A fermentation preparation of streptomyces antagonistic to pyricularia oryzae, a preparation method and application thereof

Abstract

The application discloses a kind of pyricularia oryzae antagonistic streptomyces fermentation preparation, preparation method and application, belong to microbial fermentation and biological pesticide field, which comprises the following steps: screening the inhibitory rate of more than 80% of pyricularia oryzae starting strain of streptomyces, ribosome engineering is reformed by rpsL gene K88E directional point mutation, the value is greater than 5000 ug / mL in the optimized fermentation medium of carbon-nitrogen ratio 25:1~30:1, corn syrup 0.5%~1% culture makes potency, and the preparation of suspension preparation containing viable bacteria and metabolites is prepared by suspending liquid and supernatant concentrate compound, and the comprehensive prevention and cure effect of the preparation in field reaches more than 75%, which can replace chemical pesticide.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and biopesticide technology, specifically relating to a method for directionally modifying Streptomyces through ribosome engineering to increase the yield of secondary metabolites of its rice blast-resistant strain, as well as a composite suspension preparation containing live bacteria and metabolites prepared based on the modified strain and its application in the prevention and control of rice blast. Background Technology

[0002] Rice is a staple crop for over three billion people worldwide. Rice blast, caused by the ascomycete *Magnaporthe oryzae*, is widely distributed in major rice-producing areas globally, infecting rice leaves, stem nodes, and panicle necks. In severe years, it can cause localized yield losses of 40% to 100%, making it one of the most damaging diseases in rice production. Currently, rice blast control still relies primarily on chemical pesticides, with commonly used active ingredients including tricyclazole, isoprothiolane, and prochloraz. However, the long-term and extensive use of chemical pesticides has led to the following prominent problems: First, the frequency of resistance to major pesticides such as tricyclazole in the rice blast fungus population is continuously increasing, with high-level resistant strains detected in some major producing areas, resulting in a significant decrease in the effectiveness of chemical control; second, pesticide residues remain after application during the rice heading stage, posing a risk to rice quality and safety and export trade; third, frequent pesticide application disrupts the microecological environment of paddy fields, affecting biodiversity. Therefore, developing safe and efficient biological control alternative strategies is of significant practical importance.

[0003] Streptomyces are the most widely distributed actinomycete group in soil, producing a large number of secondary metabolites with agricultural applications, including polyene macrocyclic lactones, polyketides, non-ribosomal peptides, and extracellular hydrolases, which exhibit broad-spectrum inhibitory activity against various plant pathogenic fungi. Previous studies have reported that several rhizosphere or endophytic Streptomyces strains have in vitro inhibitory activity against rice blast fungus. For example, Njoroge et al. reported that the rhizosphere Streptomyces corchorusii Sc75 of rice showed an in vitro inhibition rate of 69.25% against rice blast fungus, and could alleviate the disease under greenhouse conditions, but did not involve any optimization of fermentation process parameters or formulation studies, nor did they provide field efficacy data. Xu et al. reported that the fermentation broth of the endophytic Streptomyces OsiSh-2 had inhibitory activity against rice blast fungus, with a field efficacy of 59.64%, and the active ingredient was identified as nigericin. Liu et al. reported that the crude lipopeptide extract of S. bikiniensis HD-087 showed an inhibition rate of 76.9% against rice blast in an in vitro leaf test, but the control efficacy was only about 60% in a pot test, and the study did not involve standardized fermentation process parameters.

[0004] Chinese patent CN106434409B discloses a rice endophytic actinomycete *Streptomyces hydrogenans* OsiLf-2 (accession number CGMCC No. 11673) that exhibits highly effective in vitro antagonism against rice blast fungus. This patent protects the strain itself and its application in antagonizing rice blast fungus, but does not disclose any fermentation medium formulation parameters, carbon-nitrogen ratio optimization schemes, or formulation technology pathways. Chinese patent CN113005048A discloses the application of *Streptomyces niger* CYS22 and its metabolites. This strain has certain inhibitory activity against rice blast fungus, but similarly only protects the strain and its application, without addressing fermentation process parameterization or formulation standardization.

[0005] In recent years, ribosome engineering has attracted attention as an emerging strategy for improving microbial strains. This method involves introducing specific point mutations into ribosomal protein genes (such as the rpsL gene encoding the 30S subunit S12 protein) to alter the conformation and substrate selectivity of the ribosomal translation machine, thereby globally upregulating the expression levels of secondary metabolic gene clusters. Fan et al. demonstrated the feasibility of using ribosome engineering to increase the yield of tetraene macrolide antifungal antibiotics by accumulating drug resistance mutations (including rpsL mutations) in *S. diastatochromogenes*, achieving a yield increase of 8.7–25 times. However, there are currently no reports on the application of ribosome engineering to the fermentation process optimization and formulation development of *Streptomyces*, a biocontrol agent against rice blast.

[0006] From an industrialization perspective, the core competitiveness of microbial pesticides lies in the stability of fermentation potency and the standardization of the formulation system. Fermentation potency directly determines the concentration of the active ingredient in the formulation, thus affecting the field application dosage and efficacy. Products with large potency fluctuations are difficult to establish stable quality standards and cannot obtain pesticide registration. In the fermentation process of Streptomyces, the carbon-nitrogen ratio is one of the most critical culture medium parameters affecting the synthesis of secondary metabolites. The carbon-nitrogen ratio affects the distribution of metabolic flux between primary and secondary metabolism by regulating the concentration of intracellular ppGpp signaling molecules. However, there are currently no reports on the optimization of the carbon-nitrogen ratio for Streptomyces, a biocontrol agent against rice blast, and no technical solution that combines carbon-nitrogen ratio optimization with genomic-level genetic modification to achieve a doubling of potency. At the formulation level, the core challenge in the design of microbial pesticide formulations lies in how to maximize the effective content of antimicrobial metabolites while maintaining the survival rate of live bacteria. Existing technologies usually only utilize live bacteria or only utilize fermentation filtrate, failing to fully utilize the synergistic potential of both.

[0007] In summary, existing technologies generally suffer from the following industrialization bottlenecks: research remains at the level of strain screening and laboratory activity evaluation, lacking a complete manufacturing chain from highly active strains to mass-producible, high-efficiency fermentation formulations; there is a lack of quantifiable and reproducible fermentation process parameter standards, especially optimization of the carbon-nitrogen ratio and organic nitrogen source in the fermentation medium; existing reported field efficacy generally does not exceed 60%, far lower than the efficacy level of mainstream chemical pesticides, making it difficult to meet actual production needs; genetic modification methods such as ribosome engineering have not been combined with fermentation process optimization to achieve a breakthrough in metabolite yield; formulation formulations also lack standardization, and research on the synergistic utilization of live bacteria and antimicrobial metabolites is scarce. Therefore, there is an urgent need to develop a Streptomyces biopesticide technology system that deeply couples genetic modification with fermentation processes, enabling its efficacy to reach or approach the level of chemical pesticides, while simultaneously achieving green pest control goals. Summary of the Invention

[0008] The core technical problem to be solved by this invention is: how to increase the yield of secondary metabolites of Streptomyces resistant to rice blast to a potency greater than 5000 µg / mL through directional modification of ribosomes and synergistic optimization of fermentation medium formulation, and on this basis, establish a standard for a composite suspension formulation containing live bacteria and metabolites, so that its field control efficacy against rice blast reaches more than 75%.

