Ocean spore bacillus HMF13 and application thereof

Abstract

This invention relates to a type of Bacillus aquatilis (Bacillus cereus) Oceanobacillus sp. HMF13, with accession number CGMCC No. 30479. This invention also provides microbial inoculants containing the above-mentioned strain and their applications. The strain HMF13 of this invention has high colonization ability, high salt and alkali tolerance, and can promote plant growth. Furthermore, the strain HMF13 of this invention also has the effect of controlling soft rot disease in Chinese cabbage.

Description

TECHNICAL FIELD

[0001] The present application relates to an oceanic bacillus HMF13 and its application. BACKGROUND

[0002] Soil, as a valuable natural resource, provides a material basis for human survival and is the earliest means of production used by humans. In many regions of China, especially in the northern and coastal regions, saline-alkali soils are widely distributed. Because the saline-alkali soil contains a large amount of soluble salt, planting crops on saline-alkali soil will affect the growth and development of crops, so the yield of crops planted on saline-alkali soil is very low, or even zero. Therefore, applying new technologies to alleviate the harm of soil salinity to ensure food security has become a hot issue of concern in the world today.

[0003] At present, the main technical means for the management and development of saline-alkali soil includes physical management and chemical management. Physical management mainly focuses on building a large number of water conservancy facilities to remove a large amount of salt in the soil with water, but this technical means has the disadvantages of large engineering quantity, high capital investment cost, and unsustainability. Chemical management is to add new chemical substances to the saline-alkali soil to achieve the purpose of managing the saline-alkali soil, which has the advantage of quick effect but is easy to introduce a large amount of new chemical substances into the environment, causing secondary pollution.

[0004] There are also a large number of beneficial microorganisms in saline-alkali soil, which can effectively protect halophytes from the adverse effects of salt-alkali, drought, disease and other biological and abiotic stresses. Some salt-tolerant growth-promoting bacteria not only improve plant yield but also improve the surrounding soil properties. Therefore, isolating and applying plant rhizosphere beneficial microorganisms to enhance the salt-tolerance of plants, promote plant growth, and improve soil physical and chemical properties should be a low-cost, eco-friendly technology to improve the productivity of saline-alkali soil.

[0005] However, there are still some problems with salt-tolerant growth-promoting bacteria, such as few strains, weak salt-tolerance and poor soil colonization ability. Therefore, screening and researching salt-tolerant microorganisms that can be applied to saline-alkali soil improvement is a problem that needs to be solved by those skilled in the art.

[0006] In recent years, with the continuous expansion of Chinese cabbage cultivation area and the increase of continuous cropping period, Chinese cabbage soft rot has developed more seriously, which has seriously affected the production of Chinese cabbage. In some years in the northern region, the disease caused more than 50% reduction in yield of Chinese cabbage, and even zero yield in some plots. Chinese cabbage soft rot is a serious bacterial disease, and the main pathogen is Pectobacterium carotovorum subsp. carotovorum (Burkholder) Hauben et al. Erwinia carotovora subsp .carotovoraCurrently, the main methods for controlling this disease are a combination of breeding disease-resistant varieties and chemical pesticides, but the results are not ideal, and long-term use of chemical pesticides can easily lead to environmental pollution and pesticide residues. Summary of the Invention

[0007] The purpose of this invention is to provide a Bacillus macrocephala HMF13 with high colonization ability, high salt and alkali tolerance, and the ability to promote plant growth, as well as its application. The provided Bacillus macrocephala HMF13 can be used for the biological control of soft rot disease in Chinese cabbage, especially for the biological control of soft rot disease in Chinese cabbage cultivated in saline-alkali soil.

[0008] The present invention adopts the following technical solution:

[0009] A type of oceanic Bacillus ( Oceanobacillus sp HMF13 was deposited on April 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30479.

[0010] Furthermore, the *Bacillus oceanus* HMF13 is resistant to salt and alkali.

