CN108913622B - Preparation and application of a kind of Bacillus megaterium BM22 and its spore powder - Google Patents

Abstract

The invention discloses a bacillus megaterium BM22 and a preparation method and an application of spore powder thereof, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) in 2017, 12 months and 15 days, and the preservation number is CGMCC No. 15070. Bacillus megaterium BM22 is used as strain and is produced into spore powder through seed culture, fermentation culture and diatomite adsorption. The bacillus megatherium BM22 provided by the invention has a special effect on the photinia fraseri leaf spot, has the characteristics of stress resistance, good storability, high temperature resistance, low temperature resistance and drying resistance, is suitable for commercial production, can avoid the serious consequence of environmental pollution caused by chemical drugs, and has the characteristics of sustainability and 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 microorganisms, and in particular relates to a Bacillus megaterium BM22 strain, a spore powder thereof, and an application in the prevention and treatment of erythema red leaf heather.

Background technique

Photinia xfraseriDress (scientific name: Photinia×fraseriDress) is a hybrid species of the Rosaceae and Photinia genus. It is an evergreen small tree or shrub with a height of 5 meters for trees and 2 meters for shrubs. The crown is spherical, the leaves are leathery, oblong to obovate, lanceolate, the tip of the leaf is acuminate, the base of the leaf is cuneate, the leaf margin has glandular serrations, the flowers are numerous and dense, compound corymbs inflorescence, the flowers are white , Pear is yellow-red, blooms from May to July, September to October. Mainly distributed in the subtropical and temperate regions of southeastern and eastern Asia and North America, it has also been widely cultivated in many provinces of China. The red-leaf heather is used as a street tree, and its poles stand like torches; when used as a hedge, its shape is like a fire dragon; when trimmed and landscaping, its shapes can be varied and the landscape effect is beautiful. The red-leaf heather is named for its bright red shoots and young leaves. There are two common varieties, red robin and red lip. Among them, the red robin has bright and dazzling leaves and is more ornamental. In the spring and autumn, the new shoots and young leaves of the red-leaf heather are fiery red, the color is bright and lasting, and it is very vigorous. In the hot summer season, the leaves turn bright green, giving a fresh and cool feeling. Heather is named for its bright red shoots and young leaves.

Red leaf heather erythema [Cercospora eriobotryae (Enjojii) Saw.], mainly damages leaves. The lesions on the leaves are round to irregular, dark red in size 2-15mm, gray in the center, and dark red at the edges. In the later stage, many black dots appear on the front of the leaves, that is, the fruiting bodies of pathogenic bacteria. The bacteria mostly overwinter on dead leaves, and the conidia in the next spring carry out initial infection and re-infection by air transmission. The disease peaks from July to September every year. It is generally prone to disease in rainy seasons or when it is hot and humid. The disease is generally dominated by chemical control. However, due to the long-term application of chemical pesticides, it is easy to pollute the environment and destroy the ecology.

SUMMARY 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 control of rhododendron erythraea, aiming at the defects of the prior art in preventing and treating erythema rhododendron.

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

The Bacillus megaterium BM22 strain of the present invention was isolated from the healthy Eucommia ulmoides leaves in Yangtianwo Pharmacy, Dayi, Sichuan by the plate dilution coating method on June 15, 2017, and has been deposited in the Chinese Academy of Sciences on December 15, 2017 The General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee of the 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 cultivated on NA plate medium for 2 days is milky white, round, rough surface, corrugated edge, opaque and non-viscous.

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

The present invention uses Bacillus megaterium as the active ingredient of the spore powder, and uses the Bacillus megaterium BM22 as the strain to obtain the spore powder through seed culture, fermentation culture and diatomaceous earth adsorption.

The spore powder containing Bacillus megaterium BM22 as the strain of 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 is prepared by the following method:

1) first-grade slanted seed: make beef extract peptone slanted medium, after inoculation with Bacillus megaterium, cultivate to make first-grade seed;

2) Secondary liquid seeds: after the nutrient succulent culture liquid is autoclaved, filled in a triangular flask under the control of oxygen supply, inoculated with Bacillus megaterium slanted seeds in a sterile state, and shaken cultured to make Bacillus megaterium liquid seeds;

3) After the culture solution is autoclaved, it is placed in a triangular flask under the control of oxygen supply, and the liquid seeds of Bacillus megaterium are inoculated in a sterile state. The inoculation amount is 15% of the total volume of the liquid. For the supernatant, the spores at the bottom of the centrifuge cup were adsorbed with diatomaceous earth, and stored in a 4°C refrigerator for later use.

The components of the nutritious succulent culture solution are: 7 g of beef extract, 10 g of peptone, 5 g of NaCl, and 1000 mL of a dipping solution of rhododendron leaves.

