Streptomyces sp. and use thereof

Abstract

The application belongs to the technical field of biological control, and relates to research on plant disease control bacteria, in particular to a Streptomyces and application of the bacteria in prevention and treatment of pepper Sclerotium rolfsii. Streptomyces griseoaurantiacus The Streptomyces is classified as and is preserved in the China Center for Type Culture Collection, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and has a preservation number of CCTCC NO: M 20231358 and a preservation time of July 21, 2023. Streptomyces griseoaurantiacus XQ-29 has good antibacterial property on pepper Sclerotium rolfsii, and the antibacterial rate can reach 96.83%. The application significantly inhibits mycelium growth, sclerotium production, sclerotium germination and sclerotium growth of pepper Sclerotium rolfsii, destroys the integrity of the cell membrane of pepper Sclerotium rolfsii, increases the peroxidation degree of the plasma membrane of pepper Sclerotium rolfsii, and obviously promotes the growth of peppers.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol technology and relates to the research of biocontrol bacteria for plant diseases, specifically to a Streptomyces and its application in the control of white mold disease in peppers. Background Technology

[0002] Biocontrol microorganisms refer to beneficial microorganisms that can be used to control plant diseases. Currently, the most studied biocontrol microorganisms are fungi, bacteria, and actinomycetes. Relatively few biocontrol bacteria have been discovered for controlling white mold disease in peppers. However, there are existing applications of bacterial and fungal biocontrol bacteria for white mold disease in other plants.

[0003] For example, prior art CN 202110129025 discloses a Bacillus belye QBB3, which has a strong inhibitory ability against apple white rot fungus, with a control efficacy of about 92.75%.

[0004] Existing technology CN 202110929652 discloses a fluorescent Pseudomonas HT1 strain, which has a maximum control efficacy of up to 92% against Coptis chinensis white rot.

[0005] The prior art CN202210689788 discloses a Bacillus belye LT1, which has a maximum control efficacy of 78.41% against Coptis chinensis white rot.

[0006] However, no biocontrol bacteria used to prevent and control white mold disease in peppers have been disclosed. Summary of the Invention

[0007] The purpose of this invention is to provide a Streptomyces strain that can effectively prevent and control white mold disease in peppers.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] A Streptomyces species, taxonomically named Streptomyces griseoaurantiacus, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M20231358, and deposited on July 21, 2023.

[0010] This invention also protects the application of the Streptomyces in the prevention and control of white mold disease in peppers.

[0011] In one preferred embodiment, the Streptomyces can significantly inhibit the mycelial growth, sclerotium production, sclerotium germination and sclerotium growth of *Streptomyces capsulatum*.

[0012] In one preferred embodiment, the Streptomyces can significantly disrupt the integrity of the cell membrane of white rot of pepper and increase the degree of peroxidation of the white rot of pepper fungus cell membrane.

[0013] In one preferred embodiment, the Streptomyces is capable of producing melanin, indoleacetic acid, and siderophores.

[0014] In one preferred embodiment, the Streptomyces can significantly inhibit the size of lesions caused by white rot of pepper.

[0015] The present invention also protects the application of the aforementioned Streptomyces in promoting the growth of chili peppers.

[0016] An agent or composition for controlling white mold disease in peppers, comprising the aforementioned Streptomyces.

[0017] In one preferred embodiment, the formulation or composition contains a bacterial suspension or fermentation broth of Streptomyces griseoaurantiacus XQ-29.

[0018] In one preferred embodiment, the volume concentration of Streptomyces griseoaurantiacus XQ-29 in the fermentation broth is 1% to 20%.

[0019] In one preferred embodiment, the method for preparing the fermentation broth includes:

[0020] The Streptomyces strain was inoculated into Gao's No. 1 medium and cultured for 20-28 hours. Then, 1 mL of the bacterial suspension was inoculated into the fermentation medium and cultured at 28±5℃ and 180-200 r / min for 6-7 days with shaking to obtain the fermentation broth.

[0021] In one preferred embodiment, the fermentation medium comprises, by weight: 15-19 parts cornmeal, 8-12 parts sucrose, 1.5-2.4 parts peptone, 0.1-0.9 parts K2HPO4, 0.1-0.9 parts MgSO4·7H2O, 1-3 parts NaCl, 0.01-0.02 parts FeSO4·7H2O, 1-3 parts CaCO3, and water to a final volume of 1000 mL.

