Bacillus pumilus and application thereof

Abstract

The invention provides bacillus pumilus and application thereof, and belongs to the technical field of microorganisms, the bacillus pumilus YM3-12 provided by the invention is preserved in China General Microbiological Culture Collection Center on October 21, 2024, and the preservation number is CGMCC No. 32281; the strain is high in salt and alkali resistance, has the functions of dissolving organic phosphorus, dissolving inorganic phosphorus, dissolving potassium, degrading protein, degrading cellulose, producing iron carriers and the like, can antagonize Erwinia persicae and fusarium oxysporum, has an obvious growth promoting effect on wheat, radishes and cucumbers in a salt and alkali environment, and is very wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus pumilus and application thereof. Background Art

[0002] Soil salinization and secondary salinization are widespread worldwide, especially in arid and semi-arid regions. my country ranks third in the world in terms of saline-alkali land area, with an area of ​​about 1×10 8 hm 2 , widely distributed in Northwest, Northeast, North China and coastal areas of my country, agricultural production is facing unprecedented challenges. As one of my country's staple crops, wheat is mainly produced in areas where soil salinization occurs frequently. At the same time, as the only staple crop that survives the winter, spring is a critical period for its jointing growth and grain filling, but it is also the peak period for soil salt return. Therefore, the threat of soil salinization to wheat production safety needs to be paid special attention to.

[0003] Erwinia persica Erwinia persicina ) is a plant pathogen that can cause diseases in many crops, especially alfalfa. The fungus can spread through water, soil, seeds and other channels, and invade the plant through natural openings, causing necrosis of plant stems, leaves, flowers and pods, seriously affecting the normal growth and yield of crops.

[0004] Fusarium oxysporum Fusarium oxysporum ) is a facultative parasitic fungus that can infect plants and survive in the soil. It has a strong ability to survive. It can survive in the soil for more than ten years and maintain strong biological activity, making it difficult to control. The pathogen can infect more than 1,000 plants such as melons, Solanaceae, rice, bananas and cotton. Generally, it will cause rhizome and bulb rot, vascular bundle atrophy, plant yellowing and other symptoms, posing a great threat to agricultural production.

[0005] Plant growth-promoting rhizobacteria (PGPR) are a key type of microorganisms present in the rhizosphere soil of plants. They play a vital role in improving plant growth efficiency, reducing the incidence of pests and diseases, and increasing crop yields. PGPR has been proven to significantly improve the host plant's defense and tolerance to adversity through a special physiological mechanism. Bacillus pumilus) As an important PGPR resource, most of them exist in the soil or colonize in the roots of certain plants. They have strong tolerance and produce antagonistic substances such as antibiotics, antimicrobial proteins, monoacyl diglucosylglycerol, etc., which can inhibit a variety of pathogens. Wang Jing et al. found that the intracellular substances of Bacillus pumilus AR03 have a sustainable disease control effect on tobacco black shank disease through confrontation culture, solid-state fermentation and field plot experiments, and are very potential biocontrol strains. Kaushal et al. found that Bacillus pumilus YSPMK11 has efficient biocontrol characteristics, and its genome contains genes for two lipopeptides (Itu D and Srf C). At the same time, Bacillus pumilus can also secrete a variety of highly active enzymes, such as chitinase, lipase, alkaline serine keratase, and xylanase, which promote the degradation of macromolecules through these metabolites. Bacillus pumilus AR57 screened by Jagadeesa et al. can degrade keratin and can be used for the bioconversion of poultry solid waste. In addition, Bacillus brevis can also repair contaminated soil, reduce the toxicity of fungal metabolites, and reduce the pollution of the environment by waste. However, different strains have certain differences in their metabolic characteristics and biocontrol effects.

[0006] At present, there has been no report on a Bacillus brevis with a salt tolerance range of 0-12% (0-120 g / L), an alkali tolerance range of 7-14, and multiple functions such as phosphate solubilization, potassium solubilization, protein degradation, cellulose degradation, siderophore production, and antagonism against Erwinia persica and Fusarium oxysporum. There is also a lack of research on the application of Bacillus brevis in promoting the growth of wheat, radish and cucumber in saline-alkali soil environments. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a strain of Bacillus pumilus and applications thereof.

[0008] The strain provided by the present invention has strong salt-alkali resistance, can antagonize various pathogens, and also has multiple functions such as dissolving organic phosphorus, dissolving inorganic phosphorus, dissolving potassium, degrading protein, degrading cellulose and producing iron carriers. It has a significant promoting effect on wheat, radish and cucumber grown in a saline-alkali environment and can improve the growth state of plants under saline-alkali stress.

[0009] The technical solution of the present invention is as follows: A strain of Bacillus brevis ( Bacillus pumilus ) YM3-12, deposited on October 21, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32281.

[0010] The Bacillus pumilus ( Bacillus pumilus)The 16S rDNA gene sequence of YM3-12 is shown in SEQ ID NO.1.

