CN108913622A - The preparation and application of a kind of bacillus megaterium BM22 and its gemma pulvis - Google Patents

Abstract

The invention discloses the preparation and application of a kind of bacillus megaterium BM22 and its thallospore pulvis, which was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 15th, 2017, and deposit number is CGMCC No.15070.Using bacillus megaterium BM22 as strain, through seed culture, fermented and cultured and kieselguhr adsorption at gemma pulvis.Bacillus megaterium BM22 provided by the invention has special efficacy to photinia glabra red spot disease, and there is resistance, Storage is good, resistant to high temperatures, low temperature, resistance to drying, it is suitable for merchandized handling, it is not only avoided that the serious consequence for polluting environment caused by chemicals, but also has the characteristics that sustainable, at low cost.

Description

Preparation and application of a kind of bacillus megaterium BM22 and its spore powder

technical field

The invention belongs to the technical field of microbes, and in particular relates to a bacillus megaterium BM22 strain and its spore powder and its application in the prevention and treatment of Photinia erythema.

Background technique

Photinia (scientific name: Photinia × fraseri Dress) is a hybrid of Rosaceae and Photinia. It is an evergreen small tree or shrub. The height of the tree can reach 5 meters, and the height of the shrub can reach 2 meters. The crown is spherical, the leaves are leathery, oblong to obovate, lanceolate, the leaf tip is acuminate, the leaf base is wedge-shaped, the leaf margin has serrated glands, the flowers are many and dense, compound corymbs, white flowers , pome fruit yellow-red, blooming from May to July, and from September to October. It is mainly distributed in the subtropical and temperate regions of southeastern and eastern Asia and North America, and has been widely cultivated in many provinces of China. Photinia fragrans can be used as a street tree, and its pole stands like a torch; as a hedge, its shape is lying like a fire dragon; when pruned and landscaped, its shape can be in various poses and with different expressions, and the landscape effect is beautiful. Photinia is named for its bright red shoots and young leaves. There are two common varieties, red robin and red lip, among which red robin has bright and eye-catching leaves and better ornamental value. In spring and autumn, the new shoots and young leaves of Photinia fragrans are fiery red, with bright and lasting colors and great vitality. During the high temperature season in summer, the leaves turn bright green, giving people a fresh and cool feeling. Photinia is named for its bright red shoots and young leaves.

Photinia red spot disease [Cercospora eriobotryae (Enjojii) Saw.], mainly damages leaves. Lesions on leaves are round to irregular in shape, dark red in size 2-15mm, gray in the center, and dark red in the edge. In the later stage, many black dots are produced on the front of the leaves, which are the fruiting bodies of the pathogenic fungus. Most of the pathogens survive the winter on dead leaves, and the conidia in the following spring are transmitted by airflow for initial infection and re-infection. From July to September every year, it enters the peak period of onset. Generally, it is prone to disease during rainy season or high temperature and humidity. The disease is generally controlled by chemical methods, but since the long-term application of chemical pesticides is easy to pollute the environment and damage the ecology, the discussion of comprehensive disease control measures has yet to be carried out.

Contents of the invention

The technical problem to be solved by the present invention is to provide the application of Bacillus megaterium BM22 and its spore powder in the prevention and treatment of Photinia erythema in view of the defects of the prior art in the prevention and treatment of Photinia erythema.

The present invention is specifically realized through the following technical solutions:

The Bacillus megaterium (Bacillus megaterium) BM22 strain of the present invention was isolated on June 15, 2017 from the healthy leaves of Eucommia ulmoides in Yangtianwo Pharmacy, Dayi, Sichuan, and was preserved in the Chinese Academy of Sciences on December 15, 2017. The General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (CGMCC), Institute of Microbiology, is located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.15070.

The single colony of Bacillus megaterium BM22 of the present invention cultured on the NA plate medium for 2 days is milky white, round, rough in surface, corrugated in edge, opaque and non-sticky.

The 16S rDNA sequence of Bacillus megaterium BM22 of the present invention is shown in SEQ ID NO.1.

The spore powder with bacillus megaterium as the active ingredient of the present invention uses bacillus megaterium BM22 as the strain to obtain the spore powder through seed cultivation, fermentation cultivation and diatomite adsorption.

