Ochrobactrum teleogrylli P17 and application thereof

Abstract

The application discloses an Ochrobactrum teleogrylli P17 and application thereof, and belongs to the technical field of microorganisms. The Ochrobactrum teleogrylli P17 is preserved in the China General Microbiological Culture Collection Center on April 21, 2025, and the address of the preservation center is No. 3, Xili Beichen, Chaoyang District, Beijing, and the Institute of Microbiology of Chinese Academy of Sciences, and the preservation number is CGMCC No. 34270. The Ochrobactrum teleogrylli P17 is screened from paddy rhizosphere soil, has trivalent arsenic oxidation capacity, can improve the growth of crops under heavy metal arsenic stress, effectively blocks the migration of arsenic from soil to plants, effectively reduces the heavy metal arsenic in paddy grains by more than 50%, and increases the yield by more than 12%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a strain of Ochrobactrum teleogrylli P17 and application thereof. BACKGROUND

[0002] Arsenic (As) is a non-metallic element widely existing in nature, which is carcinogenic and is defined as a class I carcinogen by the International Agency for Research on Cancer (IARC), and can cause lung damage, peripheral nerve damage, skin disease or cardiovascular disease, etc. The arsenic in soil is derived from the release of arsenic-containing sulfide rocks and oxide rocks through weathering and rain erosion, and the pollution caused by mineral exploitation, processing and use. The arsenic in soil is transmitted upward through the crop root system, accumulated in the fruit, and transmitted through the food chain to ultimately threaten human health.

[0003] Arsenic has two main chemical forms in nature, trivalent arsenic AsIII and pentavalent arsenic AsV. The toxicity of AsIII is 100 times that of AsV, and it is more mobile in soil. Oxidizing AsIII to AsV is an effective way to remediate pollution. AsIII oxidation can be achieved by chemical oxidants such as chlorine, peroxide and ozone, but the cost is high and the soil fertility is damaged. Microbial method mainly uses arsenic-oxidizing microorganisms to oxidize AsIII to AsV to prevent crops from absorbing arsenic. It has been reported that microorganisms in alpha, beta and gamma proteobacteria can oxidize arsenite to arsenate with less toxicity. Several strains of Ochrobactrum have been used for arsenic remediation in soil. For example, Ochrobactrum ciceri SW1 reduces the background value of arsenic by secreting EPS to adsorb AsV (Potential role of bacterial extracellular polymeric substances as biosorbent material for arsenic bioremediation, 2019); Ochrobactrum anthropi EBC-SK As11 isolated from tailings soil confirms the arsenic oxidation ability (Detoxification of arsen genotypes by arsenite-oxidizing bacteria through arsenic biotransformation, 2024); Rhizosphere microorganism Ochrobactrum tritici As5 has the ability to accumulate arsenic (Arsenic accumulation by a rhizosphere bacterial strain Ochrobactrum tritici reduces rice plant arsenic levels, 2020); Ochrobactrum sp. EEELCW01 in paddy soil can couple mineralization to fix arsenic (Arsenic biomineralization by iron oxidizing strain (Ochrobactrum sp.) isolated from a paddy soil in Hunan, China, 2021). Domestic invention patents 202411247830.4, 201510532891.X, 202011393481.9, 202310754949.X also report the growth-promoting ability of strains of this genus, such as phosphorus-dissolving and potassium-dissolving, long-hormone-producing, and siderophore-producing.

[0004] The above literatures are reviewed, and no Ochrobactrum strain with arsenic oxidation function and growth promoting ability isolated from rhizosphere soil is found. SUMMARY

[0005] In view of the defects of the prior art, the purpose of the present application is to provide an Ochrobactrum teleogrylli P17 and its application.

[0006] In order to achieve the above technical effects, the present application adopts the following technical solutions:

[0007] The first aspect of the present application is to provide an Ochrobactrum teleogrylli P17, which has been preserved in the China General Microbiological Culture Collection Center on April 21, 2025, the address is No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 34270.

[0008] The second aspect of the present application is to provide the application of the Ochrobactrum teleogrylli P17 in the oxidation of trivalent arsenic.

[0009] The third aspect of the present application is to provide the application of the Ochrobactrum teleogrylli P17 in improving the resistance of crops to arsenic stress.

[0010] The fourth aspect of the present application is to provide the application of the Ochrobactrum teleogrylli P17 in blocking the absorption of arsenic by crops.

