CN113980866A

CN113980866A - Citrus aurantium endophytic bacterium and application thereof

Info

Publication number: CN113980866A
Application number: CN202111451614.8A
Authority: CN
Inventors: 李海云; 徐勤茜; 杨颖芳; 王少杨; 李子院; 赵梓伶; 陶雪婷; 郝再彬; 柴寿海
Original assignee: Guilin University of Technology
Current assignee: Guilin University of Technology
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-01-28
Anticipated expiration: 2041-11-30
Also published as: CN113980866B

Abstract

The invention discloses a sour orange endophytic bacterium and application thereof, and belongs to the technical field of nematode control. The endophytic bacteria is bacillus thuringiensis which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 1 day 2021, the preservation number is CGMCC No.23700, and the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The corrected mortality rate of the strain fermentation liquor to the second-instar nematodes is 93.37%, and the mortality rate of the nematodes is 77.89% after the strain fermentation liquor is diluted to 5 times; the highest inhibition rate of the Bt028 fermentation filtrate on the hatching of the eggs of the root-knot nematodes is 71.63% at 3 d. The Bt028 fermentation liquor can obviously reduce the activities of oocyst catalase, acetylcholinesterase and carboxylesterase of root-knot nematodes and the total sugar and protein contents of oocysts. The strain can inhibit normal metabolism of nematode, reduce nematode resistance, and cause injury or death of nematode organism.

Description

Citrus aurantium endophytic bacterium and application thereof

Technical Field

The invention relates to the technical field of nematode control, in particular to a sour orange endophytic bacterium and application thereof.

Background

Root-knot nematodes are highly specialized species of xenotrophic plant pathogenic nematodes. The plant nutrition can be absorbed through the oral needle, which causes great harm to the underground root, the overground stem, the leaves, the fruits and the like of the plant and can also transmit some fungal and bacterial diseases. At present, the commonly used nematicides are divided into chemical preparations and biological preparations, the chemical preparations have high toxicity and large residues, and along with the use of a large amount of chemical reagents for many years, the resistance of the nematodes to pesticides is continuously improved, and the actual control effect of the conventional chemical pesticides is increasingly poor. Compared with the traditional chemical reagent, the biological reagent has high efficiency and low toxicity, is safe to people and livestock, does not leave pesticide residue hidden danger, and is more green and safe. With the development and popularization of biopesticides, the number of biopesticides on the market is continuously increased, but no biological agent with good control effect on nematodes in different ages exists at present. Therefore, the search for a good microbial strain remains one of the main research directions for current nematicidal agents.

Disclosure of Invention

The invention aims to provide a sour orange endophytic bacterium and application thereof, and aims to solve the problems in the prior art, the bacterium has chitinase activity and good effect in preventing and treating meloidogyne insect pests, the lethality rate of the bacterium to the meloidogyne can reach 93.37%, and a new bacterium source is provided for preventing and treating the meloidogyne.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a sour orange endophytic bacterium, which is Bacillus thuringiensis (Bacillus thuringiensis) which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 1 days in 2021, wherein the preservation number is CGMCC No.23700, and the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

The invention also provides a fermentation method of the sour orange endophytic bacteria, which comprises the following steps:

(1) activating the sour orange endophytic bacteria, inoculating to a fermentation medium, and performing shaking culture to prepare a seed solution;

(2) inoculating the seed liquid into the fermentation medium, and performing shaking culture to prepare fermentation liquid.

Preferably, the fermentation medium comprises the following components in mass concentration: 8g/L starch, 10g/L colloidal chitin and 20g/L peptone, pH 7.0.

Preferably, in step (1), the culture conditions of the seed liquid are: carrying out shaking culture at the temperature of 30 ℃ and the rotating speed of 150r/min for 24 h;

in the step (2), the culture conditions of the fermentation liquor are as follows: the seed liquid is inoculated into the fermentation medium according to the inoculation amount of 5 percent, and is subjected to shaking culture at the temperature of 30 ℃ and the rotating speed of 150r/min for 48 hours.

