Bacillus alpine and application thereof, composite biological agent and preparation method and application thereof
By preparing the compound biological agents of Bacillus algae Y391 and Bacillus cerebellum AR156, the environmental pollution and drug resistance problems of cypress root knot nematode disease were solved, and the green and environmentally friendly production increase and growth promotion effect of cypress was achieved.
Patent Information
- Application Number
- CN202510703204.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-08-22
AI Technical Summary
The existing chemical control methods have high costs, environmental pollution and drug resistance when preventing and treating nematode disease of the leukost, and lack efficient prevention and control methods with green and environmental protection.
Complex biological agents were prepared by Bacillus algae Y391 and Bacillus cerebellum AR156, and were used in the silt building through root irrigation and seed coating. Bio-control methods were used to prevent and treat the silt nematode disease in the southern root knot, and promote the growth of the silt building.
The green and environmental protection of the silhouette has been achieved, with a 20-25% increase in yield, which has promoted the emergence of silhouette seedlings and plant growth, and reduced the damage to the environment by chemical agents.
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Figure CN120519331A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbial pesticides, and in particular relates to a strain of Bacillus subtilis and its application, a composite biological agent and its preparation method and application. Background Art
[0002] Trichosanthes kirilowii, a Cucurbitaceae crop, also known as Trichosanthes kirilowii and hanging melon, is grown in many places in my country, including Shandong, Jiangsu, Anhui, etc. It is one of my country's traditional Chinese medicinal materials. Many parts of it can be used as medicine, and its seeds can be made into edible hanging melon seeds. It has certain edible value, health value and medicinal value. The planting area of Trichosanthes kirilowii in Jiangsu region exceeds 50% of the national planting area of Trichosanthes kirilowii.
[0003] In recent years, the scale of Trichosanthes kirilowii cultivation has continued to expand, but at the same time, common pests and diseases affecting the crop have become increasingly severe. Southern root-knot nematode disease is a significant disease affecting Trichosanthes kirilowii production, occurring in all Trichosanthes kirilowii production areas in my country. This disease has a devastating impact on Trichosanthes kirilowii cultivation. When infected, the aboveground plant develops slowly, leaves turn yellow, the plant becomes weak, and the entire plant becomes dwarfed. In severe cases, the entire plant dies, with partial or even complete root rot. Root-knot nematode disease severely impacts both the yield and quality of Trichosanthes kirilowii.
[0004] Currently, chemical control is still the primary method for the prevention and control of plant parasitic nematodes. However, chemical control is associated with high costs, chemical residues, phytotoxicity caused by improper application, the development of nematode resistance through long-term application, safety concerns for humans and livestock, and severe soil pollution. The development of efficient, environmentally friendly methods for the prevention and control of root-knot nematodes remains an urgent challenge. Summary of the Invention
[0005] In light of this, the present invention aims to provide a strain of Bacillus altitudinis Y391, its applications, and a composite biological agent, as well as its preparation method and application. This invention employs a biological control method, targeting root-knot nematodes, to screen for biocontrol strains that are highly effective in controlling the disease in the habitat of Trichosanthes kirilowii plants infected with root-knot nematodes, thereby providing a resource for biological control of southern root-knot nematodes. The strain of Bacillus altitudinis Y391 obtained using this invention has the ability to control southern root-knot nematodes and promote tomato growth. Furthermore, when prepared into a composite agent with Bacillus cereus AR156, it can promote the emergence of Trichosanthes kirilowii seedlings and has a growth-promoting effect on Trichosanthes kirilowii plants, increasing yield by 20-25%.
[0006] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a strain of Bacillus subtilis Y391, which was deposited in the General Microbiology Center of the China Culture Collection Administration on March 13, 2025, with a deposit number of CGMCC NO.33810.
[0007] The present invention also provides a culture of Bacillus subtilis Y391.
[0008] Preferably, the culture comprises a fermentation liquid containing bacteria and a metabolite extract not containing bacteria; and the culture is obtained by culturing Bacillus subtilis Y391.
[0009] The present invention also provides the use of the Bacillus subtilis Y391 described in the above technical solution or the culture of the Bacillus subtilis Y391 described in the above technical solution in preventing and controlling southern root-knot nematode disease and promoting tomato growth.
[0010] Preferably, the application method comprises one or more of root irrigation, seed coating and seed soaking.
