Streptomyces cinnamoneus and its use

Abstract

This invention belongs to the field of microbial biological control, and relates to the *Streptomyces cinnamonensis* Sc-7 strain, which antagonizes rice bacterial blight and bacterial leaf streak, and promotes growth, as well as its application in plant disease control and growth promotion. This invention discloses a *Streptomyces cinnamonensis* Sc-7 strain, with the preservation number CCTCC NO: M 20231179. *Streptomyces cinnamonensis* Sc-7 has the following uses: antagonistic effect against rice bacterial blight and bacterial leaf streak, inhibitory effect against plant pathogenic bacteria and fungi, and growth-promoting effect on plants.

Description

Technical Field

[0001] This invention belongs to the field of microbial biological control, and relates to the Streptomyces cinnamonensis Sc-7 strain, which has antagonistic effects against rice bacterial blight and bacterial leaf streak and promotes growth, and its application in plant disease control and growth promotion. Background Technology

[0002] Rice is the world's most consumed food crop, and producing green and environmentally friendly rice is a fundamental guarantee for human health and the sustainable development of the ecological environment. Rice diseases seriously threaten rice yield and quality. Bacterial blight of rice (BBR) and bacterial leaf streak of rice (BLS) are the two most important bacterial diseases. BBR and BLS are caused by *Xanthomonas oryzae* pv. oryzae (Xoo) and *Xanthomonas oryzae* pv. oryzicola (Xoc), respectively, and are characterized by their epidemic, explosive, and devastating nature. They generally reduce rice yield by 20%–30%, and in severe cases, by more than 50%, and also affect rice quality, posing a serious threat to safe rice production. BLS has been listed as an important quarantine rice disease by countries such as Australia, China, and the United States.

[0003] Currently, the control of BBR and BLS mainly relies on chemical pesticides, with major fungicides including thiamethoxam, kasugamycin, chlorobromoisocyanuric acid, tetracycline, thiamethoxam, thiamethoxam, copper oxychloride, thiamethoxam, copper rosinate, and pyraclostrobin. However, long-term use of these fungicides has many drawbacks, such as environmental pollution, harm to biological health, and the development of drug resistance. Therefore, the development of novel microbial fungicides for the efficient control of BBR and BLS is urgently needed. Existing literature indicates that actinomycetes have abundant secondary metabolites with various antibacterial activities, but these have been rarely used in the control of BBR and BLS. Biocontrol bacteria are ecologically safe for soil and the environment, have no toxic side effects on humans, animals, and plants, and pathogens are unlikely to develop resistance. Biocontrol bacteria generally produce complex natural active products such as polypeptides, macrolides, and small molecule compounds, which have good inhibitory effects on plant pathogens. Some biocontrol bacteria can even promote plant growth. Screening for new antagonistic microorganisms can provide effective biological control of plant diseases.

[0004] The known uses of Streptomyces cinnamonensis are as follows:

[0005] Invention CN107779419A discloses a *Streptomyces cinnamonensis* strain ZX6 for controlling sunflower sclerotinia disease. Strain ZX6 exhibits a strong ability to inhibit the mycelial growth of *Sclerotinia cinnamonensis*. Treatment with the fermentation broth of strain ZX6 reduced the incidence of sunflower seed husk infection by 40%, the fermentation filtrate of strain ZX6 promoted seed germination by 51.1%, the control effect of the fermentation broth on detached leaves was 54%, and the control effect of the fermentation broth of strain ZX6 in pot experiments was 58.3%, effectively inhibiting the growth and reproduction of *Sclerotinia cinnamonensis*. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide Streptomyces cinnamonensis Sc-7 strain and its uses.

[0007] To solve the above-mentioned technical problems, the present invention provides a Streptomyces cinnamonensis Sc-7, which has the accession number CCTCC NO: M 20231179.

[0008] The present invention also provides the use of the above-mentioned Streptomyces cinnamonensis Sc-7: it has a high antagonistic effect on rice bacterial blight and rice bacterial leaf streak.

[0009] This invention also provides another use for the aforementioned Streptomyces cinnamonensis Sc-7: its inhibitory effect on plant pathogenic bacteria and fungi. The plant pathogenic bacteria are: Xanthomonas axonopodis pv. phaseoli (Xap), Pseudomonas syringae pv. glycinea (Psg), Xanthomonas axonopodis pv. glycines (Xag), and Dickeya zeae (Dz), the pathogenic fungi of Tomato early blight, Fusarium oxysporum f.sp. momordicae, Fusarium solani, and Colletetrichum orbiculare, the pathogen of watermelon anthracnose.

[0010] The present invention also provides another use for the aforementioned Streptomyces cinnamonensis Sc-7: promoting growth in plants (rice seedlings). This growth promotion includes increasing the height and root length of the seedlings, as well as increasing the weight of the above-ground parts of the plant (including fresh weight and dry weight) and the weight of the roots (including fresh weight and dry weight).

[0011] Given the increasing problems and shortcomings of pesticide control for bacterial diseases of rice, the use of microorganisms and their secondary metabolites for the biological control of rice diseases has become a research hotspot. This invention aims to provide a Streptomyces cinnamonensis Sc-7 strain that antagonizes both Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae epv. oryzicola (Xoc), and its application in the control of rice diseases and the promotion of rice seedling growth.