[0011] The above-mentioned Bacillus pumilus ( Bacillus pumilus ) The culture method of YM3-12 comprises the following steps: Bacillus pumilus ( Bacillus pumilus ) YM3-12 was inoculated into LB solid medium and inverted at 35-37°C until a single colony grew out; a single colony was picked and inoculated into LB liquid medium, and cultured at 35-37°C, 180-200rpm until the logarithmic growth phase, and then inoculated into LB liquid medium at an inoculum volume fraction of 2-4%, and cultured at 35-37°C, 180-200rpm for 22-24h to obtain Bacillus pumilus ( Bacillus pumilus ) YM3-12 bacterial liquid.

[0012] The above-mentioned Bacillus pumilus ( Bacillus pumilus ) Application of YM3-12 in at least one of the following: solubilizing organic phosphorus, solubilizing inorganic phosphorus, solubilizing potassium, degrading protein, degrading cellulose, and producing iron carriers.

[0013] The above-mentioned Bacillus pumilus ( Bacillus pumilus ) Use of YM3-12 in antagonizing at least one of Erwinia persica and Fusarium oxysporum.

[0014] The above-mentioned Bacillus pumilus ( Bacillus pumilus ) Application of YM3-12 in promoting plant growth in saline-alkali soil environment.

[0015] Preferably according to the present invention, the plant is wheat, radish or cucumber.

[0016] Preferably according to the present invention, the soil is saline-alkali soil with a salt content of ≤6 g / Kg and a pH of ≤9.

[0017] A microbial agent containing the above-mentioned Bacillus pumilus ( Bacillus pumilus )YM3-12.

[0018] The beneficial effects of the present invention include at least the following: The Bacillus pumilus YM3-12 provided by the present invention has strong salt-alkali resistance, and can tolerate a salt concentration of up to 12% (120 g / L) and an alkaline environment with a pH value of 14. The strain can improve the stress resistance of plants in saline-alkali environments, and has a significant growth-promoting effect on wheat, radish and cucumber. At the same time, the strain also has the ability to decompose organic phosphorus, dissolve inorganic phosphorus, decompose potassium, degrade protein, degrade cellulose, produce siderophores and antagonize multiple pathogens, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is the picture of strain YM3-12 observed under a microscope after Gram staining.

[0020] Figure 2 This is the morphology of the colonies grown by strain YM3-12 after three-zone streaking on LB solid culture medium.

[0021] Figure 3 This is a graph showing the growth of strain YM3-12 on LB solid culture medium with different salinities.

[0022] Figure 4 This is a graph showing the growth of strain YM3-12 on LB solid medium with different alkalinity; In the figure: 7 is pH=7, 8 is pH=8, 9 is pH=9, 10 is pH=10, 11 is pH=11, 12 is pH=12, 13 is pH=13, and 14 is pH=14.

[0023] Figure 5 This is a graph showing the growth of strain YM3-12 in organophosphorus bacterial culture medium.

[0024] Figure 6 This is a graph showing the growth of strain YM3-12 in inorganic phosphorus bacterial culture medium.

[0025] Figure 7 This is a graph showing the growth of strain YM3-12 in silicate bacterial culture medium.

[0026] Figure 8 This is a graph showing the growth of strain YM3-12 in casein medium.

[0027] Fig. 9 This is a graph showing the growth of strain YM3-12 in sodium carboxymethyl cellulose medium.

[0028] Fig.10 This is a graph showing the growth of strain YM3-12 in CAS medium.

[0029] Fig.11 This is a diagram showing the effect of strain YM3-12 antagonizing Erwinia persica.

[0030] Fig.12 This is a diagram showing the effect of strain YM3-12 in antagonizing Fusarium oxysporum.

[0031] Fig.13 This is a graph showing the growth of wheat in different treatment groups on the 12th day after the bacterial treatment; In the figure: A is a picture of wheat potted in different treatment groups, B is a picture of wheat morphology in different treatment groups; CK1 was the sterile water treatment group, CK2 was the LB liquid culture medium treatment group, and YM3-12 was the YM3-12 bacterial liquid treatment group.

[0032] Fig.14 These are the physiological plant height diagrams of wheat in each treatment group at the 4th, 8th and 12th day of bacterial treatment; In the figure: A is a diagram showing the measurement results of the physiological plant height of wheat on the 4th day of treatment; B is a diagram showing the measurement results of the physiological plant height of wheat on the 8th day of treatment; C is a diagram showing the measurement results of the physiological plant height of wheat on the 12th day of treatment; CK1 was the sterile water treatment group, CK2 was the LB liquid culture medium treatment group, and YM3-12 was the YM3-12 bacterial liquid treatment group.

[0033] Fig.15 These are the above-ground fresh weight, underground fresh weight, above-ground dry weight, and underground dry weight of wheat in each treatment group on the 12th day after the bacterial treatment; In the figure: A is a graph showing the above-ground fresh weight of wheat; B is a graph showing the underground fresh weight of wheat; C is a graph showing the above-ground dry weight of wheat; D is a graph showing the underground dry weight of wheat; CK1 was the sterile water treatment group, CK2 was the LB liquid culture medium treatment group, and YM3-12 was the YM3-12 bacterial liquid treatment group.

