Biocontrol streptomyces ArbiCoenn strain and application thereof

Abstract

The invention relates to a biocontrol streptomyces ArbiCoenn strain and application thereof. The preservation number of the biocontrol streptomyces ArbiCoenn strain is CGMCC No.34660, and the biocontrol streptomyces ArbiCoenn strain has a relatively strong inhibition effect on fusarium solani and can be used for preventing and treating sugarcane top rot or leaf blight.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and specifically relates to a biocontrol strain of Streptomyces abigoon and its application. Background Technology

[0002] Streptomyces abicorensis is a microorganism with significant biocontrol potential, belonging to the genus Streptomyces (Streptomyces). Streptomyces Streptomyces are the most antibiotic-producing group among actinomycetes, possessing broad-spectrum antibacterial activity. Chinese patent CN113801808A discloses a strain of *Streptomyces abigoon*. Streptomyces abikoensis ), against Fusarium solani ( Fusarium solani This bacterium has a strong inhibitory effect on root rot caused by this bacterium. Using the bacterial solution prepared by this bacterium to control root rot in oil peony can achieve a good control effect, and at the same time it can also promote the growth of oil peony plants.

[0003] Therefore, as a biocontrol bacterium with broad application prospects, exploring new applications of Streptomyces abigoon in the agricultural field is expected to provide new ideas and methods for solving the problems of disease control and drug-resistant bacteria. Summary of the Invention

[0004] The purpose of this invention is to provide a biocontrol strain of Streptomyces abicorne with broad-spectrum resistance to a variety of plant pathogens, in order to supplement existing biocontrol strain resources.

[0005] This invention is achieved through the following technical solution: This invention provides a biocontrol strain of Streptomyces abigoni, which was deposited at the China Microbial Culture Collection Center on May 23, 2025, with accession number CGMCC No. 34660.

[0006] The aforementioned biocontrol agent, Streptomyces abigoni, inhibits Fusarium rot (… Fusarium sacchari ) and / or Tainan Chilospora ( Stagonospora tainanensis Its application in the preparation of products that inhibit Fusarium rot ( ) or in the preparation of products that inhibit Fusarium rot ( ) Fusarium sacchari ) and / or Tainan Chilospora ( Stagonospora tainanensis Applications in products.

[0007] The above-mentioned biocontrol agent Streptomyces abigonis is used in the control of plant shoot rot and / or plant leaf blight.

[0008] Furthermore, the pathogen causing the top rot is *Fusarium solani* (also known as the bark rot fungus). Fusarium sacchari The pathogen causing leaf blight is *Polyspora tainanensis* (…). Stagonospora tainanensis ).

[0009] Furthermore, the plant in question is sugarcane.

[0010] Furthermore, the application involves spraying plants with the aseptic fermentation broth of the aforementioned Streptomyces abigoonii.

[0011] Furthermore, the application involves spraying plants with an extract from the sterile fermentation broth of the Streptomyces abicornis.

[0012] Furthermore, the extract of the sterile fermentation broth is at least one of lipopeptides and crude protein extracted from 75% ammonium sulfate.

[0013] Furthermore, the sterile fermentation broth is obtained through culture medium cultivation. The culture medium may be a medium containing soybean flour, corn flour, glucose, ammonium sulfate, and calcium carbonate; or a medium containing soybean flour, soluble starch, yeast extract, peptone, sodium chloride, and calcium carbonate; or a medium containing soluble starch, glycerol, corn starch, peptone, yeast extract, NaCl, and CaCO3; or a medium containing soluble starch, beef extract, glucose, yeast extract, peptone, and CaCO3; or a medium containing soluble starch, soybean flour, yeast extract, and CaCO3; or a medium containing soybean flour, soluble starch, NaCl, and CaCO3; or a medium containing soluble starch, soybean flour, yeast extract, NaCl, and CaCO3; or a medium containing Gao's No. 1 liquid medium, PDW medium, or LB medium.

[0014] Preferably, the culture medium comprises soybean flour, soluble starch, NaCl, and calcium carbonate. More preferably, each 1 L of the culture medium contains 10-20 g of soybean flour, 10-30 g of soluble starch, 3-5 g of NaCl, 3-5 g of CaCO3, and the remainder is water. In a specific preferred embodiment, the aseptic fermentation broth is obtained through culture medium F, the specific formula of which is: 15 g of soybean flour, 20 g of soluble starch, 4 g of NaCl, 4 g of CaCO3, with deionized water added to 1 L, achieving an inhibition rate of up to 95.78% against Fusarium rotundus.

[0015] Preferably, the fermentation temperature is 24-36 °C. In some preferred embodiments, the fermentation temperature is 32 °C or 36 °C, and the inhibition rate of Fusarium rotundus after filtration is higher than the initial inhibition rate.

[0016] Preferably, the shaking speed is 160 rpm to 220 rpm. In some preferred embodiments, the shaking speed is ≥180 rpm, such as 180 rpm, 200 rpm, or 220 rpm, and the inhibition rate of the fermentation broth against *Fusarium solani* is higher than 80%. Further, in some embodiments, when the shaking speed is 200 rpm, the inhibition rate against *Fusarium solani* can reach 92%.

