Salt-tolerant bacillus and application thereof

Abstract

The invention relates to a salt-tolerant bacillus and application thereof, and belongs to the technical field of microbial fertilizers, the salt-tolerant bacillus is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 10, 2024, and the preservation number is CGMCC No.31260; the strain is relatively strong in saline-alkaline tolerance, has the capabilities of dissolving potassium, decomposing cellulose, degrading protein and dissolving organophosphorus, can antagonize various plant pathogenic bacteria such as Erwinia persicae, fusarium oxysporum and fusarium fusiforme, can generate a good growth promoting effect on corn in a salt-free or salt-containing environment, and is very wide in application prospect.

Description

Technical Field

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

[0002] As an important food and feed crop in my country, corn plays an important role in agricultural production. Corn is planted in a large area and has a high yield in my country, but there is still a large gap between supply and demand. The growing conditions of corn are relatively harsh, and it is very sensitive to changes in soil, temperature, water and other conditions. There are a large number of saline-alkali lands in the arid, semi-arid and coastal areas in northern my country. The soil environment suitable for corn growth is decreasing. Corn has poor growth ability on saline-alkali land and low yield. By adding microbial agents to the soil, it is possible to promote the growth of crops such as corn in saline-alkali land and increase yield, and it can also repair the soil ecological environment of saline-alkali land.

[0003] Microbial agents are products containing specific living microorganisms. Through the microorganisms contained in them, they can improve the distribution of microbial communities and soil nutrient structure in the soil to a certain extent, so that crops can maintain normal growth or even better than normal growth conditions in harsh environments such as saline-alkali stress. Plant growth-promoting rhizobacteria (PGPR) are a commonly used strain group in microbial agents, including Bacillus spp., Pseudomonas spp., Klebsiella spp., Azotobacter spp., etc. They can directly or indirectly promote plant growth through various mechanisms such as dissolving phosphorus, potassium, fixing nitrogen, producing growth hormones, and antagonizing pathogens. However, PGPR with different growth-promoting characteristics has different growth-promoting effects on different crops. The rational application of PGPR plays an extremely important role in promoting crop growth and improving and managing saline-alkali land.

[0004] Bacillus halotolerans is an important type of PGPR. In recent years, studies have found that Bacillus halotolerans has broad application prospects in the improvement and treatment of salinized soils and the development of microbial agents. Specifically, it has biocontrol functions such as salt resistance and growth promotion for some crops and prevention and control of microbial diseases. The halotolerans KKD1 ​​isolated from cocoa powder by Wu et al. has strong salt-alkali resistance, can grow under high salinity (13%) and high alkalinity (pH 10.0), and has good antagonistic effects on some plant pathogens; through genetic screening of the strain KKD1 ​​gene, it was found that osmotic balance, membrane transport and ion balance regulation under salt and alkali stress are important factors for its salt-alkali resistance; in addition, the lipopeptides surfactin and fengycin produced by strain KKD1 ​​are the main antifungal components. Etemadzadeh et al. isolated a halotolerans SCM034, which can produce some lipopeptide antibiotics to inhibit the growth of Staphylococcus aureus and Saccharomyces cerevisiae. Thomloudi et al. isolated the salt-tolerant Bacillus Hil4 from the leaves of medicinal plants. The strain has antagonistic ability to gray mold and can promote plant growth. Zhang et al. reported the whole genome sequence of the salt-tolerant Bacillus ZB201702. Through whole genome analysis, many potential gene clusters involved in defense mechanisms were revealed. The strain ZB201702 is tolerant to abiotic stresses and has the ability to resist drought, salt stress and promote plant growth. Some researchers isolated the salt-tolerant Bacillus MS50-18A from saline soil, which can effectively prevent and control root rot and has an excellent interaction with pepper crops. Analysis of the genome draft of the strain showed 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 halotolerant 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] Halophilic Bacillus not only has application value in the stress resistance and growth promotion of some crops, but also has application potential in other aspects. For example, halophilic Bacillus has broad application prospects in removing high molecular weight hydrocarbons from polluted environments: the halophilic Bacillus 1-1 isolated from crude oil contaminated soil by Deng et al. can efficiently degrade crude oil; some halophilic Bacillus have been shown to exhibit high degradation ability for 1,2-benzophenone. At the end of 2021, Rayasam et al. found that halophilic Bacillus DSM8802 can produce biodegradable polyhydroxyalkanoates under stress conditions. El-Fakharany et al. found that halophilic Bacillus OHEM18 can produce asparaginase with clinical application value.

[0006] In summary, halotolerant Bacillus has multiple biological functions, especially the ability to promote the growth of crops in salt-alkali environments, making it a PGPR with important application value. At present, microbial agents made from halotolerant Bacillus are lacking in the salt-resistant growth-promoting effect on corn crops, and due to the complexity of the soil environment, some strains often cannot exert a good growth-promoting effect. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a salt-tolerant Bacillus and its application. The strain is isolated and screened from the sea bottom 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. It can also antagonize a variety of plant pathogens and have a good growth-promoting effect on corn in a salt-free or salty environment.

[0008] The technical solution of the present invention is 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 halotolerant Bacillus 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 at 37° C. until a single colony grew out; a single colony was picked up and placed into 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 halotolerant Bacillus DKY-9 in promoting corn growth is performed by soaking corn seeds in a bacterial solution of the halotolerant Bacillus DKY-9 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 halotolerant 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-alkali resistance, has the ability to dissolve potassium, decompose cellulose, degrade protein and dissolve organic phosphorus, and 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 a salt-free or salty environment, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[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 organophosphorus bacteria culture medium;

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

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

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

[0032] Fig.11 These are randomly selected photos of maize plants from different treatment groups after 4 days of cultivation;

[0033] Fig.12 The data of plant height, root length and fresh weight of corn in different treatment groups after 4 days of cultivation;

[0034] Fig.13 These are randomly selected photos of maize plants from different treatment groups after 8 days of cultivation;

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

[0036] Fig.15 These are randomly selected photos of maize plants from different treatment groups after 12 days of cultivation;

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

[0038] The following is explained in conjunction with specific embodiments:

[0039] Source of experimental materials:

[0040] Bacterial microbiochemical 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 Collection Biotechnology Center, catalog number SHBCC D10977;

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

[0044] Fusarium fujikura: purchased from Shanghai Collection Biotechnology Center, catalog number SHBCC D19154.

[0045] Embodiment 1:

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

[0047] The strains were isolated and screened from the sea sediments 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 were 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 grew to the logarithmic phase, the culture solution was centrifuged, the bacterial precipitate was washed with physiological saline and resuspended to obtain the OD 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. Take 10 μL of the starting strain suspension prepared in step (1) and evenly apply 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 ARTP mutagenesis conditions, and the mutagenesis was repeated twice, and then the mutagenized bacteria were 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 resistance using LB solid culture medium containing different concentrations of NaCl, and then screened for alkali resistance using LB solid culture medium of different pH values. Finally, a strain with strong salt and alkali resistance was obtained and named "DKY-9".

[0057] The colony morphology of strain DKY-9 on LB solid medium and its observation under a 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, neat at the edges, 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 does not utilize lactose, galactose, glucose and rhamnose.

[0059] The 16S rDNA gene sequence of strain DKY-9 was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing result is 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, and a phylogenetic tree was constructed using MEGA X software. The results showed that the strain DKY-9 obtained by the above screening was in a branch with Bacillus halotolerans, with 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 No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.31260.

[0061] Embodiment 2:

[0062] The specific method for cultivating the salt-tolerant Bacillus DKY-9 is as follows:

[0063] (1) The halodurable Bacillus DKY-9 strain was transferred into a test tube containing 5 mL of LB liquid medium, and the tube was placed in a shaker at 180 rpm and 37° C. for 12 h to obtain a 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 bacterial solution of halotolerant Bacillus DKY-9.

[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 of 0 to 10% were prepared, i.e., the 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 salt-tolerant Bacillus DKY-9 bacterial solution prepared in Example 2 was spread on the above LB solid culture media, inverted and cultured at 37° C. for 24 h, and the growth of salt-tolerant Bacillus DKY-9 on LB solid culture media of 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 halotolerant Bacillus DKY-9 can grow in an environment with a salinity of 0-9%, but stops growing when the salinity increases to 10%, which means that halotolerant Bacillus DKY-9 can tolerate a salt environment of up to 9% (90 g / 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 bacterial solution of the halotolerant Bacillus DKY-9 prepared in Example 2 was spread on the above LB solid culture medium, and incubated at 37° C. for 24 h to observe the growth of the halotolerant Bacillus DKY-9 on LB solid culture medium with different alkalinity.

[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 can be seen that halotolerant Bacillus DKY-9 can grow in an environment with a pH of 7 to 11, but stops growing when the pH rises to 12, which means that halotolerant Bacillus DKY-9 can tolerate an alkaline environment with a maximum pH of 11.

[0071] Experimental Example 2:

[0072] Determination of the Potassium-Solution, Cellulose-Degradation, Protein-Degradation and Organic Phosphorus-Solution Capacities of Halophilic Bacillus DKY-9

[0073] (1) The potassium-solubilizing ability of strain DKY-9 was determined by 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, pH 7.0.

[0076] Colony growth Figure 4 As shown by Figure 4 It can be seen that strain DKY-9 is a smooth and transparent oil droplet-shaped colony in silicate bacterial culture medium, indicating that strain DKY-9 has 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 on sodium carboxymethyl cellulose medium with 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 pH 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 with 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, pH 7.3.

[0084] Colony growth Figure 6 As shown by Figure 6 It can be seen that in the casein culture medium, a transparent degradation zone appeared in the strain DKY-9, which indicates that the strain DKY-9 has the ability to degrade protein.

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

[0086] The strain DKY-9 was inoculated on the Montkina organophosphate bacterial medium with 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 bacteria 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 strain DKY-9 in the culture medium of organophosphorus bacteria, indicating that strain DKY-9 has the ability to degrade organophosphorus.

[0089] Experimental Example 3:

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

[0091] The pathogenic fungus Fusarium oxysporum of cotton wilt, the pathogenic fungus Fusarium fujikuroi of rice pathogenic bacteria, and the pathogenic bacteria Erwinia peache (PeachErwinia) of garlic pathogenic bacteria were selected to study the antagonistic ability of strain DKY-9 against the above pathogenic bacteria.

[0092] (1) The antagonistic fungal ability of strain DKY-9 was determined by the plate confrontation method. The specific steps are as follows: Fusarium oxysporum and Fusarium fujikura were inoculated on PDA solid culture medium respectively, and cultured at 28°C for 4-5 days until the culture medium was full of round cake-shaped hyphae; the hyphae were punched out with a sterile puncher and transferred to a new PDA solid culture medium with tweezers; a single colony of strain DKY-9 was picked up with a sterile toothpick and inoculated on the PDA solid culture medium 2 cm away from the edge of the hyphae; 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 Fig. 9 As shown, Figure 8 The pathogen is Fusarium oxysporum. Fig. 9 The pathogen in the case is Fusarium fujikura; Figures 8-9 It can be seen that obvious inhibition zones appeared around strain DKY-9, which indicates that strain DKY-9 has the ability to antagonize Fusarium oxysporum and Fusarium fujikura.

[0095] (2) The antagonistic bacterial ability of strain DKY-9 was determined by a plate spreading method, and the specific steps were as follows: the Erwinia persica stored in a 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 to 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 the LB solid culture medium. After the bacterial solution is dry, use a sterilized inoculation loop to inoculate the strain DKY-9 onto the plate, invert and culture at 37°C for 24 hours, and observe whether an inhibition zone appears around the halotolerant Bacillus DKY-9.

[0097] The results are as follows Fig.10 As shown by Fig.10 It can be seen that an obvious inhibition zone appeared around the strain DKY-9, which indicates that the strain DKY-9 has 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; place the pretreated corn seeds in a sterile glass bottle at room temperature, away from light and kept in a moist place 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, a sterile group and a bacterial group. The corn seeds in the bacterial group were 5 ~10 9cfu / L DKY-9 bacterial solution for 30s, then immediately pour out the bacterial solution; roll up all corn seeds.

[0102] (3) Rolling seedlings: Under sterile conditions, soak the sterilized kraft paper with sterile water, wrap the corn seeds with the soaked kraft paper, and place the corn radicle 3 cm away from the edge of the kraft paper. Using the geotropism of the roots, turn the radicle of the seeds downward and the plumule upward; roll about 8 to 10 seeds per roll and secure with ropes 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 the trace element solution are as follows:

[0104] Iron salt solution: ferrous sulfate heptahydrate 5.56 g / L, disodium ethylenediaminetetraacetate 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: Add NaCl with a final concentration of 100 mmol / L to the above-mentioned ordinary Hoagland's nutrient solution to simulate a high-salt environment.

[0107] (5) The rolled corn seeds were placed in a sterile transparent glass bottle, and then 200 mL of ordinary Hoagland's nutrient solution and high-salt Hoagland's nutrient solution were added to the sterile group and the bacterial group, respectively, that is, a total of four treatment groups were set up: a salt-free sterile group, a salt-free bacterial group, a salt-free sterile group, and a salt-free bacterial group; the bottles were capped and placed in a light incubator for culture, and ordinary Hoagland's nutrient solution / high-salt Hoagland's nutrient solution were added every 4 days;

[0108] The light incubator was set with two cycles: the light cycle was 25°C, 16h, and the light intensity was 60%; the dark cycle was 25°C, 8h, and the light intensity was 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 randomly selected corn plants in different treatment groups after 4 days of cultivation Fig.11 The plant height, root length and fresh weight data of corn in different treatment groups are shown in Fig.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] The morphological photos of corn plants randomly selected from different treatment groups after 8 days of cultivation are shown in Figure 2. Fig.13 The plant height, root length and fresh weight data of corn in different treatment groups are shown in Fig.14 As shown. Figures 13-14 It can be seen that under salt-free conditions, the plant height, root length and fresh weight of corn treated with DKY-9 bacterial solution showed an upward trend, which was specifically manifested as follows: compared with the salt-free sterile group, the plant height, root length and fresh weight of corn in the salt-free bacterial group increased by 39.25% (reaching a significant difference, p<0.05), 19.60% and 30.66%, respectively. Under salt conditions, the plant height and fresh weight of corn treated with DKY-9 bacterial solution increased, while the root length was inhibited, which was specifically manifested as follows: compared with the salt-free sterile group, the plant height and fresh weight of corn in the salt-containing bacterial group increased by 18.10% and 11.89%, respectively, and the root length decreased by 9.65%.

[0112] The morphological photos of corn plants randomly selected from different treatment groups after 12 days of cultivation are shown in Figure 2. Fig.15 The plant height, root length and fresh weight data of corn in different treatment groups are shown in Fig.16 As shown. Figures 15-16 It can be seen that under salt-free and salt-containing conditions, the plant height, root length and fresh weight of corn treated with DKY-9 bacterial solution were increased. Specifically, under salt-free conditions, the plant height, root length and fresh weight of corn in the bacterial group increased by 42.82% (reaching an extremely significant difference, p<0.01), 31.42% and 44.48% (reaching a significant difference, p<0.05) respectively compared with the sterile group; under salt conditions, the plant height, root length and fresh weight of corn in the bacterial group increased by 36.82% (reaching an extremely significant difference, p<0.01), 52.67% and 41.43% respectively compared with the sterile group.

[0113] The above results show that 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 cultivation.

Claims

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

1.

2. The method for cultivating the halotolerant Bacillus DKY-9 according to claim 1, characterized in that: The steps include: The salt-tolerant Bacillus DKY-9 strain was inoculated into LB solid culture medium and inverted at 37° C. until a single colony grew out; a single colony was picked up and placed into 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.

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.

4. The use of the halotolerant Bacillus DKY-9 according to claim 1, characterized in that: Used to antagonize Erwinia persica, Fusarium oxysporum, and Fusarium fujikura.

5. The use of the halotolerant Bacillus DKY-9 according to claim 1, characterized in that: Used to promote corn growth.

6. The use according to claim 5, characterized in that Promote corn growth in a salt environment with a sodium chloride concentration below 100 mmol / L.

7. The use according to claim 5, characterized in that The application method is to soak the corn seeds in the bacterial solution of halotolerant Bacillus DKY-9 in advance.

8. The use according to claim 7, characterized in that The concentration of the salt-tolerant Bacillus DKY-9 bacterial solution is 10 5 ~10 9 cfu / L.

9. A live bacteria preparation, characterized in that: The salt-tolerant Bacillus DKY-9 described in claim 1 is used as an active ingredient.

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