[0034] Fig.16 This is a graph showing the growth of radish seedlings in different treatment groups on the 8th day after the bacterial treatment; In the figure: CK1 is the sterile water treatment group, CK2 is the LB liquid culture medium treatment group, and YM3-12 is the YM3-12 bacterial liquid treatment group.

[0035] Fig.17 These are the physiological plant height graphs of radish seedlings in each treatment group on day 0, 1, 2, 3, 4, 5, 6, 7, and 8 of bacterial treatment; In the figure: CK1 is the sterile water treatment group, CK2 is the LB liquid culture medium treatment group, and YM3-12 is the YM3-12 bacterial liquid treatment group.

[0036] Fig.18 This is the total fresh weight of radish seedlings in each treatment group on the 8th day of bacterial treatment; In the figure: CK1 is the sterile water treatment group, CK2 is the LB liquid culture medium treatment group, and YM3-12 is the YM3-12 bacterial liquid treatment group.

[0037] Fig.19 This is a graph showing the growth of cucumber seedlings in different treatment groups on the 8th day after the bacterial treatment; In the figure: A is a picture of cucumber potted plants in different treatment groups; B is a picture of cucumber morphology in different treatment groups; CK1 was the sterile water treatment group, CK2 was the LB liquid culture medium treatment group, and YM3-12 was the YM3-12 bacterial liquid treatment group.

[0038] Fig. 20These are the physiological plant height graphs of cucumber seedlings in each treatment group on day 0, 1, 2, 3, 4, 5, 6, 7, and 8 of bacterial treatment; In the figure: CK1 is the sterile water treatment group, CK2 is the LB liquid culture medium treatment group, and YM3-12 is the YM3-12 bacterial liquid treatment group.

[0039] Fig.21 This is the total fresh weight of cucumber seedlings in each treatment group on the 8th day of bacterial treatment; In the figure: CK1 is the sterile water treatment group, CK2 is the LB liquid culture medium treatment group, and YM3-12 is the YM3-12 bacterial liquid treatment group. DETAILED DESCRIPTION

[0040] The following is a description with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0041] Source of experimental materials: Bacterial microbiochemical identification tube: purchased from Qingdao Haibo Biotechnology Co., Ltd.; Erwinia persica: purchased from Shanghai Collection of Microorganisms, catalog number SHMCC D10977; Fusarium oxysporum: purchased from Shanghai Microbiological Collection Center, catalog number SHMCC D25188; Jinchun No. 6 wheat seeds: purchased from Hejian Fuyichun Seed Sales Co., Ltd.; Vermiculite: purchased from Lingshou County Xuyang Mining Co., Ltd.

[0042] Bacillus brevis ( Bacillus pumilus ) YM3-12 is referred to as "Bacillus pumilus YM3-12" or "strain YM3-12". Example 1 Isolation, Screening and Identification of Bacillus pumilus YM3-12 The specific isolation and screening methods of strains are as follows: (1) Soil samples were collected from the saline-alkali soil experimental field in Yanggu County, Liaocheng City, Shandong Province, and wheat was planted in the soil. When the wheat seeds had been in the soil for one week, 5 g of soil was taken from the rhizosphere of the wheat, added to a conical flask containing 50 mL of sterile water, and shaken evenly.

[0043] (2) Pipette 100 μL of the mixed soil suspension into a centrifuge tube containing 900 μL of sterile water, and dilute the soil suspension into 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Different gradients.

[0044] (3) Take the dilution of 10 -5 , 10 -6 , 10 -7 100 μL of each soil suspension was spread on LB solid culture medium with a pH of 8 and a salt concentration of 3%, and placed upside down in a 37°C constant temperature incubator for 1-2 days. Three plates were spread for each dilution.

[0045] (4) When colonies grow on the culture medium, pick them out with a toothpick. Purify the obtained colonies on the LB plate culture medium using the three-zone method until a single colony is obtained, and number the obtained single colony. Screen the colonies for functions such as salt-alkali resistance, organic phosphorus solubility, inorganic phosphorus solubility, potassium solubility, nitrogen fixation, protein degradation, fat degradation, cellulose degradation, and iron carrier production.

[0046] Through the above separation and screening work, the inventors finally screened out a strain that has the characteristics of salt-alkali resistance, organic phosphorus solubilization, inorganic phosphorus solubilization, potassium solubilization, protein degradation, cellulose degradation and iron carrier production, and named it "YM3-12".

[0047] The morphology of strain YM3-12 obtained by the above screening on LB solid medium is as follows: Figure 1 , Figure 2 as shown; Figure 1 As shown in the figure, after Gram staining, strain YM3-12 was observed under a 100x oil-immersed microscope and appeared to be a purple long rod, which is a Gram-positive bacterium; Figure 2 As shown, strain YM3-12 was cultured on LB solid medium with three zones for one day. The grown colonies of strain YM3-12 were off-white, irregularly round, with smooth and neat edges, and had strong toughness and density when observed.

