Bacillus altitudinis DXT2-4 and application thereof

Abstract

The invention belongs to the technical field of bioengineering. The invention discloses a bacillus altitudinis strain DXT2-4 CGMCC (China General Microbiological Culture Collection Center) NO.25810. The bacillus altitudinis strain DXT2-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC for short) on June 29, 2023, the registration number of the preservation center is CGMCC NO.25810, the preservation date is June 29, 2023, and the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacillus altitudinis strain DXT2-4 provided by the invention is simple to culture, short in period, free of ecological toxicity, rich in available carbon source variety and high in salt tolerance, can promote growth and development of a rice root system and produce cellulase to decompose cellulose through phosphorus solubilizing, siderophore producing and the like, is beneficial to soil remediation, can effectively inhibit growth of streptomyces scabies, and has a good application prospect. The resistance is good.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering. Background Art

[0002] The antibacterial substances produced by Bacillus generally have a wide antibacterial spectrum, and have the functions of binding lipopolysaccharide and neutralizing endotoxin. The metabolite of Bacillus, poly-γ-glutamic acid, can be used to degrade plastics, and can also be used as a desert water retainer to treat industrial wastewater and degrade toxic substances in the environment, such as polycyclic aromatic hydrocarbons, petroleum, organophosphorus pesticides, and organochlorine pesticides. It has high environmental compatibility and heavy metal ion adsorption, and can improve the physical and chemical properties of the soil, playing an important role in plant disease prevention and soil remediation.

[0003] Bacillus altitudinis is widely distributed in various habitats such as soil, plant surfaces, water bodies, and agricultural waste. It has a broad spectrum of antibacterial activity, high spore yield, and strong adaptability to adversity, and is considered to be one of the most promising beneficial microorganisms for application and development. Bacillus altitudinis is a new species of the genus Bacillus, which has antagonistic activity against a variety of pathogenic fungi and can effectively inhibit the growth of a variety of pathogenic fungi such as Sclerotium rolfsii, Solanum solani, Rhizoctonia solani, and Colletotrichum gloeosporioides. Summary of the invention

[0004] In view of this, the first object of the present invention is to provide a strain of Bacillus altitudinis DXT2-4, whose deposit number is CGMCC NO.25810.

[0005] The Bacillus altitudinis DXT2-4 provided by the present invention has been deposited in the General Microbiological Culture Collection Center of China General Microbiological Culture Collection Committee, with a deposit number of CGMCC No. 25810, a deposit date of June 29, 2023, and a depository referred to as CGMCC. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The second object of the present invention is to provide the use of Bacillus altitudinis DXT2-4 in the preparation of a bacterial agent for inhibiting the growth of potato scab pathogens, in the preparation of a microbial saline-alkali land improver, in the preparation of a bacterial agent for converting ineffective phosphorus into effective phosphorus that can be absorbed by plants, in the preparation of a bacterial agent for producing iron carriers, in the preparation of a preparation for promoting the growth and development of plant roots, and in the preparation of a bacterial agent for preventing and controlling potato scab.

[0007] In a specific embodiment of the present invention, in a specific embodiment of the present invention, the plant is rice.

[0008] The Bacillus altitudinis DXT2-4 provided by the present invention is simple to culture, has no ecotoxicity, is salt-alkali resistant, has a rich variety of available carbon sources, can decompose insoluble phosphorus in the soil, can produce cellulase, siderophore, etc., and can effectively inhibit the growth of pathogenic bacteria of potato scab and promote the growth and development of rice roots. It has very broad application prospects in biological control of potato scab and plant root growth-promoting preparations. Description of the drawings:

[0009] Figure 1 This is an electron micrograph of Bacillus altitudinis DXT2-4.

[0010] Figure 2 This is a staining image of Bacillus altitudinis DXT2-4.

[0011] Figure 3 It is a graph of the antagonism of Bacillus altitudinis DXT2-4.

[0012] Figure 4 This is a graph showing the results of determination of phosphate solubilization and siderophore production by Bacillus altitudinis DXT2-4.

[0013] Figure 5 This is the result of the determination of cellulase production by Bacillus altitudinis DXT2-4

[0014] Figure 6 This is a potted plant result diagram showing the control effect of Bacillus altitudinis DXT2-4 on potato scab.

[0015] Figure 7 This is a diagram of how Bacillus altitudinis DXT2-4 promotes the growth and development of rice roots. Specific implementation method:

[0016] Unless otherwise specified, the strains to be tested in the following examples all refer to Bacillus saltitudinis DXT2-4.