[0011] Furthermore, the Bacillus oceanicus HMF13 can improve the germination rate of wheat seeds under salt stress.

[0012] Furthermore, the Bacillus oceanicus HMF13 has a growth-promoting effect on wheat under saline-alkali conditions.

[0013] Furthermore, the Bacillus oceanicus HMF13 can colonize efficiently in saline-alkali environments.

[0014] Furthermore, the *Bacillus oceanicus* HMF13 can antagonize *Erwinia carotenoides* subsp. *carotenoides*.

[0015] A microbial inoculant comprising the cells, spores, fermentation broth, and / or extracellular metabolites of the aforementioned Bacillus oceanus HMF13.

[0016] Furthermore, the viable count of Bacillus oceanicus HMF13 in the microbial agent is not less than 10. 10 CFU / g.

[0017] Furthermore, the microbial agent also includes diatomaceous earth, vermiculite, activated carbon, or biochar as a carrier.

[0018] Application of the above-mentioned Bacillus macrocephala HMF13 in the control of soft rot disease in Chinese cabbage.

[0019] The application of the above-mentioned bacillus oceanised HMF13 in improving the seed germination rate of plants under salt stress environment.

[0020] The application of the above-mentioned bacillus oceanised HMF13 in promoting the growth of wheat in saline-alkali soil.

[0021] The bacillus oceanised HMF13 of the present application is collected from saline-alkali land, has strong salt-alkali tolerance and high colonization ability, and the microbial agent prepared by using the strain is suitable for saline-alkali land improvement and can promote plant growth and seed germination. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is the colony morphology of the bacillus oceanised HMF13 cultured in Gibbson improved medium for 24 h.

[0023] Figure 2 It is the morphology of the bacillus oceanised HMF13 under optical microscope after Gram staining.

[0024] Figure 3 It is the phylogenetic tree of the bacillus oceanised HMF13 constructed based on 16S rDNA.

[0025] Figure 4 It is the result of the confrontation test of the bacillus oceanised HMF13 and the erwinia carotovora subsp. carotovora in Gibbson improved medium plate. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. It should be noted that the protection scope of the present application is not limited to these embodiments. Any changes or equivalent replacements without departing from the concept of the present application are included in the protection scope of the present application.

[0027] Example 1: Isolation and purification of the strain

[0028] The rhizosphere soil of reed, a halophyte, in saline-alkali land in Gaopo village, Liutaizhen, Changli county, Qinhuangdao city, Hebei province, was selected, the rhizosphere soil was shaken gently, and 6 soil samples were collected, each of which was 100 g. Then, 10 g of the mixed soil sample was taken, 90 mL of sterile water was added, and the mixture was shaken at 180 r·min-1 for 30 min on a shaking table. After that, the mixture was gradiently diluted with 9 mL of sterile water, and 10 -1 , 10 -5 , 10 -6 , 10-7 Diluted liquid 0.1 mL was coated on Gibbson modified medium plates and placed in a 37°C incubator for 5 days. Obvious colony morphology differences were picked and purified by plate streaking method. Single colonies were selected for standby. A total of 9 strains of single colonies were purified and numbered HMCY1, HMCY2, HMCY3, HMCY4, HMCY5, HMCY6, HMCY7, HMCY8, and HMCY9.

[0029] Gibbson modified medium: casein 5.0 g, sodium citrate 3.0 g, yeast extract powder 10.0 g, KCl 2.0 g, proteose peptone 5.0 g, MgSO4·7H2O 2.0 g, NaCl 100 g, pH 9.0, agar 2%, 1000 mL water.

[0030] While maintaining the pH of the Gibbson modified medium at 9, the salinity of the medium was increased by 2% respectively. The NaCl concentration in the medium was 100 g·L -1 , 120 g·L -1 , 140 g·L -1 , 160 g·L -1 , 180 g·L -1 , and 200 g·L -1 , respectively. A plurality of media with different salinities were prepared. The single colonies of the 9 strains of bacteria purified as described above were inoculated from the medium streaks onto each of the Gibbson modified medium plates with changed salinity, and placed in a 37°C constant temperature incubator for 5 days. Whether the strains grew out was used as a standard for judging whether the strains were resistant to the corresponding salinity. Finally, 2 strains with strong salt tolerance, HMCY1 and HMCY5, were screened, both of which could grow in a medium with pH=9 and a salt concentration of 200 g·L -1 .

