A salt-tolerant Bacillus strain and its application

Abstract

The present invention relates to a salt-tolerant Bacillus and an application thereof, belonging to the technical field of microbial fertilizers. The salt-tolerant Bacillus was deposited in the General Microbiology Center of the China Culture Collection Administration on July 10, 2024, with a deposit number of CGMCC No. 31260. The strain has strong salt and alkali resistance, and has the ability to dissolve potassium, decompose cellulose, degrade protein, and dissolve organic phosphorus. At the same time, it can antagonize various plant pathogens such as Erwinia persica, Fusarium oxysporum, and Fusarium fujikura. Moreover, the strain can produce a good growth-promoting effect on corn in both salt-free and salt-containing environments, and has a very broad application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fertilizers, in particular to a salt-tolerant bacillus and application thereof. Background Art

[0002] Corn has relatively demanding growing conditions and is very sensitive to changes in soil, temperature, and moisture. Adding microbial agents to the soil can promote the growth of crops like corn in saline-alkali land, thereby increasing yields and repairing the soil ecosystem.

[0003] Microbial agents are products containing specific living microorganisms. These agents can modify the distribution of soil microbial communities and the soil's nutrient structure, thereby enabling crops to maintain normal growth, or even surpass normal growth conditions, in harsh environments such as saline-alkali stress. Plant growth-promoting rhizobacteria (PGPRs) are a commonly used group of strains in microbial agents. These include Bacillus spp., Pseudomonas spp., Klebsiella spp., and Azotobacter spp., among others. They can directly or indirectly promote plant growth through various mechanisms, including phosphate solubilization, potassium solubilization, nitrogen fixation, growth hormone production, and pathogen antagonism. However, PGPRs with different growth-promoting properties have varying effects on different crops. Therefore, the rational application of PGPRs plays a crucial role in promoting crop growth and improving and managing saline-alkali soils.

[0004] Bacillus halotolerans is an important PGPR. Recent studies have shown that halotolerans have broad application prospects in the improvement and management of salinized soils and the development of microbial agents. Specifically, they have biocontrol capabilities, such as salt-resistant growth promotion for some crops and the prevention and control of microbial diseases. Wu et al. isolated the halotolerans strain KKD1 from cocoa powder, which exhibits strong salt- and alkali-tolerant properties. It can grow under high salinity (13%) and high alkalinity (pH 10.0) conditions and exhibits excellent antagonistic effects against several plant pathogens. Genetic screening of the KKD1 strain revealed that osmotic balance, membrane transport, and ion homeostasis regulation under salt and alkali stress are key factors in its salt- and alkali-tolerant properties. Furthermore, the lipopeptides surfactin and fengycin produced by strain KKD1 are the main antifungal components. Etemadzadeh et al. isolated the halotolerans strain SCM034, which produces lipopeptide antibiotics that inhibit the growth of Staphylococcus aureus and Saccharomyces cerevisiae. Thomloudi et al. isolated the salt-tolerant Bacillus sp. Hil4 from the leaves of medicinal plants. This strain has antagonistic effects on gray mold and can promote plant growth. Zhang et al. reported the complete genome sequence of the salt-tolerant Bacillus sp. ZB201702. Whole-genome analysis revealed many potential gene clusters involved in defense mechanisms. Strain ZB201702 is tolerant to abiotic stresses and has the ability to resist drought and salt stress and promote plant growth. Some researchers isolated the salt-tolerant Bacillus sp. MS50-18A from saline soil, which can effectively prevent and control root rot and has excellent interactions with pepper crops. Analysis of the draft genome of this strain revealed that it has genes related to glycine / betaine uptake and bacteriocin biosynthesis, which can effectively resist salt stress environments. Jiménez-Gómez et al. verified that the salt-tolerant Bacillus SCCPVE07 can promote plant development under normal or salt stress environments. For example, after the strain SCCPVE07 was inoculated in the soil, not only did the potassium, iron, and magnesium content in the lettuce plants increase, but the content of protocatechuic acid and caffeic acid in the plants also increased significantly; after the strain SCCPVE07 was inoculated in the soil, the potassium, carbon, calcium, and iron content in the coriander plants increased, and the content of phenolic compounds in the plants also increased significantly.