[0048] In addition, the strain YM3-12 obtained by screening was subjected to physiological and biochemical characteristics identification, and the identification results were as follows: strain YM3-12 can utilize fructose, trehalose, xylose and sucrose; but cannot utilize galactose, sorbitol, maltose, cellobiose, raffinose, rhamnose and glucose.

[0049] The 16S rDNA gene sequence of strain YM3-12 was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. The obtained 16S rDNA sequence was subjected to BLAST analysis with the existing sequences in the NCBI database, and strains with similar homology were selected. The phylogenetic tree was constructed using MEGA X software. The results showed that the strain YM3-12 obtained by the above screening was similar to Bacillus pumilus Combined with the physiological and biochemical characteristics of the strain, it was identified as Bacillus pumilus ( Bacillus pumilus ).

[0050] A strain of Bacillus brevis ( Bacillus pumilus ) YM3-12, deposited on October 21, 2024 in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32281.

[0051] Example 2 The specific method for culturing Bacillus pumilus YM3-12 bacterial solution is as follows: The strain YM3-12 was inoculated into LB solid culture medium and inverted at 37°C until a single colony grew; a single colony was picked out and inoculated into LB liquid culture medium, and cultured at 37°C, 180 rpm in a shaking incubator for 12 h until the logarithmic growth phase to obtain the seed solution of the strain; the inoculation amount was inoculated into 50 mL LB liquid culture medium at a volume fraction of 2%, and cultured at 37°C, 180 rpm in a shaking incubator for 24 h to obtain the YM3-12 bacterial solution.

[0052] The components of the LB liquid culture medium are: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 1 L distilled water, pH 7.0; sterilized at 121°C for 20 min.

[0053] Experimental Example 1 Determination of Salt-Alkali Tolerance of Bacillus pumilus YM3-12 (1) Determination of salt tolerance of strain YM3-12: LB solid culture medium with NaCl concentration ranging from 0 to 14% was prepared, i.e., the NaCl concentration was 0 g / L (0%), 10 g / L (1%), 40 g / L (4%), 70 g / L (7%), 90 g / L (9%), 100 g / L (10%), 110 g / L (11%), 120 g / L (12%), 130 g / L (13%), and 140 g / L (14%); a single colony of Bacillus pumilus YM3-12 was picked up with an inoculation loop after burning and sterilized, and inoculated onto the above LB solid culture medium by streak method, and cultured inverted at 37°C for 24 h to 48 h, and the growth of Bacillus pumilus YM3-12 on culture medium with different salt concentrations was observed.

[0054] The results are as follows Figure 3 As shown by Figure 3 It can be seen that Bacillus pumilus YM3-12 can grow in LB plates with salinity of 0-12%, but stops growing when the salinity increases to 13%, which indicates that Bacillus pumilus YM3-12 can tolerate a salt environment of up to 12% (120 g / L).

[0055] (2) Determination of the alkali resistance of strain YM3-12: LB solid culture media with pH values ​​of 7-14 were prepared respectively, i.e., the pH values ​​were 7, 8, 9, 10, 11, 12, 13, and 14, respectively; a single colony of Bacillus pumilus YM3-12 was picked up with an inoculation loop after burning and sterilized, and inoculated onto the above LB solid culture media by streaking, and incubated inverted at 37°C for 24 h-48 h, and the growth of Bacillus pumilus YM3-12 on LB solid culture media with different pH values ​​was observed.

[0056] The results are as follows Figure 4 As shown by Figure 4 It can be seen that Bacillus pumilus YM3-12 can grow in the LB plate with a pH of 7-14, which indicates that Bacillus pumilus YM3-12 can tolerate an alkaline environment with a pH of 7-14.

[0057] Experimental Example 2 Determination of the ability of Bacillus pumilus YM3-12 to degrade organic phosphorus, inorganic phosphorus, potassium, protein, cellulose and produce siderophore (1) The phosphate solubilization ability of strain YM3-12 was determined using an organophosphate bacterial culture medium. The specific method is as follows: The strain YM3-12 was inoculated on the Montkina organophosphate bacterial medium with a sterilized toothpick and cultured at 28°C for 5 days to observe the growth of the colonies. The components of the Montgina organophosphorus bacteria culture medium are as follows: glucose 10 g / L, (NH 4 ) 2 SO 4 0.5g / L, MgSO 4 7H 2 O 0.3g / L, MnSO 4 ·4H 2 O 0.03g / L, KCl 0.3g / L, FeSO 4 7H 2 O 0.03g / L, NaCl 0.3g / L, CaCO 3 5.0g / L, lecithin 0.2g / L, agar 15g / L, pH 7.0~7.5, solvent is water.