[0022] The beneficial effects of this invention are:

[0023] The *Streptomyces griseoaurantiacus* XQ-29 provided by this invention exhibits excellent antifungal activity against white mold disease of pepper, with an inhibition rate of up to 96.83%. It also significantly inhibits mycelial growth, sclerotium formation, sclerotium germination, and sclerotium growth of the white mold pathogen. The *Streptomyces* species significantly disrupts the integrity of the white mold cell membrane and increases the peroxidation level of the white mold pathogen's plasma membrane; simultaneously, the *Streptomyces* species produces melanin, indoleacetic acid, and siderophores. Importantly, the *Streptomyces* species significantly promotes pepper growth. Attached Figure Description

[0024] Figure 1 The image shows the inhibitory effect of strain XQ-29 on white rot of pepper. The left side is the control, and the right side is the confrontation culture of strain XQ-29 and white rot of pepper.

[0025] Figure 2 A shows the qualitative test for indoleacetic acid; the left side is the water control, and the right side is XQ-29. B shows the cellulase production capacity of strain XQ-29. C shows the melanin production capacity of strain XQ-29. D shows the siderophore production capacity of strain XQ-29.

[0026] Figure 3 A phylogenetic tree constructed based on 16S rDNA gene sequences;

[0027] Figure 4 The effect of fermentation broth of strain XQ-29 on the mycelial growth of white rot fungus;

[0028] Figure 5 The effects of strain XQ-29 on sclerotium germination and growth of *Sclerotium sclerotiorum*, the causal agent of white mold on peppers; A shows the effect of different time periods and different fermentation broth concentrations on sclerotium germination; B shows the statistical dendrogram of germination rate; C shows the effect of different concentrations of fermentation broth on mycelial and sclerotium production of *Sclerotium sclerotiorum*; D shows the statistical dendrogram of mycelial and sclerotium dry weight.

[0029] Figure 6 The following are indoor control efficacy tests of strain XQ-29 against chili peppers: A shows the control efficacy test against chili pepper seedlings, where I is water treatment, II is XQ-29 fermentation broth stock treatment, III is XQ-29 bacterial suspension treatment, and IV is 0.4% thifluzamide treatment; B is a schematic diagram of inoculation for the seedling control efficacy test; C is a diagram of the control efficacy test at the six-leaf stage of chili peppers; and D is a statistical tree diagram of the measurement results of the size of lesions at the base of the stem after removing excess substrate.

[0030] Figure 7Figure A shows the growth promotion experiment of strain XQ-29; Figure A: a) Pepper treated with water, b) Pepper treated with XQ-29 fermentation broth stock solution, c) Pepper treated with XQ-29 bacterial suspension; B) Comparison of individual pepper plants of the three treatments, C) Comparison of XQ-29 bacterial suspension treatment group and water treatment group, D) Comparison of XQ-29 fermentation broth stock solution treatment group and water treatment group.

[0031] Figure 8 The effect of strain XQ-29 on the mycelial morphology of *Sclerotium affine*, the causal agent of white mold on peppers; A is the control group under an optical microscope, B is the mycelial morphology of the 10% XQ-29 fermentation broth treatment under an optical microscope, C is the control group under a scanning electron microscope, and D is the mycelial morphology of the 10% XQ-29 fermentation broth treatment under a scanning electron microscope.

[0032] Figure 9 The effect of strain XQ-29 on the cell wall structure of *Sclerotium affine*, the causal agent of white rot in peppers;

[0033] Figure 10 A represents the PI staining result, B represents the DCFH-DA staining result, C represents the conductivity measurement result, D represents the absorbance value at 280 nm, E represents the absorbance value at 260 nm, and F represents the MDA detection result. Detailed Implementation

[0034] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0035] In the following examples, “strain XQ-29” refers to Streptomyces griseoaurantiacus XQ-29, which is deposited at the China Center for Type Culture Collection (299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), with accession number CCTCC NO: M 20231358 and deposit date of July 21, 2023.

[0036] Data Analysis

[0037] One-way ANOVA was performed on all data using SPSS 23.0, and multiple comparisons and significance analysis were conducted using Duncan's test.

