Bacillus subtilis IVF-WKB2 and its application

Abstract

The present invention provides a Bacillus subtilis IVF-WKB2 and an application thereof, and belongs to the field of microbial technology. The present invention provides a Bacillus subtilis IVF-WKB2, and the deposit number of the Bacillus subtilis IVF-WKB2 is CGMCC No.28314. The Bacillus subtilis IVF-WKB2 has the function of promoting plant growth, can increase crop yield, increase the number of plant fruits and single fruit weight, etc. At the same time, it can also promote the growth of plants under low temperature and / or weak light conditions, enhance the stress resistance of plants to low temperature and / or weak light, and especially promote the growth of cucumbers that are not resistant to low temperature and / or weak light. Furthermore, the Bacillus subtilis IVF-WKB2 and chemical fertilizers are co-applied during the cucumber planting process, and the Bacillus subtilis IVF-WKB2 and chemical fertilizers can synergistically promote the growth of cucumbers and increase the yield of cucumbers.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to Bacillus subtilis IVF-WKB2 and applications thereof. Background Art

[0002] cucumber( Cucumis sativus Cucumber (L.) is a common vegetable and fruit, also known as cucumber or cucumber, which is now widely grown in temperate and tropical regions. Cucumber has become one of the essential vegetables in modern families due to its high nutritional value, so the demand is relatively large. However, cucumbers prefer warmth and are not cold-resistant. Due to the influence of the planting environment, fertilizers and human factors, the yield of cucumbers cannot reach the expected effect.

[0003] It is generally believed in the prior art that biological measures can not only reduce the pressure on agriculture, but also have important significance for promoting the healthy development of sustainable agriculture. For example, rhizosphere growth-promoting bacteria can not only colonize in the rhizosphere of plants, promote plant growth, promote the absorption and utilization of mineral nutrients, but also inhibit harmful organisms. However, due to the influence of their sources, the effects of different rhizosphere growth-promoting bacteria are not the same. At the same time, although some microbial agents have the effect of promoting crop growth, the effect of applying microbial agents alone during crop growth is often not ideal. In order to better promote crop growth, it is often necessary to apply microbial agents together with chemical fertilizers. However, various substances in chemical fertilizers often have an adverse effect on the effect of microbial agents in the process of co-acting with microbial agents, thereby reducing the effect of microbial agents. Therefore, although there are many microbial agents with growth-promoting effects reported at present, there are few microbial agents that can synergistically enhance growth with chemical fertilizers, especially microbial agents that can act together with fertilizers to synergistically promote cucumber growth. There are no related reports. Summary of the invention

[0004] In view of the defects in the prior art, the object of the present invention is to provide a Bacillus subtilis IVF-WKB2; the Bacillus subtilis IVF-WKB2 can promote the growth of cucumbers, significantly increase the yield of cucumbers, and at the same time enhance the stress resistance of cucumbers that are intolerant to low temperature and / or weak light, and promote the growth of cucumbers under low temperature and weak light conditions. At the same time, the Bacillus subtilis IVF-WKB2 can also work together with chemical fertilizers to synergistically promote the growth of cucumbers and increase the yield of cucumbers.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a Bacillus subtilis ( Bacillus subtilis ) IVF-WKB2, the deposit number of the Bacillus subtilis IVF-WKB2 is CGMCC No.28314.

[0007] Preferably, the 16S rDNA sequence of Bacillus subtilis IVF-WKB2 is shown as SEQ ID NO.1.

[0008] The present invention provides a microbial preparation, comprising the Bacillus subtilis IVF-WKB2 described in the above technical solution.

[0009] Preferably, the viable count of the Bacillus subtilis IVF-WKB2 in the microbial preparation is ≥10 7 cfu / mL.

[0010] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0011] The Bacillus subtilis IVF-WKB2 is cultured in a culture medium to obtain a microbial preparation.

[0012] Preferably, the culture temperature is 28-37° C.; the culture is accompanied by rotation; and the rotation speed is 200-220 rpm.

[0013] The present invention provides a fertilizer, comprising the Bacillus subtilis IVF-WKB2 described in the above technical solution, the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution.

[0014] The present invention provides use of the Bacillus subtilis IVF-WKB2 described in the above technical solution, the microbial preparation described in the above technical solution, the microbial preparation prepared by the preparation method described in the above technical solution, or the fertilizer described in the above technical solution in promoting plant growth and / or enhancing plant stress resistance.

[0015] Preferably, the plant comprises cucumber.