The spore powder containing Bacillus megaterium BM22 in the present invention contains no less than 1×10 ¹⁰ cfu/g of live spores.

The spore powder with Bacillus megaterium BM22 of the present invention as bacterial species is prepared by the following method:

1) First-class slant seeds: make beef extract peptone slant medium, inoculate Bacillus megaterium, and cultivate to make first-class seeds;

2) Secondary liquid seeds: After the nutrient fleshy culture liquid is sterilized by high pressure, it is filled in a triangular flask under the control of oxygen ventilation, and the slanted seeds of Bacillus megaterium are inoculated in a sterile state, and cultured by shaking to make liquid seeds of Bacillus megaterium;

3) After the culture solution was sterilized by high pressure, it was stored in a triangular flask under the control of ventilation, and the liquid seed of Bacillus megaterium was inoculated in a sterile state. The inoculation amount was 15% of the total volume of the liquid. For the supernatant, absorb the spores at the bottom of the centrifuge cup with diatomaceous earth, and store it in a 4°C refrigerator for later use.

The composition of the nutrient succulent culture solution is: 7g of beef extract, 10g of peptone, 5g of NaCl, and 1000mL of soaking solution of Photinia fragrans leaves.

The culture solution is: 0.5-0.6% of corn flour, 0.08-0.2% of corn starch, 0.9-1.2% of bran, 0.18-0.54% of glucose, 0.05-0.15% of soybean cake powder, 0.2-0.5 _% of soybean powder, KH2 PO ₄ 0.005～0.006%, (NH ₄ ) ₂ SO ₄ 0.006～0.008%, MnSO ₄ 0.004～0.005%, CaCO ₃ 0.0005～0.002%, MgSO ₄ 7H ₂ O 0.005～0.006%, and the balance red leaves Heather leaf impregnation solution, pH 7.0-7.5.

The spore powder containing Bacillus megaterium BM22 obtained by the above preparation method is also within the protection scope of the present invention.

For the application of the spore powder in the treatment of Photinia erythema, the powder is prepared at a concentration of 1×109cfu/mL, and then diluted for administration.

The application of the bacillus megaterium BM22 of the invention in the prevention and treatment of Photinia erythema.

The application of the Bacillus megaterium BM22 spore powder of the present invention in the prevention and treatment of Photinia erythema is applied by dusting at the seedling stage or during plant growth.

The method for preventing and treating Photinia erythema with Bacillus megaterium BM22 of the present invention also belongs to the scope of protection of the present invention, and the method is applied by spraying powder/mist during the seedling stage or growth process of Photinia spp.

The beneficial effects of the present invention are:

The Bacillus megaterium BM22 provided by the present invention has a special effect on Photinia erythema, and the test shows that the inhibition rate on Photinia erythema reaches 98%. The Bacillus megaterium BM22 is made into a spore powder, which also has a good control effect. It is suitable for commercial production. It can not only avoid the serious consequences of environmental pollution caused by chemical drugs, but also has the characteristics of stress resistance, good storage performance, high temperature resistance, low temperature resistance, and drying resistance. It is also in line with the national sustainable development strategy. Compared with chemical drugs, it also has the characteristics of low cost and has wide application prospects.

Description of drawings

Fig. 1 is the electrophoresis figure of the PCR amplification product of Bacillus megaterium BM22rDNA-ITS sequence of the present invention;

Fig. 2 is a phylogenetic tree of strain BM22 constructed based on 16S rDNA sequence.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and is not intended to limit the present invention to other forms. The contents are changed or modified into equivalent embodiments with equivalent changes. Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the solution content of the present invention all fall within the protection scope of the present invention.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Isolation and Identification of Bacillus megaterium BM22

1.1 Isolation, purification and preservation of endophytic bacteria

Healthy leaves of Eucommia ulmoides were collected from the black spot occurrence area of Eucommia ulmoides in Dayi, Sichuan. Rinse the collected samples with sterile water, weigh 1.0g of tissue, soak in 70% alcohol for 1-2min, then disinfect with 1-3% sodium hypochlorite solution for 3-5min, wash several times with sterile water, absorb Apply 100 μL of washing liquid for the last time to NA plate (3g beef extract, 10g peptone, 5g sodium chloride, 15-20g agar powder, 1000mL distilled water, pH 7.0, after mixing and dispensing, autoclave at 121°C for 30min), culture in the dark at 27°C 24h, used as a control for surface disinfection, to check whether the surface disinfection is thorough.

