Salt-tolerant bacillus velezensis SD13 and application thereof

Abstract

The invention provides a salt-tolerant bacillus velezensis SD13 and application of the salt-tolerant bacillus velezensis SD13, the preservation number of the salt-tolerant bacillus velezensis SD13 is CCTCC NO: M2023570, and the gene sequence of the salt-tolerant bacillus velezensis SD13 is as shown in SEQ ID NO: 1. The strain is widely applied to agricultural biological prevention and control, can be used for preventing and controlling plant diseases, and particularly has a good effect on plant diseases in saline-alkali soil, including fusarium oxysporum of cowpeas, green cucumbers, peppers, muskmelons, bitter gourds and the like. In addition, the strain SD13 can be combined with an organic fertilizer to improve the salt tolerance of plants, and the strain SD13 has a prominent effect in the aspect of improving the salt tolerance of cowpeas. The strain has good salt tolerance, provides a new effective way for preventing and treating plant diseases and improving the salt tolerance of plants in agricultural production, is beneficial to reducing the use of chemical pesticides, reducing environmental pollution and promoting agricultural sustainable development, and has important economic and ecological values.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a salt-tolerant Velez subtilis SD13 and an application thereof. Background Art

[0002] Soil salinization is a common problem in most farmlands and irrigated areas. It has become a potential threat to future crop production. Increased soil salinity has a negative impact on soil microbial communities, especially the colonization and survival of biocontrol bacteria that control soil-borne diseases. Cowpea (Vigna unguiculata) is a tropical legume plant that originated in West Africa and is cultivated in most tropical regions of the world. It is estimated that 14.5 million cowpeas are killed each year worldwide. 6 Cowpea is an important cash crop in tropical and subtropical regions of my country, with a planting area of ​​more than 6.7 million hectares. 5 hectares, with an annual output of about 1.5×10 6 Tons. Cowpea wilt is a biotic stress disease that seriously threatens cowpea production and occurs in many cowpea-growing areas. This disease can cause 30% to 100% yield loss, posing a threat to the development of cowpea industrialization.

[0003] Chinese patent CN111254093B screened a strain of Bacillus Velezii 229-15, which has antagonistic activity against cowpea wilt pathogen. Chinese patent CN111154688B screened a strain of Bacillus Velezii SF259, which has antibacterial activity against cowpea wilt pathogen; Chinese patent CN109370939B screened a strain of Bacillus Velezii 504, which has antibacterial activity against cowpea wilt pathogen; existing salt-tolerant microorganisms often have limited salt tolerance and narrow application range, and there is also a lack of Bacillus Velezii for cowpea soil pretreatment and cowpea seedling root irrigation treatment, which can effectively prevent and control the occurrence of wilt in advance and reduce the incidence of cowpea wilt. Summary of the invention

[0004] In view of this, the present invention proposes a salt-tolerant Bacillus Velez subtilis SD13 and its application to solve the above problems.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A salt-tolerant Bacillus velezensis SD13, the strain (Bacillus velezensis SD13) is deposited in the China Center for Type Culture Collection (Wuhan University), its strain collection number is CCTCCNo: M2023570, and the preservation date is April 21, 2023.

[0007] Furthermore, the gene sequence of the Bacillus velezensis SD13 is shown in SEQ ID NO:1.

[0008] Furthermore, a salt-tolerant Bacillus Velezii SD13 was used in agricultural biological control.

[0009] Furthermore, the salt-tolerant Bacillus Velez subtilis SD13 is used for plant diseases.

[0010] Furthermore, the salt-tolerant Bacillus Velez subtilis SD13 is used to prevent and control plant diseases in saline-alkali land.

[0011] Furthermore, the plant diseases include but are not limited to cowpea wilt, cucumber wilt pathogen, pepper wilt pathogen, melon wilt pathogen, and bitter melon wilt pathogen.

[0012] Furthermore, a salt-tolerant Bacillus Velez subtilis SD13 was used in combination with organic fertilizers to improve plant salt tolerance.

[0013] Furthermore, the salt-tolerant Bacillus Velez SD13 is used in combination with organic fertilizer to improve the salt tolerance of cowpea.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] First, in terms of agricultural biological control, SD13 can be used to control a variety of plant diseases, especially plant diseases in saline-alkali land. For common diseases such as cowpea wilt and cucumber wilt, SD13 can effectively inhibit the growth of pathogens, reduce the incidence of diseases, and reduce yield losses caused by diseases. Compared with existing technologies, SD13 has enriched the types of biocontrol bacteria and provided new options for agricultural disease control.