[0017] Preferably, the fermentation initiation pH is 7-11; the strains of the present invention can produce antibacterial substances under pH conditions of 3.0-12.0. However, at a pH of 7.0-11.0, the sterile filtrate exhibits an inhibition rate of over 40% against Fusarium rotundus.

[0018] Preferably, the fermentation time is 7-9 days. For example, the fermentation time is 7 days or 8 days, etc.

[0019] The present invention has the following advantages and effects: 1. The aseptic fermentation broth of the biocontrol strain of Streptomyces abigonis of the present invention has a good inhibitory effect on Fusarium solani and Chilodonella tainanensis, and can be used to develop related preparations to inhibit Fusarium solani or Chilodonella tainanensis.

[0020] 2. Based on the principle of culture medium optimization, this invention optimizes the fermentation culture medium obtained by screening by optimizing factors such as fermentation temperature, rotation speed, initial pH, liquid volume, and culture days, providing a more suitable nutrient environment for growth, reproduction, and production of active substances. The optimal fermentation conditions are further verified by using response surface methodology to improve the antibacterial ability of the fermentation broth.

[0021] 3. This invention studies the active ingredients of the sterile fermentation broth of the biocontrol strain Streptomyces abigoon, providing a foundation for the effective use of it for microbial control and the development of related formulations.

[0022] 4. The biocontrol strain of Streptomyces abigonis of the present invention can be used to control shoot rot, leaf blight, etc., and has a good prospect for development and application in the field of microbial pesticides. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the gene sequencing alignment results of strain RT-W-1-C; Figure 2 The plate inhibition effect of RT-W-1-C on Fusarium solani and Chilodonella tainanensis; Figure 3 The plate inhibition effect of fermentation filtrate of RT-W-1-C on Fusarium solani in different fermentation media was investigated. Figure 4 The inhibitory effect of different amounts of fermentation broth added on Fusarium solani was investigated. Figure 5The effect of fermentation temperature on the antibacterial activity of antagonistic strains in fermentation broth; Figure 6 The effect of liquid volume on the antibacterial activity of antagonistic bacterial strain fermentation broth; Figure 7 The effect of shaker speed on the antibacterial activity of antagonistic bacterial strain fermentation broth; Figure 8 The inhibitory effect of different amounts of fermentation broth added on Fusarium solani was investigated. Figure 9 The effect of fermentation time on the antibacterial activity of antagonistic strain fermentation broth; Figure 10 The effect of initial pH on the antibacterial activity of antagonistic strain fermentation broth; Figure 11 Comparison of the inhibition effect of fermentation broth on plate before and after single-factor optimization; Figure 12 These are the results of the PB experiment; Figure 13 The plate inhibition effect of various extracted active substances on Fusarium solani; Figure 14 The plate inhibition effect of fermentation broth with different addition amounts on the pathogens of sugarcane top rot and leaf blight was investigated. Figure 15 The effects of fermentation broths with different dilution ratios on the in vitro control of sugarcane top rot were investigated. Figure 16 The in vivo control efficacy of fermentation broths with different dilution ratios against sugarcane top rot was investigated. Figure 17 The study aimed to assess the in vivo control effects of various active substances on sugarcane top rot. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0025] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the protection scope of the present invention.

[0026] Example 1: Isolation and identification of the biocontrol strain Streptomyces abigoon RT-W-1-C (CGMCC No. 34660) This strain was isolated from the rhizosphere soil of *Passiflora edulis* in Maotang, Qinpai Village, Baiwei Town, Shanglin County, Nanning City.

[0027] 2.1. Strain culture Streptomyces RT-W-1-C were placed in PDW medium and cultured at 28℃ and 200 r / min until the logarithmic growth phase. After centrifugation at 4000 r / min for 10 min, the bacterial cells were collected, and a small amount of water was absorbed with sterile absorbent paper. The cells were then placed in cryovials and immediately flash-frozen in liquid nitrogen for 2-3 hours. They were stored at -80℃ and sent to Wuhan Bena Technology Co., Ltd. for sequencing.

[0028] 2.2. Sequencing, assembly, and annotation of the genome The sequencing for this study was commissioned to Wuhan Bena Technology Co., Ltd. Genomic DNA was extracted using the SDS method. After passing quality checks, a DNA library was constructed. Third-generation and second-generation genome sequencing were performed sequentially using Nanopore and NovaSeq 6000 (Illumina, USA) sequencing platforms. Nanopore data was used to assemble a high-quality bacterial genome contig, while Illumina second-generation data was used to correct errors in the assembly results, resulting in a high-quality assembled genome and effectively avoiding sequence contamination introduced by Nanopore data splitting errors. After sequencing, the raw data underwent quality control, filtering out low-quality and excessively short reads. The filtered reads were then assembled de novo, and the assembled draft genome was corrected for errors. Simultaneously, structural analysis and functional annotation of the assembled genome were performed using relevant databases.

