A biocontrol agent for controlling tomato gray mold and its application

Abstract

This invention provides a biocontrol bacterium for controlling tomato gray mold and its application, belonging to the field of microbial technology. The biocontrol bacterium is... Streptomyces cavourensis The CS19 strain was deposited on January 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.37282. This invention also provides a biocontrol suspension containing the CS19 strain and a biocontrol agent containing the fermentation filtrate of the CS19 strain. Furthermore, this invention provides the application of the above-mentioned CS19 strain, biocontrol suspension, or biocontrol agent in inhibiting the growth of *Botrytis cinerea*. The above-mentioned CS19 strain can effectively inhibit the elongation of *Botrytis cinerea* mycelium and the germination of spores; the propylparaben, ethylparaben, and indole-3-carboxaldehyde contained in its fermentation filtrate exhibit good antibacterial activity against *Botrytis cinerea*; moreover, the fermentation filtrate is non-pathogenic to tomato fruits and can effectively inhibit the growth and infection of *Botrytis cinerea* mycelium in leaves and fruits.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and specifically to a biocontrol bacterium for controlling tomato gray mold and its application. Background Technology

[0002] Gray mold is caused by the fungus *Botrytis cinerea* (…). Botrytis cinerea Botrytis cinerea (also known as Gracilaria) is a fungal disease caused by bacteria that spreads through air and soil. Under natural conditions, Botrytis cinerea exists in plant debris and soil as conidia, mycelium, or sclerotia. Sclerotia can overwinter in the soil, resistant to low temperatures and other adverse conditions. When temperatures rise in spring, the sclerotia germinate in the host, forming mycelium, which produces conidia that infect plant cells, inducing cell death and causing the infected plant tissue to gradually rot. Studies have shown that the optimal conditions for conidia germination are a temperature above 16°C and humidity above 95%. Conidia can be spread through various means, including air and water, and are characterized by rapid reproduction and a wide host range.

[0003] Tomatoes are an important crop for agricultural economic development, widely planted and high-yielding in my country. However, during the growth process and post-harvest storage, the stems, leaves, and fruits are susceptible to infection by gray mold, causing plant rot and death, resulting in significant economic losses. When seedlings are infected, the leaves show water-soaked rot, and the young stems initially constrict in a water-soaked manner, later forming brown lesions that are easily broken. In fruit, the disease often begins with residual withered flowers or stigmas, first causing flower rot, then spreading to the fruit surface and pedicel. Symptoms first appear near the fruit stem, pedicel, or navel; young fruits become soft and rotten throughout, and before maturity, the diseased parts of the fruit peel show grayish-white water-soaked soft rot, rapidly expanding into large, irregular spots. The fruit either becomes dehydrated and stiff or moist and soft. Diseased fruits generally do not fall off and easily spread to other parts, causing the entire bunch to rot in severe cases. Leaf infection typically begins at the leaf tip, spreading inwards in a "V" shape. Initially, the lesions are water-soaked with irregular edges, later turning light brown to yellowish-brown with alternating light and dark concentric rings. Fallen petals and stamens on leaves or branches can form circular or spindle-shaped lesions. Stems are less affected; damaged areas initially appear water-soaked, then expand into elongated oval or striped spots, eventually encircling the stem and causing the upper branches and leaves to wilt and die. In high humidity, a dense layer of grayish-brown mold will develop on the fruit, leaves, and stems affected by the disease. Severe cases can lead to premature plant death, production disruption, and significant losses.

[0004] Currently, the control of gray mold mainly includes strengthening field cultivation management, breeding highly resistant varieties, and applying pesticides. While these measures can reduce the occurrence of gray mold to some extent, they also have certain drawbacks. Therefore, biological control through the secondary metabolites of microorganisms has become a hot topic that is efficient, safe, and in line with green ecological development. Currently, various fungi, bacteria, and actinomycetes have been found to effectively control gray mold through different mechanisms of action. Among them, fungi mainly include *Trichoderma* and *Pseudomonas erythrosporum*; bacteria mainly include *Bacillus* and *Pseudomonas*; and actinomycetes are mainly *Streptomyces*. Biocontrol bacteria, due to their diversity, rapid reproduction, wide host range, and ease of cultivation, have better survival, competition, and adaptability in the natural environment, and are therefore widely used in the research and development of biological control products. Summary of the Invention

[0005] In view of this, the main objective of the present invention is to provide a biocontrol bacterium, fermentation filtrate and application of which can effectively prevent and control tomato gray mold. The biocontrol bacterium can effectively inhibit the spore germination and mycelial elongation of tomato gray mold, and is not pathogenic to tomato fruit. It is environmentally friendly and safe in practical applications.