[0038] Example 1

[0039] Isolation and identification of Streptomyces

[0040] 1. Materials and Methods

[0041] 1.1 Materials

[0042] Chili pepper variety and chemical agents: Fuxiang 15 chili pepper seeds were selected. Before sowing, a mixture of 70% alcohol and 0.1%...

[0043] Chili seeds were sterilized with NaClO. After soaking in sterile water overnight, the seeds were sown in a sterile substrate and cultured for 15 days. They were then transplanted into individual sterile pots. The agent used was 0.4% thifluzamide suspension, provided by Zhejiang Tianyi Agricultural Chemical Co., Ltd.

[0044] The tested strain of *Sclerotium affine*, the pathogen causing white mold on peppers, was isolated from pepper plants infected with white mold in the field in Ningxiang, Hunan Province. The pathogen was isolated according to the methods described by Fang Zhongda (Fang Zhongda, 1979. *Methods of Plant Disease Research*. Beijing: Agricultural Press). After morphological observation and pathogenicity verification, the isolated and purified pathogen was preserved on PDA slant.

[0045] PDA medium: glucose 20g / L -1 200g L of potatoes -1 20g L of agar -1 Add water to make up the volume, and the pH will remain natural.

[0046] Fermentation medium: 17g corn flour boiled in water (1000mL) to obtain an extract, 10g sucrose L -1 1.9g L of peptone -1 K2HPO4 0.5g L -1 MgSO4·7H2O 0.5g L -1 NaCl2 g L -1 ,

[0047] FeSO4·7H2O 0.01g L -1 ,CaCO3 1g L -1 pH is natural.

[0048] Gao's No. 1 culture medium: 20 g / L soluble starch -1 0.5g L of NaCl -1 KNO3 1g L -1 K2HPO4

[0049] 0.5g L -1 MgSO4·7H2O 0.5g L -1 FeSO4·7H2O 0.01g L -1 20g L of agar -1 Dilute to volume with water, pH 7.2-7.4.

[0050] 1.2 Isolation and Identification of Streptomyces

[0051] 1.2.1 Isolation and purification of Streptomyces

[0052] A biocontrol strain antagonistic to *Sclerotium affine*, the causal agent of white mold on peppers, was screened from the rhizosphere soil of pepper fields in Hunan Province through isolation, purification, and confrontation culture experiments. It was named XQ-29. Confrontation culture revealed that strain XQ-29 significantly inhibited the growth of *Sclerotium affine* mycelia. Figure 1 The antibacterial rate reached 96.83%. Streptomyces strains were inhibited through...

[0053] The culture was obtained using a serial dilution technique (Islam S, 2015), and the purified single colonies were transferred to Gao's No. 1 medium for storage. 1.2.2 Morphological observation and biochemical characterization analysis

[0054] Strawberry strain XQ-29 was streaked onto ISP2–7 medium and incubated at 28°C for 7–14 days. The color of aerial and substrate hyphae, the presence or absence of soluble pigments, and growth status were observed. Physiological and biochemical characteristics of strain XQ-29 were determined according to Bergey's Manual of Bacteriological Identification (Buchanan & Gibbons, 1994), including carbon source utilization, nitrogen source utilization, melanin production, and hydrogen sulfide production.

[0055] The results showed that strain XQ-29 was initially orange, turned orange after 3 days, and gradually turned red after 7 days. The colony surface was dry with water lines, and it was Gram-positive. Morphological characteristics of strain XQ-29 on ISP1-7 media showed that it produced no soluble pigment on any media except ISP4 and ISP7. Aerial hyphae were mostly yellow or white, with a few being light yellow or red. Substrate hyphae were mostly red, with a few being yellow or white. The strain grew best on ISP6 and ISP7 media, relatively well on ISP2 and ISP4 media, and moderately on ISP1, ISP3, and ISP5 media (Table 1). It could utilize glucose, lactose, xylose, fructose, and mannitol as its sole carbon source, with glucose, lactose, and fructose showing the best utilization; it could not utilize sucrose as its sole carbon source. It could utilize proline, tyrosine, yeast extract, and glutamic acid as its sole nitrogen source, with yeast extract showing the best utilization.

