1115 antagonism results show that strain CD11 is effective against the pathogen: *Fusarium oxysporum*, a watermelon-specific strain. Fusarium oxysporum f. sp. snowy ), pepper anthracnose spores ( Colletotrichum capsici ), soybean-specific Fusarium oxysporum ( Fusarium oxysporumf.sp. glycines ), Fusarium oxysporum ( Fusarium fujikuroi ), Fusarium graminearum ( Fusarium pseudograminearum ), Fusarium solani ( Fusarium solani Fusarium graminearum ( ), Fusarium graminearum ) and Ralstonia solanacearum ( Ralstonia solanacearum All of them have good antagonistic and antibacterial effects. Note: Strain CD11 has a broad antibacterial spectrum, strong antibacterial activity, and significant inhibitory effect on plant pathogens.

[0045] Example 3.

[0046] This example studies the cellulase and β-1,3-glucanase production capabilities of Streptomyces calica CD11 strain.

[0047] CD11 spores were inoculated into 100 mL of liquid TSB medium and cultured at 28°C and 180 rpm with shaking for 7 days. After centrifugation at 8000 rpm for 10 minutes, the supernatant was used for quantitative determination of enzyme activity. Cellulase and β-1,3-glucanase activities were determined using a cellulase activity assay kit (Suzhou Keming Biotechnology Co., Ltd.) and a β-1,3-glucanase kit (Suzhou Keming Biotechnology Co., Ltd.), respectively. One unit of cellulase activity, expressed as 1 μg of glucose produced per minute per mL of sample solution at 37°C, is defined as one unit of enzyme activity (U). One unit of β-1,3-glucanase activity, expressed as 1 mg of reducing sugar produced per hour per mL of sample solution at 37°C, is defined as one unit of enzyme activity (U). All experiments were repeated three times.

[0048] The cellulase detection medium consists of the following components: (NH4)2SO4 2.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, sodium carboxymethyl cellulose (CMC-Na) 20.0 g, Congo red 0.2 g, and distilled water 1000 mL, sterilized at 121℃ for 20 min. The β-1,3-glucanase detection medium consists of the following components: dextran 5.0 g, NaNO3 2.0 g, K2HPO4 1.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, Congo red 0.05 g, agar 20 g, and distilled water 1000 mL, sterilized at 121℃ for 20 min.

[0049] The results of the cellulase activity assay of strain CD11 are as follows: Figure 4As shown in Figure A: CD11 bacterial pellets were inoculated onto cellulose Congo red medium and cultured at 30°C for 5 days. Hydrolysis zones were observed on the outer edge of the colonies, indicating that this strain can produce cellulase. The cellulase activity, measured using a cellulase kit, was 25.3 U / mL.

[0050] The results of the β-1,3-glucanase activity assay of strain CD11 are as follows: Figure 4 As shown in Figure B: CD11 bacterial discs were inoculated onto β-1,3-glucanase detection medium and cultured at 30°C for 5 days. Hydrolysis zones were observed on the outer edge of the colonies, indicating that this strain can produce β-1,3-glucanase. The β-1,3-glucanase activity, measured using a β-1,3-glucanase kit, was 0.79 U / mL.

[0051] Example 4.

[0052] This example studies the siderophore production capacity of Streptomyces calica CD11 strain.

[0053] Detection of siderophore production activity of the strain: The test strain was inoculated onto siderophore selection medium (CAS detection medium) and incubated at 30℃ for 7 days. Results showed that an orange-yellow color zone appeared around the colony. Figure 5 This indicates that the strain possesses the ability to produce siderophores. Specifically, strains A4, CD08, CD11, and J4 were selected as test subjects. The control (CK) served as a blank control for siderophore production. Strain A4 was molecularly identified as belonging to the genus *Streptomyces*. Streptomyces luridus Strain CD08 was molecularly identified as belonging to the genus Streptomyces (Streptomyces). Streptomyces cyslabdanicus Strain CD11 is *Streptomyces calacana* ( Streptomyces californicus The strain J4, which is the strain for which protection is claimed in this application, has the accession number CGMCC No. 38498. Strain J4 has been molecularly identified as belonging to the genus *Streptomyces*. Streptomyces rochei Currently, strains A4, CD08, and J4 are preserved in the research group's laboratory.

