A strain of streptomyces variabilis yj-15 and its application

Abstract

The application discloses a strain of Streptomyces variabilis YJ-15 and an application thereof. The Streptomyces variabilis YJ-15 has a good bacteriostatic effect on a pathogenic Fusarium pseudograminearum of wheat basal stem rot. Disease prevention tests show that the fermentation liquor of the strain YJ-15 can reduce the occurrence of the wheat basal stem rot, and the prevention efficiency is 62.0 %. Meanwhile, the strain YJ-15 has an antagonistic capacity to nine kinds of plant pathogenic fungi such as Fusarium graminearum. The strain YJ-15 provided by the application can provide a strain resource and a theoretical basis for biological control of wheat basal stem rot and other plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a variant Streptomyces YJ-15 strain for controlling wheat stem base rot and its application. Background Technology

[0002] Wheat stem rot, a significant soil-borne disease in wheat production, has caused severe losses globally. In recent years, wheat stem rot has become widespread in the main wheat-producing areas of the Huang-Huai region of my country, posing a serious threat to the sustainable and stable production of wheat. Various Fusarium species can cause wheat stem rot, with *Fusarium pseudogranatum* being the dominant pathogen in the Huang-Huai wheat region. Fusarium pseudograminearum ).

[0003] Currently, the control of wheat stem rot mainly relies on chemical control methods, supplemented by agricultural control measures. However, the extensive use of chemical agents can disrupt the ecological balance, pollute the environment, and harm human and animal health, hindering the green and sustainable development of agriculture. Therefore, biological control has received widespread attention due to its advantages such as safety and environmental friendliness. Summary of the Invention

[0004] The purpose of this invention is to provide a variant of Streptomyces YJ-15 for the prevention and control of wheat stem base rot and its application.

[0005] A mutant Streptomyces ( Streptomyces variabilis The strain, with accession number CGMCC No.38510, was named YJ-15.

[0006] The mutant Streptomyces ( Streptomyces variabilis Application of this technology in the prevention and control of wheat stem base rot.

[0007] The mutant Streptomyces ( Streptomyces variabilis It has an antagonistic effect on Fusarium graminearum, which causes wheat stem rot.

[0008] A microbial pesticide for controlling wheat stem base rot, comprising the aforementioned *Streptomyces mutabilis* ( Streptomyces variabilis ).

[0009] A variant of Streptomyces ( Streptomyces variabilis The fermentation broth of the *Streptomyces mutabilis* ( ) was prepared by the *Streptomyces mutabilis* ( ) Streptomyces variabilis Obtained through fermentation.

[0010] A microbial fungicide for controlling wheat stem base rot, said fungicide containing the aforementioned *Streptomyces mutabilis* ( Streptomyces variabilis ) and / or its fermentation products.

[0011] The classification name of this invention's *Streptomyces griseus* YJ-15 is: *Streptomyces mutabilis* (…). Streptomyces variabilis(This entry was deposited on April 29, 2026, at the China General Microbiological Culture Collection Center, with proof of viability. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC No. 38510.)

[0012] The beneficial effects of this invention are as follows: The mutant Streptomyces YJ-15 of this invention exhibits good antagonistic activity against Fusarium graminearum. The results of this invention demonstrate that strain YJ-15 can provide strain resources and a theoretical basis for developing highly effective biocontrol microbial agents for wheat stem rot. Attached Figure Description

[0013] Figure 1 The figure shows the antagonistic effect of strain YJ-15 against *Fusarium graminearum*. In the figure, A is the control; B is the antagonistic effect of strain YJ-15 against *Fusarium graminearum*.

[0014] Figure 2 The figure shows the inhibitory effect of volatile substances from strain YJ-15 on *Fusarium graminearum*. In the figure, A represents the control; B represents the inhibitory effect of strain YJ-15 on *Fusarium graminearum*.