[0006] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides a biocontrol agent for controlling gray mold in tomatoes, namely *Streptomyces carvulgella*. Streptomyces cavourensis The CS19 strain was deposited on January 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37282.

[0007] The CS19 strain was screened from crop cultivation soil in Zhongmu County, Zhengzhou City, Henan Province, and it showed good inhibitory effects against tomato gray mold. The full-length nucleotide sequence of the strain's 16S rRNA is shown in SEQ ID NO.3, identifying it as *Streptomyces kauri*. Streptomyces cavourensis Experiments have shown that strain CS19 and the fermentation filtrate both have a significant inhibitory effect on the growth of Botrytis cinerea (gray mold) on tomatoes.

[0008] Secondly, the present invention provides a biocontrol bacterial seed solution for controlling tomato gray mold, comprising the aforementioned CS19 strain. Preferably, the seed solution is mainly prepared by inoculating the CS19 strain into NYBD medium and culturing it until the absorbance reaches 490 nm = 0.05–0.1. More preferably, the seed solution is cultured at 30 °C for 24 h. The sterile fermentation filtrate obtained by filtering the bacterial suspension after fermentation through a 0.22 μm sterile filter can effectively control tomato gray mold.

[0009] Thirdly, the present invention provides a biocontrol agent for controlling tomato gray mold, comprising the fermentation filtrate of the aforementioned CS19 strain. Preferably, the fermentation filtrate includes propylparaben, ethylparaben, and indole-3-carboxaldehyde, which are potential antibacterial substances capable of effectively inhibiting... B. cinerea active.

[0010] Furthermore, the fermentation filtrate is mainly obtained by inoculating CS19 seed culture with an absorbance of 490nm = 0.05-0.1 into NYBD medium at a volume ratio of 1:1000, fermenting at 30 °C for at least 10 days, and then filtering. Preferably, the stirring speed during the fermentation process is 180-200 r / min.

[0011] Fourthly, the present invention provides the application of the above-mentioned biocontrol bacteria, the above-mentioned biocontrol bacteria suspension, or the above-mentioned biocontrol agent in inhibiting the growth of botrytis cinerea in tomatoes.

[0012] Furthermore, the application manifests itself in at least one of the following aspects: 1) Inhibits the elongation of mycelia of Botrytis cinerea (gray mold) on tomatoes; 2) Inhibits the spore germination of tomato gray mold.

[0013] Furthermore, the application includes applications in at least one of the following aspects: 1) Inhibits the growth of Botrytis cinerea mycelium in detached tomato fruits; 2) Inhibits the infection of Botrytis cinerea mycelia in detached tomato fruits; 3) Inhibits the growth of gray mold mycelium on tomato leaves; 4) Inhibits the infection of gray mold mycelium on tomato leaves.

[0014] Experiments have shown that strain CS19 can effectively inhibit the mycelial elongation and spore germination of *Botrytis cinerea*. In the plate confrontation test, the diameter of *Botrytis cinerea* inoculated with CS19 differed from that of the control group by up to 5.88 cm, demonstrating significant antibacterial activity. Under a 40x microscope, it was found that the mycelial density of *Botrytis cinerea* in the treatment group increased and the apical branching increased, indicating that its growth was significantly inhibited. The 20% volume ratio fermentation filtrate of CS19 after 10 days of liquid fermentation showed an inhibition rate of 86.47% against *Botrytis cinerea*. Eight potential antibacterial substances were screened from the metabolites of CS19 fermentation filtrate by non-targeted detection analysis using LC-MS / MS. Among them, propylparaben, ethylparaben, and indole-3-carboxaldehyde showed good antibacterial activity. At a concentration of 200 μg / mL, propylparaben achieved an inhibition rate of 100%, and ethylparaben achieved an inhibition rate of 88.63%, which was significantly better than the commonly used preservative benzoic acid and the commonly used pesticide pyrimethanil. This provides a reference for the development and application of biocontrol agents in the later stage. The aseptic fermentation filtrate of strain CS19 was non-pathogenic to tomato fruit and could effectively inhibit the growth and infection of Botrytis cinerea mycelium in tomato leaves and fruit. Attached Figure Description