[0016] The present invention provides a method for promoting plant growth, comprising: applying the microbial preparation described in the above technical solution or the fertilizer described in the above technical solution.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a Bacillus subtilis ( Bacillus subtilis) IVF-WKB2, the deposit number of the Bacillus subtilis IVF-WKB2 is CGMCC No.28314. In the present invention, the Bacillus subtilis IVF-WKB2 or its microbial preparation has the effect of promoting plant growth, including increasing crop yield, increasing the number of fruit set of plants, increasing the weight of single fruit of plants, etc. At the same time, it also has the effect of promoting plant growth under low temperature and / or weak light conditions, and enhancing the stress resistance of plants to low temperature and / or weak light. The results of the embodiments of the present invention show that the Bacillus subtilis IVF-WKB2 can significantly increase the plant height, leaf area, whole plant fresh weight and whole plant dry weight of cucumbers that are not tolerant to low temperature and / or weak light, thereby achieving the effect of promoting cucumber growth. At the same time, the present invention applies the Bacillus subtilis IVF-WKB2 and chemical fertilizers together during the cucumber planting process, and the Bacillus subtilis IVF-WKB2 and chemical fertilizers can synergistically promote the growth of cucumbers and increase the yield of cucumbers. In summary, the Bacillus IVF-WKB2 or its microbial preparation provided by the present invention has the effects of promoting plant growth and increasing crop yield, and can be used to prepare plant growth regulators, fertilizers and other products.

[0019] Biological deposit information

[0020] The Bacillus subtilis provided by the present invention ( Bacillus subtilis ) IVF-WKB2, deposited on August 31, 2023 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.28314. 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 will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 This is a colony morphology diagram of Bacillus IVF-WKB2 in the culture medium in Example 1;

[0023] Figure 2 This is a colony morphology of Bacillus IVF-WKB2 under a microscope in Example 1;

[0024] Figure 3 This is a phylogenetic analysis diagram of Bacillus IVF-WKB2 in Example 1;

[0025] Figure 4This is a bar chart of different measurement indicators of cucumbers under different treatments in Application Example 1;

[0026] Figure 5 This is the growth status diagram of cucumber under different treatments in Application Example 1;

[0027] Figure 6 This is a graph showing the effect of the fertilizer control group treatment method on the growth of cucumber fruits in Application Example 2;

[0028] Figure 7 This is a graph showing the effect of the treatment method of adding fertilizer IVF-WKB2 group in Application Example 2 on the growth of cucumber fruit;

[0029] Figure 8 This is a graph showing the effects of different treatments on the per-acre yield of cucumber in the IVF-WKB2 group, the control group, the Y1 group, and the Y2 group in Application Example 2;

[0030] Fig. 9 This is a graph showing the effects of different treatments on the number of cucumber fruits in the IVF-WKB2 group, the control group, the Y1 group, and the Y2 group in Application Example 2;

[0031] Fig.10 This is a graph showing the effects of different treatments on per-acre yield of cucumber in IVF-WKB2 group, control group, Y1 group and Y2 group in Application Example 2;

[0032] Fig.11 This is a graph showing the effects of different treatments on the number of cucumber fruits in the IVF-WKB2 group, control group, Y1 group, and Y2 group in Application Example 2;

[0033] Fig.12 This is a graph showing the effects of different treatments on the per-acre yield of cucumber in the IVF-WKB2 group, the fertilizer control group, the Y1 group, and the Y2 group in Application Example 2;

[0034] Fig.13 This is a graph showing the effects of different treatments on the number of cucumber fruits in the IVF-WKB2 group, fertilization control group, Y1 group, and Y2 group in Application Example 2. DETAILED DESCRIPTION

[0035] The present invention provides a Bacillus subtilis ( Bacillus subtilis ) IVF-WKB2, the deposit number of the Bacillus subtilis IVF-WKB2 is CGMCC No.28314.

[0036] In the present invention, the Bacillus subtilis IVF-WKB2 is isolated from the rhizosphere soil of cucumber in the greenhouse of the Nankou pilot experimental base of the Chinese Academy of Agricultural Sciences. The Bacillus subtilis IVF-WKB2 of the present invention was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on August 31, 2023.

[0037] In the present invention, the culture medium of the Bacillus subtilis IVF-WKB2 can be selected from LB culture medium, TSB culture medium and aerobic fermentation culture medium. In the present invention, the aerobic fermentation culture medium preferably includes, by weight percentage: 0.5% to 2.0% corn flour, 0.1% to 1.0% glucose, 0.5% to 3% bean cake powder, 0.1% to 1.5% fish meal, 0.31% to 1.69% calcium carbonate, 0.05% to 0.15% ammonium sulfate, 0.01% to 0.06% dipotassium hydrogen phosphate, 0.01% to 0.06% magnesium sulfate, 0.01% to 0.06% manganese sulfate and the balance water; more preferably includes: 1% corn flour, 0.5% glucose, 1.5% bean cake powder, 0.8% fish meal, 1% calcium carbonate, 0.1% ammonium sulfate, 0.03% dipotassium hydrogen phosphate, 0.03% magnesium sulfate and 0.03% manganese sulfate, and the rest is water.