[0054] The specific detection method is as follows: 100, 50, 20 and 10 μL of the fermentation supernatant of each of the above strains were added to the CAS detection system, respectively. The change of the system color from blue to green, yellow, yellowish-brown or orange-yellow was used as the basis for judging the siderophore activity.

[0055] The results obtained are as follows Figure 6As shown in the figure, strain CD11 exhibits a significantly higher siderophore-producing capacity than other strains in the same genus, while strain J4 shows no siderophore-producing ability. This indicates that CD11 possesses a prominent siderophore-producing capacity among Streptomyces. This ability not only facilitates its competitive colonization in the rhizosphere but may also contribute to its indirect inhibition of plant pathogens. Therefore, CD11 can be used to prepare microbial agents with iron nutrient competition, biocontrol and antibacterial effects, and plant health promotion properties.

[0056] Example 5.

[0057] This example studies the IAA production capacity of Streptomyces calendula CD11 strain.

[0058] Assay for IAA production activity of the strain: The strain was inoculated into TSB medium containing tryptophan (final concentration 3 mmol / L) and cultured at 170 rpm and 28℃ for 7 days. 200 μL of the bacterial suspension was transferred to a 2 mL centrifuge tube, and an equal volume of Salkowski colorimetric solution was added (1 g FeCl3·6H2O was added to 21.485 mL of concentrated H2SO4 to bring the volume to 50 mL). A mixture of 100 μL of uninoculated TSB medium and an equal volume of Salkowski colorimetric solution was used as a control. After incubation at room temperature in the dark for 30 min, the mixture was observed. The strain turning red indicated IAA production; the deeper the red color, the stronger the IAA production ability. No color change indicated no IAA production. Specifically, strain A4 (… Streptomyces luridus ), strain CD08 ( Streptomyces cyslabdanicus ), strain CD11 ( Streptomyces californicus ) and strain J4 ( Streptomyces rochei The strains were inoculated separately into liquid medium containing L-tryptophan and cultured with shaking under the same conditions. After culture, the fermentation broth of each strain was centrifuged, and the fermentation supernatant was collected. A certain volume of the fermentation supernatant was mixed with Salkowski chromogenic solution, and after reacting at room temperature in the dark, the absorbance was measured at 450 nm using a spectrophotometer. A standard curve was established using IAA standards, and the relative yield of IAA in the fermentation supernatant of each strain was calculated according to the standard curve equation. In this experiment, the IAA standard curve equation was: y = 0.0096x + 0.2454, where y is the absorbance at 450 nm and x is the IAA concentration. The coefficient of determination of the standard curve was R. 2 =0.9867.

[0059] The results obtained are as follows Figure 7As shown, there are significant differences in IAA production capacity among different strains. Strain CD11 had the highest relative IAA production, with three replicates of 351.63 μg / mL, 378.19 μg / mL, and 382.72 μg / mL, respectively, and an average of approximately 370.85 μg / mL, which was significantly higher than other tested strains (significance marker a). Strain A4 had a moderate relative IAA production, with an average of approximately 288.26 μg / mL (significance marker b). Strains J4 and CD08 had relatively low relative IAA production, with averages of approximately 240.34 μg / mL and 212.78 μg / mL, respectively (significance marker c).

[0060] The above results indicate that, compared with other strains of the same genus, *Streptomyces calacana* CD11 has the strongest IAA production capacity among the closely related microorganisms compared. IAA is an important active substance for rhizosphere growth-promoting microorganisms to exert their plant growth-promoting effects. It can promote root elongation, lateral root development, and root hair formation, thereby improving the plant's ability to absorb water and nutrients. Therefore, CD11's high IAA production capacity can be regarded as an important growth-promoting functional characteristic that distinguishes it from other closely related microorganisms, and provides experimental evidence for its ability to promote crop growth, enhance rhizosphere vitality, and improve plant health.

[0061] Example 6.

[0062] This example demonstrates the control effect of CD11 solid fermentation biocontrol agent on wheat stem base rot.

[0063] 1. The preparation of the solid fermentation biocontrol agent of Streptomyces calica CD11 strain is as follows.

[0064] (1) Seed liquid preparation: The spores on the fresh slant of strain CD11 were inoculated into seed liquid culture medium and cultured at 30℃ and 180r / min for 2 days for later use; the seed liquid culture medium was Gao's No. 1 liquid culture medium.