[0012] This invention isolated 160 actinomycetes from soils in different habitats. After primary and secondary screening, one strain, Sc-7, was obtained, which showed strong inhibitory effects against both Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae epv. oryzicola (Xoc), the pathogen causing bacterial leaf blight of rice. This invention aims to provide new actinomycete resources for the biological control of bacterial diseases of rice.

[0013] The preservation information for strain Sc-7 is as follows:

[0014] Preservation name: Streptomyces cinnamonensis Sc-7, depositary institution: China Center for Type Culture Collection; depositary address: Wuhan University, Wuhan, China, accession number: CCTCC NO: M 20231179, deposit date: July 3, 2023.

[0015] The Sc-7 strain involved in this invention was screened from the rhizosphere soil of Pinus tabulaeformnis in Jinhua City, Zhejiang Province. Based on morphological characteristics, physiological and biochemical characteristics, and 16S rDNA sequence analysis, it was identified as Streptomyces cinnamonensis. Figure 4 ).

[0016] Streptomyces cinnamonensis strain Sc-7 exhibited strong inhibitory effects against both Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzaeepv. oryzicola (Xoc), the pathogen causing bacterial leaf blight of rice. After culturing strain Sc-7 in Gao's No. 1 medium for 7 days (28℃), 200 μL of the fermentation filtrate was used to determine its inhibitory effect on Xoo and Xoc using the Oxford cup method. The inhibition zone diameters reached 43.2±2.6 mm and 40.3±1.5 mm, respectively. Figure 5 ).

[0017] The antibacterial spectrum test results showed that the fermentation filtrate of strain Sc-7 had a good inhibitory effect on four plant pathogenic bacteria, namely, bacterial blight of bean (Xanthomonas axonopodis pv. phaseoli, Xap), bacterial spot of soybean (Pseudomonas syringae pv. glycinea, Psg), bacterial scab of soybean (Xanthomonas axonopodis pv. glycines, Xag), and bacterial basal rot of rice (Dickeya zeae, Dz) (Table 3). The strain also showed good inhibitory effects against the four plant pathogenic fungi tested. It exhibited particularly strong inhibitory effects against *Alternaria solani* (tomato early blight) and *Fusarium oxysporum* f.sp. *momordicae* (bitter melon wilt), with mycelial inhibition rates reaching 51.5% and 44.1%, respectively. The inhibition rates against *Fusarium solani* (poplar wilt) and *Colletetrichum orbiculare* (watermelon anthracnose) were 36.6% and 30.3%, respectively (Table 4). These results further demonstrate that strain Sc-7 has a broad-spectrum effect in controlling plant diseases.

[0018] Pot experiment on rice showed that the fermentation broth of strain Sc-7 had a relative control efficacy of over 70% against rice bacterial blight and bacterial leaf streak, which was higher than that of the chemical pesticide pyraclostrobin control (Table 5). Figure 8 A rice pot seedling growth-promoting experiment showed that a 10-fold dilution of the Sc-7 strain fermentation broth increased rice seedling height by 20.2%, root length by 72.13%, aboveground fresh weight and root fresh weight by 70.58% and 107.7% respectively, and aboveground dry weight and root dry weight by 93.63% and 53.08% respectively. Figure 10 This indicates that the Sc-7 strain can provide an excellent strain for the development of microbial pesticides and has good application prospects in the biological control of crops such as rice. Attached Figure Description

[0019] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] Figure 1 Colony morphology diagrams of 12 typical actinomycetes;

[0021] Figure 2 Morphological characteristics of colonies, substrate hyphae, aerial hyphae, and spore hyphae of strain Sc-7;

[0022] Figure 3 These are some of the physiological and biochemical experimental results of strain Sc-7;

[0023] Figure 4 A phylogenetic tree of Sc-7 strains constructed based on 16S rDNA sequences;

[0024] Figure 5 The figure shows the inhibitory effect of the fermentation filtrate of strain Sc-7 on the growth of rice bacterial blight pathogen Xoo and bacterial leaf streak pathogen Xoc.

[0025] Figure 6 Figure showing the effect of fermentation broth of strain Sc-7 on the cell morphology and structure of Xoo, the pathogen of rice bacterial blight.

[0026] Figure 7 Figure showing the effect of fermentation broth of strain Sc-7 on the cell morphology and structure of Xoc, a bacterial leaf streak pathogen of rice.

[0027] Figure 8 The control effect of Sc-7 strain fermentation broth on rice bacterial blight and bacterial leaf streak is shown in the figure:

[0028] Figure 8 In the middle: CK1: sterile water negative control; CK2: Xoo or Xoc positive control; T1: 2-fold diluted fermentation broth treatment group; T2: 2-fold diluted fermentation broth prevention group; T3: pesticide pyraclostrobin control;

[0029] Figure 9 The graph shows the effect of Sc-7 strain fermentation broth on rice seed germination.

[0030] Figure 9 In Chinese: CK1: Sterile water; CK2: Gao's No. 1 culture medium; T1: Fermentation stock solution; T2: 10-fold fermentation dilution; T3: 50-fold fermentation dilution; T4: 100-fold fermentation dilution;

[0031] Figure 10 The graph shows the growth-promoting effect of the fermentation broth of strain Sc-7 on rice seedlings.

