CN114214220A

CN114214220A - Bacillus thuringiensis and application thereof in promoting plant growth

Info

Publication number: CN114214220A
Application number: CN202010991981.6A
Authority: CN
Inventors: 方荣祥; 张玉满; 王海婷; 肖娜; 陈晓英
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2022-03-22
Anticipated expiration: 2040-09-18
Also published as: CN114214220B

Abstract

The invention discloses a bacillus thuringiensis strain and application thereof in promoting plant growth. The Bacillus thuringiensis is particularly Bacillus thuringiensis (FR103), and the preservation number of the Bacillus thuringiensis in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19018. Experiments prove that the growth of the plant can be promoted by spraying the bacillus thuringiensis FR103 on the leaf surface of the plant and irrigating the root, which is particularly marked by the remarkable increase of the fresh weight of the overground part. The invention has important application value.

Description

Bacillus thuringiensis and application thereof in promoting plant growth

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to bacillus thuringiensis and application thereof in promoting plant growth.

Background

Bacillus thuringiensis (Bt) is a gram-positive agrobacterium which can parasitize larvae of moths and butterflies and plant surfaces, and is an important entomopathogenic bacterium. Bt produces Insecticidal Crystal Proteins (ICPs) (mainly Cry (Crystal) Proteins) called delta-endotoxins in the spore formation process, and has specific strong poisoning effect on more than 100 kinds of plant pests such as Lepidopera (Lepidopera), Diptera (Diptera), Coleoptera (Coleopter) and animal and plant nematodes. Bt is the most widely used microbial insecticide in the world. In addition, the gene of Bt insecticidal crystal protein is transferred into crops such as tobacco, corn and cotton, so that high-insect-resistant varieties which are popularized in a large area are obtained. The Bt gene becomes an insect-resistant gene which is most widely applied in plant transgenic breeding, partially replaces the traditional chemical pesticide, and has important value for environment-friendly sustainable agriculture.

In 1901, scientists isolated Bt from silkworms suffering from "sudden fall disease" for the first time, and so far, 6 million varieties of Bt strains, which are divided into 70 serotypes, have been isolated from all over the world successively. Since the first cloning by Schnepf et al in 1981 into the delta-endotoxin encoding gene of Bacillus thuringiensis subspecies HD-1 (HD-1), more and more insecticidal crystal proteins were cloned and sequenced sequentially (Schnepf and Whiteley, 1981; Schnepf et al, 1985; Takawa et al, 1989; Guotthree et al, 1991). In addition to the classical BtCry crystal toxins, it has been discovered that cytolytic Cyt, a cytolytic enzyme, Cyt, and another insecticidal nutritional protein (Vip) that is not a delta-endotoxin, is secreted during the vegetative growth phase prior to Bt sporulation and is referred to as a second generation insecticidal protein. The diversity of insecticidal toxic proteins expands the application range of Bt, and more than 300 insecticidal crystal protein coding genes (www.lifesci.sussex.ac.uk/home/Neil _ Crickmore/Bt /) have been isolated and determined so far, including cry1-78, cyt1-3 and Vip 1-3 groups. Because Bt has plasmid polymorphism and the diversity and conservation of nuclear genes, the number and variety of genes for coding insecticidal proteins and the positioning on plasmids and chromosomes are different, the insecticidal spectrum and insecticidal toxicity of Bt are obviously different due to the difference of serotypes or subspecies (Kronstad et al, 1983), new Cry, Cyt and Vip proteins are continuously separated and identified from Bt (Panwar1and Kaur, 2019), the Bt insecticidal spectrum is further enriched, and a foundation is laid for Bt modification. The Bt biological agent promotes the development of modern agriculture by the advantages of low toxicity, high efficiency and the like and the large-area application of Bt transgenic plants. However, with the long-term large-area use of Bt and Bt genes, the resistance risk of plant pests to the Bt genes is also increased year by year, so that new Bt strains are urgently needed to be screened and discovered, new Bt functions are explored, existing Bt strains are researched and developed/modified, and resource reserve is provided for reasonably and continuously utilizing Bt.

With the rapid development of economy, the pollution of soil, water and the like is becoming more serious, and the pollution treatment needs aging accumulation, wherein the weight reduction/drug reduction is one of the important measures for developing continuous ecological agriculture. The existing microbial fertilizer (agent) mostly depends on the superposition effect of various microbial strains, and the growth promoting effect is relatively slow, so the microbial fertilizer (agent) is often mixed with a chemical fertilizer for application. In addition, when a large amount of bacterial manure is applied for a long time, plant pest groups also tend to rise along with the improvement of soil organic matters. The environment-friendly biological pesticide represented by the Bt strain only focuses on the insecticidal action, and the Bt strain reports with other functions except the insecticidal action are not seen; the prior relevant patent of Bt also focuses on the application and research of insect resistance, bactericide and biopesticide by taking insecticidal protein as the center, and other functional Bt strain patents except for insecticidal are not seen yet. Digging Bt new strains or multifunctional strains from the existing resources, and organically combining biological bacterial manure with biological pesticide to promote the process of reducing weight and reducing pesticide. Because insects are often the transmission vector of plant viruses, screening multifunctional strains for killing insects, preventing diseases and increasing yield provides important microorganism resources with independent intellectual property rights for developing ecological agriculture, and has important theoretical and application values.

Disclosure of Invention

The object of the present invention is to promote plant growth.

