CN106939290B - Bacillus subtilis HMB26553 and application thereof - Google Patents

Bacillus subtilis HMB26553 and application thereof Download PDF

Info

Publication number
CN106939290B
CN106939290B CN201710157385.6A CN201710157385A CN106939290B CN 106939290 B CN106939290 B CN 106939290B CN 201710157385 A CN201710157385 A CN 201710157385A CN 106939290 B CN106939290 B CN 106939290B
Authority
CN
China
Prior art keywords
hmb26553
bacillus subtilis
cotton
strain
microbial agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201710157385.6A
Other languages
Chinese (zh)
Other versions
CN106939290A (en
Inventor
马平
李社增
鹿秀云
郭庆港
张晓云
王莹
王培培
赵卫松
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute Of Plant Protection Hebei Academy Of Agricultural And Forestry Sciences
Original Assignee
Institute Of Plant Protection Hebei Academy Of Agricultural And Forestry Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute Of Plant Protection Hebei Academy Of Agricultural And Forestry Sciences filed Critical Institute Of Plant Protection Hebei Academy Of Agricultural And Forestry Sciences
Priority to CN201710157385.6A priority Critical patent/CN106939290B/en
Publication of CN106939290A publication Critical patent/CN106939290A/en
Application granted granted Critical
Publication of CN106939290B publication Critical patent/CN106939290B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C12R2001/125
    • C12N1/205
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor

Abstract

The invention discloses a Bacillus subtilis strain HMB26553, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 13211. The invention also discloses a microbial agent produced by using the strain and application of the microbial agent in preventing and treating plant diseases such as cotton rhizoctonia solani and the like. The bacillus subtilis HMB26553 has high control effect on cotton rhizoctonia rot, and the average control effect is more than 90.0 percent; secondly, the microbial agent has good drug effect durability and no environmental pollution problem; in addition, the HMB26553 has a broad antibacterial spectrum and has good inhibitory effects on verticillium dahliae, verticillium wilt, botrytis cinerea and cucumber target spot bacteria besides verticillium wilt.

