Application of Streptomyces rochei YHLC-1 in prevention and treatment of pepper late blight

Abstract

The application relates to the technical field of biological control, and specifically discloses application of Streptomyces rochei YHLC-1 in prevention and treatment of pepper blight. The Streptomyces rochei YHLC-1 is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No. 35491, and is preserved on August 1, 2025; the application also provides a composite biological agent which comprises the following components in the following amounts: 1-5% of Streptomyces rochei YHLC-1 spore powder, 0.1-1% of matrine, 2-8% of a surfactant, 1-5% of a stabilizer, 2-6% of an antifreezing agent, 0.5%-3% of a synergist, 10-20% of a filler, and the rest of solvent. The composite biological agent can prevent and treat pepper blight and has a remarkable promoting effect on the growth of peppers.

Description

Technical Field

[0001] This application relates to the field of biological control technology, specifically to the application of Streptomyces loucheri YHLC-1 in the control of pepper blight. Background Technology

[0002] Chili peppers are an important vegetable and economic crop in my country and even the world. During the cultivation of chili peppers, Phytophthora blight, caused by Phytophthora capsici, is a devastating soil-borne disease. This pathogen has a strong infectivity and spreads rapidly, easily causing the base of the chili pepper plant stem to rot, wilt, and even die the entire plant. The fruit may even develop water-soaked rot and be covered with a white mold layer, resulting in serious yield and economic losses.

[0003] Biocontrol microbial agents are biological agents that utilize microorganisms or their metabolites to control agricultural pests. Due to their environmental friendliness, high selectivity, low likelihood of developing resistance, and promotion of ecological cycles, they are considered sustainable green biological pesticides. *Streptomyces loucheri* is one such important biocontrol microbial agent. Currently, various *Streptomyces loucheri* strains have been reported, exhibiting rich strain diversity. They are primarily used to control bacterial diseases such as bacterial wilt and root rot, as well as fungal diseases such as powdery mildew and anthracnose in wolfberry. However, their control effect against *Phytophthora capsici* is not ideal. Furthermore, no *Streptomyces loucheri* strain has been reported to have a clearly defined inhibitory and control effect on *Phytophthora capsici*. Summary of the Invention

[0004] In order to overcome the problem that existing Streptomyces loucheri species are not effective in controlling pepper blight, this application provides a Streptomyces loucheri YHLC-1 species and its application.

[0005] In the first aspect, this application provides an application of Streptomyces loucherei YHLC-1 in the control of pepper blight, employing the following technical solution:

[0006] A type of Streptomyces loucheri ( Streptomyces rochei The application of YHLC-1 in the prevention and control of pepper blight, wherein the Streptomyces loucheri is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35491, and the deposit date is August 1, 2025.

[0007] Secondly, this application provides a compound biological agent for the prevention and control of pepper blight, comprising the aforementioned Streptomyces loucheri YHLC-1.

[0008] Optionally, the compound biological agent further includes matrine, surfactants, stabilizers, antifreeze agents, synergists, fillers, and solvents.

[0009] Optionally, the composite biological agent comprises the following components in the following proportions: Streptomyces loucherei YHLC-1 spore powder 1-5%, matrine 0.1-1%, surfactant 2-8%, stabilizer 1-5%, antifreeze 2-6%, synergist 0.5%-3%, filler 10-20%, and the balance being solvent;

[0010] The Streptomyces loucherei YHLC-1 spore powder is obtained by fermentation, drying, and pulverization of Streptomyces loucherei YHLC-1; the effective viable count of the Streptomyces loucherei YHLC-1 spore powder is ≥2.6×10⁻⁶. 11 CFU / g, moisture content ≤10%.

[0011] In some embodiments, the content of the Streptomyces loucherei YHLC-1 spore powder may be 1-3% or 3-5%.

[0012] In one specific implementation, the content of the Streptomyces loucheri YHLC-1 spore powder may also be 1%, 3% or 5%.

[0013] In some embodiments, the content of matrine may be 0.1-0.5% or 0.5-1%.

[0014] In one specific implementation, the content of matrine may also be 0.1%, 0.5% or 1%.