[0038] In the present invention, the morphological characteristics of the Bacillus subtilis IVF-WKB2 are as follows Figure 1 and Figure 2 As shown, specifically: the colony morphology is small and round, the colony surface is smooth, flat or convex, the edges are neat, opaque, wet and sticky; under an optical microscope, the bacteria are blunt-round, arranged individually, and have spores. In the present invention, the Bacillus subtilis IVF-WKB2 is positive by Gram staining.

[0039] In the present invention, the 16S rDNA sequence of the Bacillus subtilis IVF-WKB2 is 1384 bp in total, as shown in SEQ ID NO.1.

[0040] The 16S rDNA sequence of Bacillus subtilis IVF-WKB2 was obtained and compared with the sequence in the GenBank database by BLAST analysis. The molecular identification cluster map constructed was as follows Figure 3 As shown, the target strain IVF-WKB2 is at the same developmental level as Bacillus subtilis and has the closest relationship, so the target strain is Bacillus subtilis and is named Bacillus subtilis IVF-WKB2.

[0041] The present invention provides a microbial preparation, comprising the Bacillus subtilis IVF-WKB2 described in the above technical solution. In the present invention, the number of viable bacteria of the Bacillus subtilis IVF-WKB2 in the microbial preparation is preferably ≥10 7 cfu / mL, more preferably 10 7 In the present invention, the microbial preparation preferably includes any one or two or more of the culture of Bacillus subtilis IVF-WKB2, the fermentation product of Bacillus subtilis IVF-WKB2 and the metabolite of Bacillus subtilis IVF-WKB2.

[0042] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0043] The Bacillus subtilis IVF-WKB2 is cultured in a culture medium to obtain a microbial preparation.

[0044] The present invention preferably carries out expansion culture of the Bacillus subtilis IVF-WKB2 in a culture medium to obtain a Bacillus subtilis IVF-WKB2 culture fluid. The culture medium used for the expansion culture of the present invention preferably includes a TSB liquid culture medium; the inoculation amount of the expansion culture is preferably 1% to 10% of the total volume of the expansion culture medium, and more preferably 1%; the temperature of the expansion culture is preferably 28 to 37°C, and more preferably 37°C; the time of the expansion culture is preferably 6 to 8h, and more preferably 8h; the expansion culture is preferably accompanied by rotation; the rotation speed is preferably 200 to 220rpm, and more preferably 220rpm. After the expansion culture is completed, a Bacillus subtilis IVF-WKB2 culture fluid is obtained. After obtaining the Bacillus subtilis IVF-WKB2 culture fluid, the present invention preferably detects the OD of the Bacillus subtilis IVF-WKB2 culture fluid. 600 The above-mentioned expanded culture of the present invention preferably obtains OD 600 The value of Bacillus subtilis IVF-WKB2 culture fluid is 1. In the present invention, the Bacillus subtilis IVF-WKB2 culture fluid can also be referred to as the culture of Bacillus subtilis IVF-WKB2.

[0045] After obtaining the culture fluid of Bacillus subtilis IVF-WKB2, the present invention preferably centrifuges the culture fluid of Bacillus subtilis IVF-WKB2; the centrifugation preferably includes a first centrifugation and a second centrifugation; the speed of the first centrifugation is preferably 3000~6000g, more preferably 5000g; the time of the first centrifugation is preferably 5~15min, more preferably 10min. After the first centrifugation is completed, the present invention preferably rinses the precipitate obtained by the first centrifugation; the rinsing solvent preferably includes sterile water. After the rinsing is completed, the present invention preferably performs a second centrifugation; the speed and time of the second centrifugation are the same as the speed and time of the first centrifugation, which will not be repeated here. After the second centrifugation is completed, the present invention obtains a precipitate containing strain IVF-WKB2. After obtaining the precipitate containing strain IVF-WKB2, the present invention preferably resuspends the precipitate containing strain IVF-WKB2; the resuspending reagent preferably includes sterile water; the amount of sterile water used is based on the ability to obtain an OD value of 0.02 for Bacillus subtilis IVF-WKB2 at a wavelength of 600nm using an ultraviolet spectrophotometer; in this way, an effective viable cell count of ≥10 7cfu / mL of Bacillus subtilis IVF-WKB2 bacterial solution. In the present invention, the effective viable count is ≥10 7 The Bacillus subtilis IVF-WKB2 bacterial liquid with a concentration of cfu / mL can be directly applied as a microbial preparation by root irrigation; or the Bacillus subtilis IVF-WKB2 fermentation liquid can be further prepared.