Description

Bacillus subtilis HMB26553 and application thereof
Technical Field
The invention belongs to the field of biological control, and particularly relates to bacillus subtilis HMB26553, a microbial agent produced by using the bacillus subtilis, and application of the bacillus subtilis HMB26553 in control of cotton rhizoctonia solani and other diseases.
Background
The cotton seedling stage disease is caused by Rhizoctonia solani (Rhizoctonia solani K ü hn), and the main symptoms of the disease are that the cotton seedling stage disease is infected before germination to cause rotten seeds, the cotton seedling stage disease is infected before emergence to cause rotten buds, the cotton seedling is damaged after emergence, yellow brown disease spots are generated at the base part of the near soil surface in the initial stage, the disease spots gradually expand to surround the whole base part to be obviously contracted, the diseased seedling withers and withers, the diseased seedling is pulled out, the cortex below the stem base part is left in the soil, only the rat tail-shaped xylem with sharp threading is stored, after the cotyledon damage, yellow brown disease spots are generated at the middle part of the cotyledon, the disease usually falls off and perforates, the cotton seedling growth and development are seriously affected by the cotton seedling stage disease, and the cotton seedling usually dies in pieces after the disease occurs, and the problem to be solved urgently.
The cotton blight is mainly controlled chemically, such as dressing, coating or soaking seeds with chemical agents before sowing, spraying and irrigating roots with chemical agents after sowing, but the chemical control effect is poor, and the problems of environmental pollution, easy generation of drug resistance after long-term use, high cost and the like exist. 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 microorganisms currently used for controlling cotton rhizoctonia rot are mainly: trichoderma harzianum (CN104430549A), Bacillus atrophaeus (CN104531545A), Bacillus megaterium (CN103320360A), Bacillus amyloliquefaciens (CN 105176893A; CN104498386A), Bacillus subtilis (CN 105199997A; CN 104770398A; CN 104642390A; CN 105340972A), Rhizoctonia binuclear (CN1952106A), Serratia (CN103122330A), Pseudomonas chlororaphis (CN1932006A) and Pseudomonas fluorescens (CN 1058230A). From the above, the microorganism for preventing and treating cotton rhizoctonia solani 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 continuous evolution of pathogenic microorganisms, and the screening of biocontrol microorganisms having an inhibiting effect on pathogenic bacteria is the most economical and effective method for preventing and treating the pathogenic microorganisms.
Disclosure of Invention
The invention aims to provide a bacillus subtilis strain which has the advantages of high efficiency, wide bactericidal spectrum and the like.
The invention also aims to provide a microbial agent produced by utilizing the bacillus subtilis.
The third object of the present invention is to provide a method for producing the microbial agent.
The fourth purpose of the invention is to provide the application of the bacillus subtilis in preventing and treating plant diseases such as cotton rhizoctonia solani.
The fifth purpose of the invention is to provide the application of the microbial agent in preventing and controlling plant diseases such as cotton rhizoctonia solani.
The invention is realized by the following technical scheme:
a Bacillus subtilis strain HMB26553, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 13211.
The invention also provides a microbial agent produced by utilizing the bacillus subtilis HMB26553, and the active component of the microbial agent is bacillus subtilis HMB 26553; also included are extracellular metabolites thereof.
The microbial agent can be liquid preparation or powder preparation.
The preparation method of the microbial agent comprises the following steps:
(1) activating strains: activating the HMB26553 strain stored at low temperature on an LB plate culture medium, selecting a single strain to be placed on an LB slant culture medium, and culturing for 10-16 hours at 25-35 ℃ to obtain an activated strain;
(2) preparing a seed solution: scraping a ring of the activated strain obtained in the step (1) by using a sterile inoculating ring, inoculating the ring into 100mL of LB liquid culture medium, and culturing for 10-16 hours at the temperature of 25-35 ℃ and the rotating speed of a shaking table of 150-220 rpm to obtain seed liquid;
(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a corn meal and soybean meal culture medium (pH value is 7.0) according to the volume ratio of 1-3%, and performing fermentation culture for 35-40 h under the conditions of 25-35 ℃ and the rotating speed of a shaking table of 150-220 rpm to obtain a fermentation liquid;
(4) detecting the quantity of the thalli and spores in the fermentation liquid, and stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the thalli; the resulting liquid formulation is HMB 26553.
The LB plate culture medium or LB inclined plane culture medium in the step (1) of the preparation method comprises the following components in percentage by weight: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride, 12-18 g of agar powder and 1000mL of water.
The LB liquid culture medium in the step (2) of the preparation method comprises the following components in parts by weight: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride and 1000mL of water.
The LB plate culture medium, the LB slant culture medium and the LB liquid culture medium are all prepared according to a conventional method.
The corn meal and soybean meal culture medium in the step (3) of the preparation method comprises the following components in percentage by weight: 1.0-3.0% of corn flour, 1.0-3.0% of soybean flour, 0.1-0.8% of NaCl, and MnSO 4·H 20.5-1.0% of O and the balance of water.