The invention firstly protects Bacillus thuringiensis FR103, which has been preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 of No.1 Siro-Shih-1 of the morning-Yang district in Beijing) in 11 months and 27 days in 2019, and the preservation number is CGMCC No. 19018. Bacillus thuringiensis (Bacillus thuringiensis) FR103CGMCC No.19018 is called Bacillus thuringiensis FR103 for short.

The invention also protects a microbial inoculum which contains the bacillus thuringiensis FR 103.

The usage of the microbial inoculum can be a1) or a 2):

a1) regulating and controlling the growth of plants;

a2) preparing the product for regulating and controlling the growth of the plants.

The invention also provides a preparation method of the microbial inoculum, which comprises the following steps: inoculating bacillus thuringiensis FR103 to a bacterial culture medium and culturing to obtain a bacterial liquid, namely the microbial inoculum.

The bacterial culture medium can be LB liquid culture medium.

In the preparation method of the microbial inoculum, the specific culture conditions can be as follows: 30 + -2 ℃ and 200-250rpm/min (such as 200-230rpm/min, 230-250rpm/min, 200rpm/min, 230rpm/min or 250rpm/min) for 12-36h (such as 12-24h, 24-36h, 12h, 24h or 36 h).

The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of bran, soybean meal, corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The invention also protects the application of the bacillus thuringiensis or the microbial inoculum containing the bacillus thuringiensis, which can be a1) or a 2):

a1) regulating and controlling the growth of plants;

In the above application, the regulating the plant growth may be promoting the plant growth.

In the above application, the product may be a fertilizer. The fertilizer may be a microbial fertilizer.

In the above application, the bacillus thuringiensis may be bacillus thuringiensis FR 103. The microbial inoculum containing the bacillus thuringiensis can be any one of the microbial inoculants.

The invention also protects a product containing bacillus thuringiensis or a bacillus thuringiensis-containing microbial inoculum; the product can function to regulate plant growth.

In the above product, the regulating plant growth may be promoting plant growth.

Any of the above products may be a fertilizer. The fertilizer may be a microbial fertilizer.

In the above product, the bacillus thuringiensis may be bacillus thuringiensis FR 103. The microbial inoculum containing the bacillus thuringiensis can be any one of the microbial inoculants.

The invention also provides a method for promoting plant growth, which can be used for treating plants by using the bacillus thuringiensis so as to promote the plant growth.

In the above method, the treatment of the plant with Bacillus thuringiensis can be carried out by spraying the plant with the above-ground parts (such as leaves) and/or by pouring the Bacillus thuringiensis with the below-ground parts (such as roots).

In the above method, the treating the plant with bacillus thuringiensis may be treating the plant with a microbial inoculum containing bacillus thuringiensis.

In the above method, the treatment of the plant with the Bacillus thuringiensis-containing microbial inoculum can be carried out by spraying the microbial inoculum containing Bacillus thuringiensis onto the aerial parts (such as leaves) of the plant and/or pouring the microbial inoculum containing Bacillus thuringiensis onto the underground parts (such as roots) of the plant.

In any of the above methods, the bacillus thuringiensis can be bacillus thuringiensis FR 103. The microbial inoculum containing the bacillus thuringiensis can be any one of the microbial inoculants.

Any of the above-described methods for promoting plant growth may be specifically characterized by an increase in the fresh weight of the aerial parts of the plant.

Any of the plants described above may be any of the following c1) to c 8): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the Liliaceae family; c4) a cruciferous plant; c5) leek; c6) chinese leek (Allium tuberosum rottl. exspreng) jiuxian No. 18; c7) arabidopsis thaliana; c8) the wild type Arabidopsis thaliana Columbia-0 subtype.

Any one of the microbial fertilizers can be a compound microbial fertilizer and/or a biological organic fertilizer. The compound microbial fertilizer can be a fertilizer compounded by microbial inoculum, nutrient substances and organic matters. The compound microbial fertilizer has the functions of both microbes and chemical fertilizers. The biological organic fertilizer can be a fertilizer formed by compounding a microbial inoculum and a decomposed organic fertilizer. The dosage form of the compound microbial fertilizer and/or the biological organic fertilizer can be granules.

Experiments prove that the plant growth can be promoted by spraying the bacillus thuringiensis FR103 on the leaf surfaces of plants (Chinese chives or arabidopsis) and irrigating roots, and the fresh weight of the overground parts of the plants is obviously increased. The invention has important application value.

Drawings

FIG. 1 is a partial result of primary screening of plant growth-promoting bacteria. Indicates significant differences, indicates very significant differences.

FIG. 2 is a partial result of the rescreening of plant growth-promoting bacteria. Indicates significant differences, indicates very significant differences; the percentage represents the percentage of fresh weight increase compared to the corresponding control group and is calculated by the formula: (fresh weight of aerial parts of Arabidopsis plants of experimental group-fresh weight of aerial parts of Arabidopsis plants of control group)/fresh weight of aerial parts of Arabidopsis plants of control group.

FIG. 3 shows the results of morphological identification and biochemical experiments of FR 103.

FIG. 4 shows the results of morphological identification and biochemical experiments of FR 104.