[0046] After obtaining the Bacillus subtilis IVF-WKB2 bacterial liquid, the present invention preferably ferments and cultures the Bacillus subtilis IVF-WKB2 to obtain the Bacillus subtilis IVF-WKB2 fermentation liquid. In the present invention, the fermentation culture medium preferably includes an aerobic fermentation medium; the aerobic fermentation medium has been described above and will not be repeated here. The fermentation culture of the present invention preferably inoculates the Bacillus subtilis IVF-WKB2 bacterial liquid into the aerobic fermentation medium for fermentation culture. When the present invention is inoculated, the volume ratio of the Bacillus subtilis IVF-WKB2 bacterial liquid to the aerobic fermentation medium is preferably (1~2): (10~30), more preferably 1:10; the temperature of the fermentation culture is preferably 28~37°C, more preferably 37°C; the fermentation culture time is preferably 10~16h, more preferably 12h; the fermentation culture is preferably accompanied by rotation; the rotation speed is preferably 200~220rpm, more preferably 220rpm. After the fermentation culture is completed, the present invention obtains the Bacillus subtilis IVF-WKB2 fermentation liquid.

[0047] After obtaining the Bacillus subtilis IVF-WKB2 fermentation liquid, the present invention preferably dilutes the fermentation liquid; the dilution solvent preferably includes sterile water; the volume ratio of the sterile water to the aerobic fermentation liquid is preferably (10-30): (1-2), more preferably 10:1. The fermentation liquid after dilution can be directly applied to plants as a microbial preparation. 600 Preferably, the effective viable count of the fermentation broth is ≥ 10 7 cfu / mL.

[0048] The present invention provides a fertilizer, including the Bacillus subtilis IVF-WKB2 described in the above technical solution, the microbial preparation described in the above technical solution, or the microbial preparation prepared by the preparation method described in the above technical solution. In the present invention, the fertilizer preferably may also include other fertilizers; the other fertilizers preferably include chemical fertilizers; the chemical fertilizers preferably include water-soluble compound fertilizers; the mass ratio of nitrogen, phosphorus and potassium in the water-soluble compound fertilizer is preferably 20:10:20. The present invention applies the microbial preparation and the water-soluble compound fertilizer in combination, and the microbial preparation and the water-soluble compound fertilizer have a synergistic technical effect in promoting the growth of cucumbers, significantly increasing the single fruit weight and the number of fruits set of cucumbers, thereby significantly increasing the yield of cucumbers.

[0049] The present invention provides the use of Bacillus subtilis IVF-WKB2 described in the above technical solution, the microbial preparation described in the above technical solution, the microbial preparation prepared by the preparation method described in the above technical solution, or the fertilizer described in the above technical solution in promoting plant growth and / or enhancing plant stress resistance. In the present invention, the plant preferably includes cucumbers; the cucumbers preferably include cucumbers that are intolerant to low temperatures and / or weak light. In the present invention, the temperature range of the low temperature preferably includes 10~20°C, optionally, the daytime temperature range is >14°C and ≤20°C, and the night temperature range is ≥10°C and ≤14°C. In the present invention, the weak light condition range is preferably 3000lx~10000lx, and the illumination time range is preferably 6h~8h. The variety of the cucumber preferably includes any one or more of Deruit 89, Jinchun No. 4 and Zhongnongda No. 11. In the present invention, the promoting plant growth preferably includes any one or more of increasing the plant height, increasing the stem thickness of the plant, increasing the number of fruit set of the plant, increasing the single fruit weight of the plant, increasing the female flower rate of the plant, increasing the leaf area of ​​the plant, increasing the dry weight of the plant, increasing the fresh weight of the plant and increasing the root length of the plant. In the present invention, the Bacillus subtilis IVF-WKB2 can further achieve the effect of increasing plant yield by promoting plant growth. In the present invention, the enhancing the stress resistance of the plant preferably includes enhancing the stress resistance of the plant under low temperature and / or weak light conditions.

[0050] The present invention also provides a method for promoting plant growth, comprising applying the microbial preparation described in the above technical solution or the fertilizer described in the above technical solution; the method of application includes root irrigation. In the present invention, the application period of the microbial preparation or the fertilizer preferably includes the entire growth period; optionally, it includes any period or any two or more periods of the seedling period, flowering period and fruit setting period. In the present invention, the microbial preparation or the fertilizer is preferably applied at 70-120 mL / plant each time, more preferably 100 mL / plant; the frequency of application is preferably once every 1-2 weeks, more preferably once a week.

[0051] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] Example 1

[0053] Isolation and identification of Bacillus subtilis IVF-WKB2

[0054] 1. Source of Bacillus subtilis IVF-WKB2

[0055] The strains were isolated and cultured on LB solid medium using the streak method from different cucumber rhizosphere soil samples, where the cucumbers were grown in the greenhouse of the Nankou pilot experimental base of the Chinese Academy of Agricultural Sciences. The streak method was repeatedly operated on LB solid medium until a single bacterial colony with stable growth was obtained, which was named strain IVF-WKB2.