[0011] The fifth aspect of the present application is to provide the application of the Ochrobactrum teleogrylli P17 in crop growth promotion.

[0012] Further, the crops include rice and corn.

[0013] The sixth aspect of the present application is to provide a microbial agent comprising the Ochrobactrum teleogrylli P17.

[0014] A seventh aspect of the present invention provides a method for preparing the bacterial agent, comprising the following steps: picking Ochrobactrum teleogrylli P17 colonies from a preservation slant using an inoculation needle, streaking them onto an NA (Nutrient Agar) plate, and activating them at 30±1℃ for 1 day; then inoculating them into an NB (Nutrient Broth) liquid seed shake flask for expansion culture, and shaking and culturing at 30±1℃ and 150-200 r / min for 10-30 h, preferably 12-16 h; continuing the shake flask or fermenter expansion culture to obtain the Ochrobactrum teleogrylli P17 bacterial suspension. Quality control is performed using the plate gradient dilution method, and the bacterial suspension CFU is tested to be ≥2×10⁻⁶. 8 / mL, bacterial contamination rate ≤10%, after aliquoting, store at 4℃ for later use.

[0015] The eighth aspect of the present invention is to provide a method for applying Ochrobactrum teleogrylli P17 inoculant to exert the effects of Ochrobactrum teleogrylli P17 in blocking arsenic absorption and promoting crop growth during crop cultivation, comprising the following steps:

[0016] (1) Inoculum treatment: Dilute the inoculum with water to a CFU concentration of 10. 6 -10 7 / mL for seed dressing;

[0017] (2) Application of microbial agent: When the crop is transferred to the field, apply it with the water for planting. When the seedlings of direct-seeded crops emerge, apply it during the first watering. The dosage is 5-10L per mu.

[0018] Currently, the main technologies for treating arsenic-containing wastewater include chemical adsorption, ion exchange, membrane separation, and iron salt methods. However, these methods are characterized by high cost and complex operation.

[0019] Microbial remediation of arsenic-contaminated soil involves altering the bioavailability of arsenic through biochemical reactions such as oxidation-reduction, adsorption, methylation, and precipitation by microorganisms, thereby remediating the soil. There are two main microbial remediation technologies for arsenic-contaminated soil: biosorption and bio-oxidation-reduction.

[0020] Based on the results of the embodiments of the present invention, the application of Ochrobactrum teleogrylli P17 in the treatment of arsenic-containing wastewater or the remediation of arsenic-contaminated soil is provided.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1.The present application isolates and screens a strain of Ochrobactrum teleogrylli P17, which can oxidize trivalent arsenic to pentavalent arsenic, significantly reducing the toxicity and migration of arsenic;

[0023] 2.The Ochrobactrum teleogrylli P17 provided by the present application can promote crop growth, has certain arsenic blocking effect, blocks the absorption of crops to arsenic, and improves the resistance of crops to arsenic stress; at the same time, it has low dosage, low cost, high efficiency, is environment-friendly, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] The drawings constituting a part of the specification of the present application are used to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application.

[0025] Figure 1 A colony morphology diagram of the Ochrobactrum teleogrylli P17 provided by the present application.

[0026] Figure 2 A phylogenetic tree of the Ochrobactrum teleogrylli P17 provided by the present application. DETAILED DESCRIPTION

[0027] It should be pointed out that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0028] The components of each medium used in the following examples are as follows:

[0029] NB medium: 10 g of proteose peptone, 3 g of beef extract, 5 g of sodium chloride, 1000 mL of distilled water, pH 6.5-7.5, and 15-20 g of agar for solid NA.

[0030] Example 1, screening of Ochrobactrum teleogrylli P17

[0031] 1) Acclimation: 10 g of rhizosphere soil of rice (mature rice in Fengshan, Guangxi) was taken into a triangular flask containing 100 mL of sterile water, 3-5 glass rods, and mixed for 20 min at 200 r / min on a shaker. 10 mL was inoculated into NB liquid medium containing AsIII 3 mM (NaAsO2), and after 48 h of culture, it was transferred to NB liquid medium containing higher AsIII, with each transfer increasing by 3 mM, and diluted and plated on NA plates containing the corresponding AsIII concentration. The change in morphological diversity was observed until the colony diversity was significantly reduced, and the acclimation was stopped when the AsIII concentration was increased to 18 mM.