FIG. 5 shows the growth promoting effect of Bacillus thuringiensis FR103 and Bacillus thuringiensis FR104 on wild type Arabidopsis thaliana. Indicates there was a very significant difference. The percentage represents the percentage of fresh weight increase compared to the corresponding control group and is calculated by the formula: (fresh weight of aerial parts of Arabidopsis plants of experimental group-fresh weight of aerial parts of Arabidopsis plants of control group)/fresh weight of aerial parts of Arabidopsis plants of control group.

FIG. 6 shows the growth promoting effect of Bacillus thuringiensis FR103 and Bacillus thuringiensis FR104 on Chinese chives. Indicates there was a very significant difference. The percentage represents the percentage of fresh weight increase compared to the corresponding control group and is calculated by the formula: (fresh weight of aerial part of leek in experimental group-fresh weight of aerial part of leek in control group)/fresh weight of aerial part of leek in control group.

Deposit description

The strain name is as follows: bacillus thuringiensis

Latin name: bacillus thuringiensis

The strain number is as follows: FR103

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 11/2019 and 27/month

Registration number of the preservation center: CGMCC No.19018

The strain name is as follows: bacillus thuringiensis

Latin name: bacillus thuringiensis

The strain number is as follows: FR104

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 11/2019 and 27/month

Registration number of the preservation center: CGMCC No.19019

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Beef extract peptone solid medium: dissolving 10g of peptone, 3g of beef extract, 5g of sodium chloride and 15g of agar in a small amount of water, and then diluting to 1L with water; sterilizing at 121 deg.C for 30 min.

Beef extract peptone liquid medium: dissolving 10g of peptone, 3g of beef extract and 5g of sodium chloride in a small amount of water, and then diluting to 1L with water; sterilizing at 121 deg.C for 30 min.

LB solid medium: dissolving 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar in a small amount of water, and then adding water to a constant volume of 1L; sterilizing at 121 deg.C for 30 min.

LB liquid medium: dissolving 10g of tryptone, 5g of yeast extract and 10g of sodium chloride in a small amount of water, and then adding water to a constant volume of 1L; sterilizing at 121 deg.C for 30 min.

Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004, 36: 167-.

Chinese chive (Allium Tuberosum Rottll. ex Spreng) Jiuxiang No. 18 is a commercial seed of chive. Hereinafter, the Jiujiangxing Chinese chives (Allium tuberosum Rottll. ex Spreng) No. 18 is abbreviated as chives.

In the following examples, statistical analysis was performed on the average of the above-ground fresh weight of an arabidopsis individual plant and the yield of different treated leek cells, and SPSS index-sample t test was used for data significance analysis.

Example 1 isolation, identification and preservation of Bacillus thuringiensis FR103CGMCC No.19018 and Bacillus thuringiensis FR104 CGMCC No.19019

First, obtaining of the Strain

1. Insect samples and rhizosphere soil samples were collected from plant rhizosphere soil.

2. Taking an insect sample, firstly treating 15Sec with 75% (v/v) ethanol water solution, then disinfecting the surface with 15% (m/v) sodium hypochlorite solution for 15min, then cleaning with sterile water for 3 times, grinding with a sterile mortar under sterile conditions to obtain homogenate, standing for 10min, and collecting a supernatant fluid 1.

3. To 5g rhizosphere soil sample was added 45mL of sterile distilled water, stirred for 15min, left to stand for 10min, and the supernatant 2 was collected.

4. Diluting the supernatant 1and the supernatant 2 respectively (adding 9mL of sterile water into 1mL of the supernatant, mixing well), sucking 1mL of the supernatant from the test tube, adding the supernatant into another sterile test tube containing 9mL of sterile water, mixing well, and so on to obtain 10^-2、10^-3、10^-4、10^-5、10^-6、10^-7Serial dilutions of the bacterial suspension. And uniformly coating 0.1mL of each dilution on a beef extract peptone solid medium, and standing and culturing at the constant temperature of 30 ℃ for 2-3 d.

5. After the step 4 is completed, single bacterial colonies on the beef extract peptone solid medium are respectively picked up and repeatedly purified for more than 3 times. )

164 strains of bacteria were obtained in total and designated FR001-FR164, respectively.

And (3) strain preservation: respectively inoculating single colonies of 164 strains of bacteria to a beef extract peptone liquid culture medium, and culturing at 28 ℃ for 16h to obtain corresponding culture bacteria liquid. 1 part by volume of the culture broth and 1 part by volume of a 50% (v/v) aqueous solution of glycerol were mixed and stored at-80 ℃.

Second, primary screening of plant growth-promoting bacteria

The bacteria to be tested are 164 strains of bacteria obtained in the first step respectively.

Detecting the effect of each bacterium to be detected on promoting the growth of arabidopsis thaliana, wherein the repeated steps are as follows:

1. activating the strain of the bacteria to be detected, which is stored at the temperature of minus 80 ℃ in the step one, on a beef extract peptone solid medium.

2. After the step 1 is completed, selecting a single colony on a beef extract peptone solid culture medium, inoculating the single colony on a 3mL beef extract peptone liquid culture medium, and carrying out shaking culture at 28 ℃ and 230rpm/min for 12h to obtain a culture bacterial liquid 1.

3. After the step 2 is completed, inoculating 50 μ L of the culture solution 1 into 50mL of beef extract peptone liquid medium, and carrying out shaking culture at 28 ℃ and 230rpm/min overnight to obtain a culture solution 2.

4. And (3) after the step 3 is finished, taking the culture bacterial liquid 2, centrifuging at 5000rpm/min for 10min, and collecting thalli.