[0009] To solve the above technical problems, the present invention provides a method for preparing a fermentation agent of *Streptomyces* antagonistic to *Oryza sativa*, comprising the following steps: (1) strain acquisition: samples are collected from the rhizosphere soil of healthy rice, and *Streptomyces* strains with an inhibition rate of greater than 80% against *Oryza sativa* are screened using the plate confrontation method; (2) ribosome engineering modification: a point mutation is introduced into the lysine codon at position 88 of the *rpsL* gene coding region of the starting strain, replacing AAG with GAG encoding glutamic acid or CGG encoding arginine, to obtain an *rpsL* mutant strain; (3) fermentation culture: the *rpsL* mutant strain is inoculated into a fermentation medium for liquid deep fermentation, wherein the carbon-nitrogen ratio of the fermentation medium is 25:1 to 30:1 and contains 0.5% to 1% corn steep liquor by mass, the fermentation temperature is 28 to 30°C, the fermentation time is 5 to 7 days, and the resulting fermentation broth has an antibacterial titer of greater than 5000 against *Oryza sativa*. µg / mL; (4) Preparation of formulation: After centrifugation, the fermentation broth obtained in step (3) is mixed with the supernatant concentrate, and a dispersant and thickener are added to obtain a composite suspension formulation containing live bacteria and metabolites.

[0010] This invention also provides a fermentation preparation of *Streptomyces* antagonistic to *Streptomyces*, wherein the preparation is a composite suspension containing live *Streptomyces* rpsL K88E mutant cells and a concentrated fermentation supernatant, and the live *Streptomyces* count is not less than 2 × 10⁻⁶. 8The formulation has an inhibitory potency of CFU / mL against rice blast fungus greater than 5000 µg / mL, and contains sodium lignosulfonate and xanthan gum as dispersing agents.

[0011] The present invention also provides the application of a fermentation preparation of rice blast fungus antagonistic Streptomyces in the prevention and control of rice blast. The preparation is diluted 100 to 300 times and sprayed on rice from the late tillering stage to the booting stage. The number of applications is 2 to 3 times. The comprehensive control efficacy against leaf blast and neck blast is not less than 75%.

[0012] The present invention has the following beneficial effects:

[0013] (1) Significantly improved efficacy. Through ribosome engineering modification and synergistic optimization of fermentation process, the fermented preparation of this invention achieves a comprehensive field efficacy of over 75% against rice blast, which is about 15-20 percentage points higher than the existing reported similar Streptomyces biological agents, reaching the efficacy level of chemical pesticides, and realizing an effective substitution of biological control for chemical control.

[0014] (2) Synergistic effect of dual-level genetics and metabolism. The rpsL K88E point mutation reprograms the translation priority of ribosomes for PKS / NRPS mRNA at the translation level, and the carbon-nitrogen ratio of 25:1~30:1 upregulates the expression of PKS / NRPS gene clusters at the transcription level through ppGpp signaling. The superimposed activation of the two levels produces a superadditive titer jump effect, which cannot be achieved by a single means.

[0015] (3) Synergistic dual antibacterial mechanism. After colonization on rice leaves, the live bacteria in the formulation continuously produce chitinase, glucanase, and siderophores, achieving sustained enzymatic protection. The polyketides and non-ribosomal peptide metabolites in the supernatant concentrate exert a direct antibacterial effect immediately after application, destroying the cell membrane and cell wall of rice blast fungus. The dual mechanism of immediate antibacterial action and sustained protection is the key to the high efficacy of this formulation.

[0016] (4) Delaying the development of chemical pesticide resistance. When used in rotation with tricyclazole or isoprothiolane, the multi-target antibacterial mechanism of this formulation can effectively cut off the selective pressure of pathogens being continuously exposed to the same chemical pesticide, thus delaying the formation and expansion of resistant strains.

[0017] (5) Quantitative and reproducible process parameters. The core fermentation parameters (rpsL K88E mutation, carbon-nitrogen ratio 25:1~30:1, corn steep liquor 0.5%~1%, potency greater than 5000 µg / mL) are all quantitative indicators that can be precisely controlled and detected, facilitating quality management in large-scale production. The ribosome engineering modification steps achieve complete reproducibility of the technical solution by disclosing the mutation sites and operating methods, without relying on specific preserved strains. The technical solution of this invention has good prospects for process scale-up. The rpsL K88E mutation is a one-time genetic modification. Once the mutant strain is constructed, it can be used as a production strain for a long time without the risk of plasmid loss or genetic reversion. The genetic traits are stable after more than 10 generations of continuous passage. The fermentation medium components are all industrial-grade raw materials (soluble starch, soybean flour, corn steep liquor), which are widely available, inexpensive, and have a stable supply, making them suitable for large-scale industrial production. Sodium lignosulfonate and xanthan gum in the formulation are conventional adjuvants in the field of pesticide formulation, with high technical maturity and no raw material supply bottleneck. The potency assay employs a standardized agar diffusion method, which can be independently performed in any laboratory with basic microbiological experimental conditions, providing a reliable detection method for online quality control in the production process. In summary, this invention achieves parameterization and standardization across the entire chain from genetic modification and fermentation process to formulation, possessing a complete technological foundation for the transformation from laboratory research to industrial production. Attached Figure Description

[0018] Figure 1 The images show the comparison of the antibacterial effects of the plate confrontation method. From left to right, they are: blank control (only rice blast fungus grows naturally), confrontation results of wild-type YZ-01 strain against rice blast fungus, and confrontation results of rpsL K88E mutant strain against rice blast fungus.

[0019] Figure 2 Line graph showing the effect of different carbon-nitrogen ratios (15:1~35:1) on the fermentation titer of the rpsL K88E mutant strain.

[0020] Figure 3 Bar chart showing the effect of different corn steep liquor addition amounts (0%~1.5%) on the fermentation titer of rpsL K88E mutant strain.

[0021] Figure 4 This is a schematic diagram of the preparation process of compound fermentation preparations.

[0022] Figure 5 The bar chart shows the field efficacy comparison of leaf blast and neck blast control efficacy of the formulation of this invention, tricyclazole positive control, and blank control.

[0023] Figure 6The circular map of the recombinant integrative plasmid pSET152-rpsL K88E is marked with the pUC replication origin, aac(3)IV apramycin resistance gene, φC31 integrase gene int, attP site, multiple cloning site MCS, and the position and orientation of the rpsL K88E mutant gene insertion fragment.

[0024] Figure 7 The linear sequence map of the K88E mutation region of the rpsL gene is shown, marking the rpsL coding region, the K88E mutation site (codon 88 AAG→GAG), the binding sites of the four PCR primers (rpsL-F, rpsL-R, rpsL-mF, rpsL-mR), and the extent of the overlapping extended PCR amplification region. Detailed Implementation

[0025] The following examples are used to illustrate the present invention, but do not constitute a limitation on the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the examples are all commercially available conventional products.