[0005] Halophilous Bacillus not only has application value in stress-resistant growth promotion for some crops but also has potential in other areas. For example, it has broad application prospects in removing high-molecular-weight hydrocarbons from polluted environments. Deng et al. isolated Halophilous Bacillus 1-1 from crude oil-contaminated soil, which can efficiently degrade crude oil. Some Halophilous Bacillus strains have also been shown to exhibit high degradation of 1,2-benzophenone. At the end of 2021, Rayasam et al. discovered that Halophilous Bacillus DSM8802 can produce biodegradable polyhydroxyalkanoates under stress conditions. El-Fakharany et al. discovered that Halophilous Bacillus OHEM18 can produce asparaginase, which has clinical application value.

[0006] In summary, Halophilic Bacillus has multiple biological functions, especially its ability to promote growth in crops under salt and alkali conditions, making it a valuable PGPR. Currently, microbial agents derived from Halophilic Bacillus are lacking in their ability to promote growth in corn crops under salt conditions, and due to the complexity of the soil environment, some strains often fail to achieve a satisfactory growth-promoting effect. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides a salt-tolerant Bacillus and its application. The strain is isolated and screened from the seabed mud in the estuary area of Dongying City, Shandong Province, China. It has the ability to dissolve potassium, decompose cellulose, degrade protein and dissolve organic phosphorus. At the same time, it can antagonize a variety of plant pathogens and has a good growth-promoting effect on corn in both salt-free and salt-containing environments.

[0008] The technical solutions of the present invention are as follows:

[0009] A strain of Bacillus halotolerans DKY-9 was deposited on July 10, 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. 31260.

[0010] The 16S rDNA gene sequence of the halodurable Bacillus sp. DKY-9 is shown in SEQ ID NO.1.

[0011] The above-mentioned method for culturing the halotolerant Bacillus DKY-9 comprises the following steps:

[0012] The salt-tolerant Bacillus DKY-9 strain was inoculated into LB solid culture medium and inverted and cultured at 37° C. until a single colony grew; the single colony was picked and transferred to LB liquid culture medium, and shaken and cultured at 120-180 rpm and 37±1° C. to obtain a salt-tolerant Bacillus DKY-9 bacterial solution.

[0013] The salt-tolerant Bacillus DKY-9 is used in potassium solution, cellulose decomposition, protein degradation and organic phosphorus solution.

[0014] The salt-tolerant Bacillus DKY-9 is used in antagonizing Erwinia persica, Fusarium oxysporum and Fusarium fujikura.

[0015] The application of the halotolerant Bacillus DKY-9 in promoting corn growth.

[0016] Preferably, the halotolerant Bacillus DKY-9 is used to promote corn growth in a salt environment with a sodium chloride concentration of less than 100 mmol / L.

[0017] Preferably, the application of the halodurable Bacillus DKY-9 in promoting corn growth is performed by soaking corn seeds in a halodurable Bacillus DKY-9 bacterial solution in advance.

[0018] Further preferably, the concentration of the halodurable Bacillus DKY-9 bacterial solution is 10 5 ~10 9 cfu / L.

[0019] A live bacteria preparation with the halodurable Bacillus DKY-9 as an effective ingredient.

[0020] Beneficial effects of the present invention:

[0021] The salt-tolerant Bacillus DKY-9 provided by the present invention has strong salt and alkali resistance, and has the ability to dissolve potassium, decompose cellulose, degrade protein and dissolve organic phosphorus. At the same time, it can antagonize various plant pathogens such as Erwinia persica, Fusarium oxysporum, and Fusarium fujikura. Moreover, this strain can produce a good growth-promoting effect on corn in both salt-free and salty environments, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony morphology of strain DKY-9 and the bacterial body observation under a microscope after Gram staining;

[0023] Figure 2 The growth of strain DKY-9 on LB solid medium with different salinity;

[0024] Figure 3 The growth of strain DKY-9 on LB solid medium with different alkalinity;

[0025] Figure 4 The growth of strain DKY-9 in silicate bacterial culture medium;

[0026] Figure 5 The growth of strain DKY-9 in sodium carboxymethyl cellulose medium;