5. After the step 4 is finished, taking the thalli, and washing the thalli for 2 times by using PBS buffer solution; resuspending with 20mL PBS buffer to obtain OD_600nmThe value is about 1.0.

6. Adding 1.8L MS aqueous solution (containing 2.6g MS) into 8L vermiculite, and mixing to obtain culture medium.

7. Sowing wild type Arabidopsis seeds in an MS solid culture medium, and performing light-dark alternate culture (16h illumination culture/8 h dark culture) at 22 ℃ for 10 days to obtain Arabidopsis seedlings.

8. Detection of fresh weight of aerial parts of Arabidopsis plants

Experimental groups:

(1) adding 50mL of bacterial liquid to be detected and 800mL of tap water into the culture medium, fully and uniformly mixing, and filling into a culture basin (the specification is 7cm multiplied by 7 cm).

(2) After the step (1) is completed, the arabidopsis seedlings with basically consistent growth states are transplanted into a culture pot, and are alternately cultured in light and dark at the temperature of 22 ℃ (16h illumination culture/8 h dark culture) for 7 days. 2 strains per pot, for a total of 27 pots.

(3) After completion of step (2), alternate light-dark culture (16h light culture/8 h dark culture) was carried out at 22 ℃ for 20 days. During the culture period, the fungus solution to be tested is sprayed on the leaf surface and irrigated to the root. The spraying dosage of the leaf surface is 0.2mL of bacterial liquid/strain to be detected. The root irrigation dosage is 0.5mL of bacterial liquid/strain to be detected.

(4) And (4) after the step (3) is finished, weighing the overground part of the arabidopsis thaliana plant to obtain the fresh weight of the overground part of the arabidopsis thaliana plant.

(5) The average value of the fresh weight of the aerial parts of 54 Arabidopsis plants was calculated.

Control group: and replacing the bacterial liquid of the bacteria to be detected in the experimental group with PBS buffer solution, wherein the other solutions are not changed.

Part of the results are shown in FIG. 1(PBS treated as control group, strain treated as experimental group). The results show that the fresh weight average of the overground parts of the Arabidopsis thaliana plants treated by FR102, FR103, FR104, FR105, FR106, FR107, FR108, FR111, FR112, FR113, FR114, FR115, FR116 and FR118 is increased to a certain extent compared with the control group, namely the Arabidopsis thaliana plants have the function of promoting plant growth; the fresh weight of the aerial parts of Arabidopsis plants treated with other strains (e.g.FR 101, FR109, FR110, FR117, FR119) was reduced to some extent or not significantly different compared to the control group, i.e.not having a plant growth promoting function.

Third, the re-screening of plant growth-promoting bacteria

The test bacteria are FR102, FR103, FR104, FR105, FR106, FR107, FR108, FR111, FR112, FR113, FR114, FR115, FR116 or FR 118.

And detecting the effect of each bacterium to be detected on promoting the growth of arabidopsis thaliana again. The experiment was performed 4-5 biological replicates and the average was taken, with each replicate being 1-8 of step two.

Part of the results are shown in FIG. 2(PBS treated as control group, strain treated as experimental group). The result shows that compared with a control group, the fresh weight of the overground parts of the Arabidopsis plants treated by FR103 and FR104 is remarkably increased and the stability is better, namely the Arabidopsis plants have the function of remarkably promoting the growth of plants; although the fresh weight of the overground part of the Arabidopsis plant treated by FR108 is also obviously increased, the stability is poor, and subsequent experiments are not carried out.

It can be seen that FR103 and FR104 are both plant growth promoting bacteria.

Identification of tetra, FR103 and FR104

FR103 and FR104 were identified by methods described in the literature references (Dongxu bead, Chuiamiaiyin (2001) Manual of general bacterial systems identification, Beijing: scientific Press; R.E. Bukannan, N.E. Gibbs (1984) Bergey Manual of bacteria identification (eighth edition) institute of microbiology, national academy of sciences, Bogey Manual of bacteria identification, translation group, Beijing: scientific Press).

Identification of (I) FR103

1. Morphological identification

(1) Colony morphology

FR103 was inoculated into Luria-Bertani (LB) solid medium (aqueous solution containing tryptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L, pH 7.4), shake-cultured overnight at 28 ℃/200rpm, OD was adjusted_600nmTo 1.5, dilution 10⁵And doubling, and coating 45uL of diluted bacteria liquid on a plate. The morphology of single colonies was observed when cultured at 28 ℃ for 1 day or 3 days.

The results are shown in FIG. 3, panel A. The result shows that when cultured for 1d, the colony of FR103 is round, has a diameter of about 7mm, irregular edge, dry surface, light yellow color and opaque colony; when cultured for 3d, the colony is circular, has the diameter of about 1cm, irregular edge, dry surface, light yellow color and opaque colony.

(2) Electron microscopy of bacterial morphology

FR103 is inoculated to an LB liquid culture medium, shake culture is carried out at 28 ℃ for 2d, 3d or 4d, and the bacterial morphology is observed by adopting a Hitachi cold field emission scanning electron microscope (model SU 8010).

The results are shown in FIG. 3B. The results showed that FR103 cells were rod-shaped at 2 days, sporulated after 3 days, and octahedral crystals were formed after 4 days, with the octahedral crystals having a regular morphology.