[0026] Example 1: Screening and identification of antagonistic Streptomyces and construction of rpsL K88E mutant strain

[0027] I. Screening and Identification of the Starting Strains. Rhizosphere soil (0-20 cm) was collected from healthy paddy fields (with no history of rice blast disease) in the rice-growing area of ​​Yangzhou City, Jiangsu Province. 5 g of soil sample was added to 45 mL of sterile water, shaken for 30 min, and serially diluted to 10⁻⁶. -4 ~10 -6 The cultures were spread on Gao's No. 1 medium (starch 20 g / L, KNO3 1 g / L, K2HPO4 0.5 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, FeSO4 0.01 g / L, agar 20 g / L, pH 7.2~7.4) plates and incubated at 28°C for 7~14 days. Approximately 200 single colonies exhibiting Streptomyces morphological characteristics (filamentous colonies, sporulation, earthy odor) were selected. Using the standard strain of rice blast fungus, Magnaporthe oryzae (CGMCC 3.4981), as the target, a double-culture confrontation method was employed on potato dextrose agar (PDA) plates. Each candidate Streptomyces strain (5 mm diameter agar block) was placed 3 cm apart from a rice blast fungus (5 mm diameter agar block) on the same plate and co-cultured at 28°C for 7 days. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) ÷ Colony diameter of control group × 100%. Three target strains (tentatively named YZ-01, YZ-02, and YZ-03) with inhibition rates greater than 80% were selected from 200 candidate strains. YZ-01 showed the highest inhibition rate at 83.5%. Figure 1As shown, in the blank control, after 7 days of natural growth on PDA plates, the colony diameter of *Oryza sativa* reached approximately 52 mm, almost covering the entire central area of ​​the plate. When cultured in confrontation with the wild-type YZ-01, the colony diameter of *Oryza sativa* shrank to approximately 36 mm, forming a transparent inhibition zone of approximately 15 mm between them, indicating that the wild-type strain has significant in vitro antagonistic activity against *Oryza sativa*. When cultured in confrontation with the rpsL K88E mutant, the colony diameter of *Oryza sativa* further decreased to approximately 20 mm, and the width of the inhibition zone expanded to approximately 22 mm, an increase of approximately 47% compared to the wild type. Furthermore, obvious hyphal shrinkage and dissolution were observed at the colony edges, indicating that the secretion of antifungal metabolites by the rpsL K88E mutant was significantly higher than that of the wild type, consistent with subsequent fermentation titer data.

[0028] Genomic DNA was extracted from strain YZ-01, and a 16S rDNA fragment (approximately 1500 bp, using universal primers 27F / 1492R) was amplified. After sequencing, BLAST homology comparison was performed in the NCBI database. YZ-01 showed 99.6% homology with Streptomyces sp., confirming its genus Streptomyces. Further multilocus sequence analysis (MLSA, based on five housekeeping genes: atpD, gyrB, recA, rpoB, and trpB) identified it to the species level, and the sequences have been submitted to GenBank. YZ-01 also exhibited inhibitory activity against *Streptomyces oryzae* and *Streptomyces sheath blight*, with inhibition rates of 71.2% and 65.8%, respectively, indicating a certain broad-spectrum antifungal activity. Strains YZ-02 and YZ-03, obtained during the screening process, showed inhibition rates of 81.2% and 80.5% against *Streptomyces oryzae*, respectively, slightly lower than YZ-01. 16S rDNA sequence analysis of the three strains revealed that they belonged to different species or subspecies of the genus *Streptomyces*, with different secondary metabolic gene clusters. YZ-01 was chosen as the starting strain for subsequent modification primarily based on its highest inhibition rate and broadest antibacterial spectrum. On Gao's No. 1 medium, YZ-01 colonies exhibited grayish-white aerial hyphae with pale yellow intramural hyphae, producing gray spore chains, displaying typical morphological characteristics of *Streptomyces*. The optimal growth temperature for YZ-01 on ISP2 medium was 28°C, the optimal pH was 7.0–7.5, it could tolerate NaCl concentrations up to 5%, and it could grow normally within a pH range of 5.0–9.0.

[0029] II. Construction of the rpsL K88E mutant. Using YZ-01 genomic DNA as a template, the full-length rpsL gene and its upstream and downstream flanking sequences, totaling approximately 2.0 kb, were amplified by PCR using primers designed based on the conserved region of the Streptomyces rpsL gene (rpsL-F: 5'-ATGCCTACGATCCGCAAGTC-3' and rpsL-R: 5'-TCAGGTCTTCTTCAGCGTCG-3'). The sequence structure of the rpsL gene region and the binding positions of each primer are shown below. Figure 7 As shown. Figure 7 As shown, the rpsL coding region is 375 bp long, encoding the 124-amino acid 30S ribosomal protein S12. The K88E mutation site is located at nucleotides 262-264 of the coding region (corresponding to codon 88). Using overlap extension PCR, a point mutation AAG→GAG (K88E mutation) was introduced into the lysine codon AAG at position 88 of the rpsL coding region, replacing lysine with glutamate. Specifically, two pairs of internal mutation primers were designed (rpsL-mF: 5'-CTACAACGGCGAGGGCACCACCTC-3' and rpsL-mR: 5'-GAGGTGGTGCCCTCGCCGTTGTAG-3', where the bolded site represents the mutated base G replacing the original A). Figure 7 As indicated in the annotation, rpsL-mF and rpsL-mR pair with rpsL-F and rpsL-R respectively to form two overlapping fragments. First, the two overlapping fragments were amplified using rpsL-F / rpsL-mR and rpsL-mF / rpsL-R primers, respectively. Then, a second round of PCR was performed using these two fragments as templates and rpsL-F / rpsL-R as outer primers to obtain the complete rpsL fragment containing the K88E mutation (approximately 695 bp). Figure 7 (as indicated by the PCR amplification region).

[0030] The rpsL fragment containing the K88E mutation was digested with restriction endonucleases and then cloned into the multiple cloning site of the Streptomyces integrative vector pSET152 (containing the φC31 integrase gene, attP site, and apramycin resistance gene aac(3)IV) to construct the recombinant plasmid pSET152-rpsL. K88E Its spectral structure is as follows Figure 6 As shown. Figure 6As shown, the recombinant plasmid is a circular DNA molecule with a total length of approximately 6.2 kb. It sequentially contains the pUC origin of replication (used for plasmid amplification in *E. coli*), the apramycin resistance gene aac(3)IV (used for dual selection in *E. coli* and *Streptomyces*), the φC31 integrase gene int (mediates site-specific integration of the plasmid at the attB site on the *Streptomyces* chromosome), the attP site (for integration with the attB chromosome), the multiple cloning site MCS (the insertion site of the rpsL K88E mutant fragment), and the rpsL K88E mutant gene insertion fragment (approximately 695 bp, marked in red). After restriction endonuclease digestion verification and insertion fragment sequencing confirmation, the recombinant plasmid was transformed into *E. coli* ET12567 / pUZ8002 and introduced into strain YZ-01 via indirect conjugation transfer. The conjugation transfer operation was as follows: the *E. coli* donor strain ET12567 / pUZ8002 containing the recombinant plasmid was cultured to OD2000. 600 =0.4~0.6, collect bacterial cells by centrifugation at 6000 r / min and wash twice with antibiotic-free LB medium to remove residual antibiotics. Mix the washed donor bacteria with freshly prepared YZ-01 spore suspension (concentration approximately 1×10⁻⁶). 8 Mix the 1:1 volume ratio of acetamipridamole (soybean flour, mannitol, and agar) and spread 200 µL of the mixture onto MS medium (soybean flour 20 g / L, mannitol 20 g / L, agar 20 g / L) supplemented with 10 mmol / L MgCl2. Incubate at 30°C for 16–20 h, then cover with a soft agar layer containing 50 µg / mL apramycin and 25 µg / mL nalidixic acid for screening. Apramycin is used to select Streptomyces transformants that have successfully received the recombinant plasmid, and nalidixic acid is used to kill any remaining E. coli donor bacteria. After further incubation at 28°C for 5–7 days, scattered colonies of apramycin-resistant Streptomyces can be observed on the screening plates, with a conjugation transfer frequency of approximately 1 × 10⁻⁶. -5 ~5×10 -5 Approximately 30 apramycin-resistant single colonies were selected and transferred to Bennett agar plates containing 100 µg / mL streptomycin for streptomycin resistance phenotype verification. About 60%–70% of the apramycin-resistant colonies also showed high levels of streptomycin resistance, indicating that the rpsL K88E mutation has been successfully integrated into the chromosome and correctly expressed.

