Bacillus tequilensis and application thereof

Abstract

The invention discloses bacillus tequilensis P + 20 and application thereof, and relates to the technical field of microorganisms, and the preservation number of the bacillus tequilensis P + 20 is CGMCC (China General Microbiological Culture Collection Center) NO.32143. The invention further discloses a preparation method of the bacillus tequilensis P + 20. The P + 20 is separated from boxthorn rhizosphere soil, is a strain with strong comprehensive capacity, has strong salt resistance, has the functions of fixing nitrogen, dissolving inorganic phosphorus, dissolving organic phosphorus and dissolving potassium, can antagonize pathogenic bacteria such as fusarium solani, fusarium oxysporum, fusarium graminearum, fusarium moniliforme and aspergillus niger, can secrete auxin, can be developed as a microbial fertilizer for saline-alkali soil, and has a wide application prospect. The prospect is wide.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to a strain of Bacillus equilensis P+20 and application thereof. Background Art

[0002] Saline-alkali land is an important reserve arable land resource in my country. The rational development and utilization of saline-alkali land is of great significance to ensuring the supply of agricultural products, improving the ecological environment and promoting the sustainable development of regional agriculture. Compared with traditional physical and chemical improvement measures such as freshwater salt reduction and application of chemical improvers, the application of microbial fertilizers to promote crop growth in saline-alkali land is a low-cost and environmentally friendly biological improvement measure with good application prospects and is also a hot topic in current research.

[0003] The development of microbial fertilizers suitable for saline-alkali land is crucial for improving and utilizing saline-alkali land. The key lies in screening beneficial strains that have the functions of promoting plant growth, antagonizing pathogens, and adapting to saline adversity. The patent "Production method and application of a salt-alkali tolerant Siamese Bacillus and its live bacterial preparation" (application number: 202010761301.1) provides a Siamese Bacillus isolated from rice rhizosphere soil. Its bacterial agent has a high preventive effect on rice seedling disease caused by Fusarium fusae, and promotes the growth of rice in saline-alkali land. The patent "A salt-alkali tolerant Marxist Bacillus and its application" (application number: 201910931685.4) provides a Marxist Bacillus isolated from reed rhizosphere soil. The strain has strong salt-alkali tolerance, has the functions of decomposing protein, dissolving inorganic phosphorus, and antagonizing a variety of plant pathogenic fungi. Its bacterial agent promotes the growth of corn in saline soil. The patent "A strain of salt-alkali tolerant polymyxa bacillus and its application" (application number: 201810952171.2) provides a strain of polymyxa bacillus isolated from cotton rhizosphere soil. The strain has strong salt-alkali tolerance, effectively decomposes self-toxic substances, alleviates continuous cropping obstacles, and its bacterial agent promotes cotton growth in saline soil. The rhizosphere is a hot spot for the interaction between plants and soil microorganisms, and plant growth-promoting bacteria are also mainly isolated from rhizosphere soil, but related patents tend to isolate strains with strong single functions in a targeted manner, and have not yet paid attention to strains with complex functions such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and antagonism of pathogens.

[0004] Wolfberry is a halophyte with high economic value and is widely used in saline-alkali land cultivation. However, there are currently few microorganisms with comprehensive growth-promoting capabilities for wolfberry cultivation in saline-alkali land. Therefore, it is an urgent problem for those skilled in the art to explore more microbial resources that can be applied to wolfberry cultivation in saline-alkali land. Summary of the invention

[0005] In view of this, the present invention provides a strain of Bacillus tequilensis P+20 and applications thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A Bacillus tequilensis P+20, the P+20 has a deposit number of CGMCC NO. 32143. It was deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms on October 9, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and was classified and named Bacillus tequilensis.

[0008] Another object of the present invention is to provide a microbial agent comprising the above-mentioned Bacillus tergillosis P+20.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned microbial agent, wherein the terquila Bacillus P+20 is expanded and cultured in LB liquid medium, then cultured in a solid fermentation medium using apple pomace as a raw material, and then dried at 40-45°C and crushed to obtain the microbial agent;

[0010] Wherein, potassium dihydrogen phosphate is also added to the solid fermentation medium.

[0011] Preferably, the Bacillus tergrillii P+20 is cultured in a solid fermentation medium at a temperature of 37° C. for 3 days.

[0012] Preferably, the added amount of potassium dihydrogen phosphate is 0.2%.