[0015] Figure 1 For strain CS19 and B. cinerea A rendering of a two-panel standoff; Figure 2 For strain CS19 B. cinerea Microscopic image of hyphal morphology inhibition (10×40). Figure 3 The plate morphology and Gram staining of strain CS19 are shown. Figure 4 A phylogenetic tree constructed based on the full-length 16S rRNA sequence of strain CS19; Figure 5 The growth curve of strain CS19; Figure 6 The effects of different volume ratios of fermentation filtrate from strain CS19 on B. cinerea Growth inhibition; Figure 7 The results of non-targeted detection by LC-MS / MS of fermentation filtrate of strain CS19; Figure 8 For potential antibacterial substances B. cinerea Its antibacterial effect; Figure 9 The image shows the control effect of aseptic fermentation filtrate of strain CS19 on botrytis cinerea in detached tomato fruits. Figure 10 The image shows the control effect of aseptic fermentation filtrate of strain CS19 on botrytis cinerea on detached leaves. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0017] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Raw materials, equipment, or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, the reagents, culture media, etc., used in this invention are all existing conventional reagents and can be prepared using existing methods or purchased commercially.

[0018] The culture media used in the following examples of this invention are as follows: PDA medium: 200g potato, 20g anhydrous glucose, 20g agar powder, 1000mL distilled water.

[0019] Gao's No. 1 medium: 20g soluble starch (mixed with cold water first and then heated), 0.5g NaCl, 1g KNO3, 20g agar, 0.5g K2HPO4·3H2O, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 1000 mL distilled water, pH 7.4-7.6. When pouring the medium, add 5 mg potassium dichromate per 100 mL of medium (potassium dichromate cannot be autoclaved).

[0020] NYBD medium: 3g beef extract, 5g peptone, 10g glycerol, 5g mannitol, pH 7.0-7.2, 1000mL distilled water.

[0021] Example 1: Biocontrol strain Streptomyces kawuerii Streptomyces cavourensis Isolation, screening and identification of CS19 I. Isolation and Screening of Biocontrol Strains CS19

[0022] Weigh 10 g of cultivated soil sample collected from Zhongmu County, Zhengzhou City, Henan Province, and add it to a 250 mL Erlenmeyer flask containing 90 mL of sterile water. Place the flask in a shaker at 28 ℃ and shake at a constant speed of 180-200 r / min for 30 min to fully disperse the microbial cells. Let it stand for 20-30 s to obtain 10 g of the sample. -1 Suspension. After the sample separates into layers, take 1 mL from the upper layer and add it to 9 mL of sterile water. Mix well to obtain 10 mL of the suspension. -2 Suspension. Perform serial dilutions to achieve a turbid concentration of 10. -3 ~10 -7 Take 10 respectively -2 10 -3 and 10 -4 Three concentration gradient turbid solutions, 100 μL each, were inoculated into Gao's No. 1 medium. 10 μL of each solution was then taken... -5 10-6 and 10 -7 Three concentration gradient turbid solutions, 100 μL each, were inoculated into PDA and LB media. The Gao's No. 1 and LB media were incubated in the dark at 35 ℃ for 3 days, and the PDA media were incubated in the dark at 24 ℃ for 3–5 days, until colonies appeared. Typical colonies were selected based on their morphology, size, and color, and inoculated onto new media. Purification was performed 3–5 times using the streak plate method to obtain single colonies.