[0015] Figure 3 The image shows the morphological characteristics of strain YJ-15. In the figure, A is the front view of a colony of strain YJ-15 grown on Gao's No. 1 medium for 7 days; B is the back view of a colony of strain YJ-15 grown on Gao's No. 1 medium for 7 days; and C is the spore hyphae produced by strain YJ-15 on Gao's No. 1 medium.

[0016] Figure 4 A neighbor-joining phylogenetic tree for strain YJ-15 constructed based on the 16S rRNA gene sequence.

[0017] Figure 5 The inhibition rate of sterile fermentation broth from different sources of strain YJ-15 against Fusarium oxysporum is shown in the figure. In the figure, A is LB medium; B is Gao's No. 1 liquid medium; C is PDB medium; and D is fermentation medium.

[0018] Figure 6 This study demonstrates the protective effect of strain YJ-15 against wheat stem rot. In the figure, A represents the control; B represents the treatment with strain YJ-15.

[0019] Figure 7 The extracellular enzyme production capacity of strain YJ-15 is shown in the figure. In the figure, A represents the protease detection medium; B represents the cellulase detection medium; and D represents the β-1,3-glucanase detection medium. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Example 1: Isolation and Screening of Strains YJ-15 Soil samples were collected in May 2025 from wheat fields in Yanjin County, Xinxiang City, Henan Province, where wheat stem rot had occurred. 10 g of soil samples were placed in 90 mL of sterile water and shaken at 28℃ and 150 r / min for 2 h. The suspension was then serially diluted to 10... −3 10 −4 and 10 −5 For each concentration, 100 μL was plated onto Gao's No. 1 plate (containing 50 μg / mL potassium dichromate), incubated at 28℃ for 7 days, and then single colonies were picked for purification.

[0022] Streptomyces cells to be tested were symmetrically streaked and inoculated at a distance of 2.5 cm from the center of a PDA plate. The plates were incubated at 28°C for 2 days. Then, 5 mm Fusarium wilt mycelium discs were inoculated into the center of the plates. PDA plates inoculated only with Fusarium wilt were used as controls. The antagonistic effect was observed after incubation for another 3 days.

[0023] Finally, one strain with good antagonistic ability was obtained through screening, designated YJ-15, with an inhibition rate of 65.6 ± 1.1%. Figure 1 ).

[0024] First, strain YJ-15 was streaked onto yeast malt extract agar (ISP2) medium and cultured in the dark at 28°C for 7 days. Then, using a double-plate inversion method, a PDA culture dish inoculated with a 5 mm Fusarium graminearum mycelium cake was placed on top of the dish, completely sealed with sealing film, and cultured for another 3 days before observing the antibacterial effect. Figure 2 As shown, the volatile substances produced by YJ-15 also have weak antibacterial ability, with an antibacterial rate of 9.8 ± 0.1%. The formula for Gao's No. 1 culture medium is as follows: soluble starch 20.0 g, KNO3 1.0 g, K2HPO4 0.5 g, NaCl 0.5 g, MgSO4·7H2O 0.5 g, agar 15.0 g, distilled water 1000 mL, pH 7.2.

[0025] PDA culture medium formula: 200 g potato, 20 g glucose, 1000 mL distilled water.

[0026] ISP2 culture medium formula: 4 g yeast extract, 10 g malt extract, 4 g glucose, 15 g agar, 1000 mL distilled water, pH 7.2.

[0027] Example 2: Classification and Identification of Strain YJ-15 (1) Morphological characteristics of strain YJ-15 Strawberry strain YJ-15 was streaked onto Gao's No. 1 medium and incubated in the dark at 28°C for 7 days. The morphological characteristics of colonies and spore chains were then observed. Figure 3 As shown, the aerial hyphae of strain YJ-15 are grayish-white, while the basal hyphae are brownish-brown and produce soluble pigments. The spore filaments are spiral-shaped.