[0015] Optionally, the preparation method of the Streptomyces loucheri YHLC-1 spore powder is as follows: First, Streptomyces loucheri YHLC-1 is activated and cultured to obtain Streptomyces loucheri YHLC-1 seed liquid; then, Streptomyces loucheri YHLC-1 seed liquid is inoculated into a solid fermentation medium at an inoculation rate of 20-30 v / w%, and fermented for 9-11 days to obtain fermented product; then, the fermented product is dried at 40-45℃ to reduce its moisture content to below 10%, and passed through an 80-mesh sieve to obtain Streptomyces loucheri YHLC-1 spore powder.

[0016] Optionally, the surfactant is an alkyl polysaccharide and / or a fatty alcohol polyoxyethylene ether; the stabilizer is xanthan gum and / or sodium alginate.

[0017] Optionally, the antifreeze is glycerol or ethylene glycol; the synergist is an organosilicon synergist; and the solvent is water or wood vinegar.

[0018] Thirdly, this application provides an application of a compound biological agent in the prevention and control of pepper blight. The method of using the compound biological agent is as follows: 5-10 days after the pepper seedlings are transplanted, the compound biological agent is diluted to 300-1000 times, and then the diluted solution is applied to the roots of the pepper. The treatment is carried out 3 times during the entire growth period, with an interval of 30±3 days.

[0019] In summary, this application has the following beneficial effects:

[0020] 1. This application isolates and screens a new strain of Streptomyces loucheri from the rhizosphere soil of cucumber greenhouses. By applying the biological agent made from this strain to the roots of peppers, it is possible to control pepper blight and significantly promote the growth of peppers.

[0021] 2. This application describes the preparation of a compound biological agent using Streptomyces loucheri YHLC-1. When the diluted solution of the compound biological agent is applied to the roots of chili peppers, the control efficiency against chili pepper blight reaches 74.91-78.36% compared to the blank control group. Furthermore, it increases the height of chili pepper seedlings by 12.72-24.02% and the yield per chili pepper plant by 1019.1-1401.7 g / plant compared to the blank control group.

[0022] 3. By using xanthan gum, sodium alginate, glycerol, etc. as carriers, this application can not only improve the preservation stability of compound biological agents, extending the shelf life to more than 2 years, but also provide a strong buffer and protective environment for Streptomyces loucheri YHLC-1 spores, enabling them to adapt to the complex and variable soil, water, and pH conditions in the field, thereby ensuring that they can maintain a high survival rate and colonization ability after inoculation into the environment. Detailed Implementation

[0023] In this embodiment, the matrine content is ≥95%, and it is prepared into a 10% matrine mother liquor using a solvent. The raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0024] The following describes this application in further detail with reference to preparation examples, embodiments, and performance testing.

[0025] Preparation Example 1

[0026] Preparation Example 1 provides a Streptomyces loucherii YHLC-1.

[0027] A new strain, YHLC-1, was isolated and screened from the rhizosphere soil of cucumber greenhouses in Shuainan Village, Daotian Town, Shouguang City, Shandong Province, and identified as *Streptomyces loucheri*. Streptomyces rochei It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35491, and the deposit date is August 1, 2025.

[0028] Preparation Example 2

[0029] Preparation Example 2 provides a Streptomyces louchei YHLC-1 spore powder, the preparation method of which includes the following steps:

[0030] (1) Activation and seed culture preparation of Streptomyces loucheri YHLC-1: The culture preserved in glycerol tubes was activated by streaking on Gao Shi No. 1 plates and cultured in a constant temperature incubator at 30℃ for 10 days to obtain single colonies. Then, the activated Streptomyces loucheri single colonies were picked and inoculated into seed culture medium in seed bottles and cultured in a shaking incubator at 30℃ and 200rpm for 40 hours to obtain the seed culture.

[0031] The formula of the Gao's No. 1 culture medium is as follows: soluble starch 20g / L, potassium nitrate 1g / L, ferrous sulfate heptahydrate 0.01g / L, magnesium sulfate heptahydrate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, and sodium chloride 0.5g / L.

