Endophyte bacillus SE34 and application thereof

Abstract

The invention relates to an endophyte bacillus SE34, which is classified and named as bacillus sp., and is preserved in China General Microbiological Culture Collection Center on November 20, 2024, the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 32723. The strain is applied to antagonizing various phytopathogens and repairing various heavy metals, polycyclic aromatic hydrocarbons and heavy metal and polycyclic aromatic hydrocarbon combined polluted soil. The strain has the functions of dissolving phosphorus, producing iron carriers, producing cellulase, producing arginine decarboxylase, resisting acid, resisting salt, resisting multiple heavy metals, polycyclic aromatic hydrocarbons and combined pollution of the heavy metals and the polycyclic aromatic hydrocarbons and the like, can promote the growth of sudangrass under the stress of chromium, can antagonize multiple phytopathogens, and has practical popularization and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to an endophyte Bacillus sp. Bacillus SE34 and its application. BACKGROUND

[0002] Soil micro-ecological health is crucial to ensure high and stable crop yields, agricultural product quality and green transformation of agriculture. Due to unreasonable irrigation, application of chemical fertilizers and unreasonable development of land, soil quality is declining, crop yield is decreasing and certain environmental pollution is caused, which seriously threatens the homeostasis, function and resilience of soil micro-ecology. In addition, organic and inorganic combined pollution interacts through synergy, antagonism or changes in environmental behavior, and its environmental effect and ecological toxicity far exceeds the simple superposition of single pollutants, thereby producing complex systematic effects on soil micro-ecology, causing soil fertility degradation, soil-borne disease aggravation, crop yield reduction and weakening of specific ecosystem service functions such as carbon sequestration, and directly threatening the sustainability of agricultural production and regional ecological safety. Furthermore, soil salinization leads to a significant decrease in soil fertility, which is one of the major obstacles to the expansion of crop planting areas and yield improvement. Therefore, exploring environmentally friendly and efficient biological prevention and repair technologies has important scientific value for improving soil microstructure, enhancing soil fertility, balancing soil pH, repairing contaminated soil and promoting plant growth.

[0003] Plant growth-promoting bacteria generally have the ability to produce auxins, produce siderophores, solubilize phosphorus, solubilize potassium, fix nitrogen, or at least one of the above. Inoculation of plant growth-promoting bacteria can make soil nutrients available, promote plant root absorption of soil water and nutrients, and promote plant growth. Bacillus is one of the most common groups of plant growth-promoting bacteria, which provides a sustainable and environmentally acceptable strain for promoting plant growth through hormone synthesis, nutrient solubilization and plant biological protection, and occupies an important position in plant growth-promoting bacteria due to its strong stress resistance and easy survival and colonization in soil environment. Therefore, screening of plant growth-promoting bacteria with multiple pollution resistance and resistance to harsh environments can further expand the role of plant growth promotion and colonization under stress conditions, so as to improve plant stress resistance, promote plant growth, and even repair and improve the stress environment. However, there is no public report so far. SUMMARY

[0004] In view of the above situation, in order to overcome the deficiencies of the prior art, the purpose of the present application is to provide an endophyte Bacillus sp. SE34, which is classified and named as Bacillus sp. BacillusThe strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2024, and the address is No. 1, Huayuancun, Beijing, China, and the preservation number is CGMCC No. 32723.

[0005] The application of the strain in antagonizing various plant pathogenic bacteria.

[0006] The application of the strain in repairing various heavy metals (Cr 6+ , Pb 2+ , Mn 2+ , As 3+ ) and polycyclic aromatic hydrocarbon (pyrene, benzo[a]pyrene) and heavy metal and polycyclic aromatic hydrocarbon compound contaminated soil.

