Bacillus velezensis HY54 and application thereof

Abstract

The invention relates to the technical field of agricultural microorganisms, and provides bacillus velezensis HY54 and application thereof. The preservation number of the bacillus velezensis HY54 provided by the invention is CCTCC (China Center For Type Culture Collection): M 20242819, and the bacillus velezensis HY54 has strong adaptability to the environment and shows good growth capability under the conditions of high salt and drought. In addition, the strain also has the capabilities of low temperature resistance, high temperature resistance, growth promotion, disease prevention and control, cellulose degradation and soil heavy metal cadmium and lead pollution resistance, and when the strain is combined with nitrogen-fixing bacteria for use, the crop yield can be increased by 12%, which is significantly superior to that of a single strain treatment group.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and particularly to a Bacillus velezensis HY54 and its applications. Background Art

[0002] Agricultural production is facing increasingly serious problems of soil degradation, crop diseases, and straw treatment. Although traditional chemical means can solve problems in the short term, long-term use will lead to the deterioration of the ecological environment. Microbial technology provides a new way for the green development of agriculture, and especially Bacillus velezensis has attracted much attention due to its versatility. Existing studies have shown that Bacillus velezensis has the abilities of disease resistance, growth promotion, and cellulase secretion, but the existing strains still have deficiencies in tolerance and actual application effects. Therefore, screening out a Bacillus velezensis with more excellent characteristics is of great significance for solving the above problems.

[0003] The existing Bacillus velezensis strains have problems of insufficient salt tolerance, limited stress resistance, and single function in agricultural applications. Some strains are restricted in growth under extreme environments (such as high-salt and high-drought conditions) and are difficult to play a stable role in complex agricultural environments. Therefore, developing a Bacillus velezensis that can adapt to high-salt and high-drought extreme environments is of great significance in the application of saline-alkali land. Summary of the Invention

[0004] In view of this, the present invention provides a Bacillus velezensis HY54 that can adapt to high-salt and high-drought extreme environments and its applications.

[0005] The technical solution of the present invention is realized as follows: In the first aspect, the present invention provides a Bacillus velezensis HY54, which was deposited at the China Center for Type Culture Collection on December 16, 2024. Address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M20242819, and the culture collection center identified the strain as being in a viable state on December 23, 2024.

[0006] In the second aspect, the present invention provides the application of Bacillus velezensis HY54 in promoting plant growth.

[0007] On the basis of the above technical solutions, preferably, the plants include plants under drought and salt stress.

[0008] In the third aspect, the present invention provides the application of Bacillus velezensis HY54 in degrading straw cellulose.

[0009] Fourthly, the present invention provides the application of Bacillus velezensis HY54 in inhibiting plant diseases.

[0010] Based on the above technical solutions, preferably, the plant disease is one of the mycelial blocks of Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, Magnaporthe oryzae, and Phytophthora capsici.

[0011] A Bacillus velezensis HY54 and its application of the present invention have the following beneficial effects compared with the prior art:

[0012] (1) The Bacillus velezensis HY54 of the present invention has strong environmental adaptability and shows good growth ability under high-salt and drought conditions.

[0013] (2) The Bacillus velezensis HY54 of the present invention can adapt to the environment of 5°C - 50°C and is suitable for extreme environments such as deserts and the Qinghai-Tibet Plateau, providing technical support for global climate-adaptive agriculture.

[0014] (3) After the Bacillus velezensis HY54 of the present invention is applied to the soil, it still maintains a high activity survival ability after 6 months, reducing the application frequency of the microbial agent and improving the economy and convenience of agricultural production.

[0015] (4) The Bacillus velezensis HY54 of the present invention also has the abilities of promoting growth, disease prevention and control, cellulose degradation, and resisting soil heavy metal cadmium and lead pollution. In addition, when used in combination with nitrogen-fixing bacteria, it can increase the crop yield by 12%, which is significantly better than the single-strain treatment group. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 It is the electron microscope image of Bacillus velezensis HY54 of the present invention. Detailed Embodiments

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0019] Example 1 Strain Isolation and Identification

[0020] The strain of the present invention was isolated from the surface soil of farmland in Hubei Province. The isolation steps are as follows: Collect soil samples covered with decomposed straw from the Sanhua Cross-river Industrial Park in Xishui, Huanggang City, Hubei Province. Take 5 g of soil, add 45 mL of sterile water, and prepare gradient dilution solutions (10 -2 to 10 -6 ). Spread the dilution solution on the CMC-Na medium plate and culture at 28 °C for 3 days. Screen single colonies according to the cellulose degradation clear zone. Select the dominant colonies on the LB medium and continuously streak for purification to obtain strain HY-54 and store it.