[0029] 2.3 Functional annotation analysis of the whole genome of Streptomyces To obtain comprehensive gene function information, eight major databases were used for gene function annotation, including: UniProt (Universal Protein), KEGG Pathway (Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), Pfam (Protein families database), COG (Cluster of Orthologous Groups of proteins), TIGERfam, NR (Non-Redundant Protein Sequence Database), and RefSeq (Reference Sequences). The predicted gene sequences were BLAST+ (Version: 2.11.0+) aligned with functional databases such as COG, KEGG, UniProt, and RefSeq to obtain gene function annotation results. The software hmmer (Version: 3.3.2) was used for functional annotation based on databases such as Pfam and TIGERfam.

[0030] The 16S sequencing results are as follows: BLAST comparison analysis showed that this bacterium was similar to... Streptomyces abikoensis (NR 118287.1) shows the highest homology of up to 97%, and is related to Streptomyces abikoensi s(OK 576708.1) has a homology of up to 96% ( Figure 4 Based on the above characteristics, strain RT-W-1-C was named *Streptomyces abicornis*. Streptomyces abikoensis This strain was deposited on May 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101), with accession number CGMCC No. 34660.

[0031] Example 2 Antibacterial Experiment Fusarium solani was used in the antibacterial experiment. Fusarium sacchari Tainan Chilospora ( Stagonospora tainanensis (This information comes from the Guangxi Key Laboratory of Sugarcane Biology, College of Agriculture, Guangxi University.)

[0032] 2.1 Culture medium Potato dextrose agar (PDA): 6 g potato starch, 20 g anhydrous glucose, 20 g agar, add deionized water to 1 L.

[0033] Potato glucose water culture medium (PDW): 6 g potato flour, 20 g anhydrous glucose, add deionized water to 1 L.

[0034] Luria-Bertani medium (LB for short): 10 g tryptone, 5 g yeast extract, 10 g NaCl, add deionized water to 1 L. (LB solid medium is LB medium with 18 g agar added.) 2.2 Antibacterial test Take fungal discs (Ф=6 mm) cultured for 5-7 days and inoculate them in the center of PDA medium. Place three 6 mm diameter rhizosphere fungal discs 2.2 cm to each side of the central pathogen (three discs on the same line). The control group consists of PDA plates with pathogen discs only in the center. Each treatment is repeated three times. Incubate at 28℃ for 9 days (mainly to observe whether the fungus in the control group has completely covered the entire plate; if so, take a picture). Measure the colony diameter of the pathogen using the cross-sectional method.

[0035] The inhibition rate (I%) is calculated using the following formula: I% = (CT) / C × 100% Where I represents the inhibition rate, C represents the lateral growth of the control group, and T represents the lateral growth of the experimental group.

[0036] 2.3. Results: like Figure 2 As shown, the biocontrol bacterium RT-W-1-C isolated in Example 1 has an antagonistic effect of more than 50% against Fusarium solani and Chilodonella tainanensis.

[0037] Example 3: Optimization of fermentation conditions for RT-W-1-C strain

[0038] 3.1 Preparation of RT-W-1-C seed culture Take 5 RT-W-1-C mycelial cakes that have been cultured for 5-7 days and place them in 150 mL LB, then place them in a shaker at 28℃ and 200 rpm for 24 h to obtain RT-W-1-C seed culture.

[0039] 3.2 Screening of the optimal fermentation medium 500 μL of seed culture was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of each of the 10 different fermentation media, and cultured with shaking at 200 r / min and 28℃ for 7 days. After 7 days, the fermentation broth was centrifuged at 4℃ and 10000 r / min for 10 min to obtain the supernatant, which was then filtered once through a 0.22 μm bacterial filter to obtain the fermentation filtrate. The fermentation filtrate was mixed with PDA medium at approximately 60℃ at a volume fraction of 10%, and 15 mL was poured into each petri dish. For the control, different unshaken fermentation media were mixed with PDA medium at approximately 60℃ at a volume fraction of 10%, and 15 mL was poured into each petri dish. Each treatment was repeated in triplicate. After cooling, a 6 mm Fusarium rotundifolia K5-3 mycelial cake was inoculated into the center of the plate. The plates were incubated at 28℃ for 9 days, and the diameter of the pathogen colonies was measured using a cross-sectional method to calculate the inhibition rate.

[0040] The culture medium AI formulation is as follows: Fermentation medium A: 20 g soybean flour, 30 g corn flour, 20 g glucose, 4 g ammonium sulfate, 4 g calcium carbonate, add deionized water to 1 L.

[0041] Fermentation medium B: 15 g soybean flour, 20 g soluble starch, 5 g yeast powder, 2 g peptone, 4 g sodium chloride, 4 g calcium carbonate, add deionized water to 1 L. Fermentation medium C: 10 g soluble starch, 10 mL glycerol, 2.5 g corn starch, 5 g peptone, 2 g yeast powder, 1 g NaCl, 4 g CaCO3, add deionized water to 1 L.

[0042] Fermentation medium D: 15 g soluble starch, 2 g beef extract, 1 g glucose, 3 g yeast powder, 2 g peptone, 3 g CaCO3, plus deionized water to 1 L.

[0043] Fermentation medium E: 25 g soluble starch, 15 g soybean flour, 2 g yeast powder, 4 g CaCO3, add deionized water to 1 L.

[0044] Fermentation medium F: 15 g soybean flour, 20 g soluble starch, 4 g NaCl, 4 g CaCO3, plus deionized water to 1 L.

