A cold-resistant Bacillus IVF-WKB47 and its use

Abstract

The present invention relates to the field of microbial technology, and in particular to a cold-resistant Bacillus IVF-WKB47 and its use. The present invention provides a cold-resistant Bacillus ( Peribacillus frigoritolerans ) IVF-WKB47, with a deposit number of CGMCC No.28315. The Bacillus IVF-WKB47 or its microbial preparation has the effect of promoting plant growth and increasing crop yield, including increasing the number of fruits set in the plant, increasing the weight of a single fruit in the plant, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a cold-resistant Bacillus IVF-WKB47 and a use thereof. Background Art

[0002] cucumber( Cucumis sativusL. ) is an annual climbing herb of the Cucurbitaceae family, with slender stems and rough bristles; leaves are broadly ovate-cordate or lobes are triangular; flowers are slightly pubescent and yellow-white; fruits are oblong or cylindrical and yellow-green when ripe; seeds are narrowly ovate and white; flowering and fruiting period is summer. Cucumbers are native to northern India at the southern foot of the Himalayas and are now distributed throughout the world. They like warmth, are intolerant to cold, like humidity, are intolerant to drought, and have strong light. Cucumbers require a deep soil layer, fertile soil, rich organic matter, good ventilation, and slightly acidic to neutral soil. Due to the above-mentioned likes of warmth, intolerant to cold, like humidity, intolerant to drought, strong light, and cucumbers require a deep soil layer, fertile soil, rich organic matter, good ventilation, and slightly acidic to neutral soil, the planting conditions of cucumbers are high, and they can only be planted in some areas. Cucumbers cannot be widely planted, which affects the yield of cucumbers.

[0003] However, there are currently no reports on the ability of cold-tolerant Bacillus to significantly promote plant growth, especially cucumber yield. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a cold-resistant Bacillus IVF-WKB47 and its use. The cold-resistant Bacillus IVF-WKB47 has the advantages of promoting plant growth and significantly increasing plant yield, especially can improve the stress resistance of cucumbers that are not tolerant to low temperature and / or weak light, and promote the growth of cucumbers under low temperature and weak light conditions.

[0005] To this end, the present invention provides the following technical solutions:

[0006] A cold-resistant Bacillus ( Peribacillus frigoritolerans )IVF-WKB47 has been deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, the deposit date is August 31, 2023, and the deposit number is CGMCC No.28315.

[0007] The cold-resistant Bacillus IVF-WKB47 was isolated from the rhizosphere soil of greenhouse cucumbers in the Nankou pilot experimental base of the Chinese Academy of Agricultural Sciences; the colony characteristics of the cold-resistant Bacillus IVF-WKB47 on the LB solid culture medium are: smooth surface, neat edges, opaque, and wet and sticky.

[0008] A microbial preparation contains the cold-resistant Bacillus IVF-WKB47, its culture, its fermentation product and / or at least one metabolite thereof.

[0009] The present invention discloses a microbial preparation, which includes the aforementioned cold-resistant Bacillus IVF-WKB47 strain, at least one metabolite thereof, culture, and fermentation broth. The "metabolites", "cultures", and "fermentation broth" used herein include any components, compounds, substances, or byproducts (including but not limited to small molecule secondary metabolites) produced by the cold-resistant Bacillus IVF-WKB47 strain described herein and having any beneficial effects disclosed in the present invention. The beneficial effects include resistance to low temperatures, resistance to weak light, and promotion of plant growth, and the promotion of plant growth includes but is not limited to increasing per-acre yield, single fruit weight, number of fruits, plant stem thickness, plant fresh weight of the whole plant, and plant leaf area.

[0010] As an practicable manner, in the microbial preparation, the viable count of the cold-resistant Bacillus IVF-WKB47 is ≥10 7 cfu / mL, as a more preferred practicable form, the viable count is ≥10 8 cfu / mL, ≥10 9 cfu / mL, viable count ≥10 10 cfu / mL, or even higher.

[0011] A method for preparing the microbial preparation comprises: expanding and culturing the cold-resistant Bacillus IVF-WKB47.