(3) Mycoderm formation experiment

Inoculating FR103 into beef extract peptone medium, standing at 30 deg.C for 24h, observing bacterial membrane formation condition and shaking for membrane disruption.

The results are shown in FIG. 3C. The results show that FR103 forms a sterile membrane on the surface of the liquid, and has a band-like aggregation of similar biofilms inside the medium, which break up after shaking.

2. Results of homology analysis of 16S rRNA

(1) Using a single colony of FR103 as a template, 27F: 5 '-AGAGTTTGATCMTGGCTCAG-3' and 1492R: and carrying out PCR amplification on the primer pair consisting of 5 '-TACGGYTACCTTGTTACGACTT-3' to obtain a PCR amplification product.

The reaction system was 50. mu.L, including 5uL 10 XBuffer, 5uL 2mM dNTP, 2uL 25mM MgSO₄1.5. mu.L of primers 10 pmol/. mu.L of 27F and 1492R, 1uL of 1.0U/uLKOD-Plus DNA polymerase (TOYOBO, Cat. No. KOD-201).

The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; 15s at 94 ℃, 30s at 56 ℃, 90s at 68 ℃ and 30 cycles; 10min at 68 ℃.

(2) Sequencing the PCR amplification product.

The result shows that the PCR amplification product contains SEQ ID NO: 1.

Converting SEQ ID NO: 1 are aligned at the NCBI. The results showed that FR103 has up to 100% homology with Bacillus thuringiensis (Bacillus thuringiensis).

3. Biochemical experiments

(1) Hydrolysis test of soluble starch

Solid plate: prepared from an aqueous solution containing 5g/L, NaCl 5g/L beef extract, 5g/L soluble starch and 10g/L agar.

FR103 is inoculated on a solid plate, after 3d of culture at 28 ℃, Lugol iodine solution (standard iodine solution) is poured on the surface of the plate, and transparent rings are arranged around colonies to hydrolyze starch.

The results are shown in FIG. 3D. The results show that there are clear circles of FR103, indicating that FR103 can hydrolyze starch.

(2) Lecithase assay

Yolk flat plate: taking fresh egg yolk under aseptic condition, adding equivalent physiological saline for dilution and shaking up, taking 10ml, adding into 200ml of LB solid culture medium with temperature of 50-55 ℃ and sterility, mixing evenly, pouring into a culture dish to prepare an egg yolk flat plate, and standing overnight for use.

And (3) inoculating the FR103 cultured for 18-24 h on a yolk plate, culturing at room temperature for 1d or 2d, observing whether an opaque region appears around the lawn of the fungus, and judging whether the fungus produces the lecithinase.

The results are shown in FIG. 3, panel E. The results show that FR103 forms a clear white opaque ring (regular and smooth boundary) at 1d, which indicates that the bacterium has strong capability of producing lecithinase.

(3) V.p. test

Culture medium: an aqueous solution containing peptone 5g/L, glucose 5g/L and sodium chloride 5 g/L.

FR103 is inoculated to the culture medium by 1 per mill, and is subjected to shake culture at 37 ℃ and 230rpm for 6d to obtain a culture solution. Then 600ul of 5% alpha-naphthol ethanol solution was added to 2.5ml of the culture solution, and after mixing, 200ul of 40% KOH solution was added, and after mixing again, color change was observed, and the positive bacteria appeared red.

The results are shown in FIG. 3, panel F. The result showed that FR103 was positive in v.p. reaction, indicating that the bacterium was able to decompose glucose to produce pyruvic acid, and further decarboxylate pyruvic acid to acetylmethylmethanol, which was oxidized to diacetyl in an alkaline environment and further combined with guanidino groups contained in arginine and the like in the medium to form a red compound.

The results show that FR103 has a strong ability to decompose glucose.

In conclusion, according to the morphological identification, molecular identification and biochemical experiment results of FR103, FR103 was identified as Bacillus thuringiensis (Bacillus thuringiensis).

Identification of (di) FR104

1. Morphological identification

(1) Colony morphology

Replacing FR103 with FR104 according to the method of 1(1) in the step (one), and keeping the other steps unchanged.

The results are shown in FIG. 4A. The result shows that when the bacterial colony is cultured for 1d, the bacterial colony is circular, the diameter is about 6mm, the edge is irregular, the surface is dry, the color is light yellow, and the bacterial colony is opaque; when cultured for 3d, the colony is circular, has the diameter of about 1.1cm, irregular edge, dry surface, light yellow color and opaque colony.

(2) Electron microscopy of bacterial morphology

Replacing FR103 with FR104 according to the method of 1(2) in the step (one), and keeping the other steps unchanged.

The results are shown in FIG. 4B. The results showed that FR104 cells at 2 days had a rod-like shape, spores were produced after 3 days, and crystals having an octahedral structure were produced after 4 days, with the octahedral crystal morphology being irregular.

(3) Mycoderm formation experiment

Replacing FR103 with FR104 according to the method of 1(3) in the step (one), and keeping the other steps unchanged.

The results are shown in FIG. 4C. The results show that FR104 forms a pellicle on the surface of the culture solution, and the pellicle is easily broken after shaking.

2. Results of homology analysis of 16S rRNA

(1) Using a single colony of FR104 as a template, 27F: 5 '-AGAGTTTGATCMTGGCTCAG-3' and 1492R: and carrying out PCR amplification on the primer pair consisting of 5 '-TACGGYTACCTTGTTACGACTT-3' to obtain a PCR amplification product.