[0045] Fermentation medium G: 20 g soluble starch, 15 g soybean flour, 5 g yeast powder, 4 g NaCl, 4 g CaCO3, add deionized water to 1 L.

[0046] Fermentation medium H: Gao's No. 1 liquid medium: soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, pH 7.2, deionized water to a final volume of 1 L.

[0047] Fermentation medium I: PDW medium: PDW powder.

[0048] Fermentation medium J:LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and deionized water to a final volume of 1000 mL.

[0049] The inhibition rate (I%) is calculated using the following formula: I% = (CT) / (C - 0.5) × 100% Where I represents the inhibition rate, C represents the lateral growth of the control group, and T represents the lateral growth of the experimental group.

[0050] The results of the antibacterial rate of different culture media are as follows: Figure 3 As shown, It can be seen that medium F is the optimal fermentation medium for RT-W-1-C. Its inhibition rate is as high as 95.78%. To adapt to future production applications and to better study its antagonistic mechanism, its addition amount will be reduced for further optimization.

[0051] 3.3 Effects of different amounts of fermentation broth added on Fusarium solani K5-3 750 μL of seed culture was added to 150 mL of fermentation medium F. After shaking at 28℃ and 200 rpm for 7 days, the fermentation broth was obtained. After centrifugation for 10 min, the supernatant was filtered through a 0.22 μm bacterial filter to obtain sterile fermentation broth. The control group consisted of 15 mL of PDA inoculated with *Fusarium solani* cultured for 5-7 days in the center of a petri dish. The experimental groups consisted of 250 μL, 500 μL, 750 μL, 1 mL, 1.25 mL, and 1.5 mL of sterile fermentation broth mixed with PDA to form 15 mL of PDA containing sterile fermentation broth. The plates were then inoculated, and *Fusarium solani* cultured for 5-7 days was inoculated again in the center of the petri dish. Each group was repeated in triplicate. After incubation at 28℃ for 9 days, the colony size was measured and photographed, using the same method as in section 3.2.

[0052] The results are as follows Figure 4 As shown, the inhibition rates were all higher than 60% when the addition amounts were 750 μL, 1 mL, 1.25 mL, and 1.5 mL. The PDA culture dish containing 500 μL of sterile fermentation broth showed an inhibition rate of 52.2% against Fusarium rotundus. To better observe the subsequent optimization effect, an addition amount of 500 μL of sterile fermentation broth was used to optimize the fermentation conditions.

[0053] 3.4 Effect of fermentation temperature on the antibacterial activity of antagonistic strains' fermentation broth The optimal fermentation medium selected was used as the test medium. 750 μL of seed culture was inoculated into 250 mL Erlenmeyer flasks containing 150 mL of the optimal fermentation medium. After shaking for 7 days at 24℃, 28℃, 32℃ and 36℃ at 200 rpm, the antibacterial activity of the crude fermentation extracts obtained at different fermentation temperatures was measured. The measurement method was the same as in 3.2.

[0054] The results are as follows Figure 5 As shown, it can be seen that the fermentation broth produced by strain RT-W-1-C at these four temperatures showed higher inhibition rates against Fusarium rotundifolia after filtration only at fermentation temperatures of 32℃ and 36℃ compared to the initial inhibition rate. Among them, the inhibition rate of the fermentation broth at 32℃ was 69.45%, so subsequent fermentation conditions were optimized at this temperature.

[0055] 3.5 Effect of liquid volume on the antibacterial activity of antagonistic strain fermentation broth The optimal liquid culture medium selected during the experiment was used as the test medium. The optimal fermentation medium was set to be 50 mL, 75 mL, 100 mL, 125 mL and 150 mL. The seed liquid was inoculated at a rate of 0.5% and fermented in a shaker at 32℃ and 200 rpm for 7 days. The antibacterial activity of the crude fermentation extract obtained with different medium loading was measured. The measurement method was the same as in 3.2.

[0056] The results are as follows Figure 6 As shown in the results, the inhibitory effect of the aseptic fermentation broth produced by strain RT-W-1-C on Fusarium solani increases with decreasing liquid volume. When the liquid volume is 50 mL, the inhibition rate of the aseptic fermentation broth against Fusarium solani increases to 84.05%. Therefore, a liquid volume of 50 mL was used for subsequent optimization of fermentation conditions.

[0057] 3.6 Effect of shaking speed on the antibacterial activity of antagonistic strains' fermentation broth The optimal fermentation medium selected during the experiment was used as the test medium. 250 μL of seed culture was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of the optimal fermentation medium. The flasks were shaken at 32 °C for 7 days at rotation speeds of 140 rpm, 160 rpm, 180 rpm, 200 rpm and 220 rpm, respectively. The antibacterial activity of the crude fermentation extracts obtained at different rotation speeds was then measured using the same method as in 3.2.

[0058] The results are as follows Figure 7 The results showed that when the shaking speed was 180 rpm, 200 rpm, and 220 rpm, the inhibition rate of the antagonistic bacteria in the fermentation broth was higher than 80%. Among them, the inhibition rate was the highest at 200 rpm, reaching 92%. Therefore, a shaking speed of 200 rpm was selected for further optimization of fermentation conditions.