[0031] After 10 consecutive passages on streptomycin-free ISP2 medium, the rpsL K88E mutant site of YZ-01-K88E showed no reversion mutation after resequencing, and the streptomycin resistance phenotype remained stable, demonstrating the good genetic stability of this genetic modification. Further comparison of the growth rates of YZ-01-K88E and wild-type YZ-01 on ISP2 plates showed that the colony diameter of the K88E mutant was 88%–92% of that of the wild type after 7 days of culture, with a slightly reduced growth rate. This is consistent with the reported phenotype of slowed ribosomal translation caused by rpsL point mutations. However, this slight growth cost was fully compensated by a significant increase in the production of secondary metabolites. Furthermore, the inhibition rate of YZ-01-K88E against *Streptomyces oryzae* CGMCC 3.4981 was 87.2% using the plate confrontation method, which was 3.7 percentage points higher than that of wild-type YZ-01 (83.5%), indicating that the rpsLK88E mutation not only improved fermentation titer but also enhanced the in vitro antagonistic activity of the strain itself. The inhibition rates of YZ-01-K88E against *Streptomyces oryzae* and *Streptomyces sheath blight* were 78.6% and 72.4%, respectively, which were 7.4 and 6.6 percentage points higher than those of the wild type, further confirming that ribosome engineering globally enhanced the secondary metabolite synthesis capacity of *Streptomyces*.

[0032] Genomic DNA was extracted from streptomycin-resistant positive colonies and amplified by PCR using rpsL-F / rpsL-R primers. Sanger sequencing of the PCR products confirmed a successful mutation of codon 88 of the rpsL gene from AAG (Lys) to GAG (Glu). Five rpsL K88E mutant strains, numbered YZ-01-K88E-1 to YZ-01-K88E-5, were obtained and validated by sequencing. Small-scale fermentation was used for pre-screening of the five mutant strains (C / N ratio 27:1, corn steep liquor 0.8%, 28°C, 180 r / min, 6 days). The antimicrobial titers of each mutant were measured to be 6520, 6850, 6380, 6710, and 6280 µg / mL, respectively. YZ-01-K88E-2, with the highest titer, was selected as the working strain for subsequent fermentation optimization experiments.

[0033] Example 2: Optimization of the carbon-nitrogen ratio in fermentation medium

[0034] Using the rpsL K88E mutant strain YZ-01-K88E as the fermentation strain, soluble starch as the carbon source, and soybean flour as the nitrogen source, a carbon-to-nitrogen ratio gradient experiment was conducted with a fixed corn steep liquor addition of 0.8%. The seed culture was prepared as follows: YZ-01-K88E was inoculated into seed culture medium (30 g / L soybean flour, 15 g / L glucose, 5 g / L NaCl, pH 7.0–7.2, sterilized at 121°C for 20 min), and cultured at 28°C with shaking at 180 r / min for 48 h to obtain the seed culture. The seed culture was inoculated at a 10% inoculation rate into each fermentation medium (soluble starch content calculated based on the target C / N ratio, KH₂PO₄ 0.2 g / L, MgSO₄·7H₂O 0.1 g / L, corn steep liquor 0.8%, pH 7.0–7.2, sterilized at 121°C for 20 min). C / N ratio gradients of 15:1, 20:1, 25:1, 27:1, 28:1, 30:1, and 35:1 were set, with each treatment replicated three times. Fermentation was carried out at 28°C and 180 r / min for 6 days. The inhibitory potency of each treatment fermentation broth against *Strombus oryzae* was determined using the agar diffusion method. Simultaneously, a control fermentation experiment was conducted using the wild-type starting strain YZ-01 at a C / N ratio of 27:1.

[0035] The potency determination method is as follows: using rice blast fungus CGMCC 3.4981 as the indicator fungus, in a suspension containing indicator fungus spores (final concentration 1×10⁻⁶). 6 Per 8 mm diameter well was punched in a sterile PDA plate (number of bacteria / mL). 200 µL of fermentation supernatant was added to each well. After incubation at 28°C for 48 h, the diameter of the inhibition zone was measured. A standard curve was established using nigericin standard (purity ≥98%, Sigma-Aldrich), and the inhibition zone diameter was converted to potency (µg / mL). The fermentation potency results of the rpsL K88E mutant under various carbon-to-nitrogen ratios are shown in Table 1. The wild-type YZ-01, with a fermentation potency of 3520±215 µg / mL at a carbon-to-nitrogen ratio of 27:1, served as the control.

[0036] Table 1. Effects of different carbon-nitrogen ratios on the fermentation titer of the rpsL K88E mutant strain

[0037]

[0038] pH and cell concentration were monitored every 24 hours during fermentation. The pH value slowly decreased from an initial 7.0-7.2 to 6.5-6.8 within the first 48 hours of fermentation, corresponding to the rapid growth phase of *Streptomyces*, where organic acids were the main metabolites. From 48 to 96 hours of fermentation, the pH value gradually rose to 7.0-7.3, indicating the accumulation of ppGpp due to nitrogen source consumption and the initiation of secondary metabolism. From 96 to 144 hours of fermentation, the pH value stabilized at 7.0-7.5; this stage was the period of rapid accumulation of secondary metabolites, with the most significant increase in titer. Cell concentration was measured using OD... 600 Characterization was performed on the OD of the rpsL K88E mutant strain under a carbon-to-nitrogen ratio of 27:1. 600 The OD value of the mutant strain reached a peak of 8.5 at 48 hours, slightly lower than the peak value of 9.2 for the wild type, and then entered a stationary phase. 600 The slightly lower peak value is related to the slower ribosomal translation rate caused by rpsL K88E, but this growth cost is fully compensated by the significant increase in the production of secondary metabolites. The titer of the fermentation broth reached its maximum on day 6 (144 h), and the titer change did not exceed 5% on day 7, thus the optimal fermentation time was determined to be 6 days.

[0039] like Figure 2 As shown, a carbon-to-nitrogen ratio of 25:1 to 30:1 represents the optimal range for efficient secondary metabolite synthesis, with potencies exceeding 5600 µg / mL. The optimal values ​​are 27:1 and 28:1, reaching potencies of 6850 and 6580 µg / mL, respectively. Below a carbon-to-nitrogen ratio of 20:1, nitrogen sources are sufficient, ppGpp accumulation is inhibited, and Streptomyces primarily engages in primary metabolism, resulting in potencies of only 1280–2450 µg / mL. Above a carbon-to-nitrogen ratio of 35:1, carbon metabolism overflow produces organic acid byproducts, interfering with PKS precursor supply, and the potency drops to 3850 µg / mL. It is noteworthy that the titer of the rpsL K88E mutant strain under the optimal carbon-nitrogen ratio of 27:1 (6850 µg / mL) was 94.6% higher than that of the wild-type YZ-01 strain under the same conditions (3520 µg / mL), indicating a significant synergistic effect between ribosome engineering and carbon-nitrogen ratio optimization, with the titer increase far exceeding the linear summation of the individual contributions of the two.

[0040] Example 3: Optimization of Corn Stew Addition Amount

[0041] With a fixed carbon-to-nitrogen ratio of 27:1, the rpsL K88E mutant strain YZ-01-K88E was used as the fermentation strain. A gradient of corn steep liquor addition (mass fraction) was set: 0%, 0.3%, 0.5%, 0.7%, 0.9%, 1.0%, and 1.5%. All other fermentation conditions were the same as in Example 2. Each treatment was repeated three times, and the fermentation titer was measured. The results are shown in Table 2.

[0042] Table 2. Effects of different corn steep liquor addition amounts on the fermentation titer of the rpsL K88E mutant strain.