[0023] The plate confrontation method was used to determine the effect of isolated strains on... B. cinerea Antagonistic activity. Mature spores in PDA plates were washed with sterile PDB and collected into centrifuge tubes. Adjustment B. cinerea The spore concentration was 1×10 5 cfu / mL. Place a 6 mm sterile filter paper in the center of a PDA plate and inoculate with 5 μL of spore suspension. Inoculate the strain at four symmetrical points 2 cm from the center. Use plates inoculated only with spore suspension as a control. Repeat each plate three times. After incubating at 24 ℃ for 5 days, photograph plates with inhibition zones. Figure 1 As shown, the diameter is measured and the antibacterial rate is calculated using the cross-cross method.

[0024] Antibacterial rate = [(Coronavirus diameter in control group - Coronavirus diameter in treatment group) / Coronavirus diameter in control group] × 100% Figure 1 The Actinomycete CS19 shown exhibited good antagonistic activity, with a colony diameter difference of up to 5.88 cm compared to the control group. Therefore, the Actinomycete CS19 strain was identified as the target biocontrol bacterium.

[0025] Actinomycetes CS19 and B. cinerea The confrontation plates showed antibacterial effects after 3 days of incubation, with the antibacterial effect becoming more pronounced on the 4th day. The confrontation plates after these two days were observed under a 40x microscope. B. cinerea The morphological changes of the outermost creeping hyphae were obtained as follows: Figure 2 The results shown are from Figure 2 The results show that Actinomycete CS19 has a good inhibitory effect on the growth and elongation of Botrytis cinerea hyphae and changes the hyphal morphology. The control group has a lower hyphal density, longer hyphae, and extensive elongation and growth in all directions. After treatment with Actinomycete CS19, the hyphal density of Botrytis cinerea increased, the hyphal tips branched more, and the growth was significantly inhibited.

[0026] Therefore, through extensive screening, this invention has obtained an actinomycete strain CS19 that has a significant inhibitory effect on Botrytis cinerea. It can alter the morphology of Botrytis cinerea and effectively inhibit the growth of Botrytis cinerea mycelia. It has broad research value and application prospects and can be used for the research and development of biocontrol agents for Botrytis cinerea. II. Morphological and molecular biological identification of biocontrol strain CS19

[0027] 1. Morphological observation and identification The CS19 strain, which has good antibacterial activity, was inoculated into Gao's No. 1 plates and cultured at 35 ℃ for 7 days. The morphology, size, color, and pigment production of the colonies were observed, and Gram staining was performed.

[0028] The specific procedure for Gram staining includes: sampling, drying, and fixing single colonies cultured for 7 days using the smear method; adding crystal violet staining solution, staining for 1 minute, and rinsing with water; adding iodine solution, staining for 1 minute, and rinsing with water; adding destaining solution, shaking the slide, and destaining for about 20-60 seconds depending on the thickness of the smear, rinsing with water, and absorbing excess water; adding safranin staining solution, staining for 1 minute, and rinsing with water; absorbing excess water with filter paper or air-drying, and then examining under a 100x oil immersion microscope.

[0029] Microscopic examination results of strain CS19 are shown below Figure 3 , Figure 3 Blue-purple indicates positive bacteria, and red indicates negative bacteria. From Figure 3 As can be seen, the CS19 colony morphology on Gao's No. 1 agar plates is characterized by dry, opaque, round, wrinkled, and radial colonies. The hyphae and aerial hyphae are white or gray, while the basal hyphae are pale yellow and pigment-producing. Gram staining is purple, indicating Gram-positive bacteria. The spores are chain-like and linear. Therefore, strain CS19 exhibits the typical morphology of the Streptomyces genus.

[0030] 2. Molecular biological identification The 16S rRNA gene of strain CS19 was amplified and sequenced. The primer nucleotide sequences were: 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The PCR amplification program was: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 55 ℃ annealing for 20 s, 72 ℃ extension for 1 min, for 34 cycles, followed by a 72 ℃ extension for 10 min, and storage at 4 ℃. The PCR product was analyzed by 1% agarose gel electrophoresis and then sent to Sangon Biotech Co., Ltd. for sequencing. The sequencing results, compared with NCBI sequence alignment, showed high homology with various Streptomyces. However, based on partial 16S rRNA gene sequences, identification is often limited to the genus level, making it difficult to pinpoint the specific species.