[0058] Colony growth Figure 5 As shown by Figure 5 It was found that a transparent degradation zone appeared around strain YM3-12 in the culture medium of organophosphorus bacteria, indicating that strain YM3-12 has the ability to degrade organophosphorus.

[0059] (2) The phosphate solubilizing ability of strain YM3-12 was determined using inorganic phosphorus bacterial culture medium. The specific method is as follows: The strain YM3-12 was inoculated on the Montkina inorganic phosphate bacterial medium with a sterilized toothpick and cultured at 28°C for 5 days to observe the growth of the colonies. The components of the Montgina inorganic phosphorus bacterial culture medium are as follows: glucose 10 g / L, (NH 4 ) 2 SO 4 0.5g / L, MgSO 4 7H 2 O 0.3g / L, MnSO 4 ·4H 2 O 0.03g / L, KCl 0.3g / L, FeSO 4 7H 2 O 0.03g / L, NaCl 0.3g / L, Ca 3 (PO 4 ) 2 10.0g / L, agar 15g / L, pH 7.0~7.5, solvent is water.

[0060] Colony growth Figure 6 As shown by Figure 6 It was found that a transparent degradation zone appeared around strain YM3-12 in the inorganic phosphorus bacterial culture medium, indicating that strain YM3-12 has the ability to dissolve inorganic phosphorus.

[0061] (3) The potassium-solubilizing ability of strain YM3-12 was determined using silicate bacterial culture medium. The specific method is as follows: The strain YM3-12 was inoculated onto silicate bacterial culture medium using a sterilized toothpick and cultured at 28°C for 4 days to observe the growth of the colonies. The components of the silicate bacterial culture medium are as follows: sucrose 5 g / L, MgSO 4 0.5g / L, CaCO 3 0.1g / L, Na 2 HPO 4 2g / L, FeCl 3 0.005g / L, glass powder 1g / L, agar 15g / L, pH 7.0, solvent is water.

[0062] Colony growth Figure 7 As shown by Figure 7 It can be seen that strain YM3-12 is a smooth and transparent oil droplet-shaped colony in the silicate bacterial culture medium, indicating that strain YM3-12 has the ability to solubilize potassium.

[0063] (4) The ability of strain YM3-12 to degrade protein was determined using casein medium. The specific method is as follows: The strain YM3-12 was inoculated on casein medium with a sterilized toothpick and cultured at 37°C for 3 days to observe the growth of the colonies. The components of the casein culture medium are as follows: casein (casein) 10 g / L, beef extract 3.0 g / L, NaCl 5.0 g / L, K 2 HPO 4 2.0g / L, agar 15-20g / L, bromothymol blue 0.05g / L, pH 7.3-7.5, solvent is water.

[0064] Colony growth Figure 8 As shown by Figure 8 It can be seen that in the casein culture medium, a transparent degradation zone appeared in strain YM3-12, which indicates that strain YM3-12 has the ability to degrade protein.

[0065] (5) The ability of strain YM3-12 to degrade cellulose was determined using sodium carboxymethyl cellulose culture medium. The specific method is as follows: Use a sterilized toothpick to inoculate strain YM3-12 on sodium carboxymethyl cellulose medium and culture at 37°C for 3 days. Then add 1 mg / 100 mL Congo red solution to submerge strain YM3-12 for staining for 30 minutes. Finally, wash off the stain with 1 mol / L NaCl solution and observe the growth of the colony. The components of the sodium carboxymethyl cellulose culture medium are as follows: 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L disodium hydrogen phosphate, 20.0 g / L sodium carboxymethyl cellulose, 2.0 g / L peptone, 0.5 g / L yeast extract powder, 14.0 g / L agar, pH 7.2, and the solvent is water.

[0066] Colony growth Fig. 9 As shown by Fig. 9 It can be seen that in the sodium carboxymethyl cellulose culture medium, strain YM3-12 showed a very obvious yellow degradation zone, which shows that strain YM3-12 has a strong ability to degrade cellulose.

[0067] (6) The ability of strain YM3-12 to produce siderophores was determined using CAS medium. The specific method is as follows: Use a sterilized toothpick to inoculate strain YM3-12 onto CAS medium, culture at 37°C for 3 days, and observe the growth of the colonies; The CAS medium components are as follows: chrome azurol 0.06 g / L, hexadecyltrimethylammonium bromide 0.073 g / L, ferric chloride hexahydrate 0.0026 g / L, sodium dihydrogen phosphate dihydrate 0.295 g / L, disodium hydrogen phosphate dodecahydrate 1.213 g / L, ammonium chloride 0.125 g / L, potassium dihydrogen phosphate 0.037 g / L, sodium chloride 0.0625 g / L, agar 20 g / L, pH 6.7-6.9, and the solvent is water.

[0068] Colony growth Fig.10 As shown by Fig.10 It can be seen that in the CAS medium, a transparent degradation zone appeared in strain YM3-12, which indicates that strain YM3-12 has the ability to produce siderophores.