[0012] As an practicable manner, in the expansion culture step, the inoculation amount of the cold-resistant Bacillus IVF-WKB47 is 1 v / v% to 10 v / v% of the total volume of the expansion culture medium, preferably 1 v / v%, 3 v / v%, 5 v / v%, 7 v / v% or 8 v / v%, and more preferably 1 v / v%.

[0013] As an practicable manner, in the expansion culture step, the time condition for the expansion culture is the conventional culture time of cold-resistant Bacillus, and the culture time is preferably 6 to 8 hours, more preferably 8 hours.

[0014] As an practicable manner, in the enlarged culture step, the rotation speed of the enlarged culture is the rotation speed of conventional culture of cold-resistant Bacillus, preferably 200-220 rpm, more preferably 220 rpm.

[0015] As an practicable manner, in the enlarged culture step, the temperature condition of the enlarged culture is the conventional culture temperature of cold-resistant Bacillus, and the temperature is preferably 28-37°C, and more preferably 37°C.

[0016] As an practicable method, in the step of expanding the culture, the culture medium for the expansion culture can be a conventional culture medium suitable for cold-resistant Bacillus, preferably TSB liquid culture medium, which is a conventional commercially available product and has no special requirements on the components and dosage. After the above expansion culture, the obtained bacterial liquid is detected by an ultraviolet spectrophotometer, and the OD is measured at a wavelength of 600nm. 600 The value is 0.5~1.5, and OD is more preferred 600 The value is 1.

[0017] As an practicable method, after the expansion culture, the obtained bacterial solution is further centrifuged to obtain a bacterial cell precipitate of cold-resistant Bacillus IVF-WKB47. As a preferred embodiment, the OD 600 The bacterial solution with a value of 1 is subjected to secondary centrifugation, including the first centrifugation and the second centrifugation. The speed and time of the centrifugation are conventional techniques in the art. The speeds of the first centrifugation and the second centrifugation can be 3000~6000g, preferably 4000~5000g, more preferably 5000g, and the centrifugation time can be 5~15min, preferably 8~13min, more preferably 10min. After the first centrifugation, it also includes a step of washing with sterile water to obtain a bacterial precipitate after the first centrifugation. There are no special requirements for the amount of sterile water and the method of washing. After the above-mentioned centrifugation step, a precipitate containing strain IVF-WKB47 is obtained.

[0018] As an practicable method, after the centrifugation step, the precipitate of strain IVF-WKB47 obtained in the centrifugation step is resuspended with sterile water, and the obtained bacterial suspension is detected by ultraviolet spectrophotometer, and the OD value of cold-resistant Bacillus IVF-WKB47 at a wavelength of 600nm is 0.02, so that the effective viable count ≥10 7 cfu / mL of Bacillus frostbitus IVF-WKB47 bacterial solution.

[0019] As an practicable method, the cold-resistant Bacillus IVF-WKB47 bacterial solution obtained by the above-mentioned expanded culture is preferably selected from OD 600 The cold-resistant Bacillus IVF-WKB47 bacterial solution with a value of 0.02 can be directly applied to plants, and the application methods include but are not limited to root irrigation, coating, seed soaking, composting, field flooding, drip irrigation of plants or plant organs, etc.

[0020] As an practicable manner, the cold-resistant Bacillus IVF-WKB47 bacterial solution obtained by the above-mentioned expanded culture is used to prepare the cold-resistant Bacillus IVF-WKB47 fermentation broth. As a more preferred embodiment, the OD 600The cold-resistant Bacillus IVF-WKB47 bacterial liquid with a value of 0.02 was used as a seed liquid, which was inoculated into a fermentation medium for aerobic fermentation to obtain a fermentation liquid of cold-resistant Bacillus IVF-WKB47.

[0021] As an practicable manner, the fermentation medium for aerobic fermentation can be a conventional fermentation medium suitable for cold-resistant Bacillus in the art. As a preferred embodiment, the fermentation medium for aerobic culture can include the following components (by weight): 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 and 0.01% to 0.06% manganese sulfate, and the balance is water.