[0021] CMC-Na medium: CMC-Na (sodium carboxymethyl cellulose) 10 g / L, yeast extract 2 g / L, NaCl 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.2 g / L, make up to 1 L with distilled water, and adjust the pH to 6.8 with NaOH or HCl.

[0022] LB medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, make up to 1 L with distilled water, and adjust the pH to 7.0 with NaOH or HCl.

[0023] LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, adjust the pH to 7.0.

[0024] Strain identification: Identify HY-54 by 16S rDNA sequence analysis and morphological observation.

[0025]

[0026] The screened strain HY-54 was inoculated into LB medium and cultured at 28 °C for 2 days. The colony morphology was as follows Figure 1 shown. The colonies were milky white, with wrinkles on the surface, slightly convex in the middle, and uneven edges.

[0027] After comparison, the sequence homology of this strain with Bacillus velezensis reached 99.8%. Therefore, it was named Bacillus velezensis HY-54 and deposited in the China Center for Type Culture Collection on December 16, 2024. Address: Wuhan University, Wuhan, China. The deposit number was CCTCC NO: M 20242819. The culture collection center identified the strain as viable on December 23, 2024.

[0028] Example 2 Preparation of Bacterial Agent

[0029] The microbial bacterial agent of Bacillus velezensis HY-54 was prepared by fermentation method as follows:

[0030] S1, Activation: The strain HY-54 was inoculated onto LB plate medium and cultured at 30 °C for 2 days to obtain an activated strain.

[0031] S2, Fermentation: The activated strain was inoculated into a liquid medium (formula: tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L), and cultured at 25 °C and 150 rpm for 48 h to obtain a fermentation broth.

[0032] S3, Bacterial Agent: A powder was prepared by spray drying, and the viable bacteria concentration reached 5×10 11 CFU / g.

[0033] Example 3 Salt and Drought Tolerance of Bacillus velezensis HY-54

[0034] 1. Salt Tolerance of Bacillus velezensis HY-54

[0035] Bacillus velezensis HY-54 was inoculated into LB medium containing different mass concentrations (1%, 6%, 12%) of NaCl. The LB liquid medium without NaCl was used as the control group. It was cultured in a constant temperature incubator at 30 °C and shaken at 120 rpm for 48 h. The OD600 value of the culture solution was measured every 12 h using a spectrophotometer (wavelength 600 nm) to evaluate the growth of the strain. The results are shown in the following table.

[0036] Table 1 Salt Tolerance of Bacillus velezensis HY-54

[0037]

[0038] The results showed that Bacillus velezensis strain HY-54 could grow at NaCl concentrations of 1% - 12%. Among them, at 6% NaCl, the OD600 value reached 1.15 ± 0.04 after 48 hours, indicating strong salt tolerance.

[0039] 2. Drought tolerance of Bacillus velezensis HY-54

[0040] Using PEG-6000 to simulate drought conditions, LB liquid media containing 10%, 15%, and 20% (by mass) of PEG-6000 (polyethylene glycol) were prepared, and the LB liquid medium without PEG-6000 was used as the control group. Bacillus velezensis HY-54 was inoculated into each medium and cultured at 30 °C and 120 rpm for 48 hours. The OD600 value of the culture broth was measured every 12 hours to evaluate the growth ability of the strain. The results are shown in the following table.

[0041] Table 2 Drought tolerance of Bacillus velezensis HY-54

[0042] PEG-6000 Concentration (%) OD600 Value (24 h) OD600 Value (48 h) 0 (Control Group) 1.20±0.05 1.50±0.04 10 0.98±0.04 1.30±0.05 15 0.85±0.03 1.10±0.03 20 0.65±0.03 0.85±0.02

[0043] The results showed that at PEG-6000 concentrations of 10% - 20%, Bacillus velezensis strain HY-54 showed strong growth ability. Especially at a PEG-6000 concentration of 15%, the OD600 value reached 1.10 ± 0.03, which was significantly higher than the drought tolerance of most existing strains, proving its good adaptability under simulated drought conditions.