(2) Sequencing the PCR amplification product.

The result shows that the PCR amplification product contains SEQ ID NO: 2.

Converting SEQ ID NO: 2 are aligned at NCBI. The results showed that FR104 has up to 100% homology with Bacillus thuringiensis (Bacillus thuringiensis).

3. Biochemical experiments

(1) Hydrolysis test of soluble starch

Replacing FR103 with FR104 according to the method of 3(1) in the step (one), and keeping the other steps unchanged.

The results are shown in FIG. 4D. The results show that there are clear circles of FR104, indicating that FR104 can hydrolyze starch.

(2) Lecithase assay

Replacing FR103 with FR104 according to the method of 3(2) in the step (one), and keeping the other steps unchanged.

The results are shown in FIG. 4, E. The results show that FR104 forms a clear white opaque ring (irregular border) at 2d, indicating that the bacterium has a weak ability to produce lecithinase.

(3) V.p. test

Replacing FR103 with FR104 according to the method of 3(3) in the step (one), and keeping the other steps unchanged.

The results are shown in FIG. 4, panel F. The result showed that FR104 was positive in v.p. reaction, indicating that the bacterium was able to decompose glucose to produce pyruvic acid, and further decarboxylate pyruvic acid to acetylmethylmethanol, which was oxidized to diacetyl in an alkaline environment and further combined with guanidino groups contained in arginine and the like in the medium to form a red compound.

The results show that FR104 has a strong ability to decompose glucose.

In conclusion, according to the morphological identification, molecular identification and biochemical experiment results of FR104, FR104 was identified as Bacillus thuringiensis (Bacillus thuringiensis).

Fifth, preservation

The FR103 separated in the first step is preserved in the China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu No.1 Beijing, Chaoyang, and the like) in 27.11 months in 2019, and the preservation number is CGMCC No. 19018. FR103 is called Bacillus thuringiensis FR103CGMCC No.19018, simply Bacillus thuringiensis FR 103.

The FR104 separated in the first step is preserved in the China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Xilu No.1 Beijing, Chaoyang, and the like) in 2019, 11 months and 27 days, and the preservation number is CGMCC No. 19019. FR104 is called Bacillus thuringiensis FR104 CGMCC No.19019, abbreviated as Bacillus thuringiensis FR 104.

Example 2 growth promoting Effect of Bacillus thuringiensis FR103 and Bacillus thuringiensis FR104 on wild type Arabidopsis thaliana

The experimental site is an arabidopsis greenhouse in the institute of microbiology and biotechnology laboratory of the Chinese academy of sciences. A pot experiment is adopted to carry out a growth promotion experiment of wild type arabidopsis, the experiment is repeated three times to obtain an average value, and the specific steps of each experiment are as follows:

1. preparation of Bacillus thuringiensis FR103 bacterial liquid 1and Bacillus thuringiensis FR103 bacterial liquid 2

(1) The strain of Bacillus thuringiensis FR103 stored at-80 ℃ in step one of example 1 was activated on LB solid medium.

(2) After the step (1) is completed, selecting a single colony on an LB solid culture medium, inoculating the single colony on 3mL of an LB liquid culture medium, and carrying out shaking culture at 28 ℃ and 230rpm/min for 12h to obtain an activated bacterial liquid.

(3) After the step (2) is completed, 50 mul of the activated bacterium solution is inoculated in 50mL of LB liquid culture medium, and shaking culture is carried out at 28 ℃ and 230rpm/min overnight, so as to obtain the cultured bacterium solution.

(4) And (4) after the step (3) is finished, taking the culture bacterial liquid, centrifuging at 5000rpm/min for 10min, and collecting thalli.

(5) After the step (4) is finished, taking the thalli and using ddH₂O resuspending to obtain OD_600nmBacillus thuringiensis FR103 strain 1and OD having a value of about 1.0_600nmBacillus thuringiensis FR103 strain 2 with a value of about 0.5.

2. Sowing wild type Arabidopsis seeds in an MS solid culture medium, and performing light-dark alternate culture (16h illumination culture/8 h dark culture) at 22 ℃ for 10 days to obtain Arabidopsis seedlings.

3. Detection of fresh weight of aerial parts of Arabidopsis plants

A. Experimental groups:

(1) adding 50mL of Bacillus thuringiensis FR103 bacterial solution 1and 800mL of tap water into 8L of nutrient soil, mixing well, and placing into a culture pot (specification is 7cm multiplied by 7 cm).

(2) After the step (1) is completed, the arabidopsis seedlings with basically consistent growth states are transplanted into a culture pot, and are alternately cultured in light and dark at the temperature of 22 ℃ (16h light culture/8 h dark culture) for 2 weeks. 2 strains per pot, for a total of 3 pots.

(3) After completion of step (2), alternate light-dark culture (16h light culture/8 h dark culture) was carried out at 22 ℃ for 7 days. During the culture period, the bacillus thuringiensis FR103 bacterial liquid 2 is sprayed on the leaf surface and irrigated to the root. The spraying dosage of the leaf surface is 0.5mL of Bacillus thuringiensis FR103 bacterial liquid 2/strain. The root irrigation dosage is 1mL of Bacillus thuringiensis FR103 bacterial liquid 2/strain.

(5) The average value of the fresh weight of the aerial parts of 6 Arabidopsis plants was calculated.