[0031] While maintaining the NaCl concentration of the Gibbson modified medium at 100 g·L -1 , the pH of the medium was increased by 1 unit respectively. The pH of the medium was 9, 10, 11, and 12, respectively. A plurality of media with different pH values were prepared. The single colonies of the 9 strains of bacteria purified as described above were inoculated from the medium streaks onto each of the Gibbson modified medium plates with changed pH, and placed in a 37°C constant temperature incubator for 5 days. Whether the strains grew out was used as a standard for judging whether the strains were resistant to the corresponding alkalinity. Finally, 2 strains with strong alkali tolerance, HMCY4 and HMCY5, were screened, both of which could grow in a medium with pH=12 and a salt concentration of 100 g·L -1 .

[0032] HMCY1, HMCY4, HMCY5 were put into Gibbson modified medium with increasing salt concentration and pH, and it was found that strain HMCY5 could still grow in Gibbson modified medium with pH 12 and salt concentration 200 g·L -1 -1, and was named HMF13.

[0033] Example 2 Identification of strain HMF13

[0034] (1) Microbiological characteristics

[0035] As shown in Figure 1 , colony morphology observation found that strain HMF13 showed round shape, small colony, milk yellow color, rough surface and irregular edge on Gibbson modified medium. Microscopic observation (as shown in Figure 2 ) showed that the cell was rod-shaped, 1-2 μm x (0.5-1 μm) in size, aggregated in short chains or bead-like arrangement, spores were oval, mesosporous, and gram-positive. According to the "Berger's Bacterial Identification Manual", the strain was preliminarily judged to belong to Bacillus.

[0036] (2) Physiological and biochemical characteristics

[0037] The partial physiological and biochemical characteristics of strain HMF13 are shown in Table 1.

[0038] Table 1 Partial physiological and biochemical characteristics of strain HMF13

[0039] .

[0040] (3) Molecular biological characteristics

[0041] The genomic DNA of strain HMF13 was extracted, and used as a template for PCR amplification with bacterial 16S rDNA universal primers. The amplified product was recovered and sequenced to obtain a DNA sequence containing 1500 bp as shown in SEQ ID No. 1. The sequencing results were input into GeneBank database for BLAST comparison analysis, and the 16S rDNA sequence was compared in NCBI database, and the identification result at the molecular level showed that the strain belonged to Ocean Bacillus, and the phylogenetic tree result is shown in Figure 3 . According to the morphological, sequencing analysis and physiological and biochemical results, it was identified as Ocean Bacillus ( Oceanobacillus sp ).

[0042] Oceanospirillum sp. HMF13 was preserved on April 30, 2024 at the China General Microbiological Culture Collection Center, located at No. 1, Yikhina Street, No. 3, Chaoyang District, Beijing, China, and managed by the Institute of Microbiology of the Chinese Academy of Sciences, with a postal code of 100101, and a preservation number of CGMCC No. 30479.

[0043] Example 3: Pot experiment to test the colonization ability of Oceanospirillum sp.

[0044] Four bacterial strains Hh-01, Hh-03, Hh-04, and Hh-06, which can well colonize in saline-alkali soil (soil salt content of 0.3%), preserved in the applicant's laboratory, were compared with HMF13. The colonization ability of Oceanospirillum sp. HMF13 was evaluated through a pot experiment test.

[0045] (1) The soil used in the pot experiment was taken from the test base set up by the applicant in Huanghua, which was saline-alkali soil. After air-drying, it was sterilized at high temperature to make sterile soil, and the salt content was tested to be 0.7%.