[0011] The present invention also provides a composite biological agent, characterized in that it comprises a first component and a second component; The first component includes Bacillus sp. Y391 and / or its metabolites; The second component includes Bacillus cereus AR156 and / or its metabolites; The Bacillus subtilis Y391 was deposited in the General Microbiology Center of China Culture Collection Administration on March 13, 2025, with the deposit number CGMCC NO.33810; The Bacillus cereus AR156 was deposited in the General Microbiology Center of China Culture Collection Administration on January 26, 2007, with the deposit number CGMCC NO.1929. Preferably, the metabolite of Bacillus albus Y391 and the metabolite of Bacillus cereus AR156 are obtained by culturing Bacillus albus Y391 and Bacillus cereus AR156, respectively.
[0012] The present invention also provides a method for preparing the composite biological agent, comprising the following steps: Bacillus albus Y391 and Bacillus cereus AR156 are fermented and cultured respectively to obtain Bacillus albus Y391 and Bacillus cereus AR156 fermentation broths; the two fermentation broths are diluted to predetermined concentrations and then mixed and compounded in a predetermined ratio to obtain the composite biological agent.
[0013] Preferably, the fermentation medium formula of the Bacillus cereus Y391 is: NaCl 10 g, soy peptone 4 g, tryptone 10 g, yeast extract powder 5 g, ultrapure water to 1 L, pH 7.0, and fermentation culture conditions are: 28 ° C, 200 rpm, and fermentation time 30 h; the fermentation medium formula of the Bacillus cereus AR156 is: maltose 0.25%, corn flour 0.5%, soybean flour 0.5%, tryptone 0.5%, CaCl2·2H2O 0.05%, MnSO4·H2O 0.05%, K2HPO4 0.1%, pH 7.0, and fermentation culture conditions are: 28 ° C, 200 rpm, and fermentation time 48 h. The total viable bacteria concentration of the composite biological agent is 5×10 9 ~1×10 10 CFU / mL.
[0014] Preferably, the preparation ratio of the Bacillus altaica Y391 and the Bacillus cereus AR156 is 3:1 to 1:3 based on the number of viable bacteria.
[0015] The present invention also provides the use of the composite biological microbial agent or the composite biological microbial agent obtained by the above preparation method in promoting the emergence and growth of Trichosanthes kirilowii and improving soil.
[0016] Preferably, the application method comprises one or more of root irrigation, seed coating and seed soaking.
[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention isolates a strain from the rhizosphere soil of a Trichosanthes kirilowii plant suffering from root-knot nematode disease, and identifies it as Bacillus altitudinis, named Y391. The Bacillus altitudinis Y391 obtained by the present invention and / or its culture have obvious effects in preventing and controlling southern root-knot nematode disease and promoting tomato growth.
[0018] The present invention prepares a composite biological agent containing Bacillus cereus Y391 and Bacillus cereus AR156. The composite biological agent obtained by the present invention can promote the emergence of Trichosanthes kirilowii seedlings and has a growth-promoting effect on Trichosanthes kirilowii plants, increasing the yield of Trichosanthes kirilowii by 20-25%. The composite biological agent can be used as a green and environmentally friendly biocontrol agent for effectively preventing and treating Trichosanthes kirilowii root-knot nematode disease and promoting the growth of Trichosanthes kirilowii, effectively reducing the damage to the environment caused by chemical agents and the threat to humans.
[0019] Biodeposit Information: Bacillus altitudinis Y391 was deposited on March 13, 2025 at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.33810.
[0020] Bacillus cereus AR156 was deposited on January 26, 2007 at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.1929. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.
[0022] Figure 1 Schematic diagram of the colony morphology of Bacillus subtilis Y391; Figure 2 Schematic diagram of Gram staining results of Bacillus subtilis Y391; Figure 3 This is a schematic diagram of the greenhouse experimental efficacy of Bacillus subtilis Y391; Figure 4 This is a schematic diagram of the growth promotion effect of Bacillus subtilis Y391 in a greenhouse experiment; Figure 5 Schematic diagram of the effect of composite biological agents on the emergence rate of Trichosanthes kirilowii plants (30 days); Figure 6 Schematic diagram of the growth-promoting effect of the composite biological agent on Trichosanthes kirilowii plants (60 days); Figure 7 Schematic diagram of the yield-increasing effect of the composite biological agent on Trichosanthes kirilowii plants (150 days). DETAILED DESCRIPTION
[0023] The present invention provides a strain of Bacillus altitudinis Y391, which was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on March 13, 2025, with the deposit number CGMCC No. 33810. Bacillus altitudinis Y391 grows on a plate and forms milky white, nearly circular colonies with a slight depression in the center. In the early stages of growth, the surface is smooth and wrinkle-free, but wrinkles develop after a period of continued growth. Gram staining and observation under an optical microscope revealed Gram-positive bacteria, and Bacillus altitudinis Y391 appears as short rods.