The preparation method of the corn flour and soybean meal culture medium comprises the steps of mixing corn flour, soybean meal, NaCl and MnSO in percentage by weight 4·H 2And O, mixing, adding water, adjusting the pH value, and uniformly stirring.
The viable count of the bacillus subtilis HMB26553 is more than 17.0 × 10 8cfu/mL。
The bacillus subtilis HMB26553 is used for preventing and treating cotton verticillium wilt, cotton wilt, tomato gray mold or cucumber target spot disease.
The microbial agent is applied to prevention and treatment of cotton verticillium wilt, cotton fusarium wilt, tomato gray mold or cucumber target spot disease.
The use method of the microbial agent comprises the following steps: diluting the obtained microbial agent with water until viable cell number is 10 7cfu/mL, soaking seeds for half an hour before cotton sowing; or adsorbing the obtained microbial agent with calcium carbonate to obtain Bacillus subtilis HMB26553Powder, before cotton sowing, according to the seed ratio of 1: 10 seed dressing.
Screening and separating process of HMB26553 strain
Collecting soil samples from southern Yigou county forest of Milin county of Rizhi City, Xizang autonomous region, 7 Yue Hebei province, of plant protection institute in 2012, taking the soil samples back to the laboratory, weighing 1g of the soil samples, putting the soil samples into a 250mL sterile triangular flask, adding 100mL of sterile water, putting the flask on a shaking table, oscillating the flask at 170rpm for 30min, standing the flask for 2h, taking 10mL of supernatant, adding the supernatant into a 50mL sterile centrifuge tube, carrying out constant-temperature water bath at 80 ℃ for 30min, taking 1mL of sterile water, and adding 9mL of sterile water into the flask to obtain 10mL10 -3Multiplying the soil microorganism suspension, and then diluting the soil suspension to 10 -4、10 -5、10 -6Taking 200 mu L of microbial suspension with each concentration of the dilution liquid, coating the microbial suspension on an LB culture medium plate, repeating each concentration for 3 times, culturing for 1 d-3 d at the constant temperature of 30 ℃, and separating and purifying bacteria. And screening biocontrol bacteria by using cotton rhizoctonia solani as a target through a flat plate confronting method and a pot experiment method. And screening a strain with good control effect on cotton rhizoctonia rot from the strain, and naming the strain as HMB 26553.
Taxonomic identification of the HMB26553 strain:
(1) morphological characterization
The thallus is in a rod shape and cultured on an LB culture medium for 10 hours to generate spores which are Zhongsheng, oval, no cyst expansion, negative acid-fast staining, no parasporal crystal, motion and flagellum perigenesis. On a nutrient agar plate, the colony is light milky white, purulent, round and neat in edge at the initial culture stage, the colony is raised into a steamed bun shape, and the surface is moist; the bacterial colony in the later culture period is light yellow, the edge is irregular, and the surface is dry and has wrinkles; 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. These morphological characteristics were substantially identical to those of Bacillus described in the Manual of identification of common bacterial systems (east elegans bead et al, science publishers, 2001), and it was preliminarily judged that the strain HMB26553 belongs to Bacillus.
(2) Identification and classification using 16S rDNA sequences
Performing PCR amplification by using genome DNA of HMB26553 as a template and universal primers F27 and R1492 as primers to obtain a PCR amplification product; wherein the primer sequence is as follows:
27F:5’-AGAGTTTGATCATGGCTCAG-3’(SEQ ID No.1),
R1492:5’-GGCTACCTTGTTACGACTT-3’(SEQ ID No.2)。
the 16S rDNA PCR reaction system (50. mu.L) was 10 XPCR Buffer (Mg) 2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; 1 μ L of Taq (5U/. mu.L), 1 μ L of 27F (10 μmol/L), and 1 μ L of R1492(10 μmol/L); HMB26553 genomic DNA 50 ng; ddH 2O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1.5 min; 10min at 72 ℃. And carrying out gel electrophoresis on the obtained PCR amplification product, and sending the product to Shanghai biological engineering Co., Ltd for sequencing to obtain a 16S rDNA sequence (shown as SEQ ID No.3) of HMB 26553. Homology comparison is carried out on the obtained 16S rDNA sequence of the HMB26553 in Genbank, and the result shows that the homology of the strain HMB26553 and the 16S rDNA sequence of the bacillus reaches 98 percent; meanwhile, a phylogenetic tree is constructed by using MEGA software (Molecular evolution Genetics Analysis), and as a result (see FIG. 1), HMB26553 is aggregated with Bacillus, which indicates that HMB26553 belongs to Bacillus.
(3) Identification and classification using gyrB gene sequences
Carrying out PCR amplification by using HMB26553 genome DNA as a template and using degenerate primers gyrB-F and gyrB-R of a gyrB gene of bacillus as primers to obtain a PCR amplification product; wherein the sequences of the gyrB-F primers and the gyrB-R primers are as follows:
gyrB-F:5’-TTGRCGGHRGYGGHTATAAAGT-3’(SEQ ID No.4),
gyrB-R:5’-TCCDCCSTCAGARTCWCCCTC-3’(SEQ ID No.5)。
the PCR amplification reaction system (50. mu.L) of gyrB was 10 XPCR Buffer (Mg) 2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; 1 μ L of Taq (5U/. mu.L); 1 μ L of gyrB-F (10 μmol/L), 1 μ L of gyrB-R (10 μmol/L); HMB26553 genomic DNA 50 ng; ddH 2O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 45s, and 72 ℃ for 1 min; 10min at 72 ℃. The amplification product is sent to Shanghai biological engineering Co., LtdAnd obtaining the gyrB gene sequence of the HMB26553 strain (shown as SEQ ID No. 6). The gyrB gene sequences of the obtained HMB26553 strain were subjected to homology comparison in Genbank. The result shows that the homology of the HMB26553 and the gyrB gene sequence of the bacillus subtilis is the highest and reaches 97.5 percent; meanwhile, MEGA software (Molecular evolution Genetics Analysis) is utilized to construct a phylogenetic tree, and the result (shown in figure 2) shows that the HMB26553 strain and the Bacillus subtilis are polymerized together, so that the strain HMB26553 is the Bacillus subtilis and is a new strain.
