CN107164285B - A strain of Bacillus subtilis BBD012 and its application in controlling tomato diseases - Google Patents

Abstract

The invention relates to a bacillus subtilis BBD012, which is classified and namedBacillus subtilisThe preparation is Bacillus subtilis BBD012 culture solution, and the preparation method comprises inoculating Bacillus subtilis BBD012 single colony in L B liquid culture medium, and culturing in 28 deg.C constant temperature shaking culture box at 160r.min^‑1Culturing for 24h, and diluting bacterial colony of the cultured bacterial liquid to 4 × 10 with sterile water⁸cfu·mL^–1The control method is to spray the tomato plant with the culture solution of bacillus subtilis BBD 012. The bacillus subtilis BBD012 has good antagonistic effect on botrytis cinerea and phyllosticta cinerea, has the prevention and treatment effects on the botrytis cinerea and the phyllosticta cinerea of 64.9% and 58.0% respectively, and has good disease prevention effect.

Description

A strain of Bacillus subtilis BBD012 and its application in controlling tomato diseases

technical field

The invention relates to the technical field of microorganisms, in particular to Bacillus subtilis BBD012 and its application in preventing and treating tomato diseases.

Background technique

Tomato gray mold is a serious and common disease on tomatoes, which occurs in all vegetable areas. In addition to harming tomatoes, it can also harm more than 20 kinds of crops such as eggplant, pepper, cucumber, and gourd. Years with low temperature and continuous rainy weather are seriously harmful. When the disease is serious, the stems and leaves will die, and a large number of rotten flowers and rotten fruits will directly affect the yield. Tomato gray mold has become the main disease of tomato production in protected areas in my country. Gray mold is caused by the infection of the fungus Botrytis cinerea.

Tomato leaf mold disease is caused by the infection of tomato leaf mold fungus ( Fulvia fulva ), and symptoms will appear on the leaves, stems, flowers and fruits of tomato, but the common symptoms are on the leaves, and some regression occurs on the back of the leaves in the early stage. Green spots, which turn into gray or black-purple irregular mold layers in the later stage. The front of the leaves turns green and yellow at the corresponding parts. In severe cases, the leaves often dry and curl. From the perspective of the order of incidence, it often spreads upward from the lower part of the plant, and the temperature ranges from 9°C to 34°C. The pathogen can grow and develop, and the optimum temperature for development is 20 to 25°C. At the optimum temperature and high humidity (relative humidity above 80%), it only takes 10 days to half a month to develop the disease.

At present, there is no breakthrough in tomato disease-resistant breeding, and domestic control of tomato gray mold and leaf mold mainly relies on chemical agents. However, long-term and continuous drug use has made Botrytis cinerea and leaf mold resistant, resulting in a decline in the control effect of chemical agents year by year, and chemical residues seriously pollute agricultural products and the environment, endangering the health of humans and animals. Biological control has become an important means of disease control, and endophyte bacteria have unique advantages as potential resource bacteria for plant disease biological control, and have shown good prospects for utilization. Bacillus subtilis is a mesophilic, aerobic, spore-producing, gram-positive rod-shaped bacterium capable of producing endospores resistant to heat, drought, ultraviolet light and organic solvents, which inhibit the growth of plant pathogens. It has a wide range, including root diseases, branch diseases, leaf and flower diseases and diseases of harvest products. It is an ideal biocontrol microorganism.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Bacillus subtilis BBD012, which is classified and named as Bacillus subtilis BBD012, and the preservation number is CCTCC NO: M 2017097.

Another object of the present invention is to provide a preparation for controlling tomato gray mold and/or leaf mold.

The Bacillus subtilis BBD012 can be used for inhibiting tomato Botrytis cinerea and tomato leaf mold.

The preparation for controlling tomato gray mold and/or leaf mold is a Bacillus subtilis BBD012 culture solution.

The Bacillus subtilis BBD012 culture solution contained 4×10 ⁸ cfu·mL ⁻¹ of colonies.

The preparation method of the Bacillus subtilis BBD012 culture solution is as follows: inoculate a single colony of Bacillus subtilis BBD012 in a LB liquid medium, cultivate in a constant temperature shaking incubator at 28° C. for 160 r·min ^-1 for 24 hours, and use sterile water for culturing. After culturing, the colony of the obtained bacterial solution was diluted to 4×10 ⁸ cfu·mL ^{- 1} .