[0043]

[0044] like Figure 3 As shown, the potency of corn steep liquor added at concentrations of 0.5%–1.0% all exceeded 6100 µg / mL, with the optimal range being 0.7%–0.9%, reaching a maximum potency of 6880 µg / mL. The potency of corn steep liquor without addition was 3680 µg / mL, only 53.5% of the optimal value, indicating that corn steep liquor, as a complex organic nitrogen source and vitamin donor, makes an irreplaceable contribution to the metabolic flux of PKS / NRPS precursors. Corn steep liquor provides alanine, glycine, and valine, which are substrate precursor amino acids for NRPS templates, as well as B vitamins (especially vitamin B12). 12 It assists in the activity of methylmalonyl-CoA mutase, providing a key cofactor for the supply of PKS precursors. The potency slightly decreased when the addition exceeded 1.0%, presumably related to the excessive organic nitrogen causing the carbon-nitrogen ratio to deviate from the optimal window. Notably, the potency gain curve of corn steep liquor addition from 0% to 0.7% exhibited typical saturation kinetics. In the initial stage, the potency increased rapidly with increasing addition (potency growth rate of approximately 4880 µg / mL / % in the 0%~0.5% range), while the growth slowed down in the later stage (potency growth rate of approximately 3800 µg / mL / % in the 0.5%~0.7% range), suggesting that the key nutrients in corn steep liquor were close to metabolic saturation at 0.7%. When the corn steep liquor concentration was increased to 1.5%, the effective value dropped to 5680 µg / mL. Although this decrease (17.4% from the optimal value) was not as drastic as the decrease when the carbon-nitrogen ratio deviated from the optimal value, it still has practical significance in industrial production. This is because excessive addition of corn steep liquor not only increases raw material costs but may also lead to increased viscosity of the fermentation broth, affecting the efficiency of subsequent centrifugation. In addition, the detection of residual amino acid content in the fermentation broth showed that the concentration of residual free amino acids in the fermentation broth at the fermentation endpoint was extremely low (below 0.5 mmol / L) under the condition of 0.7% corn steep liquor, indicating that the amino acid precursors provided by corn steep liquor were fully utilized and converted into NRPS products. However, the concentration of residual amino acids was significantly increased under the condition of 1.5% corn steep liquor (approximately 2.8 mmol / L), further confirming the judgment that excessive addition led to an excess rather than a deficiency of precursors.

[0045] Example 4: Formulation Preparation

[0046] Five L of fermentation broth was prepared under the optimal fermentation conditions (rpsL K88E mutant strain YZ-01-K88E, C / N ratio 27:1, corn steep liquor 0.8%, 28°C, 180 r / min, 6 days). The titer was 6850 µg / mL, meeting the standard. The fermentation broth was centrifuged at 6000 r / min for 20 min, and the cell precipitate and supernatant were collected separately. The cells were resuspended in an equal volume of sterile water, and the viable count was determined to be 4.2 × 10⁻⁶. 8 CFU / mL. The supernatant was concentrated under reduced pressure at 45°C to 1 / 4 of its original volume to obtain a supernatant concentrate. The temperature was controlled below 50°C during the concentration process to avoid degradation and inactivation of the heat-sensitive antibiotics.

[0047] To verify the physical and biological stability of the formulation, accelerated stability testing was conducted on the finished product. The formulation samples were stored at 4°C (refrigerated conditions), 25°C (room temperature conditions), and 37°C (accelerated conditions), and samples were taken monthly to test viable cell count, potency, and physical appearance. After 12 months of storage at 4°C, the viable cell count was 3.2 × 10⁻⁶. 8 The CFU / mL concentration was 6280 µg / mL, and all indicators remained above 90% of their initial values. After storage at 25°C for 12 months, the viable count was 2.8 × 10⁻⁶. 8 The CFU / mL count (80% of the initial value) and potency of 5800 µg / mL (89% of the initial value) still meet the quality standard requirements. After 6 months of storage at 37°C accelerated conditions, the viable count decreased to 1.5 × 10⁻⁶. 8 The CFU / mL concentration decreased to 4200 µg / mL, no longer meeting the quality standards, indicating a limited shelf life of the formulation under high-temperature conditions. Prolonged exposure to high temperatures should be avoided during actual storage and transportation. Sodium lignosulfonate, as a dispersant, effectively prevented the aggregation of bacterial cells and metabolite particles, while xanthan gum, as a thickener, provided suitable suspension stability. The synergistic effect of these two agents is key to maintaining the formulation's stability for more than 12 months at room temperature. The pH value of the formulation fluctuated by no more than 0.3 pH units during the 12-month storage period under various temperature conditions, indicating a reasonable pH buffer system design.

[0048] like Figure 4 As shown, the bacterial suspension and supernatant concentrate were mixed at a mass ratio of 1:4, and 1.0% sodium lignosulfonate (dispersant / wetting agent) and 0.3% xanthan gum (thickener / suspension stabilizer) were added. The mixture was homogenized at 3000 r / min for 10 min using a high-speed homogenizer, the pH was adjusted to 6.8, and the solution was passed through a 200-mesh stainless steel sieve and dispensed into 50 mL amber glass bottles to obtain the finished compound suspension formulation. The finished product test results are as follows: viable count 3.5 × 10⁻⁶. 8The product had a concentration of CFU / mL, a potency of 6520 µg / mL, a pH of 6.8, and was a uniform brownish-yellow suspension. After standing for 48 hours, the volume fraction of the supernatant was less than 5%, meeting the quality standards for suspension preparations. The finished product was stored at 25°C, and samples were taken every 3 months to test the viable count and potency. After 12 months, the viable count remained at 2.8 × 10⁻⁶. 8 The concentration of CFU / mL is above 5800 µg / mL, and the potency is maintained above 5800 µg / mL, indicating satisfactory storage stability.

[0049] Example 5: Field efficacy trial

[0050] The experiment was conducted at a rice planting base in Yangzhou City, Jiangsu Province, using Nanjing 46 (a rice blast-susceptible variety). The experimental design included four treatment groups: the formulation of this invention (100-fold dilution of the fermentation preparation of the rpsL K88E mutant), the fermentation preparation of wild-type YZ-01 (100-fold dilution, control biological agent), tricyclazole 750 g / L wettable powder (positive chemical control, recommended dosage), and a blank control (sprayed with water). Each treatment was replicated three times, with a plot area of ​​30 m². 2 The mixture was randomly arranged in a block design. The pesticide was sprayed once each at the late tillering stage, early booting stage, and 5-7 days before heading. The efficacy was calculated using the leaf blast disease index and neck blast incidence rate.

[0051] Table 3 Field control efficacy results for each treatment group

[0052]

[0053] Analysis of variance and Duncan's multiple comparison test were performed on the field efficacy data. The results showed that the differences in efficacy among the treatment groups were highly significant (F=42.68, P<0.001). The efficacy difference between the K88E mutant formulation treatment group and the blank control group was highly significant (P<0.001), the efficacy difference between the K88E mutant formulation treatment group and the wild-type YZ-01 formulation treatment group was significant (P<0.05), and the efficacy difference between the K88E mutant formulation treatment group and the tricyclazole positive control group was not significant (P>0.05). The 95% confidence interval of the comprehensive efficacy of the formulation of this invention is 76.9%, which is 74.0%~79.8%. The lower limit of 74.0% is close to but not lower than the target threshold of 75%. Considering the variability of the efficacy of biological agents in different years and ecological zones, it is recommended to adjust the application frequency and concentration appropriately according to the local climate and the occurrence pattern of rice blast in subsequent promotion. In terms of yield assessment, the actual yield of rice in the treatment area of ​​this invention increased by 28.5% compared with the blank control area, and decreased by 2.3% compared with the tricyclazole treatment area (the difference was not significant), indicating that the yield-preserving effect of this invention's formulation is basically equivalent to that of chemical pesticides. Rice quality analysis showed that the head rice rate, chalkiness, and amylose content of rice in the treatment area of ​​this invention's formulation were not significantly different from those in the blank control area, indicating that the formulation has no adverse effects on rice quality.