[0027] Figure 6 The growth of strain DKY-9 in casein medium;

[0028] Figure 7 The growth of strain DKY-9 in organophosphate bacterial culture medium;

[0029] Figure 8 The antagonistic effect of strain DKY-9 on Fusarium oxysporum;

[0030] Figure 9 The antagonistic effect of strain DKY-9 on Fusarium fujikuraensis;

[0031] Figure 10 The antagonistic effect of strain DKY-9 on Erwinia persica;

[0032] Figure 11 These are morphological photos of randomly selected corn plants from different treatment groups after 4 days of cultivation;

[0033] Figure 12 Data on plant height, root length and fresh weight of maize in different treatment groups after 4 days of cultivation;

[0034] Figure 13 These are morphological photos of randomly selected corn plants from different treatment groups after 8 days of cultivation;

[0035] Figure 14 The data of plant height, root length and fresh weight of corn in different treatment groups after 8 days of cultivation;

[0036] Figure 15 These are morphological photos of corn plants randomly selected from different treatment groups after 12 days of cultivation;

[0037] Figure 16 Data on plant height, root length and fresh weight of corn in different treatment groups after 12 days of cultivation. DETAILED DESCRIPTION

[0038] The following describes the process in conjunction with specific embodiments:

[0039] Source of experimental materials:

[0040] Bacterial microbiological identification tube: purchased from Haibo Biological Company;

[0041] Corn seeds: Zhengdan 958, purchased from Shandong Grain Industry Socialized Service Group Co., Ltd.

[0042] Erwinia persica: purchased from Shanghai Biotechnology Collection Center, catalog number SHBCC D10977;

[0043] Fusarium oxysporum: purchased from Shanghai Biotechnology Collection Center, catalog number SHBCC D25188;

[0044] Fusarium fujikura was purchased from Shanghai Biotechnology Collection Center with the catalog number SHBCC D19154.

[0045] Example 1:

[0046] Isolation, Screening and Mutagenesis of Halotolerant Bacillus DKY-9

[0047] The strains were isolated and screened from the seabed mud in the estuary area of Dongying City, Shandong Province, China, and the screened strains were continuously purified by the three-zone plate streaking method until a single colony was obtained.

[0048] The above single colony was selected as the starting strain and subjected to ARTP mutagenesis treatment. The specific mutagenesis steps are as follows:

[0049] (1) Preparation of starting strain suspension:

[0050] The starting strain was inoculated into LB liquid medium and cultured at 37°C and 180 rpm. When the bacteria reached the logarithmic phase, the culture solution was centrifuged, the bacterial pellet was washed with physiological saline and resuspended to obtain the OD value. 600 The starting strain bacterial suspension is 0.4.

[0051] (2) Mutagenesis process:

[0052] Heat the iron slide with an alcohol burner and let it cool in a sterile environment. Pipette 10 μL of the starting strain suspension prepared in step (1) and evenly spread it on the surface of the iron slide.

[0053] ARTP mutagenesis conditions: set the instrument output power to 120 W, the gas flow rate to 10 slm, and perform the treatment at a distance of 2 mm from the emission source;

[0054] The iron slide containing the bacterial suspension of the starting strain was transferred to the ARTP mutagenesis chamber, and the mutagenesis was carried out for 22 seconds under the above-mentioned ARTP mutagenesis conditions, and the mutagenesis was repeated twice. The mutagenized bacteria were then rinsed into an appropriate amount of physiological saline to obtain the mutagenized strain.

[0055] (3) Screening of mutagenic strains:

[0056] The strain induced in step (2) was screened for salt tolerance using LB solid culture medium containing different concentrations of NaCl, and then screened for alkali tolerance using LB solid culture medium at different pH values. Finally, a strain with strong salt and alkali tolerance was obtained and named "DKY-9".

[0057] The colony morphology of strain DKY-9 on LB solid medium and its observation under microscope after Gram staining are shown in the figure below. Figure 1 shown by Figure 1 As shown in Figure A, the colony morphology of strain DKY-9 is round, convex in the middle, with neat edges, and milky white and opaque. Figure 1 As shown in Figure B, the strain is a typical rod-shaped Gram-positive bacterium.