[0046] (2) The bacterial strains Hh-01, Hh-03, Hh-04, Hh-06, and HMF13 were inoculated into LB liquid medium, respectively, at a temperature of 30°C and a rotation speed of 160 r·min -1 -1, and cultured for 48 h to obtain fermentation broth. Then the fermentation broth was prepared into a bacterial suspension with a bacterial content of 1.0×10 9 CFU / mL.

[0047] (3) The sterile soil and the solid fermentation medium were uniformly mixed at a ratio of 4:1, and were filled into flowerpots with a diameter of 10 cm and a height of 11 cm to a near-full degree.

[0048] (4) 200 mL of the prepared bacterial suspension was irrigated, the flowerpots were placed in a tray filled with water, and the water was absorbed through the holes in the bottom of the flowerpots to moisten the surface soil. The water in the tray was removed, and the flowerpots were placed in a greenhouse with a temperature of 20-28°C. After 15 days, 1 g of soil was sampled from 6 cm to detect the number of viable bacteria, with 5 repetitions for each strain. Water was irrigated every 4 days during this period.

[0049] The solid fermentation medium (mass ratio): bran: chaff: 3% glucose water = 7:3:3, urea 2.16%, potassium dihydrogen phosphate 2.77%, uniformly mixed, bagged, 121°C high-pressure wet heat sterilization for 60 min, intermittent sterilization for 2 times.

[0050] The colonization number of each strain in each gram of soil was detected, and the colonization rate was calculated. As shown in Table 2.

[0051] Colonization rate (%) = colonization number ÷ inoculation number × 100.

[0052] Table 2: Colonization rate detection results of strains

[0053] .

[0054] As shown in Table 2, the ocean Bacillus HMF13 has excellent colonization ability in saline-alkali soil, indicating that the strain can be well colonized and grown in saline-alkali soil.

[0055] Example 4 Promoting growth test of ocean Bacillus HMF13 on wheat germination

[0056] The strain HMF13 was inoculated in LB liquid medium, the temperature was 30℃, the rotation speed was 160r / min, and the culture was carried out for 48h to obtain the fermentation liquor, and then the fermentation liquor was prepared into a bacterial suspension with a bacterial content of 1.1×10 9 CFU / mL.

[0057] The above prepared bacterial suspension and sterile water were respectively prepared into 7 concentrations according to the volume ratio of 1:0, 1:10, 1:20, 1:30, 1:40, 1:50, 0:1, which were respectively recorded as T0, T1, T2, T3, T4, T5, CK, a total of 7 treatments. The wheat seeds (dry alkali wheat variety Jetmace 19) were sterilized with 75% ethanol for 30s, and the seeds were washed with sterile water for 3 times; then the seeds were sterilized with 1% sodium hypochlorite for 10~15min, and then washed with sterile water for 4 times, and then the wheat seeds were soaked with the bacterial suspension with different concentrations for 4~6h. Two layers of filter paper were placed on the culture dish, 15mL sterile water was added to wet, and then placed in a 24℃ incubator, and the seed germination rate was measured after 24h, and the seed germination rate was also measured after 3d, 5d, 7d.

[0058] Table 3 Effect of ocean Bacillus HMF13 fermentation dilution on wheat seed germination rate

[0059] .

[0060] As shown in Table 3, the ocean Bacillus HMF13 fermentation dilution has a certain promoting effect on the wheat seed germination. In the case of no dilution, i.e. T0 treatment, the wheat seeds soaked in the fermentation liquor had a germination rate of 95.0% after 7d, which was increased by 10% compared with the control CK. With the expansion of the dilution ratio of the fermentation liquor, the wheat seed germination rate showed different changes, among which T1 and T2 were increased by 12% and 11% compared with the control CK.