[0032] 2) Single bacteria separation and purification: a plate with about 50-200 colonies diluted and plated was selected, and morphologically different strains were picked for 3 times of separation and purification to obtain pure culture, numbered and preserved in a 4°C refrigerator.

[0033] 3) Screening: potassium permanganate colorimetry was used for screening, and KMnO4 reacted with AsIII to become light yellow during colorimetry. Arsenic-oxidizing microorganisms can oxidize AsIII to AsV, and KMnO4 does not react with AsV during colorimetry, showing purple color; as a result, the P17 strain was obtained.

[0034] It can be seen that the Ochrobactrum teleogrylli P17 strain provided by the present application has high arsenic resistance, and can at least survive under the condition of AsIII 18 mM.

[0035] Example 2, strain identification

[0036] The purified strain was sent to BioMeasure for 16S rDNA sequence determination, and the returned data was compared with the existing sequences in the EzBioCloud database (Database ver.2023.8.23), and a strain with similar homology was selected, MEGA7.0 software was used, Neighbor-Joining method was selected to construct a phylogenetic tree, as shown in Figure 2 The results show that the P17 selected above has a similarity of 99.78% with Chrobactrum teleogrylli LCB8, and is relatively close in evolutionary distance, and in combination with the morphological characteristics of the colonies Figure 1 shown in the table, it is identified as Ochrobactrum teleogrylli, named as Ochrobactrum teleogrylli P17.

[0037] The Ochrobactrum teleogrylli P17 is isolated from the rhizosphere soil of rice, and the 16S rDNA gene sequence is as follows (SEQ ID NO: 1):

[0038] >P17

[0039]

[0040] The Ochrobactrum teleogrylli P17 strain has been deposited with the China General Microbiological Culture Collection Center on April 21, 2025, at an address of No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, with a preservation number of CGMCC No. 34270.

[0041] Example 3, preparation of Ochrobactrum teleogrylli P17 liquid inoculant

[0042] 1) A small amount of the Ochrobactrum teleogrylli P17 strain preserved in the NA slant was dipped with an inoculation needle to the NA plate, and cultured at 30±1℃ for 1d.

[0043] 2) Two well-grown colonies were selected from the activated NA plate and inoculated into a 100-250mL triangular flask containing NB, with a liquid volume of ≤50%, and cultured at 30℃, 200r / min for 12-16h.

[0044] 3) Inoculate 1-20% according to the inoculation amount into the fermentation flask and culture for 20-30h, preferably 20-26h. The fermentation flask has a liquid volume of ≤50%, a temperature of 30±1℃, and a rotation speed of 150-200r / min; the fermentation tank has a liquid volume of ≤80%, and the rotation speed and ventilation are adjusted to a dissolved oxygen of ≥5%, and an appropriate amount of soybean oil defoaming agent is added to control the foam during the stable period of fermentation.

[0045] 4) Quality inspection: the plate dilution method was used to determine that the CFU of the bacterial liquid was ≥2×10 8 / mL, the bacterial impurity rate was ≤10%, and after dispensing, it was stored at 4℃ for standby use.

[0046] Example 4, verification of arsenic oxidation ability of Ochrobactrum teleogrylli P17

[0047] The bacterial agent was prepared according to Reference Example 3, with the company's own Ochrobactrum daejeonense MG35 (CGMCC No. 19745) and Pseudomonas fluorescens M321 as controls, and NB medium as blank control. During fermentation, 0.5 mM (75 mg / L) of AsIII-containing NB medium was added to a 100 mL fermentation flask, and the OD (600 nm) value was tested at the end of fermentation. After centrifugation at 5000 r / min for 10 min, the supernatant was sent to Shanghai Detection Technology Co., Ltd. for detection of total inorganic arsenic, AsIII and AsV. The results, as shown in Table 1, confirmed that Ochrobactrum teleogrylli P17 can convert all AsIII in the culture medium to AsV, and has arsenic oxidation ability.

[0048] Table 1

[0049]

[0050] Based on the oxidation ability of Ochrobactrum teleogrylli P17 to trivalent arsenic, it can be used not only to improve the growth of crops, but also to improve the growth environment of crops and reduce the content of trivalent arsenic in water or soil, thereby achieving the effect of wastewater treatment and soil remediation.