[0013] Preferably, the apple pomace is the residue after fresh apples are crushed and squeezed to produce juice and pectin is further extracted, and 0.15% slaked lime is added to the apple pomace, and then physically squeezed to a moisture content of 70%, further dried, and crushed through a 20-mesh sieve to obtain the obtained product.

[0014] Another object of the present invention is to provide the use of the above-mentioned Bacillus tergillosis P+20 or the above-mentioned microbial agent in promoting the growth of plants in saline soil.

[0015] Another object of the present invention is to provide the use of the above-mentioned Bacillus tergillosis P+20 or the above-mentioned microbial agent in saline soil improvement, wherein the soil improvement is one or more of nitrogen fixation, inorganic phosphorus decomposition, organic phosphorus decomposition, and potassium decomposition.

[0016] Another object of the present invention is to provide the use of the above-mentioned Bacillus tergillosis P+20 or the above-mentioned microbial agent in the preparation of a soil pathogen antagonist, wherein the soil pathogen is one or more of Fusarium solani, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme, Phytophthora capsici and Aspergillus niger.

[0017] Another object of the present invention is to provide a method for promoting the growth of wolfberry in saline soil, using the above-mentioned Bacillus tergillium P+20 or the above-mentioned microbial agent as base fertilizer applied to the root soil or diluted 200-1000 times for root irrigation, root dipping or seed mixing.

[0018] Beneficial effects: The Bacillus tergillosis P+20 of the present invention is separated from the rhizosphere soil of wolfberry, is a strain with strong comprehensive ability, has strong salt tolerance, and has the functions of nitrogen fixation, inorganic phosphorus decomposition, organic phosphorus decomposition and potassium decomposition. It can antagonize pathogens such as Fusarium solani, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme and Aspergillus niger, and can secrete auxin. It can be developed as a microbial fertilizer for saline-alkali land and has broad prospects.

[0019] The present invention successfully prepared the tekila bacillus P+20 bacterial agent using organic waste apple pomace as raw material, and the number of live bacteria reached 10 11 / g, which is helpful for the resource utilization of waste and protection of the environment. In addition, the application of this fungus agent can effectively promote the growth of wolfberry in saline soil, and then serve the cultivation of wolfberry in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The colony morphology (left) and microscopic morphology (right) of Bacillus tequila P+20 on LB solid medium.

[0022] Figure 2 Bacillus tequila P+20 can maintain growth on LB solid medium containing 10% NaCl.

[0023] Figure 3 This is the growth status of Bacillus tequila P+20 inoculated in nitrogen-free Ashby medium, inorganic phosphorus medium, organic phosphorus medium and silicate medium.

[0024] Figure 4 This is the antagonistic effect of Bacillus tequila P+20 on pathogens. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1 Isolation, identification and preservation of strain P+20

[0027] 1. Separation

[0028] Weigh 10 g of Lycium barbarum rhizosphere soil from the coastal saline-alkali land of the Yellow River Delta, place it in a triangular flask containing glass beads and 90 mL of sterile water, shake it vigorously on a culture shaker for 30 min, draw 1 mL of the suspension, and dilute it with sterile water in a gradient manner, with the dilution concentrations being 10 -1 , 10 -2 , 10 -3 , 100 μL of three gradient dilutions were respectively spread on solid nitrogen-free Ashby medium, inorganic phosphorus medium, organic phosphorus egg yolk medium and silicate medium, and inverted in a 37°C incubator for culture. After 4 days, single colonies of different shapes on the four culture media were picked up, and they were streaked and purified on the corresponding culture media until no foreign bacteria grew. A total of 108 strains of bacteria were purified, and then inoculated into various screening plates, and 29 strains of bacteria with the comprehensive ability of nitrogen fixation, organic / inorganic phosphorus and potassium solubilization were obtained.

[0029] Ashby nitrogen-free medium: mannitol 10.0 g, KH2PO4 0.2 g, MgSO4 0.2 g, NaCl 0.2 g, CaSO4 0.1 g, CaCO3 5 g, deionized water 1 L, pH 6.8-7.0, when used for solid culture medium, add 1.5% agar.

[0030] Inorganic phosphorus culture medium: glucose 10g, (NH4)2SO40.5g, NaCl 0.3g, KCl0.3g, FeSO4·7H2O0.003g, MnSO4·4H2O 0.003g, MgSO4·7H2O 0.3g, Ca(PO4)25g, deionized water 1L, pH 7.2, when used for solid culture medium, add 1.5% agar.