[0032] The seed culture medium is formulated as follows: 25 g / L glucose, 15 g / L medium-temperature soybean meal, 2.5 g / L yeast powder, 1 g / L calcium carbonate, and pH adjusted to 7.5-7.8.

[0033] (2) Preparation and sterilization of solid fermentation medium: Mix wheat bran and rice husk at a mass ratio of 8:2, and add the following components according to the total weight of solid matrix: 1% glucose, 0.5% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO4·7H2O; then adjust the moisture content of the matrix with deionized water, stir thoroughly to make the material moisture uniform, and achieve the state of "can be squeezed into a ball by hand and can be broken when touched"; finally, divide the mixed material into shallow heat-resistant trays (material layer thickness 2-3 cm), and sterilize in a high-pressure steam sterilizer at 121℃ for 60 min to completely kill miscellaneous bacteria.

[0034] (3) Inoculation and fermentation: Cool the sterilized solid fermentation medium to room temperature (about 30°C); in a sterile operating table, spray the seed liquid prepared in step (1) evenly onto the cooled solid medium at an inoculation rate of 20%-30% (v / w, i.e., 20-30 mL of seed liquid per 100 g of dry medium). Stir thoroughly to ensure that the inoculum and the material are mixed evenly. Place the inoculated material in a constant temperature incubation room, spread it evenly in a sterilized shallow dish (material layer thickness 3-5 cm), cover it with 1-2 layers of sterile damp gauze to maintain humidity, and carry out static fermentation at a temperature of 30±1°C and a relative humidity of 60%-70%. After fermentation for about 48 hours, stir for the first time to break the mycelial clumping, replenish oxygen, and dissipate heat. Thereafter, stir 1-2 times as needed according to the material temperature. The total fermentation time is 9-11 days. The early stage (2-3 days) is the stage of massive mycelial growth, and the later stage (4-10 days) is the stage of massive spore formation. When the material changes from white to grayish-white or gray and has a typical actinomycete spore aroma, it indicates that the fermentation is complete.

[0035] (4) Drying and pulverizing: After fermentation, the solid material is immediately dried at a low temperature in a circulating air oven or dryer at 40-45℃ to rapidly reduce the moisture content of the material to below 10% in order to terminate fermentation and maintain spore activity. The material is then pulverized using a universal pulverizer and passed through an 80-mesh sieve to obtain uniform Streptomyces loucheri YHLC-1 spore powder.

[0036] The spore powder of *Streptomyces loucherei* YHLC-1 was tested according to the method in GB20287-2006. The results showed that the effective viable count of *Streptomyces loucherei* YHLC-1 spore powder was ≥2.6 × 10⁻⁶. 11 CFU / g, moisture content ≤10%, and contamination rate ≤5%.

[0037] Comparative Preparation Example 1

[0038] Comparative preparation example 1 provides a Streptomyces louchei 26B spore powder.

[0039] The above-mentioned Streptomyces loucherii 26B spore powder was prepared using Streptomyces loucherii 26B with preservation number CGMCC No. 24171, and the preparation method was the same as that in Preparation Example 2. Example 1

[0040] Example 1 provides a compound biological agent.

[0041] The preparation method of the above-mentioned compound biological agent includes the following steps: Weigh out 30g of Streptomyces loucherei YHLC-1 spore powder, 50g of 10% matrine mother liquor, 30g of alkyl polysaccharide glycoside, 20g of fatty alcohol polyoxyethylene ether, 10g of xanthan gum, 20g of sodium alginate, 40g of glycerol, 15g of organosilicon synergist, and 150g of kaolin. First, premix the Streptomyces loucherei YHLC-1 spore powder and kaolin in a mixer for 15 minutes to ensure uniform spore dispersion and no clumping. Then, add the other components sequentially, and add wood vinegar to make up to 1000g, stirring thoroughly until homogeneous. Finally, homogenize using a homogenizer to obtain the compound biological agent.

[0042] Examples 2-5

[0043] Examples 2-5 each provide a compound biological agent.

[0044] The difference between the above embodiments and Embodiment 1 is that the content of Streptomyces loucheri YHLC-1 spore powder and matrine is shown in Table 1 below.