[0022] As an practicable manner, during the aerobic fermentation, the inoculation amount of the cold-resistant Bacillus IVF-WKB47 bacterial solution is 1 v / v% to 20 v / v%, preferably 3 v / v%, 6 v / v%, 10 v / v% or 20 v / v%, and more preferably 10 v / v%.

[0023] As an practicable manner, the aerobic fermentation time is the conventional fermentation culture time of cold-resistant Bacillus, preferably 10 to 16 hours, preferably 11 hours, 12 hours, 14 hours, and more preferably 12 hours.

[0024] As an practicable manner, the rotation speed of the aerobic fermentation is the rotation speed of conventional fermentation culture of cold-resistant Bacillus, preferably 200-220 rpm, more preferably 220 rpm.

[0025] As an practicable manner, the temperature of the aerobic fermentation is the temperature of conventional fermentation culture of cold-resistant Bacillus, preferably 28-37°C, more preferably 37°C.

[0026] As an practicable manner, the obtained fermentation broth of Bacillus frostbitus IVF-WKB47 can be diluted with sterile water, and the sterile water used is 5 to 30 times the volume of the fermentation broth, preferably 5 times, 10 times, 15 times or 30 times, and more preferably 10 times.

[0027] As an practicable manner, the diluted fermentation liquid can be directly applied to plants, and the application methods include but are not limited to root irrigation, coating, seed soaking, composting, field flooding, drip irrigation of plants or plant organs, etc.

[0028] Use of the cold-resistant Bacillus IVF-WKB47, the microbial preparation or the microbial preparation prepared by the preparation method in promoting plant growth.

[0029] Optionally, the promoting plant growth comprises increasing at least one of the yield per mu of the plant, the weight of a single fruit, the number of fruits set, the stem diameter of the plant, the fresh weight of the whole plant of the plant and the leaf area of ​​the plant;

[0030] Optionally, the promoting plant growth includes enhancing the stress resistance of plants under low temperature and / or weak light conditions;

[0031] Optionally, the low temperature has a temperature range of 10°C to 20°C; Optionally, the daytime temperature range is >14°C and ≤20°C, and the nighttime temperature range is ≥10°C and ≤14°C;

[0032] Optionally, the weak light condition ranges from 3000lx to 10000lx, and the illumination time ranges from 6h to 14h.

[0033] Optionally, the plant includes cucumber ( Cucumis sativusL .);

[0034] Optionally, the cucumber is a cucumber that is intolerant to low temperature and / or weak light;

[0035] Optionally, the cucumber is Deruit 89, Jinchun No. 4 or Zhongnongda No. 11.

[0036] A plant growth regulator, seed coating agent, root irrigation agent, seed soaking agent, microbial pesticide or microbial fertilizer, comprising the cold-resistant Bacillus IVF-WKB47, the microbial preparation or the microbial preparation prepared by the preparation method.

[0037] A method for promoting plant growth comprises applying the cold-resistant Bacillus IVF-WKB47, the microbial preparation, the microbial preparation prepared by the preparation method, or the plant growth regulator or fertilizer.

[0038] As an practicable manner of the present invention, the application method is but not limited to root irrigation, coating, seed soaking, composting, field flooding, drip irrigation of plants or plant organs, etc., and root irrigation is more preferred.

[0039] As an exemplified embodiment of the present invention, when applying a bacterial solution of cold-resistant Bacillus IVF-WKB47, the application amount of the bacterial solution is 10-100 mL / strain each time, and the application frequency is once every 1 to 2 weeks; preferably, the application amount of the bacterial solution is 50 mL / strain each time, and the application frequency is once a week; preferably, the effective viable count of cold-resistant Bacillus IVF-WKB47 in the bacterial solution is ≥10 7 cfu / mL;

[0040] As an exemplified embodiment of the present invention, when applying the fermentation dilution of cold-resistant Bacillus IVF-WKB47, the application amount of the fermentation dilution is 70-120 mL / strain each time, and the application frequency is once every 1 to 2 weeks; preferably, the application amount of the fermentation dilution is 100 mL / strain each time, and the application frequency is once a week; the effective viable count of cold-resistant Bacillus IVF-WKB47 in the fermentation dilution is ≥10 7 cfu / mL;

[0041] As an practicable mode of the present invention, the application period includes the entire growth period of the cucumber, and the application period can be selected from the seedling stage, flowering stage and / or fruit setting stage;

[0042] As an embodied mode of the present invention, the plant includes cucumber.