[0024] In the present invention, the culture includes a fermentation broth containing bacteria or a metabolite extract without bacteria; the concentration of viable bacteria of Bacillus subtilis Y391 in the fermentation broth is 5×10 9 ~1×10 10 CFU / mL; the bacterial-free metabolite extract is the fermentation filtrate obtained by centrifuging the fermentation broth of Bacillus subtilis Y391; the centrifugation speed is 8000 r / min, and the centrifugation time is 5 min.
[0025] The present invention provides a method for preparing the culture, which specifically comprises the following steps: activating the strain, inoculating the activated strain into a liquid culture medium for fermentation, and obtaining a Bacillus subtilis Y391 culture capable of preventing and treating southern root-knot nematode disease and promoting tomato growth.
[0026] In the present invention, the root-knot nematode control method is to kill the second-instar larvae of the root-knot nematode; the root-knot nematode is Meloidogyne incognita. The Trichosanthes kirilowii variety is Wanluo No. 7, hybridized and bred by the Horticulture Institute of Anhui Academy of Agricultural Sciences; and the application method includes one or more of root irrigation, seed coating, and seed soaking.
[0027] The present invention also provides a composite biological agent, comprising a first component and a second component; The first component includes Bacillus sp. Y391 and / or its metabolites; The second component includes Bacillus cereus AR156 and / or its metabolites; The Bacillus subtilis Y391 was deposited in the General Microbiology Center of China Culture Collection Administration on March 13, 2025, with the deposit number CGMCC NO.33810; The Bacillus cereus AR156 was deposited in the General Microbiology Center of China Culture Collection Administration on January 26, 2007, with the deposit number CGMCC NO.1929. The present invention also provides a method for preparing the composite biological agent, comprising the following steps: fermenting and culturing Bacillus cerevisiae Y391 and Bacillus cereus AR156 respectively to obtain fermentation broths of Bacillus cerevisiae Y391 and Bacillus cereus AR156; and diluting the two fermentation broths to predetermined concentrations and then mixing and compounding them in a predetermined ratio to obtain the composite biological agent.
[0028] In the present invention, the step of fermenting and culturing the Bacillus cereus Y391 and Bacillus cereus AR156 preferably includes: the fermentation medium formula of Bacillus cereus Y391 is: 10 g NaCl, 4 g soy peptone, 10 g tryptone, 5 g yeast extract powder, ultrapure water to 1 L, pH value 7.0, and fermentation culture conditions are: 28°C, 200 rpm, and fermentation time 30 h; the fermentation medium formula of Bacillus cereus AR156 is: 0.25% maltose, 0.5% corn flour, 0.5% soybean flour, 0.5% tryptone, 0.05% CaCl2·2H2O, 0.05% MnSO4·H2O, 0.1% K2HPO4, pH value 7.0, and fermentation culture conditions are: 28°C, 200 rpm, and fermentation time 48 h.
[0029] The total concentration of live bacteria in the composite biological agent is 5×10 9 ~1×10 10 CFU / mL. In terms of viable bacteria count, the ratio of Bacillus albopictus Y391 to Bacillus cereus AR156 is 3:1 to 1:3.
[0030] The present invention also provides the use of the composite biological microbial agent or the composite biological microbial agent obtained by the above preparation method in promoting the emergence and growth of Trichosanthes kirilowii and improving soil.
[0031] To further illustrate the present invention, a strain of Bacillus subtilis and its application, a composite biological agent and its preparation method and application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0032] Example 1, Isolation, Screening and Identification of Bacillus subtilis Y391: (1) Strain isolation and screening: Strain source: Bacillus subtilis Y391 was isolated from the rhizosphere soil of a Trichosanthes kirilowii plant (variety: Wanlou No. 7) infected with root-knot nematode disease collected from a Trichosanthes kirilowii cultivation base in Anhui Province.