[0069] Experimental Example 3 Determination of the antagonistic ability of Bacillus pumilus YM3-12 against plant pathogens Select the pathogenic bacteria Erwinia persicae ( Erwinia persicina ) and Fusarium oxysporum, the pathogen of cotton wilt ( Fusarium oxysporum ), the antagonistic ability of strain YM3-12 against the above pathogens was studied.

[0070] (1) The plate spreading method was used to determine whether strain YM3-12 could antagonize Erwinia persica. The specific steps were as follows: the Erwinia persica stored in the glycerol tube was inoculated on LB solid culture medium and cultured at 37°C until a single colony grew; the single colony was inoculated into 5 mL LB liquid culture medium and cultured at 37°C and 180 rpm for 10-12 h to obtain the Erwinia persica bacterial liquid for later use; Take 100 μL of the above-mentioned peach-colored Erwinia bacterial solution and spread it on the LB solid culture medium. After the bacterial solution is dry, use a sterilized inoculation loop to inoculate the strain YM3-12 onto the plate, invert and culture at 37°C for 24 h, and observe whether there is an inhibition zone around Bacillus pumilus YM3-12.

[0071] The results are as follows Fig.11 As shown by Fig.11 It can be seen that an obvious inhibition zone appeared around strain YM3-12, which indicates that strain YM3-12 has the ability to antagonize Erwinia persica.

[0072] (2) The plate confrontation method was used to determine whether strain YM3-12 could antagonize Fusarium oxysporum. The specific steps were as follows: Fusarium oxysporum was inoculated on PDA solid culture medium and cultured at 28°C for 4-5 days until the culture medium was full of round mycelium; the mycelium was punched out with a sterile puncher and transferred to a new PDA solid culture medium with tweezers; a single colony of strain YM3-12 was picked up with a sterile toothpick and inoculated on the PDA solid culture medium 2 cm away from the edge of the mycelium; the culture was continued at 28°C for 2-5 days to observe whether an inhibition zone appeared; The components of the PDA solid culture medium are as follows: 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, and natural pH.

[0073] The results are as follows Fig.12 Indicated by Fig.12 It can be seen that inhibition zones appeared around strain YM3-12, indicating that strain YM3-12 has the ability to antagonize Fusarium oxysporum.

[0074] Experimental Example 4 Determination of the wheat growth-promoting effect of Bacillus pumilus YM3-12 in saline-alkali soil environment (1) Vernalization of wheat: Wrap the cleaned wheat seeds with wet gauze for 12 hours. After the seeds germinate, place them in a 4°C refrigerator for 3-7 days for vernalization.

[0075] (2) Prepare 15 cultivation pots for cultivating wheat. The 15 cultivation pots were evenly divided into three groups, namely CK1 group (watered with sterile water), CK2 group (watered with sterilized LB liquid culture medium), and experimental group (watered with YM3-12 bacterial solution diluted with sterile water). Each group was set up with 5 replicates.

[0076] The soil used was taken from the saline-alkali soil experimental field in Yanggu County, Liaocheng City, Shandong Province, with a salt content of 3.45-6 g / Kg and a pH of 8.5-9. The soil was mixed with vermiculite in a volume ratio of 2:1 and then used.

[0077] Among them, the amount of sterile water applied in the CK1 group was 150 mL / Kg; the LB liquid culture medium in the CK2 group was applied by diluting it 100 times with sterile water, and the application amount was 150 mL / Kg; the application method of the Bacillus pumilus YM3-12 bacterial solution added to the experimental group was: the bacterial solution prepared in Example 2 was diluted 100 times with sterile water, and the application amount was 150 mL / Kg.

[0078] (3) Select wheat seeds of uniform size after vernalization in step (1) and plant them in cultivation pots. Thin out the wheat seedlings after they emerge on the second day of cultivation so that there are 3 wheat seedlings in each cultivation pot. Add bacteria after thinning. Measure the physiological plant height of wheat (the maximum distance after straightening) on ​​the 4th, 8th and 12th day after the bacterial treatment, and take photos of the growth of wheat on the 12th day. Then harvest the seedlings and take photos to record the morphological photos of wheat after root washing in different treatment groups, and measure the agronomic traits such as aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight.

[0079] After 12 days of bacterial treatment, the wheat potted plants and morphological photos of different treatment groups are shown in the figure. Fig.13 As shown by Fig.13 It can be seen that after the 12th day of cultivation, the wheat plants in the experimental group grew more vigorously than those in the control group, with a significant increase in both the above-ground and underground parts. The results of wheat physiological plant height measurement on the 4th, 8th and 12th days after bacterial treatment are as follows: Fig.14 As shown by Fig.14 It was found that on the 4th day, the wheat plant height of the experimental group increased by 37.55% compared with that of CK1 (reaching a significant difference, p<0.05), and increased by 32.42% compared with that of CK2; on the 8th day, the wheat plant height of the experimental group increased by 43.32% compared with that of CK1 (reaching a significant difference, p<0.05), and increased by 36.85% compared with that of CK2; on the 12th day, the wheat plant height of the experimental group increased by 38.31% compared with that of CK1 (reaching an extremely significant difference, p<0.01), and increased by 28.42% compared with that of CK2 (reaching an extremely significant difference, p<0.01).