[0056] 2. Morphological identification

[0057] Pick a small amount of the target strain (strain IVF-WKB2) with an inoculation loop and streak it on LB solid medium. After culturing at 28°C for 20 hours, observe the morphology, shape, size, edge, surface, ridge shape, transparency, color of the colony and medium, etc. Figure 1 ; After 24 hours of culture, examine under a microscope and record the bacterial morphology. Figure 2 .

[0058] Depend on Figure 1 and Figure 2 It can be seen that after culturing in LB medium for 24 hours, the colony morphology of the target strain is small and round, the colony surface is smooth, flat or convex, the edge is neat, opaque, wet and sticky. Gram staining is positive, and under an optical microscope, the bacteria are blunt and round, arranged individually, and have spores.

[0059] 3. Molecular Biology Identification

[0060] (1) DNA extraction

[0061] The genomic DNA of the target strain IVF-WKB2 was extracted using the TIANamp Bacteria DNA kit; the TIANamp Bacteria DNA kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0062] (2) PCR amplification and sequencing

[0063] Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using universal primers:

[0064] Upstream primer: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO. 2);

[0065] Downstream primer: 5′-AAGGAGGTGATCCAGCCGCA-3′ (SEQ ID NO. 3);

[0066] The PCR reaction system was 25 μL, including 1 μL template DNA, 1 μL 10 μmol / L upstream primer, 1 μL 10 μmol / L upstream primer, 12.5 μL Norveg 2× Taq PCR Master Mix, and ddH2 O to make up to 25 µL;

[0067] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 1 min, extension at 72°C for 2 min, 30 cycles; and extension at 72°C for 10 min.

[0068] The obtained PCR products were detected by agarose gel electrophoresis under standard conditions and then sent to Beijing Ruiboxingke Biological Co., Ltd. for sequencing. The sequencing results were processed and compared with GenBank.

[0069] The sequencing result of 16S rDNA is shown in SEQ ID NO.1, specifically:

[0070]

[0071] (3) Construction of phylogenetic tree

[0072] The sequencing results were compared in NCBI to preliminarily determine the species, and the standard sequences of each known Bacillus genus were obtained from GenBank. The sequences were aligned using Readseqn software, and the sequence similarity was calculated using MEGA5.01 and phylogenetic analysis was performed. The results are shown in Figure 3 .

[0073] Depend on Figure 3 It can be seen that the target strain IVF-WKB2 is at the same developmental level as Bacillus subtilis and has the closest relationship. Therefore, the target strain is Bacillus subtilis, named Bacillus subtilis IVF-WKB2, and then deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on August 31, 2023. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 28314.

[0074] Example 2

[0075] The preparation method of Bacillus subtilis IVF-WKB2 bacterial liquid is as follows:

[0076] Bacillus subtilis IVF-WKB2 was cultured in TSB (Tryptic Soy Broth) liquid medium with an inoculation volume of 1% v / v; the speed of the culture was 220 rpm, the temperature was 37°C, and the time was about 6 hours. After the culture was completed, the OD was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer. 600 The bacterial solution was centrifuged at 5000g for 10 min, the bacterial precipitate was washed with 25 mL of sterile water, and then centrifuged at 5000g for 10 min. After the waste liquid was discarded, the bacterial solution was resuspended with sterile water and the OD was measured at a wavelength of 600 nm using an ultraviolet spectrophotometer. 600 The bacterial solution is 0.1.

[0077] Example 3

[0078] The preparation method of Bacillus subtilis IVF-WKB2 fermentation dilution liquid comprises the following steps:

[0079] (1) A fermentation medium was prepared having the following composition, by weight percentage: 1% corn flour, 0.5% glucose, 1.5% soybean meal, 0.8% fish meal, 1% calcium carbonate, 0.1% ammonium sulfate, 0.03% dipotassium hydrogen phosphate, 0.03% magnesium sulfate and 0.03% manganese sulfate, with the remainder being water.

[0080] (2) Fermentation: Add 20 mL of Bacillus subtilis IVF-WKB2 OD = 0.02 bacterial solution to 200 mL of fermentation medium in step (1) (using a 500 mL Erlenmeyer flask as the culture container), and culture overnight (12 h) at 37°C and 220 rpm. Dilute the cultured fermentation solution with 2 L of sterile water to obtain a Bacillus subtilis IVF-WKB2 fermentation dilution solution. The OD of the fermentation dilution solution is 600 0.02, effective viable count ≥ 10 7 cfu / mL.

[0081] Application Example 1

[0082] A method for promoting cucumber growth, specifically a method for promoting cucumber growth in a plug tray, comprising the following steps:

[0083] Experimental materials: Deruit 89, a main commercial cucumber variety grown in the north and not tolerant to low temperatures or weak light, was used as the experimental material.