[0059] 3.7 Effect of a second reduction in fermentation broth addition on the antibacterial activity of the antagonistic strain's fermentation broth The optimal fermentation medium selected through screening was used as the test medium. After shaking at 32℃ and 200 rpm for 7 days, the fermentation broth was obtained. After centrifugation for 10 min, the supernatant was filtered through a 0.22 μm bacterial filter to obtain sterile fermentation broth. The control group consisted of 15 mL of PDA followed by inoculation of *Fusarium solani* cultured for 5-7 days into the center of a petri dish. The experimental groups consisted of 31.3 μL, 62.5 μL, 125 μL, 250 μL, and 500 μL of sterile fermentation broth mixed with PDA to prepare 15 mL of PDA containing sterile fermentation broth. The plates were then inoculated, and *Fusarium solani* cultured for 5-7 days was inoculated into the center of each petri dish. Each group was repeated in triplicate. After incubation at 28℃ for 9 days, the colony size was measured and photographed.

[0060] The results are as follows Figure 8 The results showed that when the added amount was 250 μL, the inhibition rate was 46.78%. Further reduction in the added amount resulted in an inhibition rate below 30%, which was not conducive to subsequent optimization experiments. Therefore, an added amount of 250 μL was chosen for subsequent optimization.

[0061] 3.8 Effect of fermentation time on the antibacterial activity of antagonistic strains' fermentation broth The optimal fermentation medium selected during the experiment was used as the test medium. 250 μL of seed culture was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of the optimal fermentation medium, and then placed in a shaker at 32℃ and 200 r / min for 2 d, 3 d, 4 d, 5 d, 6 d, 7 d and 8 d respectively. The antibacterial activity of the sterile fermentation filtrate obtained after different shaking times against F. solani was measured.

[0062] The results are as follows Figure 9 The results showed that the antibacterial activity of the sterile fermentation broth produced by the antagonistic strains increased with fermentation time, and the increase in inhibition rate tended to plateau at fermentation times of 7 and 8 days. Therefore, to save production costs, a fermentation time of 7 days was selected for further optimization.

[0063] 3.9 Effect of initial pH on the antibacterial activity of antagonistic strain fermentation broth The optimal fermentation medium selected was used as the test medium. 250 μL of seed liquid was inoculated into 250 mL Erlenmeyer flasks containing 50 mL of the optimal fermentation medium adjusted to pH 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 respectively. The antibacterial activity of the crude extract at different initial pH was measured after the optimal fermentation temperature, optimal shaking speed and optimal fermentation time.

[0064] The results are as follows Figure 10 The results showed that strain RT-W-1-C exhibited strong pH adaptability, producing antibacterial substances under pH conditions ranging from 3.0 to 12.0. At pH values ​​of 7.0 to 11.0, the sterile filtrate showed an inhibition rate of over 40% against *Fusarium solani*, indicating that strain RT-W-1-C prefers an alkaline environment and has a strong tolerance to extreme alkalinity. The sterile filtrate showed the best inhibitory effect against *Fusarium solani* at an initial pH of 9.0, with an inhibition rate of 57.49%. Therefore, pH 9.0 was used as the initial pH for subsequent experiments.

[0065] Example 4: Changes in inhibition rate before and after single-factor optimization of fermentation conditions for RT-W-1-C strain Take 750 μL of seed culture and add it to 150 mL of fermentation medium F. After shaking in a shaker at 28℃ and 200 rpm for 7 days, the fermentation broth is obtained. After centrifugation for 10 min, the supernatant is filtered through a 0.22 μm bacterial filter to obtain sterile fermentation broth.

[0066] After single-factor optimization, the culture medium was prepared in a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium F at pH 9. After shaking in a shaker at 32℃ and 200 rpm for 7 days, the fermentation broth was centrifuged for 10 min, and the supernatant was filtered through a 0.22 μm bacterial filter to obtain sterile fermentation broth.

[0067] The experimental group consisted of 250 μL of sterile fermentation broth mixed with PDA to make 15 mL of PDA containing sterile fermentation broth. The mixture was then poured onto a petri dish, and Fusarium solani cultured for 5-7 days was inoculated into the center of the dish. The control group consisted of 1.5 mL of fermentation medium mixed with PDA and poured onto a petri dish. Each group was repeated in triplicate. After incubation at 28℃ for 9 days, colony size was measured and photographed. (Images are shown below.) Figure 11 As shown in Table 1, after single-factor optimization, the inhibition rate was 30.45% higher than before optimization.

[0068] Table 1. Inhibition rate of fermentation broth before and after single-factor optimization

[0069] Example 5: Response surface methodology optimization of fermentation conditions for RT-W-1-C strain.

[0070] 5.1 Plackett-Burman experimental design In the Plackett-Burmam experimental design (PB design, refer to Tables 2 and 3), each factor was assigned two levels, coded as -1 and 1. The low level represented the original culture conditions, and the high level was 1.25 times the low level. A Plackett-Burmam experimental design with N=12 trials was set up, with A, B, C, D, and E representing time (h), rotation speed (r / min), inoculum size (%), temperature (°C), sample volume (mL / 250 mL Erlenmeyer flask), and pH, respectively.