[0044] Example 4 Temperature tolerance of Bacillus velezensis HY-54

[0045] 1. Low-temperature and high-temperature tolerance of Bacillus velezensis HY-54

[0046] The HY-54 strain was inoculated into LB liquid medium with an initial bacterial liquid concentration of 1×106 CFU / mL.

[0047] Culture conditions: 5 °C (low temperature), 30 °C (normal temperature control), and 50 °C (high temperature) were set respectively, and the cultures were shaken at 120 rpm (to ensure uniform medium and sufficient dissolved oxygen) for 48 h. Then the viable cell concentration was detected. The results are shown in the following table.

[0048] Table 3 Temperature adaptability of Bacillus velezensis HY-54

[0049] Temperature (°C) Viable Bacteria Concentration (CFU / mL) 5 <![CDATA[5×10 8 ±0.2]]> 30 <![CDATA[1×10 12 ±0.1]]> 50 <![CDATA[3×10 8 ±0.3]]>

[0050] As can be seen from Table 3, the Bacillus velezensis HY-54 strain can maintain a high viable cell concentration at both 5°C and 50°C, while most of the control strains are completely inactivated at 50°C and can only grow stably within the neutral temperature range (20 - 40°C). This shows that the Bacillus velezensis HY-54 strain of the present application has strong adaptability to high and low temperatures and can be applied to extreme environments such as deserts and the Qinghai-Tibet Plateau, providing technical support for global climate-adaptive agriculture.

[0051] 2. Cross-tests of temperature tolerance, salt tolerance, and drought tolerance

[0052] Prepare LB liquid media containing different mass fractions of NaCl and PEG-6000, inoculate Bacillus velezensis HY-54 respectively, and culture at different temperatures (5°C, 30°C, 50°C) under the condition of 120 rpm for 48 hours. Measure the OD600 value of the culture solution every 12 hours to evaluate the growth ability of the strain. The results are shown in the following table.

[0053] Table 4 Cross-tests of salt tolerance, drought tolerance, and temperature tolerance of Bacillus velezensis HY-54

[0054]

[0055] The results show that the treatment group at 30°C has a better effect. Among them, the treatment group with 1% NaCl + 10% PEG-6000 at 30°C has the best effect, indicating that Bacillus velezensis HY-54 can adapt to the environment of high salt and high drought.

[0056] Example 5 Growth promotion test of Bacillus velezensis HY-54

[0057] The test plot is located in the Xishui Sanhua Cross-River Industrial Park, Huanggang City, Hubei Province. Treat rice seedlings (plant height 10.5 ± 0.5 cm) with the Bacillus velezensis HY-54 bacterial agent. The control group is sprayed with sterile water, and the test group is sprayed with the HY-54 bacterial solution at 5×10 8 CFU / mL. There are 30 plants in each group, and the experiment is repeated 3 times. Standardized water and fertilizer management is carried out during the test period. After 30 days, measure the plant height, root length, and dry weight of the roots of the rice. The results are shown in the following table.

[0058] Table 5 Rice growth indicators

[0059] Treatment Group Plant Height (cm) Root Length (cm) Root Dry Weight (g) Control Group 45.2±0.8 10.5±0.4 1.8±0.2 HY-54 Group 52.6±0.9** 12.8±0.5** 2.3±0.3**

[0060] Note: ** indicates significantly higher than the control group (P < 0.05).

[0061] As can be seen from Table 5, the plant height, root length, and dry weight of the roots of the treatment group sprayed with the Bacillus velezensis HY-54 bacterial solution are significantly higher than those of the control group (P < 0.05), indicating that the Bacillus velezensis HY-54 strain has a significant growth promotion effect.

[0062] Example 6: Cellulose Degradation Test of Bacillus velezensis HY-54

[0063] To verify the cellulose degradation ability of strain HY-54, a straw compost experiment was conducted in this example for testing.