[0058] In addition, the physiological and biochemical characteristics of the screened strain DKY-9 were identified using bacterial microbiochemical identification tubes. The identification results are as follows: the strain DKY-9 can utilize raffinose, trehalose, xylose, sucrose, maltose and D-cellobiose; but cannot utilize lactose, galactose, glucose and rhamnose.

[0059] The 16S rDNA gene sequence of strain DKY-9 was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results are shown in SEQ ID NO. 1. The obtained 16S rDNA sequence was compared with existing sequences in the NCBI database by BLAST analysis, and strains with similar homology were selected. A phylogenetic tree was constructed using MEGA X software. The results showed that the strain DKY-9 obtained above was in a branch with Bacillus halotolerans, showing the highest homology. Combined with the physiological and biochemical characteristics of the strain, it was identified as Bacillus halotolerans.

[0060] Bacillus halotolerans DKY-9 was deposited on July 10, 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. 31260.

[0061] Example 2:

[0062] The specific method for culturing halotolerant Bacillus DKY-9 is as follows:

[0063] (1) Transfer the halodurable Bacillus subtilis DKY-9 strain into a test tube containing 5 mL of LB liquid medium and culture it in a shaker at 180 rpm and 37°C for 12 h to obtain the DKY-9 activated bacterial solution;

[0064] (2) The activated bacterial solution in step (1) was transferred to a conical flask containing 50 mL of LB liquid culture medium at a 1% inoculum volume (0.5 mL), and cultured at 180 rpm and 37°C for 24 h to obtain a halodurable Bacillus DKY-9 bacterial solution.

[0065] Experimental Example 1:

[0066] Determination of salt-alkali tolerance of halotolerant Bacillus sp. DKY-9

[0067] (1) Determination of salt tolerance of strain DKY-9: LB solid culture media with NaCl concentrations ranging from 0 to 10% were prepared, i.e., NaCl concentrations were 0 g / L (0%), 20 g / L (2%), 70 g / L (7%), 90 g / L (9%), and 100 g / L (10%), respectively. 100 μL of the halophilic Bacillus DKY-9 bacterial solution prepared in Example 2 was spread on the above LB solid culture media, and cultured in an inverted manner at 37° C. for 24 h. The growth of halophilic Bacillus DKY-9 on LB solid culture media with different salinities was observed.

[0068] The results are as follows Figure 2 As shown, Figures A, B, C, D, and E are the growth conditions of strain DKY-9 on LB solid medium with NaCl concentrations of 0%, 2%, 7%, 9%, and 10%, respectively; Figure 2 It can be seen that the halogen-tolerant Bacillus DKY-9 can grow in an environment with a salinity of 0-9%, but stops growing when the salinity increases to 10%, which shows that the halogen-tolerant Bacillus DKY-9 can tolerate a salt environment of up to 9% (90g / L).

[0069] (2) Determination of alkali resistance of strain DKY-9: LB solid culture medium with a pH of 7 to 12 was prepared respectively; 100 μL of the halogen-tolerant Bacillus DKY-9 bacterial solution prepared in Example 2 was spread on the above LB solid culture medium, and incubated inverted at 37°C for 24 h. The growth of halogen-tolerant Bacillus DKY-9 on LB solid culture medium with different alkalinity was observed.

[0070] The results are as follows Figure 3 As shown, Figures A, B, C, D, E, and F are the growth conditions of strain DKY-9 on LB solid medium with pH values of 7, 8, 9, 10, 11, and 12, respectively; Figure 3 It was found that the halodurable Bacillus DKY-9 could grow in an environment with a pH of 7 to 11, but stopped growing when the pH increased to 12, which indicated that the halodurable Bacillus DKY-9 could tolerate an alkaline environment with a maximum pH of 11.

[0071] Experimental Example 2:

[0072] Determination of Potassium-Solution, Cellulose-Decomposition, Protein-Degradation and Organophosphorus-Solution Capacities of Haloperidol Bacillus DKY-9

[0073] (1) The potassium-solubilizing ability of strain DKY-9 was determined using silicate bacterial culture medium. The specific method is as follows:

[0074] The strain DKY-9 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.