[0051] As for wastewater treatment, the conversion of trivalent arsenic can be achieved by adding Ochrobactrum teleogrylli P17, and those skilled in the art can determine the appropriate dosage according to the concentration of arsenic in the wastewater.

[0052] As for soil remediation, Ochrobactrum teleogrylli P17 can be added for arsenic-contaminated soil remediation based on the treatment conditions in the water body and according to a certain liquid-soil ratio. As for pH control and medium selection, conventional technical solutions in the art can be used, and therefore will not be described here.

[0053] Example 5, rice hydroponics test

[0054] Rice variety japonica zhongzao 39, the bacterial agent prepared according to Reference Example 3, CFU was 9×10 8 / mL, diluted 100 times with water, so that the CFU was 10 6 -10 7 / mL. Hydroponics used Hoggland culture solution (Coolaber company, dry powder + concentrated liquid). The hydroponic cup is 90mm in upper diameter, 57mm in lower diameter and 175mm in height. The planting basket is 80mm in inner diameter, 110mm in outer diameter and 70mm in height. According to the final concentration of sodium arsenite, the test groups are divided into 0, 0.10mM arsenic groups. NB blank control, Ochrobactrum dajeonense (preserved number CGMCC No. 19745), Pseudomonas fluorescens M321 treatment are set. Each treatment has 4 replicates. The seeds after seed dressing are placed in the hydroponic cup with nutrient solution. The temperature of the climate room is kept at 26℃, and the light intensity is about 3000lux. The light time is day / night: 15 / 9h. The plant height and root length are measured at 20d, and the total arsenic content of plant heavy metal is determined. The results shown in Table 2 show that Ochrobactrum teleogrylli P17 can reduce the total arsenic of plant by 74.8% under 0.1mM hydroponics, and has obvious promoting effect on the growth under 0, 0.1mM AsIII hydroponics, and has certain growth promoting effect.

[0055] Table 2

[0056]

[0057] Example 6, application on rice potting

[0058] Rice variety japonica zhongzao 39, the inoculum prepared according to reference example 3, CFU is 6×10 8 / mL, diluted 50 times with water, so that CFU is 10 6 -10 7The bacterial agent prepared in Reference Example 3 has a CFU of 10 x 1010 / mL, which is diluted 80 times with water to make the CFU 10 x 1010 / mL, and then used for seed dressing. The soil is collected from Changsha, Hunan (total arsenic 93 mg / kg soil), and has the following physicochemical properties: total nitrogen (%), 0.22 ± 0.00; total potassium (%), 1.46 ± 0.03, total phosphorus (%), 0.13 ± 0.02; alkali-hydrolyzable nitrogen (mg / kg), 122.02 ± 5.26; available phosphorus (mg / kg), 269.00 ± 10.87; available potassium (mg / kg), 164.52 ± 4.27; pH, 5.00 ± 0.03; organic matter (g / kg), 19.21 ± 0.57. Ochrobactrum daejeonense (preserved number CGMCC No. 19745) and Pseudomonas fluorescens M321 are used as comparative treatments, and there are a total of 4 treatments and 5 repetitions, a total of 20 pots, with 1.5 kg of soil in each pot. When the rice is transferred from the seedling tray to the pot, the bacterial agent (CFU about 10 billion / mL) is added to the planting water, and the amount is 10 L / acre. The blank is added with the corresponding amount of NB medium. After 40 days of culture, the plant growth and total arsenic content are determined, and the results are shown in Table 3. It can be seen that Ochrobactrum teleogrylli P17 can reduce the total arsenic in the root system by 64.6%. And it has obvious promoting effect on the growth.

[0059] Table 3

[0060]

[0061] Example 7, application in rice test in Hechi, Guangxi

[0062] The bacterial agent prepared in Reference Example 3 has a CFU of 10 x 1010 8 / mL, which is diluted 80 times with water to make the CFU 10 x 1010 6 -10 7 / mL for seed dressing. The conventional rice variety is selected from a rice planting plot with serious pollution of total arsenic (98.75 mg / kg) in Hechi, Guangxi. Ochrobactrum daejeonense MG-35 (preserved number CGMCC No. 19745) and Pseudomonas fluorescens M321 are used as comparative treatments, and the blank is added with NB medium without inoculation. There are a total of 4 treatments and 5 repetitions. When the rice is transferred from the seedling tray to the test field, the bacterial agent (CFU about 10 billion / mL) is added to the planting water, and the amount is 10 L / acre. The blank is added with the corresponding amount of NB medium. Other agronomic conditions are the same. The yield results are shown in Table 4. Application of Ochrobactrum teleogrylli P17 seed dressing + flush application can significantly reduce the accumulation of total arsenic in the grain by 54.5%, and increase the yield by 13.2%.