[0065] (2) Preparation of CD11 solid-state fermented biocontrol agent: The fermentation broth obtained in the above steps was inoculated into a solid-state fermentation medium for shallow-tray solid-state fermentation. The fermentation substrate thickness was 1.5-2 cm, the inoculation amount was 10%, and the fermentation temperature was 28-30℃. Samples were taken daily to test the number of viable bacteria. Fermentation was completed after 7-10 days. The substrate was covered with CD11 conidia, and CD11 solid-state fermented biocontrol agent was obtained, in which the spore count reached 10. 9 More than one per gram.

[0066] The solid culture medium formula is as follows: 45% wheat bran, 45% silkworm excrement, and 10% rice husk, with a material-to-water ratio of 1:0.8, pH=7.2-7.5, and sterilization at 121℃ for 30 minutes.

[0067] 2. Construction of plants susceptible to wheat seedling stem rot, as detailed below.

[0068] (1) Germination: Select wheat seeds of uniform size and without damage to the skin, disinfect them with 3% sodium hypochlorite solution for 3 minutes, rinse them 5 times with sterile water, and incubate them at 25℃ for 2 days to germinate the wheat.

[0069] (2) Preparation of pathogens: The pathogenic bacterium *Fusarium graminearum* (… Fusarium pseudograminearum (Fp) was activated on PDA medium, and the spores were washed off with sterile water, filtered through sterile absorbent cotton, and the spore suspension was collected. The spore concentration in the filtrate was adjusted to 10-1 by microscopic counting. 6 Units / mL are available for use.

[0070] (3) Inoculation: Wheat that has been germinated for 2 days was sown in pots (200g soil per pot), with 9 seeds per pot. Experimental design: Two treatments were set up in the pot experiment: a control treatment and a CD11 solid inoculant treatment. CD11 solid inoculant treatment: 2% CD11 solid fermentation biocontrol agent was added to each pot and mixed with the soil. Germinated wheat was then sown. Seven days later, when the wheat had one bud and one leaf, the pathogen (10g of the agent) was added. 6 (Number of seedlings / mL, 20mL). All seedlings were uniformly placed in a greenhouse and cultured under conditions of 25℃ and alternating 12h light / 12h dark. After 30 days of growth, the wheat seedlings were pulled out, and the roots were rinsed clean with water. The fresh weight, plant height, and stem diameter of different treatments were measured. The incidence of stem rot in wheat seedlings was investigated, and the incidence rate and disease index were calculated. The control effect was calculated based on the disease index. Six pots were planted for each treatment in each experiment.

[0071] (4) Grading standards for wheat seedling stem base rot disease: 0: No disease in the plant; 1: Browning of the root or stem in the ground, or slight browning of the first leaf sheath; 3: The first leaf sheath is obviously brown, but the leaf sheath is not black; 5: The first leaf sheath is obviously black or the second leaf sheath is obviously brown; 7: The third leaf sheath shows browning symptoms, or the plant is slow to develop or close to death due to the disease; 9: The plant dies due to the disease.

[0072] Disease index = [Σ(each disease grade × number of plants affected by that disease grade) / (number of highest grade in the grading standard × total number of plants surveyed)] × 100.

[0073] Prevention and control efficacy (%) = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100%

[0074] The results of the pot experiment obtained using the above method are shown in Table 2 and Figure 8 , Figure 9 As shown.

[0075]

[0076] Note: "*" in the table indicates significant differences in data within the same column.

[0077] As shown in Table 2, the plant height, stem diameter, and fresh weight of wheat treated with strain CD11 were significantly higher than those of the control group (CK); the disease index was significantly lower than that of CK, and the control efficacy reached 60.67%. This indicates that the inoculant prepared from strain CD11 has a good growth-promoting effect on wheat and a good control effect on wheat seedling stem rot.

[0078] Depend on Figure 8 It can be seen that, in addition to the above indicators, strain CD11 also has a good promoting effect on the SPAD value and tiller number of wheat; Figure 9 It is evident that wheat inoculated with strain CD11 has a more developed root system, produces more tillers, and exhibits better growth. This demonstrates that strain CD11 has a good growth-promoting effect on wheat and provides effective control against wheat seedling stem rot.

[0079] Example 7.

[0080] This example demonstrates the control effect of CD11 bacterial solution on bacterial wilt of tomatoes.