[0016] Secondly, regarding the problem of soil salinization, SD13 can survive in high-salt environments, overcoming the limited salt tolerance of existing salt-tolerant microorganisms. Its gene sequence is clear, providing a basis for further research and application. It has outstanding performance in improving plant salt tolerance in combination with organic fertilizers, especially in improving cowpea salt tolerance, which helps to stabilize the cultivation of cowpea and other crops in salinized soils.

[0017] Furthermore, by pre-treating the cowpea soil and irrigating the seedlings, the occurrence of wilt disease can be effectively prevented in advance. This method can reduce the incidence of cowpea wilt disease and ensure the industrial development of cowpea. Cowpea is widely planted around the world, and the application of SD13 is of great significance for increasing cowpea yield and stabilizing market supply.

[0018] Finally, the use of SD13 for biological control reduces the use of chemical pesticides and reduces environmental pollution, which meets the requirements of sustainable agricultural development. At the same time, its wide range of applications and good salt tolerance provide a comprehensive solution to soil salinization and plant disease problems, and has high economic and ecological value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the phylogenetic tree of strain SD13 based on 16S rDNA sequence.

[0020] Figure 2 The growth of strain SD13 in LB solid culture medium containing different sodium chloride contents.

[0021] Figure 3 The plant growth status and root disease area of ​​cowpea wilt treated with different treatments;

[0022] In the figure, A, a: CK; B, b: F; C, c: F+B; D, d: F+B+W; E, e: F+B+S; F, f: F+B+W+S; lowercase letters represent different treatments under 0 mM NaCl stress; uppercase letters represent different treatments under 50 mM NaCl stress. DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0026] Example 1 - Determination of salt tolerance of strains

[0027] Bacillus Velez SD13 was isolated from a cowpea field in Sanya, Hainan. The soil was taken from rice sludge after healthy cowpea crops.

[0028] The purified SD13 was transferred to LB plates containing different sodium chloride gradients (0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 13%, 15%) to observe whether it can grow normally. The SD13 single colony on the LB plate was inoculated into LB liquid culture medium and cultured at 29°C and 180r / min for 24h to obtain a bacterial suspension. The bacterial suspension was inoculated into 200mL LB liquid culture medium with a 1% inoculation amount and fermented at 29°C and 180r / min for 24h. Then it was inoculated into 200mL LB liquid culture medium containing different sodium chloride gradients (1% to 9%) at a 1% inoculation amount, cultured at 29°C and 180r / min, and the OD600 value was measured using a spectrophotometer at 12h, 24h, 48h, 73h, and 96h. After 3 days of inoculation, SD13 could grow on 10% sodium chloride solid LB medium, and SD13 could still grow normally on 9% sodium chloride LB liquid medium (as shown in Table 1, Figure 2 ), has very strong salt tolerance and has good application potential.

[0029] Table 1: OD values ​​of SD13 in LB liquid medium containing different sodium chloride

[0030]

[0031] Note: The data in the table are mean values ​​and standard deviations. Different lowercase letters in the same column indicate significant differences at the 0.05 level.

[0032] Example 2- Plate confrontation experiment of strains against pathogens

[0033] 1) Streak the Bacillus Velez strain SD13 on an LB plate and culture it in a 30°C incubator for 24 hours, then use an inoculation loop to pick a single colony and add it to 50 mL of liquid LB medium, and culture it at 30°C in a shaker at 200 rpm for 24 hours to prepare a stock solution;

[0034] 2) Add 1% by volume of the mother solution to 250 ml of liquid LB medium, and culture at 30°C and 180 rpm for 24 h to prepare a fermentation broth;

[0035] 3) After the pathogens to be tested were cultured in a 28°C incubator for 48 hours, a 5mm sterile punch was used to punch out the pathogen cakes and inoculated into the center of a potato glucose medium plate. 0.2L of fresh bacterial solution of the antagonistic bacteria to be tested was inoculated 2.5cm away from the mycelium block. The plates were slightly dried and cultured in a 28°C incubator. Each treatment was repeated 3 times. The pathogens were inoculated alone as the control. When the control just covered the culture dish, the inhibition rate of the antagonistic strains on the pathogens was measured and calculated.

[0036] The antibacterial rate calculation formula is as follows:

[0037] Bacterial inhibition rate (%) = (control pathogen colony diameter - treatment pathogen colony diameter) / control pathogen colony diameter × 100%

[0038] Table 2: Inhibition rate of SD13 against five pathogens

[0039]

[0040] Note: The data in the table are mean values ​​and standard deviations. Different lowercase letters in the same column indicate significant differences at the 0.05 level.