[0032] Figure 10 middle:

[0033] A: Growth of rice seedlings under different treatments; B: Plant height and root length of rice seedlings under different treatments; C: Fresh weight and dry weight of rice seedlings under different treatments;

[0034] CK: sterile water; T1: 10-fold fermentation dilution; T2: 50-fold fermentation dilution. Different lowercase letters indicate significant differences between treatments (P < 0.05). Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0036] Example 1: Screening and identification of Streptomyces cinnamonensis Sc-7 strain

[0037] 1 strain

[0038] (1) Actinomycete strains: Actinomycete strains isolated, purified and screened from soils in different habitats.

[0039] (2) Pathogens: Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) are both common pathogens. For example, PXO99 from the Chinese Academy of Agricultural Sciences can be used. A Strains and RS105 strain.

[0040] 2 Culture media

[0041] (1) Gao's No. 1 solid culture medium: soluble starch 20g, KNO3 1g, K2HPO4 0.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, agar 20g, water 1000mL, pH 7.4. Used for the isolation and purification of actinomycete strains.

[0042] (2) Gao's No. 1 liquid culture medium: The formula is the same as (1), but without agar, and it is used for liquid fermentation culture of actinomycete strains.

[0043] (3) NA solid medium: 3g beef extract, 5g peptone, 5g NaCl, 20g agar, 1000mL water, pH 7.2. Used for the culture of Xoo and Xoc pathogens.

[0044] (4) NA liquid culture medium: The formula is the same as (3), but without agar, and is used for the activation culture of Xoo and Xoc.

[0045] (5) PDA solid culture medium: 200g potato, 20g glucose, 18g agar, 1000mL water, pH 6.0~6.5.

[0046] 3 Experimental Methods

[0047] 3.1 Isolation, purification and preservation of actinomycete strains

[0048] Separation was performed using a serial dilution coating method. 1 g of dried soil sample was added to 99 mL of sterile water and shaken on a shaker at 120 rpm for 30 min. The supernatant was then serially diluted (10... -2 ~10 -5 Choose an appropriate concentration (10) -4 Spread 100 μL of the culture evenly onto a Gao's No. 1 agar plate containing 50 μg / mL potassium dichromate, and incubate at 28°C for 7 days until colonies appear. Select a single colony exhibiting typical actinomycete colony characteristics (i.e., dense texture, small and non-spreading, dry, radial surface, and difficult to pick up with an inoculating loop) and transfer it to another Gao's No. 1 agar plate. Purify the strain by streaking three times. Number the pure strain and store it at 4°C for later use.

[0049] 3.2 Screening of actinomycete strains antagonistic to Xoo and Xoc

[0050] 3.2.1 Initial screening of antagonistic actinomycetes using co-culture method

[0051] The collected actinomycete strains were streaked onto Gao's No. 1 medium plates and incubated at 28°C for activation before use. The test actinomycete mycelium was obtained by punching holes with a 5 mm diameter sterile punch.

[0052] Xoo and Xoc were inoculated into NA liquid medium and activated by shaking (160 r / min, 28 ℃) until OD. 600 When the concentration of bacterial culture reaches 0.6, 5 mL of bacterial culture is added to 100 mL of NA solid medium, mixed well, and poured into plates. Then, the tested actinomycete mycelial discs are inoculated into the NA medium containing the pathogen, and incubated at 28°C for 48 hours. The inhibition zones are then observed. Actinomycete strains with antagonistic effects are preliminarily screened based on the presence and size of the inhibition zones, and preserved using 25% glycerol.

[0053] 3.2.2 Oxford Cup Method for Secondary Screening of Antagonistic Actinomycetes

[0054] (1) Fermentation culture of actinomycete strains and preparation of fermentation filtrate: Actinomycetes that antagonize both Xoo and Xoc were inoculated into Gao's No. 1 medium plates and cultured at 28℃ for 3 days. Using a sterile punch with a diameter of 5 mm, holes were punched in the actinomycete plates to obtain bacterial blocks, which were then inoculated into Gao's No. 1 medium. After culturing in a shaker at 160 r / min and 28℃ for 7 days, 100 mL of fermentation broth was centrifuged at 12000 r / min for 10 min and filtered through a 0.22 μm filter membrane to obtain fermentation filtrate for use.

[0055] (2) Cultivation and rescreening of antagonistic actinomycetes using Xoo and Xoc: Xoo and Xoc were inoculated separately into NA liquid medium and activated by shaking (160 r / min, 28 ℃). When the bacterial density reached OD 600 When the concentration of Xoo and Xoc is 0.6, add 5 mL of bacterial culture to 100 mL of NA solid medium and pour into a plate. Take 200 μL of the fermentation filtrate of the actinomycete strain obtained in step (1) above, and use the Oxford cup method to determine the inhibitory effect of the fermentation filtrate of each actinomycete strain on Xoo and Xoc. Select the actinomycete strain with stronger antagonistic effect based on the size of the inhibition zone.

[0056] 3.2.3 Scanning electron microscopy observation of morphological changes in Xoo and Xoc cells after treatment with fermentation filtrate of the target strain

[0057] (1) Cultivation of Xoo and Xoc: Xoo and Xoc were inoculated into NA liquid medium and activated by shaking (160 r / min, 28 ℃) until the bacterial density reached OD. 600 =0.6.