[0031] Organophosphorus egg yolk culture medium: glucose 10g, (NH4)2SO40.5g, NaCl 0.3g, KCl 0.3g, FeSO4·7H2O 0.03g, MnSO4·4H2O 0.03g, egg yolk lecithin 0.2g, CaCO35g, yeast extract 0.4g, deionized 1L, pH 7.0-7.5, add 1.5% agar when used for solid culture medium.

[0032] Silicate culture medium: sucrose 5g, Na2HPO42 g, MgSO4·7H2O 0.005g, FeCl30.005 g, CaCO30.1g, potassium feldspar powder 1g (potassium feldspar powder needs to pass through a 1000 mesh sieve, then soak in deionized water overnight, then rinse repeatedly several times, and finally dry in the shade for later use), deionized water 1L, pH7.0-7.5, add 1.5% agar when used for solid culture medium.

[0033] 2. Screening and identification

[0034] The strains with the ability to fix nitrogen, decompose organic / inorganic phosphorus and decompose potassium were inoculated into 8ml LB liquid medium, and cultured overnight at 30℃ and 180r / min with shaking to prepare seed liquid. LB medium: 10g peptone, 5g yeast extract, 10g NaCl, 1L deionized water, and 1.5% agar when used for solid culture medium. 1.5ml of the seed liquid was inoculated into 100ml conical flasks containing 50ml nitrogen-free Ashby liquid medium at an inoculation rate of 3%, and cultured at 30℃ and 180r / min with shaking for 7d. After the fermentation liquid was taken, 4mL 6% H2O2 was added, and the high temperature digestion treatment was carried out for 20min, and the supernatant was taken to determine its absorbance value, and finally the nitrogen fixation amount was calculated according to the N standard curve.

[0035] The strains with the ability to fix nitrogen, decompose organic / inorganic phosphorus and decompose potassium were inoculated into 8ml LB liquid medium, cultured at 30℃ and 180r / min overnight to prepare seed solution. According to the inoculation amount of 3%, 1.5ml of seed solution was inoculated into 100ml conical flasks containing 50ml Ca3(PO4)2 and lecithin liquid medium, and cultured at 30℃ and 180r / min for 7 days. After the culture, the fermentation liquid was transferred to the centrifugation at 4℃ and 8000r / min for 10min. After centrifugation, the supernatant was collected and the OD was determined by molybdenum blue colorimetry. 700 Finally, the amount of phosphorus dissolved was calculated according to the available phosphorus standard curve, and the content of available phosphorus in the supernatant was determined.

[0036] The strains with the ability to fix nitrogen, decompose organic / inorganic phosphorus and decompose potassium were inoculated into 8ml LB liquid medium, and cultured overnight at 30℃ and 180r / min with shaking to prepare seed solution. According to the inoculation amount of 3%, 1.5ml seed solution was inoculated into a 100ml conical flask containing 50mL potassium-deficient liquid medium, and cultured in a shaking incubator at 180r / min and 30℃ for 7 days. After the fermentation, 20mL of the fermentation liquid was taken, 4mL of 6% H2O2 was added, and the mixture was digested at high temperature for 20min, centrifuged at 8000r / min for 10min, and the supernatant was taken to determine the K in the supernatant using a flame atomic spectrophotometer. + The content.

[0037] The principal component analysis method in SPSS17.0 software was used to extract two principal components based on the nitrogen fixation, organic / inorganic phosphorus and potassium solubilization of the strains. The variance explanation rates were 38.791% and 26.953%, respectively, and the cumulative variance explanation rate was 65.744%. The "component score coefficient matrix" was used to establish the relationship equation between the principal components and the research items. The variance explanation rate was multiplied by the component score and then accumulated to obtain the comprehensive score. The strain with the highest comprehensive score was the strain P+20 with the strongest combined ability of nitrogen fixation, organic / inorganic phosphorus and potassium solubilization.

[0038] The morphological characteristics of the strain on LB solid medium are that the colonies are gray and smooth, white and wrinkled, with irregular and sticky edges, high elasticity, can be pulled into filaments, and are difficult to pick up. The microscopic morphology of the bacteria is rod-shaped and Gram-positive (see Appendix Figure 1 ). Through 16S rDNA sequencing and sequence alignment, combined with the above morphological characteristics, the strain was identified as Bacillus equilensis.

[0039] 16S rDNA:

[0040]

[0041] 3. Preservation

[0042] On October 9, 2024, strain P+20 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was classified and named Bacillus equilensis, with the deposit number CGMCC No.32143.