[0045] Table 1. Amounts of Streptomyces loucherei YHLC-1 spore powder and matrine added in Examples 1-5

[0046] Comparative Example 1

[0047] Comparative Example 1 provides a compound biological agent.

[0048] The difference between the above comparative example and Example 1 is that the Streptomyces loucherii YHLC-1 spore powder in Example 1 was replaced with Streptomyces loucherii 26B spore powder provided in Comparative Preparation Example 1. Comparative Example 2

[0049] Comparative Example 2 provides a compound biological agent.

[0050] The difference between the above comparative example and Example 1 is that matrine was replaced with 5g of Streptomyces louchei YHLC-1 spore powder. Comparative Example 3

[0051] Comparative Example 3 provides a compound biological agent.

[0052] The difference between the above comparative example and Example 1 is that 30g of Streptomyces loucheri YHLC-1 spore powder was replaced with 300g of 10% matrine mother liquor matrine.

[0053] Performance testing

[0054] (I) Effects of pepper blight control

[0055] 1. Preparation of chili seedlings: After germinating the chili seeds (Xiangyan No. 15, a variety susceptible to blight), sow them in sterilized seedling pots. When the seedlings grow to about 8-10 leaves, select healthy and uniformly growing chili seedlings and randomly divide them into 100 groups (experimental groups 1-5, control groups 1-4 and blank control group), with 10 pots in each group, and mark them.

[0056] 2. Preparation of Phytophthora capsici spore suspension: After inoculating Phytophthora capsici onto oat agar medium and culturing for 10 days, mycelial blocks were scraped off and placed in sterile water to induce the release of zoospores. The concentration of the spore suspension was adjusted to 1×10⁻⁶ using a hemocytometer. 6 1 spore / mL, for later use.

[0057] 3. Application Method: The following treatments were applied to each group of chili seedlings using the root drenching method: Experimental groups 1-5 were treated with an 800-fold dilution of the compound biological agent from Examples 1-5; Control groups 1-3 were treated with an 800-fold dilution of the compound biological agent from Comparative Examples 1-3; Control group 4 was treated with a 1500-fold dilution of (commercially available) chemical control agent: 250 g / L azoxystrobin suspension; The blank control group was treated with plain water.

[0058] First, drench the roots of each plant with 30 mL of the diluted solution from each of the above treatment groups. Add an equal amount of water to the control treatment. After 3 days, puncture the base of the pepper roots and inoculate with 10 mL of Phytophthora capsici spore suspension. Place each treatment group in a greenhouse for cultivation, keep it moist, and observe the disease situation daily.

[0059] 4. Experimental Results: Disease incidence was investigated on days 7 and 14 after vaccination, and the disease index and control effect were calculated. The results are shown in Table 2 below.

[0060] (1) Disease grading criteria: Grade 0 - No obvious symptoms; Grade 1 - A few lesions on the leaves, or water-soaked spots on the stem; Grade 3 - Less than 1 / 3 of the leaves or lateral branches are affected; Grade 5 - 1 / 3 to 2 / 3 of the leaves or lateral branches are affected; Grade 7 - More than 2 / 3 of the leaves or lateral branches are affected, or obvious constriction of the stem; Grade 9 - The whole plant dies.

[0061] (2) The formulas for calculating the disease index and prevention and control effect are as follows:

[0062] Disease index = [∑(number of diseased plants at each level × corresponding level) / (total number of plants surveyed × 9)] × 100

[0063] Prevention and control efficacy (%) = [(Disease index of blank control group - disease index of treatment group) / disease index of blank control group] × 100%

[0064] Table 2. Disease index and prevention and treatment effect of experimental groups 1-5, control group 1-4 and blank control group

[0065]

[0066] As shown in Table 2, the compound biological agents obtained in Examples 1-5 of this application (containing *Streptomyces loucherei* YHLC-1 spore powder and matrine) exhibited significantly higher control efficacy at 7 days and 14 days than the compound biological agents in Comparative Examples 2-3 (containing only *Streptomyces loucherei* YHLC-1 spore powder or matrine), and their efficacy was comparable to that of the chemical agent azoxystrobin. This indicates that the compound biological agents provided in this application have a significant control effect against *Phytophthora capsici*, and their control level can reach or even surpass that of chemical biological control agents.