The invention also provides a microbial inoculum comprising the endophytic bacteria and metabolites thereof. The metabolite may be a fermentation broth prepared according to the fermentation method, or a chemical component extracted from the fermentation broth, but is not limited thereto.

The invention also provides application of the sour orange endophytic bacteria or the metabolites thereof in preparation of a meloidogyne incognita killing preparation.

The invention also provides application of the sour orange endophytic bacteria or the metabolites thereof in preventing and treating meloidogyne incognita diseases.

The invention discloses the following technical effects:

the invention provides a bacillus thuringiensis Bt028 strain screened from sour orange. Experiments prove that the bacillus thuringiensis Bt028 not only has the activity of chitinase, but also has good potential in preventing and controlling root-knot nematode pests. The corrected lethality rate of Bt028 fermentation liquor obtained under the optimal fermentation condition to Meloidogyne incognita reaches 93.37% at the highest point in 24h, the fermentation liquor is diluted to 2 times and 5 times, the mortality rate after nematode treatment is still higher than 77.89%, and the incubation inhibition rate of Bt028 fermentation filtrate to Meloidogyne incognita eggs reaches more than 70% when the treatment time is 3 h.

The invention utilizes the biological control means, namely utilizes the sour orange endophytic bacterium Bacillus thuringiensis Bt028 or the metabolite thereof to effectively control plant diseases, is more environment-friendly compared with other traditional chemical control, has no pesticide residue and no drug resistance, provides a theoretical basis for the development and the effective utilization of microorganism resources for control, and thus lays a long-term development foundation for green organic agriculture.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a diagram showing the morphology of Bacillus thuringiensis Bt028 on a chitinase-producing screening plate;

FIG. 2 is the effect of carbon source species on the thread killing activity of Bt028 fermentation broth;

FIG. 3 is a graph showing the effect of starch concentration on the thread killing activity of Bt028 fermentation broth;

FIG. 4 is a graph showing the effect of nitrogen source species on the thread killing activity of Bt028 fermentation broth;

FIG. 5 is a graph showing the effect of peptone concentration on the killing activity of Bt028 fermentation broth;

FIG. 6 is a graph of the effect of initial pH on the thread killing activity of Bt028 fermentation broths;

FIG. 7 is a graph showing the effect of culture temperature on the thread killing activity of Bt028 fermentation broth;

FIG. 8 is a graph showing the effect of inoculum size on the killing activity of Bt028 fermentation broth;

FIG. 9 is a graph showing the effect of liquid loading on the thread-killing activity of Bt028 fermentation broth;

FIG. 10 shows the effect of the rotational speed of the rocking bed on the thread killing activity of Bt028 fermentation broth;

FIG. 11 is a graph showing the effect of culture time on the thread killing activity of Bt028 fermentation broth;

FIG. 12 is a graph of the effect of Bt028 strain fermentation broth on root-knot nematode oocyst catalase activity;

FIG. 13 is a graph of the effect of Bt028 strain fermentation broth on root-knot nematode oocyst carboxylesterase activity;

FIG. 14 shows the effect of Bt028 strain fermentation broth on the activity of root-knot nematode oocyst acetylcholinesterase;

FIG. 15 is a graph of the effect of Bt028 strain fermentation broth on total protein of meloidogyne oocysts;

FIG. 16 is a graph showing the effect of Bt028 strain fermentation broth on root-knot nematode oocyst total sugar.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