By combining the morphological characteristics and the results of homology comparison analysis of 16S rDNA and gyrB gene sequences, HMB26553 is known to belong to Bacillus subtilis and is a new Bacillus subtilis strain different from the existing Bacillus strains.
The invention has the advantages and beneficial effects that: (1) the bacillus subtilis HMB26553 has good prevention and treatment effects on cotton rhizoctonia solani, the average prevention effect is more than 90.0 percent, (2) the bacillus subtilis HMB26553 has strong specificity on the cotton rhizoctonia solani, is not easy to generate drug resistance and has good drug effect durability; (3) the microbial agent is safe to people and livestock, and has no problem of environmental pollution; (4) the bacillus subtilis HMB26553 has a wide antibacterial spectrum, and has good inhibitory effects on verticillium wilt bacteria of cotton, fusarium wilt bacteria of cotton, botrytis cinerea and cucumber target spot bacteria besides verticillium wilt bacteria of cotton; (5) the preparation method is simple, low in cost and simple to use.
And (3) biological preservation: the Bacillus subtilis strain HMB26553 of the invention is preserved in the China general microbiological culture Collection center in 2016, 10 and 28 days, and the preservation addresses are as follows: the microbial research institute of China academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, with the collection number of CGMCC No. 13211.
Drawings
FIG. 1 is a phylogenetic tree of the HMB26553 strain obtained from the 16S rDNA sequence.
FIG. 2 is a phylogenetic tree of the HMB26553 strain obtained based on the gyrB gene sequence.
Detailed Description
The invention is further illustrated by the following specific examples, which are not to be construed as limiting in any way. The experimental methods in the following examples are all conventional methods unless otherwise specified; the percentages in the following examples are by weight unless otherwise specified.
Example 1 preparation of HMB26553 microbial Agents
The method comprises the following steps:
(1) activating strains: activating (30 ℃) a Bacillus subtilis strain HMB26553(HMB26553 is preserved at minus 80 ℃ and is preserved in China general microbiological culture Collection center in 2016, 10, 28 and 28 days, with the preservation number of CGMCC No.13211) on an LB plate culture medium (the components and the weight ratio of the components are 10g of tryptone, 5g of yeast extract, 5g of sodium chloride, 15g of agar powder and 1000mL of water), and picking single bacteria to fall on an LB slant culture medium (the components and the weight ratio of the components are 10g of tryptone, 5g of yeast extract, 5g of sodium chloride, 15g of agar powder and 1000mL of water) to culture for 12 hours at 30 ℃ to obtain an activated strain;
(2) preparing a seed solution: preparing an LB liquid culture medium (the components and the weight ratio of the components are 10g of tryptone, 5g of yeast extract, 5g of sodium chloride and 1000mL of water) according to a conventional method, filling 100mL of LB culture solution into a 250mL triangular flask, sterilizing by high-pressure moist heat, inoculating the activated strain in the inoculating loop step (1) into each flask after the temperature is reduced to room temperature, and performing shaking culture for 12 hours at the temperature of 30 ℃ and the rotating speed of a shaking table of 180rpm to obtain a seed solution;
(3) preparing a corn flour and soybean flour culture medium: according to the weight percentage, 1.5 percent of corn flour, 2.0 percent of soybean meal, 0.5 percent of NaCl and MnSO 4·H 2Adding 0.6% of O into water, and stirring and mixing uniformly to obtain a corn flour and soybean meal culture medium; subpackaging in 500mL triangular bottles, 200mL each; sterilizing the corn flour and soybean meal culture medium at 121 ℃ for 30 minutes, and cooling to 30 ℃ for later use;
(4) fermentation culture: inoculating 2mL of the seed solution obtained in the step (2) into 200mL of the culture medium of each bottle of corn flour and soybean flour prepared in the step (3); carrying out fermentation culture at 30 ℃ and a shaking table rotating speed of 180rpm for 36 hours, sampling from a triangular flask at intervals of 30 minutes, carrying out microscopic examination, counting spores and total thallus in a visual field, and calculating the spore rate (%) -mature spore number/(mature spore number + thallus number) × 100); stopping fermentation culture when the spore rate reaches 90%; co-fermentation and culturing for 48 hours to obtain the liquid preparation of HMB 26553.
Example 2 test of inhibitory Effect of HMB26553 Strain of the present invention on the hyphal growth of Rhizoctonia gossypii
Firstly, testing rhizoctonia solani RH-2: the strain of Rhizoctonia solani RH-2 is collected from a strain of Rhizoctonia solani in Pianhandan county of Hebei province, is separated and purified by a plant protection research institute of the institute of agriculture and forestry academy of sciences of Hebei province, and the plant pathological system of the plant protection academy of Hebei agriculture university is identified as Rhizoctonia solani (Rhizonicota solani), and the pathogenicity determination shows strong pathogenicity.
(II) test method:
plate determination test: first, Rhizoctonia solani (RH-2) was activated and cultured on a PDA plate for 3 days, and then in the edge area of the colony, a punch was used Punching to obtain bacterial tablet, transferring the cotton rhizoctonia solani bacterial tablet to the center of another PDA plate, then dotting the activated bacillus subtilis HMB26553 at a position 2.0 cm away from the indicator bacterial tablet, and setting a blank control (the growth condition of the cotton rhizoctonia solani without dotting the HMB26553 strain). Culturing at constant temperature of 25 deg.C, measuring control growth amount (colony radius) and treatment growth amount (growth inhibiting radius after inoculating HMB 26553) of cotton rhizoctonia solani when blank control is about to grow on the whole culture dish, and expressing antagonistic action by bacteriostatic rate. The calculation formula is as follows:
the bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100.
Results (see table 1): the inhibition rate of the bacillus subtilis HMB26553 to the cotton rhizoctonia solani reaches 69.2 percent; the bacillus subtilis HMB26553 has obvious inhibition effect on the growth of cotton rhizoctonia solani hyphae and has biocontrol potential for preventing and treating cotton rhizoctonia solani.
TABLE 1 results of experiments on the antagonistic action of the HMB26553 strain of the invention against rhizoctonia solani
Strain name Control growth volume (mm) Treatment of growth (mm) Bacteriostatic ratio (%)
HMB26553 39.0 12.0 69.2
Example 3 comparative test for controlling the Effect of the HMB26553 Strain of the invention on the Ralstonia gossypii
(I) test treatment:
(1) and (3) microbial agent: the HMB26553 liquid formulation prepared in example 1 was diluted 50 fold with water.
(2) Blank control: clean water
(II) test method:
mixing vermiculite with cotton field soil according to the proportion of 1: 1 proportion, then sterilizing for 1 hour under the high pressure and moist heat at 121 ℃, and sterilizing for 1 time under the same conditions every other day. A suspension of Rhizoctonia solani RH-2 mycelial fragments (about 10) was inoculated into 500 g of soil 7One/ml) of the mixture is 10ml, and the mixture is filled into a plastic flowerpot with the diameter of 20 cm after being uniformly stirred and is compacted. Selecting Jifeng 106 cotton variety, soaking the cotton variety with 50 times of water diluent of the HMB26553 liquid preparation prepared in the example 1 for half an hour before sowing; seeds are soaked in clear water for half an hour as a blank control. Each pot after seed soaking treatmentSowing 10 seeds, covering with sterilized soil, and culturing in greenhouse. Investigating and recording the number of the plants which are treated and subjected to the blank control of the damping-off after emergence of seedlings day by day, and calculating the morbidity and the control effect when the blank control of the damping-off is fully attacked.
Results (Table 2) in the first pot experiment, the control effect of Bacillus subtilis HMB26553 of the present invention on cotton rhizoctonia solani was 89.65%. In the second batch of pot culture test results, the control effect of the bacillus subtilis HMB26553 on cotton rhizoctonia solani is 87.99%. The bacillus subtilis HMB26553 and the microbial agent thereof have good prevention and treatment effects on cotton rhizoctonia rot.
TABLE 2 comparative test results of the prevention of cotton blight using HMB 26553A liquid formulation of the present invention
Example 4 comparative test for controlling the Effect of the HMB26553 Strain of the invention on the Ralstonia gossypii
(I) test treatment:
(1) and (3) microbial agent: the HMB26553 liquid formulation prepared in example 1 was diluted 50 times with water.
(2) Blank control: clean water
(II) test method:
preparing seedling culture soil: mixing vermiculite with cotton field soil according to the proportion of 1: 1 proportion, sterilizing for 1 hour under high pressure and moist heat at 121 ℃, sterilizing for 1 time under the same conditions every other day, and naturally cooling for later use. Culturing the rhizoctonia solani strain: 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 4-5 hypha blocks of the cotton rhizoctonia solani RH-2 with each pipe after cooling, uniformly mixing, and culturing in a constant-temperature incubator at 25 ℃ for 10 days for later use. Filling prepared seedling raising soil into a 50ml centrifugal tube with the height of 10 cm to a position 2cm away from the bottom of the tube, placing a piece of cultured small rice grains carrying rhizoctonia solani RH-2 on the soil surface, continuously filling the seedling raising soil to a position 7 cm away from the bottom of the tube, compacting and sowing. Selecting Jifeng 106 cotton variety, soaking the cotton variety with 50 times of water diluent of the HMB26553 liquid preparation prepared in the example 1 for half an hour before sowing; seeds are soaked in clear water for half an hour as a blank control. Covering with seedling soil after seeding, slightly pressing with hand, covering with a centrifuge tube cover, culturing in a dark temperature-controlled light-controlled thermostatic chamber with 12 hours of illumination at 25 deg.C, and quantitatively adding water to adjust soil humidity. The centrifuge tube cap was removed when the seeds came out of the earth. Investigating and recording the number of the plants which are treated and subjected to the blank control of the damping-off after emergence of seedlings day by day, and calculating the morbidity and the control effect when the blank control of the damping-off is fully attacked.
As a result (see Table 3), the control effect of Bacillus subtilis HMB26553 of the present invention on cotton rhizoctonia solani was 84.18%. The bacillus subtilis HMB26553 and the microbial agent thereof have good prevention and treatment effects on cotton rhizoctonia rot.
TABLE 3 comparative test results of the control effect of HMB26553 strain of the invention on cotton rhizoctonia rot
Treatment of Investigation of plant number Number of diseased plants Incidence (%) Control effect (%)
HMB26553 liquid formulations 107 3 2.8b 84.18
Blank control 96 17 17.7a --
Example 5 comparative test of the prevention of Cotton Ralstonia blight with the HMB26553 Strain of the invention
(I) test treatment:
(1) HMB26553 powder: according to the following steps of 1: 1 to the HMB26553 liquid preparation prepared in example 1 calcium carbonate was added, obtaining a HMB26553 powder.
(2) Blank control: clean water
(II) test method:
