CN104195061B - Bacillus subtilis and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology, and in particular relates to a bacillus subtilis and an application thereof. The Bacillus subtilis is Bacillus subtilis HS5B5, which is preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms, with the preservation number: CGMCC No.6088. The bacillus subtilis HS5B5 of the present invention has a strong inhibitory effect on the pathogenic bacteria Cladosporium peony of the tree peony brown spot, and can effectively prevent and treat the tree peony brown spot. The bacillus subtilis HS5B5 of the present invention is harmless to the ecological environment and will not cause drug resistance of pathogens. The culture condition of the bacillus subtilis HS5B5 of the invention is simple, easy to preserve, easy to industrialized production, and has good development and application prospects.

Description

A kind of bacillus subtilis and application thereof

technical field

The invention belongs to the field of biotechnology, and in particular relates to a bacillus subtilis and an application thereof.

Background technique

There are few pests on peony, but there are many types of diseases and serious damage. The disease is that peony is infected by pathogenic microorganisms or affected by adverse environmental conditions during its growth and overwintering process, causing a series of pathological changes from physiological mechanism to morphological structure. Among the more than 20 kinds of peony diseases that have been identified, most of them are fungal diseases, such as brown spot, gray mold, rust, white silkworm and purple feather disease. They cause partial necrosis of leaves or the death of whole leaves, harming the leaves, branches and rhizomes of peony, causing the peony to fall leaves in advance and affecting the vitality. Peony brown spot is one of the most serious diseases. This disease mainly damages leaves, and can also infect stems and flowers. It mostly occurs before and after flowering, and continues until the dead leaves. The pathogenic fungus is Cladosporium paeoniae , which belongs to the subphylum Deuteromycota, the class Hyphomycetes, the order Polyphyllales, and the genus Cladosporium. The pathogen survives the winter in dead leaves or soil with mycelium or conidia, spreads by wind and rain, and begins to occur in early summer. In autumn, the damage is serious, high temperature and high humidity, insufficient light, poor ventilation, and continuous cropping are all conducive to the occurrence of the disease.

The prevention and treatment of peony brown spot mainly depends on the use of chemical drugs, which is not only harmful to human health and ecological environment safety, but also easily causes pathogen resistance. Biological control is a prevention and control method that uses organisms or their products to control the occurrence of agricultural and forestry diseases and insect pests. Because it is safe for humans and animals, has no pollution to the environment, is not easy to produce drug resistance, has high efficiency and broad spectrum, and can promote crop growth, it is increasingly favored by people. However, the research and application of biological control in the defense of peony diseases is still very little. In order to accelerate the rapid development of my country's peony industry, it is imminent to develop high-efficiency and broad-spectrum biological control agents that are harmless to human health and ecological environment, and are not easy to cause pathogenic drug resistance.

Contents of the invention

The object of the present invention is to provide a kind of bacillus subtilis capable of inhibiting the causative bacterium Cladosporium paeoniae of tree peony brown spot.

The technical solution adopted by the present invention is: a Bacillus subtilis, said Bacillus subtilis is Bacillus subtilis HS5B5, which was preserved at No. 1 Beichen West Road, Chaoyang District, Beijing on May 9, 2012 General Microbiology Center of China Committee for Culture Collection of Microorganisms No. 3, the preservation number is: CGMCC No. 6088.

The colonies of the Bacillus subtilis HS5B5 strain on the beef extract peptone agar medium are white, rod-shaped, dry and wrinkled on the surface, flagella are middle-grown, the colony diameter is 0.8-2.3 μm, and Gram staining is positive.

The 16S rDNA sequence of the Bacillus subtilis HS5B5 strain is shown in SEQ ID NO:1.

The Bacillus subtilis ( Bacillus subtilis ) HS5B5 strain is screened by a confrontation culture method.

The antagonism and antibacterial effect of the bacillus subtilis ( Bacillus subtilis ) HS5B5 strain on the pathogen Cladosporium paeoniae of peony brown spot.

