CN111925945B - Smoke tube bacterium G14 and application thereof - Google Patents

Abstract

The invention discloses a smoke tube bacterium G14 and application thereof, and the smoke tube bacterium G14 (Bjerkandera adustaG14) is preserved in China general microbiological culture Collection center (CGMCC) at 7/6/2020 with the preservation number of CGMCC No.20218. The smoke tube bacterium G14 has various application functions, has good functions of biocontrol, growth promotion and decoloration, and has higher development and utilization values for organisms and environment.

Description

Smoke tube bacterium G14 and application thereof

Technical Field

The invention belongs to the technical field of environmental microorganisms, and relates to a smoke tube bacterium G14 and application thereof.

Background

Endophytic fungi (Plant endophytic fungi) are ubiquitous in healthy Plant tissues, and refer to those fungi that spend a certain or all of their life history in various tissues and organs of healthy plants, without the host showing any symptoms. Is considered to be one of the important sources of biological control factors for plant diseases. It can directly attack pathogenic bacteria or pathogenic factors to degrade and generate antagonistic substances or induce plants to generate systemic resistance to inhibit pathogens and the like in the face of infection of some pathogenic bacteria, and the genes of the plants still maintain natural properties and are a natural resource library for biological control of plant diseases. The endophytic fungi exist in most plants, are rich in resources, have the characteristics of wide distribution, multiple varieties and the like, have multiple varieties of secondary metabolites, can be separated from the endophytic fungi to obtain terpenoids, saponins, alkaloids, aromatics, steroids, peptides and other antibacterial substance structure types, are a hot point for developing antibacterial drugs, have the effects of inhibiting the growth of pathogenic bacteria, improving the plant resistance, promoting the plant growth, degrading chemical pollutants and the like, and are widely applied to the aspects of agriculture and forestry, medicine, environmental protection and the like.

Blueberry (A)Vacciniumspp.) is a fruit tree of the genus Vaccinium of the family Ericaceae, also known as blueberry, etc., and is a perennial shrub or small shrub. The fruit flavor is unique, the color is blue, the color is beautiful, the blue color is wrapped by the white fruit powder, the pulp is fine and smooth, the sweetness and the sourness are palatable, the fragrance is refreshing and pleasant, and the fruit flavor is favored by a plurality of consumers. The blueberry can be eaten fresh, can be processed into fruit juice beverage, blueberry fruit wine and the like, and is praised as 'golden berry' and 'king of world fruits'. Since the market of blueberries is started, people keep a hot attitude for the needs of the people, and the biggest limiting factor influencing the benefits of the blueberries is the soil problem. Phosphorus plays an important role in the growth process of crops and is one of mineral nutrient elements essential to plants. In the recent period, because the unreasonable cultivation mode of people causes severe soil environment, most of crops are seriously lack of phosphorus element, and the yield of the crops is greatly reduced.

Fusarium oxysporum and Fusarium verticillarum can cause blueberry root rot, harm the roots of plants, and are one of the more serious diseases in blueberry production at present. Alternaria alternata can cause blueberry leaf spot disease, and can seriously occur in main blueberry production areas. The main pathogenic bacteria of pepper anthracnose is bacillus thuringiensis which can cause rotten leaves and rotten fruits of peppers and even death of plants, and the disease is commonly generated in Guizhou pepper producing areas. Sclerotium rolfsii is a main pathogenic bacterium of southern blight, can cause diseases of various plants such as peanuts, sweet potatoes, apples and the like, the damage to the peanuts often occurs in the mature period of the plants, and the pathogenic bacterium can directly invade the roots or stem bases of the peanuts or invade the roots or stem bases of the peanuts from wounds to cause diseases and rot of roots, fruit stalks and pods. Sclerotinia can infect crops and vegetables, is one of important diseases of sclerotinia rot of rape, and can reduce the yield of diseased plants by half.

China's dyeing and finishing enterprises are continuously developing, and dyes can generate some additional products besides raw materials due to the influence of chemical reaction in the production and manufacturing processes, and the two products are added together to form the dye production wastewater. Some untreated or overproof dye wastewater is directly discharged into a natural water body, so that the water body is polluted in a large area, a large amount of aquatic organisms die due to poor biodegradability and high toxicity, and the water body has poor light transmission strength, high degradation difficulty and reduced photosynthesis.