[0017] Unless otherwise specified, the activation in the following examples refers to the expansion culture of Bacillus altitudinis DXT2-4 on PDA solid medium for three times.

[0018] Example 1

[0019] Isolation and identification of bacterial species

[0020] 1. Culture medium:

[0021] Potato Dextrose agar (PDA) medium (g / L): 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, natural pH.

[0022] 2. Source of strain:

[0023] Bacillus altitudinis DXT2-4 was isolated by the inventor from potato soil in Baise, Guangxi on November 15, 2022. Take 2g of soil sample and put it into a 50ml centrifuge tube, add 20ml of sterile water and shake evenly, and let it stand until it is clear. The prepared soil suspension was diluted in a clean bench, and 100μL 10 -5 The 100-fold dilution was spread on LB solid medium and cultured at 37°C for 24 hours. The obtained pure single colony was transferred to the test tube slant medium. Figure 1 and Figure 2 As shown, the strain classification characteristics: the colony morphology is round and milky white. The strain is a Gram-positive bacterium with a rod-shaped cell shape. It can decompose starch to produce sugar as a carbon source and decompose gelatin to produce styrene and ethylene glycol, which is consistent with the physiological and biochemical characteristics of Bacillus strains and can utilize mannitol, mannose and starch. The strain was named DXT2-4.

[0024] 3. Bacterial strain identification method:

[0025] The TIANGEN bacterial genomic DNA extraction kit was used to extract genomic DNA. The genomic DNA of the strain to be tested was used as a template, and the bacterial 16S rRNA gene universal primer 27F / 1492R was used to amplify the target fragment. The PCR product was detected by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Bomed Biotechnology Co., Ltd. for sequencing. 16S rRNA gene sequence analysis, after sequence comparison and identification, strain DXT2-4 was Bacillus altitudinis DXT2-4 with NPB34B (MT598007.1 NCBI) as the standard strain, and the sequencing result was shown in SEQ ID NO.1.

[0026] This strain has been deposited in the General Microbiology Center of China General Microbiological Culture Collection Committee, with the deposit number: CGMCC No.25810, the deposit date: June 29, 2023, the depository is abbreviated as CGMCC, and the depository address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] Example 2

[0028] Physiological and biochemical analysis of strains

[0029] The physiological and biochemical indicators of strain DXT2-4 were tested using the BioMerieux API 20NE non-fermenting G+ bacillus identification kit. Bacillus altitudinis DXT2-4 can utilize arabinose, maltose, glucose, etc. Glucose acidification test, glucose assimilation test, oxidase test, catalase test, starch hydrolysis, and tryptophan test were all positive. VP test and oxalic acid test were both negative. The detailed results are as follows:

[0030] Table 1 Analysis of physiological and biochemical properties

[0031]

[0032] Note: +: positive reaction; -: negative reaction

[0033] Example 3

[0034] Inhibitory effect of strains on Streptomyces scab

[0035] Potato scab pathogen 4.1765 (purchased from China General Microbiological Culture Collection Center (CGMCC), with the collection number: CGMCC NO.4.1765) was cultured on potato culture medium at 28°C for 5 days; spores and mycelium were scraped to make a pathogen suspension of a certain concentration. The activated strain DXT2-4 and 100 μl 4.1765 were used for plate confrontation experiment, cultured at 28°C, and observed after 3 days. Figure 3 The experimental results shown are as follows: Bacillus altissitudinis DXT2-4 can effectively inhibit the growth of the experimental potato scab pathogen 4.1765. The diameter of the inhibition zone is 28.4 mm.

[0036] Example 4

[0037] Salt tolerance test of strains

[0038] The basic culture medium was LB liquid culture medium. NaCl was added to the LB solid culture medium to adjust the salt concentration to 1%, 3%, 5%, 9%, and 11%, and the pH was adjusted to 7.0. The medium was sterilized by high pressure at 121° C. for 20 min.

[0039] The activated strain DXT2-4 to be tested was inoculated in LB liquid culture medium with five different salt concentrations, and the culture medium without inoculation was used as a blank control. It was placed in a constant temperature incubator at 37°C for 24 hours to observe the growth of strain DXT2-4. The OD value was measured. The results are as follows. With the increase of NaCl concentration, the concentration of strain DXT2-4 first increased and then decreased, indicating that DXT2-4 is more suitable for a saline environment. When the salt concentration reaches 9%, it can still grow normally. It proves that the strain can be used in saline-alkali land improvement, has salt tolerance, and is suitable as a microbial saline-alkali land improver.