Isolation and identification of Bacillus thuringiensis Bt028

Separation of the strains: collecting fresh fructus Citri Junoris with healthy surface (collected in Suqiao town of Yongfu county, Guangxi, 9 months in 2020), washing with water, soaking in 70% ethanol under aseptic condition for 40 s, soaking in 2.5% sodium hypochlorite for 2min, washing with sterile water for 6 times, and removing disinfectant attached to the surface of the material. And (3) coating the sterile water washed for the last time on a solid plate culture medium under a sterile condition, and culturing to ensure that no microorganism grows out, thereby indicating that the surface is thoroughly disinfected. Taking a proper amount of fresh sour orange fruits with surfaces disinfected under the aseptic condition, crushing the fresh sour orange fruits into fruit pulp, preserving heat at 80 ℃ for 20 minutes, taking 1mL of the fresh sour orange fruits, inoculating the fruit pulp into 100mL of beef extract peptone liquid culture medium (500mL of a triangular flask), carrying out shake culture at 37 ℃ and 120r/min for 2d, diluting the culture solution according to 10 < -2 >, 10 < -3 >, 10 < -4 >, 10 < -5 > and 10 < -6 >, respectively taking 20 mu L of the diluted culture solution, coating the 20 mu L of the diluted culture solution on the beef extract peptone flat culture medium by using a flat plate coater, carrying out constant temperature culture at 37 ℃ for 24-48 h, selecting colonies with better growth vigor according to the growth condition of the colonies, carrying out separation and purification for multiple times by adopting a scribing method, obtaining pure cultures, inoculating the strains on a test tube slant culture medium, carrying out constant temperature culture at 37 ℃ for 24h, and then storing the strains in a refrigerator at 4 ℃.

Identification of the strains: the extraction of the strain DNA genome and the PCR amplification reaction are respectively extracted by a bacterial genome DNA extraction kit and a bacterial genome PCR amplification kit provided by the biological engineering (Shanghai) Co., Ltd, and the specific extraction and amplification steps are carried out according to the kit specifications. The PCR amplification primers are 1492R and 27F,

1492R：GGTTACCTTGTTACGACTT

27F：AGAGTTTGATCCTGGCTCAG；

the amplification procedure was: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, extension at 72 ℃ for 5min, and 30 cycles. After completion of the PCR amplification reaction, the PCR product was subjected to agarose gel electrophoresis using 1% agarose gel, 0.5 XTBE as electrophoresis buffer, and 80V voltage. The PCR product is detected by agarose gel electrophoresis and then sent to bioengineering (Shanghai) company Limited for detection. The detection result (the nucleotide sequence is shown as SEQ ID NO. 1) is registered to NCBI website (http:// Blast. NCBI. nlm. nih. gov /) in the United states to carry out Blast comparison of DNA, and MEGA5.0 software is adopted to construct a phylogenetic tree to determine that the strain belongs to Bacillus thuringiensis.

SEQ ID NO.1 is as follows:

agcgaatggattgagagcttgctctcaagaagttagcggcggacgggtgagtaacacgtgggtaacctgcccataagactgggataactccgggaaaccggggctaataccggataacattttgaactgcatggttcgaaattgaaaggcggcttcggctgtcacttatggatggacccgcgtcgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggctttcgggtcgtaaaactctgttgttagggaagaacaagtgctagttgaataagctggcaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggaattattgggcgtaaagcgcgcgcaggtggtttcttaagtctgatgtgaaagcccacggctcaaccgtggagggtcattggaaactgggagacttgagtgcagaagaggaaagtggaattccatgtgtagcggtgaaatgcgtagagatatggaggaacaccagtggcgaaggcgactttctggtctgtaactgacactgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagagggtttccgccctttagtgctgaagttaacgcattaagcactccgcctggggagtacggccgcaaggctgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgaaaaccctagagatagggcttctccttcgggagcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccatcattaagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacggtacaaagagctgcaagaccgcgaggtggagctaatctcataaaaccgttctcagttcggattgtaggctgcaactcgcctacatgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtggggtaacctttttgga

example 2

Optimization of fermentation process of bacillus thuringiensis Bt028

Preparation of fermentation liquor of bacillus thuringiensis Bt028 strain: after the bacillus thuringiensis Bt028 preserved on the inclined plane is subjected to plate culture and activation for 24 hours, a loop is selected and inoculated into 100mL of fermentation medium (250mL of triangular flask), and the shaking culture is carried out for 24 hours on a shaking table with the temperature of 30 ℃ and the rotating speed of 150r/min, thus obtaining the seed liquid. The seed solution was inoculated into a 250mL Erlenmeyer flask containing 100mL of a pH7.0 fermentation medium at an inoculum size of 5%, and shake-cultured for 48 hours on a shaker at a temperature of 30 ℃ and a rotation speed of 150 r/min. After the culture broth was sterilized by filtration, the filtrate was collected for subsequent experiments.