[0058] (2) Fermentation culture of actinomycete strains: Fermentation filtrate of actinomycetes with antagonistic effects was prepared using the method in 3.2.2(1).

[0059] (3) Scanning electron microscopy observation: 2 mL of fermentation filtrate of different concentrations was cultured together with 2 mL of Xoo and Xoc bacterial cultures on a shaker. Xoo and Xoc bacterial cultures treated with Gao Shi No. 1 filtrate were used as control groups. After 4 h of culture, the bacterial cells were collected by centrifugation at 12000 r / min for 10 min. The collected bacterial cells were gently washed 3 times with phosphate buffer, fixed overnight in 2.5% glutaraldehyde at 4℃, and then soaked and washed 3 times with phosphate buffer for 10 min each time. They were dehydrated once each with a gradient of 30%, 50%, 70%, and 90% ethanol, and twice with 100% ethanol for 15 min each time. They were soaked twice with tert-butanol for 30 min each time, and then placed in a vacuum freeze dryer for vacuum drying. The samples were sputter-coated with gold and then observed under a scanning electron microscope.

[0060] 3.3 Identification of antagonistic Xoo and Xoc strains Sc-7

[0061] 3.3.1 Observation of morphological characteristics

[0062] One mycelial disc (5 mm in diameter) of strain Sc-7 was inoculated onto Gao's No. 1 medium plates and activated for 3 days; then transferred to fresh Gao's No. 1 medium plates and incubated at 28℃ for 7 days. The size, morphology, texture, surface, and color of the colonies were observed. Strain Sc-7 was cultured using the slide insertion and embedding methods. The morphological characteristics of the mycelia, aerial hyphae, and spore hyphae of strain Sc-7 were observed under optical and electron microscopes and photographed.

[0063] 3.3.2 Physiological and Biochemical Characteristic Experiments

[0064] Physiological and biochemical tests were conducted on strain Sc-7, including starch hydrolysis, gelatin liquefaction, and carbon and nitrogen source utilization.

[0065] 3.3.3 16S rDNA Sequence Analysis

[0066] The selected strains were plated and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The 16S rDNA sequence of the strains was submitted to GenBank, and BLAST was used for alignment. A phylogenetic tree was constructed using MEGA7 software and the Neighbor-Joining method to determine their species and genus.

[0067] 4. Experimental Results

[0068] 4.1 Obtaining pure strains of actinomycetes

[0069] A total of 160 pure actinomycete strains were isolated and purified from soil samples from different habitats. The colony counts of 12 representative pure actinomycete strains after 7 days of culture on Gao's No. 1 medium are shown below. Figure 1 As shown, different strains exhibit variations in colony morphology, texture, and pigment production, indicating the rich diversity of actinomycetes.

[0070] 4.2 Screening of antagonistic Xoo and Xoc actinomycete strains

[0071] The inhibitory effects of co-culture and the Oxford cup method on Xoo and Xoc inhibitory effects were initially and subsequently screened in 160 actinomycete strains. One strain, designated Sc-7, was obtained, exhibiting strong antagonistic effects against both Xoo and Xoc. The inhibition zone diameters against Xoo and Xoc, as determined by the Oxford cup method, were 43.2 ± 2.6 mm and 40.3 ± 1.5 mm, respectively. Figure 5 The Sc-7 strain was isolated from the rhizosphere soil of Pinus tabulaeformnis collected in Jinhua City, Zhejiang Province.

[0072] The Sc-7 strain has been deposited, and the deposit information is as follows:

[0073] Preservation name: Streptomyces cinnamonensis Sc-7, depositary institution: China Center for Type Culture Collection; depositary address: Wuhan University, Wuhan, China, accession number: CCTCC NO: M 20231179, deposit date: July 3, 2023.

[0074] 4.3 Changes in cell morphology of Xoo and Xoc cells after treatment with fermentation filtrate of strain Sc-7

[0075] Depend on Figure 6 , Figure 7 It can be seen that after culturing in the fermentation filtrate for 4 hours, compared with the control (CK), it affected the cell morphology of pathogens Xoc and Xoo to some extent. After treating Xoo cells with the fermentation filtrate of strain Sc-7, Xoo cells exhibited large-area swelling, shrinkage, perforation, and rupture. Figure 6 After Xoc cells were treated with the fermentation filtrate of strain Sc-7, the Xoc cells exhibited swelling, shrinkage, folding, adhesion, and rupture. Figure 7 Therefore, the Sc-7 strain can ultimately inhibit the growth of Xoc and Xoo pathogens.

[0076] 4.4 Identification results of Sc-7 strain

[0077] The Sc-7 strain cultured on Gao's No. 1 medium initially appeared as white, millet-like colonies. As the number of days of growth increased, the color did not change significantly; the colony surface remained milky white with white, velvety protrusions at the edges. The mycelia within the medium were densely interwoven, situated within the medium. The aerial hyphae were robust, with a few pale pink spores appearing in the center, and the edges were white. After one week, they turned pale pinkish-white, and the spore hyphae became slender and flexible. The colony surface dried, adhered tightly to the medium, was velvety, raised in the center, opaque, and had irregular, radial edges. Figure 2 ).