At present, most of plant endophytic fungi are mainly separated from rhizosphere soil, roots, stems and leaves of plants, so that a larger plant endophytic fungi resource is obtained, and fewer endophytic fungi are separated and identified from fruits, particularly edible fruits with certain nutritional and medicinal values. And most of the separated strains have single function, and the strains with multiple functions are fewer.

The problems of pesticide residue and environmental pollution cause wide attention of the public, and green and pollution-free agricultural products are more and more favored by people. Therefore, the content of available phosphorus in the soil is increased, the absorption of phosphorus by crops is increased, and the usage amount of phosphate fertilizer is reduced; biological control of plant diseases and reduction of the use of chemical pesticides; the green degradation of dye wastewater is particularly important.

Disclosure of Invention

The invention aims to provide a smoke tube bacterium G14 and application thereof. The smoke tube bacterium G14 has various application functions, has good functions of biocontrol, growth promotion and decoloration, and has higher development and utilization values for organisms and environment.

The technical scheme of the invention is as follows: a strain of Clausidophora G14, clausidophora G14 (Bjerkandera adustaG14) is preserved in China general microbiological culture Collection center (CGMCC) at 7-month and 6-month in 2020, with the preservation number of CGMCC No.20218.

In the Formica Fusca G14, the ITS rDNA nucleotide sequence of Formica Fusca G14 is shown in SEQ ID NO. 1.

In the smoke tube bacterium G14, the smoke tube bacterium G14 is applied to biocontrol.

In the smoke tube bacterium G14, the biocontrol agent is: the smoke tube bacterium G14 shows bacteriostatic activity on plant pathogenic bacteria.

In the foregoing Shigella damascena G14, the plant pathogenic bacteria include blueberry root rot pathogenic bacteria (A)Fusarium oxysporum) Pathogenic bacteria of blueberry root rot: (F. verticillioide) Pathogenic bacteria of blueberry leaf spot disease: (Alternaria alternata) Pathogenic bacteria of pepper anthracnose: (Colletotrichum scovillei) Pathogenic bacteria of southern blight: (Sclerotium rolfsii) And Sclerotinia sclerotiorum (A) and (B)Sclerotinia sclerotiorum）。

In the smoke tube bacterium G14, the smoke tube bacterium G14 is used for promoting plant growth.

In the nicotiana tabacum G14, the plants of the nicotiana tabacum G14 capable of promoting growth include Chinese cabbage and corn.

In the smoke tube bacterium G14, the smoke tube bacterium G14 is applied to degradation of dye.

In the foregoing smoke tube bacterium G14, the degradation dye is: the smoke tube bacterium G14 secretes lignin degrading enzyme to decolor the dye.

In the smoke tube bacterium G14, the lignin degrading enzyme includes laccase, lignin peroxidase and manganese peroxidase.

Compared with the prior art, the invention has the following beneficial effects: the invention starts from blueberry endophytes, and obtains a multifunctional endophyte-tobacco strain with good phosphorus dissolving function and antagonistic and degradation functions by a method of separating from the epidermis of immature blueberry fruits through targeted screeningPipefish G14 (Bjerkandera adustaG14), identified as nicotiana species by morphology and molecular biology, and deposited in the China general microbiological culture Collection center. The strain can be used for treating two blueberry root rot pathogenic bacteria: (A), (B) and (C)Fusarium oxysporum）、（F. verticillioide) Pathogenic bacteria of blueberry leaf spot: (a)Alternaria alternata) Pathogenic bacteria of pepper anthracnose: (Colletotrichum scovillei) Pathogenic bacteria of southern blight: (Sclerotium rolfsii) And Sclerotinia sclerotiorum (A), (B), (C)Sclerotinia sclerotiorum) 6 pathogenic bacteria show bacteriostatic activity, have a biocontrol function and have strong development and utilization values in preventing and treating blueberry root rot. In addition, the fertilizer also has strong phosphorus dissolving capacity, can promote the growth of Chinese cabbages and corns, has the function of promoting growth, and provides reference for the research of safe and effective soil phosphorus dissolving medicaments by integrating various factors. Meanwhile, the invention can also secrete laccase, lignin peroxidase and manganese peroxidase 3 lignin degrading enzymes, has a decoloring function on various dyes, and lays a foundation for application in dye wastewater treatment. In general, the strain of the invention is one of the endophytic strains, has multiple functions and has higher development and utilization values for human, organisms and environment.