[0056] Table 1. Growth and culture characteristics of strain XQ-29

[0057]

[0058] Note: +++: Excellent growth; ++: Good growth; +: Average growth

[0059] 1.2.3 Biological Functional Analysis of XQ-29

[0060] The cellulase, pectinase, and glucanase activities of strain XQ-29 were analyzed. The analytical methods are as follows: Pectinase production was measured using pectinase-producing medium (Liu Junzhu et al., 2022), and glucanase production was measured using Avicel medium (Li Jiajia et al., 2020). Strain XQ-29 was inoculated onto these media and cultured at 28℃ for 7 days. The presence of a clear zone around the colony was observed; if a clear zone appeared, it indicated that XQ-29 possessed the corresponding activity. Cellulase production was measured using sodium carboxymethyl cellulose medium (Fang et al., 2022). Strain XQ-29 was inoculated onto the corresponding medium and cultured at 28℃ for 3 days. The strain was then immersed in 0.1% Congo red dye solution for 10 min, followed by decolorization with 1 mol / L NaCl solution for 5 min. The formation of a clear zone around the colony was observed.

[0061] The melanin-producing and hydrogen sulfide-producing abilities of strain XQ-29 were determined. Referring to Bergey's Manual of Bacteriological Identification (Buchanan & Gibbons, 1994), the biocontrol bacteria were inoculated into tyrosine medium and Chesna agar medium, with uninoculated medium serving as a control. The cultures were incubated at 27°C for 7 days, and the colony periphery was observed to determine if it was stained blackish-brown.

[0062] The Salkowski colorimetric method was used to determine the indoleacetic acid (IAA) production capacity of strain XQ-29. Fresh XQ-29 bacterial culture was cultured, and 5 mL was added to 100 mL of sterile King medium (Li et al., 2020). King medium with added sterile water was used as a control. The cultures were incubated at 28℃ and 180 rpm on a shaker. After 12 days, the culture was centrifuged at 1000 rpm for 10 min, and 4 mL of the supernatant was added to an equal volume of colorimetric reagent. The cultures were incubated in the dark for 30 min. If the treated group turned pink, it indicated that XQ-29 could produce IAA. A standard curve was prepared according to the method of Feng et al. (Feng et al., 2013), i.e., y = 0.0364x + 0.1134 (R²). 2 =0.9994). The absorbance of the sample at 530 nm was measured, and the sample was zeroed using a colorimetric solution containing an equal volume of sterile water as a blank control. Each treatment was repeated three times.

[0063] The siderophore-producing capacity of strain XQ-29 was determined using the CAS assay. XQ-29 bacterial discs were inoculated onto CAS medium and cultured at 28°C, with uninoculated medium serving as a control. After 7 days, the presence of a yellow halo around the colonies was observed.

[0064] The ability of biocontrol bacteria to produce hydrolytic enzymes is a key factor in their biocontrol effects. This study evaluated the hydrolytic enzyme production ability of strain XQ-29. The results showed that strain XQ-29 exhibited clear circles on sodium carboxymethyl cellulose medium. Figure 2 B) indicates that it can produce cellulase. The absence of transparent circles on pectinase-producing media and Avicel medium suggests poor pectinase and dextran hydrolysis capabilities. After 7 days of cultivation on tyrosine medium, the colonies of strain XQ-29 were stained dark brown. Figure 2 C) indicates that it can produce melanin.

[0065] In the colorimetric reaction, the treatment group turned pink. Figure 2 A) indicates that strain XQ-29 can produce indoleacetic acid, and the concentration of indoleacetic acid was calculated to be 4.82 mg / L based on the standard curve.

[0066] Siderophores secreted by actinomycetes and other fungi can control plant diseases through iron competition and other mechanisms, while simultaneously promoting nutrient absorption. Strain XQ-29 developed a distinct yellow halo after 7 days of culture on CAS medium. Figure 2 D) indicates that it can produce ferrocarriers.

[0067] 1.2.4 Molecular Identification

[0068] Total DNA was extracted from strain XQ-29 using the CTAB method (Terefework et al., 2001). The 16S rDNA gene fragment of strain XQ-29 was amplified using primers 27F / 1492R (5'-AGAGTTTGATTGGCTCAG-3'; 5'-TACGGGCTACCTGTTACGATT-3') (Wang et al., 2021).