[0033] Strain isolation method: Weigh 3 g of soil around the roots of Trichosanthes kirilowii and put it into a 50 mL centrifuge tube. Add 27 mL of sterile water and shake to mix. Place the centrifuge tube in a 28°C shaker at 200 rpm for 30 min, take it out, let it stand at room temperature for 10 min, and take the supernatant as 10 -1 dilution, 10 -1 The diluent was gradually diluted to 10 -2 ~10 -6 Different concentration gradient dilutions, take 10 -4 ~10 -6A concentration gradient of dilutions was plated onto LB plates, with 100 μL per plate in triplicate. The plates were incubated at 28°C for 48 hours. Single colonies were picked from the plates for purification. The LB liquid culture was incubated on a shaker at 28°C and 200 rpm for 16 hours. The bacterial solution was mixed with equal amounts of 80% glycerol and stored in a -80°C freezer. The isolated and purified strains were screened using the spot-grafting method on LB medium. Strains with distinct inhibition zones were selected through primary and secondary screening.
[0034] (2) Observation of strain morphology: The strain Y391 was activated by streaking on LB solid medium plates using the three-line streak method and cultured in a 28°C incubator for 24 h. Figure 1 As shown, the colony morphology of strain Y391 growing on the plate is milky white, nearly round, with a slight depression in the center. In the early stage of growth, the surface of the colony is smooth and without wrinkles. After continuing to grow for a period of time, wrinkles are formed on the surface of the colony.
[0035] (3) Physiological and biochemical characteristics: Take the activated strain Y391 for Gram staining and observe the Gram staining results and bacterial morphology under an optical microscope. Figure 2 As shown, Gram staining is positive and the strain Y391 bacteria are short rod-shaped.
[0036] The physiological and biochemical characteristics of strain Y391 are shown in Table 1. Strain Y391 can utilize carbon sources such as glucose, sucrose, and D-mannitol; does not produce hydrogen sulfide gas; and is positive in MR reaction, VP reaction, and catalase reaction.
[0037] The physiological and biochemical characteristics of strain Y391 are shown in the following table:
[0038] Note: “+” indicates that the strain is positive for this reaction, and “-” indicates that the strain is negative for this reaction.
[0039] (4) Molecular identification:
[0040] Analysis of the 16S rDNA sequence of strain Y391: A single colony of MTH5-1 was selected and its 16S rDNA sequence was amplified using the following primers: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1) 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).
[0041] After the PCR amplification products were tested by 1% agarose gel electrophoresis, the gel block containing the target band was excised and sent to a testing company for sequencing. The 16S rDNA sequence fragment of strain Y391 was sequenced to be 1420 bp in length, as shown in SEQ ID NO. 3:
[0042] The measured 16S rDNA sequence was subjected to homology analysis using bioinformatics, and it was found that the 16srRNA sequence of strain 391 was 100.00% similar to that of Bacillus altitudinis strain 41KF2bT.26 (MN543858.1). Combined with the characteristics of the colonies on the culture dish, the isolated strain Y391 of the present invention was identified as Bacillus altitudinis and named Bacillus altitudinis Y391. It was deposited in the General Microbiology Center of the China Culture Collection Administration on March 13, 2025, with the deposit number CGMCC NO.33810.
[0043] Example 2, Indoor bioassay experiment of Bacillus subtilis Y391 fermentation broth against second-instar nematodes of the southern root-knot nematode: Preparation of Bacillus subtilis Y391 fermentation broth: The Bacillus subtilis Y391 obtained in Example 1 was cultured on LB solid medium at 28°C for 2 days, and a single colony was inoculated into LB liquid medium and cultured in a constant temperature shaker at 28°C and 200 rpm for 24 hours to obtain a fermentation seed liquid. The fermentation seed liquid was inoculated into LB liquid medium at an inoculum size of 2%, and cultured in a constant temperature shaker at 28°C and 200 rpm for 48 hours. The concentration of all bacterial broths was adjusted to 8.0×10 8 The bacterial fermentation liquid used in the greenhouse experiment was obtained by averaging CFU / ml. The bacterial fermentation liquid was diluted with sterile water by 2, 5, and 10 times, respectively, to prepare the bacterial suspension used in the final experiment.