[0063] Table 4

[0064]

[0065] Example 8, application in Fujian Youxi rice test

[0066] The bacterial agent prepared in Reference Example 3 has a CFU of 12 x 10 8 / mL, which is diluted 100 times with water to make the CFU 10 6 -10 7 / mL for seed dressing. A conventional rice variety is selected from a rice planting plot in Youxi, Fujian, which is seriously polluted by total arsenic (130.5 mg / kg). Ochrobactrum daejeonense MG-35 (CCTCC No. 19745) and Pseudomonas fluorescens M321 are used as comparative treatments, and the blank control is the NB culture medium without inoculation. There are a total of 4 treatments and 5 repetitions. When the rice seedlings are transferred to the test field, the bacterial agent (CFU about 10 x 10 8 / mL) is added to the planting water at a dosage of 10 L / acre, and the blank is added with the corresponding amount of NB culture medium. Other agronomic conditions are the same. The yield results are shown in Table 5, and the application of Ochrobactrum teleogrylli P17 seed dressing + flushing can significantly reduce the accumulation of total arsenic in the grain by 52% and increase the yield by 12.2%.

[0067] Table 5

[0068]

[0069] Example 9, growth-promoting application on other crops, corn

[0070] Test variety: Taiyu No. 2; conventional brown soil, taken from the test field in Cunwen Scenic Area, Tai'an City, Shandong Province, naturally air-dried, and sieved through a 20-mesh screen; potting test was carried out in an artificial climate chamber at a temperature of 25°C, humidity of 50%-60%, light for 16 h, darkness for 8 h, and light intensity of 8000 lux. There are a total of 4 treatments, each repeated 5 times, for a total of 20 pots (5 plants per pot). The bacterial agent prepared in Reference Example 3 has a CFU of 7 x 10 8 / mL, which is diluted 50 times with water to make the CFU 10 6 -10 7 / mL for seed dressing. The seeds are sown in 25 cm x 25 cm x 25 cm pots, and the bacterial agent is flushed once on the 3rd day after germination (the first time to water) according to the dosage in Reference Example 6. The growth index is measured on the 30th day of the seedling stage, and the results are shown in Table 6, indicating that the strain also shows certain growth-promoting effect on other crops.

[0071] Table 6

[0072]

[0073] The above description is merely that of the preferred embodiments of the application and is not intended to limit the application. One skilled in the art can make various modifications and variations without departing from the spirit and scope of the application. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the application shall be included in the protection scope of the application.

Claims

1. A strain of Ochrobactrum teleogrylli P17, characterized in that, The Ochrobactrum teleogrylli P17 was deposited with China General Microbiological Culture Collection Center on April 21, 2025. The deposit address is No. 1, Yitian Street, Beichen West Road, Haidian District, Beijing, China, and the deposit number is CGMCC No. 34270.

2. The method of claim 1 Ochrobactrum teleogrylli Use of P17 in the oxidation of trivalent arsenic.

3. The method of claim 1 Ochrobactrum teleogrylli Use of P17 in improving resistance of rice to trivalent arsenic stress.

4. The method of claim 1 Ochrobactrum teleogrylli Use of P17 in blocking the uptake of trivalent arsenic by rice.

5. An inoculant characterized in that, comprising the compound of claim 1 Ochrobactrum teleogrylli P17.

6. A kind Ochrobactrum teleogrylli The method for applying P17 microbial agent is characterized by... Comprising the following steps: (1) Seed dressing with bacterial agent: the bacterial agent of claim 5 is diluted with water to a CFU concentration of 10 Ochrobactrum teleogrylli P17 bacterial agent is diluted with water to a CFU concentration of 10 6 -10 7 / mL for seed dressing; (2) Bacterial agent flush: when the rice is transferred to the field, it is applied with water for planting, and the first time for water supplement after the seedlings of direct seeding rice emerge, flush is applied, and the dosage is 5-10 L per mu.

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