[0054] During field application, the following phenomena were observed: Within 24 hours of spraying the formulation of this invention, signs of mycelial shrinkage and deformation of rice blast fungus on the leaf surface were observed, which is consistent with the immediate antibacterial effect of polyketides and cyclic lipopeptides metabolites in the supernatant concentrate. Within 7-10 days after spraying, colonization traces of white powdery Streptomyces colonies were observed on the rice leaves in the treated areas, indicating that the live bacteria in the formulation successfully colonized the leaf surface and began to continuously produce extracellular hydrolases and siderophores. Regarding the evaluation of neck blast, the final spraying of the formulation of this invention 5-7 days before heading showed a particularly significant protective effect against neck blast, presumably related to the bioprotective layer formed by the continuous colonization and enzyme production of live bacteria at the neck of the panicle. Furthermore, a preliminary survey of the arthropod community in the rice fields of each treatment area was conducted during the experiment, and no significant adverse effects of the formulation of this invention on the population of natural enemy insects (such as spiders and parasitic wasps) in the rice fields were observed, preliminarily indicating that the formulation has good compatibility with the rice field ecosystem. After harvest, pesticide residue tests were conducted on the rice from each treatment area. No chemical pesticide residues were detected in the treatment area of ​​the formulation of this invention, which meets the quality and safety requirements of green food and organic agricultural products.

[0055] like Figure 5 As shown, field trial results indicate that the rpsL K88E mutant fermentation preparation of this invention achieved a comprehensive control efficacy of 76.9% against rice leaf blast and neck blast, exceeding the 75% control efficacy threshold. While this is approximately 4 percentage points lower than the tricyclazole positive control, it remains within the same efficacy range and has practical application value. Compared to the wild-type YZ-01 fermentation preparation (comprehensive control efficacy 57.0%), the rpsL K88E mutant fermentation preparation showed a 19.9 percentage point increase in efficacy, which was statistically significant (P<0.05) according to Duncan's multiple comparison test, fully demonstrating the crucial role of ribosome engineering in enhancing the field control efficacy of biocontrol Streptomyces.

[0056] Comparative Example 1: Wild-type strain (without rpsL K88E mutation)

[0057] Except for not performing rpsL K88E ribosome engineering, the fermentation and formulation preparation parameters were the same as in Example 4 (C / N ratio 27:1, corn steep liquor 0.8%). The potency of wild-type YZ-01 under optimal fermentation conditions was 3520±215 µg / mL, only 51.4% of the potency of the K88E mutant (6850 µg / mL). The overall field efficacy of the prepared formulation was 57.0%, failing to meet the 75% threshold requirement. This comparative example demonstrates that rpsL K88E ribosome engineering plays an indispensable role in achieving the target potency and field efficacy. Further analysis showed that although the potency of wild-type YZ-01 under a C / N ratio of 27:1 was 329% higher than that under a C / N ratio of 15:1 (820 µg / mL), it was still far below the target threshold of 5000 µg / mL. This indicates that relying solely on fermentation process optimization without genetic modification cannot overcome the ceiling limitation of the wild-type regulatory network on the synthesis of secondary metabolites. The expression levels of the PKS / NRPS gene cluster in wild-type strains are strictly constrained by endogenous regulatory factors. Even under optimal carbon-to-nitrogen ratio conditions, when ppGpp signaling fully activates and upregulates the gene cluster at the transcriptional level, the ribosomal translational machine still prioritizes the synthesis of growth-related proteins, resulting in limited translation efficiency of secondary metabolic mRNAs. The rpsL K88E mutation removes this translational bottleneck, enabling the efficient translation of sufficient mRNA provided at the transcriptional level into PKS / NRPS proteins, thereby achieving a doubling leap in titer.

[0058] Comparative Example 2: Low C / N ratio fermentation (C / N ratio 20:1)

[0059] Except for the carbon-to-nitrogen ratio being set to 20:1, all other parameters were the same as in Example 4 (rpsL K88E mutant strain, 0.8% corn steep liquor). The resulting fermentation broth potency was 2450±180 µg / mL, only 35.8% of the potency under the optimal carbon-to-nitrogen ratio conditions. The field efficacy of the prepared formulation was 48.5%, far below the 75% threshold. Under a carbon-to-nitrogen ratio of 20:1, the nitrogen source was sufficient, ppGpp accumulation was inhibited, Streptomyces mainly engaged in primary metabolism, and the expression level of the PKS / NRPS gene cluster was low. This comparative example demonstrates that a carbon-to-nitrogen ratio range of 25:1–30:1 is indispensable for achieving the target potency through the ppGpp signaling pathway.

[0060] Comparative Example 3: No corn steep liquor added (0% corn steep liquor)

[0061] Except for the absence of corn steep liquor, all other parameters were the same as in Example 4 (rpsL K88E mutant strain, C / N ratio 27:1). The resulting fermentation broth potency was 3680±220 µg / mL, and the field efficacy of the prepared formulation was 60.2%, which did not meet the 75% threshold requirement. The potency was significantly lower without the addition of corn steep liquor, indicating that corn steep liquor, as a complex organic nitrogen source and vitamin donor, provides NRPS substrate amino acid precursors and PKS cofactors (especially vitamin B1). 12 Methylmalonyl-CoA mutase plays an irreplaceable role in maintaining efficient metabolite synthesis.

[0062] Comparative Example 4: Pure bacterial suspension (without supernatant concentrate)

[0063] The bacterial cells obtained by centrifuging the fermentation broth in Example 4 were resuspended separately to prepare a bacterial suspension (viable count 4.2 × 10⁻⁶). 8 A control suspension was prepared by adding sodium lignosulfonate and xanthan gum (CFU / mL) without adding supernatant concentrate, and field efficacy was evaluated using the same method. The overall efficacy was 62.8%, lower than the compound formulation of this invention (76.9%), a difference of 14.1 percentage points. This comparative example shows that the synergistic combination of the immediate antibacterial components (polyketides and non-ribosomal peptide metabolites) in the supernatant concentrate and the live bacteria (continuous protective components) is indispensable for improving efficacy to over 75%. A single protective mechanism relying solely on enzyme production by live bacteria colonization is insufficient to achieve the level of chemical pesticide substitution.

[0064] Comparative Example 5: External fermentation at the carbon-to-nitrogen ratio ends (carbon-to-nitrogen ratios of 24:1 and 31:1)

[0065] To verify the rationality of the selection of the end values ​​of the C / N ratio range of 25:1 to 30:1 in the independent claims, two experiments were conducted outside the claim range, with C / N ratios of 24:1 and 31:1. The remaining parameters were the same as in Example 4 (rpsLK88E mutant strain, 0.8% corn steep liquor). The fermentation potency under the C / N ratio of 24:1 was 4520 ± 260 µg / mL, lower than the target threshold of 5000 µg / mL; the fermentation potency under the C / N ratio of 31:1 was 4680 ± 270 µg / mL, also lower than the target threshold. At a C / N ratio of 24:1, although approaching the nitrogen limitation condition, ppGpp accumulation was insufficient, and the degree of secondary metabolic activation was inadequate; at a C / N ratio of 31:1, the organic acid byproducts generated by the excess carbon source began to interfere with the normal supply of PKS precursors propionyl-CoA and methylmalonyl-CoA. The two out-of-range data points contrast sharply with the in-range data of 25:1 (potency 5620 µg / mL) and 30:1 (potency 5780 µg / mL), demonstrating that the selection of the carbon-nitrogen ratio range of 25:1 to 30:1 has a clear potency threshold boundary basis, rather than being an arbitrarily selected numerical range.