[0084] (1) Pre-seed treatment: Soak cucumber seeds, the low temperature and weak light intolerant variety Deruit 89, in warm water with an initial temperature of 50°C for 30 minutes, then soak in 75% ethanol by volume for 15 seconds, rinse once with sterile water, and then soak in 6% sodium hypochlorite for 15 minutes, shake from time to time, pour out the sodium hypochlorite, and rinse with sterile water 6 to 8 times. Then put the seeds in a 100×100mm culture dish, add 2 layers of filter paper at the bottom, add an appropriate amount of sterile water, and then place in a dark incubator at 30°C to wait for the seeds to germinate; after the seeds germinate, select seeds with consistent growth for use;

[0085] (2) Fertilization: The germinated seeds selected in step (1) with consistent growth were sown in a 6-hole plug tray (the substrate was a sterilized Flora gard peat soil seedling substrate), with one seed sown in each hole, and each plant was a biological replicate. The culture was carried out in an artificial climate chamber, and the day and night temperatures of the culture were 20°C and 10°C, respectively. The daylight hours were 16 hours per day, the light intensity was 3000lx~10000lx, and the soil moisture was 80%. When the cucumber was in the seedling stage and the cotyledons were unfolded, the experimental group was applied with the Bacillus subtilis IVF-WKB2 bacterial solution of Example 2 by root irrigation, and the application amount was 100 mL / plant each time, and the application frequency was once a week until three leaves and one heart. At the same time, a blank control group was set up, and the blank control group was applied with an equal volume of sterile water. The experimental group and the blank control group were managed normally according to the planting needs without applying other fertilizers. The cultivation conditions during the period were (day and night temperatures were 20°C and 10°C, daylight hours were 16 hours per day, light intensity was 3000lx~10000lx, and soil moisture was 80%). Six biological replicates were performed in each of the experimental group and the blank control group.

[0086] (3) Results and analysis:

[0087] When the cucumbers grow to the seedling stage with three leaves and one heart (when there is a significant difference in the standard), the plant height, stem diameter, leaf area, dry weight, fresh weight and other indicators of the cucumber seedlings in the experimental group and the blank control group were measured to evaluate the growth-promoting effect of the test strains in the seedling stage. The data statistical method was T-test. The results are shown in Table 1 and Figure 4 Table 1 shows the cucumber growth-promoting effects of the experimental group and the blank control group; Figure 4 The bar graphs of different measurement indicators of cucumber under different treatments are shown in Figure 2. Figure 5 shown. Figure 5 The sterile treatment in the table corresponds to the blank control group treatment; Figure 5 The bacterial control in the figure corresponds to the experimental group treatment.

[0088] The specific measurement standards are as follows:

[0089] Plant height: The height from the surface of the plug tray substrate to the growth point shall prevail;

[0090] Stem diameter: measured with a vernier caliper just below the cotyledon node;

[0091] Leaf area: Spread out the largest true leaf and take a photo, then use image J software to count the leaves;

[0092] Fresh weight: Wash the whole plant, shake off excess water, and weigh it with an electronic balance;

[0093] Dry weight: The whole plant was withered at 105℃ for 15min and dried at 70℃ for more than 48h until constant weight was achieved. The plant height and stem diameter were measured 8 times to ensure the accuracy of the data. Unreasonable data were eliminated during the statistical process. Three of the eight replicates were randomly selected for leaf area, whole plant fresh weight and whole plant dry weight for data statistics and analysis.

[0094] Table 1 Growth-promoting effects of cucumber in the experimental group and the blank control group

[0095]

[0096] From Table 1 and Figure 4 It can be seen that the application of the Bacillus subtilis IVF-WKB2 bacterial solution of Example 2 can significantly increase the plant height, leaf area, whole plant fresh weight and whole plant dry weight of cucumbers, and can also increase the stem diameter of cucumbers to a certain extent. The above shows that the Bacillus subtilis IVF-WKB2 bacterial solution provided by the present invention can promote the growth of cucumbers.

[0097] Application Example 2

[0098] Growth-promoting effect of Bacillus subtilis IVF-WKB2 on cucumber in greenhouse

[0099] 1. Experimental materials: Deruit 89, a main commercial cucumber variety grown in the north and intolerant to low temperature and weak light, was used as the experimental material.

[0100] 2. Experimental methods and grouping:

[0101] 2.1 Grouping

[0102] Treatment group 1: Apply the fermentation dilution of Bacillus subtilis IVF-WKB2 in Example 3, starting from the seedling stage of cucumber, and once a week during the entire growth period of cucumber, 100 mL / plant each time, and additionally apply a water-soluble compound fertilizer (purchased from Stanley) with a nitrogen, phosphorus and potassium mass ratio of 20:10:20, and the application amount is determined according to the instructions of the water-soluble compound fertilizer. The water-soluble compound fertilizer and the microbial agent are applied at the same time, once a week. Recorded as: fertilized IVF-WKB2 group.