[0078] Physiological and biochemical results showed that strain Sc-7 could decompose cellulose, starch, urea, and fat, producing melanin and hydrogen sulfide, but could not produce catalase or gelatinase. The MR and VP tests were negative (Table 1). Figure 3 It can utilize a variety of carbon and nitrogen sources (Table 2).

[0079] Table 1. Physiological and biochemical test results of strain Sc-7

[0080]

[0081] Note: +++: Strong ability; ++: Medium ability; +: Weak ability; -: None

[0082] Table 2 Results of carbon and nitrogen source utilization experiments of strain Sc-7

[0083]

[0084]

[0085] Note: ++: vigorous growth; ++: good growth; +: average growth; -: no growth

[0086] The nucleotide sequence of the 16S rDNA sequence is shown in SEQ ID NO:1 of the sequence listing.

[0087] It is most closely evolutionarily distant from Streptomyces cinnamonensis. Figure 4 The similarity was 100%. Based on the morphological characteristics, physiological and biochemical test results, and 16S rDNA sequence analysis of strain Sc-7, combined with the species identification key and phylogenetic analysis of the genus Streptomyces, strain Sc-7 was identified as Streptomyces cinnamonensis.

[0088] Note: The *Streptomyces cinnamonensis* ZX6 described in the background section above showed that the diameter of the inhibition zone against Xoo and Xoc was less than 10 mm according to the Oxford cup method described above, indicating that it had virtually no inhibitory effect.

[0089] Example 2: Determination of the antibacterial spectrum of Streptomyces cinnamonensis Sc-7 strain

[0090] 1 strain

[0091] (1) Streptomyces cinnamonensis Sc-7 strain.

[0092] (2) Four representative plant pathogenic bacteria: Xanthomonas axonopodis pv. phaseoli (Xag), Pseudomonas syringaepv. glycinea (Psg), Xanthomonas axonopodis pv. glycines (Xag), and Dickeya zeae (Dz) of rice, are shown in Table 3. They were used to determine the antibacterial spectrum of the fermentation filtrate of strain Sc-7.

[0093] (3) Four representative plant pathogenic fungi: Alternaria solani (tomato early blight), Fusarium oxysporum f.sp. momordicae (bitter melon wilt), Fusarium solani (poplar wilt), and Colletetrichum orbiculare (watermelon anthracnose) were used to determine the antifungal spectrum of the fermentation filtrate of strain Sc-7.

[0094] 2 Culture media

[0095] (1) NA culture medium (base): 3g beef extract, 5g peptone, 5g NaCl, 1000mL water, pH 7.2 (solid culture medium with 20g agar), used for the culture of plant pathogenic bacteria.

[0096] (2) Gao's No. 1 culture medium: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 1000mL water, pH 7.2~7.4. Used for fermentation broth culture of Sc-7 strain.

[0097] (3) PDA solid culture medium: 200g potato, 20g glucose, 18g agar, 1000mL water, pH 6.0~6.5, used for the culture of plant pathogenic fungi.

[0098] 3 Experimental Methods

[0099] 3.1 Cultivation of Sc-7 strain and preparation of fermentation filtrate

[0100] One mycelial cake (5 mm in diameter) of strain Sc-7 was taken using a punch and inoculated into Gao's No. 1 culture medium at a volume of 50 / 250 mL, initial pH 7.3, and cultured at 160 rpm in a constant temperature shaker at 28 °C for 72 h to obtain the seed culture. The seed culture was then inoculated into another Gao's No. 1 culture medium at a 4% inoculation rate and fermented in a shaker for 7 days. The fermentation broth was then centrifuged in a 50 mL centrifuge tube at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane to remove residual spores, yielding the fermentation filtrate for later use.

[0101] 3.2 Activation of plant pathogenic bacteria and fungi

[0102] Four pathogenic bacteria were inoculated into NA medium and activated at 28°C for 2 days. They were then transferred to fresh NA medium and incubated at 28°C for 2 days for bacterial antibacterial spectrum determination.

[0103] Take a small amount of preserved fungal hyphae and inoculate them onto PDA medium. Incubate at 28°C for 5 days. When the hyphae are growing vigorously, take the edge hyphae and transfer them to a new PDA medium for 5 days of incubation.

[0104] 3.3 Determination of the antibacterial and antifungal spectrum of strain Sc-7

[0105] Four types of plant pathogenic bacteria that had already been activated and cultured were inoculated into NA culture medium and cultured on a shaker (160 r / min, 28℃). When the bacterial density reached OD... 600 When the concentration of bacterial culture reached 0.6, 5 mL of bacterial culture was added to 100 mL of NA solid medium and plated. Separately, 200 μL of the fermentation filtrate of strain Sc-7 was taken, and the inhibitory effect of the Sc-7 fermentation filtrate on four plant pathogenic bacteria was determined using the Oxford cup method.

[0106] Four types of pathogenic fungi were activated on PDA solid medium using sterile forceps and cultured at 28°C for 7 days for later use. A 5 mm actinomycete cake was placed upside down in the center of the PDA solid medium. Using Gao's No. 1 medium as a blank control, the pathogenic fungi cake was inoculated at the four corners about 5 cm away from the actinomycete cake. Each treatment was repeated 3 times and cultured at 28°C. The results were recorded when there was no obvious growth of pathogenic fungi.