[0075] The components of the silicate bacterial culture medium are as follows: sucrose 5g / L, MgSO4 0.5g / L, CaCO3 0.1g / L, Na2HPO4 2g / L, FeCl3 0.005g / L, glass powder 1g / L, agar 15g / L, and pH 7.0.

[0076] Colony growth Figure 4 As shown by Figure 4 It was found that strain DKY-9 formed smooth and transparent oil-drop-shaped colonies in silicate bacterial culture medium, indicating that strain DKY-9 had the ability to solubilize potassium.

[0077] (2) The ability of strain DKY-9 to degrade cellulose was determined using sodium carboxymethylcellulose culture medium. The specific method is as follows:

[0078] The strain DKY-9 was inoculated onto sodium carboxymethyl cellulose medium using a sterilized toothpick and cultured at 37°C for 2 days. Then, a 1 mg / mL Congo red solution was added to submerge the strain DKY-9 for staining for 30 minutes. Finally, the stain was washed off with a 1 mol / L NaCl solution, and the colony growth was observed.

[0079] 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, and a pH of 7.2.

[0080] Colony growth Figure 5 As shown by Figure 5 It can be seen that in the sodium carboxymethyl cellulose culture medium, the strain DKY-9 showed a very obvious yellow degradation zone, which shows that the strain DKY-9 has a strong ability to degrade cellulose.

[0081] (3) The ability of strain DKY-9 to degrade protein was determined using casein culture medium. The specific method is as follows:

[0082] The strain DKY-9 was inoculated on casein medium using a sterilized toothpick and cultured at 37°C for 2 days to observe the growth of the colonies.

[0083] 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, K2HPO4 2.0 g / L, agar 15 g / L, bromothymol blue 0.05 g / L, and pH 7.3.

[0084] Colony growth Figure 6 As shown by Figure 6 It was found that in casein culture medium, strain DKY-9 showed a transparent degradation zone, which indicated that strain DKY-9 had the ability to degrade protein.

[0085] (4) The phosphate solubilization ability of strain DKY-9 was determined using an organophosphorus bacterial culture medium. The specific method is as follows:

[0086] The strain DKY-9 was inoculated onto the Montkina organophosphate bacterial culture medium using a sterilized toothpick and cultured at 30°C for 3 days to observe the growth of the colonies.

[0087] The components of the Montgina organophosphorus bacterial culture medium are as follows: glucose 10 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.3 g / L, MnSO4·4H2O 0.03 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, NaCl 0.3 g / L, CaCO3 5.0 g / L, lecithin 0.2 g / L, agar 15 g / L, and pH 7.0.

[0088] Colony growth Figure 7 As shown by Figure 7 It was found that a transparent degradation zone appeared around the strain DKY-9 in the organophosphorus bacteria culture medium, indicating that the strain DKY-9 had the ability to degrade organophosphorus.

[0089] Experimental Example 3:

[0090] Determination of the antagonistic ability of halotolerant Bacillus sp. DKY-9 against various plant pathogens

[0091] The antagonistic ability of strain DKY-9 against the cotton wilt pathogen Fusarium oxysporum, rice pathogen Fusarium fujikuroi, and garlic pathogen Erwinia were studied.

[0092] (1) The antagonistic fungal activity of strain DKY-9 was determined using the plate confrontation method. The specific steps were as follows: Fusarium oxysporum and Fusarium fujikura were inoculated on PDA solid culture medium, 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 punch and transferred to a new PDA solid culture medium with tweezers; a single colony of strain DKY-9 was picked 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, and the appearance of an inhibition zone was observed;

[0093] 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.

[0094] The results are as follows Figure 8 and Figure 9 As shown, Figure 8 The pathogen is Fusarium oxysporum. Figure 9 The pathogen in the case is Fusarium fujikura; Figures 8-9 It can be seen that obvious inhibition zones appeared around the strain DKY-9, which indicated that the strain DKY-9 had the ability to antagonize Fusarium oxysporum and Fusarium fujikura.