[0031] To determine which species within the *Streptomyces* genus CS19 belongs to, strain CS19 was inoculated into NYBD medium and cultured at 30°C and 180-200 r / min for 36 h. Bacterial cells were collected in 2 mL centrifuge tubes, washed 1-2 times with PBS buffer to remove residual culture medium components, and centrifuged to collect at least 0.3 g of bacterial cells. The centrifuge tubes containing the collected cells were labeled and numbered, flash-frozen in liquid nitrogen for at least 10 min, stored on dry ice, and sent to Wuhan Benatech Co., Ltd. for whole-genome sequencing.

[0032] Gene sequences of 16S rRNA, trpB, atpD, gryB, and recA were selected from the whole genome sequencing results of Streptomyces CS19 and performed BLAST alignment on NCBI. Sequences with high homology were selected, and a Neighbor Joining phylogenetic tree was constructed using MEGA11. Figure 4 As shown. The results indicate that *Streptomyces CS19* and *Streptomyces carvulgella*... Streptomyces cavourensis The strains clustered in the same branch and showed high homology. Combining morphological and molecular identification, strain CS19 was ultimately identified as *Streptomyces kawuerii*. Streptomyces cavourensis The full-length 16S rRNA nucleotide sequence of strain CS19 is shown in SEQ ID NO.3. This strain was deposited on January 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37282, located in Beijing, China. III. Growth curve changes of strain CS19

[0033] Streptomyces is an aerobic bacterium that readily forms indestructible mycelial spheres during liquid culture, resulting in uneven distribution within the culture medium. Traditional methods for plotting growth curves by measuring the OD value of single-celled bacteria are not suitable. Therefore, this study references a patented method for determining the growth curve of Streptomyces strain CS19 published by the Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences. The specific procedure is as follows: (1) The bacterial strain stored in a -80 ℃ refrigerator was spread on Gao's No. 1 solid plate medium and cultured in a 35 ℃ incubator for 7 days to obtain the plate bacterial strain.

[0034] (2) Take a 1 cm sample from a well-grown plate. 2 The spores were inoculated into 10% NYBD liquid medium and cultured at 30 °C and 180-200 r / min. Samples were taken every 12 h after inoculation. After the mycelium was evenly dispersed, 1 mL of bacterial solution was aspirated into a 2 mL centrifuge tube. Three replicates were set up for each time gradient. (3) Weigh 0.1 g of 2,3,5-triphenyltetrazolium chloride powder into a light-protected centrifuge tube, add 10 mL of PBS with a concentration of 10 mg / mL stored at room temperature to dissolve it, filter to sterilize and obtain TTC solution.

[0035] (4) Add TTC solution at a ratio of 1% to the samples taken in step (2) at different time periods, and react at room temperature in the dark for 30 min.

[0036] (5) Centrifuge the reaction solution, remove the supernatant and collect the precipitate; then add 500 μL of DMSO solution to the precipitate and dissolve it at room temperature in the dark for 30 min to form a red solution. After centrifuging the red solution, take 200 μL of the supernatant and add it to a 96-well plate. Use DMSO solution as a blank control and measure its absorbance at 490 nm wavelength using an enzyme-linked immunosorbent assay reader.

[0037] The absorbance of the samples at each time point was set up in triplicate. Using Graphpad Prism software, growth curves were plotted with the measurement time as the X-axis and the absorbance at 490 nm as the Y-axis, as shown below. Figure 5 As shown, CS19 is in the logarithmic growth phase from 0 to 24 hours, during which the cell proliferation increases rapidly and the activity is good. Therefore, the seed culture time during fermentation is about 24 hours. When the culture time reaches 36 hours, the respiration of Streptomyces weakens and the growth curve tends to be relatively stable. Example 2 Application of strain CS19