[0069] Each 50 μL PCR reaction mixture contained 25 μL FineTaq PCR Super, 2 μL forward μ primers (10 μM), 2 μL reverse μ primers (10 μM), 4 μL DNA template, and 17 μL H2O. The PCR program was: 95℃ pre-denaturation for 3 min; 35 amplification cycles (95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1.5 min); 72℃ extension for 5 min. The amplified products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Homology of sequences obtained from the NCBI database was compared using BLASTn. Related gene sequences from other actinomycetes were obtained from the GenBank database, and the sequences were compared using the ClustalW algorithm on MEGA6 software. A phylogenetic tree was constructed using the maximum likelihood method (Tamura et al., 2013).

[0070] The results are as follows Figure 3 As shown, XQ-29 is Streptomyces griseoaurantiacus.

[0071] Example 2

[0072] The effect of Streptomyces on white mold disease of pepper

[0073] Antibacterial evaluation of fermentation broth of strain XQ-29

[0074] Strain XQ-29 was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of Gao's No. 1 medium and cultured on a shaker at 28°C and 180 rpm for 24 h. Then, 1 mL of the culture medium was transferred to a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium and cultured on a shaker at 28°C and 180 rpm for 6 days. The culture was centrifuged, and the supernatant was filtered through a 0.22 μm bacterial filter to obtain the fermentation broth. The antibacterial effect of the fermentation broth was tested using the toxic medium method (Ruan Hongchun et al., 2021). The fermentation broth was added to PDA medium to obtain media with final concentrations of 20%, 10%, 5%, 2%, and 1% (v / v). Mycelial cakes (d = 8 mm) of *Sclerotium typhimurium* were inoculated in the center of the plates with the mycelium facing down. PDA plates with the corresponding volume of fermentation broth were used as controls. Each treatment was repeated three times. After culturing in a constant temperature incubator at 28°C for 3 days, the colony diameter was measured using the cross-cross method, and the inhibition rate was calculated. Inhibition rate (%) = [(Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - 8mm) × 100, where 8mm is the diameter of the mycelial cake.

[0075] The results showed that the growth of *Sclerotium affine* was significantly inhibited after adding different volumes of fermentation broth to the PDA medium. Figure 4 When the fermentation broth concentration was 10% and 20%, the inhibition rates against *Sclerotium affine* reached 74.03% ± 0.00% and 91.78% ± 0.03%, respectively (Table 2). These results indicate that strain XQ-29 has a significant inhibitory effect on *Sclerotium affine*.

[0076] Table 2. Inhibition rate of fermentation broth of strain XQ-29 against *Sclerotium affine*, the causal agent of white rot in peppers.

[0077]

[0078]

[0079] Note: All data in the table are mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level according to Duncan's new multiple range test.

[0080] Biological control potential assessment

[0081] The impact of mycelial and sclerotial formation of white mold disease in peppers

[0082] Two mycelial cakes of *Sclerotium affine*, the causal agent of pepper white mold, were cultured on PDA plates for 2 days. Two mycelial cakes were then added to PDA plates containing 5%, 10%, and 20% fermentation broth, respectively. PDA plates without added fermentation broth served as a control. Each group was repeated three times. The treated PDA plates were cultured in a shaker at 180 rpm and 28°C for 2 days. After 2 days, the PDA plates were removed and allowed to stand at room temperature. After 14 days, mycelia and sclerotia were collected by filtering through gauze, drying off the excess water, and weighing.

[0083] Impact on the germination of sclerotia of pepper white mold disease

[0084] Sclerotia were collected from *Sclerotium affine*, the causal agent of white mold on peppers, cultured on PDA plates for 14 days and divided into four groups of 27 sclerotia per group. Three groups were immersed in 100 μL of 5%, 10%, and 20% fermentation broth, respectively, for 20 min, with water immersion serving as a control. The four groups of sclerotia were inoculated onto plates with final fermentation broth concentrations of 5%, 10%, and 20%, respectively, with PDA plates without added fermentation broth serving as a control. Nine sclerotia were inoculated onto each plate. All sclerotia were incubated at 28°C. Germination rates were calculated and observed 12 h, 24 h, and 36 h after inoculation.