[0043] The technical solution of the present invention has the following advantages:

[0044] 1. A cold-resistant Bacillus provided by the present invention ( Peribacillus frigoritolerans ) IVF-WKB47, with a deposit number of CGMCC No.28315. The cold-resistant Bacillus IVF-WKB47 or its microbial preparation has the effect of promoting plant growth, including increasing per-acre yield, single fruit weight, number of fruits, etc. At the same time, it also promotes plant growth under low temperature and / or weak light conditions, enhances the plant's resistance to low temperature and / or weak light, and especially promotes the growth of cucumbers that are not resistant to low temperature and / or weak light. Therefore, the cold-resistant Bacillus IVF-WKB47 or its microbial preparation can promote plant growth and increase crop yield.

[0045] 2. The present invention provides a plant growth regulator, seed coating agent, root irrigation agent, seed soaking agent, microbial pesticide or microbial fertilizer, comprising the cold-resistant Bacillus IVF-WKB47, the microbial preparation or the microbial preparation prepared by the preparation method; since the cold-resistant Bacillus IVF-WKB47 or its microbial preparation has the function of promoting plant growth and increasing crop yield, it can be used to prepare products such as plant growth regulators, seed coating agents, root irrigation agents, seed soaking agents, microbial pesticides or microbial fertilizers.

[0046] Biological deposit information

[0047] The cold-resistant Bacillus provided by the present invention ( Peribacillus frigoritolerans ) IVF-WKB47, deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on August 31, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit number is CGMCC No.28315. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 This is a colony morphology diagram of the cold-resistant Bacillus IVF-WKB47 in the culture medium in Example 1;

[0050] Figure 2 This is a colony morphology of the cold-resistant Bacillus IVF-WKB47 under a microscope in Example 1;

[0051] Figure 3 This is a phylogenetic analysis diagram of cold-resistant Bacillus IVF-WKB47 in Example 1;

[0052] Figure 4 The effects of the control and IVF-WKB47 treatments on the growth of cucumber fruits in Experimental Example 1;

[0053] Figure 5 The effects of different treatments on per-acre yield of cucumber in IVF-WKB47, control, Y1 and Y2 in Experimental Example 1;

[0054] Figure 6 The effects of different treatments on the number of cucumber fruits in IVF-WKB47, control, Y1 and Y2 in Experimental Example 1;

[0055] Figure 7 The effects of different treatments on per-acre yield of cucumber in IVF-WKB47, fertilizer control, Y1 and Y2 in Experimental Example 1;

[0056] Figure 8 The effects of different treatments on the number of cucumber fruits in IVF-WKB47, fertilizer control, Y1 and Y2 in Experimental Example 1;

[0057] Fig. 9 It is a bar chart of different measurement indicators of cucumbers under different treatments in Experimental Example 2; among each indicator, compared with the blank control (or sterile control), Indicates p < 0.05, Indicates p < 0.01, indicates p < 0.001;

[0058] Fig.10 This is the growth diagram of cucumber seedlings under different treatments in Experimental Example 2. DETAILED DESCRIPTION

[0059] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0060] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0061] Example 1 Isolation and identification of cold-resistant Bacillus IVF-WKB47

[0062] 1. Source of cold-resistant Bacillus IVF-WKB47

[0063] Bacillus frostbite ( Peribacillus frigoritolerans )IVF-WKB47 was isolated from the cucumber rhizosphere soil in the greenhouse of the Nankou Pilot Experimental Base of the Chinese Academy of Agricultural Sciences.

[0064] The collected cucumber rhizosphere soil was resuspended in sterile water and diluted to 10 by ten-fold dilution method. -3 ~10 -8 g / ml gradient concentration of bacterial suspension, aspirate 0.1ml of the bacterial suspension and use the streak method to separate and culture on LB solid medium; the formula of LB solid medium is: NaCl (10 g / L) + peptone (10 g / L) + yeast extract (5 g / L) + agar (15 g / L).