[0041] The results of the plate confrontation experiment showed that the inhibition rate of SD13 against five pathogens, including cowpea wilt, cucumber wilt, pepper wilt, melon wilt, and bitter melon wilt, reached 66.7% to 82.3%, indicating that SD13 has a broad-spectrum antibacterial activity and strong antibacterial ability.

[0042] Example 3- Effect of SD13 combined with different organic fertilizers on salt tolerance of cowpea

[0043] There are 8 treatments in total:

[0044] (1) Blank control (CK);

[0045] (2) 50 mM NaCl (50 mM);

[0046] (3) 50mM NaCl+ strain SD13 (50mM+B);

[0047] (4) 50 mM NaCl + 10% earthworm manure (50 mM + W);

[0048] (5) 50 mM NaCl + strain SD13 + 10% earthworm manure (50 mM + B + W);

[0049] (6) 50 mM NaCl + 10% sheep manure (50 mM + S);

[0050] (7) strain SD13 + 10% sheep manure (50 mM + B + S);

[0051] (8) 50 mM NaCl + strain SD13 + 5% earthworm manure + 5% sheep manure (50 mM + B + W + S).

[0052] Experimental steps: Mix earthworm manure and sheep manure with soil evenly. Transplant cowpea seedlings into pots, add 800mL of 50mmol / LNaCl, set clear water as blank control, ensure that the soil is completely soaked and no water overflows from the pot. Add 5mL of 13 bacterial solution. Cultivate in a greenhouse. Water once every 3 days, 200mL / pot each time, place a tray at the bottom of the pot, if water overflows from the bottom of the pot, pour it back into the pot in time, finally add 1×106CUF / mLSD if water overflows from the bottom of the pot and pour it back into the pot in time. Water cowpea seedlings once a day after 5 true leaves. After 45 days, the aboveground dry weight, underground fresh weight, chlorophyll content, relative water content, electrolyte permeability, malondialdehyde, and proline of cowpea plants were measured.

[0053] Table 3: Effects of strain SD13 combined with different organic fertilizers on salt tolerance of cowpea

[0054]

[0055] Note: The data in the table are mean values ​​and standard deviations. Different lowercase letters in the same column indicate significant differences at the 0.05 level.

[0056] Table 3 shows that compared with the 50mMNaCl treatment, the aboveground dry weight and underground dry weight of cowpea plants in the 50mM+B treatment can increase by 19% and 7%, respectively. However, the 50mM+B+W treatment or 50mM+B+S treatment further significantly increased the aboveground dry weight and fresh weight of cowpea plants; compared with the blank control, the relative water content of cowpea after 50mMNaCl treatment decreased significantly, by 2%; compared with the 50mMNaCl treatment, the relative water content of cowpea after 50mM+B treatment, 50mM+W treatment, 50mM+S treatment and 50mM+B+S treatment was higher, but the difference was not significant. However, the relative water content of cowpea increased significantly by 25% after 50mM+B+W treatment; as shown in Figure 18, compared with the blank control, the plasma membrane permeability of cowpea treated with 50mMNaCl was higher, but the difference was not significant; compared with the 50mMNaCl treatment, the 50mM+B, 50mM+W, 50mM+S, 50mM+B+S and 50mM+B+W treatments could reduce the electrolyte permeability, but the effect was not significant, among which the 50mM+B treatment had the best effect; compared with the blank control, the total chlorophyll of cowpea leaves treated with 50mMNaCl decreased by 41%; compared with the 50mMNaCl treatment, the total chlorophyll of 50mM+B, 50mM+W, 50mM+B+W, 50mM+S and 50mM+B+S treatments increased by 1.3% and 1.5%, respectively. 88%, 70%, 71%, 95% and 75% higher, and there was no significant difference between them; compared with the blank control, the MDA content of cowpea treated with 50mMNaCl increased significantly by 149%; compared with the 50mMNaCl treatment, the 50mM+B treatment could reduce the MDA content of cowpea by 53%; compared with the 50mM+B treatment, the 50mM+B+W treatment could further reduce the MDA content to 81%; compared with the blank control, the MDA content of cowpea treated with 50mMNaCl increased significantly by 149%; compared with the 50mMNaCl treatment, the 50mM+B treatment could reduce the MDA content of cowpea by 53%; compared with the 50mM+B treatment, the 50mM+B+W treatment could further reduce the MDA content to 81%. The results show that strain SD13 can promote plant growth and improve plant salt tolerance under salt stress, and can be used to prepare bio-organic fertilizer to further improve plant salt tolerance.