the test is carried out in the cotton test field of Bacun in Gaoyang county of Baoding City of Hebei province. According to the following steps of 1: 1 ratio to the HMB26553 liquid formulation prepared in example 1 calcium carbonate was added to produce a HMB26553 powder. Selecting Jifeng 106 cotton varieties, and performing sowing by using HMB26553 powder according to the ratio of medicinal seeds of 1: 10 seed dressing; untreated seeds were used as a blank. Each treatment was repeated 4 times, randomly arranged, and the cell area was 40 square meters. And (4) investigating the number of seedlings per unit area and the number of seedlings died due to damping-off after seedling emergence, investigating once every 3 days, accumulatively counting the incidence rate of cotton rhizoctonia rot, and calculating the control effect.
Results (see table 4): the control effect of the bacillus subtilis HMB26553 on cotton rhizoctonia solani is 100.0%. The bacillus subtilis HMB26553 and the microbial agent thereof have very good prevention and treatment effects on cotton rhizoctonia rot.
TABLE 4 comparative test results of the control effect of HMB26553 strain of the invention on cotton damping-off
Treatment of Investigation of plant number Number of diseased plants Incidence (%) Control effect (%)
HMB26553 174 0 0.0b 100.0
Blank control 186 14 7.5a --
Example 6 test of the inhibitory Effect of the HMB26553 Strain of the invention on 4 phytopathogenic fungi
(one) test 4 kinds of pathogenic fungi and their sources:
verticillium dahliae (Verticillium dahliae) VD-1, Fusarium oxysporum (Fusarium oxysporum f.sp.vasicinfectam) FOV-1, Botrytis cinerea (Botrytis cinerea) BC-32 and Corynespora cucumerinum (Corynespora cassiicola) CC-1.
The 4 pathogenic fungi strains come from the plant disease biological control laboratory of the plant protection research institute of agriculture and forestry academy of sciences of Hebei province.
(II) test method:
the inhibition effect of the bacillus subtilis HMB26553 strain on 4 plant pathogenic fungi is detected by adopting a confrontation culture method, and the HMB26553 strain is activated for 24 hours at 30 ℃ on an LB slant culture medium to obtain the activated HMB26553 strain. Activating 4 pathogenic fungi on a PDA culture medium for 5 days, punching a bacterial disc with consistent bacterial age at the edge of the bacterial colony by using a puncher with the diameter of 6mm, transferring hypha downwards to the center of another PDA flat plate, inoculating HMB26553 strains in a shape of a Chinese character 'pin' at a position 2cm away from the bacterial disc, repeatedly culturing at the constant temperature of 25 ℃ for 5 days by taking non-inoculated HMB26553 as a control after 3 times of treatment, measuring the radius of the treated bacterial colony and the distance (bacterial inhibition zone) between a bacterial inoculation point and the edge of the bacterial colony of the pathogenic bacteria, determining the bacteriostatic degree, and calculating the bacteriostatic rate.
The bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100.
TABLE 5 test results of the inhibition of 4 phytopathogenic fungi by the HMB26553 strain of the invention
The result (see table 5) shows that the HMB26553 strain of the invention has the bacteriostasis rate of 65.83-94.43% to 4 tested pathogenic fungi, wherein the bacteriostasis rate to verticillium dahliae of cotton reaches 84.43%, and the bacteriostasis rate to fusarium wilt of cotton is the lowest of 65.83%, which indicates that the HMB26553 strain of the invention has a broad bacteriostasis spectrum.
SEQUENCE LISTING
<110> institute of plant protection of academy of agriculture, forestry and science of Hebei province
<120> Bacillus subtilis HMB26553 and application thereof
<160>6
<170>PatentIn version 3.5
<210>1
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223>27F
<400>1
agagtttgat catggctcag 20
<210>2
<211>19
<212>DNA
<213>Artificial Sequence
<220>
<223>R1492
<400>2
ggctaccttg ttacgactt 19
<210>3
<211>969
<212>DNA
<213>Bacillus subtilis
<400>3
cggagtggcg ggtgctatac atgcagtcga gcggacagat gggagcttgc tccctgatgt 60
tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg atggttgttt gaaccgcatg gttcaaacat aaaaggtggc 180
ttcggctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaatggctc 240
accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa 420
gaacaagtgc cgttcgaata gggcggcacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540
cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600
agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660
cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720
actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780
gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840
cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900
ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960
gtcttgaca 969
<210>4
<211>22
<212>DNA
<213>Artificial Sequence
<220>
<223>gyrB-F
<400>4
ttgrcgghrg ygghtataaa gt 22
<210>5
<211>21
<212>DNA
<213>Artificial Sequence
<220>
<223>gyrB-R
<400>5
tccdccstca gartcwccct c 21
<210>6
<211>959
<212>DNA
<213>Bacillus subtilis
<400>6
ctccttggat cggtgtagtg cgtcggtcgt aacgcactat caacagagct tgatgtgacg 60
gttcaccgtg acggtaaaat tcaccgccaa acctataaac gcggagttcc ggttacagac 120
cttgaaatca ttggcgaaac ggatcataca ggaacgacga cacattttgt cccggaccct 180
gaaattttct cagaaacaac cgagtatgat tacgatctgc ttgccaaccg cgtgcgtgaa 240
ttggcctttt taacaaaggg cgtaaacatc acgattgaag ataaacgtga aggacaagag 300
cgcaaaaatg aataccatta cgaaggcgga attaaaagtt atgtagagta tttaaaccgc 360
tctaaagagg ttgtccatga agagccgatt tacattgaag gcgaaaagga cggcattacg 420
gttgaagtgg ctttgcaata caatgacagc tacacaagca acatttactc gtttacaaac 480
aacattaaca cgtacgaagg cggtacccat gaagctggct tcaaaacggg cctgactcgt 540
gttatcaacg attacgccag aaaaaaaggg cttattaaag aaaatgatcc aaacctaagc 600
ggagatgacg taagggaagg gctgacagcg attatttcaa tcaaacaccc tgatccgcag 660
tttgagggcc aaacgaaaac aaagctgggc aactcagaag cacggacaat caccgatacg 720
ttattttcta cggcgatgga aacatttatg ctggaaaatc cagatgcggc caaaaaaatt 780
gtcgataaag gcttaatggc ggcaagagca agaatggctg cgaaaaaagc gcgtgaacta 840
acacgccgta agagtgcttt ggaaatttca aacttgcccg gtaagttagc ggactgctct 900
tcaaaagatc cgagcatttc cgagttatat atcgtagagg gagactcttg acggcggaa 959