B. Control group

Replacing H for the bacillus thuringiensis FR103 bacterial liquid 1and the bacillus thuringiensis FR103 bacterial liquid 2 in the experimental group ₂0, and the others are not changed.

After completion of step (3), the growth state of Arabidopsis thaliana is shown in the left panel of A in FIG. 5 (CK is a control group).

The average of the fresh weight of the aerial parts of Arabidopsis plants is shown as B in FIG. 5 (H)₂0 is control group and strain treatment is experimental group). Results show that compared with a control group, the fresh weight of the overground part of an arabidopsis thaliana plant treated by bacillus thuringiensis FR103 is obviously increased by 100.7%.

According to the steps, the bacillus thuringiensis FR103 is replaced by the bacillus thuringiensis FR104, and other steps are not changed.

The growth state of Arabidopsis thaliana is shown in the right panel of A in FIG. 5 (CK is control).

The average of the fresh weight of the aerial parts of Arabidopsis plants is shown as B in FIG. 5 (H)₂0 is control group and strain treatment is experimental group). Results show that compared with a control group, the fresh weight of the overground part of the Arabidopsis plant treated by the Bacillus thuringiensis FR104 is obviously increased by 129.0%.

The results show that both bacillus thuringiensis FR103 and bacillus thuringiensis FR104 can remarkably promote the growth of wild arabidopsis; the growth of the wild type arabidopsis is promoted by increasing the fresh weight of the overground part of the wild type arabidopsis plant.

Example 3, bacillus thuringiensis FR103 and bacillus thuringiensis FR104 were tested on the growth promoting effect of leeks on organic agricultural farms, beijing tianan.

The preparation methods of bacillus thuringiensis FR103 bacterial liquid 2 and bacillus thuringiensis FR104 bacterial liquid 2 were the same as in example 2.

And 9, 9 and 11 days in 2019, leveling the chives planted in the greenhouse, and then uniformly spraying bacterial manure on the ground. Randomly selecting 7 cells, wherein each cell has an area of 8m²。

The experiment was set up with three treatments, a blank Control (CK), bacillus thuringiensis FR103 treatment (FR103) and bacillus thuringiensis FR104 treatment (FR104), each treatment selecting 2 cells. Different treatment rooms are separated by ridge stems. 1 isolation plot was set between the placebo and strain treatments (including bacillus thuringiensis FR103 treatment and bacillus thuringiensis FR104 treatment). And managing the greenhouse according to a unified routine.

The following experiments were performed, respectively:

blank Control (CK): on the 1 st day of experiment, spraying tap water on leaf surfaces and irrigating roots; on the 10 th day of the experiment, spraying tap water on the leaf surfaces and irrigating roots; on the 14 th day of the experiment, the aerial parts of the leeks were harvested. The foliar spray dose was about 4mL of tap water per plant. The root irrigation dose was about 1mL tap water per plant.

Bacillus thuringiensis FR103 treatment (FR 103): on the 1 st day of the experiment, spraying the leaf surface with bacillus thuringiensis FR103 bacterial solution 2 and irrigating the root; on the 10 th day of the experiment, spraying the leaf surface with bacillus thuringiensis FR103 bacterial solution 2 and irrigating the root; on the 14 th day of the experiment, the aerial parts of the leeks were harvested. The spraying dosage of the leaf surface is about 4mL of Bacillus thuringiensis FR103 bacterial liquid 2/strain. The root irrigation dose is about 1mL of Bacillus thuringiensis FR103 strain liquid 2/strain.

Bacillus thuringiensis FR104 treatment (FR 104): on the 1 st day of the experiment, spraying the bacillus thuringiensis FR104 bacterium liquid 2 on leaf surfaces and irrigating roots; on the 10 th day of the experiment, spraying the bacillus thuringiensis FR104 bacterium liquid 2 on the leaf surface and irrigating the root; on the 14 th day of the experiment, the aerial parts of the leeks were harvested. The spraying dosage on the leaf surface is about 4mL of Bacillus thuringiensis FR104 bacterial liquid 2/strain. The root irrigation dose is about 1mL of Bacillus thuringiensis FR104 bacterial liquid 2/strain.

When the Chinese chives are harvested, 2 harvesting belts (4 harvesting cells are obtained by processing each harvesting belt through 2 harvesting belts) are uniformly divided, and the total harvesting area is 0.6m²。

The aerial parts of the leeks in each of the 4 treated harvesting plots were weighed (fresh weight) to obtain the fresh weight of the aerial parts of the leeks, i.e., the yield of the leeks.

The statistical result of the fresh weight of the aerial parts of the Chinese chives is shown in figure 6. The results show that compared with the blank control, the fresh weight of the aerial parts of the Chinese chives (namely the yield of the Chinese chives) treated by the bacillus thuringiensis FR103 and the bacillus thuringiensis FR104 is obviously increased by 163.5 percent and 166 percent respectively.

The results show that both bacillus thuringiensis FR103 and bacillus thuringiensis FR104 can obviously promote the growth of the Chinese chives; the growth of the Chinese chives is promoted by increasing the fresh weight of the overground parts of the Chinese chives.

Thus, it can be seen that both bacillus thuringiensis FR103 and bacillus thuringiensis FR104 can significantly promote plant growth. The invention has important application value.