[0107] Mycelial growth inhibition rate = (average diameter of control colonies – average diameter of treated colonies) / average diameter of control colonies × 100%

[0108] 4. Experimental Results

[0109] The antibacterial spectrum test results showed that strain Sc-7 had a certain inhibitory effect on four plant pathogenic bacteria: Xanthomonas axonopodis pv. phaseoli (Xap), Pseudomonas syringae pv. glycinea (Psg), Xanthomonas axonopodis pv. glycines (Xag), and Dickeya zeae (Dz), which causes bacterial blight of soybean (see Table 3).

[0110] Table 3. Inhibitory effects of Sc-7 strain against four representative plant pathogenic bacteria.

[0111]

[0112] Note: Different lowercase letters indicate significant differences between treatments, P < 0.05

[0113] The effects of strain Sc-7 on the growth of plant pathogenic fungi were determined using the plate confrontation method. The results are shown in Table 4. Strain Sc-7 exhibited inhibitory effects on all four tested plant pathogenic fungi. It showed particularly strong inhibitory effects against *Alternaria solani* (early blight pathogen of tomato) and *Fusarium oxysporum* f.sp. *momordicae* (wilt pathogen of bitter melon), with mycelial inhibition rates of 51.5% and 44.1%, respectively. The inhibition rates against *Fusarium solani* (wilt pathogen of poplar) and *Colletetrichum orbiculare* (anthracnose pathogen of watermelon) were 36.6% and 30.3%, respectively. These results further demonstrate that strain Sc-7 possesses a broad antifungal spectrum for controlling plant diseases.

[0114] Table 4. Inhibitory effects of Sc-7 strain against four representative plant pathogenic fungi.

[0115]

[0116] Note: Different lowercase letters indicate significant differences between treatments, P < 0.05

[0117] Example 3: Study on the control efficacy of Sc-7 strain fermentation broth against bacterial leaf blight and bacterial streak in rice.

[0118] 1. Pathogens and rice varieties

[0119] (1) Actinomycetes used for prevention: Streptomyces cinnamonensis Sc-7 strain.

[0120] (2) Pathogens: Xoo and Xoc.

[0121] (3) Rice variety: Zhenhui 084 (medium indica rice), from Jinhua Plant Protection Station, Zhejiang Province.

[0122] 2 Culture media

[0123] (1) Gao's No. 1 culture medium (base): 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 1000mL water, pH 7.4 (Gao's No. 1 solid culture medium with 20g agar), used for the preparation of fermentation broth for Sc-7 strain.

[0124] (2) NA medium: 3g beef extract, 5g peptone, 5g NaCl, 20g agar, 1000mL water, pH 7.2 (NA medium without agar). Used for the culture of Xoo and Xoc pathogens.

[0125] 3 Experimental Methods

[0126] 3.1 Rice cultivation

[0127] Select plump, regularly shaped Zhenhui 084 seeds, soak them in 2% sodium hypochlorite for 30 minutes, rinse with sterile water, germinate them in a plate containing sterile water, and then transfer them to seedling trays for cultivation. Let the rice seedlings grow in an artificial climate chamber (40,000 lx light for 16 hours, temperature 28℃; darkness for 8 hours, temperature 26℃), water them every two days, and change the nutrient solution once a week. When the rice leaves are more than 20 cm long, they are ready for use.

[0128] 3.2 Culture of Xoo and Xoc pathogens

[0129] Xoo and Xoc pathogens were inoculated separately onto NA medium plates and activated at 28°C for 2 days. Then, Xoo and Xoc were inoculated into NA medium and cultured for 2 days. The supernatant was discarded by centrifugation (12000 rpm, 5 min), and sterile water was added to prepare OD (diethyltoluene) solution. 600 Xoo and Xoc bacterial suspensions at a concentration of 0.6 were used for efficacy testing.

[0130] 3.3 Preparation of fermentation broth for strain Sc-7

[0131] Using a sterile punch with a diameter of 5 mm, holes were punched in the Sc-7 actinomycete plate to obtain bacterial blocks. These blocks were then inoculated into a 250 mL Erlenmeyer flask containing 100 mL of Gao's No. 1 culture medium. After culturing on a shaker at 160 r / min and 28 °C for 7 days, 100 mL of the fermentation broth was centrifuged at 12000 r / min for 10 min and filtered through a 0.22 μm filter membrane to obtain the fermentation filtrate for later use.

[0132] The fermentation filtrate was diluted with an equal volume of sterile water to obtain a 2-fold dilution of the Sc-7 strain fermentation solution.

[0133] 3.4 Pot control efficacy experiment of rice bacterial leaf blight (BBR) and rice bacterial leaf streak (BLS)

[0134] 3.4.1 Regarding rice bacterial blight (BBR):

[0135] (1) Sterile water control group (CK1): Leaves were cut using only sterile scissors dipped in sterile water, and the cut leaves were kept moist for 1 day after being bagged.

[0136] (2) Xoo control (CK2): Leaves were cut using sterile scissors dipped in Xoo bacterial suspension (OD600=0.6), and the cut leaves were bagged for 1 day to keep the cut leaves moist.

[0137] (3) Treatment 1 (T1): Use sterile scissors to dip the leaves in the Xoo bacteria suspension and cut them. After 3 hours, spray the Sc-7 strain 2 times fermentation dilution evenly on both sides of the leaves (dosage: 10 mL per plant) for a total of 3 times (1 time per hour). Cover the cut leaves with bags for 1 day to keep them moist.