Cut the surface-sterilized bark tissue into pieces, put them into a sterile mortar, add sterilized quartz sand and 10mL sterile water, grind them thoroughly, let stand for 30min, dilute 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , a total of 5 gradients, absorb 100 μL of 10 ^-3 , 10 ^-4 , 10 ^-5 gradient grinding solution and spread them on NA plates (3g of beef extract, 10g of peptone, 5g of sodium chloride , agar powder 15-20g, distilled water 1000mL, pH 7.0, after mixing and dispensing, autoclave at 121°C for 30min), repeat each treatment 3 times, incubate at 27°C for 48h, pick single colonies with obvious differences in colony morphology and enter the primary screening .

According to the differences in colony size, color, protrusion, edge characteristics, surface smoothness, transparency, etc., pick a single colony and streak it on the NA plate for purification. A total of 36 endophytic bacteria were obtained.

1.2 Plate antagonism of endophytic bacteria on Cercospora eriobotryae (Enjojii) Saw.

Plate confrontation method: inoculate 7 days of Cercosporaeriobotryae (Enjojii) Saw. bacteria cake (diameter 6mm) in the center of the petri dish, about 3cm away from the bacteria cake, and use the inoculation loop to dip the endophytic bacteria cultured for 2 days respectively , draw a thin line on each symmetrical side of the bacteria, culture at 28°C, take the plate only inoculated with the bacteria as a control, measure the colony growth diameter of the bacteria when the plate is full of the control, and repeat 3 times for each treatment. Calculate the colony growth inhibition rate according to the following formula:

Colony growth inhibition rate = (net growth diameter of control colony - net growth diameter of treated colony) / net growth diameter of control colony × 100%.

Table 1 shows the strains that have good antagonistic effect on Photinia eriobotryae (Enjojii) Saw. among the 32 strains of endophytic bacteria, and the inhibition rate of BM22 reaches 98%.

Table 1 Antagonistic effect of endophytic bacteria on Photinia erythema (7d)

1.3 Identification of endogenous antagonistic bacteria

The endophyte BM22 was identified as Bacillus megaterium by morphological observation combined with physiological and biochemical indicators (Table 2) and molecular biology.

Table 2 Physiological and biochemical indicators of strain BM22

The single colony of the BM22 strain cultured on the NA plate medium for 2 days was milky white, round, with rough surface, corrugated edges, opaque and non-sticky.

Molecular biology identification (16S rDNA): PCR amplification and electrophoresis were carried out by extracting bacterial DNA, and the recovered product was sent to a biotechnology company for sequencing; homology BLAST analysis was performed between the measured sequence and the sequence reported in the GenBank database, and Clustalx (1.83) software for multiple sequence comparisons, and then use the neighbor-joining method in Mega4.0 software to construct a phylogenetic tree to determine the status of BM22 strain in microbial phylogeny. A DNA fragment with a length of 949bp was identified by molecular biology (Figure 1). The 16S rDNA sequence of the BM22 strain was submitted to the GenBank database for BLAST analysis, and the bacterial 16S rDNA sequence with high homology was selected, and multiplexed with Clustalx software. For matching and permutation analysis, a phylogenetic tree was constructed using Mega analysis software (Fig. 2). It can be seen that the BM22 strain and JX286698 are clustered into one branch with 99% 16SrRNA gene nucleotide sequence similarity and high bootstrap value, and are far away from other bacillus, indicating that BM22 is related to Bacillus megaterium The closest relationship.

Based on the above characteristics, the above-mentioned BM22 strain is classified as Bacillus megaterium BM22, and it has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, Institute of Microbiology, Chinese Academy of Sciences on December 15, 2017, and the preservation number is CGMCC No. .15070.

The preparation of embodiment 2 bacillus megaterium BM22 spore powder

The bacillus megaterium BM22 obtained according to embodiment 1 is made into spore powder preparation through seed culture and fermentation culture, specifically through the following methods:

The conventional method is to prepare beef extract peptone slant medium (beef extract 5g, peptone 10g, NaCl 3.5g, agar 18g, water 1000mL), inoculate Bacillus megaterium, and cultivate to make first-class seeds.