[0085] Depend on Figure 5 As shown in A and B, the germination rate of *Sclerotium sclerotium* gradually decreased with increasing fermentation broth concentration. As illustrated in the figure, after 36 hours, all sclerotia in the control group had germinated and the mycelium had completely covered the plate. In the treatment group, even with sclerotia germination at a 20% concentration, mycelial growth was significantly inhibited. Simultaneously, the production of both *Sclerotium sclerotium* and mycelium was significantly inhibited under XQ-29 fermentation broth treatment. Figure 5 (C and D). It can be observed that the XQ-29 fermentation broth not only inhibits the production of sclerotia but also prevents their maturation. At a 20% fermentation broth concentration, *Sclerotium oxysporum* no longer produces sclerotia.

[0086] In vivo control efficacy against white mold disease of pepper

[0087] The in vivo efficacy of strain XQ-29 was determined at the seedling stage and the 6-leaf stage of peppers.

[0088] Seedling stage control efficacy test: A 3-day cultured mycelial cake (d=8mm) of *Sclerotium affine* var. *pepigeninii* was inoculated into Erlenmeyer flasks containing barley grains. After incubation at 28℃ for 7 days, inoculum containing the pathogen was obtained. Pepper seedlings 15 days after sowing were selected and treated with either fermentation broth or mycelial suspension of active strain XQ-29 for root immersion. After 30 minutes, the seedlings were transplanted into pots containing 85g of sterilized substrate and then treated with either fermentation broth or mycelial suspension for root drenching, 10mL per pot. One barley grain containing *Sclerotium affine* mycelium was inoculated at four points around the base of the pepper seedling stem in four directions. Disease incidence was observed and counted after 3 days. Disease incidence % = number of infected plants / total number of plants. Treatment with 0.4% thifluzamide and sterile water served as positive and negative controls, respectively.

[0089] Efficacy test at the 6-leaf stage: Peppers of the same growth stage and at the 6-leaf stage were divided into two groups. The roots were irrigated with XQ-29 fermentation broth and water, respectively, with 10 mL per pot. A fungal cake (d = 8 mm) of *Sclerotium typhimurium* was inoculated at the base of the pepper stem and cultured at 28℃ for 3 days. The disease incidence was observed and statistically analyzed after 3 days. To more clearly observe the disease situation, the substrate inoculated at the pepper roots was removed, excess impurities were washed off with water, and the peppers were allowed to air dry. The length of the lesions at the base of the pepper stem was then calculated.

[0090] like Figure 6 As shown, barley grains containing the mycelium of *Sclerotium cirrhifolium* were inoculated in four directions about 1 cm from the base of the pepper seedling stem. Figure 6 (A and B). Five days after transplanting, the disease rate of pepper seedlings in the control group reached 40%, while the pepper seedlings treated with XQ-29 fermentation broth and bacterial suspension were similar to those treated with 0.4% thiofuran suspension, with no obvious symptoms observed.

[0091] In an experiment determining the resistance of XQ-29 to white mold disease in peppers during different growth stages, peppers in the control group showed lodging and leaf blight symptoms 4 days after inoculation at the stem base, while peppers treated with XQ-29 fermentation broth did not exhibit these symptoms. Figure 6 C). Measure the length of the lesion at the base of the diseased pepper stem, such as... Figure 6 As shown in Figure D, the lesion size in the control group was 27.46±0.46 mm, which was significantly larger than that in the treatment group.

[0092] Growth-promoting effect on chili pepper plants

[0093] Pepper seedlings 14 days after sowing were divided into three groups of 20 plants each. Seven days after transplanting, the roots of each seedling were drenched with either XQ-29 bacterial solution or its stock solution, 10 mL per plant, every 7 days for a total of 3 drenchings. A control group was also drenched with plain water. Two months later, the number of pepper fruits, plant height, stem length, root length, dry weight, fresh weight, and chlorophyll content (SPAD) of each group were measured and statistically analyzed.

[0094] like Figure 7 As shown in ABCD, it can be seen that the pepper plants treated with strain XQ-29 were generally taller than those treated with water, with the changes being more pronounced in the original solution treatment.

[0095] To further understand the growth-promoting effect of XQ-29, the number of pepper fruits, maximum leaf length, maximum leaf width, plant height, stem length, root length, dry weight, fresh weight, and chlorophyll content (SPAD) of the three treatment groups were measured and statistically analyzed. The results are shown in Table 3. The data indicate that the pepper plants treated with XQ-29 fermentation broth produced more fruits, grew more vigorously, and produced more chlorophyll.

[0096] Table 3. Growth-promoting effects of strain XQ-29 on pepper plants.