[0040] Table 2 Salt tolerance results of DXT2-4

[0041] NaCl 1％ 3％ 5％ 7％ 9％ 11％ <![CDATA[OD 600 ]]> 1.142 1.212 1.399 1.326 1.296 0.048

[0042] Example 5

[0043] Determination of the sensitivity of strains to different pH

[0044] The basic culture medium LB liquid culture medium was adjusted to pH 3, 5, 7, 9, 11, and 13 respectively. The activated strain DXT2-4 to be tested was inoculated on the sensitivity test medium of six pH values, placed in a constant temperature incubator at 37°C for 24 hours, and the growth of the strain DXT2-4 to be tested was observed. If the strain DXT2-4 can grow normally, it means that the strain can tolerate the pH; otherwise, it means that the strain cannot tolerate the pH. The concentration OD value was measured, and the results were as follows. When the pH was less than 5 and greater than 11, the growth of the strain DXT2-4 was significantly inhibited, and it could continue to grow normally at a pH of 11. It proves that the strain can be used in saline-alkali land improvement, has alkali resistance, and is suitable as a microbial saline-alkali land improver.

[0045] Table 3 DXT2-4 pH sensitivity determination

[0046] pH 1 3 5 7 9 11 <![CDATA[OD 600 ]]> 0 0 0.159 1.321 1.317 0

[0047] Example 6

[0048] Results of the test on the ability of strains to solubilize phosphate and produce siderophores

[0049] Phosphate-dissolving medium (g / L): 10 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate, 0.03 g manganese sulfate, 0.3 g potassium sulfate, 0.03 g ferrous sulfate, 5.0 g tricalcium phosphate, and 15 g agar.

[0050] MSA basal medium (1 L): 4 g glucose, 5 g peptone, 0.5 g potassium chloride, 0.5 g magnesium sulfate, dilute to volume with ddH2O, and adjust the pH to 7.0.

[0051] Phosphate buffer (100 mL): 590.5 mg of sodium dihydrogen phosphate, 2.427 g of disodium hydrogen phosphate, 250 mg of ammonium chloride, 75 mg of potassium dihydrogen phosphate, 125 mg of sodium chloride, make up to 100 mL with ddH2O.

[0052] CAS dye solution (100mL):

[0053] Solution A: weigh 60.5 mg of chrome azurol S, dilute to 50 mL with ddH2O, then add 10 mL of 1 mmol / L ferric chloride (prepared with 10 mmol / L hydrochloric acid);

[0054] Solution B: weigh 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA) and dilute to 40 mL with ddH2O;

[0055] Mix liquid A and slowly add it into liquid B along the wall of the cup.

[0056] MSA-CAS medium: Add 50 mL of phosphate buffer and 50 mL of CAS dye solution to 1 L of MSA basal medium and mix well.

[0057] 6 μL of strain DXT2-4 was dropped onto the phosphate-solubilizing medium. After 5 days of growth, a phosphate-solubilizing circle was produced (e.g. Figure 4 A), the size of the phosphate-solubilizing zone is 6.5 mm. This proves that the strain has the ability to fix nitrogen and solubilize phosphate, that is, to convert the ineffective phosphorus that cannot be absorbed by plants into effective phosphorus that can be absorbed by plants, and promote the absorption of a large number of nutrients by plants. The application of phosphate-solubilizing bacteria can promote plant growth and improve the microecological environment of plant roots.

[0058] The strain was inoculated into MSA-CAS solid medium and cultured at 28°C for 2 days. The bacterial culture medium turned from blue to yellow, indicating that the strain could produce iron carriers (such as Figure 4 B).

[0059] Example 7

[0060] Test results of the ability of strains to produce cellulase and siderophore

[0061] Sodium carboxymethyl cellulose (CMC-Na) culture medium: CMC-Na 15 g, disodium hydrogen phosphate 2.5 g, potassium dihydrogen phosphate 1.5 g, peptone 2.5 g, agar powder 20 g, dilute to 1000 mL with distilled water, pH 7.2-7.5.

[0062] The purified strains were inoculated on sodium carboxymethyl cellulose (CMC-Na) medium, inverted and cultured at 30°C for 48 hours, then stained with 0.5% Congo red for 30 minutes and decolorized with 1 mol / L NaCl for 30 minutes. Congo red can form a red complex with sodium carboxymethyl cellulose, but it does not react with hydrolyzed cellobiose and glucose. The relative level of bacterial cellulase production can be preliminarily judged based on the size of the transparent circle on each bacterial plate. Figure 5 As shown, the diameter of the decellulose circle of DXT2-4 is 27 mm, which has a strong decellulose ability.