[0064] Specific method: Cut corn straw into sections (3 - 5 cm), mix it with the microbial agent (concentration 5×10 11 CFU / g) at a mass ratio of 1:10, adjust the moisture content to 60% - 70%, and stack it under ventilated conditions. Compost for 30 days at 25°C, sample every 10 days to measure the degradation rate of cellulose content, and at the same time use the Congo red staining method to measure the cellulase activity (represented by the ratio of the diameter of the clear zone / colony diameter D / d). The results are shown in the following table.

[0065] Table 6: Cellulose Degradation Ability of Bacillus velezensis HY-54

[0066] Time (d) Cellulose Degradation Rate (%) D / d Value 0 0 1.0±0.1 10 18.5±1.2 2.4±0.2 20 35.2±1.5 2.9±0.3 30 52.0±1.8 3.2±0.2

[0067] As can be seen from Table 6, during the 30-day composting process, the cellulose degradation rate of Bacillus velezensis HY-54 strain reached 52.0 ± 1.8%, significantly higher than that of the ordinary composting process (the degradation rate is usually 30% - 40%). In addition, the cellulase activity (D / d value) of Bacillus velezensis HY-54 strain reached 3.2 ± 0.2 at 30 days, significantly better than that of the control strain (Bacillus subtilis, D / d value is 2.5 ± 0.2), fully proving its high efficiency in straw degradation.

[0068] Example 7: Pest and Disease Resistance Test of Bacillus velezensis HY-54

[0069] To verify the inhibitory ability of strain HY-54 against different pathogenic bacteria, the plate confrontation method was used for the experiment. Inoculate blocks of Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum, Magnaporthe oryzae, and Phytophthora capsici on PDA medium (PDE 200 g / L, glucose 20 g / L, agar 15 g / L, made up to 1 L with distilled water, pH adjusted to 5.6 - 5.8). After the mycelia of the pathogenic bacteria grow to the center of the plate, inoculate HY-54 bacterial solution (concentration 5×10 8 CFU / mL) on the opposite side. After culturing at 28°C for 5 days, measure the diameter of the inhibition zone and calculate the inhibition rate at the same time. In addition, a field experiment was used to test the in vivo control effect of strain HY-54 against the above-mentioned pathogenic bacteria. The results are shown in the following table.

[0070] Control effect (%) = (disease index of the control group - disease index of the treatment group) / disease index of the control group × 100%. Calculate the in vivo control effect according to this formula.

[0071] Table 7 Disease resistance of Bacillus velezensis HY-54

[0072] Pathogen Species Inhibition Zone Diameter (mm) Inhibition Rate (%) In Vivo Control Efficacy (%) Rhizoctonia solani Kühn 21.3±1.2 75±3 85±3 Botrytis cinerea 18.6±1.0 68±2 80±4 Fusarium graminearum 19.8±1.1 70±3 83±3 Magnaporthe oryzae 20.3±1.2 72±4 84±2 Phytophthora capsici 23.0±1.2 78±3 87±4

[0073] As can be seen from Table 7, the HY-54 strain showed significant inhibitory effects on 5 important agricultural pathogenic bacteria. Among them, the diameter of the inhibition zone against Phytophthora capsici was 23.0±1.2 mm, the inhibition rate reached 78%, and the in vivo control effect was 87%. This indicates that it has the ability to control a wide range of diseases, can effectively inhibit the growth of pathogenic bacteria and reduce the incidence of diseases.

[0074] Example 8 Experiment on the combination of Bacillus velezensis HY-54 and nitrogen-fixing bacteria

[0075] Set up the following three groups of experiments to verify the combined effect of Bacillus velezensis HY-54 and nitrogen-fixing bacteria:

[0076] Control group: Sprayed with sterile water.

[0077] Single HY-54 group: Sprayed once on the 7th, 14th, and 21st days respectively, and sprayed with HY-54 bacterial solution (5×10 8 CFU / mL).

[0078] Combined use group: Sprayed once on the 7th, 14th, and 21st days respectively, and sprayed with L HY-54 bacterial solution (5×10 8 CFU / m) and nitrogen-fixing bacterial solution (1×10 8 CFU / mL). After 50 days, the plant height and yield of rice were measured, and the results are shown in the following table.