[0067] (II) Verification of control effects in the field

[0068] 1. Test crop: chili pepper, variety Xiangyan 15; Target disease: chili pepper blight.

[0069] 2. Experimental Site Conditions: The experiment was conducted in a greenhouse in Shangkou Town, Shouguang City, Weifang City. This greenhouse had a history of severe disease outbreaks, with an incidence rate exceeding 90%. The ridges were 1.5m wide, with 2 rows per ridge, a plant spacing of 50cm, and furrow width of 30cm. Management practices were consistent with local cultivation conditions.

[0070] 3. Experimental Design and Survey Methods: This experiment designed four treatments: 500-fold and 1000-fold dilution of the compound biological agent from Example 1, 1000-fold dilution of azoxystrobin suspension, and a water control. Pepper seedlings were treated 5 days after transplanting (3 rows per treatment). The roots of the peppers were irrigated with the diluted solutions of the above treatments. Plant height was measured 30 days after each treatment. A total of three treatments were conducted throughout the growth period, with a 30-day interval. Harvesting and yield calculations were performed, and disease incidence was recorded. The disease index and control effect were calculated, using the same statistical methods as described in the section on pepper blight control effects. The results are shown in Table 3 below.

[0071] Table 3 Results of field efficacy tests

[0072]

[0073] According to the test results in Table 3, the incidence rate and disease index of the compound biological agent provided in this application decreased significantly after treatment with 500-fold and 1000-fold dilutions. The 500-fold dilution showed a relatively low incidence rate and a control efficacy of 78.36%, while the 1000-fold dilution showed a control efficacy of 74.91%, comparable to the control efficacy of the chemical agent treatment group. This demonstrates that the compound biological agent can effectively control the occurrence of disease and reduce its harm in the field. Furthermore, by comparing the above data, it can be seen that the compound biological agent provided in this application has a certain growth-promoting effect on chili peppers. After transplanting and applying the agent, the plants in the compound biological agent treatment group were more robust than those in the control and chemical agent treatment groups, and the plant growth rate was also faster than that in the control and chemical agent treatment groups. After 30 days, the plant height in the compound biological agent treatment group was 9.48%~20.46% higher than that in the chemical agent treatment group, and 12.72%~24.02% higher than that in the control group. Moreover, the yield was 915.9g~1298.5g higher than that in the chemical agent treatment group, and 1019.1g~1401.7g higher than that in the control group, showing a significant effect in ensuring yield and increasing income.

[0074] (III) Stability of compound biological agents

[0075] 1. Test formulation

[0076] ①The formulation of this invention: the compound biological agent provided in Example 1;

[0077] ② Control formulation group: Compared with Example 1, diatomaceous earth was used as filler, water was used instead of wood vinegar, agricultural emulsion 600# was used as surfactant, and xanthan gum, sodium alginate and antifreeze were not added.

[0078] 2. Experimental Methods

[0079] (1) Storage stability test

[0080] Sample preparation: Seal both formulations separately in 500mL brown glass bottles, with three replicates for each formulation. Place the samples in a constant temperature incubator at 54±2℃ for accelerated storage testing (according to the guidelines for environmental safety evaluation of chemical pesticides, storage at 54℃ for 14 days is approximately equivalent to storage at room temperature (25℃) for 2 years). Samples were taken and tested on day 0 (initial), day 7, and day 14 of storage.

[0081] Detection indicators: The number of viable bacteria was determined according to the method in GB20287-2006.

[0082] Appearance: Observe whether the preparation shows signs of layering, precipitation, clumping or mold growth.

[0083] (2) pH adaptability test

[0084] Sample preparation: Take the formulation of this invention and the control formulation, and dilute them 10 times with sterile water. Adjust the pH of the diluted solution to 4.0, 5.0, 6.0 (control, original pH), 7.0, 8.0, and 9.0 respectively with dilute HCl or NaOH solution. Place the samples with different pH values ​​in a constant temperature shaker at 30℃ (100 rpm) for 24 hours to simulate stress under different acid and alkaline environments.