[0097]

[0098] Note: All data in the table are mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences at the P<0.05 level according to Duncan's new multiple range test.

[0099] Example 3

[0100] Mechanism of action study

[0101] Effects on the mycelial morphology of white mold fungus of pepper

[0102] Two mycelial cakes (d=8mm) of *Sclerotium affine*, the causal agent of pepper rot, were selected and cultured on PDA plates for 3 days. The cakes were then added to PD liquid medium and cultured at 28℃ and 180 rpm for 3 days. The mycelia were obtained by filtration through sterile gauze and ground into a homogenate using a mortar. This homogenate was then added at a 1:100 ratio to both ordinary PD medium and PD medium containing 10% XQ-29 fermentation broth. After 12 hours of culture on a shaker at 28℃ and 180 rpm, the mycelia were filtered through gauze to obtain young mycelia. The mycelial morphology was observed under an optical microscope after being prepared onto slides. The morphological changes of the mycelia under a scanning electron microscope (SEM) were also observed.

[0103] The results showed that strain XQ-29 had a significant inhibitory effect on the mycelium of the pathogen. Mycelial hyphae of *Sclerotium typhimurium* were co-cultured with a 10% XQ-29 fermentation broth for 12 hours, and mycelial morphology was observed. The results indicated that compared with the control group, the mycelium ( Figure 8 Compared to A), the mycelia treated with fermentation broth were deformed, twisted, and had more branches. Figure 8 B). Under scanning electron microscopy, hyphae were observed to be significantly different from those in the control group ( Figure 8 C), the mycelia treated with the fermentation broth showed more perforations. Figure 8 D).

[0104] Effects on the cell wall structure of white rot fungus

[0105] Calcofluor White Stain (CFW) is a non-specific fluorescent dye that binds to cellulose and chitin in the cell wall. CFW staining (Yanqiu Zhao, 2019) was used to analyze the effect of the original XQ-29 fermentation broth on the cell wall structure of *Sclerotium affine*.

[0106] The mycelia of *Sclerotium affine*, the causal agent of white mold on peppers, were cultured using the previous method. The obtained mycelia were washed with 0.1% PBS buffer. After blotting on lens paper, one drop of staining agent and one drop of 10% potassium hydroxide were added. A coverslip was placed on the sample, and staining was performed for 1 minute. The fluorescence of the mycelia was observed under a fluorescence microscope after 12–24 hours.

[0107] like Figure 9 As shown, after 12 hours of CFW staining, the hyphae in the control group exhibited bright blue fluorescence, while the fluorescence in the XQ-29 fermentation broth treated group was slightly weaker, and the blue fluorescence emitted by the hyphae in the treated group gradually weakened with increasing treatment time. The experimental results indicate that the original XQ-29 fermentation broth can affect the cell wall structure of *Sclerotium affine* hyphae.

[0108] Effects on the cell membrane integrity of white rot fungus

[0109] The effect of XQ-29 fermentation broth on the cell membrane integrity of *Sclerotium typhimurium* was observed using propidium iodide (PI, Sigma-Aldrich) staining (Xiao et al., 2021). *Sclerotium typhimurium* hyphae under different treatments were cultured and obtained according to the previous method. The hyphae were washed with 0.1% PBS buffer and collected in 1.5 mL centrifuge tubes. 1 mL of 0.1% PBS buffer and 5 μL (10 mg L⁻¹) of PI dye were added, and the tubes were incubated in the dark for 30 min. The hyphae were rinsed twice with 0.1% PBS buffer and prepared as slides for fluorescence observation under a fluorescence microscope (Olympus Co., Tokyo, Japan).

[0110] The results showed that after treatment with 10% XQ-29 strain fermentation broth for 12 hours, the mycelium of *Sclerotium oxysporum* var. *pepifolium* emitted obvious red fluorescence, but no obvious fluorescence was detected in the control group. Figure 10 A).

[0111] To further confirm the effect of strain XQ-29 on the membrane permeability of *Sclerotium affine*, the biofilm causal agent of pepper white mold, the conductivity of *Sclerotium affine* treated with different concentrations of XQ-29 fermentation broth was measured using a conductivity meter (DDSJ-308F, INESA Instrument, Shanghai, China; Ma et al., 2019). The absorbance at 260 and 280 nm of the *Sclerotium affine*-treated bacterial solutions was measured using a microplate reader. Measurements were taken at five time points, with three replicates for each treatment.