Drawings

FIG. 1 is a colony morphology of strain G14; wherein A is the front of a colony morphology map; b is the back of the colony morphology chart;

FIG. 2 is a phylogenetic tree of the ITS sequence of strain G14;

FIG. 3 shows the inhibitory effect of strain G14 on 6 pathogenic bacteria;

FIG. 4 is a graph showing the effect of bacterial strain G14 on phosphorus solubilization in PKO (left) and Monkina (right);

FIG. 5 shows the growth promoting effect of strain G14 on Chinese cabbage (left: plant height; right: root)

FIG. 6 shows the activity changes of laccase, lignin peroxidase and manganese peroxidase during the liquid culture of strain G14;

FIG. 7 shows the decolorization of 5 dyes by crude enzyme solution of manganese peroxidase of smoke bacteria G14 of strain G14.

Detailed Description

The invention is further described with reference to the following figures and examples, which are not to be construed as limiting the invention.

Example 1: fungus of tobacco pipe (Bjerkandera adustaG14) isolation, identification and preservation

1.1 Culture medium and preparation thereof

PD medium: peeling fresh potato 200g, boiling distilled water 1.1L for 30min, filtering with 4 layers of gauze to obtain potato liquid, adding chloramphenicol 0.05g into the potato liquid, and dissolving in distilled water to 1L;

PDB culture medium: adding 20g/L glucose into PD culture medium;

PDA culture medium: 20g/L agar powder is added into the PDB culture medium;

carbon source screening culture medium: adding 20g/L carbon source (glucose, maltose, sucrose, soluble starch, fructose) based on PD culture medium, and naturally adding pH;

nitrogen source screening culture medium: on the basis of PDB culture medium, 1g/L nitrogen source (ammonium nitrate, ammonium chloride, ammonium sulfate, urea and peptone) has natural pH;

metal ion screening culture medium: adding 0.5mmol/L metal source (CuS) based on PDB culture medium

、MnS

、CaC

、MgS

、ZnS

And FeC

) And the pH is natural.

PKO inorganic phosphorus Medium Ca ₃ ( PO4) ₂ 3.0 g, sucrose 10.0 g, naCl 0.5g, KCl 0.2 g, (NH) ₄ )2 SO ₄ 0.1g，MgSO ₄ •7H2O 0.1 g，MnSO ₄ 0.004g, yeast extract 0.5g ₄ 0.004g, 1 000 mL of distilled water, 15.0 g of agar, and pH 7.0.

10g of Monatina organophosphorus medium, (NH) ₄ ) ² SO ₄ 0.5g, naCl 0.3 g, KCl 0.3 g, lecithin 0.2 g, mgSO 0 ₄ •7H ₂ O 0.3 g，FeSO ₄ •7H ₂ O 0.03g，MnSO ₄ •4H ₂ O 0.03 g，CaCO ₃ 5g, yeast extract 0.4 g, agar 20g, distilled water 1 000 mL, pH 7.0.

1.2 Separating and purifying strain

The collected blueberry fruits attached with the strain G14 are soaked in 75% alcohol solution for 10s by using an ultra-clean bench in a laboratory, then transferred into 3% (V/V) NaClO solution for disinfection for 3min, washed by sterile water, and then dried on sterile absorbent paper. Cutting pericarp with diameter of about 1cm, inoculating into PDA solid culture medium, culturing in 28 deg.C constant temperature incubator for 4d, inoculating fresh mycelium at pericarp edge into new PDA culture medium, culturing, and repeating the transfer for multiple times to obtain purified strain G14.