[0038] This embodiment mainly aims to understand the effect of the fermentation filtrate of strain CS19 on... B. cinerea Inhibition effect I. Effects of different volume ratios of fermentation filtrate at different cultivation times on B. cinerea Inhibition effect The bacterial strain stored at -80 °C was inoculated into 50 mL centrifuge tubes containing 5 mL of NYBD medium at a volume ratio of 1:1000. The culture was prepared by incubating at 30 °C and 180–200 r / min for 24 h. After the mycelium in the seed culture was evenly dispersed, it was inoculated into 100 mL Erlenmeyer flasks containing 50 mL of NYBD medium at the same ratio and incubated at 30 °C and 180–200 r / min for 1, 3, 5, 7, 10, and 15 days. The obtained bacterial culture was filtered through a 0.22 μm filter to obtain sterile fermentation filtrate.

[0039] Fermentation filtrates from different fermentation times were added to the PDA medium to be solidified at volume ratios of 5%, 10%, 15%, and 20% to prepare drug-containing plates. Blank PDA plates were used as controls. A 6 mm sterile filter paper disc was placed at the center of each treatment medium, and 5 μL of a 1×10⁻⁶ concentration was inoculated onto it.5 cfu / mL B. cinerea Spore suspension. Photographs were taken after 5 days of incubation at 24 ℃. Colony diameters for each treatment were measured using the cross-hatching method, and the inhibition rate was calculated. The experiment was repeated three times. Results are shown below. Figure 6 .

[0040] Antibacterial rate = [(Coronavirus diameter in control group - Coronavirus diameter in treatment group) / Coronavirus diameter in control group] × 100% from Figure 6 As can be seen, the fermentation filtrate of strain CS19 began to show significant antibacterial activity after 5 days of fermentation, and the antibacterial activity continued to strengthen with the increase of the volume fraction of the fermentation filtrate. After 10 days of fermentation, the inhibition rate reached 74.67% at a volume fraction of 10%, and 86.47% at a volume fraction of 20%. II. Metabolomics Analysis of Fermentation Filtrate from Strains CS19

[0041] Based on the growth curve of strain CS19, secondary metabolites began to be synthesized in large quantities after 36 h of culture. The bacterial culture, cultured at 30℃ and 180-200 r / min for 36 h, was filtered through a 0.22 μm filter, and the sterile fermentation filtrate was collected, cryopreserved in liquid nitrogen, and sent to Wuhan Bena Technology Co., Ltd. for non-targeted metabolomics analysis to comprehensively detect metabolites in the fermentation filtrate. The detection results are shown below. Figure 7 Four major classes of metabolites were detected in the samples. Among them, 1259 substances matched the public databases MS1 and MS2, and 332 substances matched the standards in MS1, MS2, and RT. Based on the MS2 score, eight substances with potential antibacterial properties and one commonly used preservative were screened from the 332 substances, as shown in Table 1.

[0042] Table 1 Potential antibacterial substances in the fermentation filtrate of strain CS19

[0043] III. Effects of different concentrations of potential antibacterial substances on B. cinerea Antibacterial effect The effects of different concentrations of potential antibacterial substances on mycelial growth inhibition were determined using a mycelial growth inhibition test. B. cinerea The antibacterial activity was determined. 0.25 g of the standard was weighed into a 10 mL centrifuge tube, dissolved in 5 mL of DMSO to prepare a 50 mg / mL stock solution, which was then filtered through a 0.22 μm filter. The stock solution was added to 40 mL of PDA medium to prepare plates with concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL. A 6 mm sterile filter paper was placed in the center of each plate, and 5 μL of the solution was inoculated with 1×10⁻⁶ mol / L of the medium. 5 cfu / mL B. cinereaSpore suspension. Commonly used pesticides for controlling gray mold, pyrimethanil, and benzoic acid, a common preservative, were used as controls. The control (CK) was a blank control without any added substances. Since the potential antimicrobial substance is soluble in DMSO but not in water, DMSO solution was added to the plates as an additional control (DMSO-CK) to determine the effect of the solvent on the antimicrobial activity of the potential antimicrobial substance. Each plate was replicated three times. After incubation at 24 °C for 5 days, photographs were taken, and colony diameters were measured using the cross-hatching method. The calculated inhibition rates are shown in Table 2.