[0071] The Lenth method was used to identify significant effects, and regression analysis was performed on the results (Table 4). The resulting linear regression equation was: Y = 60.81 + 3.64A + 0.67767B - 0.0433C + 2.59D - 3.62E - 2.46AB + 2.56BD - 2.42BE + 1.39DE. The analysis showed that the model p-value was 0.0154 < 0.05, reaching a significant level. The model coefficient of determination R² = 0.9966, indicating good correlation. The corrected coefficient of determination Radj² = 0.9811, indicating that 98.11% of the variability in the experimental data could be explained by this regression model. Furthermore, generally, the lower the coefficient of variation (CV), the higher the reliability and accuracy of the experiment. The CV value of the RT-W-1-C strain was 1.56%, indicating good reliability and accuracy of the Plackett-Burman experiment. Precision, defined as the ratio of effective signal to noise (Adeq Precisior), is considered reasonable if it is greater than 4.0. The experimental precision of strain RT-W-1-C reached 25.9196 (Table 4). The results show that... Figure 12 According to Table 3, the fermentation conditions that significantly affect the production of active substances by the RT-W-1-C strain are fermentation temperature, initial pH, and fermentation time.

[0072] Table 2. Plackett-Burman Experimental Factor Level Design

[0073] Table 3. Plackett-Burma Experimental Design and Results

[0074] Table 4 Regression Analysis of Culture Conditions

[0075] 5.2 Experimental Design of the Steepest Climbing Path Based on the evaluation effect of the Plackett-Burman design, the optimal values ​​of key factors were determined: the gradient direction of the experimental value change was taken as the climbing direction, and the climbing direction of other factors was determined according to the sign of the effect coefficient. Higher values ​​were selected for positive coefficients and lower values ​​were selected for negative coefficients. At the same time, the antibacterial activity of the crude extract of fermentation broth under different treatments was measured to determine the center point of the key factors.

[0076] Based on the Plackett-Burman experiment, the significant factors were screened, and the direction and step size of change were designed according to the effect coefficients of each factor and the actual situation. The steepest climbing experiment was conducted by selecting high and low levels according to the positive and negative effects. Among them, taking into account factors such as cost and workload, the culture medium formula of RT-W-1-C was fixed, the liquid volume was fixed at 62.5 mL, and the shaking speed was fixed at 250 r / min.

[0077] The regression equation from RT-W-1-C: Given Y = 60.81 + 3.64A + 0.67767B - 0.0433C + 2.59D - 3.62E - 2.46AB + 2.56BD - 2.42BE + 1.39DE, we know that the partial regression coefficients of factors A, D, and E are 3.64, 2.59, and -3.62, respectively. Since factor E is the most significant, we use E as the unit of measurement for the rate of change.

[0078]

[0079] According to the calculation results, E The step size is set to 1 based on the actual experimental conditions. A The step size is set to 4°C based on the actual experimental conditions. D The step size was set to 0.5 days based on the actual experimental conditions. As shown in Table 5, the second treatment group, with a culture temperature of 32℃, a fermentation time of 8.75 days, and an initial pH of 9.0, showed that the RT-W-1-C strain had the highest inhibition rate on Fusarium rotundifolia mycelial growth, at 69.95%. Therefore, the second group of experimental conditions was selected as the center point of the response surface methodology.

[0080] Table 5 Results of the steepest climb test

[0081] Experimental Example 6. Extraction and Preliminary Analysis of Antibacterial Active Components from RT-W-1-C Strains 6.1 Extraction of secondary metabolites from antagonistic strains and determination of their antibacterial activity The seed culture of the antagonistic strain was inoculated into the optimal fermentation medium at an inoculum of 0.5%, and fermentation was carried out under optimal conditions. The obtained fermentation broth was mixed with ethyl acetate at a 1:1 ratio, and the extraction was repeated twice. The organic phases were combined, and the extract was concentrated to a paste under reduced pressure at 40°C using a rotary evaporator. The paste was dissolved in methanol at a 10:1 ratio, filtered through a 0.22 μm filter membrane to obtain a crude extract of secondary metabolites, which was stored at -20°C for later use.

[0082] Take 500 µL of crude extract and mix thoroughly with 14.5 mL of PDA medium. Pour the mixture onto agar plates and inoculate the center of each plate with a 6 mm *Fusarium solani* spore. Use a PDA plate containing an equal volume of methanol as a control. Perform three replicates for each treatment. Incubate at 28°C for 9 days. Observe the mycelial growth of *Fusarium solani* and measure the colony diameter to calculate the inhibition rate.

[0083] 6.2 Extraction of crude protein from antagonistic strains and determination of their antibacterial activity Antimicrobial proteins from antagonistic strains were extracted using a fractional salting-out precipitation method with ammonium sulfate. The seed culture of the antagonistic strain was inoculated at a rate of 0.5% into the optimal fermentation medium, and fermentation was carried out under optimal conditions. The obtained fermentation broth was centrifuged, the bacterial cells were discarded, and the supernatant was collected. (NH₄)₂SO₄ was added to the supernatant to saturation levels of 25%, 50%, and 75%, respectively, and the mixture was stirred. The mixture was incubated at 4°C for 12 h, then centrifuged at 4000 r / min for 20 min. The precipitate was collected and dissolved in TE buffer at a ratio of 10:1. The precipitate was dialyzed overnight at 4°C using a dialysis bag. After centrifugation at 4000 r / min for 20 min, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm bacterial filter to obtain a sterile crude protein extract, which was stored at -20°C for later use.