[0007] The endophyte SE34 of the application has the functions of phosphorus solubilization, iron carrier production, cellulase production, arginine decarboxylase production, acid tolerance, salt tolerance, tolerance to various heavy metals (Cr 6+ , Pb 2+ , Mn 2+ , As 3+ ) and polycyclic aromatic hydrocarbon (pyrene, benzo[a]pyrene) and heavy metal and polycyclic aromatic hydrocarbon compound, can promote sudangrass growth under chromium stress, and can also antagonize various plant pathogenic bacteria. The strain is expected to be developed into a biological agent, and has wide application prospects in improving soil, repairing contaminated soil, promoting plant growth and preventing plant diseases, and has practical popularization and application value. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 The figure is a comparison chart of the arginine decarboxylase production capacity of the strain of the application; Figure 2 The figure is a comparison chart of the antagonistic ability of the strain of the application to pathogenic bacteria. DETAILED DESCRIPTION

[0009] The specific embodiments of the application will be described in detail below in combination with examples and specific cases.

[0010] The application is given in the following examples in the specific implementation.

[0011] The application is a strain of endophyte Bacillus SE34 and its application. The strain is classified and named as Bacillus sp., and was preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2024, and the address is No. 1, Huayuancun, Beijing, China, and the preservation number is CGMCC No. 32723. Bacillus

[0012] ​NB medium: Beef extract powder 3 g, peptone 10 g, sodium chloride 5 g, distilled water 1000 mL, pH 7.2 ± 0.2, sterilized at 121 °C for 20 min. NA medium: Beef extract powder 3 g, peptone 10 g, sodium chloride 5 g, agar powder 15-20 g, distilled water 1000 mL, pH 7.2 ± 0.2, sterilized at 121 °C for 20 min. LB liquid medium: Tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, distilled water 1000 mL, pH 7.2 ± 0.2, sterilized at 121 °C for 20 min. Organophosphorus bacterial medium: Glucose 10 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium sulfate 0.3 g, ferrous sulfate 0.03 g, calcium phosphate 5 g, lecithin 0.2 g, agar 15 g, distilled water 1000 mL, pH 7.0-7.5. CAS detection medium: Chrome azurol (CAS) 60.5 mg, hexadecyl trimethyl ammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate 2.465 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg, distilled water 1000 mL, pH 6.8 ± 0.2. Cellulase plate medium: Peptone 10 g, yeast powder 10 g, carboxymethyl cellulose sodium 10 g, sodium chloride 5 g, potassium dihydrogen phosphate 1 g, agar powder 15 g, distilled water 1000 mL, pH 7.0. Arginine decarboxylase (ADC) blank medium: Peptone 5.0 g, beef extract 5.0 g, D-glucose 0.5 g, vitamin B6 (pyridoxal) 5 mg, bromocresol purple (1.6%) 0.625 mL, phenol red 0.02 g, distilled water 1000 mL, agar powder 18 g, pH 6.0. ADC medium: Peptone 5.0 g, beef extract 5.0 g, glucose 0.5 g, pyridoxal 5.0 mg, bromocresol purple (1.6%) 0.625 mL, phenol red 0.02 g, distilled water 1000 mL, agar powder 18.0 g, L-arginine hydrochloride 10.0 g, adjust pH 6.0-6.3.

[0013] Preparation of inorganic salt medium: MgSO4·7H2O 0.2 g, CaCl2·2H2O 0.01 g, FeSO4·7H2O 0.01 g, K2HPO4 0.4 g, MnSO4·H2O 0.02 g, NH4NO3 1.0 g, Na2HPO4 0.6 g, distilled water 1000 mL, pH 7.2-7.4.

[0014] The endophyte SE34 was isolated from the leaves of a healthy Sorghum sudanense plant growing in farmland in Xingyang City, Henan Province. The plant surface soil was washed with sterile water in a clean bench, then separated according to different tissues. After the surface of the plant organs was sterilized, they were placed in a sterile mortar with a small amount of sterile water and ground. The grinding liquid was the original liquid. The original liquid was gradiently diluted, and 100 μL of 10 -3 ,10 -4 ,10 -5 dilutions were spread on NA solid plates and cultured at 30°C for 24 h. Morphologically different colonies were picked and purified by streaking. The obtained pure single colonies were transferred to test tube slant medium and stored in a 4°C refrigerator for standby use.