[0061] Example 5 Promoting growth test of ocean Bacillus HMF13 on wheat germination under salt stress

[0062] 1) Preparation of bacterial suspension: the strain HMF13 was inoculated in LB liquid medium, the temperature was 30℃, the rotation speed was 160r / min, and the culture was carried out for 48h to obtain the fermentation liquor, and then the fermentation liquor was prepared into a bacterial suspension with a bacterial content of 1.1×10 9CFU / mL of bacterial suspension. The bacterial suspension was diluted with sterile distilled water, and the bacterial suspension and sterile water were mixed at a volume ratio of 1:20,

[0063] Put on standby.

[0064] 2) Test seeds and treatment: The test material was the seed of wheat variety Nongda 212. Uniformly sized wheat seeds were selected, surface sterilized with 0.1% mercuric chloride for 3 min, rinsed with sterile distilled water 3 times, and soaked in sterile water (CK) or the bacterial suspension prepared in step 1) for 2 h at room temperature. After soaking, the seeds were rinsed with distilled water 2 to 3 times, and arranged in Petri dishes (2 layers of moist sterile filter paper were placed on the bottom of the dish), 20 seeds per Petri dish, 5 replicates per group. The seeds were irrigated with 0%, 0.4% (low salt concentration), or 0.8% (high salt concentration) NaCl solution, and incubated in an artificial climate chamber. The light / dark cycle was 12 h / 12 h, the day / night temperature was 28°C / 23°C, and the relative humidity was 70%.

[0065] The number of germinated seeds was observed and recorded every day from the first day of salt stress, and the germination rate was calculated. The radicle was considered to have germinated when it reached half the length of the seed. The number of germinated seeds after 7 d was recorded as the final number of germinated seeds. The germination rate was calculated according to the following formula: Germination rate (%) = (final number of germinated seeds / test seed number) x 100. The test results are shown in Table 4.

[0066] Table 4 Germination rate of wheat seeds soaked in fermentation broth of Bacillus oceanae HMF13 under different salt stresses

[0067] .

[0068] As can be seen from Table 4, the germination rate of wheat seeds soaked in the bacterial suspension of HMF13 under salt stress was 12% (salt concentration 0%), 14% (salt concentration 0.4%), and 53% (salt concentration 0.8%) higher than that of wheat seeds soaked in sterile water, respectively. That is, after HMF13 seed soaking treatment, when irrigated with a 0.4% salt concentration NaCl solution, the wheat germination rate of the treatment using HMF13 seed soaking was 14% higher than that of the treatment without HMF13 seed soaking; when irrigated with a 0.8% salt concentration NaCl solution, the wheat germination rate of the treatment using HMF13 seed soaking was 53% higher than that of the treatment without HMF13 seed soaking. This shows that HMF13 seed soaking can alleviate the damage of salt stress to wheat seeds.

[0069] Example 6 Antagonistic effect test of Bacillus oceanae HMF13 on Erwinia carotovora subsp. carotovora.

[0070] The specific steps are as follows:

[0071] 1) Prepare a bacterial suspension of the pathogenic bacteria Erwinia carotovora subsp. carotovora at a concentration of 107 CFU / mL; 1 mL of the pathogenic bacteria suspension was added to the surface of the LB medium plate, and the bacteria liquid was evenly coated with a spreader.

[0072] The pathogenic bacteria E. carotovora subsp. carotovora strain was isolated and identified from stored Chinese cabbage and preserved in the laboratory.

[0073] Preparation of pathogenic bacteria suspension: The E. carotovora subsp. carotovora strain was inoculated in LB liquid medium, the temperature was 30°C, the rotation speed was 160 r·min -1 , and the culture was incubated for 48 h to obtain the fermentation broth, and then the fermentation broth was prepared into a bacteria suspension containing 1.0×10 7 CFU / mL of bacteria.

[0074] 2) A 5mm diameter hole was punched on the above-mentioned pathogenic bacteria coated medium with a 1000μL pipette tip.

[0075] 3) Add the bacteria suspension of B. oceanicum HMF13 to the hole, and fill it without spilling.