Study of culture conditions of bacillus thuringiensis Bt028 strain: on the basis of a basic fermentation medium (10g/L glucose, 20g/L peptone, 1.3g/L dipotassium hydrogen phosphate, 0.08g/L calcium chloride, 0.2g/L magnesium sulfate, 0.08g/L manganese sulfate, pH 7.0), the types of carbon sources (10g/L colloidal chitin, sucrose, starch, maltose, glucose, lactose) and the concentrations of carbon sources (4, 8, 12, 16, 20g/L), the types of nitrogen sources (20g/L ammonium sulfate, tryptone, yeast powder, beef extract powder, sodium nitrate, urea) and the concentrations (10, 15, 20, 25, 30g/L), the temperatures (25, 30, 35, 40 ℃), the pH values (6, 6.5, 7, 7.5, 8), the inoculation amounts (1%, 2%, 5%, 10%, 15%, the contents (40, 60 liquid amounts), the amounts of fermentation broth, the fermentation broth, and the like were examined by a single-factor test, 80. 100, 120mL/250mL), the rotating speed of a shaking table (90, 120, 150, 180, 220r/min), the culture time (12, 24, 36, 48, 60, 72h) and the like have influence on the nematicidal activity of the fermentation liquor of the Bt028 strain and chitinase production. When the nematode contact killing experiment is carried out, the filtrate of the fermentation liquor is diluted by 5 times; the original fermentation liquor filtrate is adopted when the activity of the chitinase is determined.

As shown in fig. 2-11, the results show that: the culture conditions with the strongest killing activity of the fermentation liquor of the bacillus thuringiensis Bt028 strain are as follows: adding 10g/L colloidal chitin and 20g/L peptone to 8g/L starch, wherein the initial pH is 7.0, the culture temperature is 30 ℃, the liquid loading amount is 80mL/250mL, the rotating speed of a shaking table is 150r/min, and the culture time is 48 h.

In the embodiment, the influence of chitinase production and nematode killing activity of different fermentation culture medium compositions is inspected by using a single-factor test, the optimal fermentation process of the bacillus thuringiensis Bt028 is finally obtained, and the subsequent experiments of nematode killing activity and inhibition of egg hatching are carried out on the basis.

Example 3

Application of bacillus thuringiensis Bt028 in killing meloidogyne incognita and inhibiting egg hatching

Contact killing experiment of bacillus thuringiensis Bt028 fermentation liquor on root-knot nematode: taking a sterile 12-hole plate, and adding 1mL of strain Bt028 fermentation liquor and 200 mu L of nematode suspension after filtration sterilization into each hole. Each treatment was repeated 3 times with a blank liquid medium as a control. After the 12-well plate was incubated in a 25 ℃ incubator for a certain period of time, the survival of the second instar larvae was observed. The judgment standard of the nematode death and activity is as follows: dead nematode is still and still, and the nematode is still when the needle is touched, and live nematode is in bending and wriggling state or after the needle is touched. The mortality and corrected mortality of the fermentation broth to the second instar larvae were calculated according to the following two formulas:

inhibition experiment of bacillus thuringiensis Bt028 fermentation liquid on hatching of root knot nematode eggs: taking a sterile 12-hole plate, adding 1mL of Bacillus thuringiensis Bt028 fermentation liquor and 200 mu L of 200 granules/mL nematode egg suspension into each hole, filtering, sterilizing, and then respectively diluting by 0, 2, 5 and 10 times. Each treatment was repeated 3 times with a blank liquid medium as a control. After placing the 12-hole plate in a constant-temperature incubator at 25 ℃ for a period of time, observing the hatching condition of the eggs, and calculating the relative inhibition rate of the fermentation liquor to the hatching of the eggs according to the following formula:

experiments prove that as shown in table 1, the bacillus thuringiensis Bt028 not only has chitinase activity, but also has good potential in preventing and controlling root-knot nematode pests. The corrected lethality rate of Bt028 fermentation liquor obtained under the optimal fermentation condition on Meloidogyne incognita reaches 93.37% at the highest point in 24h, the fermentation liquor is diluted to 2 times and 5 times, the mortality rate after nematode treatment is still higher than 77.89%, and the hatching inhibition rate of Bt028 fermentation filtrate on eggs of Meloidogyne incognita reaches more than 70% when the treatment time is 3d (Table 2).

As can be seen from the table 2, the inhibition rate of the fermentation filtrate of the bacillus thuringiensis Bt028 on the hatching of the eggs of the root-knot nematodes reaches 71.63% at 3d, and the inhibition rate of the fermentation filtrate is still 56.76% after the fermentation filtrate is diluted five times. The inhibition ratio of the original fermentation liquor at 9 days is 58.59 percent, and the inhibition ratio of the fermentation liquor diluted by 5 times is 43.43 percent.

TABLE 1 lethal effect of fermentation liquor of strain Bt028 on Meloidogyne incognita

TABLE 2 inhibition of root knot nematode egg hatching by strain Bt028 fermentation broth

The result analysis of the embodiment shows that the biological control means, namely, the organisms or the metabolites thereof are used for effectively controlling the plant diseases, so that the obvious technical effect is obtained, and other traditional chemical control methods are more environment-friendly, have no pesticide residue and do not generate drug resistance, thereby providing a theoretical basis for the development and the effective utilization of the microorganism resources for control. Thereby laying a long-term development for green organic agriculture.

Example 4

Preliminary research on biocontrol mechanism of bacillus thuringiensis Bt028

The effect of bacillus thuringiensis Bt028 fermentation broth on root-knot nematode oocyst catalase, carboxylesterase, acetylcholinesterase, total sugars and total protein was performed according to the methods of zhangjie et al: putting 50 meloidogyne oocysts with consistent sizes into a centrifuge tube, adding 500 mu L of Bt028 strain fermentation filtrate which is diluted by 0, 2, 5 and 10 times into each tube, sealing, culturing at 25 ℃ for a certain time, adding 1mL of phosphate buffer solution with the concentration of 0.02mol/L and the pH value of 6.0, homogenizing at low temperature, centrifuging at 4 ℃ and 10000r/min for 10min, and taking supernatant to respectively determine catalase, acetylcholinesterase, carboxylesterase activity and total sugar and total protein contents by a kit method. Blank liquid medium was used as a control in place of fermentation broth. The results are shown in FIGS. 12 to 16.

The results show that after Bt028 fermentation filtrate is used for treating root-knot nematode oocysts for 24h, the activity of the root-knot nematode oocyst catalase is 128.23nmol/mg, and the activity of the nematode oocyst catalase is reduced by 42.94% at 96h when the activity of the nematode oocyst catalase is 90.57nmol/mg (see figure 12).

After the oocysts are treated by the Bt028 fermentation filtrate, the carboxylesterase activity of the oocysts is gradually reduced after 48 hours, the carboxylesterase activity of the oocysts is always lower than that of the oocysts treated by the Bt028 fermentation filtrate, and the carboxylesterase activity of the Bt028 fermentation filtrate is reduced by 90.63% than that of the oocysts treated by the control after 96 hours (see figure 13).

The activity of the acetylcholinesterase treated by the Bt028 fermentation filtrate at 96h is 0.18nmol/mg, and the acetylcholinesterase activity of the control group is reduced by 67.86% by 0.56nmol/mg (see figure 14).