[0015] The endophyte SE34 strain was cultured on NA medium for 24 h. The colonies were yellowish, Gram-positive, methylation-negative, V-P test-positive, oxidase-negative, contact enzyme-positive, starch hydrolysis-negative, citrate utilization test-negative, glucose oxidation and fermentation acid-producing, and gelatin liquefaction test-negative. The 16S rDNA sequencing was Bacillus SE34, and the classification and naming was Bacillus sp. It was preserved in the China General Microbiological Culture Collection Center on November 20, 2024, at address: No. 1 Yard, West Beichen Road, Beijing City, Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences, No. 3, with preservation number CGMCC No. 32723. Bacillus

[0016] 4.1, determination of salt-tolerant ability and pH sensitivity of SE34: SE34 seed liquid was inoculated in LB liquid medium containing NaCl (0, 1, 2, 4, 8, 10, 11, 12, 13, 14, 15%) with pH 7 at an inoculation amount of 1%, and cultured at 30°C in a 180 rpm shaker for 24 h. The OD600 value of the bacterial liquid was determined. The results showed that SE34 could grow under NaCl stress of 0-11%, proving that the strain could be applied in saline-alkali land improvement; ​SE34 seed liquid was inoculated in LB liquid medium with pH 4, 5, 6, 7, 8, 9, 10, 11, 12 respectively, and cultured in a 30°C, 180 rpm shaker for 24 h. The OD600 value of the bacterial liquid was measured. It was found that SE34 could still grow normally in the pH range of 5-9. It showed that the strain had a wide pH range.

[0017] SE34 single colonies were respectively inoculated on organic phosphorus solid medium, CAS medium and cellulase medium, and cultured at 30°C for 48-72 h. The size of the ratio of transparent circle diameter D to colony diameter d was calculated to represent the size of its phosphorus solubilizing ability. The measured ratio was 1.73-1.89, 1.23-1.32, and 1.47-1.59 respectively, indicating that SE34 had good phosphorus solubilizing, siderophore producing and cellulase producing abilities.

[0018] The test strains were inoculated into ADC blank medium without arginine hydrochloride and ADC medium with arginine hydrochloride, and cultured in a 28°C constant temperature incubator for 5 d. If the color of the blank control tube changed to yellow, and the experimental tube changed to magenta, it was determined that the strain had arginine decarboxylase activity, and the result was positive. If there was no color change, it was determined to be negative, see Figure 1 ; the results showed that SE34 had the ability to produce arginine decarboxylase.

[0019] SE34 seed liquid was inoculated into LB liquid medium containing different concentrations of Pb 2+ (1000, 1100, 1200, 1300, 1400, 1500, 1600 mg / L), Mn 2+ The concentration of As 3+ was set as (5, 10, 15, 20, 25 mg / L), and the concentration of Cd 2+ was set as (5, 10, 15, 20, 25 mg / L), and the concentration of Cr 6+ was set as (0, 20, 40, 60, 80, 100 mg / L). The culture was cultured in a 30°C, 180 rpm shaker for 2-3 d. The LB medium containing different concentrations of different heavy metals without inoculation was used as a control to observe whether the bacterial liquid was turbid.

[0020] SE34 seed liquid was inoculated into inorganic salt medium containing 50 mg / L of pyrene and 50 mg / L of benzo[a]pyrene respectively, and cultured in a 30°C, 180 rpm shaker for 24-48 h. The inorganic salt medium containing 50 mg / L of pyrene and 50 mg / L of benzo[a]pyrene without inoculation was used as a control to observe whether the bacterial liquid was turbid.

[0021] Results showed that SE34 could tolerate 1400 mg / L Pb 2+ , 500 mg / L Mn 2+ , 10 mg / L As 3+ , 10 mg / L Cd 2+ , 60 mg / L Cr 6+ , 50 mg / L pyrene and 50 mg / L benzo[a]pyrene.