[0085] Detection indicators: The number of viable bacteria in each treatment was determined according to the method of GB20287-2006, and the relative survival rate relative to the pH 6.0 control treatment was calculated.

[0086] (3) The test results are as follows:

[0087] Table 4 Storage stability test results

[0088]

[0089] As shown in Table 4, after 14 days of accelerated storage at 54°C, the spore survival rate of the formulation group of this invention reached 87.7%, significantly higher than the 42.3% of the control formulation group. This indicates that the carrier used in this invention (containing wood vinegar, xanthan gum, sodium alginate, glycerol, etc.) can greatly slow down the decay rate of spores during storage, effectively maintaining the biological activity of the formulation, and the expected shelf life can reach more than 2 years.

[0090] In terms of appearance, the formulation of this invention is a uniform suspension with slight precipitation that disperses easily when shaken; while the control formulation group has severe stratification, with the bottom clumping and lumps remaining even after shaking, indicating that the composite biological agent provided in this application has excellent physical stability.

[0091] Table 5 pH adaptation results

[0092]

[0093] As shown in Table 5, the relative survival rate of spores in the formulation group of this invention was significantly higher than that in the control formulation group within the pH range of 4.0-9.0. Under strong acid (pH 4.0) and strong alkaline (pH 9.0) stress conditions, the effective viable count of the formulation of this invention remained above 81%, while that of the control group was below 50%. The results indicate that the carrier system of this invention provides a strong buffer and protective environment for *Streptomyces louchei* spores, enabling them to adapt to the complex and variable soil, water, and pH conditions in the field, thereby ensuring that they maintain a high survival rate and colonization ability after inoculation into the environment.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A compound biological agent for the prevention and control of pepper blight, characterized in that, The components include the following contents: Streptomyces loucheri YHLC-1 spore powder 1-5%, matrine 0.1-1%, surfactant 2-8%, stabilizer 1-5%, antifreeze 2-6%, synergist 0.5%-3%, filler 10-20%, and the balance is solvent; The Streptomyces loucherei YHLC-1 spore powder is obtained by fermentation, drying, and pulverization of Streptomyces loucherei YHLC-1; the effective viable count of the Streptomyces loucherei YHLC-1 spore powder is ≥2.6×10⁻⁶. 11 CFU / g, moisture content ≤10%; The Streptomyces loucherii strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35491, and the deposit date is August 1, 2025.

2. The compound biological agent according to claim 1, characterized in that, The compound biological agent comprises the following components in the following proportions: 3% Streptomyces loucheri YHLC-1 spore powder, 0.5% matrine, 5% surfactant, 3% stabilizer, 4% antifreeze, 1.5% synergist, 15% filler, and the balance being solvent.

3. The compound biological agent according to claim 1, characterized in that, The preparation method of Streptomyces loucheri YHLC-1 spore powder is as follows: First, Streptomyces loucheri YHLC-1 is activated and cultured to obtain Streptomyces loucheri YHLC-1 seed liquid; then, Streptomyces loucheri YHLC-1 seed liquid is inoculated into solid fermentation medium at an inoculation rate of 20-30 v / w %, and fermented for 9-11 days to obtain fermented product; then, the fermented product is dried at 40-45℃ to reduce its moisture content to below 10%, and passed through an 80-mesh sieve to obtain Streptomyces loucheri YHLC-1 spore powder.

4. The compound biological agent according to any one of claims 1-3, characterized in that, The surfactant is an alkyl polysaccharide and / or a fatty alcohol polyoxyethylene ether; the stabilizer is xanthan gum and / or sodium alginate.

5. The compound biological agent according to any one of claims 1-3, characterized in that, The antifreeze is glycerol or ethylene glycol; the synergist is an organosilicon synergist; and the solvent is water or wood vinegar.

6. The application of the compound biological agent as described in any one of claims 1-5 in the prevention and control of pepper blight, characterized in that, The method of using the compound biological agent is as follows: 5-10 days after the chili seedlings are transplanted, the compound biological agent is diluted to 300-1000 times, and then the diluted solution is applied to the roots of the chili peppers. The treatment is repeated 3 times during the entire growth period, with an interval of 30±3 days.

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