[0081] 1. For the specific preparation method of the solid fermentation biocontrol agent of Streptomyces calica CD11 strain, please refer to Example 6.

[0082] 2. Construction of a tomato bacterial wilt susceptible plant model: Surface-sterilized tomato seeds were germinated on water agar plates for 3 days, then transplanted into seedling trays containing commercial substrate. At the three-leaf stage, seedlings were transplanted into six-cell seedling trays, each cell containing approximately 120g of experimental soil. Six technical replicates were set up for each treatment; each replicate corresponded to one seedling tray containing six seedlings (36 seedlings per treatment). Plants were grown in a greenhouse under natural temperature (25-35°C) and relative humidity (60-80%) conditions, irrigated daily with sterile water. The seedling tray positions were randomly changed every 3 days to minimize the position effect. Seven days after transplanting, the seedlings were irrigated with CD11 bacterial solution (12.0mL per plant) to achieve a final soil density of Streptomyces CD11 of 10-1. 7 CFU / g. 14 days after transplanting, Ralstonia solanacearum (… Ralstonia solanacearum RS1115 was inoculated into the soil, with a final density of 10. 6 CFU / g soil. The experiment lasted 42 days, with the disease index (DI) recorded daily from the first visible wilting symptom.

[0083] DI uses a 0-4 grade rating system: Grade 0, no wilting; Grade 1, 1-25% wilting; Grade 2, 26-50% wilting; Grade 3, 51-75% wilting; Grade 4, 76-100% wilting or plant death.

[0084] The disease index (DI, %) is calculated as follows: DI(%) = [Σ(number of diseased plants at each severity level × di) / (total number of plants × maximum di)] × 100%.

[0085] The area under the disease progression curve (AUDPC) is calculated based on the DI values ​​at different time points to summarize the disease progression and for statistical comparison. Figure 10 It is evident that the severity of bacterial wilt in tomato potted plants treated with CD11 bacterial solution was significantly reduced compared to the control group.

[0086] Example 8.

[0087] This example describes the isolation, purification, and identification of purpuric acid, a CD11 antibacterial metabolite.

[0088] The crude extract of Streptomyces CD11 metabolites was separated and purified using semi-preparative liquid chromatography (HPLC). An Agilent XDB-C18 (Analytical 4.6 x 250 mm 5-Micron) column was used. The mobile phases were A: methanol + 1‰ formic acid and B: H₂O + 1‰ formic acid. The column temperature was 30.0°C, and the flow rate was 1 mL / min. The preliminary HPLC separation program is shown in Table 3, and the separation chromatograms are shown in [Table 3]. Figure 11 a.

[0089]

[0090] Examples are based on chromatograms ( Figure 11 Metabolite chromatographic peaks in (a) were combined with time intervals to separate CD11 antibacterial metabolites, and the antibacterial activity of each separated component was verified using filter paper discs; the results showed that the target peak with an elution time of 20.531 min was the potential antibacterial active component ( Figure 11 (b) Subsequently, the target peak of the antibacterial metabolite with an elution time of 20.531 min was recovered using liquid chromatography. After separation, the antibacterial metabolite was used for purity verification using the liquid chromatography identification procedure (Table 4). The pure antibacterial metabolite had an elution time of 15.031 min. Figure 11 c), the antibacterial activity of the isolated and purified CD11 antibacterial metabolites was verified using filter paper discs. Figure 11 (d). Subsequently, high-resolution mass spectrometry analysis was performed on the antibacterial active component. The primary mass spectrometry results showed that the molecular weight of component A was [A+H]⁺ = 686.3331 ( ). Figure 11 (e). Secondary mass spectrometry results are shown in […]. Figure 11 f, combined with molecular weight data and secondary mass spectrometry results ( Figure 11 e, Figure 11 f), analysis of secondary metabolite gene clusters in strain CD11 genome (f) Figure 11(h), and the antibacterial compound was finally identified as Viomycin, whose structure is shown in [reference needed]. Figure 11 g.

[0091]

[0092] Example 9

[0093] This example is a comparative verification of the purpuric acid production capacity and antibacterial effect of strains of the same genus.