[0057] Example 4 - Control effect of strain SD13 combined with organic fertilizer on cowpea wilt under NaCl stress A total of 8 treatments were set:

[0058] (1) Control (CK);

[0059] (2) Pathogen (F);

[0060] (3) Pathogen + strain SD13(F+B);

[0061] (4) pathogens + 10% earthworm manure (F+W);

[0062] (5) pathogen + strain SD13 + 10% earthworm manure (F+B+W);

[0063] (6) pathogens + 10% sheep manure (F+S);

[0064] (7) pathogen + strain SD13 + 10% sheep manure (F+B+S);

[0065] (8) Pathogen + strain SD13 + 5% earthworm manure + 5% sheep manure (F+B+W+S).

[0066] Experimental steps: Add 2×10 5 CUF / mL cowpea wilt pathogen spore suspension, then add earthworm manure and sheep manure, and mix evenly with the soil. Cut the 0.5cm root tip of the cowpea seedling and plant it in the pot, add 50mM NaCl 800mL, set clear water as a blank control, and ensure that the soil is completely soaked and no water overflows from the pot. Place a tray at the bottom of the pot, and if water overflows from the bottom of the pot, pour it back into the pot in time, and finally add 1×10 6 5mL of CUF / mLSD13 bacterial solution. Cultivate in a greenhouse. Water once every 3 days, 200mL / pot each time. If water overflows from the bottom of the pot, pour it back into the pot in time. Water the cowpea seedlings once a day after they have 5 true leaves. Observe and record the disease situation after 45 days, and calculate the disease index and control effect.

[0067] Table 4: Evaluation criteria for identification of resistance to Fusarium wilt in cowpea

[0068]

[0069]

[0070] Incidence rate (%) = number of diseased plants / total number of plants × 100%

[0071] Disease index = ∑ (number of diseased plants at each level × value of the disease level) / (total number of plants × highest level value) × 100%

[0072] Control effect (%) = (CK disease index - treatment disease index) / CK disease index × 100%

[0073] Table 5: Control effect of strain SD13 combined with organic fertilizer on cowpea wilt under NaCl stress

[0074]

[0075] Note: The data in the table are mean values ​​and standard deviations. Different lowercase letters in the same column indicate significant differences at the 0.05 level.

[0076] The results showed that under 0mMNaCl stress, compared with the pathogen control treated with F, all treatments significantly reduced the disease index of cowpea wilt, among which the disease index of treatments containing strain SD13 F+B, F+B+W, F+B+S and F+B+W+S was the lowest, with control effects of 86%, 78%, 75% and 78%, respectively. Under 50mMNaCl stress, compared with the F treatment, the cowpea wilt disease index of treatments F+B, F+B+W and F+B+W+S with strain SD13 significantly decreased, and the control effect was the best, with control effects of 78.0%, 76.6% and 80.8%, indicating that strain SD13 can also effectively prevent and control cowpea wilt under salt stress.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A salt-tolerant Bacillus Velez subtilis SD13, characterized in that: The Bacillus velezensis SD13 is deposited in the China Center for Type Culture Collection, and its strain collection number is CCTCC No: M2023570, and the collection date is April 21, 2023.

2. The salt-tolerant Bacillus Velez subtilis SD13 according to claim 1, characterized in that: The gene sequence of Bacillus velezensis SD13 is shown in SEQ ID NO:

1.

3. Application of a salt-tolerant Bacillus Velez subtilis SD13 in agricultural biological control.

4. The use according to claim 3, characterized in that: The salt-tolerant Bacillus Velez SD13 is used for plant diseases.

5. The use according to claim 4, characterized in that: The salt-tolerant Bacillus Velez SD13 is used for preventing and controlling plant diseases in saline-alkali land.

6. The use according to claim 4 or 5, characterized in that: The plant diseases include but are not limited to cowpea wilt, cucumber wilt, pepper wilt, melon wilt, and bitter melon wilt.

7. Application of a salt-tolerant Bacillus Velez subtilis SD13 in combination with organic fertilizers to improve plant salt tolerance.

8. The use according to claim 7, characterized in that: The salt-tolerant Bacillus Velez SD13 is used in combination with organic fertilizer to improve the salt tolerance of cowpea.

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