1.3 Identification of the species

And (3) identifying the strain morphology: in a clean bench, fresh clumps of strain G14 were picked up by a sterile punch of 6.0mm diameter, placed in the center of PDA medium and cultured at a constant temperature of 28 ℃. And observing the growth condition, morphology, color and the like of the bacterial colony in time, and recording the transverse and longitudinal diameters of the bacterial colony. During the culture at the 4 th day, hyphae at the edges of colonies were picked and placed on a glass slide, and morphological characteristics of the hyphae were observed with an optical microscope to perform morphological identification. The morphological identification is referred to in the handbook of fungal identification. During the culture period of the strain of the smoke tube bacterium G14, hyphae of 2d and 4d colonies grow rapidly, and the 4d strain is paved on a culture dish. The colony is characterized by a circular shape, ring lines at the edge of the colony, milky floccule at the center, developed hyphae and radial growth (figure 1).

And (3) identifying the strain in molecular biology: scraping new living thingsAnd (3) melting hypha of the strain G14, extracting genomic DNA of the strain G14 by using a Fungal DNA Midi Kit Fungal DNA extraction Kit as a template, and performing PCR amplification on the strain by using a Fungal universal primer. The amplified PCR amplification product is sent to Chongqing Kyongke encyclopedia biotechnology company for sequencing, and simultaneously the sequence is compared with B.adusta sequences with different sources in the comparison result of BlastN and ITS sequences of different species in the same genus for multi-sequence comparison analysis, clustalW in MEGA7.0 software is used for multi-sequence comparison, and then a phylogenetic tree is constructed by adopting an MEGA7.0 middle adjacency method (Neighbor junction, NJ). After the total DNA of the strain G14 is amplified by ITS1 and ITS4 primers, a PCR product is sent to Chongqing KyogKongshi biotechnology limited company for sequencing to obtain a sequence with the length of 570 bp. After obtaining the sequencing result, the sequence is registered in GenBank to obtain a registration number MT601950. Meanwhile, the sequence similarity comparison is carried out by Blast in NCBI, MEGA7.0 software is used for manufacturing strains similar to the strain to construct a phylogenetic tree (figure 2), and the result shows that the strains of the smoke tube bacterium G14 and the smoke tube bacteriumB.adustaKJ668570.1 has the highest similarity and the closest relationship.

1.4 Strain preservation of strain G14:

the preservation unit: china general microbiological culture Collection center (CGMCC), address: the microbial research institute of Chinese academy of sciences, no. 3 Xilu-Beijing, chaoyang, beijing area, and China academy of sciences, is classified and named as: fungus of tobacco pipe (Bjerkandera adustaG14), preservation number is CGMCC No.20218, preservation date: year 2020, month 07, and day 06.

Example 2: tobacco pipe bacterium (A), (B)Bjerkandera adustaG14) determination of the bacteriostatic Properties

The bacteriostatic activity of the strain on 6 plant pathogenic bacteria to be tested is determined by adopting a plate confronting method. Firstly, activating all endophytic fungi strains and pathogenic bacteria, then punching holes on fresh hyphae at the edges of the endophytic fungi strains and the pathogenic bacteria activated in a PDA (personal digital assistant) plate by using a puncher with the diameter of 6mm in a superclean workbench, respectively inoculating fungus cakes obtained by punching the holes on the PDA plate with the diameter of 90 mm, setting 3 times as experimental groups on two sides of a culture medium and the same diameter of 20mm away from the edge of a dish, and inoculating the pathogenic bacteria only on the right side of the plate as a control groupThe same 3 replicates were set, and then both the experimental and control groups were incubated in a constant temperature incubator at 28 ℃ with the front side first and the top side after 1 day. After 5-7 days, the diameter of the pathogenic bacteria colony of the control group plant and the diameter of the pathogenic bacteria colony of the experimental group plant are photographed and recorded when the pathogenic bacteria colony of the control group grows to about two thirds of the culture dish. And (3) calculating an inhibition rate: inhibition (%) = ((control group plant pathogenic bacteria colony diameter-6 mm) - (experimental group plant pathogenic bacteria colony diameter-6 mm)/(control group plant pathogenic bacteria colony diameter-6 mm)) × 100. The strain G14 is measured to be used for treating two blueberry root rot pathogenic bacteria (A), (B) and (C)Fusarium oxysporum）、（F. verticillioide) The inhibition rate reaches 60.84 percent and 55.69 percent. Pathogenic bacteria of blueberry leaf spot: (A)Alternaria alternata) The inhibition ratio of (2) was 44.44%. For sclerotium rolfsii pathogenic bacteria: (Sclerotium rolfsii) Pathogenic bacteria of pepper anthracnose: (Colletotrichum scovillei) And Sclerotinia sclerotiorum (A) and (B)Sclerotinia sclerotiorum) The inhibition rates of (a) were 56.92%, 45.71% and 45.45%, respectively. The inhibition rate of the strain G14 on 6 pathogenic bacteria is shown in figure 3.