Claims (5)

1. A Bacillus subtilis strain HMB26553, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 13211.
2. The microbial agent produced by using the bacillus subtilis HMB26553 of claim 1, characterized in that the active ingredient is bacillus subtilis HMB 26553.
3. The microbial inoculant according to claim 2, wherein the microbial inoculant is a liquid or a powder.
4. The use of the bacillus subtilis HMB26553 of claim 1 for the control of cotton verticillium wilt, cotton fusarium wilt, tomato gray mold and cucumber target spot disease.
5. The use of the microbial inoculant of claim 2 for the control of cotton verticillium wilt, cotton wilt, tomato gray mold and cucumber target spot.
CN201710157385.6A 2017-03-16 2017-03-16 Bacillus subtilis HMB26553 and application thereof Active CN106939290B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710157385.6A CN106939290B (en) 2017-03-16 2017-03-16 Bacillus subtilis HMB26553 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710157385.6A CN106939290B (en) 2017-03-16 2017-03-16 Bacillus subtilis HMB26553 and application thereof

Publications (2)

Publication Number Publication Date
CN106939290A CN106939290A (en) 2017-07-11
CN106939290B true CN106939290B (en) 2020-02-11

Family

ID=59469646

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710157385.6A Active CN106939290B (en) 2017-03-16 2017-03-16 Bacillus subtilis HMB26553 and application thereof

Country Status (1)

Country Link
CN (1) CN106939290B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108315284B (en) * 2018-04-27 2021-02-02 山东农业大学 Bacillus safensis capable of producing protease and resisting botrytis cinerea and application thereof
CN108707554A (en) * 2018-05-21 2018-10-26 河北省农林科学院植物保护研究所 A pair of of Ustilago maydis combination and its application
CN108795829A (en) * 2018-07-11 2018-11-13 金福赛(北京)生物科技有限公司 Bacillus subtilis and its application in microbial inoculum and pork pig feed
CN109486707A (en) * 2018-11-23 2019-03-19 北京林业大学 A kind of bacillus subtilis strain and its application