[0095] (2) The antagonistic bacterial ability of strain DKY-9 was determined by the plate spreading method. The specific steps were as follows: Erwinia persica stored in a glycerol tube was inoculated on LB solid medium and cultured at 37°C until a single colony grew; the single colony was inoculated into 5 mL of LB liquid medium and cultured at 37°C and 180 rpm for 10-12 h to obtain a Erwinia persica bacterial solution for later use;

[0096] Take 100 μL of the above-mentioned peach-colored Erwinia bacterial solution and evenly spread it on LB solid culture medium. After the bacterial solution is dry, use a sterilized inoculation loop to inoculate the strain DKY-9 onto the plate. Incubate it upside down at 37°C for 24 hours and observe whether there is an inhibition zone around the halodurable Bacillus DKY-9.

[0097] The results are as follows Figure 10 As shown by Figure 10 It can be seen that an obvious inhibition zone appeared around the strain DKY-9, which indicated that the strain DKY-9 had the ability to antagonize Erwinia persica.

[0098] Experimental Example 4:

[0099] The specific steps for determining the growth-promoting effect of halotolerant Bacillus DKY-9 on corn are as follows:

[0100] (1) Pretreatment of corn seeds: Select Zhengdan 958 corn seeds with full grains and uniform size, soak them in 75% (v / v) alcohol for 5 minutes, rinse them with sterile water 2 to 3 times to wash away the toxic substances on the surface until they are colorless, then soak them in 3% (v / v) NaClO solution with shaking for 10 minutes, and finally rinse them repeatedly with sterile water until they are colorless to complete the pretreatment; the pretreated corn seeds are placed in a sterile glass bottle and stored in a dark and moist place at room temperature for 20 hours for later use.

[0101] (2) Treatment of corn seeds with strain DKY-9: The pretreated corn seeds were divided into two groups, namely the sterile group and the bacterial group. The corn seeds in the bacterial group were 5 ~10 9cfu / L DKY-9 bacterial solution for 30 seconds, then immediately discard the bacterial solution; all corn seeds were rolled.

[0102] (3) Rolling seedlings: Under sterile conditions, soak the sterilized kraft paper with sterile water, wrap the corn seeds with the soaked kraft paper, place the corn radicle 3 cm away from the edge of the kraft paper, and use the root's geotropism to roll the seeds with the radicle downward and the plumule upward; each roll contains about 8 to 10 seeds, and secure them with a rope to prevent the seeds from falling off.

[0103] (4) Preparation of ordinary Hoagland nutrient solution: The components are as follows: 945 mg / L calcium nitrate tetrahydrate, 506 mg / L potassium nitrate, 80 mg / L ammonium nitrate, 136 mg / L potassium dihydrogen phosphate, 493 mg / L magnesium sulfate, 2.5 mL / L iron salt solution, 5 mL / L trace element solution, pH 6.0; wherein the components of the iron salt solution and trace element solution are as follows:

[0104] Iron salt solution: ferrous sulfate heptahydrate 5.56 g / L, disodium EDTA 7.46 g / L, pH 5.5;

[0105] Trace element solution: potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L.

[0106] Preparation of high-salt Hoagland's nutrient solution: NaCl with a final concentration of 100 mmol / L was added to the above-mentioned ordinary Hoagland's nutrient solution to simulate a high-salt environment.

[0107] (5) Place the rolled corn seeds in a sterile transparent glass bottle, and then add 200 mL of ordinary Hoagland's nutrient solution and high-salt Hoagland's nutrient solution to the sterile group and the bacterial group, respectively. That is, a total of four treatment groups are set up: salt-free sterile group, salt-free bacterial group, salt-free sterile group, and salt-free bacterial group; cover the bottle cap and place it in a light incubator for cultivation, and add ordinary Hoagland's nutrient solution / high-salt Hoagland's nutrient solution every 4 days;

[0108] The light incubator was set with two cycles: a light cycle of 25°C, 16 h, and a light intensity of 60%; and a dark cycle of 25°C, 8 h, and a light intensity of 0%.

[0109] (6) The growth morphology of corn was photographed on the 4th, 8th and 12th day of growth, and then the seedlings were harvested and the agronomic traits of corn such as plant height (maximum distance after straightening), root length and fresh weight were measured.