[0080] The weight of wheat after 12 days of bacterial treatment is as follows: Fig.15 As shown by Fig.15 It was found that: in terms of aboveground fresh weight, the experimental group increased by 57.92% compared with CK1 (reaching an extremely significant difference, p<0.01), and increased by 53.67% compared with CK2 (reaching an extremely significant difference, p<0.01); in terms of aboveground dry weight, the experimental group increased by 58.54% compared with CK1 (reaching an extremely significant difference, p<0.01), and increased by 47.73% compared with CK2 (reaching a significant difference, p<0.05); in terms of underground fresh weight, the experimental group increased by 118.13% compared with CK1 (reaching an extremely significant difference, p<0.01), and increased by 80.99% compared with CK2 (reaching an extremely significant difference, p<0.01); in terms of underground dry weight, the experimental group increased by 86.59% compared with CK1 (reaching an extremely significant difference, p<0.01), and increased by 63.26% compared with CK2 (reaching an extremely significant difference, p<0.01). Through data comparison, it was concluded that in saline-alkali soil environment, Bacillus pumilus YM3-12 has a significant growth-promoting effect on wheat, especially helping wheat to increase the accumulation of root biomass.

[0081] Experimental Example 5 Determination of the growth-promoting effect of Bacillus pumilus YM3-12 on radish seedlings in saline-alkali soil (1) Radish seed pretreatment.

[0082] (2) Prepare 9 cultivation pots and divide them into three groups: CK1 group (watered with sterile water), CK2 group (watered with sterilized LB liquid culture medium), and experimental group (watered with YM3-12 bacterial solution diluted with sterile water). Set up 3 replicates for each group.

[0083] The soil used was taken from the saline-alkali soil experimental field in Yanggu County, Liaocheng City, Shandong Province, with a salt content of 3.45-6.00 g / Kg and a pH of 8.5-9. The soil was mixed with vermiculite in a volume ratio of 2:1 and then used.

[0084] Among them, the amount of sterile water applied in the CK1 group was 200 mL / Kg; the LB liquid culture medium applied in the CK2 group was diluted 100 times with sterile water, and the application amount was 200 mL / Kg; the application method of the Bacillus pumilus YM3-12 bacterial solution added to the experimental group was: the bacterial solution prepared in Example 2 was diluted 100 times with sterile water, and the application amount was 200 mL / Kg.

[0085] (3) Select radish seeds of uniform size after pretreatment in step (1) and plant them in cultivation pots. Thin out the radish seedlings after they emerge on the second day of cultivation so that there are 3 radish seedlings in each cultivation pot. Add bacteria after thinning. The plant height on the day of adding bacteria is taken as the plant height on day 0. The physiological plant height of radish on days 0-8 and the growth status and total fresh weight of each plant of radish seedlings in different treatment groups at the time of harvesting on day 8 are recorded respectively.

[0086] When the seedlings were harvested on the 8th day, the radish morphology photos of the different treatment groups were as follows: Fig.16 As shown by Fig.16 It can be seen that the radish seedlings in the experimental group grew more vigorously than those in the control group when harvested. The results of the physiological plant height measurement of radish on days 0-8 are as follows Fig.17 As shown by Fig.17It was found that on the 0th day, the height difference of radish seedlings in the three groups was small, and the physiological height of radish seedlings in the experimental group was 10.64% higher than that in the CK1 group and 4.00% higher than that in the CK2 group. After the bacterial treatment, the physiological height of radish seedlings in the experimental group on the 1st to 8th day increased by 5.43%, 8.36%, 15.42% (extremely significant difference, p<0.01), 14.62% (extremely significant difference, p<0.01), 11.38% (significant difference, p<0.05), 9.82% (significant difference, p<0.05), 8.68% (significant difference, p<0.05), and 5.95% respectively compared with the CK1 group; compared with CK2, it increased by 2.34%, 6.58%, 7.09%, 7.56%, 5.83%, 5.84%, 6.17%, and 3.55% respectively.

[0087] The total fresh weight of each radish plant at the time of seedling harvest is as follows: Fig.18 As shown by Fig.18 It was found that the fresh weight of radish seedlings in the experimental group increased by 33.52% compared with CK1 (reaching a significant difference, p<0.05), and increased by 20.27% compared with CK2. The above data analysis showed that Bacillus pumilus YM3-12 has a growth-promoting effect on radish in the early stages of growth and development.

[0088] Experimental Example 6 Determination of the growth-promoting effect of Bacillus pumilus YM3-12 on cucumber in saline-alkali soil environment (1) Pretreatment of cucumber seeds.