[0112] like Figure 10 As shown in Figure C, the conductivity of the bacterial culture treated with XQ-29 fermentation broth was higher than that of the control group, and significantly higher at a treatment concentration of 20%. After 3 hours of treatment, the cells of *Sclerotium affine*, the causal agent of pepper white mold, showed leakage of nucleic acids and proteins, which gradually increased over time. Figure 10 (D, E). This indicates that strain XQ-29 can disrupt the integrity of the cell membrane of *Sclerotium cirrhifolium*, the causal agent of white rot in peppers.

[0113] Effects of reactive oxygen species and lipid peroxidation on white rot fungus

[0114] The effect of XQ-29 fermentation broth on ROS accumulation in mycelia was observed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich) staining (Ma et al., 2019). Mycelia of *Sclerotium affine* under different treatments were cultured and obtained according to method 1.4.1. Mycelia were washed with 0.1% PBS buffer and collected in 1.5 mL centrifuge tubes. 100 μL of DCFH-DA dye was added to immerse the mycelia, and the tubes were incubated at 37°C in the dark for 30 min. The mycelia were then washed with 0.1% PBS buffer until no further fading occurred. Mycelia were then prepared as slides and observed under a fluorescence microscope (Olympus Co., Tokyo, Japan) to observe their fluorescence.

[0115] Malondialdehyde (MDA) is a major product of lipid peroxidation. To assess the degree of lipid peroxidation in the plasma membrane, MDA content was measured using the thiobarbituric acid (TBA) method (Pereira et al., 2003). Fresh *Scleroderma gracilis* mycelia cultured in PD medium for 2 days were thoroughly ground and added to fermentation broth of strain XQ-29 at final concentrations of 5%, 10%, and 20%, respectively. MDA content was measured after incubation for 3 h, 6 h, 9 h, and 12 h.

[0116] like Figure 10As shown in Figure B, using DCFH-DA to detect ROS production in mycelium, after treatment with 10% XQ-29 fermentation broth for 12 hours, the mycelium of *Sclerotium oxysporum* var. *pepifolium* emitted significant green fluorescence, while the control group showed relatively weaker fluorescence. The intensity of the green fluorescence was related to the amount of ROS accumulation; the more ROS accumulated, the greater the fluorescence intensity. Therefore, the results indicate that strain XQ-29 can induce ROS accumulation in *Sclerotium oxysporum* var. *pepifolium* mycelium.

[0117] Meanwhile, after 3 hours of treatment with the original XQ-29 fermentation broth, the MDA content remained almost unchanged. However, between 9 and 12 hours, the MDA content in the treated groups increased sharply, and the higher the concentration, the greater the MDA content. Figure 10 F).

[0118] In summary, the *Streptomyces* sp. XQ-29 provided by this invention exhibits excellent antifungal activity against pepper white mold, with an inhibition rate of up to 96.83%. It also significantly inhibits the mycelial growth, sclerotium formation, sclerotium germination, and sclerotium growth of the *Streptomyces* sp. 2Q. The *Streptomyces* sp. 2Q ...

Claims

1. A type of Streptomyces griseoaurantiacus XQ 29, characterized in that, The Streptomyces XQ 29 is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20231358, and deposited on July 21, 2023.

2. The application of Streptomyces according to claim 1 in the prevention and control of white mold disease in peppers.

3. The application of Streptomyces according to claim 1 in promoting pepper growth.

4. A preparation or composition for controlling white mold disease in peppers, characterized in that, It includes the Streptomyces as described in claim 1.

5. The formulation or composition according to claim 4, characterized in that, The formulation or composition contains a bacterial suspension or fermentation broth of Streptomyces griseoaurantiacus XQ-29.

6. The formulation or composition according to claim 5, characterized in that, The volume concentration of Streptomyces griseoaurantiacus XQ-29 in the fermentation broth is 1% to 20%.

7. The formulation or composition according to claim 5, characterized in that, The preparation method of the fermentation broth includes: inoculating the Streptomyces strain into Gao's No. 1 medium, culturing for 20-28 h, taking 1 mL of bacterial suspension and inoculating it into the fermentation medium, and shaking it at 23-33℃ and 180-200 r / min for 6-7 days to obtain the fermentation broth.

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