In the method for treating and preventing tomato gray mold and/or leaf mold with the preparation, the tomato plants are sprayed with the Bacillus subtilis BBD012 culture solution, that is, the Bacillus subtilis BBD012 culture solution is sprayed on the front and back of the tomato leaves.

beneficial effect

The Bacillus subtilis BBD012 of the present invention is screened from white lentil plants, and the strain forms a stable ecological relationship in the process of co-evolution with the host plant, and the endophytic bacteria can produce or induce the host to produce some antibacterial substances by itself, or by interacting with the host plant to produce some antibacterial substances. Plant pathogens compete for niches and nutrients to control plant diseases. Bacillus subtilis BBD012 has a good antagonistic effect on tomato botrytis cinerea and leaf mold, and the strain also has a certain antagonistic effect on three kinds of osmanthus osmanthus pathogens (Cyphaecystis sp., Vitis vinifera, anthracnose). The control effect of Bacillus BBD012 on tomato gray mold and leaf mold reached 64.9% and 58.0%, respectively, which had a good disease control effect.

Preservation of biological material

A strain of Bacillus subtilis BBD012, classified as Bacillus subtilis BBD012, has been deposited in the China Center for Type Culture Collection, the deposit unit is referred to as CCTCC, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2017097, deposited on March 8, 2017.

Description of drawings

Fig. 1 is the bacteriostatic effect diagram of Bacillus subtilis BBD012 of the present invention to tomato leaf mold;

Fig. 2 is the figure of the bacteriostatic effect of Bacillus subtilis BBD012 of the present invention to tomato botrytis cinerea;

In the figure: the left is the control, the right is the antibacterial effect.

Detailed ways

Example 1

Controlling potted tomatoes with preparations for preventing tomato botrytis:

Step 1. Inoculate a single colony of Bacillus subtilis BBD012 into 200 mL of LB liquid medium, cultivate it in a constant temperature shaking incubator at 28°C for 160 r.min ^-1 for 24 hours, and dilute the cultured bacterial solution to 4×10 ⁸ cfu.mL ^{– 1.} A preparation for preventing botrytis cinerea of tomato is prepared;

Step 2: Take potted tomatoes that have grown to 5-6 leaves and have the same growth pattern, spray the front and back sides of tomato leaves with preparations for treating and preventing tomato gray mold, and use clear water as a control. After 24 hours, the spore suspension of tomato gray mold 1×10 ⁷ spores/mL (inject a little sterile water into the PDA plate that has been covered with Botrytis cinerea, scrape off the spores on the surface of the agar, pour it into a sterile container, and filter it with sterilized gauze after fully shaking , counted in a hemocytometer) spray inoculated on it. Tomato plants were cultured in an artificial climate chamber (23°C, r≧90%). 3 pots per treatment, 3 plants per pot, repeated 3 times. 7 days after inoculation, the incidence of tomato was investigated, and the disease was graded according to the 5-level standard. Grade 0: no disease spots, no symptoms on the leaves, healthy plants; grade 1: the area of disease spots accounts for less than 10% of the total leaves; grade 2: the area of disease spots accounts for 10%-50% of the total leaves; grade 3: the area of disease spots More than 50% of the total leaves; Grade 4: Plant death.

Disease index=100×[Σ(number of diseased leaves at all levels×number of disease grades)]/(total number of leaves under investigation×4) Control effect (%)=[(control disease index-disease index after treatment with antagonistic strains)]/control Disease index]×100

The results showed that the disease index of the tomato plants treated with spray treatment and prevention of tomato gray mold was 23.1 lower than that treated with water, and the control effect was 64.9% (see Table 1).

Table 1 Control effect of Bacillus subtilis BBD012 on tomato gray mold

Example 2

Controlling Potted Tomatoes with Preparations for the Prevention of Tomato Leaf Mildew:

Step 1. Inoculate a single colony of Bacillus subtilis BBD012 into 200 mL of LB liquid medium, cultivate it in a constant temperature shaking incubator at 28°C for 160 r.min ^-1 for 24 hours, and dilute the cultured bacterial solution to 4×10 ⁸ cfu.mL ^{– 1.} A preparation for preventing botrytis cinerea of tomato is prepared;

Step 2: Take potted tomatoes that have grown to 5-6 leaves and have the same growth pattern, spray the front and back sides of tomato leaves with preparations for treating and preventing tomato gray mold, and use clear water as a control. After 24 hours, the spore suspension of tomato gray mold 1×10 ⁷ spores/mL (inject a little sterile water into the PDA plate that has been covered with Botrytis cinerea, scrape off the spores on the surface of the agar, pour it into a sterile container, and filter it with sterilized gauze after fully shaking , counted in a hemocytometer) spray inoculated on it. Tomato plants were cultured in an artificial climate chamber (23°C, r≧90%). 3 pots per treatment, 3 plants per pot, repeated 3 times. 7 days after inoculation, the incidence of tomato was investigated, and the disease was graded according to the 5-level standard. Grade 0: no disease spots, no symptoms on the leaves, healthy plants; grade 1: the area of disease spots accounts for less than 10% of the total leaves; grade 2: the area of disease spots accounts for 10%-50% of the total leaves; grade 3: the area of disease spots More than 50% of the total leaves; Grade 4: Plant death.

Disease index=100×[Σ(number of diseased leaves at all levels×number of disease grades)]/(total number of leaves under investigation×4) Control effect (%)=[(control disease index-disease index after treatment with antagonistic strains)]/control Disease index]×100

The results showed that the disease index of tomato plants inoculated with BBD012 strain was 17.7 lower than that treated with water, and the control effect was 58.0% (see Table 2).

Table 2: Control effect of Bacillus subtilis BBD012 on tomato leaf mold

Example 3

Screening, isolation and purification of Bacillus subtilis strain BBD012:

Step 1. Preparation of culture medium:

LB medium for bacterial plate culture (tryptone 10g/L; yeast extract 5g/L; sodium chloride 10g/L; agar powder 18g/L), LB liquid medium for liquid culture (without agar powder), fungi PDA medium for culture and plate confrontation test (potato 200g/L; glucose 20g/L; agar powder 18g/L). Step 2: Collect 3g of leaves of healthy white lentil plants, soak in 75% alcohol for 30s, then disinfect with 1% sodium hypochlorite for 3min, rinse with sterile distilled water for 5 times, and apply 200 μL of the last rinse solution to LB The sterilized leaves were ground in a sterile mortar, mixed with 5 ml of sterile distilled water, and 200 μL of the grinding liquid was taken and spread on the LB medium plate. Cultivate at 28°C and observe daily. For example, there are no colonies in the medium coated with the last rinse solution, and the colonies grown in the medium coated with the grinding solution are endophytes. After purification and culture, store them in a refrigerator at 4°C. spare.

Step 3. Determination of antagonistic activity

Tomato Botrytis cinerea, tomato leaf mold, osmanthus leaf spot fungus (thorn ball shell), osmanthus anthracnose fungus and osmanthus leaf spot fungus (Vitis vinifera ) were activated and cultured, and the 8mm cake was placed in the center of the PDA medium. Four 8mm filter paper sheets containing endophytic bacteria were placed at 40mm around, and cultured in a 28°C incubator for 4 to 5 days. The radius of the inhibition zone was measured and the inhibition rate was calculated.

The results showed that a total of 19 endophytic bacteria were isolated from the leaves of healthy white lentils. Among them, the isolated BBD012 had the strongest antibacterial activity, and the antibacterial rate against tomato leaf mold was 65.32%, Figure 1; The rate was 79.52%, Figure 2; it also had a certain degree of inhibition on the other three pathogens, Table 3.

Table 3 Bacteriostatic rate of fermentation broth of strain BBD012 against five pathogenic fungi

Step 4. Identification of strongly antagonistic endophytic strains

1. Morphological, physiological and biochemical identification

Methods refer to "Berger's Bacterial Identification Manual" (Buchanan & Gibbons, 1984) and "Common Bacterial System Identification Manual" (Dong Xiuzhu and Cai Miaoying, 1999).

1) Morphological features

The cells of strain BBD012 were rod-shaped, solitary, and periflagellated, and were Gram-positive. Colonies are white and opaque with irregular edges.

2) Physiological and biochemical characteristics

Strain BBD012 is aerobic and cannot exercise, can use sucrose, fructose, glucose, maltose, mannitol, but not lactose, can hydrolyze starch and gelatin, can decompose milk to produce acid, citrate test, V-P test, nitrate Reduction test and contact enzyme test were positive, phenylalanine dehydrogenase test, methyl red test and oxidase test were negative, the optimum growth pH was 7.0, and the optimum growth temperature was 30°C.

2. 16S rDNA and gyrA gene sequencing

The bacterial genome DNA rapid extraction kit (REF B518225-0050 of Shanghai Sangon Bioengineering Co., Ltd.) was used as the template for bacterial strain BBD012 genomic DNA. 16S rDNA PCR amplification was performed using primer 16SL: 5'-ACGGCTACCTTGTTACGACCT-3' (whose sequence is as SEQ ID NO: 1) and primer 16SR: 5'-AGAGTTTGATCCTGGCTCAG-3' (whose sequence is as SEQ ID NO: 2). The gyrA gene was amplified by PCR using primers gyrA-f: CGATCAGGGAAATGCGTACGTCCTT (its sequence is as SEQ ID NO: 3) and primer gyrA-r: CAAGGTAATGCTCCAGGCATTGCT (its sequence is as SEQ ID NO: 4)). Reaction system (50 μL): 10×buffer 5.0 μL, dNTPs ( 2.5 mmol·L ⁻¹ ) 4.0 μL, primer 16SR (10 pmol·L ⁻¹ ) 2.0 μL, primer 16SL ( 10 pmol·L ⁻¹ ) 2. 0 μL, Taq enzyme ( 5 U·μL ^-1 ) 0.5 μL, template DNA ( 50 ng·μL ^-1 ) 2 μL, ddH ₂ O 34.5 μL. PCR reaction program: 94°C for 5 min, 94°C for 1 min, 50°C for 1 min, 72°C for 2 min, 30 cycles; 72°C for 10 min. The PCR products were purified and then sent to Shanghai Sangon Bioengineering Company for sequencing. According to the sequencing results, the 16S rDNA sequence and the gyrA gene sequence of the relevant strains were called out from the GenBank database with Blast software, and the multiple sequence alignment was carried out with the ClustalWV1.82 software respectively, and then the sequence analysis was carried out with the Mega 4.1 software, and the neighbor-joining method was adopted. (Neighbor-joining, NJ method) for phylogenetic analysis and homology comparison.

The results showed that the 16S rDNA (gene sequence shown in SEQ ID NO: 5) and gyrA gene sequence (shown in SEQ ID NO: 6) of strain BBD012 had a homology of up to 99% with Bacillus subtilis . Combined with its morphological characteristics, physiological and biochemical characteristics, 16S rDNA and gyrA gene sequence analysis, the strain was identified as B. subtilis BBD012.

SEQUENCE LISTING

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Claims (5)

1. A bacillus subtilis BBD012, its characterized in that: classificationIs named as Bacillus subtilis (Bacillus subtilis) The culture medium is preserved in China center for type culture Collection, the preservation unit is CCTCC for short, and the preservation number is CCTCC NO: m2017097, preservation date 3, 8 days 2017.

2. A preparation for preventing and treating tomato gray mold and/or leaf mold is characterized in that the preparation is a culture solution of the bacillus subtilis BBD012 of claim 1, and the culture solution contains a bacterial colony 4 × 10⁸cfu·mL^{– 1}。

3. The preparation of claim 2, wherein the Bacillus subtilis BBD012 is prepared by inoculating a single colony of Bacillus subtilis BBD012 in L B liquid medium, and culturing at 28 deg.C for 160r min^-1Culturing for 24h, and diluting bacterial colony of the cultured bacterial liquid to 4 × 10 with sterile water⁸cfu·mL^{– 1}。

4. A method for the treatment and prevention of tomato gray mold and/or leaf mold using the formulation according to any one of claims 2-3, characterized in that: and (3) spraying the tomato plant by using a bacillus subtilis BBD012 culture solution.

5. The method of claim 4, wherein: spraying the culture solution of the bacillus subtilis BBD012 on the front and back surfaces of the tomato leaves.

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