[0065] 2. Morphological identification

[0066] Pick a small amount of target strain with an inoculation loop and streak it on LB solid medium. After culturing at 37℃ for 20h, observe the morphology, shape, size, edge, surface, ridge shape, transparency, color of colonies and medium, etc. (see the results for details). Figure 1 ), culture for 24 hours and then examine under a microscope to record bacterial morphology (see results in Figure 2 ).

[0067] 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 rod-shaped, arranged individually, and have spores.

[0068] 3. Molecular Biology Identification

[0069] (1) DNA extraction

[0070] The genomic DNA of the target strain IVF-WKB47 was extracted using the TIANamp Bacteria DNA kit; the kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0071] (2) PCR amplification and sequencing

[0072] Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using universal primers:

[0073] Upstream primer: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO. 1);

[0074] Downstream primer: 5′-AAGGAGGTGATCCAGCCGCA-3′ (SEQ ID NO. 2);

[0075] The PCR reaction system was 25 µL, including 1 µL of template DNA, 1 µL of 10 µmol / L upstream primer, 1 µL of 10 µmol / L downstream primer, 12.5 µL of Novagen 2× Taq PCR Master Mix, and ddH2O to make up to 25 µL;

[0076] 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; extension at 72°C for 10 min;

[0077] 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.

[0078] (3) Construction of phylogenetic tree

[0079] 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 ).

[0080] Depend on Figure 3It can be seen that the target strain Bacillus cold-resistant IVF-WKB47 is at the same developmental level as Bacillus cold-resistant and has the closest relationship, which is consistent with the results of physiological and biochemical identification. Therefore, the target strain is Bacillus cold-resistant and is named Bacillus cold-resistant IVF-WKB47. It was then deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on August 31, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit number is CGMCC No.28315.

[0081] Example 2 Cold-resistant Bacillus IVF-WKB47 bacterial liquid

[0082] This embodiment provides a method for preparing a cold-resistant Bacillus IVF-WKB47 bacterial liquid, comprising the following steps:

[0083] The cold-resistant Bacillus IVF-WKB47 was inoculated into Tryptic Soy Broth (TSB) liquid culture medium (a conventional commercial product) for expansion culture. The inoculation amount of cold-resistant Bacillus IVF-WKB47 was 1% v / v. The expansion culture conditions were: the culture speed was 220 rpm, the culture temperature was 37°C, and the culture time was about 6 hours. 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, washed with 25 mL of sterile water, and then centrifuged at 5000g for 10 min. After discarding the waste liquid, 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 effective viable count is ≥10 7 cfu / mL.

[0084] Example 3 Cold-resistant Bacillus IVF-WKB47 fermentation dilution

[0085] This embodiment provides a method for preparing a fermentation dilution of cold-resistant Bacillus IVF-WKB47, comprising the following steps:

[0086] (1) A fermentation medium is prepared, comprising the following components (by weight): 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, 0.03% manganese sulfate, and the remainder water.

[0087] (2) Fermentation: The cold-resistant Bacillus IVF-WKB47 culture liquid (OD 6000.1) 20ml, add to the fermentation medium 200ml in step (1) (use 500mL Erlenmeyer flask as culture container), culture overnight (12h) at 37℃ and 220rpm. After the fermentation liquid is diluted with 2L sterile water, the effective viable count is ≥10 7 cfu / mL or OD 600 is 0.02.

[0088] Example 4 A method for promoting cucumber growth

[0089] This embodiment provides a method for promoting the growth of cucumbers in a plug tray, comprising the following steps:

[0090] (1) Pre-seed treatment: Soak cucumber seeds (Derrite 89, a variety that is not resistant to low temperature and weak light) in 50°C warm water for 30 minutes, then soak in 75% alcohol for 15 seconds, rinse once with sterile water, and then soak in 6% sodium hypochlorite for 15 minutes. Shake occasionally during this period, 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 it in a dark incubator at 30°C and wait for the seeds to germinate; after the seeds germinate, select seeds with consistent growth for use;