[0103] Treatment group 2: Only water-soluble compound fertilizer (purchased from Stanley) with a nitrogen, phosphorus and potassium mass ratio of 20:10:20 was applied, and the application amount was determined according to the instructions of the water-soluble compound fertilizer. This is recorded as the fertilized control group.

[0104] Treatment group 3: Apply commercial bacterial liquid Y1 to irrigate cucumber seedlings. Among them, commercial bacterial liquid Y1 is a microbial agent purchased from Hebei Devoduo Biotechnology Co., Ltd. The method of applying the microbial agent is to dissolve 2g of commercial bacterial liquid Y1 in 1L of water, and apply it from the time the cucumber enters the seedling stage. The cucumber is applied once a week during the entire growth period, 100mL / plant each time. At the same time, a water-soluble compound fertilizer with a nitrogen, phosphorus and potassium mass ratio of 20:10:20 (purchased from Stanley Company) is additionally applied, and the application amount is determined according to the instructions of the water-soluble compound fertilizer. The water-soluble compound fertilizer and the microbial agent are applied at the same time, once a week. Recorded as: Fertilizer Y1 group. (The effective strains of commercial bacterial liquid Y1 are Bacillus subtilis and Bacillus licheniformis, and the effective viable count (cfu) is ≥10 billion / g, the same below).

[0105] Treatment group 4: Apply commercial bacterial liquid Y2 to irrigate cucumber seedlings. Among them, commercial bacterial liquid Y2 is 360 bacterial housekeeper purchased from Zhengzhou Zhongke Chemical Products Co., Ltd. The application method of the 360 ​​bacterial housekeeper is to dissolve 7.5g of commercial bacterial liquid Y2 in 1L of water, and apply it from the time the cucumber enters the seedling stage. The cucumber is applied once a week during the entire growth period, 100mL / plant each time. At the same time, a water-soluble compound fertilizer with a nitrogen, phosphorus and potassium mass ratio of 20:10:20 (purchased from Stanley Company) is additionally applied, and the application amount is determined according to the instructions of the water-soluble compound fertilizer. The water-soluble compound fertilizer and the microbial agent are applied at the same time, once a week. Recorded as: fertilization Y2 group. (The microbial composition of 360 bacterial housekeeper is Bacillus subtilis, Bacillus licheniformis and Bacillus jelly-like; the effective viable count of 360 bacterial housekeeper is ≥50 billion / gram. The same below).

[0106] Treatment group 5: The fermentation dilution of Bacillus subtilis IVF-WKB2 in Example 3 was applied, and no other fertilizer was applied. The fermentation dilution of Bacillus subtilis IVF-WKB2 was applied from the beginning of the cucumber seedling stage, and once a week throughout the growth period of the cucumber, 100 mL / plant each time. Recorded as: IVF-WKB2 group.

[0107] Treatment group 6: No substance was added. Recorded as: control group.

[0108] Treatment group 7: Apply commercial bacterial solution Y1 to irrigate cucumber seedlings. Commercial bacterial solution Y1 is a microbial agent purchased from Hebei Devodo Biotechnology Co., Ltd. No other fertilizers are applied. The application method of the microbial agent is to dissolve 2g of commercial bacterial solution Y1 in 1L of water, and apply it from the time the cucumber enters the seedling stage. It is applied once a week throughout the growth period of the cucumber, 100mL / plant each time. Recorded as: Y1 group.

[0109] Treatment group 8: Apply commercial bacterial solution Y2 to irrigate cucumber seedlings. The commercial bacterial solution Y2 is 360 Bacteria Manager purchased from Zhengzhou Zhongke Chemical Products Co., Ltd. No other fertilizers are applied at the same time. The application method of the 360 ​​Bacteria Manager is to dissolve 7.5g of commercial bacterial solution Y2 in 1L of water, and apply it from the time the cucumber enters the seedling stage. It is applied once a week throughout the growth period of the cucumber, 100mL / plant each time. Recorded as: Y2 group.

[0110] 2.2 Experimental methods

[0111] 2.2.1 Species planting: Cucumbers were planted in greenhouses using local planting methods and in accordance with the area of ​​the experimental plot;

[0112] Experimental plot description: The experimental plots were located in the same plot of the plastic greenhouse of the Nankou pilot base of the Chinese Academy of Agricultural Sciences (day and night temperatures were 20°C and 10°C, respectively, light intensity 6000-10000 lx, light duration 8 h, soil moisture 80%), that is, one plot was divided into 8 plots, of which plot 1 was used for treatment group 1, plot 2 was used for treatment group 2, plot 3 was used for treatment group 3, plot 4 was used for treatment group 4, plot 5 was used for treatment group 5, plot 6 was used for treatment group 6, plot 7 was used for treatment group 7, and plot 8 was used for treatment group 8. Cucumber 9930 was cultivated between adjacent plots as protection rows; the area of ​​each plot was 7m 2 20 cucumbers were planted in each plot with a spacing of 35 cm between cucumbers. Except for the different types of microbial agents in each treatment and whether additional fertilizers were applied, other management was the same.