[0044] Isolation of southern root-knot nematodes: Mince the root tissue of Trichosanthes kirilowii into approximately 1 × 1 cm pieces. Use the Baermann funnel method to isolate and collect pathogenic nematodes from both the plant tissue and the rhizosphere soil. Every 24 hours, 5–10 mL of the suspension at the bottom of the funnel is collected to observe the nematode population and replace the water. The resulting nematode suspension is placed in a 50 mL centrifuge tube and held vertically for 10 hours. After the nematodes settle, remove the supernatant to obtain a highly concentrated nematode suspension, which is then stored in a laboratory refrigerator at 4°C.
[0045] The collected second-instar nematodes were diluted with sterile water to a nematode suspension of 500 nematodes / mL. In vitro experiments were performed in 96-well microplates. 100 μL of the nematode suspension was added to each well, with approximately 50 nematodes per well. 100 μL of the bacterial suspension diluted 2-, 5-, and 10-fold was also added. Clear water was used as a control. After 24 hours of treatment, the number of nematode deaths was counted and the corrected mortality rate was calculated. The corrected mortality rate was calculated as follows: Corrected nematode mortality (%) = (nematode mortality in the treatment group - nematode mortality in the control group) / (1 - mortality in the control group) × 100%; The in vitro killing effect of Bacillus subtilis Y391 fermentation liquid on southern root-knot nematodes is shown in the following table: .
[0046] Example 3, Greenhouse Disease Prevention Experiment of Bacillus subtilis Y391 Fermentation Broth Prepare a fermentation broth of Bacillus subtilis Y391 and a nematode suspension according to the method of Example 2, respectively, and set aside. Select tomato plants (variety: Hezuo 903) and soak the tomato seeds in a 3% sodium hypochlorite solution for 30 minutes to disinfect their surface. Rinse the seeds with clean water to remove the sodium hypochlorite solution from their surface. Germination is accelerated at 26°C, and seeding is performed in plug trays. Select tomato seedlings that have uniform growth and reach the four-leaf stage and transplant them into individual pots.
[0047] Seven days after transplanting, each tomato seedling was inoculated with 20 mL of 8.0×10 8 CFU / ml of Bacillus subtilis Y391 fermentation liquid was used as a control, and the culture was continued for 7 days before inoculation with nematode suspension, with 500 nematodes per plant. 50 days after transplanting, the disease status of tomato plants in the treatment group and the control group was statistically analyzed. The root length, fresh root weight, number of root egg masses and number of root knots of the diseased tomato plants were counted. The results are as follows: Figure 3 Refer to the table below for the disease classification method of tomato root knot nematode disease to classify the tomato root knot nematode disease-infected plants: .
[0048] Calculate the disease index and relative prevention effect: Disease occurrence index (%) = Σ (disease level × number of diseased plants at that level) / (total number of plants surveyed × number of plants with the highest disease level) × 100%; Relative control effect (%) = (disease severity of the control group - disease severity of the treatment group) / disease severity of the control group × 100%.
[0049] The test results of the greenhouse experimental efficacy (50 days) of the fermentation liquid of Bacillus subtilis Y391 are shown in the following table:
[0050] Among them, the root length of tomato plants treated with the fermentation liquid of Bacillus subtilis Y391 increased by 32.64% compared with the control group, the fresh root weight of tomatoes increased by 87.69%, the number of root knots on the roots of tomatoes decreased by 43.88%, and the number of nematode eggs on the roots of tomatoes decreased by 41.35%. The prevention efficiency of the fermentation liquid of Bacillus subtilis Y391 reached 59.74%.
[0051] Example 4, greenhouse growth promotion experiment of Bacillus subtilis Y391 fermentation broth: A fermentation broth of Bacillus subtilis Y391 was prepared as in Example 2. Tomato plants (variety: Hezuo 903) that had been transplanted for 7 days were prepared as in Example 3.
[0052] Each tomato seedling was inoculated with 20 mL of bacterial fermentation liquid, and water treatment was used as the control. After 30 days of cultivation, the growth indicators such as plant height, stem diameter, number of leaves, and chlorophyll content of leaves of the control and treatment groups were measured and statistically analyzed. Figure 4 .