[0022] 4.5, Determination of tolerance to two or more mixed pollutants: SE34 seed liquid was inoculated into inorganic salt medium containing different concentrations of Pb 2+ (800, 1000, 1200, 1400 mg / L), Cd 2+ (5, 10 mg / L), Cr 6+ (30, 40, 50 mg / L), pyrene (30, 40, 50 mg / L), and benzo[a]pyrene (30, 40, 50 mg / L), and cultured at 30°C in a 180 rpm shaker for 24-48 h. The inorganic salt medium containing the corresponding mixture without inoculation was used as a control, and the turbidity of the bacterial liquid was observed. The results showed that SE34 could grow in 1400 mg / L Pb 2+ and 50 mg / L pyrene or 50 mg / L benzo[a]pyrene; could grow in 50 mg / L Cr 6+ and 50 mg / L pyrene or 50 mg / L benzo[a]pyrene; could grow in 10 mg / L Cd 2+ and 50 mg / L pyrene or 50 mg / L benzo[a]pyrene; and could grow in 30 mg / L Cr 6+ , 1400 mg / L Pb 2+ and 50 mg / L pyrene or 50 mg / L benzo[a]pyrene.

[0023] The pathogenic fungi of Sclerotium rolfsii, Pythium aphanidermatum, Alternaria alternata, Botrytis cinerea, Phytophthora infestans, and Fusarium oxysporum were placed in the center of PDA medium, and SE34 single colonies were picked and inoculated on both sides of the pathogenic fungal colonies at a distance of 2 cm, and cultured at 28°C for 5 days. The confrontation was observed, and the results are shown in Figure 2 ; indicating that SE34 has obvious inhibitory effect on a variety of plant diseases: peanut white thread disease, corn basal rot, kiwi black spot disease, cucumber gray mold disease, potato late blight, and watermelon wilt. The endophytic strain from healthy plant tissue is harmless to the ecological environment, easy to reproduce and prepare into biological pesticides, and is a safe, broad-spectrum new biocontrol microorganism for preventing and treating plant fungal diseases.

[0024] The activated SE34 seed liquid was centrifuged to obtain bacterial cells, which were diluted with sterile water to obtain a bacterial suspension with a concentration of 10 7 cfu / mL.

[0025] The chromium-contaminated soil was loaded into pots, 1 kg per pot, and 50 mL of bacterial suspension was inoculated into each pot before and after the sudan grass was planted, respectively, and 200 seeds of sudan grass were planted in each pot. After 20 days, the relevant indicators of plants and soil were measured, respectively. The results showed that under chromium stress, inoculation of endophytic bacteria SE34 could promote the growth of sudan grass, which was manifested as the plant height, root length, aboveground fresh weight and underground fresh weight of sudan grass being significantly higher than the control without inoculation.

[0026] Table 1 Effect of SE34 inoculation on the growth of sudan grass under chromium stress Treatment Plant height (cm) Root length (cm) Fresh weight of aboveground part (g / plant) Fresh weight of underground part (g / plant) CK 7.83±1.36b 0.95±0.04b 0.01±0.0003b 0.005±0.0009b SE34 9.79±1.44a 1.52±0.23a 0.03±0.004a 0.008±0.0003a In summary, the above-mentioned strain is the first endophytic bacteria Bacillus SE34 screened by the applicant, which has the functions of phosphorus solubilization, iron carrier production, cellulase production, arginine decarboxylase production, acid and salt tolerance, and can tolerate various heavy metals (Cr 6+ , Pb 2+ , Mn 2+ , As 3+ ) and polycyclic aromatic hydrocarbons (pyrene, benzo[a]pyrene), and combined pollution of heavy metals and polycyclic aromatic hydrocarbons, and can antagonize various plant pathogenic fungi, and can promote the growth of sudan grass under chromium stress. The strain is expected to be developed as a biological agent, and has broad application prospects in improving soil, remediating contaminated soil, promoting plant growth and preventing plant diseases, and has practical application value.

Claims

1. An endophytic bacterium, Bacillus SE34, which is classified and named Bacillus. Bacillus sp. was deposited on November 20, 2024 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32723.

2. Use of the endophyte Bacillus SE34 of claim 1 in antagonizing a variety of plant pathogens.

3. The endophytic Bacillus SE34 described in claim 1 in the remediation of multiple heavy metals Cr 6+ Pb 2+ Mn 2+ As 3+ Applications in soils contaminated with polycyclic aromatic hydrocarbons (PAHs) such as pyrene, benzo[a]pyrene, heavy metals, and PAHs.

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