[0089] (2) Prepare 9 cultivation pots and divide them into three groups: CK1 group (watered with sterile water), CK2 group (watered with sterilized LB liquid culture medium), and experimental group (watered with YM3-12 bacterial solution diluted with sterile water). Set up 3 replicates for each group.

[0090] The soil used was taken from the saline-alkali soil experimental field in Yanggu County, Liaocheng City, Shandong Province, with a salt content of 3.45-6 g / Kg and a pH of 8.5-9. The soil was mixed with vermiculite in a volume ratio of 2:1 before use.

[0091] Among them, the amount of sterile water applied in the CK1 group was 200 mL / Kg; the LB liquid culture medium applied in the CK2 group was diluted 100 times with sterile water, and the application amount was 200 mL / Kg; the application method of the Bacillus pumilus YM3-12 bacterial solution added to the experimental group was: the bacterial solution prepared in Example 2 was diluted 100 times with sterile water, and the application amount was 200 mL / Kg.

[0092] (3) Select cucumber seeds of uniform size after pretreatment in step (1) and plant them in cultivation pots. Thin out the cucumber seedlings after they emerge on the third day of cultivation so that there are two cucumber seedlings in each cultivation pot. Add bacteria after thinning. The plant height on the day of adding bacteria is taken as the plant height on day 0. The physiological plant height of cucumbers on days 0-8 and the growth status and total fresh weight of cucumber seedlings in different treatment groups at the time of harvesting on day 8 are recorded respectively.

[0093] When the seedlings were harvested on the 8th day, the cucumber pots and morphological photos of the different treatment groups were as follows: Fig.19 As shown by Fig.19 It can be seen that the cucumber seedlings in the experimental group grew more vigorously than those in the control group when harvested. The physiological plant height measurement results of cucumber on days 0-8 are as follows Fig. 20 As shown by Fig. 20 It can be seen that on day 0, the height difference of cucumber seedlings in the three groups was small, and the cucumber seedlings in the CK1 group were the tallest. Specifically, the physiological height of cucumber seedlings in the experimental group was 0.93% higher than that in the CK2 group and 1.36% lower than that in the CK1 group. After the bacterial treatment, the plant height of the cucumber seedlings in the experimental group on the 1st to 8th day increased by 4.86%, 14.63%, 22.50% (extremely significant difference, p<0.01), 28.78% (extremely significant difference, p<0.01), 26.37% (extremely significant difference, p<0.01), 24.18% (extremely significant difference, p<0.01), 25.43% (extremely significant difference, p<0.01), and 24.41% (extremely significant difference, p<0.01) compared with the plant height of the cucumber seedlings in the CK2 group; compared with CK1, it increased by 3.19%, 8.01%, 9.19%, 8.39%, 4.54%, 4.75%, 6.15%, and 7.78%, respectively.

[0094] The total fresh weight of cucumber plants at harvest time is as follows: Fig.21 As shown by Fig.21 It was found that the fresh weight of individual cucumber seedlings in the experimental group increased by 43.49% compared with CK2 (reaching a very significant difference, p<0.01), and increased by 7.69% compared with CK1.

[0095] The above data analysis showed that Bacillus pumilus YM3-12 has a growth-promoting effect on the early growth and development of cucumber.

Claims

1. A strain of Bacillus brevis ( Bacillus pumilus ) YM3-12, deposited on October 21, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 32281.

2. The Bacillus pumilus according to claim 1 ( Bacillus pumilus ) A method for culturing YM3-12, characterized in that: The steps include: Bacillus pumilus ( Bacillus pumilus ) YM3-12 was inoculated into LB solid medium and inverted at 35-37°C until a single colony grew out; a single colony was picked and inoculated into LB liquid medium, and cultured at 35-37°C, 180-200rpm until the logarithmic growth phase, and then inoculated into LB liquid medium at an inoculum volume fraction of 2-4%, and cultured at 35-37°C, 180-200rpm for 22-24h to obtain Bacillus pumilus ( Bacillus pumilus ) YM3-12 bacterial liquid.

3. The Bacillus pumilus according to claim 1 ( Bacillus pumilus ) Application of YM3-12 in at least one of the following: solubilizing organic phosphorus, solubilizing inorganic phosphorus, solubilizing potassium, degrading protein, degrading cellulose, and producing iron carriers.

4. The Bacillus pumilus according to claim 1 ( Bacillus pumilus ) Use of YM3-12 in antagonizing at least one of Erwinia persica and Fusarium oxysporum.

5. The Bacillus pumilus according to claim 1 ( Bacillus pumilus ) Application of YM3-12 in promoting plant growth in saline-alkali soil environment.

6. The use according to claim 5, characterized in that The plant is wheat, radish or cucumber.

7. The use according to claim 5, characterized in that The soil is saline-alkali soil with a salt content of ≤6g / Kg and a pH of ≤9.

8. A microbial agent, characterized in that: Containing the Bacillus pumilus according to claim 1 ( Bacillus pumilus )YM3-12.

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