[0043] Example 2 Functional assay of strain P+20

[0044] 1. Salt tolerance

[0045] The strain P+20 was inoculated on LB solid medium containing 10% NaCl and cultured at 37°C, and it was still able to maintain growth, indicating that the strain has a high salt tolerance and can adapt to saline-alkali environments (see Appendix Figure 2 ).

[0046] 2. Ability to fix nitrogen, release phosphorus and release potassium

[0047] After inoculating Bacillus tequila P+20 into nitrogen-free Ashby liquid medium and culturing for 7 days, the nitrogen content in the fermentation broth was 0.165 mg / L.

[0048] When Bacillus tekila P+20 was inoculated into inorganic phosphorus and organic phosphorus liquid culture media and cultured for 7 days, the phosphorus contents in the fermentation broth were 120 mg / L and 116 mg / L, respectively.

[0049] After inoculating Bacillus tekila P+20 into silicate liquid culture medium and culturing for 7 days, the potassium content in the fermentation broth was 11.7 mg / L.

[0050] The growth of Bacillus tekila P+20 inoculated on solid nitrogen-free Ashby medium, inorganic phosphorus medium, organic phosphorus medium and silicate medium at 37°C for 3 days is shown in the attached figure. Figure 3 .

[0051] 3. Ability to secrete growth hormone

[0052] Bacillus tekila P+20 was inoculated into 8 ml LB liquid culture medium, cultured overnight at 30°C and 180 r / min to prepare seed solution, which was inoculated into LB liquid culture medium containing L-tryptophan (200 mg / L), and cultured for 4 days at 30°C and 180 r / min. After 4 days, the culture solution was taken and centrifuged for 10 min at 4°C and 8000 r / min. 2 mL of supernatant was taken into a centrifuge tube, and an equal volume of Salkowski colorimetric solution was added. After standing for 30 min in a dark environment to allow color to develop, the absorbance was quickly measured at 530 nm, and a blank was used as a control. Finally, the IAA production was calculated to be 1.1 mg / L according to the IAA standard curve.

[0053] 4. Ability to antagonize pathogens

[0054] The confrontation culture method was used. The pathogenic fungi cake (Fusarium solani, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme, Phytophthora capsici and Aspergillus niger) was inoculated in the center of the PDA plate (potato extract powder 10g, glucose 20g, agar 20g, distilled water 1L), and the P+20 strain was inoculated on both sides of the circle with a radius of 3cm with the center of the cake as the center. The plate was cultured at 28℃ for 7 days, and the inhibition zone was observed and the inhibition radius was determined (see Appendix). Figure 4 ). The results showed that the inhibition radius of the P+20 strain against Fusarium solani, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme, Phytophthora capsici and Aspergillus niger was 0.78cm, 1.0cm, 0.60cm, 0.80cm, 0.78cm and 0.70cm, respectively, which was effective in antagonizing the above pathogens.

[0055] Example 3 Preparation and growth promotion verification of P+20 bacterial agent

[0056] 1. Preparation of bacterial agent using apple pomace as raw material

[0057] Apple pomace is the residue after fresh apples are crushed and squeezed to produce juice and further extract pectin. A small amount of slaked lime (0.15%) is added to the apple pomace, and the apple pomace is physically squeezed to a moisture content of about 70% using a filter press dehydrator to reduce viscosity and increase dispersibility, and then further dried and crushed through a 20-mesh sieve for later use.

[0058] Weigh the crushed apple pomace, add sterile water dissolved with potassium dihydrogen phosphate and P+20 bacterial solution (fermentation 12h, OD 600 =1.091), stir evenly, spread in a sterile container (thickness of about 1-2 cm), ferment at 37°C, dry at 40-45°C until the moisture content is ≤30%, and crush to 60 mesh to prepare a bacterial agent.

[0059] The inoculation amount, potassium dihydrogen phosphate addition amount, and fermentation days were the three main factors. Each factor was set at three levels. The L9 (33) orthogonal table was used for orthogonal experiments. The number of viable bacteria was determined by the dilution plate method. Each combination was repeated three times and the average value was taken (Table 1). The results of the range analysis showed that the optimal conditions were 0.2% potassium dihydrogen phosphate application, 2.5% inoculation amount, and 3 days of fermentation. The number of viable bacteria reached 1.72×10 11 Pieces / g.

[0060] The method of using the live bacteria preparation of Bacillus tequila P+20 is to use about 200 grams of the agent per mu. It can be applied directly to the roots as a base fertilizer, or diluted 200-1000 times with water before root irrigation, root dipping, and seed mixing.