[0044] When preparing pyrimethanil stock solution, the mass of the drug should be calculated based on the content of the active ingredient.

[0045] Table 2: Effects of different concentrations of potential antibacterial substances on... B. cinerea Antibacterial rate

[0046] Note: Mycelial growth inhibition rate = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100 Different concentrations of potential antibacterial substances B. cinerea Antibacterial plates can be found Figure 8 ,from Figure 8 As shown in Table 2, the antibacterial rate of several potential antibacterial substances gradually increases with increasing concentration. Among them, propylparaben, ethylparaben, and indole-3-carboxaldehyde exhibit better antibacterial activity. At a concentration of 200 μg / mL, propylparaben achieved a 100% antibacterial rate, while ethylparaben achieved an 88.626% antibacterial rate, significantly better than the commonly used preservative benzoic acid and the commonly used pesticide pyrimethanil. At concentrations above 200 μg / mL, indole-3-carboxaldehyde showed slightly better antibacterial activity than pyrimethanil, but no significant difference compared to benzoic acid. Therefore, propylparaben and ethylparaben, as potential antibacterial substances in fermentation broth, provide a reference for the later development and application of biocontrol agents. IV. Effects of CS19 fermentation filtrate on postharvest tomato fruit

[0047] Select healthy tomatoes of uniform size, color, and ripeness. Soak them in 84 disinfectant solution for 2 minutes, then rinse three times with sterile water and air dry. Use a PDB rinsing plate... B. cinerea Spores and adjust the concentration to 1×10 6CFU / mL, using a sterile pipette tip, holes approximately 2 mm deep and 1-2 mm in diameter were made at the equator of the fruit. Different treatment solutions were inoculated at the wounds: control group: 2 μL spore suspension + 2 μL sterile water; prevention group: 2 μL CS19 sterile fermentation filtrate + 2 μL spore suspension (treated with CS19 sterile fermentation filtrate first, then inoculated with spore suspension); control group: 2 μL spore suspension + 2 μL CS19 sterile fermentation filtrate (inoculated with spore suspension first, then treated with CS19 sterile fermentation filtrate). The interval between two inoculations was 4 h, with 9 replicates for each treatment, and this experiment was independently replicated 3 times. The fruits were placed in disposable plastic boxes and incubated at room temperature. Fruit disease incidence was observed and recorded 1-5 days after inoculation. The CS19 sterile fermentation filtrate used in this experiment was prepared by fermentation on NYBD liquid medium at 30 ℃ and 180-200 r / min for 10 days, followed by filtration through a 0.22 μm filter.

[0048] See results Figure 9 As shown, the control group of tomatoes developed obvious lesions and mycelia 48 hours after inoculation with the pathogen. Over the next 5 days, the lesion area and mycelia expanded rapidly, with depressions appearing around the lesions, and the color changing from light to dark. At 5 days after inoculation, the average lesion area was 2.73 cm². 2 The tomato fruits treated with CS19 aseptic fermentation filtrate showed differences compared to the control group. A small amount of mycelium appeared on the inoculated tomato fruits 48 hours after inoculation, and the mycelial growth rate was lower than that of the control group in subsequent observations. Therefore, CS19 aseptic fermentation filtrate can effectively inhibit the development of tomato rot caused by Botrytis cinerea. V. The efficacy of fermentation filtrate of strain CS19 in detached leaves

[0049] Tomato leaves of the same age and size were harvested, washed three times with sterile water, and dried with sterile gauze. The leaves were then placed face down on moist filter paper and inoculated with different treatment solutions according to the inoculation method described in "IV. Effects of CS19 Fermentation Filtrate on Postharvest Tomato Fruits". The interval between two inoculations was 4 hours, and each treatment was replicated 6 times. The treated tomato leaves were placed in disposable plastic containers and incubated at room temperature. Disease development and lesion area were observed and measured for 5 consecutive days. Results are as follows: Figure 10 As shown.