[0063] Example 8

[0064] Potted experiment on the control of potato scab by strains

[0065] The potato variety Solanum sphaeroides tissue culture seedlings were transplanted into vermiculite pots (17 cm in diameter). After 15 days of growth, the seedlings with the same growth were selected and inoculated with 100 mL of Streptomyces scab spore suspension (1×10 7 CFU / mL) in a flower pot; after another 20 days of growth, 100 mL of fermentation broth of Bacillus altitudinis strain DXT2-4 (1×10 8 CFU / mL), the control group did not add biocontrol bacteria, and the treatment without any inoculation was used as the blank control, and each treatment was repeated 3 times. After the potatoes were harvested, the incidence rate, disease index and control effect were calculated according to the scab disease classification standard.

[0066] Statistical results show that the morbidity of potatoes treated with pathogens alone reached 100%, and the disease index reached 0.7, while the morbidity of potatoes treated with DXT2-4 was 14.29%, and the disease index was only 0.07. The strain DXT2-4 had a 63% efficacy in preventing potato scab.

[0067] Potato scab incidence statistics:

[0068] Grading Standard:

[0069] Level 0: potato skin is healthy, no disease spots

[0070] Level 1: The area of ​​the diseased spot is 0-1 / 6

[0071] Level 2: The area of ​​the diseased spots is 1 / 6-1 / 3

[0072] Level 3: The area of ​​the diseased spots is 1 / 3-1 / 2

[0073] Level 4: The area of ​​diseased spots is more than 1 / 2

[0074] Incidence rate (%) = number of diseased tubers / total number of investigated tubers × 100

[0075] Disease index = (number of tubers at each disease level × representative value of the disease level) / (total number of surveyed individuals × highest disease level) × 100

[0076] Relative efficacy (%) = (disease index of control group – disease index of treatment group) × 100 / disease index of control group

[0077] Statistics

[0078]

[0079]

[0080] Example 9

[0081] Experiment on promoting rice root development by bacterial strains

[0082] Two treatments were set up in greenhouse pots:

[0083] CK: Conventional watering:

[0084] T: After conventional fertilization, add Bacillus altitudinis DXT2-4 for irrigation. Each ml of bacterial solution contains 1×10 8 CFU live bacteria.

[0085] Each treatment was repeated 3 times. After the rice had grown for two months, the root growth was observed. The results showed that strain DXT2-4 had a significant promoting effect on the growth of rice roots (e.g. Figure 7 As shown), it can be used in growth-promoting bacteria.

[0086] The Bacillus altitudinis DXT2-4 provided by the present invention has the advantages of simple cultivation, short cycle, no ecotoxicity, salt tolerance, rich types of available carbon sources, ability to decompose insoluble phosphorus in soil, ability to produce cellulase, siderophore, etc., and ability to effectively inhibit the growth of pathogenic bacteria of potato scab and promote the growth of rice root system, and has very broad application prospects in biological control of potato scab, improvement of saline-alkali land and growth-promoting bacterial agents.

Claims

1. A strain of Bacillus altitudinis DXT2-4, whose deposit number is CGMCCNO.25810.

2. The Bacillus altitudinis according to claim 1 Application of DXT2-4 in the preparation of a bacterial agent for inhibiting the growth of pathogenic bacteria of potato scab.

3. The Bacillus altitudinis according to claim 1 The application of DXT2-4 in the preparation of a microbial saline-alkali land improver is characterized in that: The salt mass concentration of the saline-alkali land is not higher than 9%, and the pH value of the saline-alkali land is 5-9.

4. The Bacillus altitudinis according to claim 1 The application of DXT2-4 in the preparation of a bacterial agent for converting ineffective phosphorus into effective phosphorus that can be absorbed by plants.

5. The Bacillus altitudinis according to claim 1 Application of DXT2-4 in the preparation of bacterial agents producing siderophores.

6. The Bacillus altitudinis according to claim 1 Application of DXT2-4 in the preparation of cellulose-degrading bacterial agent.

7. The Bacillus altitudinis according to claim 1 Application of DXT2-4 in the preparation of preparations for promoting plant root growth and development.

8. Use of Bacillus altitudinis DXT2-4 according to claim 1 in the preparation of a bacterial agent for preventing and treating potato scab.

9. The use according to claim 7, characterized in that: The plant is rice.

Images