[0053] The results of the greenhouse experiment on the growth promotion effect of Bacillus subtilis Y391 fermentation liquid (30 days) are shown in the following table:
[0054] Among them, the plant height of the group treated with Bacillus subtilis Y391 fermentation liquid increased by 39.40% and the number of leaves increased by 20.88% compared with the control group.
[0055] Example 5, preparation of composite biological agent: In this example, a composite biological agent was prepared using the Bacillus cereus Y391 screened in Example 1 and the Bacillus cereus AR156 screened in the laboratory in the early stage.
[0056] The Bacillus cereus AR156 was deposited in the General Microbiology Center of China Culture Collection Administration on January 26, 2007, with the deposit number being CGMCC NO.1929.
[0057] The preparation method of the composite biological agent comprises the following specific steps: culturing Bacillus cerevisiae Y391 and Bacillus cereus AR156 on LB solid medium at 28°C for 2 days, inoculating a single colony of Bacillus cerevisiae Y391 into a fermentation medium for cultivation (the fermentation medium formula is: NaCl 10 g, soy peptone 4 g, tryptone 10 g, yeast extract powder 5 g, ultrapure water to 1 L, pH 7.0, fermentation culture conditions are: 28°C, 200 rpm, fermentation time 30 h), and inoculating a single colony of Bacillus cereus AR156 into a fermentation medium for cultivation (the fermentation medium formula is: maltose 0.25%, corn flour 0.5%, soybean flour 0.5%, tryptone 0.5%, CaCl2·2H2O 0.05%, MnSO4·H2O 0.05%, K2HPO4 0.1%, pH The fermentation conditions were: 28°C, 200 rpm, and 48 h. The two fermentation broths were diluted to a total viable bacterial concentration of 5 × 10 9 ~1×10 10CFU / mL and then mixed and compounded in a ratio of 3:1 to 1:3 to obtain the composite biological agent.
[0058] Example 6, application of composite biological agent: This example uses the composite biological agent prepared in Example 5 at a ratio of 1:1 to verify the growth promotion and soil improvement of Trichosanthes kirilowii. The specific steps include: Female tubers of Wanluo No. 7 (a hybrid bred at the Horticulture Institute of the Anhui Academy of Agricultural Sciences) were cut into 5-8 cm long segments. The treatment group soaked the seeds in a 100-fold dilution of a compound biological agent for 5 minutes before planting; the control group was mixed with plant ash and then planted. The roots were placed flat in a hole and irrigated with a 200-fold dilution of the compound biological agent at a rate of 2-3 liters per mu (approximately 200 times the original volume). The soil was then covered and the bed surface was raked and leveled. The control group used plain water for root irrigation as a control. After the Trichosanthes kirilowii seedlings emerged, the treatment group used a 200-fold dilution of the compound biological agent at a rate of 3-5 liters per mu (approximately 200 times the original volume). The aboveground parts of the Trichosanthes kirilowii plants were sprayed with the compound biological agent once a month for three times throughout the growing season. The control group was watered with plain water; the aboveground parts of the Trichosanthes kirilowii plants were sprayed with plain water as a control.
[0059] (1) Growth index determination: The emergence rate of Trichosanthes kirilowii plants was counted one month after planting ( Figure 5 ), the height, stem diameter, number of leaves and chlorophyll content of the Trichosanthes kirilowii plants were counted 2 months after planting ( Figure 6 ), after the fruiting of the Trichosanthes kirilowii, the number of fruiting plants per treatment was counted ( Figure 7 ); The effects of the composite biological agent on the growth indicators of Trichosanthes kirilowii are shown in the following table:
[0060] Note: The seedling emergence rate in the table is the data 30 days after planting, the plant height, stem diameter, chlorophyll and leaf number are the data 60 days after planting, and the number of fruits per plant is the data 150 days after planting.
[0061] The results are shown in the table above. The seedling emergence rate of Trichosanthes kirilowii treated with the composite biological agent increased by 11.17% compared with the control group; after 3 months of planting, the plant height, stem diameter, chlorophyll content SPAD value and leaf number of the Trichosanthes kirilowii treated group increased by 25.21%, 9.59%, 19.71% and 25.94% respectively compared with the control group; when the Trichosanthes kirilowii fruited, the number of fruits per plant in the composite biological agent treated group increased by 22.40% compared with the control group.