[0066] Comparative Example 6: External fermentation of corn steep liquor (0.4% and 1.1% corn steep liquor)

[0067] To verify the rationality of selecting the extreme values ​​of the corn steep liquor addition range of 0.5%–1.0%, two experiments were conducted outside these extreme values: 0.4% and 1.1%. All other parameters were the same as in Example 4 (rpsL K88E mutant strain, C / N ratio 27:1). The fermentation titer at 0.4% corn steep liquor was 4650 ± 255 µg / mL, lower than the target threshold of 5000 µg / mL, indicating that a 0.4% corn steep liquor addition was insufficient to provide adequate NRPS substrate amino acid precursors and vitamin B1. 12 Cofactors. The fermentation potency of corn steep liquor at 1.1% was 5950±300 µg / mL, which, although still above the 5000 µg / mL threshold, was 13.5% lower than the optimal value of 0.7% (6880 µg / mL). Considering the raw material costs and batch fluctuations in industrial production, setting the upper limit of corn steep liquor addition to 1.0% rather than a higher value provides a reasonable engineering safety margin. Comparison of the two out-of-range data with the in-range data at 0.5% (potency 6120 µg / mL) and 1.0% (potency 6520 µg / mL) confirms the technical rationality of the 0.5%~1.0% range.

[0068] The detection methods and data are analyzed in detail below:

[0069] I. Potency Determination Method. The agar diffusion method (cup-and-dish method) was used, with a spore suspension of rice blast fungus CGMCC 3.4981 as the indicator (final concentration 1×10⁻⁶). 6 The sample was prepared by punching wells (8 mm diameter) in PDA plates and adding 200 µL of the supernatant of the fermentation broth to each well. After incubation at 28°C for 48 h, the diameter of the inhibition zone was measured. A series of concentrations (100, 500, 1000, 2000, 5000, 10000 µg / mL) of nigericin standard were prepared to establish a standard curve. The regression equation was Y = 5.23X + 2.15 (R²). 2 =0.9983), where Y is the diameter of the inhibition zone (mm) and X is lg [potency (µg / mL)].

[0070] Summary analysis of potency and control efficacy of each comparative example. The potency and field control efficacy data of Example 4 and all comparative examples are summarized in Table 4.

[0071] Table 4 Summary of potency and field efficacy under various experimental conditions

[0072]

[0073] Table 4 clearly shows that the potency and efficacy of Example 4 of this invention are significantly superior to those of the comparative examples. Compared with Comparative Example 1 (removal of rpsL mutation), the potency increased by 94.6% and the efficacy increased by 19.9 percentage points, proving that ribosome engineering modification is the decisive factor for the potency leap. Compared with Comparative Example 2 (removal of C / N ratio optimization), the potency increased by 179.6%, indicating that C / N ratio optimization is an irreplaceable control parameter at the fermentation process level. Compared with Comparative Example 3 (removal of corn steep liquor), the potency increased by 86.1%, proving that the precursors and cofactors provided by corn steep liquor are crucial for the efficient synthesis of metabolites. Compared with Comparative Example 4 (removal of supernatant compound), the efficacy increased by 14.1 percentage points, verifying the synergistic effect of the two-component formulation design. The out-of-range data of Comparative Examples 5 and 6, from the perspective of numerical range boundaries, prove the technical rationality of the selection of the C / N ratio range of 25:1~30:1 and the corn steep liquor range of 0.5%~1.0% in the independent formulation. Based on the above analysis, the four innovative features in the technical solution of this invention (rpsL K88E mutation, carbon-nitrogen ratio of 25:1 to 30:1, corn steep liquor of 0.5% to 1%, and compounding of bacterial cells and supernatant) are all essential technical features that are indispensable for achieving the target potency and target efficacy. Removing any one of these features will prevent the achievement of the preset technical effect threshold.

[0074] II. Quantitative Demonstration of Nonlinear Synergistic Effect. To verify whether there is an additive synergistic effect between ribosome engineering and C / N ratio optimization, the data from Example 2 and Comparative Example 1 were analyzed as follows. The baseline potency was defined as 820 µg / mL for wild-type YZ-01 at a C / N ratio of 15:1 (minimum fermentation conditions). The potency gain of wild-type at a C / N ratio of 27:1 was 3520 - 820 = 2700 µg / mL (denoted as ΔA, i.e., the individual contribution of C / N ratio optimization). The potency gain of rpsL K88E mutant at a C / N ratio of 15:1 was 1280 - 820 = 460 µg / mL (denoted as ΔB, i.e., the individual contribution of rpsL mutant, the marginal effect under unoptimized C / N ratio conditions). If the two are simply linearly superimposed, the expected combined potency = 820 + 2700 + 460 = 3980 µg / mL. The actual combined potency was 6850 µg / mL, exceeding the expected value of 2870 µg / mL for linear superposition. The superadditive ratio was 6850 / 3980 = 1.72, meaning the actual potency was 1.72 times the expected linear potency. The mechanism of this superadditive effect lies in the following: the rpsL K88E mutation reprograms the ribosome at the translational level, enabling it to have higher translation efficiency for PKS / NRPS mRNA; and the 27:1 carbon-to-nitrogen ratio upregulates the mRNA abundance of the PKS / NRPS gene cluster at the transcriptional level through ppGpp signaling. When both levels are activated simultaneously, the abundant mRNA provided by the transcriptional layer is efficiently translated into protein by the translational layer, achieving a synergistic effect that transcends linear superposition.

[0075] The high efficiency of this invention stems from the systematic integration of the following multi-level synergistic mechanisms. At the genetic modification level, the rpsL K88E point mutation alters the tertiary structure of the 30S ribosomal subunit S12 protein, switching the ribosome from the wild-type high-speed, low-fidelity translation mode to a low-speed, high-fidelity mode. This conformational change effectively mimics the translation deceleration effect after ppGpp binds to RNA polymerase, allowing secondary metabolism-related mRNAs more ribosome occupancy time and significantly improving translation efficiency. At the fermentation process level, nitrogen restriction conditions with a carbon-to-nitrogen ratio of 25:1 to 30:1 activate the intracellular RelA protein to catalyze ppGpp accumulation. ppGpp, as a critical response signaling molecule, binds to the β' subunit of RNA polymerase, inhibiting the transcription of growth-related genes such as rRNA / tRNA, while simultaneously upregulating the transcriptional levels of PKS-I polyketide synthase and the NRPS gene cluster through pathway-specific regulatory factors. Corn steep liquor, as a complex organic nitrogen source and vitamin donor, provides substrate precursor amino acids such as alanine, glycine, and valine for the NRPS template, while also providing vitamin B1. 12 It assists in the activity of methylmalonyl-CoA mutase, ensuring an adequate supply of PKS precursor methylmalonyl-CoA.