[0084] Take 500 µL of crude extract and mix thoroughly with 14.5 mL of PDA medium. Pour the mixture onto agar plates and inoculate the center of each plate with a 6 mm *Fusarium solani* spore. Use a PDA plate containing an equal volume of methanol as a control. Perform three replicates for each treatment. Incubate at 28°C for 9 days. Observe the mycelial growth of *Fusarium solani* and measure the colony diameter to calculate the inhibition rate.

[0085] 6.3 Extraction of lipopeptides from antagonistic strains and determination of their antibacterial activity Lipopeptides from antagonistic bacterial strains were extracted using acid precipitation and methanol extraction. The seed culture of the antagonistic strains was inoculated at a rate of 0.5% into the optimal fermentation medium, and fermentation was carried out under optimal conditions. The resulting fermentation broth was adjusted to pH 2.0 with 6 mol / L HCl and allowed to precipitate at 4°C for 24 h. The precipitate was collected by centrifugation at 4000 r / min for 20 min, dissolved in methanol at a ratio of 10:1, allowed to stand at 4°C, and then centrifuged at 12000 r / min for 20 min. The precipitate was discarded, and the supernatant was filtered through a 0.22 μm bacterial filter to obtain sterile lipopeptide extracts.

[0086] Take 500 µL of crude extract and mix thoroughly with 14.5 mL of PDA medium. Pour the mixture onto agar plates and inoculate the center of each plate with a 6 mm *Fusarium solani* spore. Use a PDA plate containing an equal volume and concentration of methanol as a control. Perform three replicates for each treatment. Incubate at 28℃ for 9 days. Observe the mycelial growth of *Fusarium solani* and measure the colony diameter to calculate the inhibition rate.

[0087] 6.4 Determination of the antibacterial activity of volatile metabolites from antagonistic strains The antagonistic activity of volatile products from antagonistic strains against *Fusarium solani* was determined using the plate-on-plate method. 100 μL of the seed culture of the antagonistic strain was spread onto PDA agar plates. Simultaneously, a 6 mm *Fusarium solani* mycelial cake was inoculated into the center of another PDA agar plate. The two plates were then inverted face-to-face, and the outer seams of both plates were sealed with sealing film. The plates were incubated at 28°C. PDA plates inoculated only with *Fusarium solani* and untreated PDA plates were used as controls. Each treatment was repeated three times. After 9 days of incubation, the growth of both the antagonistic strain and *Fusarium solani* was observed, colony diameter was measured, and mycelial growth inhibition rate was calculated.

[0088] 6.5 Results like Figure 13 As shown in Table 6, the main antibacterial active substances produced by the fermentation broth of strain RT-W-1-C were crude protein and lipopeptides, while the secondary metabolites produced had no antibacterial effect. The crude protein, the main antibacterial active substance produced by the fermentation broth, showed an inhibition rate of 47.52% against *Fusarium solani*, and the lipopeptides showed an inhibition rate of 40.91% against *Fusarium solani*. However, the volatile products produced by the strain showed an inhibition rate of only 5.25% against *Fusarium solani*, indicating almost no inhibitory effect. Therefore, further research is needed on the crude protein and lipopeptides.

[0089] Table 6. Inhibition rate of various extracted active substances against Fusarium solani

[0090] Experimental Example 7. Control of Sugarcane Top Rot and Leaf Blight by RT-W-1-C 7.1 Plate inhibition effect of different concentrations of RT-W-1-C fermentation broth on sugarcane top rot pathogen and sugarcane leaf blight pathogen The RT-W-1-C fermentation broth, which had undergone single-factor optimization, was mixed with PDA at dosages of 500 μL, 1 mL, and 1.5 mL, respectively, and then plated into 15 mL agar plates containing the fermentation broth. *Fusarium sacchari*, the pathogen of sugarcane top rot, and *Stagonospora tainanensis*, the pathogen of sugarcane leaf blight, which had been cultured for 5-7 days, were inoculated in the center of each plate. The plates were incubated at 28°C for 5 days and 9 days, respectively, with three replicates for each treatment. The control group consisted of 15 mL PDA plates inoculated with the pathogens of sugarcane top rot and sugarcane leaf blight, which had been cultured for 5-7 days, and incubated at 28°C for 5 days and 9 days, respectively, with three replicates for each treatment.

[0091] The results are as follows Figure 14 As shown, the plate inhibition rates of 500 μL RT-W-1-C fermentation broth against sugarcane top rot pathogens and sugarcane leaf blight pathogens were 45.40% and 14.54%, respectively. The plate inhibition rates of 1 mL RT-W-1-C fermentation broth were 58.61% and 36.01%, respectively, and the plate inhibition rates of 1.5 mL RT-W-1-C fermentation broth were 70.11% and 39.15%, respectively. Therefore, the next step in the control of sugarcane top rot is in vitro control.