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1766091A (en) * 2005-09-23 2006-05-03 中国农业大学 Bacillus subtilis and its uses
CN1924005A (en) * 2006-09-11 2007-03-07 河北省农林科学院植物保护研究所 Bacillus subtilis, bacterium agent and application thereof
CN101990907A (en) * 2010-08-06 2011-03-30 湖北省农业科学院植保土肥研究所 Composite biological antimicrobial for preventing wilt and greensickness of cotton
CN102515954A (en) * 2011-12-16 2012-06-27 上海绿乐生物科技有限公司 Special biological organic fertilizer for verticillium wilt and production method for same
CN102524303A (en) * 2011-08-31 2012-07-04 南京农业大学 Mixture for preventing blight and verticillium wilt of cotton
CN103772008A (en) * 2013-12-27 2014-05-07 上海绿乐生物科技有限公司 Compound microbial fertilizer with efficient disease prevention function and production method of compound microbial fertilizer
CN105199997A (en) * 2015-11-03 2015-12-30 河北省农林科学院植物保护研究所 Bacillus subtilis for controlling cotton rhizoctonia and application of bacillus subtilis

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1766091A (en) * 2005-09-23 2006-05-03 中国农业大学 Bacillus subtilis and its uses
CN1924005A (en) * 2006-09-11 2007-03-07 河北省农林科学院植物保护研究所 Bacillus subtilis, bacterium agent and application thereof
CN101990907A (en) * 2010-08-06 2011-03-30 湖北省农业科学院植保土肥研究所 Composite biological antimicrobial for preventing wilt and greensickness of cotton
CN102524303A (en) * 2011-08-31 2012-07-04 南京农业大学 Mixture for preventing blight and verticillium wilt of cotton
CN102515954A (en) * 2011-12-16 2012-06-27 上海绿乐生物科技有限公司 Special biological organic fertilizer for verticillium wilt and production method for same
CN103772008A (en) * 2013-12-27 2014-05-07 上海绿乐生物科技有限公司 Compound microbial fertilizer with efficient disease prevention function and production method of compound microbial fertilizer
CN105199997A (en) * 2015-11-03 2015-12-30 河北省农林科学院植物保护研究所 Bacillus subtilis for controlling cotton rhizoctonia and application of bacillus subtilis

Also Published As

Publication number Publication date
CN106939290A (en) 2017-07-11

Similar Documents

Publication Publication Date Title
Ji et al. Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars
Jha et al. The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth-promoting potential
US10212912B2 (en) Endophytic microbial symbionts in plant prenatal care
Pedraza et al. Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop
Li et al. The endophytic bacteria isolated from elephant grass (Pennisetum purpureum Schumach) promote plant growth and enhance salt tolerance of Hybrid Pennisetum
CN102010835B (en) Streptomyces corchorusii strain NF0919, purpose and preparation method of active zymotic fluid thereof
Hiltunen et al. Interactions and biocontrol of pathogenic Streptomyces strains co‐occurring in potato scab lesions
Lee et al. Isolation and characterization of endophytic actinomycetes from Chinese cabbage roots as antagonists to Plasmodiophora brassicae
JP4417998B2 (en) Plant disease control method using Bacillus subtilis strain having antagonistic action
Lin et al. Potential biocontrol Bacillus sp. strains isolated by an improved method from vinegar waste compost exhibit antibiosis against fungal pathogens and promote growth of cucumbers
CN102433282B (en) Bacillus subtilis NB12, as well as culture method and application thereof
CN102965314B (en) Bacillus subtilis and preparation and application of microbial inoculum thereof
CN104894010B (en) A kind of composite microbiological fertilizer of antagonism silborne fungal diseases and its preparation method and application
CN104164394B (en) The bacterial strain of one strain antagonistic phytopathogen and application thereof
CN106635870B (en) Bacillus and its application
Jha et al. Evaluation of Multispecies Plant-Growth-Promoting Consortia for the Growth Promotion of Jatropha curcas L.
Zeng et al. Use of Coniothyrium minitans and other microorganisms for reducing Sclerotinia sclerotiorum
CN106754484B (en) One plant of rhizobium leguminosarum and its fermentation culture method and application
Amaresan et al. Isolation and characterization of plant growth promoting endophytic bacteria and their effect on tomato (Lycopersicon esculentum) and chilli (Capsicum annuum) seedling growth
CN103184162B (en) Trichoderma asperellum and applications thereof
CN108271340A (en) For the compound endophyte composition and method by design of improved plant trait
CN101993836B (en) Bacillus subtilis strain YB-81, fungicide and preparation method and application thereof
CN101760438B (en) Broad-spectrum antifungal plant endophytic bacillus subtillis and application thereof
CN1952116A (en) Bacillusamyloliquefaciens strain and application thereof
CN101760439B (en) Bacillus pumilus NMCC 46 and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant