CN112458012A - Bacillus belgii microbial agent and application thereof - Google Patents
Bacillus belgii microbial agent and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus velezensis TK2019 microbial agent and application thereof, the Bacillus velezensis TK2019 microbial agent is obtained by separating 44 groups of cotton field rhizosphere soil in Tosuke city of Xinjiang, the microbial agent prepared by the Bacillus velezensis TK2019 is applied to prevention and control of cotton verticillium wilt, cotton wilt, tomato gray mold and red date black spot, the inhibition rate of the strain TK2019 on cotton verticillium dahliae reaches 94.37%, the inhibition rate on 4 pathogenic fungi to be tested is 84.83% -93.73%, the prevention effect of the Bacillus velezensis TK2019 microbial agent stock solution and dilution are 50 times highest, respectively 92.86% and 91.97%, the experiment shows that the TK2019 microbial agent has the prevention effect on cotton wilt and the sterilization effect of the Bacillus velezensis TK2019 is 92.69%, and the invention provides advantages of the Bacillus velezensis TK and the microbial agent (TK) such as broad spectrum, has wide value for expanding the application field of microbial strains.
Description
Technical Field
The invention relates to the technical field of microorganisms and application thereof, in particular to a Bacillus velezensis TK2019 microbial agent and application thereof.
Background
The cotton Verticillium wilt is a cotton soil-borne disease caused by Verticillium dahliae Kleb, and has the main symptoms: the verticillium wilt of cotton can occur in the whole growing period of cotton. Generally, symptoms begin to appear in the 3-5 true leaf stage, and a large amount of diseases occur in the field after the buds of the cotton appear and bloom in the middle and later growth stages. The cotton verticillium wilt was first reported in 1914 in Virginia, and then spread to dozens of countries and regions including China, causing great loss of cotton yield, and becoming a main obstacle to high and stable cotton yield. The cotton verticillium wilt seriously affects the growth and development of cotton, cotton buds and bolls are rare after the disease occurs, cotton bolls are cracked in advance, and yield reduction is caused, so that the problem to be solved urgently is solved. Xinjiang is the most important cotton production base in China, the yield accounts for about 85% of the whole country, and verticillium wilt caused by Verticillium dahliae seriously hinders the sustainable development of the Xinjiang cotton industry.
The main prevention and cure methods of cotton verticillium wilt are breeding disease-resistant variety, chemical prevention and cure, biological prevention and cure, etc. The method is characterized in that a disease-resistant variety is cultivated, because of lack of effective disease resistance sources, a high-disease-resistant variety which is successfully cultivated at present does not exist, an effective bactericide is not available for chemical prevention and treatment, environmental pollution is easily caused after long-term use, and the chemical prevention and treatment is limited by pesticide effect and use mode and is difficult to play a role; in the aspect of biological control, a plurality of microorganisms are proved to have biological control effect, and some microorganisms are gradually applied. Many biocontrol microorganisms can produce active substances such as various antibiotics, extracellular enzymes and the like, and the spores have strong stress resistance and better application prospect in the aspect of preventing and treating soil-borne diseases,
biological control has been receiving more and more attention in recent years because of its advantages such as strong specificity, high control effect, good durability of drug effect, and no environmental pollution. The main microorganisms currently used for preventing and treating cotton verticillium wilt are: trichoderma harzianum (CN110846243A), Bacillus methylotrophicus (CN111187727A), Bacillus atrophaeus (CN105695366A), Bacillus megaterium (CN109180352A), Bacillus amyloliquefaciens (CN 107043720A; CN107043719A), Pseudomonas fluorescens (CN 110643538A; CN 110684686A; CN106754469A), Streptomyces Abutigerus (CN106305793A), Streptomyces galnardus (CN106434406A) and the like. From the above, the microorganism for preventing and treating cotton verticillium wilt is mainly Bacillus (Bacillus) because the Bacillus has the characteristics of wide distribution, easy separation and culture, capability of generating spores with strong stress resistance, long storage period, convenient use and the like; in addition, the bacillus can generate endogenic spores, has strong stress resistance, and is more favorable for the production and transportation of the microbial inoculum compared with other types of biocontrol bacteria, such as survival, colonization and propagation in a dosage form processing environment. Thus, bacillus is an ideal biocontrol microorganism.
Due to the biodiversity and the co-evolutionary characteristics of organisms, more microorganisms of different species need to be screened for coping with different species and the continuous evolution of each microorganism, and the screening of biocontrol microorganisms having an inhibitory effect on pathogenic bacteria is the most economical and effective method for preventing and treating pathogenic microorganisms.
Disclosure of Invention
The invention aims to solve the technical problems that different types and continuous evolution of pathogenic microorganisms have poor effect of preventing cotton verticillium wilt by using the existing microorganisms, and applicable microbial strains are not rich enough, and the like, and provides a Bacillus belezii TK2019 microbial agent and application thereof. The method comprises the steps of separating a new strain Bacillus velezensis (Bacillus velezensis) TK2019 which has bright characteristics and is subjected to serial identification from rhizosphere soil of a cotton field of 44 groups of Tupist city of Xinjiang, preparing a microbial agent by utilizing the obtained new strain Bacillus velezensis TK2019, and applying the microbial agent to preparation of medicines for preventing and controlling cotton verticillium wilt, cotton wilt, tomato gray mold and red date black spot, wherein the bacterial inhibition rate of the Bacillus velezensis TK2019 on 4 pathogenic fungi for testing is 84.83-93.73%, the bacterial inhibition rate of the Bacillus velezensis TK2019 has a remarkable inhibition effect on the cotton verticillium wilt morbidity, the microbial agent stock solution and the diluent are 50 times as high as 92.86% and 91.97%, and the microbial agent for preventing cotton verticillium wilt is 92.69% and the microbial agent for cotton verticillium wilt is wide as indicated by field tests, and the microbial agent for cotton verticillium wilt is wide as 92.69% and the bacterial spectrum of the bacterial agent for TK2019 And the method has wide value in expanding the application field of microbial strains.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a Bacillus subtilis TK2019 microbial agent, which comprises a bacterial liquid obtained by fermenting and metabolizing a new strain Bacillus subtilis TK2019, wherein the viable count of the Bacillus subtilis TK2019 is not less than 108CFU/mL。
The invention provides a Bacillus velezensis TK2019 microbial inoculum which is a microbial liquid preparation or a microbial powder preparation.
In the invention, a strain Bacillus velezensis TK2019 is separated and screened from rhizosphere soil of 44 cotton fields in Shuke City in Xinjiang, and the molecular level identification of a well-known and accepted strain system and the test and verification of a physiological and biochemical system of the strain prove that the obtained Bacillus (Bacillus) belongs to a typical new strain in the category, the strain Bacillus velezensis TK2019 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No. 20810, preservation date: 09/27 days 2020.
The gene sequence of the Bacillus velezensis TK2019 strain is shown in SEQ ID NO: 1.
In the invention, the NA culture medium for separating and purifying Bacillus velezensis TK2019 comprises the following components: 3g/L of beef extract, 7g/L of peptone, 5g/L of NaCl, 17g/L of agar and 7.0 of pH value.
In the invention, the corn flour-enriched soybean meal culture medium of Bacillus velezensis TK 2019: corn flour 2.0g/L, soybean flour 2.5g/L, NaCl 0.5g/L, MnSO4·H2O0.7 g/L, pH 7.0.
Meanwhile, the invention provides a preparation method of a Bacillus velezensis TK2019 microbial agent, which specifically comprises the following steps:
(1) activating the Bacillus subtilis TK2019 stored at a low temperature on an NA plate culture medium, selecting a single colony on an NA slant culture medium, and culturing at 20-37 ℃ for 6-12 hours to obtain the activated Bacillus subtilis TK 2019.
(2) And (2) scraping a ring of the activated Bacillus velezensis TK2019 strain in the step (1) by using a sterile inoculating ring, inoculating the ring into a 100mL NA liquid culture medium, and culturing for 6-12 hours at the temperature of 20-37 ℃ and the rotating speed of a shaking table of 120-180 rpm to obtain a Bacillus velezensis TK2019 seed liquid.
(3) Inoculating the seed liquid of the Bacillus velezensis TK2019 obtained in the step (2) into a corn flour and soybean meal culture medium with the pH value of 7.0 according to the inoculation amount of 1-3% of the mass-volume ratio, and performing fermentation culture for 30-36 hours at the temperature of 20-37 ℃ and the rotating speed of a shaking table of 120-180 rpm to obtain the Bacillus velezensis TK2019 fermentation liquid.
(4) And (3) detecting the quantity of the bacteria and spores in the fermentation liquid obtained in the step (3), stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the bacteria, and preparing a liquid preparation or a powdery microbial agent of the Bacillus velezensis TK2019 microbial agent according to needs.
The invention provides an application of a Bacillus velezensis TK2019 microbial agent in preparation of a drug for preventing and treating cotton verticillium wilt, cotton wilt and red date black spot.
Further, the invention provides an application of a Bacillus velezensis TK2019 microbial agent in preparation of a medicament for preventing and treating cotton verticillium wilt, cotton wilt and red date black spot, wherein the liquid microbial agent is prepared by diluting the microbial agent with water until the viable count is more than 107cfu/mL, directly soaking the cotton seeds for 0.5h before sowing; the powder is prepared by adsorbing the microbial agent by calcium carbonate to prepare Bacillus welfare (Bacillus velezensis) TK2019 powder, and mixing the powder with cotton seeds according to the mass ratio of 1: 10 and mixing uniformly and then applying.
By implementing the content of the invention through the implementation of the above specific technical scheme provided by the invention, the following beneficial effects can be obtained:
(1) the strain Bacillus velezensis TK2019 provided by the invention is verified to belong to a typical new strain with the strain number of TK2019 in the obtained Bacillus (Bacillus) category through well-known and accepted strain system molecular level identification and strain physiological and biochemical system test verification, and further needs to be preserved according to legal requirements.
(2) The application of the Bacillus velezensis TK2019 and the microbial agent thereof in prevention and treatment of cotton verticillium wilt, cotton wilt and red date black spot has the advantages that the average prevention effect is over 80.0%, the specificity of the Bacillus velezensis TK2019 to cotton verticillium wilt is strong, the drug resistance is not easy to generate, the drug effect durability is good, the Bacillus velezensis TK2019 is safe to people and livestock, and the problem of environmental pollution is avoided.
(3) The Bacillus velezensis TK2019 provided by the invention has a wide antibacterial spectrum, and has a good inhibition effect on cotton verticillium wilt, cotton wilt, tomato botrytis cinerea and red date black spot in addition to cotton verticillium wilt.
Drawings
FIG. 1 is a phylogenetic tree of Bacillus velezensis TK2019 constructed based on the 16S rDNA gene.
FIG. 2 shows the colony morphology of Bacillus belgii (Bacillus velezensis) TK2019 on NA medium.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.
Isolation and purification of NA Medium from Bacillus velezensis TK 2019: 3-6g/L of beef extract, 7-13g/L of peptone, 4-6g/L of NaCl, 13-19g/L of agar and 7.0 of pH value.
Corn meal-enriched soybean meal medium of Bacillus belgii (Bacillus velezensis) TK 2019: 1.0-3.0g/L corn flour, 1.0-3.0g/L soybean flour, 0.1-0.8g/L NaCl, and MnSO4·H2O0.5-1.0 g/L, pH 7.0.
Example 1: isolation, screening and identification of Bacillus velezensis TK2019
(I) separation and purification
Weighing 1.0g of soil sample from a soil sample, adding 100mL of sterile water into a 250mL sterilized triangular flask, placing the flask on a shaker, oscillating at 150rpm for 30min, standing for 2h, taking 10mL of supernatant into a sterilized centrifuge tube, carrying out 80 ℃ constant temperature water bath for 30min, then adding 1mL of supernatant into a sterilized centrifuge tube9mL of sterile water, namely 10mL of sterile water-3The soil microorganism suspension is continuously diluted with sterile water in a gradient manner from 10-4、10-5、10-6And (3) coating 200 mu L of each of the three dilutions on an NA culture medium plate, and culturing in a constant-temperature incubator at 30 ℃ for 1-3 d. Single colonies were picked from the plates and inoculated into new separation plates for purification, repeating 3 times for each concentration. Inoculating the purified Bacillus velezensis TK2019 on a culture medium, culturing at constant temperature of 30 ℃ for 3-4d in an incubator, and storing at 4 ℃ for later use.
(II) classification and identification:
sequencing and analysis of Bacillus velezensis TK2019 (hereinafter referred to as "strain TK 2019") 16S rDNA.
(1) Extraction of PCR template DNA
Inoculating the strain TK2019 into an NA culture medium, culturing at 30 ℃ for 72h to obtain bacterial cells, and extracting the genome DNA by adopting a novel plant genome DNA rapid extraction kit.
(2) PCR amplification
Primer:
F16S-27：5’-AGAGTTTGATCATGGCTCAG-3’；
R16S-1492：5’-GGCTACCTTGTTACGACTT-3’。
reaction system:
| composition of | Volume/. mu.L |
| 10×PCR Buffer(Mg2+) | 5 |
| dNTP Mixture(2.5mM) | 5 |
| Taq(5U/μL) | 1 |
| 27F(10μm/μL) | 1 |
| 1492R(10μm/μL) | 1 |
| Fungus sample template | 50ng |
| ddH2O | Make up to 50. mu.L |
The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min for 30s, and 30 cycles; extension 72 ℃ for 10 min.
(3) Sequence determination
And sequencing the PCR amplification product after electrophoresis detection and purification, wherein the sequence of the PCR amplification product is shown as SEQ ID NO:1 is shown. The obtained sequence results were subjected to BLAST search at the National Center for Biotechnology Information (NCBI) of the United states, a model strain having high similarity was selected as a reference strain, and a 16S rDNA gene phylogenetic tree was constructed using the Neighbor-joining method (Neighbor-joining) in MEGA5.0 software, with a self-developed value (Bootstrap) of 1000. The results are shown in FIG. 1. The homology of the strain TK2019 and the 16S rDNA of the Bacillus reaches 98%, the strain TK2019 and the Bacillus are polymerized together, the 16S rDNA gene sequence has higher similarity with a model strain of Bacillus velezensis CR-502(AY603658), the similarity reaches 97.04%, and the strain number TK2019 in the obtained Bacillus (Bacillus) genus category belongs to a typical new strain through comprehensive judgment of the similarity and the homology, molecular level identification of a well-known strain system in the field and test verification of a physiological and biochemical system combining the strain.
Based on the biological characteristics, the strain TK2019 is identified as a new strain in a Bacillus velezensis branch. The strain has been deposited in the Budapest treaty International Collection of microorganisms: china general microbiological culture Collection center (CGMCC), Address: west road No. 1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101, date of deposit: 9 and 27 days in 2020, the preservation number is CGMCC No: 20810.
example 2: bacillus belgii (Bacillus velezensis) TK2019 CGMCC No:20810 measurement of physiological and biochemical characteristics
The colony morphology and physiological and biochemical characteristics of the strain TK2019 on the NA medium are shown in the attached figure 2 and Table 1.
The strain TK2019 is in a rod shape after being cultured on an NA culture medium, spores are generated after 12 hours of culture, the spores are grown in the middle of the rod shape, the cysts are not expanded, the bacteria are negative in acid-fast staining, no parasporal crystal exists, the bacteria can move, the flagellum is periphytic, gram staining is gram positive bacteria, the strain TK2019 under a microscope is gram positive bacteria [ (1-1.5) Mumx (3-5) Mum ], and the strains are arranged in a single or double or short chain shape. On a nutrient agar plate, the colony at the initial culture stage is milky white, has a rough surface, and has irregular and opaque edges and corrugated bulges; carrying out streak culture on a nutrient agar inclined plane to form a straight line shape; the white mycoderm is formed on the surface of the culture medium by static culture in the liquid culture medium. The morphological characteristics are basically consistent with those of Bacillus described in a handbook of identifying common bacteria systems, and the strain TK2019 is preliminarily judged to belong to Bacillus.
The analysis of physiological and biochemical characteristics of the TK2019 such as enzyme activity, carbon source assimilation and carbon source acid production shows that the TK2019 can utilize carbon sources such as D-mannose, D-mannitol, D-glucose, D-xylose and L-arabinose, can not utilize D-arabinose, tyrosine and phenylalanine, is positive in V-P test, catalase test, oxidase test and methyl red reaction, can hydrolyze casein, reduce nitrate, secrete gelatinase, can not produce indole and can not produce H2S, according to the morphological characteristics, physiological and biochemical characteristics and Bergey details of the strain TK2019The analysis is carried out by a manual of bacteria identification, and the physiological and biochemical characteristics have greater similarity with gram-positive bacteria bacillus.
Table 1: determination of physiological and biochemical characteristics of strain TK2019
By integrating the results of 16S rDNA gene sequence homology comparison analysis and biochemical test comparison, the strain TK2019 provided by the invention has clear difference with the strains in the same genus range of common Bacillus velezensis (Bacillus velezensis), and has the characteristics of new strains in the same genus of Bacillus velezensis (Bacillus velezensis).
Example 3: preparation of Bacillus velezensis TK2019 microbial agent
In this embodiment, on the basis of the embodiment 1-2, the preparation method of the microbial agent of Bacillus belgii (Bacillus velezensis) TK2019 specifically includes the following steps:
(1) activating Bacillus subtilis TK2019 stored at low temperature on an NA plate culture medium, selecting a single colony on an NA slant culture medium, and culturing at 20-37 ℃ for 6-12 h to obtain the activated Bacillus subtilis TK 2019.
(2) And (2) scraping a ring of the activated Bacillus velezensis TK2019 strain in the step (1) by using a sterile inoculating ring, inoculating the ring into a 100mL NA liquid culture medium, and culturing for 6-12 h at the temperature of 20-37 ℃ and the rotating speed of a shaking table of 120-180 rpm to obtain a Bacillus velezensis TK2019 seed liquid.
(3) Inoculating the seed liquid of the Bacillus velezensis TK2019 obtained in the step (2) into a corn flour and soybean meal culture medium with the pH value of 7.0 according to the inoculation amount of 1-3% of the mass-volume ratio, and performing fermentation culture for 30-36 hours at the temperature of 20-37 ℃ and the rotating speed of a shaking table of 120-180 rpm to obtain the Bacillus velezensis TK2019 fermentation liquid.
(4) And (3) detecting the quantity of the bacteria and spores in the fermentation liquid obtained in the step (3), stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the bacteria, and preparing a liquid preparation or a powdery microbial agent of the Bacillus velezensis TK2019 microbial agent according to needs.
Example 4: preparation of Bacillus velezensis TK2019 microbial agent
The embodiment provides a preparation method of a Bacillus belgii (Bacillus velezensis) TK2019 microbial agent on the basis of embodiments 1-3, wherein the inoculation amount of a Bacillus belgii (Bacillus velezensis) TK2019 seed liquid is 2%, the fermentation temperature is 30 ℃, and the fermentation time is 32 hours.
Example 5: preparation of Bacillus velezensis TK2019 microbial agent
In this embodiment, on the basis of embodiments 1 to 3, a preparation method of a Bacillus belgii (Bacillus velezensis) TK2019 microbial inoculum is provided, wherein the inoculation amount of the Bacillus belgii (Bacillus velezensis) TK2019 seed solution is 1%, the fermentation temperature is 20 ℃, and the fermentation time is 30 hours.
Example 6: preparation of Bacillus velezensis TK2019 microbial agent
The embodiment provides a preparation method of a Bacillus belgii (Bacillus velezensis) TK2019 microbial agent on the basis of embodiments 1-3, wherein the inoculation amount of a Bacillus belgii (Bacillus velezensis) TK2019 seed liquid is 3%, the fermentation temperature is 37 ℃, and the fermentation time is 36 hours.
Example 7: preparation of Bacillus velezensis TK2019 microbial agent
The embodiment provides a preparation method of a Bacillus belgii (Bacillus velezensis) TK2019 microbial agent on the basis of embodiments 1-3, wherein the inoculation amount of a Bacillus belgii (Bacillus velezensis) TK2019 seed liquid is 2%, the fermentation temperature is 32 ℃, and the fermentation time is 34 hours.
Example 8: inhibition of hypha growth of Bacillus velezensis TK2019
Verticillium wilt of cotton 2015: the Verticillium wilt bacterium 2015 strain is collected from 143 groups of stone river cotton Verticillium wilt disease strains in Uygur autonomous area of Xinjiang, is separated and purified by a microorganism application research institute of Xinjiang agricultural academy of sciences, is identified as Verticillium dahliae by a microorganism application research institute of Xinjiang agricultural academy of sciences, and shows strong pathogenicity in pathogenicity measurement.
Firstly, Verticillium dahliae (Verticillium dahliae) VD2015 is activated and cultured on a PDA (personal digital assistant) plate for 3d, then a puncher is used for punching a bacterial sheet in the edge area of a bacterial colony to prepare a bacterial sheet, the cotton Verticillium dahliae bacterial sheet is transferred to the center of another PDA plate, the activated strain TK2019 is spotted at a position 2.0cm away from the indicator bacterial sheet, and a blank control (the growth condition of the cotton Verticillium dahliae of the strain TK2019 is not spotted) is set. Culturing at constant temperature of 25 ℃, measuring the control growth amount (colony radius) and the treatment growth amount (growth inhibition radius after inoculating the strain TK 2019) of the cotton verticillium dahliae when the blank control is about to grow over the whole culture dish, and expressing the antagonistic action by the bacteriostatic rate. The calculation formula is as follows: the bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100. See table 2 for results.
Table 2: antagonistic action of strain TK2019 on cotton verticillium dahliae
| Strain name | Control growth volume (mm) | Treatment of growth (mm) | Bacteriostatic ratio (%) |
| TK2019 | 30.0 | 4.69 | 94.37 |
As can be seen from Table 2, the inhibition rate of the Bacillus velezensis TK2019 provided by the invention on verticillium dahliae is 94.37%, which indicates that the Bacillus velezensis TK2019 has an obvious inhibition effect on the growth of verticillium dahliae hyphae and has biocontrol potential for preventing and treating cotton verticillium wilt.
Example 9: application of Bacillus velezensis TK2019 microbial agent in cotton verticillium wilt prevention and treatment
(1) Comparison test of Bacillus velezensis (Bacillus velezensis) TK2019 microbial agent on cotton verticillium wilt control effect
In the experiment, on the basis of example 4, the prepared Bacillus belief (Bacillus velezensis) TK2019 microbial agent is diluted by 50 times, clear water is set as a blank control sample, and the prepared Bacillus belief (Bacillus velezensis) TK2019 microbial agents of examples 5 to 7 are investigated.
Culturing verticillium dahliae strains of cotton: completely and plump millet granules purchased in the market are soaked in clear water for 4 hours, the water is drained, the millet granules are respectively filled in 50ml centrifuge tubes, each tube is 20 g, and the millet granules are subjected to high-pressure moist heat sterilization for half an hour at the temperature of 121 ℃; and (3) connecting 10-15 mycelia of the cotton verticillium dahliae after cooling, uniformly mixing, and culturing for 7 days in a constant-temperature incubator at 25 ℃ for later use.
Mixing vermiculite and cotton field soil according to a mass ratio of 1: 1, mixing, sterilizing at 121 deg.C for 1 hr under high pressure and moist heat, and sterilizing again for 1 time every other day under the same conditions. 300g of soil was taken and inoculated with a suspension of the mycelial fragment of Verticillium dahliae (about 10)7piece/mL) 10mL, and filling the mixture into a plastic flowerpot with the diameter of 20cm after uniformly stirring, and compacting the mixture for later use. Selecting a cotton variety of Xinluzao 36, and soaking the cotton variety with a Bacillus velezensis TK2019 microbial inoculum stock solution prepared in example 4 and diluent diluted by different times for 0.5h before sowing; soaking seeds in clear water for 0.5h as blank control. After seed soaking treatment, sowing 10 seeds in each pot, covering with sterilized soil, heatingAnd (4) normally culturing indoors. After emergence of seedlings, the microbial agent treatment of Bacillus velezensis TK2019 and the number of verticillium wilt disease-causing strains in blank control are investigated and recorded day by day, when the verticillium wilt in blank control is fully developed, the disease incidence and the prevention and control effect are calculated, and specific results are shown in table 3.
Table 3: the invention relates to a control effect comparison test result of Bacillus velezensis TK2019 microbial agent on cotton verticillium wilt
As can be seen from the data statistics in table 3, in the first batch of potting test results, the original liquid of the microbial inoculum of Bacillus belief (Bacillus velezensis) TK2019 provided in embodiment 4 of the present invention and the diluted 50 times thereof have high control effects on cotton verticillium wilt, which are 93.16% and 92.48%, respectively. In the second batch of pot culture test results, the control effects of the Bacillus subtilis TK2019 microbial inoculum stock solution and the diluted 50 times on cotton verticillium wilt are respectively 92.86% and 91.97%, which shows that the Bacillus subtilis TK2019 microbial inoculum has a good control effect on cotton verticillium wilt.
(2) Field contrast test of Bacillus velezensis TK2019 microbial agent for cotton verticillium wilt control
The test was carried out in the cotton test field of darettdele west village, bleeker, municipality, viygur autonomous area, Xinjiang, using Bacillus beijerinckii (Bacillus velezensis) TK2019 microbial inoculum prepared in example 4. In field test, each biocontrol bacterium is diluted to 100 hundred million/mL viable bacteria before test, and is applied to 75 L.hm with seedling emergence water after cotton sowing-2The biocontrol bacteria are applied with water for the second time and the third time respectively in 75 L.hm-2And (4) biocontrol bacteria. And taking adjacent cotton without the applied inoculants as a control. Except the application of bacteria, the other field management measures are the same. The bacteria application mode is a drip irrigation bacteria application mode. The drip irrigation method adopts a movable pressurized drip irrigation mode, is carried out within about 2 hours before the end of dripping water, and adds the medicament into the drip irrigation systemIn the medicine box, the bactericide is dripped along with water to finish the application. The cotton variety is selected from Xinluzao 36. Each treatment was repeated 4 times, randomly arranged, and the cell area was 66.7 square meters. The disease onset in cotton fields was investigated and recorded from the onset of disease in each treatment. The diseased leaves and the boll opening period of the cotton are observed in the cotton bud boll period, the number of diseased plants is counted by observing the color change of the xylem of the cotton stalk, the disease incidence, the disease index and the relative disease prevention effect are calculated, and the specific result statistics are shown in a table 4.
Table 4: the control effect comparison result of the microbial agent on the cotton verticillium wilt
| Strain name | Investigation of plant number (strain) | Number of disease plants (plant) | Incidence (%) | Control effect (%) |
| The invention | 208 | 6 | 2.88 | 92.69 |
| Blank control | 203 | 80 | 39.41 | —— |
As can be seen from the data in Table 4, the prevention effect of the Bacillus belief (Bacillus velezensis) TK2019 microbial agent on cotton verticillium wilt is 92.69%. The Bacillus subtilis TK2019 microbial inoculum has a very good prevention and treatment effect on cotton verticillium wilt.
Example 5: application of Bacillus velezensis TK2019 in control of plant pathogenic fungi
In this example, 4 pathogenic fungi tested in the laboratory of beneficial microorganisms from institute for microorganism application, institute of agricultural sciences, Xinjiang, were: verticillium dahliae (Verticillium dahliae) VD2016, Fusarium oxysporum f.sp.vasinfectum F2015, Botrytis cinerea (Botrytis cinerea) B2018, and myrothecium grisea (Alternaria alternata) A2015.
And (3) detecting the inhibiting effect of the strain TK2019 on 4 plant pathogenic fungi by adopting a confronting culture method, and activating the strain TK2019 on an NA slant culture medium at 34 ℃ for 24h to obtain the activated strain TK 2019. Activating 4 pathogenic fungi on a PDA culture medium for 5 days, punching a bacterial disc with consistent bacterial age on the edge of a bacterial colony by using a puncher with the diameter of 7mm, transferring hypha downwards to the center of another PDA flat plate, inoculating a strain TK2019 in a shape of a Chinese character 'pin' at a position 2cm away from the bacterial disc, taking the non-inoculated strain TK2019 as a reference, culturing at the constant temperature of 25 ℃ for 7 days, measuring the radius of the treated bacterial colony and the distance (a bacterial inhibition zone) between a bacterial inoculation point and the edge of the bacterial colony of the pathogenic bacteria, determining the bacteriostatic degree, calculating the bacteriostatic rate, and repeating the treatment for 3 times. The bacteriostatic ratio (%) (control growth amount-treated growth amount)/control growth amount × 100, and the specific results are shown in table 5.
Table 5: inhibition effect of strain TK2019 on 4 plant pathogenic fungi
As can be seen from the data in Table 5, the bacterial strain TK2019 has the bacteriostasis rate of 84.83-93.73% for 4 pathogenic fungi to be tested, wherein the bacteriostasis rate of the bacterial strain TK2019 on verticillium dahliae of cotton reaches 84.83%, the bacteriostasis rate on fusarium wilt of cotton, botrytis cinerea and red date black spot pathogen is more than 90%, the bacteriostasis rate on fusarium wilt of cotton is the highest and is 93.73%, and the bacterial strain TK2019 disclosed by the invention has broad bacteriostasis spectrum.
In summary of the embodiments, the invention separates and screens a strain of Bacillus velezensis TK2019 from a 44-group cotton field soil sample in Tushuke City of Uygur autonomous region of Xinjiang, and the microbial agent of the strain has a significant inhibiting effect on the incidence rate of cotton verticillium wilt. According to the invention, the strain TK2019 has high control effect on cotton verticillium wilt when the microbial inoculum is diluted by different times, wherein the control effect of a microbial inoculum stock solution and the microbial inoculum diluted by 50 times is the highest, and is respectively 92.86% and 91.97%, through field tests, the control effect of the TK2019 on cotton verticillium wilt is 92.69%, the inhibition effect of the strain TK2019 on 4 plant pathogenic fungi is further considered, and the result shows that the bacterial inhibition rate of the strain TK2019 on the tested 4 pathogenic fungi is 84.83% -93.73%, wherein the bacterial inhibition rate on the cotton verticillium wilt reaches 84.43%, and the bacterial inhibition rate on the cotton wilt is 93.73%, which indicates that the strain Bacillus velezensis TK2019 has a wide bacterial inhibition spectrum and has wide application value in the technical field of microbial inoculum application.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
Sequence listing
<110> institute of microorganism application of Sinkiang academy of agricultural sciences (Xinjiang-Yameiya bioengineering research and development center, China)
<120> Bacillus belgii microbial agent and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1474
<212> DNA
<213> Bacillus belgii (Bacillus velezensis)
<400> 1
gggcatggcg ggtgctatac atgcagtcga gcggacagat gggagcttgc tccctgatgt 60
tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcagacat aaaaggtggc 180
ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240
accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
acggagcaac gcccgcgtga gtgatgaarg attttcrgat cgtaaagctc tgttgttagg 420
gaagaacaag tgccgttcaa atagrgcggc accttgacgg tacctaaccc agaaagccac 480
ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat 540
tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc 600
ggggagggtc attggaaact ggggaacttg arwgcagaag aggagagtgg aattccacgt 660
gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc 720
tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 780
ccacgccgta aacgatgagt gctawgtgtt agggggtttc cgccccttag tgctgcagct 840
aacgcattaa gcactccgyc tggggagtac ggtcgcaaga ctgaaactca aaggaattga 900
cgggggcccg cacaagcggt ggagcatgtg gtytaaattc gaagcaacgc gaagaacctt 960
accaggtctt gacatcctct gacaatccta gagataggac gtcccctttc agggggcasa 1020
cttgacatgg tgagtgcatg gttgtcgtca gcttcgtgtc gatgagatgc twgggtwaar 1080
tccygcaacg agccgcarcc sttgaatkmt tagcttgacc agcattcagt tgggcactct 1140
aaggtgagag ccggtgacaa accggaggaa ggtggatatg acgtcaaatc atcatgcccc 1200
ttatgacctg ggctacacac gtggaacaat ggacagaaca aagggcagcg aaaccgcgag 1260
gttaagttaa tcccacaaat ctgttctcag aacggatcgc agtctataac tcgactgcgt 1320
gaagctggaa tgtctagtaa tcgcggatca atatgccgcg gtgaatacgt tcccgcgcct 1380
tgtacacacc gcccgtaaca ccacgagagt ttggcacacc cgaagtcggt gaggtaacct 1440
tttaggagcc agccgccgaa ggtgaccaga agtt 1474
Claims (8)
1. The Bacillus subtilis TK2019 microbial agent is characterized by being obtained by fermenting Bacillus subtilis TK 2019.
2. The Bacillus subtilis TK2019 according to claim 1, wherein the Bacillus subtilis TK2019 is deposited in China general microbiological culture Collection center (CGMCC) with the deposit number of CGMCC No: 20810.
3. the microbial agent of Bacillus velezensis TK2019 of claim 1, wherein the gene sequence of the Bacillus velezensis TK2019 is shown as SEQ ID NO: 1.
4. The Bacillus velezensis TK2019 microbial agent according to claim 1, wherein the microbial agent is a liquid preparation or a powder preparation.
5. The microbial agent of Bacillus beilis (Bacillus velezensis) TK2019 as claimed in claim 1, wherein the isolated and purified NA medium of Bacillus beilis (Bacillus velezensis) TK 2019: 3-6g/L of beef extract, 7-13g/L of peptone, 4-6g/L of NaCl, 13-19g/L of agar and 7.0 of pH value.
6. The Bacillus subtilis TK2019 microbial inoculant according to claim 1, wherein the enriched maize meal flour culture medium of Bacillus subtilis TK 2019: 1.0-3.0g/L of corn flour, 1.0-3.0g/L of soybean flour, 0.1-0.8g/L of NaCl0, MnSO4·H2O0.5-1.0 g/L, pH 7.0.
7. A preparation method of a Bacillus velezensis TK2019 microbial agent is characterized by comprising the following steps:
(1) activating a preserved Bacillus belezii (Bacillus velezensis) TK2019 CGMCC No:20810 strain on an NA plate culture medium, selecting a single strain to be cultured on an NA slant culture medium at 20-37 ℃ for 6-12 hours to obtain an activated Bacillus belezensis (Bacillus velezensis) TK2019 strain;
(2) scraping a ring of the activated Bacillus velezensis TK2019 strain obtained in the step (1) by using a sterile inoculating ring, inoculating the bacterial strain into a 100mLNA liquid culture medium, and culturing for 6-12 hours at the temperature of 20-37 ℃ and the rotating speed of a shaking table of 120-180 rpm to obtain a Bacillus velezensis TK2019 seed liquid;
(3) inoculating the seed liquid of Bacillus velezensis TK2019 obtained in the step (2) into a corn flour culture medium according to an inoculation amount of 1-3% of the mass-volume ratio, and performing fermentation culture for 30-36 h at the temperature of 20-37 ℃ and the rotating speed of a shaking table of 120-180 rpm to obtain a Bacillus velezensis TK2019 fermentation liquid;
(4) and (3) detecting the quantity of the bacteria and spores in the fermentation liquid obtained in the step (3), stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the bacteria, and preparing a liquid or powdery microbial agent of the Bacillus velezensis TK2019 microbial agent according to needs.
8. The application of the Bacillus beili (Bacillus velezensis) TK2019 microbial inoculum according to claim 1 in preparation of medicines for preventing and treating cotton verticillium wilt, cotton verticillium wilt and red date black spot.
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Application publication date: 20210309 Assignee: XINJIANG ZHIKU SILK ROAD INTELLECTUAL PROPERTY BIG DATA CENTER (Co.,Ltd.) Assignor: INSTITUTE OF MICROBIAL APPLICATIONS, XINJIANG ACADEMY OF AGRICULTURAL SCIENCES (CHINA XINJIANG-ARMENIA BIOENGINEERING RESEARCH AND DEVELOPMENT CENTER) Contract record no.: X2024980045065 Denomination of invention: A microbial agent of Bacillus thuringiensis and its application Granted publication date: 20221011 License type: Open License Record date: 20250106 |