[0028] (2) Physiological and biochemical characteristics of strain YJ-15 The physiological and biochemical indicators of strain YJ-15 were tested according to "Rapid Identification and Systematic Classification of Actinomycetes". The specific results are shown in Table 1.

[0029] Table 1. Physiological and biochemical characteristics of strain YJ-15

[0030] (3) 16S rRNA gene sequence analysis of strain YJ-15 Genomic DNA was extracted from strain YJ-15, and the 16S rRNA gene sequence was amplified using primers 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ). The amplified product was purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the gene sequence is shown in SEQ ID NO.1. The sequencing results were compared with the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software. Figure 4 As shown, strain YJ-15 and Streptomyces mutagenesis ( S. variabilis Herb, T14, NRRL B-3984, and Streptomyces lauryleri ( S. labedae NBRC15864 AJR1 clustered together. Based on morphological and physiological-biochemical characteristics, strain YJ-15 was ultimately identified as Streptomyces mutabilis.

[0031] Example 3: Inhibitory effect of sterile fermentation broth of strain YJ-15 on Fusarium oxysporum. (1) Preparation of sterile fermentation broth of strain YJ-15 Fresh YJ-15 bacterial blocks (1 cm) were selected and placed in 100 mL of PDB, LB, Gao's No. 1 liquid, and fermentation medium, respectively, and cultured at 28℃ and 180 r / min for 5 days with shaking. The fermentation broth was centrifuged at 12000 r / min for 15 min, and the supernatant was then filtered through a 0.22 μm bacterial filter to remove bacterial cells, thus obtaining sterile fermentation broths of strain YJ-15 from different sources.

[0032] PDB culture medium formula: 200.0 g potato, 20.0 g glucose, 1000 mL distilled water.

[0033] LB medium formula: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 1000 mL distilled water.

[0034] Fermentation medium formula: glucose 25.0 g, peptone 25.0 g, yeast extract 5.0 g, MgSO4·7H2O 2.0 g, K2HPO4 2.0 g, KH2PO4 2.0 g, CaCO3 5.0 g, distilled water 1000 mL, pH 7.2.

[0035] (2) Effect of sterile fermentation broth of strain YJ-15 on the colony growth of Fusarium pseudograss Aseptic fermentation broths of strain YJ-15 from different sources were mixed with melted PDA medium at a ratio of 1:9. After the medium solidified, *Fusarium graminearum* was inoculated into the center of the plates. PDA medium without aseptic fermentation broth served as a control. After incubation at 28°C for 3 days, colony size was measured and the inhibition rate was calculated. Figure 5 As shown, sterile fermentation broths from different sources all inhibited the growth of Fusarium pseudograss. The sterile fermentation broth obtained from fermentation medium had the strongest antibacterial ability, at 34.4%, while the sterile fermentation broth obtained from LB medium had the weakest antibacterial ability, at only 19.1%.

[0036] Example 4: Analysis of the disease resistance ability of strain YJ-15 (1) Preparation of fermentation broth of strain YJ-15 Preparation method of fermentation broth of strain YJ-15: Pick a 1 cm piece of strain YJ-15 and put it into 100 mL of fermentation medium. Incubate at 28℃ and 150 r / min for 5 days to obtain the fermentation broth of strain YJ-15.

[0037] (2) Analysis of the disease resistance ability of strain YJ-15 Germinated wheat was planted in flowerpots and cultured at room temperature for one week. A 5 mm diameter *Fusarium graminearum* mycelium cake was then inoculated at the base of each wheat plant, and 1 mL of fermentation broth from strain YJ-15 was poured over the soil. A control group inoculated only with the pathogen was used. After another three weeks of culture, the length of the lesions was measured. Figure 6 As shown, compared with the control, the incidence of stem rot was reduced after treatment with fermentation broth of strain YJ-15, with a control efficacy of 62.0±3.4%. This indicates that strain YJ-15 has the ability to control wheat stem rot.