[0138] (4) Treatment 2 (T2): First, spray the Sc-7 strain 2 times fermentation dilution evenly on the front and back of the leaves (dosage: 10 mL per plant), spray 3 times in total (1 time per hour). After 3 hours, use sterile scissors to dip the Xoo strain suspension to cut the leaves, and cover the cut leaves with a bag for 1 day to keep the cut leaves moist.

[0139] (5) Treatment 3 (T3): First, spray the 1000-fold dilution of 25% pyraclostrobin evenly on both sides of the leaves (dosage: 10 mL per plant), spray 3 times in total (1 spray per hour). After 3 hours, use sterile scissors to dip in the Xoo bacteria suspension to cut the leaves, and cover the cut leaves with a bag for 1 day to keep the cut leaves moist.

[0140] Five plants were used in each treatment, with eight leaves (isotopic leaves) per plant, and each treatment was repeated three times.

[0141] 3.4.2 Targeting Bacterial Leaf Streak (BLS) in Rice

[0142] The potted plant control treatment is similar to that for rice bacterial blight mentioned above, except that the Xoo bacterial suspension in "3.4.1" is replaced with Xoc bacterial suspension.

[0143] When the disease stops progressing, the incidence rate is recorded. Disease severity is assessed on a leaf-by-leaf basis, with rice bacterial blight classified into 0-9 levels.

[0144] Grade 0: No lesions at the cut; Grade 1: Lesions covering less than 10% of the leaf area; Grade 3: Lesions covering 11%–25% of the leaf area; Grade 5: Lesions covering 26%–45% of the leaf area; Grade 7: Lesions covering 46%–65% of the leaf area; Grade 9: Lesions covering more than 65% of the leaf area.

[0145] Rice bacterial leaf streak disease is classified into 0-10 levels:

[0146] Grade 0: No lesions on leaves; Grade 1: Lesions less than 10% of the total leaf length; Grade 2: Lesions 10%–20% of the total leaf length; Grade 3: Lesions 20%–30% of the total leaf length; Grade 4: Lesions 30%–40% of the total leaf length; Grade 5: Lesions 40%–50% of the total leaf length; Grade 6: Lesions 50%–60% of the total leaf length; Grade 7: Lesions 60%–70% of the total leaf length; Grade 8: Lesions 70%–80% of the total leaf length; Grade 9: Lesions 80%–90% of the total leaf length; Grade 10: Lesions more than 90% of the total leaf length.

[0147] Based on the severity of the disease, the disease index and control effectiveness are calculated using the following formula:

[0148] Disease index = ∑(number of diseased leaves at each level × corresponding level value) / (total number of leaves surveyed × highest level representative value) × 100

[0149] Relative control effect (%) = [(disease index in control area - disease index in treatment area) / disease index in control area] × 100;

[0150] Lesion inhibition rate (%) = (CK2 lesion length - treated lesion length) / CK2 lesion length × 100

[0151] 4 Experimental Results

[0152] Through pot experiment on rice control efficacy (Table 5 and...) Figure 8 It was found that for rice bacterial leaf blight and bacterial leaf streak, both spraying with a 2-fold dilution of Sc-7 strain before inoculation and inoculating with the pathogen before spraying the fermentation broth showed good control effects, with relative control efficacy and lesion inhibition rate both exceeding 70%, which was superior to the control effect of spraying the chemical pesticide pyraclostrobin. The preventive effect was better than the control effect.

[0153] Table 5. Disease index and relative control efficacy of Sc-7 strain fermentation broth against rice bacterial blight and bacterial leaf streak.

[0154]

[0155]

[0156] Note: CK1: sterile water; CK2: Xoo or Xoc; T1: 2x (1 / 2) fermentation broth for treatment; T2: 2x (1 / 2) fermentation broth for prevention; T3: pyraclostrobin (diluted 1000 times) for prevention. Different lowercase letters indicate significant differences between treatments, P < 0.05.

[0157] Example 4: Effects of Sc-7 strain fermentation broth on rice seed germination and seedling growth promotion

[0158] 1. Rice variety: Zhenhui 084 (medium-grain indica rice), from Jinhua Plant Protection Station, Zhejiang Province.

[0159] 2 Culture media

[0160] Gao's No. 1 culture medium (base): 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl, 0.01g FeSO4·7H2O, 1000mL water, pH 7.4 (Gao's No. 1 solid culture medium with 20g agar), used for the culture and fermentation broth preparation of Sc-7 strain.

[0161] 3 Experimental Methods

[0162] 3.1 Preparation of fermentation broth for strain Sc-7

[0163] Using a sterile punch with a diameter of 5 mm, holes were punched in the actinomycete plate to obtain bacterial blocks. These blocks were then inoculated into 250 mL Erlenmeyer flasks containing 100 mL of Gao's No. 1 liquid culture medium. The flasks were incubated on a shaker at 160 rpm and 28 °C for 7 days to obtain the fermentation broth (Sc-7 fermentation stock broth) for later use. The fermentation broth was diluted 10, 50, and 100 times with sterile water and stored at 4 °C for later use.