Application of the Bacillus subtilis ( Bacillus subtilis ) HS5B5 strain preparation in preventing and treating brown spot disease of peony.

The application of the bacillus subtilis ( Bacillus subtilis ) HS5B5 strain to chrysanthemum verticillium wilt, apple powdery mildew, oleifera blight, and rose black spot.

The beneficial effects of the present invention are:

1. The Bacillus subtilis ( Bacillus subtilis ) HS5B5 of the present invention has a strong inhibitory effect on the pathogen Cladosporium peony, the pathogen of tree peony brown spot, and can effectively prevent and treat tree peony brown spot.

2. The Bacillus subtilis HS5B5 of the present invention is harmless to the ecological environment and will not cause drug resistance of pathogens.

3. The culture condition of Bacillus subtilis HS5B5 of the present invention is simple, easy to preserve, easy to industrialized production, and has good development and application prospects.

Description of drawings

Figure 1 is the inhibition zone of Bacillus subtilis ( Bacillus subtilis ) HS5B5;

Fig. 2 is the colony characteristic of Bacillus subtilis ( Bacillus subtilis ) HS5B5.

detailed description

The present invention screens and isolates Bacillus subtilis HS5B5, which is preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms, with a preservation number of CGMCC No. 6088. The bacterial strain has a strong inhibitory effect on the pathogenic bacteria Cladosporium paeoniae of tree peony brown spot, and can be used for preparing biological preparations for preventing and treating tree peony brown spot.

1. Screening and isolation method of strains

1. Isolation of Bacillus in samples

(1) In the peony cultivation area of Luoyang International Peony Garden, the surface soil of peony roots was collected, and 100 samples were collected at different locations, bagged and brought back to the laboratory for air-drying. After the air-dried samples are ground, weigh 10g from each sample and add them to a triangular flask filled with 90mL sterile water and 5 glass beads, shake fully for 20-30min, so that the sample and sterile water are evenly mixed. , to prepare a sample suspension.

(2) Heat the prepared sample suspension in a water bath at 80°C for 10 minutes to kill bacteria and other microorganisms that do not produce spores, and then pipette 1 mL of the sample suspension into a container containing In a test tube of 9mL sterile water, shake and mix to prepare 10 ^-2 sample dilution.

The ^dilutions of 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and 10 ^-7 were sequentially prepared according to the above method ^{, and} 0.1 mL was spread on the corresponding beef extract peptone plate, and cultured upside down at 30°C for 48h. According to the characteristics of the colonies, the colonies with obvious differences in morphology and other traits were picked, and the colonies were repeatedly streaked and purified, and numbered and stored for later use.

2. Screening of antagonistic bacteria against peony brown spot disease

For the screening of antagonistic bacillus, Cladosporium paeoniae , the pathogen of peony brown spot disease, was used as the target, and the method was the confrontation culture screening method.

Cladosporium paeoniae used for screening was purchased from China Agricultural Microorganism Culture Collection Management Center, and the strain preservation number is ACCC 36063.

Cladosporium peony was inoculated in the center of the PDA plate, cultured at 28°C for 8 days, then inserted into the Bacillus strains obtained from the preliminary screening, and cultured at a constant temperature of 28°C for 3 days to observe the antibacterial situation, measure the diameter of the inhibition zone, and repeat 3 times for each group of experiments . According to the size of the antibacterial diameter, the strains with obvious antibacterial effect were selected, and transferred to beef extract peptone slant medium for storage for later use.

3. Bacteria identification method

(1) Observation of morphological characteristics

The observation of morphological characteristics includes the observation of colony morphology and the observation of individual bacterial morphology, mainly using the experimental methods in the "Common Bacterial System Identification Manual".

①Observation of colony morphology

Stratify the bacteria on the plate for 24 hours, observe and record the colony shape, texture, color, edge of the colony, optical properties, and secretion of soluble pigments.

②Observation of individual form

Gram staining: use strains with a bacterial age of 10-14 hours. After smearing, first stain with crystal violet, then mordant with iodine solution, decolorize with 95% ethanol, then counterstain with safranin, wash with water, and blot dry. Gram-positive bacteria are purple in microscopic examination, and Gram-negative bacteria are red in color.

Spore staining: After smearing, stain with malachite green for 15 minutes, rinse with water, air-dry, counterstain with safranin for 2 minutes, wash with water, and blot dry. Microscopic examination showed that the spores were green, and the bacteria and cysts were reddish.

Measurement of the cell size: use a micrometer to measure the cell size on Gram-stained smears, measure the width and length of more than 10 cells for each bacterial strain, and the average value is the general size of the cell.

(2) Physiological and biochemical identification

Physiological and biochemical identification In accordance with the methods in the "Common Bacteria System Identification Manual" and "Bacillus", each strain was subjected to contact enzyme reaction, aerobic test, methyl red reaction, V.P reaction test, nitrate reduction test, Identification of physiological and biochemical characteristics such as sugar alcohol fermentation test, indole production test, citrate utilization test, starch hydrolysis test, casein hydrolysis test, hydrogen sulfide production test and salt tolerance test.

(3) 16S rDNA sequence analysis

Bacterial genomic DNA was extracted using a bacterial genome extraction kit as a template for PCR reactions. Primers are universal primers for full-length amplification of 16S rDNA. 30 μL of PCR amplification reaction system: 15 μL of PCR premix, 1 μL of template DNA, 0.5 μL of each primer, 13 μL of double distilled water. PCR reaction conditions: 95°C for 3min; 95°C for 50s, 52°C for 50s, 72°C for 2min, 30 cycles; 72°C for 10min. The PCR product was recovered with a PCR gel recovery kit, detected by 1.5% gel electrophoresis, ligated with the cloning vector pGM-T under the action of T4 ligase at 16°C overnight, and transformed into Escherichia coli DH5α competent cells. The primers and sequencing of positive clones were completed by Shanghai Sangon Bioengineering Co., Ltd. Using Blast, the 16S rDNA sequence of the strain was compared with the known sequence in the GenBank database to analyze its homology.

2. Screening and separation results

1. Isolation and purification of Bacillus in the sample

From the samples, a total of 79 strains of bacteria were obtained through dilution, separation and purification, numbered and preserved respectively, and used for screening of antagonistic bacteria. Preliminary microscopic examination with a microscope found that 68 strains were bacillus, accounting for 86.1% of the total number of bacteria obtained. This shows that the sample suspension was first heated in a water bath at 80°C for 10 minutes before dilution and separation in the test. It can effectively kill the microbial cells in the sample, so as to obtain the bacillus to the maximum extent, and is suitable for the isolation of the bacillus.

2. Screening of antagonistic bacteria for the pathogen of peony brown spot disease

The pathogenic bacteria Cladosporium paeoniae of peony brown spot was used as the target, and the confrontation culture method was used to screen the antagonistic bacillus based on the antibacterial effect, that is, the size of the inhibition zone. The results showed that among the 68 strains of bacillus isolated from the samples, 11 strains had inhibitory effect on peony brown spot disease, accounting for about 16.2% of the total, of which 1 strain had an inhibition zone diameter of more than 6mm, which was bacteriostatic. The diameter of the circle is 6.18mm, as shown in Figure 1, named HS5B5, and the microbial strains were identified.

3. Observation results of individual and colony morphology

It can be seen that the bacterium of strain HS5B5 is rod-shaped, Gram staining is positive, mesospores, perinatal flagella, and from the irregular, dry, opaque characteristics of the colony, it can be preliminarily inferred that the strain HS5B5 may belong to spores Bacillus ( Bacillus ).

Table 1 Morphological characteristics of strain HS5B5

4. Identification results of physiological and biochemical characteristics

HS5B5 is a strict aerobic bacteria, methyl red reaction and VP reaction are both positive, all have catalase, nitrate reductase, starch hydrolase and casein hydrolase, can use citrate as the only carbon source, all have It can ferment glucose and mannitol to produce acid, and can grow under the conditions of 10% NaCl, 7% NaCl, 5% NaCl, and 2% NaCl salt concentration, and can grow at 45°C but not at 65°C. Its physiological and biochemical characteristics are the same as the standard strain of Bacillus subtilis. According to the morphological characteristics and physiological and biochemical characteristics of the strain HS5B5, and referring to the "Common Bacterial System Identification Manual" and "Bacillus", it can be determined that the antagonistic strain HS5B5 belongs to the genus Bacillus .

Table 2 Physiological and biochemical characteristics of strain HS5B5

Note: + indicates positive reaction; - indicates negative reaction.

16S rDNA sequencing results

Using the bacterial genome as a template, after PCR amplification, the product was detected by 1.5% agarose gel electrophoresis and a fluorescent band of about 1500bp appeared. According to the sequencing of the target region of the plasmid of the positive clone, the size of the 16S rDNA fragment is 1470bp, and the 16S rDNA sequence is shown in SEQ ID NO:1. The results of sequence analysis showed that it was the closest to the known strain Bacillus subtilis L520 (FJ906822.1) and other strains, with a homology of 93%. Combined with the results of strain individual morphology, colony morphology, and physiological and biochemical identification, it can be determined that the strain HS5B5 is Bacillus subtilis .

3. Inhibitory effect of strain HS5B5 on peony brown spot

On May 20, 2013, 200 peonies in the peony cultivation area of Luoyang International Peony Garden were selected as the test objects, and the peonies were divided into two groups A and B, and the peony seedlings in the two groups were inoculated with the same amount of peony Cladosporium, then group A was sprayed with Bacillus subtilis HS5B5 bacterial solution every two weeks; group B was sprayed with water every two weeks.

Inspection results on July 20, 2013: only 4 tree peonies in group A had brown spot disease, and 92 tree peonies in group B had brown spot disease.

The results show that: Bacillus subtilis HS5B5 in the present invention can effectively inhibit the generation of brown spot of peony, and the inhibition rate reaches 91.67%.

4. Antagonistic effect of Bacillus subtilis HS5B5 on various pathogenic bacteria

Using the confrontation culture method, use a puncher with a diameter of 6mm to take bacterial blocks from the edge of the cultivated colonies of chrysanthemum verticillium wilt, apple powdery mildew, oil tea leaf blight, and rose black spot, and inoculate the bacterial blocks on the PDA plate In the center, Bacillus subtilis ( Bacillus subtilis ) HS5B5 was inoculated at the same distance from the center of the plate, repeated three times, and then cultured in an incubator with a temperature of 28°C to calculate the growth inhibition rate of pathogenic bacteria. The results are shown in Table 3.

Table 3 Inhibitory effect of Bacillus subtilis HS5B5 on various pathogenic bacteria

The results show that: Bacillus subtilis HS5B5 in the present invention can effectively inhibit chrysanthemum verticillium wilt, apple powdery mildew, oleifera blight, and rose black spot.

SEQUENCE LISTING

<110> Henan University of Science and Technology

<120> A Bacillus subtilis and its application

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<160> 1

<170> PatentIn version 3.3

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<212>DNA

<213> Bacillus subtilis

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Claims (4)

1. A Bacillus subtilis, characterized in that: said Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis ) HS5B5. . 2. the application of a kind of bacillus subtilis as claimed in claim 1 in suppressing the pathogenic bacteria Cladosporium paeoniae of tree peony brown spot. 3. the application of a kind of bacillus subtilis as claimed in claim 1 in the preparation for preventing and treating brown spot disease of peony. 4. the application of a kind of bacillus subtilis as claimed in claim 1 in suppressing Verticillium wilt of chrysanthemum, apple powdery mildew, Fusarium oleifera, black spot of rose.