[0050] Figure 10 The results showed that tomato leaves developed distinct water-soaked lesions with irregular edges 48 hours after inoculation with the pathogen. The lesion area gradually expanded. Five days after inoculation, the average lesion diameter on the control group tomato leaves was 10.18 mm, while leaves treated with CS19 aseptic fermentation filtrate showed obvious lesions on the third day, and the lesion area did not significantly expand over time. Therefore, CS19 aseptic fermentation filtrate can not only inhibit... B. cinerea The growth of mycelium can also control the development of gray mold disease.

[0051] In summary, the actinomycetes screened from cultivated soil samples in Zhongmu County, Zhengzhou City, Henan Province in this invention are *Streptomyces carvulneratus*. Streptomyces cavourensis The CS19 strain effectively inhibited the mycelial elongation and spore germination of *Botrytis cinerea*. In the plate confrontation test, the diameter of *Botrytis cinerea* inoculated with strain CS19 differed from that of the control group by up to 5.88 cm, demonstrating significant antifungal activity. Under a 40x microscope, it was found that the mycelial density of *Botrytis cinerea* inoculated with strain CS19 increased and the apical branching increased, indicating significant inhibition of growth. The 20% volume ratio fermentation filtrate of strain CS19 after 10 days of liquid fermentation showed an inhibition rate of 86.47% against *Botrytis cinerea*. Eight potential antibacterial substances were screened from the fermentation filtrate metabolites of strain CS19 by non-targeted detection analysis using LC-MS / MS. Among them, propylparaben, ethylparaben, and indole-3-carboxaldehyde showed good antibacterial activity. At a concentration of 200 μg / mL, propylparaben achieved a 100% inhibition rate, and ethylparaben achieved an 88.626% inhibition rate, which were significantly better than the commonly used preservative benzoic acid and the commonly used pesticide pyrimethanil. The aseptic fermentation filtrate of strain CS19 was non-pathogenic to tomato fruits and could effectively inhibit the growth and infection of Botrytis cinerea mycelium in leaves and fruits.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A biocontrol agent for controlling gray mold in tomatoes, characterized in that, Streptomyces carvulgii Streptomyces cavourensis The CS19 strain was deposited on January 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37282.

2. A biocontrol bacterial seed solution for controlling tomato gray mold, characterized in that, Includes the CS19 strain as described in claim 1.

3. The biocontrol bacterial seed solution according to claim 2, characterized in that, The CS19 strain was inoculated into NYBD medium and cultured until the absorbance reached 490 nm = 0.05–0.

1.

4. A biocontrol agent for controlling gray mold in tomatoes, characterized in that, The fermentation filtrate includes the fermentation filtrate of the CS19 strain as described in claim 1 or the fermentation filtrate obtained by liquid fermentation culture of the biocontrol bacteria seed liquid as described in any one of claims 2 to 3.

5. The biocontrol agent according to claim 4, characterized in that, The fermentation filtrate is obtained by inoculating CS19 seed culture with an absorbance of 490nm = 0.05-0.1 into NYBD medium at a volume ratio of 1:1000, fermenting at 180-200 r / min and 30 ℃ for at least 10 days, and then filtering.

6. The biocontrol agent according to claim 4 or 5, characterized in that, The fermentation filtrate contains propylparaben, ethylparaben, and indole-3-carboxaldehyde, which are potential antibacterial substances.

7. The application of the biocontrol bacteria of claim 1, or the seed liquid of the biocontrol bacteria of any one of claims 2-3, or the biocontrol agent of any one of claims 4-6, in inhibiting the growth of Botrytis cinerea on tomatoes.

8. The application according to claim 7, characterized in that, It manifests itself in at least one of the following aspects: 1) Inhibits the elongation of mycelia of Botrytis cinerea (gray mold) on tomatoes; 2) Inhibits the spore germination of tomato gray mold.

9. The application according to claim 7 or 8, characterized in that, Including applications in at least one of the following aspects: 1) Inhibits the growth of Botrytis cinerea mycelium in detached tomato fruits; 2) Inhibits the infection of Botrytis cinerea mycelia in detached tomato fruits; 3) Inhibits the growth of gray mold mycelium on tomato leaves; 4) Inhibits the infection of gray mold mycelium on tomato leaves.

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