Example 3: fungus of tobacco pipe (Bjerkandera adustaG14) determination of growth-promoting Properties

And (3) endophytic fungi phosphorus dissolving screening:

the method comprises the steps of performing activated culture on separated and purified blueberry endophytic fungi, selecting activated endophytic fungi blocks (D = 0.6 cm) to be respectively inoculated on a PKO inorganic phosphorus culture medium and a Monkina organic phosphorus culture medium, placing a inoculated disposable culture dish in a constant-temperature incubator at 28 ℃ to be cultured for 72 h, judging whether a bacterial strain has a phosphorus dissolving effect by observing whether a transparent phosphorus dissolving ring appears or not, and primarily judging whether the bacterial strain has the phosphorus dissolving capacity or not by measuring and calculating the ratio of the diameter D value of the phosphorus dissolving ring appearing in the bacterial strain to the diameter D value of a bacterial colony. Experiments show that the D/D value of G14 in the Monkina culture medium is 1.7 at most, and the Monkina culture medium shows better phosphorus dissolving property. In the PKO culture medium, the D/D value of G14 is maximally 1.7, and the growth speed of the strain in the PKO culture medium and Monkinna culture medium is different from that of the phosphorus dissolving ring. The map of the phosphorus solubilizing effect of strain G14 is shown in FIG. 4.

The growth promoting effect of the blueberry phosphorus-dissolving endophytic fungi on different crops is as follows: selecting 3 crops of commercial corns, chinese cabbages and wheat as test materials, selecting soil under Pinus massoniana after Chong building of Guizhou university as test soil, sterilizing the surfaces of the seeds of the commercial Chinese cabbages, wheat and corn for 5min by using 3% sodium hypochlorite, washing the seeds by sterile water, accelerating germination under the dark condition of 28 ℃, sowing the seeds, and breeding in a seedbed. And 5d later, selecting seedlings with basically consistent growth vigor for testing. And (3) carrying out liquid fermentation on the screened phosphorus-dissolving strains by using a PDA (PDA) culture medium, and culturing for 7 d at 28 ℃ at the rotating speed of a shaking table of 180 r/min to prepare a bacterial suspension. Control 1 and treatment 2 groups were set for each plant, each group was 3 replicates and each replicate was treated with 10 plants each. The bacterial suspension is placed in a seedling raising hole basin by adopting a root irrigation mode. And (3) keeping the soil moist in the whole experiment period, harvesting plants after 30d, and measuring the height, fresh weight, root system area, leaf area and the like of the plants. In the actual test process, the strain G14 is observed to have unobvious plant height growth promoting effect on corn and wheat, has certain growth promoting effect on the roots of the corn, and has obvious growth promoting effect on white vegetables, as shown in figure 5.

Example 4: fungus of tobacco pipe (Bjerkandera adustaMeasurement of decolorization characteristic of G14)

Determination of Activity of Grifola Frondosa G14 laccase, lignin peroxidase and manganese peroxidase

And (2) filling 70mLPDB into 250mL triangular bottles, sterilizing for 30min at the high temperature of 121 ℃, inoculating 4 blocks of newly activated mycelium blocks with the diameter of 9mm into each triangular bottle on an ultra-static workbench, placing the triangular bottles in a constant-temperature incubator at 28 ℃ for light-proof culture, respectively absorbing 1.0mL of fermentation liquor on inoculated 2 nd, 4 th, 6 th, 8 th, 10 th and 15 th days, centrifuging for 5min at 7000r/min, and taking supernatant to measure the enzyme activity by using an ultraviolet visible spectrophotometer.