[0076] At the level of siderophore competition, the rpsL K88E mutant showed a significantly enhanced siderophore synthesis capacity compared to the wild type. The Streptomyces genome typically contains multiple siderophore biosynthesis gene clusters, encoding deferoxamine and isohydroxamic acid siderophores. The rpsL K88E mutation globally improves the translation efficiency of secondary metabolic gene clusters while simultaneously enhancing the expression levels of genes related to siderophore biosynthesis. This results in live bacteria in the formulation exhibiting a stronger ferric ion chelating capacity after foliar colonization, further intensifying the competitive deprivation of iron nutrition from blast fungus. Iron is an essential cofactor for key iron-sulfur proteins such as cytochrome oxidase and cis-aconitase in the mitochondrial respiratory chain of blast fungus. Limited iron supply directly inhibits aerobic respiration and the TCA cycle of blast fungus, significantly weakening its infection and spread capabilities. Siderophore competition, as an indirect antibacterial mechanism, forms a triple synergistic multi-target protection network with the direct antibacterial effect of polyketides / non-ribosomal peptides and the cell wall lysis inhibition of extracellular enzymes, making it difficult for rice blast fungus to develop complete resistance to this preparation through single-gene mutation.

[0077] The technical solution of this invention achieves breakthroughs in three dimensions compared to existing technologies. The first dimension is the deep coupling of genetic modification and fermentation process. In existing technologies, ribosome engineering and fermentation medium optimization are usually carried out as two independent research directions. This invention, for the first time, systematically integrates the two into a synergistic technical system, utilizing the rpsL K88E mutation at the translational level and carbon-nitrogen ratio optimization at the transcriptional level to implement dual activation of the same PKS / NRPS gene cluster. Experimental data show that the synergistic potency improvement (over-addition ratio 1.72) far exceeds the simple summation of their individual contributions. The second dimension is the complete transformation from laboratory activity to field efficacy. Existing reports on Streptomyces resistance to rice blast generally remain at the stage of in vitro inhibition rate or greenhouse trials, with extremely scarce field efficacy data not exceeding 60%. This invention, through systematic optimization of process parameters and standardization of formulations, for the first time increases the field efficacy of Streptomyces biological agents to 76.9%, reaching the practical level of chemical pesticide substitution. The third dimension is the two-component synergistic design of the formulation system. Unlike traditional biological pesticide formulations that use only live bacteria or fermentation supernatant as single-component systems, this invention combines live bacteria (continuous protection function) with supernatant concentrate (immediate antibacterial function) in an optimized ratio. Experimental verification in Comparative Example 4 confirmed that the two-component combination achieved a 14.1 percentage point increase in efficacy compared to the single-component formulation, thus constructing a dual-protection system that combines immediate antibacterial effect with continuous protection.

[0078] At the formulation level, the combination of live bacteria and supernatant concentrate achieves antibacterial protection on two timescales. The polyketide macrolides enriched in the supernatant concentrate possess ion-carrier activity, embedding into the lipid bilayer of the rice blast fungus cell membrane and disrupting the transmembrane proton gradient. NRPS-encoded cyclic lipopeptides bind to the chitinous layer of the rice blast fungus cell wall, interfering with chitin synthase activity and inhibiting appressorium formation. Both components exert direct antibacterial effects immediately after application. After colonization on rice leaves, the live bacteria continuously secrete chitinase and β-1,3-glucanase to directly hydrolyze the rice blast fungus cell wall, while simultaneously secreting siderophores to chelate ferric ions in the environment, depriving the rice blast fungus of the iron nutrients it needs during the infection stage. This synergistic effect of immediate chemical inhibition and continuous biological protection is the core mechanism by which this formulation achieves a field control efficacy of over 75%.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Various modifications, substitutions, and changes made to the present invention by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention are also within the scope of protection of the present invention. Technical contents not described in detail in this invention are all common knowledge in the art. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims

1. A fermentation preparation of *Streptomyces* antagonistic to *Streptomyces*, characterized in that: The formulation is a composite suspension containing live *Streptomyces* cells of *rpsL* K88E or K88R mutant strains and a concentrate of fermentation supernatant, wherein the number of live *Streptomyces* cells is not less than 2 × 10⁻⁶. 8 The formulation has an inhibitory potency of CFU / mL against rice blast fungus greater than 5000 µg / mL, and contains sodium lignosulfonate and xanthan gum as dispersing agents.

2. The fermentation preparation according to claim 1, characterized in that, The formulation has a pH of 6.0 to 7.0 and a storage stability period of not less than 12 months at 25°C.

3. The method for preparing a fermentation agent of *Streptomyces* antagonistic to *Streptomyces*, according to claim 1 or 2, characterized in that, The process includes the following steps: (1) Strawberry strain acquisition: obtaining a Streptomyces strain with an inhibition rate of greater than 80% against Magnaphorthe oryzae, the causal agent of rice blast; (2) Ribosome engineering modification: introducing a point mutation in the 88th lysine codon of the rpsL gene coding region of the starting strain, replacing AAG with GAG encoding glutamic acid or CGG encoding arginine, to obtain an rpsL mutant strain; (3) Fermentation culture: inoculating the rpsL mutant strain into a fermentation medium for liquid deep fermentation, wherein the carbon-nitrogen ratio of the fermentation medium is 25:1 to 30:1 and contains 0.5% to 1% corn steep liquor by mass, the fermentation temperature is 28 to 30°C, the fermentation time is 5 to 7 days, and the resulting fermentation broth has an inhibitory potency of greater than 5000 µg / mL against Magnaphorthe oryzae; (4) Formulation preparation: after centrifugation of the fermentation broth obtained in step (3), the bacterial suspension and the supernatant concentrate are mixed, and a dispersant and a thickener are added to obtain a composite suspension formulation containing live bacteria and metabolites.

4. The preparation method according to claim 3, characterized in that, The point mutation described in step (2) was introduced into the coding region of the rpsL gene by overlapping extension PCR, and introduced into the starting strain via conjugation transfer using the integrative vector pSET152. The mutant strain was obtained by screening with streptomycin 100 µg / mL plates.

5. The preparation method according to claim 3, characterized in that, Step (2) also includes further screening for rifampicin-resistant mutants based on the rpsL mutant to obtain rpsL and rpoB dual mutants.

6. The preparation method according to claim 3, characterized in that, The carbon-to-nitrogen ratio of the fermentation medium in step (3) is 27:1 to 28:1, the amount of corn steep liquor added is 0.7% to 0.9% by mass, the fermentation medium uses soluble starch as carbon source and soybean flour as nitrogen source, and also contains 0.1 to 0.3 g / L potassium dihydrogen phosphate and 0.05 to 0.15 g / L magnesium sulfate heptahydrate.

7. The preparation method according to claim 3, characterized in that, The fermentation culture in step (3) also includes a seed culture preparation step: the rpsL mutant is inoculated into a seed culture medium, which uses 2% to 4% soybean powder and 1% to 2% glucose as the main carbon and nitrogen sources, and is cultured at 28 to 30°C and 150 to 200 r / min for 48 h to obtain the seed culture. The inoculation amount of the seed culture is 8% to 12% of the fermentation culture medium volume.

8. The preparation method according to claim 3, characterized in that, In step (4), the mass ratio of the bacterial suspension to the supernatant concentrate is 1:3 to 1:5, the dispersant is sodium lignosulfonate, and the amount added is 0.5% to 2.0% of the total mass of the preparation, and the thickener is xanthan gum, and the amount added is 0.2% to 0.5% of the total mass of the preparation.

9. The application of the rice blast fungus antagonistic Streptomyces fermentation agent according to claim 1 or 2 in the control of rice blast, characterized in that, The preparation is diluted 100 to 300 times and sprayed on rice from the late tillering stage to the booting stage, with 2 to 3 applications. The comprehensive control efficacy against leaf blast and neck blast is not less than 75%.

10. The application according to claim 9, characterized in that, The formulation is used alternately with a chemical fungicide, which is tricyclazole or isoprothiolane, and is used alternately every 1 to 2 application cycles to delay the development of resistance to the chemical fungicide by rice blast fungus.

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