Nutritious succulent culture solution (beef extract 7g, peptone 10g, NaCl 5g, magnolia officinalis root bark soaking solution 1000mL) was autoclaved, and under oxygen control, bottled according to the proportion of 100mL liquid in a 300mL triangular flask, Inoculate Bacillus megaterium slant seeds in the state, inoculate 2 slant seeds per bottle, vibrate and cultivate, and make Bacillus megaterium liquid seeds. Wherein, 10 g of Photinia fragrans leaves are boiled in 1000 mL of water for 10 minutes, filtered, and the obtained filtrate is impregnation solution of Photinia frondosa leaves.

Liquid fermentation and diatomite adsorption into powder: corn flour 0.5-0.6%, corn starch 0.08-0.2%, bran 0.9-1.2%, glucose 0.18-0.54%, soybean cake powder 0.05-0.15%, soybean flour 0.2-0.5% , KH ₂ PO ₄ 0.005～0.006%, (NH ₄ ) ₂ SO ₄ 0.006～0.008%, MnSO ₄ 0.004～0.005%, CaCO ₃ 0.0005～0.002%, MgSO ₄ 7H ₂ O 0.005～0.006%, and the balance water (leaf impregnation solution of Photinia frondosa), pH 7.0-7.5. After the culture solution is sterilized by high pressure, it is stored in a triangular flask under the control of oxygen ventilation, and the liquid seed of Bacillus megaterium is inoculated in a sterile state. The inoculation amount is 15% of the total volume of the liquid. After 4 days of shaking culture, centrifuge for 10 minutes, and discard the supernatant solution, absorb the spore spores at the bottom of the centrifuge cup with diatomaceous earth, and store them in a 4°C refrigerator for later use.

Preservation of preparations: The bottled preparations can be stored at room temperature for 1 year or at low temperature (4°C) for 2 years without affecting the preventive and therapeutic effects.

The powder containing Bacillus megaterium BM22 prepared by the above method contains no less than 1×10 ¹⁰ cfu/g of live spores.

When the bacterial agent is used, the spore powder preparation dilution is sprayed (powdered) on the seedlings.

Example 3 Bacillus megaterium BM22 spore powder preparation spray method control test

According to the occurrence degree of erythema, it is divided into 4 occurrence areas (I: 5-10%; II: 11-30%; III: 31-60%; IV: >60%). In 4-5 months, the inoculum prepared in Example 2 adopts the concentration of spray method: 50 times of dilution, 100 times of dilution, 200 times of dilution, 400 times of dilution, 800 times of dilution, 1600 times of dilution, 2400 times of dilution to red leaves The leaves of Photinia were treated, sprayed evenly along the leaves, 100mL per plant, and sterile water was used as a control, repeated 3 times, and the incidence of Photinia fragrans was counted. 20 plants per treatment.

15 days after treatment, conduct disease investigation and statistics according to the disease grading standards in Table 3, and calculate the control effect.

Table 3 Grading standard of Photinia erythema

Incidence rate (%)=number of diseased plants/total number of plants×100;

Disease index=[Σ(number of disease-grade plants×representative series)/(total number of plants×highest representative series of disease)]×100;

Control effect (%)=(control disease index-treatment disease index)/control disease index×100.

Table 4 shows the control effect of BM22 endophytic bacteria after 15 days of spraying test, and the biocontrol bacteria showed good control effect on Photinia fragrans anthracnose. The control effect of the 50-fold dilution in the four types of occurrence areas is more than 80%, which can basically control the spread of the disease. The control effect decreases with the increase of the dilution multiple, and the control effect varies greatly when the dilution is 1600-2400 times. Generally speaking, when the disease occurs below 10%, 800 times of liquid is the economic concentration, when the disease occurs 10-30%, 400 times of the liquid is the economic concentration, when the disease occurs 30-60%, 200 times of the liquid is the economic concentration. When greater than 60%, the economic concentration is 100 times.