<110> institute of microbiology of Chinese academy of sciences

<120> bacillus thuringiensis and application thereof in promoting plant growth

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1422

<212> DNA

<213> Artificial sequence

<400> 1

agtcgagcga atggattaag agcttgctct tatgaagtta gcggcggacg ggtgagtaac 60

acgtgggtaa cctgcccata agactgggat aactccggga aaccggggct aataccggat 120

aacattttga actgcatggt tcgaaattga aaggcggctt cggctgtcac ttatggatgg 180

acccgcgtcg cattagctag ttggtgaggt aacggctcac caaggcaacg atgcgtagcc 240

gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300

cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360

tgaaggcttt cgggtcgtaa aactctgttg ttagggaaga acaagtgcta gttgaataag 420

ctggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480

taatacgtag gtggcaagcg ttatccggaa ttattgggcg taaagcgcgc gcaggtggtt 540

tcttaagtct gatgtgaaag cccacggctc aaccgtggag ggtcattgga aactgggaga 600

cttgagtgca gaagaggaaa gtggaattcc atgtgtagcg gtgaaatgcg tagagatatg 660

gaggaacacc agtggcgaag gcgactttct ggtctgtaac tgacactgag gcgcgaaagc 720

gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780

tgttagaggg tttccgccct ttagtgctga agttaacgca ttaagcactc cgcctgggga 840

gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900

tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgaaaacc 960

ctagagatag ggcttctcct tcgggagcag agtgacaggt ggtgcatggt tgtcgtcagc 1020

tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1080

atcattaagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140

acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca 1200

aagagctgca agaccgcgag gtggagctaa tctcataaaa ccgttctcag ttcggattgt 1260

aggctgcaac tcgcctacat gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320

gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380

cgaagtcggt ggggtaacct ttttggagcc agccgcctaa gg 1422

<210> 2

<211> 1421

<212> DNA

<213> Artificial sequence

<400> 2

gtcgagcgaa tggattaaga gcttgctctt atgaagttag cggcggacgg gtgagtaaca 60

cgtgggtaac ctgcccataa gactgggata actccgggaa accggggcta ataccggata 120

acattttgaa ctgcatggtt cgaaattgaa aggcggcttc ggctgtcact tatggatgga 180

cccgcgtcgc attagctagt tggtgaggta acggctcacc aaggcaacga tgcgtagccg 240

acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300

agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat 360

gaaggctttc gggtcgtaaa actctgttgt tagggaagaa caagtgctag ttgaataagc 420

tggcaccttg acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt 480

aatacgtagg tggcaagcgt tatccggaat tattgggcgt aaagcgcgcg caggtggttt 540

cttaagtctg atgtgaaagc ccacggctca accgtggagg gtcattggaa actgggagac 600

ttgagtgcag aagaggaaag tggaattcca tgtgtagcgg tgaaatgcgt agagatatgg 660

aggaacacca gtggcgaagg cgactttctg gtctgtaact gacactgagg cgcgaaagcg 720

tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt 780

gttagagggt ttccgccctt tagtgctgaa gttaacgcat taagcactcc gcctggggag 840

tacggccgca aggctgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900

gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgaaaaccc 960

tagagatagg gcttctcctt cgggagcaga gtgacaggtg gtgcatggtt gtcgtcagct 1020

cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080

tcattaagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140

cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa 1200

agagctgcaa gaccgcgagg tggagctaat ctcataaaac cgttctcagt tcggattgta 1260

ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320

tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380

gaagtcggtg gggtaacctt tttggagcca gccgcctaag g 1421

Claims

1. Bacillus thuringiensis FR103 with the preservation number of CGMCC No.19018 is provided.

2. A microbial preparation comprising the Bacillus thuringiensis FR103CGMCC No.19018 according to claim 1.

3. The method for preparing the microbial inoculum according to claim 2, which comprises the following steps: inoculating the Bacillus thuringiensis (Bacillus thuringiensis) FR103CGMCC No.19018 of claim 1 to a bacterial culture medium and culturing to obtain a bacterial liquid, namely a microbial inoculum.

4. The application of the bacillus thuringiensis or the bacillus thuringiensis-containing microbial inoculum is a1) or a 2):

a1) regulating and controlling the growth of plants;

5. A product comprising bacillus thuringiensis or a bacillus thuringiensis-containing inoculant; the product has the function of regulating and controlling the growth of plants.

6. The use of claim 4 or the product of claim 5, wherein: the regulating plant growth is promoting plant growth.

7. A method for promoting plant growth comprises treating plant with Bacillus thuringiensis to promote plant growth.

8. The use of claim 4 or 6, or the product of claim 5 or 6, or the method of claim 7, wherein: the Bacillus thuringiensis is the Bacillus thuringiensis (Bacillus thuringiensis) FR103CGMCC No.19018 of claim 1.

9. The method of claim 7 or 8, wherein: the "treating a plant with Bacillus thuringiensis" is treating a plant with the microbial preparation of claim 2.

10. The use according to claim 4, 6 or 8, or the product according to claim 5, 6 or 8, or the method according to any one of claims 7 to 9, wherein: the plant is any one of the following c1) to c 8): c1) a dicotyledonous plant; c2) a monocot plant; c3) a plant of the Liliaceae family; c4) a cruciferous plant; c5) leek; c6) chinese leek (Allium tuberosum rottl. exspreng) jiuxian No. 18; c7) arabidopsis thaliana; c8) the wild type Arabidopsis thaliana Columbia-0 subtype.