[0094] strain CD11 ( Streptomyces californicus ), closely related control strain YG01 ( Streptomyces laurentii (CGMCC No. 27519) and strain HT10 ( Streptomyces rubellomurinus The strains were inoculated into the same fermentation medium and cultured under the same shaking conditions. After cultivation, the fermentation broth of each strain was collected, and the fermentation supernatant was obtained. The fermentation supernatant was extracted with an organic solvent and concentrated to obtain crude extracts. Subsequently, high-performance liquid chromatography (HPLC) was used to detect the crude extracts of each strain at a detection wavelength of 280 nm. Currently, strains YG01 and HT10 are preserved in the research group's laboratory.

[0095] Figure 12 Detection of characteristic peaks of purpuricin in the crude extracts of strain CD11 and related strains. CD11 is *Streptomyces calacana* (…). Streptomyces californicus YG01 is Streptomyces roseum ( Streptomyces laurentii HT10 is *Streptomyces rubrum* ( Streptomyces rubellomurinus High-performance liquid chromatography (HPLC) was used to detect the crude fermentation extract at a wavelength of 280 nm.

[0096] from Figure 12 As can be seen, the crude extract of strain CD11 showed a distinct purpuricin characteristic peak at a retention time tR = 20.53 min, while the closely related control strains YG01 and HT10 did not show a purpuricin characteristic peak in the same retention time region (see the red dashed box for details). These results indicate that, compared with closely related microorganisms YG01 and HT10, CD11 has a more pronounced purpuricin-producing ability. Purpuricin, as a secondary metabolite with antibacterial activity, can serve as an important material basis for CD11's control of plant pathogens and provide experimental evidence for its application in the preparation of biocontrol agents or antibacterial metabolite formulations.

[0097] In summary, the *Streptomyces calcareae* CD11 screened by the applicant possesses both biocontrol and growth-promoting functions, and can be applied to the preparation of microbial fertilizers. Furthermore, its inoculant preparation process integrates the recycling of agricultural waste such as silkworm excrement, providing an environmentally friendly solution for soil-borne fungal diseases of wheat and bacterial wilt of tomatoes, and has significant application value in reducing chemical pesticide pollution and promoting green agriculture. Moreover, a method for isolating and purifying the CD11 antibacterial metabolite purpuricin is provided, offering technical support for the commercialization of its antibacterial substance.

[0098] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Streptomyces calacana ( Streptomycescalifornicus strain CD11, characterized in that, The California Streptomyces ( Streptomycescalifornicus The strain CD11 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 29, 2026; the accession number is CGMCC No. 38498.

2. The Streptomyces calacana as described in claim 1 ( Streptomycescalifornicus Application of strain CD11 in the preparation of antibacterial agents; the pathogen inhibited by the antibacterial agent is: *Fusarium oxysporum* var. *hypogon* (watermelon-specific type). Fusarium oxysporum f. sp. niveum ), pepper anthracnose bacteria ( Colletotrichum capsici ), soybean-specific Fusarium oxysporum ( Fusarium oxysporum f. sp. glycines ), Fusarium oxysporum ( Fusarium fujikuroi ), Fusarium graminearum ( Fusarium pseudograminearum Fusarium solani ( ) Fusarium solani Fusarium graminearum ( ), Fusarium graminearum ) or Ralstonia solanacearum ( Ralstonia solanacearum ).

3. The *Streptomyces calacana* as described in claim 1 (… Streptomycescalifornicus Application of strain CD11 in the preparation of biopesticides; the biopesticide controls Fusarium pseudograss (… Fusarium pseudograminearum Wheat stem rot caused by Ralstonia solanacearum (Ralstonia solanacearum) or by Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum Bacterial wilt of tomatoes caused by ( ).

4. The Streptomyces calacana as described in claim 1 ( Streptomycescalifornicus Application of strain CD11 in the preparation of bio-organic fertilizer.

5. The *Streptomyces calacana* as described in claim 1 ( Streptomycescalifornicus Application of strain CD11 in promoting wheat growth.

6. The Streptomyces calacana as described in claim 1 ( Streptomycescalifornicus Application of strain CD11 in increasing wheat tiller number.

7. The Streptomyces calacana as described in claim 1 ( Streptomycescalifornicus Application of strain CD11 in the production of IAA, siderophore, β-1,3-glucanase, cellulase and / or purpuricin.

8. Application of Streptomyces calacana as described in claim 1 ( Streptomycescalifornicus A method for cultivating plants using strain CD11, characterized in that, The method involves adding a bacterial suspension containing strain CD11 and / or conidia to the seedling stage of the plant; the plant is wheat or tomato.

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