[0164] 3.2 Rice seed germination treatment

[0165] Select plump rice seeds and soak them in sterile water at 28℃ for 24 hours to promote germination.

[0166] 3.3 Effects of Fermentation Broth Soaking on Rice Seed Germination

[0167] Select plump, regularly shaped seeds, soak them in 2% sodium hypochlorite solution for 30 minutes, rinse with sterile water, and place them in petri dishes. Add equal volumes of sterile water, Gao's No. 1 culture medium, Sc-7 fermentation stock solution, and 10x, 50x, and 100x dilutions respectively for soaking. After 1 day, pour off the liquid, wash with sterile water, and germinate in a light incubator. Change the sterile water daily. Seeds showing signs of germination (white sprouts) are used as the criterion for judging germination. Germination potential is measured on day 3; germination rate is measured after day 8. Sprout and root lengths are measured using vernier calipers. Each dish contains 50 seeds, with 3 dishes per treatment, and the experiment is repeated 3 times.

[0168] The formula is as follows:

[0169] Germination potential (%) = (Number of seeds germinated in 3 days / Number of seeds tested) × 100;

[0170] Germination rate (%) = (Number of seeds germinated in 7 days / Number of seeds tested) × 100

[0171] 3.4 Effects of Fermentation Liquid Irrigation on Rice Seedling Growth

[0172] The cultivation method is the same as in 3.3. When the seedlings grow to 15cm, select seedlings with uniform growth and transplant them into flowerpots filled with sterilized soil, with 3 seedlings per pot and 8 pots per treatment. Keep the soil moist by spraying water in the morning and evening. Every 5 days, treat the seedlings with 200mL of sterile water, 10-fold and 50-fold fermentation dilution of Sc-7 strain. After 30 days, remove the rice plants and measure their plant height, root length and other morphological indicators.

[0173] 4 Experimental Results

[0174] 4.1 Effects of Fermentation Broth Soaking on Rice Seed Germination

[0175] The experimental results are shown in Table 6: Both 50-fold and 100-fold dilutions of the fermentation solution improved germination potential, while the germination rates of the other treatment groups showed no significant changes compared to the control group. Among the treatment groups, the original fermentation solution, 10-fold, and 50-fold dilutions all increased the sprout and root length of rice seeds. The 10-fold and 50-fold dilutions showed the most significant effects. In terms of sprout length, the 10-fold and 50-fold dilutions increased the average sprout length by 37.26% and 65.54%, respectively; in terms of root length, the 10-fold and 50-fold dilutions increased the average root length by 176.9% and 194.53%, respectively. Figure 9 ).

[0176] Table 6. Effects of Sc-7 strain fermentation broth on rice seed germination (experiment)

[0177]

[0178] Note: CK1: Sterile water; CK2: Gao's No. 1 culture medium; T1: Fermentation stock broth; T2: 10-fold fermentation dilution; T3: 50-fold fermentation dilution; T4: 100-fold fermentation dilution. Different lowercase letters indicate significant differences between treatments (P < 0.05).

[0179] 4.2 The growth-promoting effect of Sc-7 strain fermentation broth irrigation on rice seedling growth

[0180] Experimental results are as follows Figure 10 As shown, irrigation with a 10-fold dilution of Sc-7 strain fermentation solution increased rice seedling height by 20.2%, root length by 72.13%, above-ground fresh weight and root fresh weight by 70.58% and 107.7% respectively, and above-ground dry weight and root dry weight by 93.63% and 53.08% respectively. Rice seedlings treated with the fermentation solution were thicker and had more differentiated and developed root systems.

[0181] Note: The Streptomyces cinnamonensis ZX6 described in the background section above was tested for its growth-promoting effect on rice seedlings as described above, and it was found that it had almost no effect on the height of rice seedlings.

[0182] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Streptomyces cinnamon ( Streptomyces cinnamonensis Sc-7, characterized by: The accession number is CCTCC NO: M 20231179.

2. Streptomyces cinnamon ( Streptomyces cinnamonensis The purpose and characteristics of Sc-7 are: For rice bacterial blight ( Xanthomonas oryzae pv oryzae ) and rice bacterial leaf streak ( Xanthomonas oryzae pv .oryzicola It has an antagonistic effect.

3. The Streptomyces cinnamon according to claim 2 ( Streptomyces cinnamonensis The purpose and characteristics of Sc-7 are: It has an inhibitory effect on plant pathogenic bacteria and plant pathogenic fungi; The plant pathogenic bacteria is: *Phytophthora bean* (a bacterium). Xanthomonas axonopodis pv . phaseoli ), soybean bacterial spot disease ( Pseudomonas syringae pv. glycinea ), soybean bacterial spot disease ( Xanthomonas axonopodis pv. glycines ) and bacterial basal rot of rice ( Dickeya zeae) ; The plant pathogenic fungus is *Phytophthora indicum*, also known as the tomato early blight pathogen. Alternaria solani ), Fusarium wilt of bitter melon ( Fusarium oxysporum f. sp. momordicae ), Poplar wilt pathogen ( Fusarium solani ) and watermelon anthracnose fungus ( Colletetrichum orbiculare ).

4. The Streptomyces cinnamon according to claim 2 or 3 ( Streptomyces cinnamonensis The purpose and characteristics of Sc-7 are: It has a growth-promoting effect on rice.

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