[0076] Preparation of B. oceanicum HMF13 bacteria suspension: B. oceanicum HMF13 was inoculated in LB liquid medium, the temperature was 30°C, the rotation speed was 160 r·min -1 , and the culture was incubated for 48 h to obtain the fermentation broth, and then the fermentation broth was prepared into a bacteria suspension, and the bacteria content of the bacteria suspension was detected to be 1.1×10 9 CFU / mL.

[0077] 4) Incubate at 37°C for 3 days, observe the results, and measure the diameter of the inhibition zone with a vernier caliper, and repeat the test 3 times.

[0078] Judgment standard: Inhibition zone diameter ≥ 15mm, indicating that the pathogenic bacteria E. carotovora subsp. carotovora is extremely sensitive to the fermentation broth of the antagonistic bacteria strain; 10mm≤inhibition zone diameter≤15mm, indicating moderate sensitivity; 6mm≤inhibition zone diameter≤10mm, indicating low sensitivity, and no inhibition zone indicating no sensitivity.

[0079] The test results show that the average diameter of the inhibition zone of the HMF13 strain fermentation broth on the E. carotovora is 15.4mm (as shown in Figure 4 ).

[0080] Example 7 Preparation of B. oceanicum HMF13 bacterial agent

[0081] The B. oceanicum HMF13 bacterial agent was prepared with diatomite as the carrier, and the specific steps were as follows:

[0082] (1) Preparation of LB liquid medium: beef extract 3 g, proteose peptone 10 g, sodium chloride 5 g, placed in a 1000 mL beaker, 900 mL distilled water was heated to dissolve, adjust pH to 7.2-7.4, constant volume to 1 L with distilled water, 121 ℃ sterilization for 30 min, ready for use.

[0083] (2) Activation of strains: pick a ring of Bacillus HMF13 colonies, inoculate in a 150 mL flask containing 50 mL of LB liquid medium, 160 r·min -1 , 37℃ constant temperature oscillation culture for 24 h for activation.

[0084] (3) Preparation of seed liquid: take 4 mL of activated bacterial liquid and inoculate in a 1000 mL flask containing 200 mL of LB liquid medium, 160 r·min -1 , 37℃ constant temperature oscillation culture for 24 h to obtain the seed liquid.

[0085] (4) Preparation of fermentation broth: take 180 mL of prepared seed liquid and inoculate in a 6L small fermentation tank containing 3.5L of LB liquid medium, 160 r·min -1 , 37℃ constant temperature oscillation culture for 48 h to obtain the fermentation broth, the effective viable count is 2.26×10 9 CFU / mL.

[0086] (5) Mix the fermentation broth with diatomite at a mass ratio of 10:1, spray through the freeze-drying machine to obtain the Bacillus oceanisediminis powder, the effective viable count is 4.12×10 10 CFU / g after detection.

[0087] Example 8 Field application test of Bacillus oceanisediminis inoculant

[0088] Test variety: wheat variety Jetmace 19.

[0089] Test time: from October 10, 2023 to June 10, 2024.

[0090] Test site: the applicant's dry and alkaline wheat planting base planted for many years in Huanghua City, Hebei Province.

[0091] Treatment group: two blocks of the treatment group are set up, which are north test field and core test field. Each test field is provided with one treatment, and the Bacillus oceanisediminis inoculant prepared in Example 7 is applied in the soil at the time of sowing together with other base fertilizers according to the application amount of 10 kg per mu. Other management measures are carried out according to the local dry and alkaline wheat planting management and production technical specifications.

[0092] Control group (CK): the control group CK, for not using the preparation of Bacillus oceani inoculant of Example 7 treatment. The rest of the management measures are in accordance with the local dry saline wheat planting management production technology specification implementation.

[0093] The treatment group and the control group of this embodiment are each treated 3 times, all treatments are randomly arranged, and the area of each treatment plot is 120m 2 .

[0094] On May 29, 2024, the wheat growth was investigated, and the yield was measured in the field. The test results are shown in Table 5.

[0095] Table 5 Economic property test results of Huanghua saline land wheat field

[0096] .

[0097] On June 7, 2024, the wheat in the north test field, the north test field control, the core test field, and the core field control were harvested. The yield of the north test field increased by 33.02% compared with the control, and the yield of the core test field increased by 30.01% compared with the control.

[0098] The results of Table 5 and the field harvesting results clearly show that the preparation of Bacillus oceani HMF13 can effectively improve the yield of dry saline wheat.

[0099] Example 9 Application of Bacillus oceani HMF13 in the prevention and treatment of Chinese cabbage soft rot.

[0100] This test was set up in the Chinese cabbage planting base in Liutaizhuang Town, Changli County, Qinhuangdao City. The plot is a saline land plot, and the soil salt content is 0.33%. The planted Chinese cabbage variety is Beijing No. 3.

[0101] The Bacillus oceani HMF13 bacterial suspension prepared in Example 6 (containing 1.1×10 9 CFU / mL) was prepared into two gradients of bacterial-containing liquid with bacterial content of 1.0×10 5 CFU / mL and 1.0×10 7 CFU / mL.

[0102] The specific test treatment is shown in the table.

[0103] Table 6 Substances applied in each treatment of the test

[0104] .

[0105] Each treatment 30m 2, 3 times, randomized block arrangement. The above-mentioned treatment is applied to spray white cabbage at the stage of seedling and hearting, and each treatment is sprayed twice at the stage of seedling and hearting. The spraying process is to spray the liquid or the liquid medicine or the water to the cabbage leaf base part to wet it by using a backpack sprayer to spray the front and back of the cabbage leaf conventionally. The control effect is investigated 10 days after the last spraying, 20 cabbages in each treatment are randomly investigated, the disease is graded in units of plants, and the incidence and disease index are calculated.

[0106] Disease grading standards:

[0107] 0 level: no disease;

[0108] 1 level: a small amount of disease spots on 1-2 leaves of the lower part of the plant;

[0109] 2 level: 3 or more leaves of the lower part of the plant have disease spots, and other infected parts are slightly heavier;

[0110] 3 level: all leaves are infected, and other infected parts are heavier;

[0111] 4 level: all leaves are infected, the whole plant is inclined, the leaves are drooping and cannot recover to normal, but still survive;

[0112] 5 level: whole plant death.

[0113] Incidence (%) = total number of diseased plants / total number of investigated plants x 100;

[0114] Disease index (%) = ∑(diseased plant number x grade value of the diseased plant) / (total number of investigated plants x highest grade value) x 100;

[0115] Control effect (%) = (disease index of the control area-disease index of the treatment area) / disease index of the control area x 100.

[0116] The control effect is shown in Table 7, and the application of 1.0 x 10 7 CFU / mL of Bacillus megaterium HMF13 liquid spray to prevent and control cabbage soft rot has the best effect, and the control effect reaches 81.7%, which is close to the control effect of 72% of 3000 times of chemical control method of 72% of 3000 times of liquid of agricultural streptomycin wettable powder.

[0117] Table 7 Control effect of different treatments on cabbage soft rot

[0118] .

[0119] The above-mentioned is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A type of Bacillus aquatilis ( Oceanobacillus sp HMF13, characterized in that, The preservation number is CGMCC No. 30479.

2. A microbial inoculant, characterized in that, It comprises the bacterial body of the Oceanobacillus HMF13 as claimed in claim 1 and / or the fermentation liquor prepared therefrom.

3. The microbial inoculant of claim 2, wherein, It also includes diatomite, vermiculite, activated carbon or biochar.

4. The Oceanobacillus HMF13 as claimed in claim 1 is applied to the prevention and control of Chinese cabbage soft rot.

5. The Oceanobacillus HMF13 as claimed in claim 1 is applied to the improvement of wheat seed germination rate under salt stress environment.

6. The Oceanobacillus HMF13 as claimed in claim 1 is applied to the promotion of wheat growth in saline-alkali soil.

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