Table 4 The effect of BM22 spraying on the prevention and treatment of Photinia erythema after 15 days (%)

concentration I (5-10%) II (11-30%) III (31-60%) IV>60% 50 97.1a 95.1a 84.1a 81.4a 100 95.7a 91.4b 78.2b 70.2b 200 89.1b 85.1c 70.2c 51.1c 400 87.5b 73.2d 60.1d 40.8d 800 74.1c 60.3e 38.1e 21.5e 1600 55.1d 42.1f 18.5f 9.8f 2400 21.1e 12.3g 8.2g 1.2g

In general, the Bacillus megaterium BM22 strain isolated by the present invention has special effects on Photinia erythema, has strong environmental suitability and good affinity, is suitable for commercial production, meets the strategic requirements of sustainable development, and has broad market prospects.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Sichuan Agricultural University

<120> Preparation and application of a kind of Bacillus megaterium BM22 and its spore powder

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caggctgcgg ggctatacat gcagtcgagc gaactgatta gaagcttgct tctatgacgt 60

tagcggcgga cgggtgagta aacacgtgggc aacctgcctg taagactggg ataacttcgg 120

gaaaccgaag ctaataccgg ataggatctt ctccttcatg ggagatgatt gaaagatggt 180

ttcggctatc acttacagat gggcccgcgg tgcattagct agttggtgag gtaacggctc 240

accaaggcaa cgatgcatag ccgacctgag agggtgatcg gccaacactgg gactgagaca 300

cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360

cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt tgttagggaa 420

gaacaagtac gagagtaact gctcgtacct tgacggtacc taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540

gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600

gggtcattgg aaactgggga acttgagtgc agaagagaaa agcggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcggctttt tggtctgtaa 720

ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaaga 949

Claims (10)

1. A Bacillus megaterium BM22, which is characterized in that the strain has been preserved, and the depository unit is: General Microorganism Center of China Committee for the Collection of Microorganisms, and the preservation number is CGMCC No.15070. 2. A kind of Bacillus megaterium BM22 according to claim 1, characterized in that, the 16S rDNA sequence of said Bacillus megaterium BM22 is as shown in SEQ ID NO.1. 3. a kind of preparation method taking bacillus megaterium BM22 as the spore powder of active ingredient, is characterized in that, comprises the following steps: 1) First-class slant seeds: make beef extract peptone slant medium, inoculate Bacillus megaterium, and cultivate to make first-class seeds; 2) Secondary liquid seeds: After the nutrient fleshy culture liquid is sterilized by high pressure, it is filled in a triangular flask under the control of oxygen ventilation, and the slanted seeds of Bacillus megaterium are inoculated in a sterile state, and cultured by shaking to make liquid seeds of Bacillus megaterium; 3) After the fermentation culture liquid is sterilized by high pressure, it is stored in a triangular flask under the control of ventilation, and the liquid seed of Bacillus megaterium is inoculated in a sterile state. The inoculation amount is 15% of the total volume of the liquid. For the supernatant, absorb the spores at the bottom of the centrifuge cup with diatomaceous earth, and store it in a 4°C refrigerator for later use. 4. The preparation method according to claim 3, characterized in that, the fermentation medium is: 0.5-0.6% cornmeal, 0.08-0.2% cornstarch, 0.9-1.2% bran, 0.18-0.54% glucose, Soybean cake powder 0.05～0.15%, soybean powder 0.2～0.5%, KH ₂ PO ₄ 0.005～0.006%, (NH ₄ ) ₂ SO ₄ 0.006～0.008%, MnSO ₄ 0.004～0.005%, CaCO ₃ 0.0005～0.002%, MgSO ₄ ·7H ₂ O 0.005-0.006%, and the balance of water, pH 7.0-7.5. 5. The preparation method according to claim 3, wherein the composition of the beef extract peptone slant medium is: beef extract 5g, peptone 10g, NaCl 3.5g, agar 18g, and water 1000mL. 6 . The preparation method according to claim 3 , wherein the composition of the nutrient succulent culture solution is: 7 g of beef extract, 10 g of peptone, 5 g of NaCl, and 1000 mL of soaking solution of Photinia fruticosa leaves. 7. the described preparation method of claim 3 prepares with Bacillus megaterium spore powder preparation. 8. The spore powder preparation according to claim 7, characterized in that the preparation contains no less than 1×10 ¹⁰ cfu/g of viable spores. 9. The application of Bacillus megaterium BM22 according to claim 1 in Photinia erythema. 10. The application of the Bacillus megaterium BM22 spore powder preparation according to claim 7 in the prevention and treatment of Photinia erythema, characterized in that the powder/fog application is carried out at the seedling stage or during the plant growth process.