[0091] (2) The germinated seeds selected in step (1) with uniform growth were sown in an 8-hole plug tray (the substrate was a sterilized Flora gard peat soil seedling substrate), with one seed sown in each hole, and each plant being a biological replicate. The seeds were cultured in an artificial climate chamber (day and night temperature of 20°C and 10°C, 8 h of light per day, and soil moisture of 80%). When the cucumbers were in the seedling stage and the cotyledons were unfolded, the cold-resistant Bacillus IVF-WKB47 bacterial solution in Example 2 was applied by root irrigation, with an application amount of 50 mL per plant each time, and the application frequency was once a week until the fruit setting period ended.

[0092] Example 5

[0093] The difference between this example and Example 4 is that the application amount is 100 mL / plant each time and the application frequency is once every two weeks.

[0094] Example 6

[0095] The difference between this example and Example 4 is that the application amount is 10 mL / plant each time and the application frequency is once a week.

[0096] Example 7

[0097] This embodiment provides a method for promoting cucumber growth in a greenhouse, comprising the following steps:

[0098] (1) Cucumbers (Derrite 89, a variety that is not tolerant to low temperatures and weak light) were planted using local planting methods and the area of ​​the experimental plot.

[0099] (2) When the cucumbers enter the seedling stage, the fermentation dilution of the cold-resistant Bacillus IVF-WKB47 in Example 3 is applied by root irrigation, with an application amount of 100 mL per plant each time and a frequency of once a week until the fruit setting period ends.

[0100] Example 8

[0101] The difference between this embodiment and embodiment 7 is that the application amount is 70 mL / plant each time and the application frequency is once a week.

[0102] Example 9

[0103] The difference between this example and Example 7 is that the application amount is 120 mL / plant and the application frequency is once every two weeks.

[0104] Experimental Example 1 Growth-promoting effect of cold-resistant Bacillus IVF-WKB47 on cucumber in greenhouse

[0105] 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.

[0106] 2. Experimental methods and grouping:

[0107] 2.1 Grouping

[0108] Treatment group 1 (IVF-WKB47): The fermentation dilution of the cold-resistant Bacillus IVF-WKB47 in Example 3 was applied, and no other fertilizers were added.

[0109] Treatment group 2 (control): no substance was added.

[0110] Treatment group 3 (Y1): The commercial bacterial solution Y1 was applied to irrigate the cucumber seedlings in plot 3. The commercial bacterial solution Y1 was a microbial agent purchased from Hebei Devodo Biotechnology Co., Ltd., and no other fertilizers were applied.

[0111] Treatment group 4 (Y2): The commercial bacterial solution Y2 was applied to irrigate the cucumber seedlings in plot 4. The commercial bacterial solution Y2 was 360 Bacteria Manager purchased from Zhengzhou Zhongke Chemical Products Co., Ltd. No other fertilizers were applied.

[0112] Treatment group 5 (fertilizer-added control): only a 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.

[0113] 2.2 Experimental methods

[0114] 2.2.1 Species planting: Cucumbers were planted in greenhouses using local planting methods combined with the area planting arrangements of the experimental plots;

[0115] Fertilization method: The cucumbers in the corresponding plots of treatment groups 1, 3, and 4 were irrigated from the seedling stage. Each seedling in each treatment group was irrigated with 100 mL of the corresponding IVF-WKB47 fermentation dilution or bacterial solution. It was applied once a week during the entire growth period of the cucumber, 100 mL / plant each time. At the same time, no other fertilizers were applied, and normal management was carried out according to local planting needs. Treatment group 2 did not apply any substance. Treatment group 5 only applied a water-soluble compound fertilizer with a nitrogen, phosphorus, and potassium mass ratio of 20:10:20. The application of the compound fertilizer was in accordance with its instruction manual.

[0116] Experimental plot description: The experimental plot is located in the same plot of the plastic greenhouse of the Nankou pilot base of the Chinese Academy of Agricultural Sciences (temperature is 20℃ (maintained for 10 hours) and night temperature is 10℃ (maintained for 14 hours), alternating, light intensity 6000-10000lx, light time 8h, the rest of the time is dark state, soil moisture 80%), that is, one plot is divided into 5 plots, of which plot 1 is used for treatment group 1, plot 2 is used for treatment group 2, plot 3 is used for treatment group 3, plot 4 is used for treatment group 4, and plot 5 is used for treatment group 5. Cucumber 9930 is cultivated between adjacent plots as protection rows; the area of ​​each plot is 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.

[0117] 3. Results and Analysis

[0118] The cucumbers planted in each treatment group were statistically analyzed, that is, the yield per plant and per mu of cucumbers at the seedling stage, flowering stage, and fruiting stage were counted (20 plants per furrow, about 3,500 plants per mu). The data processing method was T-test. The results are shown in Table 1. Figure 4~Figure 8 . Without applying other fertilizers, the per mu yield of the fermentation dilution with cold-resistant Bacillus IVF-WKB47 increased by 29.1%, 11.3% and 4.0% compared with the control, Y1 and Y2, respectively, and the number of fruits per mu increased by 16.7%, 10.5% and 4.1%, and the single fruit weight increased by 10.7% compared with the control. The fermentation dilution of cold-resistant Bacillus IVF-WKB47 also has a more significant effect in promoting cucumber growth. Without applying other fertilizers, the per mu yield and the number of fruits per mu of IVF-WKB47 are comparable to those of the fertilized control, indicating that the fermentation dilution of cold-resistant Bacillus IVF-WKB47 of the present invention can replace chemical fertilizers and is more environmentally friendly.

[0119] Table 1 Effects of different treatments on average per-acre yield, average single fruit weight and average number of fruits set of cucumber

[0120]

[0121] Experimental Example 2 Effect of Cold-tolerant Bacillus IVF-WKB47 in Plug Tray on Growth Promotion of Cucumber Seedlings

[0122] 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.

[0123] 2. Experimental methods and grouping:

[0124] (1) Pre-seed treatment: Soak cucumber seeds (Derrite 89, a variety that is not resistant to low temperature and weak light) in 50°C warm water for 30 minutes, then soak in 75% alcohol for 15 seconds, rinse once with sterile water, and then soak in 6% sodium hypochlorite for 15 minutes. Shake occasionally during this period, 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 it in a dark incubator at 30°C and wait for the seeds to germinate; after the seeds germinate, select seeds with consistent growth for use;

[0125] (2) Fertilization: The seeds selected in step (1) that germinated and grew uniformly were sown in a 6-hole plug tray (the substrate was a sterilized Flora gard peat soil seedling substrate), one seed was sown in each hole, and each plant was a biological replicate. The seeds were cultured in an artificial climate chamber (the day and night temperatures were 20° C. (maintained for 16 hours) and 10° C. (maintained for 8 hours), respectively, alternating between the day and night temperatures, the daylighting time was 16 hours per day, and the rest of the time was in a dark state, the light intensity was 3000 lx~10000 lx, and the soil moisture was 80%). When the cucumbers were in the seedling stage and the cotyledons were unfolded, the experimental group was applied with the cold-resistant Bacillus IVF-WKB47 bacterial solution of Example 2 by root irrigation, with an application amount of 100 mL / plant each time and the application frequency once a week. 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. Without applying any additional fertilizers, normal management is carried out according to planting needs. The cultivation conditions during this period are (day and night temperatures are 20℃ (maintained for 16 hours) and 10℃ (maintained for 8 hours), alternating between day and night, 16 hours of daylight per day, and the rest of the time is in darkness, light intensity is 3000lx~10000lx, and soil moisture is 80%).

[0126] 3. Results and analysis:

[0127] 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 are measured to evaluate the growth-promoting effect of the test strains in the seedling stage. The data statistical method is the T-test. The results are shown in 2. Fig. 9 and Fig.10 ,Table 2 shows the specific values ​​of different measurement indicators of cucumber growth promotion effect under different treatments, Fig. 9 This is a bar chart of different measurement indicators of cucumbers under different treatments. Fig.10 This is the growth diagram of cucumber seedlings under different treatments. The specific measurement standards are as follows:

[0128] Plant height: The height from the surface of the plug tray substrate to the growth point shall prevail;

[0129] Stem diameter: measured with a vernier caliper just below the cotyledon node;

[0130] Leaf area: Spread out the largest true leaf and take a photo, then use image J software to count the leaves;

[0131] Fresh weight: Wash the whole plant, shake off excess water, and weigh it with an electronic balance;

[0132] Dry weight: The whole plant was sterilized at 105℃ for 15min and dried at 70℃ for more than 48h until constant weight was achieved. Among them, plant height, stem diameter, leaf area, whole plant fresh weight and whole plant dry weight were analyzed and statistically analyzed with 6 replicates.

[0133] Table 2. Growth-promoting effects of different treatments on cucumber seedlings

[0134]

[0135] From the above table, Fig. 9 and Fig.10 It can be seen that compared with the blank control group, the application of the IVF-WKB47 bacterial solution of the present invention can significantly increase the stem diameter, whole plant fresh weight and leaf area of ​​the cucumber plants, and there is a significant difference (p < 0.05, p < 0.001, p < 0.001), indicating that the IVF-WKB47 bacterial solution of the present invention can promote the growth of stem diameter, whole plant fresh weight and leaf area of ​​cucumber plants.

[0136] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A cold-resistant Bacillus ( Peribacillus frigoritolerans )IVF-WKB47, the deposit number is CGMCCNo.28315.

2. A microbial preparation, characterized in that: Containing the cold-resistant Bacillus IVF-WKB47 according to claim 1 or a culture thereof.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the viable count of the cold-resistant Bacillus IVF-WKB47 is ≥10 7 cfu / mL.

4. A method for preparing a microbial preparation as claimed in claim 2 or 3, characterized in that: include: The cold-resistant Bacillus IVF-WKB47 is cultured in an expanded manner.

5. Use of the cold-resistant Bacillus IVF-WKB47 according to claim 1 or the microbial preparation according to claim 2 or 3 in promoting the growth of cucumbers, wherein the cucumbers are cucumbers that are intolerant to low temperatures and / or weak light.

6. The use according to claim 5, characterized in that The method for promoting cucumber growth comprises increasing at least one of per-acre yield of cucumber, single fruit weight, number of fruits, stem thickness of cucumber, whole-plant fresh weight of cucumber and leaf area of ​​cucumber.

7. The use according to claim 6, characterized in that The cucumber is Deruit 89.

8. A plant growth regulator, seed coating agent, root irrigation agent, seed soaking agent, microbial pesticide or microbial fertilizer, characterized in that: The method comprises the cold-resistant Bacillus IVF-WKB47 according to claim 1 or the microbial preparation according to claim 2 or 3.

9. A method for promoting cucumber growth, characterized in that: The method comprises applying the cold-resistant Bacillus IVF-WKB47 according to claim 1, the microbial preparation according to claim 2 or 3, or the plant growth regulator, seed coating agent, root irrigation agent, seed soaking agent, microbial pesticide or microbial fertilizer according to claim 8; the application method comprises root irrigation; When applying the bacterial solution of cold-resistant Bacillus IVF-WKB47, the application amount of the bacterial solution is 10-100 mL / strain each time, and the application frequency is once every 1-2 weeks; When applying the fermentation dilution of cold-resistant Bacillus IVF-WKB47, the application amount of the fermentation dilution is 70-120 mL / strain each time, and the frequency of application is once every 1 to 2 weeks; The period of application includes the entire growth period of the plant; The cucumber is a cucumber that is intolerant to low temperature and / or weak light.

10. The method for promoting cucumber growth according to claim 9, characterized in that: The application amount of the bacterial solution is 50 mL / strain each time, and the application frequency is once a week; the effective viable bacteria count of cold-resistant Bacillus IVF-WKB47 in the bacterial solution is ≥10 7 cfu / mL; The application amount of the fermentation dilution is 100 mL / strain each time, and the application frequency is once a week; the effective viable count of cold-resistant Bacillus IVF-WKB47 in the fermentation dilution is ≥10 7 cfu / mL; The application period includes the seedling stage, flowering stage and fruit setting stage.

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