[0061] Table 1 Orthogonal table of fermentation test conditions

[0062]

[0063] Note: K is the average number of viable bacteria at different levels of each factor (individuals / g). The inoculum volume is the ratio of its volume to the mass of apple pomace (ml / g), and the amount of potassium dihydrogen phosphate added is the ratio of its volume to the mass of apple pomace (g / g).

[0064] 2. The effect of bacterial agents on the growth of wolfberry

[0065] Soil was collected from the coastal saline-alkali land of the Yellow River Delta and potted experiments were carried out. The soil conductivity and pH value were 1.6dSm -1 and 7.23, and the soil total phosphorus, total nitrogen, total carbon, and organic carbon were 0.10, 0.16, 11.69, and 1.62 g·kg, respectively. -1 , nitrate nitrogen, ammonium nitrogen, available phosphorus, and available potassium were 2.31, 17.03, 1.39, and 107.79 mg·kg, respectively. -1 .

[0066] Select wolfberry branches about 20 cm long and plant them in a hydroponic tank. Replace the Hoagland nutrient solution (pH 5.7) every 7 days to promote rooting and germination of the branches and maintain plant growth and development. The hydroponic tank is ventilated and placed in a culture room with a light cycle of 12 h each for light / darkness. The temperature during light and dark periods is set at 25 and 18 °C, and the light intensity is 200 μmol m -2 s -1, the air humidity was 70%. After 30 days, healthy seedlings with consistent growth were selected and transplanted into plastic pots (10 cm in diameter and 15 cm in height). Each pot was filled with 300 g of soil (Yellow River Delta coastal saline-alkali soil), one plant per pot, and divided into 2 groups, with 5 pots in each group. One group of seedlings was applied with 0.1 g of Bacillus tekila P+20 bacterial agent to the roots, and the other group was applied with 0.1 g of inactivated bacterial agent as a control. The plants were collected after 35 days, dried and weighed. As shown in Table 2, the dry weight of the leaves and aboveground parts of the seedlings applied with the live bacterial preparation of Bacillus tekila P+20 were significantly higher than those of the control group, indicating that the bacterial agent can effectively promote the growth of wolfberry in saline soil.

[0067] Table 2 Biomass of potted plants in saline soil

[0068]

[0069] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0070] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Bacillus tequilensis P+20, characterized in that The deposit number of the P+20 is CGMCC NO.32143.

2. A microbial agent, characterized in that: It comprises the Bacillus terequina P+20 described in claim 1.

3. The method for preparing the microbial agent according to claim 2, characterized in that: After the tekila bacillus P+20 is expanded and cultured in LB liquid medium, it is transferred to a solid fermentation medium with apple pomace as raw material for culture, and then dried at 40-45°C and crushed to obtain; Wherein, potassium dihydrogen phosphate is also added to the solid fermentation medium.

4. The preparation method according to claim 3, characterized in that: The tequila bacillus P+20 is cultured in a solid fermentation medium at a temperature of 37° C. for 3 days.

5. The preparation method according to claim 3, characterized in that: The added amount of potassium dihydrogen phosphate is 0.2%.

6. The preparation method according to claim 3, characterized in that: The apple pomace is the residue after fresh apples are crushed and squeezed to produce juice and pectin is further extracted. 0.15% slaked lime is added to the apple pomace, and then the pomace is physically squeezed to a moisture content of 70%, further dried, and crushed to pass through a 20-mesh sieve.

7. Use of the Bacillus tequila P+20 described in claim 1 or the microbial agent described in claim 2 in promoting plant growth in saline soil.

8. Use of the Bacillus tequila P+20 of claim 1 or the microbial agent of claim 2 in improving saline soil, characterized in that: The soil improvement is one or more of nitrogen fixation, inorganic phosphorus solubilization, organic phosphorus solubilization, and potassium solubilization.

9. Use of the Bacillus tergillosis P+20 of claim 1 or the microbial agent of claim 2 in the preparation of an antagonist of soil pathogens, characterized in that: The soil pathogenic bacteria are one or more of Fusarium solani, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme, Phytophthora capsici and Aspergillus niger.

10. A method for promoting the growth of wolfberry in saline soil, characterized in that: The Bacillus tequila P+20 described in claim 1 or the microbial agent described in claim 2 is applied to the root soil as a base fertilizer or diluted 200-1000 times for root irrigation, root dipping or seed mixing.

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