[0110] Morphological photos of corn plants randomly selected from different treatment groups after 4 days of cultivation are shown in the figure below. Figure 11 The plant height, root length and fresh weight data of corn in different treatment groups are shown in Figure 12 As shown. Figures 11-12 It can be seen that under salt or no-salt conditions, the plant height, root length and fresh weight of corn treated with DKY-9 bacterial solution showed an upward trend, specifically: the plant height, root length and fresh weight of corn in the no-salt and bacteria group increased by 20.10% (reaching a significant difference, p<0.05), 25.68% and 20.84% respectively compared with the no-salt and sterile group; the plant height, root length and fresh weight of corn in the salt and bacteria group increased by 24.04%, 19.07% and 7.87% respectively compared with the salt and sterile group.

[0111] Morphological photos of corn plants randomly selected from different treatment groups after 8 days of cultivation are shown in the figure below. Figure 13 The plant height, root length and fresh weight data of corn in different treatment groups are shown in Figure 14 As shown. Figures 13-14 Under salt-free conditions, plant height, root length, and fresh weight of corn treated with DKY-9 bacterial solution all showed an upward trend. Specifically, compared with the salt-free, sterile control, the plant height, root length, and fresh weight of corn in the salt-free, sterile control group increased by 39.25% (significant difference, p < 0.05), 19.60%, and 30.66%, respectively. Under salt-containing conditions, plant height and fresh weight of corn treated with DKY-9 bacterial solution increased, while root length was inhibited. Specifically, compared with the salt-free, sterile control group, the plant height and fresh weight of corn in the salt-containing, sterile control group increased by 18.10% and 11.89%, respectively, while root length decreased by 9.65%.

[0112] Morphological photos of corn plants randomly selected from different treatment groups after 12 days of cultivation are shown in the figure below. Figure 15 The plant height, root length and fresh weight data of corn in different treatment groups are shown in Figure 16 As shown. Figures 15-16 The results showed that under both salt-free and salt-containing conditions, corn plant height, root length, and fresh weight increased after treatment with DKY-9 bacterial solution. Specifically, under salt-free conditions, the plant height, root length, and fresh weight of corn in the bacterial group increased by 42.82% (extremely significant difference, p < 0.01), 31.42%, and 44.48% (significant difference, p < 0.05), respectively, compared to the sterile group. Under salt-containing conditions, the plant height, root length, and fresh weight of corn in the bacterial group increased by 36.82% (extremely significant difference, p < 0.01), 52.67%, and 41.43%, respectively, compared to the sterile group.

[0113] The above results show that the halotolerant Bacillus DKY-9 can produce a certain growth-promoting effect on corn under both salt-free and salt-containing conditions, and the growth-promoting effect on corn is more significant in the later stage of culture.

Claims

1. A strain of halotolerant Bacillus ( Bacillus halotolerans ) DKY-9, characterized in that, On July 10, 2024, it was deposited in the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 31260. The 16S rDNA gene sequence of the halodurable Bacillus sp. DKY-9 is shown in SEQ ID NO.

1.

2. The method for cultivating the halotolerant Bacillus DKY-9 according to claim 1, wherein The steps include: The halotolerant Bacillus DKY-9 strain was inoculated into LB solid medium and inverted and cultured at 37°C until a single colony grew; a single colony was picked and transferred to LB liquid medium, and shaken and cultured at 120-180 rpm and 37±1°C to obtain a halotolerant Bacillus DKY-9 bacterial solution.

3. The use of the halotolerant Bacillus DKY-9 according to claim 1, characterized in that It is used to dissolve potassium, decompose cellulose, degrade protein, and dissolve organic phosphorus; antagonize Erwinia persica, Fusarium oxysporum, and Fusarium fujikura; and promote corn growth in a salt environment with a sodium chloride concentration below 100 mmol / L.

4. The use according to claim 3, characterized in that The application method for promoting corn growth in a salt environment with a sodium chloride concentration of less than 100 mmol / L is as follows: soaking corn seeds in a salt-tolerant Bacillus DKY-9 bacterial solution in advance.

5. The use according to claim 4, characterized in that The concentration of the salt-tolerant Bacillus DKY-9 bacterial solution is 10 5 ~10 9 cfu / L.

6. A live bacteria preparation, characterized in that The salt-tolerant Bacillus DKY-9 according to claim 1 is used as the active ingredient.

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