[0079] Table 8 Combined effect of Bacillus velezensis HY-54 and nitrogen-fixing bacteria

[0080] Treatment Group Plant Height (cm) Yield (kg / mu) Control Group 50.2±1.0 450.3±12.5 Single HY-54 Group 56.4±1.2** 508.6±15.2** Combined Use Group 62.3±1.5** 570.5±18.7**

[0081] As can be seen from Table 8, in the treatment group using Bacillus velezensis HY-54 and nitrogen-fixing bacteria in combination, the plant height and yield of rice were significantly higher than those in the control group and the single HY-54 group, indicating that the application of composite microorganisms has a synergistic effect.

[0082] Example 9 Experiment on Bacillus velezensis HY-54 alleviating soil heavy metal pollution

[0083] Prepare soil containing cadmium and lead (containing 50 mg / kg Cd and 100 mg / kg Pb), and apply Bacillus velezensis HY-54 (HY-54 group), adding 5 g of bacterial agent powder per kilogram of soil (viable bacteria concentration 5×10 11(CFU / g), with the humidity maintained at 60%-70%. The control group was not applied with the microbial agent. The initial plant height of maize was 8.5±1.0 cm and the dry weight was 0.25±0.05 g. After 30 days of treatment, the available cadmium and lead contents in the soil, as well as the plant (maize) height and root dry weight, were measured. The results are shown in the following table.

[0084] Table 9 Effects of Bacillus velezensis HY-54 on alleviating soil heavy metal pollution

[0085] Treatment Group Reduction in Effective Lead Content (%) Plant Height of Plant (cm) Root Dry Weight (g) Control Group 0 28.5±1.2 1.8±0.2 HY-54 Group 38±2 36.2±1.5 2.6±0.3

[0086] As shown in Table 9, the Bacillus velezensis HY-54 strain significantly reduced the available cadmium and lead contents in the soil (decreased by 45% and 38% respectively). The plant height and root dry weight of the HY-54 treatment group were significantly higher than those of the control group (P<0.05), indicating that it can improve the polluted soil environment and promote plant growth. The principle is that Bacillus velezensis HY-54 effectively reduces the bioavailability of heavy metals in the soil by secreting specific extracellular polysaccharides and metal-chelating proteins, thereby promoting the growth of plants in the polluted environment.

[0087] Example 10 Long-term survival experiment of Bacillus velezensis HY-54 strain in soil

[0088] The actual environmental temperature of the farmland was simulated in the experiment, the soil temperature was set at 25℃±2℃, and the experimental soil was stored in a constant temperature incubator to maintain the temperature stability.

[0089] The HY-54 microbial agent was applied to the farmland soil, with an initial concentration of 1×10 8 CFU / g soil. The viable bacteria concentration was measured every 30 days for 180 days. The results are shown in the following table.

[0090] Table 10 Long-term viability of Bacillus velezensis HY-54

[0091] Time (d) Viable Bacteria Concentration (CFU / g) 0 <![CDATA[1×10 8 > 30 <![CDATA[9×10 7 > 90 <![CDATA[6×10 7 >

[0092] As can be seen from Table 10, the Bacillus velezensis HY-54 strain can survive in the soil for a long time, and the viable bacteria concentration still remains within the effective range after 6 months. The existing strains usually have a short activity maintenance time in the soil (1-3 months). Compared with the existing strains, the Bacillus velezensis HY-54 strain of the present invention reduces the application frequency of the microbial agent, improving the economy and convenience of agricultural production.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus velezensis HY54, characterized in that: The deposit number of this strain is CCTCC NO: M 20242819.

2. Use of Bacillus velezensis HY54 as claimed in claim 1 in promoting plant growth.

3. The use according to claim 2, characterized in that: The plants include plants under drought and salt stress.

4. Use of Bacillus velezensis HY54 as claimed in claim 1 in degrading straw cellulose.

5. Use of Bacillus velezensis HY54 as claimed in claim 1 in inhibiting plant diseases.

6. The use according to claim 5, characterized in that The plant disease is one of the fungus masses of soybean sheath blight, tomato gray mold, wheat fusarium rust, rice blast and pepper phytophthora.

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