Laccase (Lac) activity assay: a3.0 mL reaction system contained 1.95mL of citric acid-disodium hydrogen phosphate buffer (pH 5.0), 1.0mL of LABTS (1.0 mmol/L), and 0.05mL of the supernatant fermentation broth, and after a water bath at 30 ℃ for 3min, the change in absorbance at 420nm within 3min was measured. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmol ABTS substrate to product per minute [ ABTS:ε ₄₂₀ =3.6×10 ⁴ L/(mol·cm)]. Each of the above treatments was repeated 3 times.

Manganese peroxidase (manganese peroxidase) activity assay: 0.84mL of sodium malonate (0.05 mol/L) and 0.05mL of SnSO ₄ (0.01 mol/L), 0.05mL2, 6-dimethoxyphenol (2, 6-DMP) (0.01 mol/L), 0.05mL of supernatant fermentation broth, and 0.01mLH ₂ O ₂ (0.01 mol/L) was placed in a cuvette, and the change in absorbance at 468nm was measured for 1min with distilled water as a control. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1. Mu. Mol of 2,6-DMP substrate per minute. [2,6-DMP:ε ₄₆₈ =4.96×10 ⁴ L/(mol·cm)]. Each of the above treatments was repeated 3 times.

Lignin peroxidase (LiP) activity assay: mixing 1.7mL of potassium sodium tartrate tetrahydrate solution (0.25 mol/L) and 0.05mL of Veratryl Alcohol (VA) solution (0.1 mol/L), preheating in a 30 deg.C water bath, adding 0.05mL of supernatant fermentation broth, and adding 0.05mL of LH ₂ O ₂ (0.1 mol/L), and the change in absorbance at 310nm within 3min was measured ^[26] . One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 μmolVA substrate per minute [ VA:ε ₃₁₀ =9.3×10 ³ L/(mol·cm)]. Each of the above treatments was repeated 3 times.

Strain G14 was monitored for 3 ligninolytic enzyme activities from day 2 and 3 enzyme activities increased with the time of culture. Wherein, the laccase activity reaches the peak value of 1.67U/L on the 8 th day, and then gradually decreases; the activity of the manganese peroxidase also reaches a peak value of 12.5U/L on the 6 th day and then gradually decreases; the lignin peroxidase activity gradually declined after reaching a large value of 4.38U/L on day 6. The results of the change over time in the enzyme-producing activity of Shigella G14 are shown in FIG. 6.

Decolorizing 5 dyes by crude enzyme liquid of the manganese peroxidase of the smoke tube bacteria G14: 5 dyes of Congo red, malachite green, crystal violet, chrome black T and methyl orange are respectively added into a PDB culture medium to ensure that the final concentration of the dyes is 50mg/L. Sterilizing at 121 deg.C under high temperature and high pressure for 30min, and inoculating 4 newly activated smoke tube bacteria G14 with diameter of 1cm into each triangular flask under aseptic condition. Culturing the inoculated triangular flask in a constant temperature incubator at 28 ℃ in the dark, absorbing 1mL of fermentation liquor when the smoke tube bacterium G14 produces enzyme for a large number of days, detecting the light absorption value of the dye fermentation liquor by using an ultraviolet visible spectrophotometer under the maximum absorption wavelengths of different dyes, and taking the dye culture liquor which is not inoculated as a reference. And scanning the maximum absorption wavelength of the dye in the range of 300-800nm by using an ultraviolet spectrophotometer. The formula for calculating the decolorization ratio of the dye is as follows. The dye decolorization rate is calculated as follows: r = (A0-A1)/A0 × 100% by weight, R is decolorization ratio, A0 is absorbance of control, and A1 is absorbance under treatment conditions.

The crude enzyme solution of chaelia G14 manganese peroxidase was decolorized with respect to five dyes as shown in fig. 7, but the decolorization effect was varied, and the decolorization ability with respect to dyes was ranked as congo red > nobelian black T > methyl orange > crystal violet > malachite green. The different decolorizing effects for different dyes were analyzed due to the different chemical compositions of the dyes.

Sequence listing

<110> Guizhou university

<120> smoke tube bacterium G14 and application

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<170> PatentIn version 3.3

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Sequence listing

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Claims (1)

1. A strain of Cladophora (A. Borealis: (A. Borealis)Bjerkandera adusta ) G14, characterized in that: the fumarose G14 is preserved in China general microbiological culture Collection center (CGMCC) at 7-6.2020, with the preservation number of CGMCC No.20218.

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