[0092] 7.2. In vitro control efficacy of RT-W-1-C fermentation broth at different dilution ratios against sugarcane top rot Leaves 8 cm in length were cut, and approximately 1.0 cm cuts were made on both sides. For each treatment, a 6 mm diameter mycelium of sugarcane top rot pathogen FF001 was placed on the left side of the leaf as a control group, and the right side of the leaf represented each treatment group, as shown below: Blank control: PDA mycelium cake without FF001 connection Positive control: First spray with Bacillus subtilis diluted 1000 times or Bacillus vibrio diluted 500 times, then inoculate with FF001 bacterial cake, and finally spray with Bacillus subtilis diluted 1000 times or Bacillus vibrio diluted 500 times.

[0093] Treatment group: First spray with fermentation liquid diluted 10 times, 50 times, or 100 times, then inoculate with FF001 bacterial cake, and finally spray with fermentation liquid diluted 10 times, 50 times, or 100 times.

[0094] Each treatment was repeated in triplicate and incubated at 25°C.

[0095] The results are as follows Figure 15 As shown, the fermentation liquid diluted 10 times has a certain preventive effect on sugarcane top rot, and the fermentation liquid has no toxic effect on sugarcane.

[0096] 7.3 In vivo control effect of RT-W-1-C fermentation broth at different dilution ratios on sugarcane top rot Add 10 mL of sterile water to an FF001 culture dish that has been cultured for 5-7 days. Use a sterilized spreader to scrape off the mycelium to release the spores. Filter the spore solution through four layers of sterile lens paper and adjust the spore concentration to 10. 5 500 μL of spore solution was injected into the sugarcane stalk at a depth of approximately 2.0 cm above the ground using a 1 mL syringe. The syringe should be inserted into the midrib of the plant, and the optimal concentration is achieved when the bacterial solution overflows from the top of the plant. The next day, sterile fermentation broth of various concentrations was sprayed onto the top of the plants, while the control group was sprayed with water. A blank control was also included, injected with water. Each treatment was repeated in triplicate, with 20 plants per replicate.

[0097] The results are shown in Table 7 and Figure 16 As shown, fermentation broths of different dilution ratios all have a certain control effect on sugarcane top rot, but the control effect is not very good. The best control effect of fermentation broth diluted 10 times is 24.93%.

[0098] Table 7. In vivo control efficacy of fermentation broth at different dilution ratios against sugarcane top rot

[0099] 7.4 In vivo control of sugarcane top rot by various active substances Add 10 mL of sterile water to an FF001 culture dish that has been cultured for 5-7 days. Use a sterilized spreader to scrape off the mycelium to release the spores. Filter the spore solution through four layers of sterile lens paper and adjust the spore concentration to 10. 5 500 μL of spore solution was injected into the sugarcane stalk at a depth of approximately 2.0 cm above the ground using a 1 mL syringe. The syringe should be inserted into the midrib of the plant; optimal results are achieved when the bacterial solution oozes from the plant tip. The next day, various active substances were sprayed onto the plant tips. The control group was sprayed with water. A blank control was also included, injected with water. Each treatment was repeated in triplicate, with 20 plants per replicate.

[0100] The results are as follows Figure 17 As shown in Table 8, these three active substances have certain preventive and control effects on sugarcane top rot, among which crude protein has the best preventive and control effect, at 20.1%.

[0101] Table 8. In vivo control efficacy of various active substances against sugarcane top rot

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0103] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] 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 biocontrol strain of Streptomyces abigouli, with accession number CGMCC No. 34660.

2. The biocontrol agent *Streptomyces abigonis* as described in claim 1, in inhibiting *Fusarium solani* (… Fusarium sacchari ) and / or Tainan Chilospora ( Stagonospora tainanensis Its application in the preparation of products that inhibit Fusarium rot ( ) or in the preparation of products that inhibit Fusarium rot ( ) Fusarium sacchari ) and / or Tainan Chilospora ( Stagonospora tainanensis Applications in products.

3. The application of the biocontrol Streptomyces abigoni as described in claim 1 in the control of plant shoot rot and / or plant leaf blight.

4. The application as described in claim 3, characterized in that: The pathogen causing the top rot is *Fusarium solani* (also known as the bark rot fungus). Fusarium sacchari The pathogen causing leaf blight is *Polyspora tainanensis* (…). Stagonospora tainanensis ).

5. The application as described in claim 3, characterized in that: The plant in question is sugarcane.

6. The application as described in any one of claims 3-5, characterized in that: It uses the sterile fermentation broth of the aforementioned Streptomyces abigonis to spray plants.

7. The application as described in any one of claims 3-5, characterized in that: It uses an extract from the sterile fermentation broth of the aforementioned Streptomyces abigonis to spray plants.

8. The application as described in claim 7, characterized in that, The extract of the sterile fermentation broth is at least one of lipopeptides and crude protein extracted from 75% ammonium sulfate.

9. The application as described in claim 6 or 7, characterized in that: The sterile fermentation broth was obtained by culturing in a culture medium containing soybean flour, soluble starch, NaCl, and calcium carbonate.

10. The application as described in claim 9, characterized in that: The fermentation temperature of the sterile fermentation broth is 24-36℃; the shaking speed is 160 rpm to 220 rpm; the initial fermentation pH is 7-11; and the fermentation time is 7-9 days.

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