[0038] Example 5: Analysis of the extracellular enzyme production capacity of strain YJ-15 Strains of strain YJ-15 were streaked onto protease, cellulase, and β-1,3-glucanase media, respectively, and incubated at 28°C for 7 days. The presence of a clear zone around the colonies was then observed. Figure 7 As shown, strain YJ-15 can grow well on all three culture media, and a clear zone appears around the colony, indicating that the strain has the ability to produce protease, cellulase and β-1,3-glucanase.

[0039] Protease culture medium formula: 100.0 mL skim milk, 15.0 g agar, 1000 mL distilled water.

[0040] Cellulase culture medium formula: 10.0 g carboxymethyl cellulose, 1.0 g MgSO4, 0.5 g NaCl, 0.3 g K2HPO4, 2.0 g yeast extract, 1.0 g KNO3, 15.0 g agar, 1000 mL distilled water, pH 7.2.

[0041] β-1,3-glucanase culture medium formula: 0.12 g aniline blue, 5 g Poria cocos powder, 6.7 g yeast extract, 6.8 g KH2PO4, 10.46 g K2HPO4, 15 g agar, 1000 mL distilled water, pH 7.2.

[0042] Example 6: Antibacterial spectrum analysis of strain YJ-15 Using a plate confrontation method, strain YJ-15 was first symmetrically inoculated 2.5 cm from the center of a PDA plate and cultured at 28°C for 2 days. Then, Fusarium graminearum was inoculated into the center of the plate. Fusarium graminearum Nine plant pathogenic fungi, including *Fusarium graminearum*, were cultured for 3-7 days, and their antagonistic effects were observed. As shown in Table 2, strain YJ-15 exhibited the strongest antagonistic ability against *Fusarium graminearum*, with an inhibition rate of 74.0%; followed by *Helicobacter zedoaria*. Bipolaris maydis The antibacterial rate was 70.9%; against Staphylococcus aureus (…). Botryosphaeria dothidea ) and Siamese anthrax ( Colletotrichumsiamense The antibacterial rate is around 60%; against Colletotrichum candida (… Colletotrichum gloeosporioides ) and Rhizoctonia graminearum ( Rhizoctonia cerealis The antibacterial rate is around 56%; against Alternaria (… Alternaria alternata ) and Fusarium moniliformes ( Fusarium proliferatum The antibacterial rates of ) were 52.1% and 48.2%, respectively; and the antibacterial rates of *Sclerotium truncatum* (…) were… Sclerotium rolfsii The antibacterial rate of strain YJ-15 was relatively low, at 46.4%. Therefore, strain YJ-15 has a good antibacterial spectrum.

[0043] Table 2. Inhibition rate of strain YJ-15 against different plant pathogenic fungi

[0044] Note: Different lowercase letters indicate significant differences at the 0.05 level.

[0045] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. 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 all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mutant Streptomyces strain ( Streptomyces variabilis The strain, with accession number CGMCC No.38510, was named YJ-15.

2. The *Streptomyces mutabilis* as described in claim 1 ( Streptomyces variabilis Application of this technology in the prevention and control of wheat stem base rot.

3. The application according to claim 2, characterized in that, The mutant Streptomyces ( Streptomyces variabilis It has an antagonistic effect on Fusarium graminearum, which causes wheat stem rot.

4. A microbial pesticide for controlling wheat stem base rot, characterized in that, Contains the *Streptomyces grayi* as described in claim 1 ( Streptomyces variabilis ).

5. A variant of Streptomyces ( Streptomyces variabilis The fermentation broth of ) is characterized in that, The mutant Streptomyces as described in claim 1 ( Streptomyces variabilis Obtained through fermentation.

6. A microbial fungicide for controlling wheat stem base rot, characterized in that, The bactericide contains *Streptomyces mutabilis* as described in claim 1. Streptomyces variabilis ) and / or its fermentation products.

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