[0113] 3. Results and Analysis

[0114] The cucumbers planted in each treatment group were statistically analyzed, that is, the single plant yield and per mu yield of cucumbers in the seedling stage, flowering stage, and fruiting stage were counted respectively (Note: 20 plants per bed, about 3500 plants per mu). The data processing method was T-test. The results are shown in Table 1. Figures 6 to 13 . Figure 6 The effect of the fertilizer control group treatment in Application Example 2 on the growth of cucumber fruit; Figure 7 This is the effect of the treatment method of adding fertilizer IVF-WKB2 group in Application Example 2 on the growth of cucumber fruit; Figure 8 The effects of different treatments on the per-acre yield of cucumber in the IVF-WKB2 group, the control group, the Y1 group, and the Y2 group in Application Example 2; Fig. 9 The effects of different treatments on the number of cucumber fruits in the IVF-WKB2 group, the control group, the Y1 group, and the Y2 group in Application Example 2; Fig.10 The effects of different treatments on the per-acre yield of cucumber in IVF-WKB2 group, control group, Y1 group and Y2 group in Application Example 2; Fig.11 The effects of different treatments on the number of cucumber fruits in IVF-WKB2 group, control group, Y1 group and Y2 group in Application Example 2; Fig.12 The effects of different treatments on the per-acre yield of cucumber in IVF-WKB2 group, fertilization control group, Y1 group and Y2 group in Application Example 2; Fig.13 This is the effect of different treatments on the number of cucumber fruits in the IVF-WKB2 group, fertilization control group, Y1 group and Y2 group in Application Example 2.

[0115] Table 2 Effects of different treatments on average per-acre yield, average single fruit weight and average number of cucumber fruits

[0116]

[0117] From Table 2, Figures 6 to 13 It can be seen that under normal fertilizer and water management, the per-acre yield of the fermented dilution of Bacillus IVF-WKB2 increased by 15.5% compared with the control, and the single fruit weight increased by 16.6%, indicating that the fermented dilution of Bacillus IVF-WKB2 has the effect of promoting cucumber growth. Compared with the fertilized control, fertilized Y1, and fertilized Y2, the per-acre yield of the fermented dilution of Bacillus IVF-WKB2 with fertilization increased by 10.9%, 30.9%, and 10.6%, the single fruit number increased by 10.1%, 3.8%, and 3.2%, and the number of fruits per acre increased by 0.7%, 26.2%, and 7.1%, respectively. The fermented dilution of Bacillus IVF-WKB2 with fertilization has a more significant effect in promoting cucumber growth. Compared with the control, the per-acre yield, single fruit weight and number of fruits of the fermented dilution of Bacillus fertilizer IVF-WKB2 increased by 45.3%, 16.2% and 25.0%, respectively, and the increase in yield was particularly significant, indicating that the fermented dilution of Bacillus fertilizer IVF-WKB2 of the present invention can more significantly increase the yield compared with ordinary fertilizers.

[0118] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A Bacillus subtilis IVF-WKB2, characterized in that: The deposit number of the Bacillus subtilis IVF-WKB2 is CGMCC No.28314.

2. The Bacillus subtilis IVF-WKB2 according to claim 1, characterized in that The 16S rDNA sequence of the Bacillus subtilis IVF-WKB2 is shown in SEQ ID NO.

1.

3. A microbial preparation, characterized in that: It comprises the Bacillus subtilis IVF-WKB2 according to claim 1 or 2.

4. The microbial preparation according to claim 3, characterized in that: The viable count of the Bacillus subtilis IVF-WKB2 in the microbial preparation is ≥10 7 cfu / mL.

5. The method for preparing the microbial preparation according to claim 3 or 4, characterized in that: include: The Bacillus subtilis IVF-WKB2 is cultured in a culture medium to obtain a microbial preparation.

6. The preparation method according to claim 5, characterized in that: The culture temperature is 28-37°C; the culture is accompanied by rotation; and the rotation speed is 200-220rpm.

7. A fertilizer, characterized in that It includes the Bacillus subtilis IVF-WKB2 according to claim 1 or 2, the microbial preparation according to claim 3 or 4, or the microbial preparation prepared by the preparation method according to claim 5 or 6.

8. Use of the Bacillus subtilis IVF-WKB2 according to claim 1 or 2, the microbial preparation according to claim 3 or 4, the microbial preparation prepared by the preparation method according to claim 5 or 6, or the fertilizer according to claim 7 in promoting plant growth; The plant is cucumber.

9. A method for promoting plant growth, characterized in that: include: Applying the microbial preparation according to claim 3 or 4 or the fertilizer according to claim 7; The plant is cucumber.

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