[0062] (2) Testing of various soil indicators: 90 days after planting, soil sections (0–25 cm) were collected from the field at the Trichosanthes kirilowii planting site, dried, crushed, and mixed. Soil organic matter, available nitrogen, available phosphorus, and available potassium were measured using a TPY-6A soil nutrient rapid analyzer. The effects of the composite biological agent on the physical and chemical properties of the Trichosanthes kirilowii soil (90 days) are shown in the following table:
[0063] The results are shown in the table above. The contents of soil ammonium nitrogen, available potassium, organic matter and available phosphorus in the composite biological agent treatment group were 36.50 mg / kg, 253.76 mg / kg, 12.78 g / kg and 62.71 mg / kg, respectively, which were 32.53%, 44.56%, 57.58% and 43.51% higher than those in the control group.
[0064] It can be seen from the above examples that the composite biological agent prepared by the present invention can be used as a green, environmentally friendly, and highly effective biocontrol agent for preventing and controlling southern root-knot nematode disease and promoting the growth of Trichosanthes kirilowii.
[0065] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A strain of Bacillus altitudinis Y391, characterized in that: The Bacillus subtilis Y391 was deposited in the General Microbiology Center of the China Culture Collection Administration on March 13, 2025, with the deposit number CGMCC NO.33810.
2. Use of the Bacillus sp. Y391 and / or its culture according to claim 1 in preventing and controlling southern root-knot nematode disease and promoting tomato growth.
3. The culture according to claim 2, characterized in that The culture comprises a fermentation liquid containing bacteria and a metabolite extract liquid not containing bacteria; the culture is obtained by culturing Bacillus subtilis Y391.
4. The use according to claim 2, characterized in that The application method comprises one or more of root irrigation, seed coating and seed soaking.
5. A composite biological agent, characterized in that: comprising a first component and a second component; The first component includes Bacillus sp. Y391 and / or its metabolites; The second component includes Bacillus cereus AR156 and / or its metabolites; The Bacillus subtilis Y391 was deposited in the General Microbiology Center of China Culture Collection Administration on March 13, 2025, with the deposit number CGMCC NO.33810; The Bacillus cereus AR156 was deposited in the General Microbiology Center of China Culture Collection Administration on January 26, 2007, with the deposit number CGMCC NO.1929. The Bacillus albus Y391 metabolite and the Bacillus cereus AR156 metabolite are obtained by culturing Bacillus albus Y391 and Bacillus cereus AR156, respectively.
6. The method for preparing the composite biological agent according to claim 5, characterized in that: The following steps are involved: Bacillus albus Y391 and Bacillus cereus AR156 are fermented and cultured respectively to obtain Bacillus albus Y391 and Bacillus cereus AR156 fermentation broths; the two fermentation broths are diluted to predetermined concentrations and then mixed and compounded in a predetermined ratio to obtain the composite biological agent.
7. The preparation method according to claim 6, characterized in that The fermentation medium formula of the Bacillus cereus Y391 is as follows: 10 g of NaCl, 4 g of soy peptone, 10 g of tryptone, 5 g of yeast extract powder, and ultrapure water to 1 L, with a pH value of 7.
0. The fermentation culture conditions are: 28°C, 200 rpm, and a fermentation time of 30 h. The fermentation medium formula of the Bacillus cereus AR156 is as follows: 0.25% maltose, 0.5% corn flour, 0.5% soybean flour, 0.5% tryptone, 0.05% CaCl2·2H2O, 0.05% MnSO4·H2O, 0.1% K2HPO4, and a pH value of 7.
0. The fermentation culture conditions are: 28°C, 200 rpm, and a fermentation time of 48 h.
8. The preparation method according to claim 6, characterized in that The total concentration of live bacteria in the composite biological agent is 5×10 9 ~1×10 10 CFU / mL. In terms of viable bacteria count, the ratio of Bacillus albopictus Y391 to Bacillus cereus AR156 is 3:1 to 1:
3.
9. Use of the composite biological agent according to claim 5 or the composite biological agent obtained by the preparation method according to any one of claims 6 to 8 in promoting the emergence and growth of Trichosanthes kirilowii and improving soil.
10. The use according to claim 9, characterized in that The application method comprises one or more of root irrigation, seed coating and seed soaking.