After the Bt028 fermentation filtrate is used for treating the root-knot nematode oocysts, the total protein of the root-knot nematode oocysts is 32.25 mu g/mu L at 24h, which is 46.73 percent lower than 60.54 mu g/mu L of the total protein of a control group; total protein was 16.92. mu.g/. mu.L at 96h, which was 70.54% lower than 57.43. mu.g/. mu.L for the control group (see FIG. 15).

After the Bt028 fermentation filtrate is used for treating the root-knot nematode oocysts, the total sugar content is continuously reduced, the total sugar content is 5.14 mu g/mu L at 96h, and is reduced by 61.24% compared with the total sugar content of 13.26 mu g/mu L in a control group (see figure 16).

The experimental result of the embodiment shows that the fermentation filtrate of the bacillus thuringiensis Bt028 can obviously reduce the activities of oocyst catalase, acetylcholinesterase and carboxylesterase of root-knot nematodes and the total sugar and protein contents of oocysts.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> Guilin university of science

<120> orange endophytic bacterium and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1402

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agcgaatgga ttgagagctt gctctcaaga agttagcggc ggacgggtga gtaacacgtg 60

ggtaacctgc ccataagact gggataactc cgggaaaccg gggctaatac cggataacat 120

tttgaactgc atggttcgaa attgaaaggc ggcttcggct gtcacttatg gatggacccg 180

cgtcgcatta gctagttggt gaggtaacgg ctcaccaagg caacgatgcg tagccgacct 240

gagagggtga tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca 300

gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg agtgatgaag 360

gctttcgggt cgtaaaactc tgttgttagg gaagaacaag tgctagttga ataagctggc 420

accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 480

cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg tggtttctta 540

agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg ggagacttga 600

gtgcagaaga ggaaagtgga attccatgtg tagcggtgaa atgcgtagag atatggagga 660

acaccagtgg cgaaggcgac tttctggtct gtaactgaca ctgaggcgcg aaagcgtggg 720

gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 780

gagggtttcc gccctttagt gctgaagtta acgcattaag cactccgcct ggggagtacg 840

gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 900

tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga aaaccctaga 960

gatagggctt ctccttcggg agcagagtga caggtggtgc atggttgtcg tcagctcgtg 1020

tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatcttag ttgccatcat 1080

taagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1140

aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggacg gtacaaagag 1200

ctgcaagacc gcgaggtgga gctaatctca taaaaccgtt ctcagttcgg attgtaggct 1260

gcaactcgcc tacatgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1320

tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta acacccgaag 1380

tcggtggggt aacctttttg ga 1402

Claims

1. The sour orange endophytic bacterium is characterized by being Bacillus thuringiensis (Bacillus thuringiensis), which is preserved in China general microbiological culture Collection center (CGMCC) at 11 months and 1 day 2021, wherein the preservation number is CGMCC No.23700, and the preservation address is as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.

2. A fermentation method of sour orange endophytic bacteria is characterized by comprising the following steps:

(1) activating the citrus aurantium endophytic bacteria of claim 1, inoculating into a fermentation medium, and performing shaking culture to prepare a seed solution;

3. The fermentation method of the endophytic bacteria of sour orange according to claim 2, wherein the fermentation medium comprises the following components in mass concentration: 8g/L starch, 10g/L colloidal chitin and 20g/L peptone, pH 7.0.

4. The fermentation method of sour orange endophytic bacteria according to claim 2, wherein in step (1), the culture conditions of the seed solution are as follows: carrying out shaking culture at the temperature of 30 ℃ and the rotating speed of 150r/min for 24 h;

5. A bacterial agent comprising the endophytic bacterium according to claim 1 and a metabolite thereof.

6. Use of the citrus aurantium endophyte according to claim 1 or a metabolite thereof in the manufacture of a meloidogyne incognita preparation.

7. Use of the citrus aurantium endophytic bacterium or metabolite thereof according to claim 1 for controlling meloidogyne incognita disease.