The culture medium is: corn flour 0.5-0.6%, corn starch 0.08-0.2%, bran 0.9-1.2%, glucose 0.18-0.54%, soybean flour 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 of red leaves Heather leaf dipping solution, pH 7.0 to 7.5.

The spore powder with Bacillus megaterium BM22 as the active ingredient obtained by the above preparation method also falls within the protection scope of the present invention.

For the application of the spore powder in the treatment of erythema rhododendron, the powder is prepared at a concentration of 1×10 9 cfu/mL, and then diluted for application.

The application of the Bacillus megaterium BM22 of the present invention in preventing and treating erythema rhododendron.

The application of the Bacillus megaterium BM22 spore powder of the present invention in the prevention and treatment of erythema rhododendron is carried out in the seedling stage or in the process of plant growth by spraying powder.

The method of Bacillus megaterium BM22 of the present invention for preventing and controlling erythema of rhododendron also belongs to the protection scope of the present invention.

The beneficial effects of the present invention are:

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

Description of drawings

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

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

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may utilize the technology disclosed above 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 content of the solution of the present invention fall within the protection scope of the present invention.

The materials, reagents, etc. 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 taken from the black spot of Eucommia ulmoides in Yangtianwo Pharmacy, Dayi, Sichuan. The collected samples were rinsed with sterile water, 1.0 g of tissue was weighed, soaked in 70% alcohol for 1-2 minutes, then disinfected with 1-3% sodium hypochlorite solution for 3-5 minutes, washed several times with sterile water, and then sucked up. The last washing solution 100μL was applied to NA plate (beef extract 3g, peptone 10g, sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7.0, after mixing and aliquoting, autoclave at 121°C for 30min), incubate in the dark at 27°C 24h, for the control of surface disinfection, check whether the surface disinfection is thorough.

Cut the surface-sterilized bark tissue into pieces, put it into a sterile mortar, add sterilized quartz sand and 10 mL of sterile water, grind it fully, let it stand for 30 minutes, and dilute it by 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 a total of 5 gradients, draw 100 μL of 10 ^-3 , 10 ^-4 , 10 ^-5 these 3 gradient grinding liquids and coat NA plates (beef extract 3g, peptone 10g, sodium chloride 5g respectively) , agar powder 15-20g, distilled water 1000mL, pH 7.0, after mixing and packaging, autoclave sterilization at 121°C for 30min), each treatment was repeated 3 times, cultured at 27°C for 48h, and single colonies with obvious differences in colony morphology were picked and entered into the primary sieve .

According to the differences in colony size, color, protrusion, edge characteristics, smooth surface, transparency, etc., pick a single colony on the NA plate for streak purification, and then transfer to the NA slant for storage at 4 °C after purification. A total of 36 endophytic bacteria were obtained.

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

Plate confrontation method: inoculate the 7d-dish Cercosporaeriobotryae (Enjojii) Saw. bacteria cake (diameter 6mm) in the center of the petri dish, at about 3cm away from the bacteria cake, respectively use the inoculation ring to pick up the 2d-cultivated endophyte , draw a thin line on each symmetrical side of the bacteria, cultivate at 28°C, take the plate only inoculated with bacteria as the control, measure the colony growth diameter of the bacteria when the control plate is full, and repeat 3 times for each treatment. Calculate the colony growth inhibition rate as follows:

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

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

Table 1 Antagonistic effect of endophytic bacteria on P. rhodozyma (7d)

1.3 Identification of species of endogenous antagonistic bacteria

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

Table 2 Physiological and biochemical indicators of strain BM22

The single colony of BM22 strain cultured for 2d on NA plate medium is milky white, round, rough surface, corrugated edge, opaque, non-sticky.

Molecular biological identification (16S rDNA): PCR amplification and electrophoresis were performed by extracting bacterial DNA, and the recovered product was sent to a biotechnology company for sequencing; homology BLAST analysis was performed between the detected sequence and the sequence reported in the GenBank database, and Clustalx (1.83) The software performs multiple sequence comparison, and then uses the neighbor-joining method in the Mega4.0 software to construct a phylogenetic tree to determine the position of the BM22 strain in the microbial phylogeny. Molecular biology identified a DNA fragment with a length of 949 bp (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. Matching permutation analysis was performed, and a phylogenetic tree was constructed with Mega analysis software (Fig. 2). It can be seen that the BM22 strain and JX286698 clustered into 1 branch with 99% 16SrRNA gene nucleotide sequence similarity and high bootstrapping value, while they were far away from other Bacillus, indicating that BM22 was related to Bacillus megaterium relationship recently.

Based on the above characteristics, the above-mentioned BM22 strain is classified as Bacillus megaterium (Bacillus megaterium) BM22, which has been preserved in the General Microbiology Center of the China Microbial 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

According to the Bacillus megaterium BM22 obtained in Example 1, spore powder preparation is made through seed culture and fermentation culture, specifically by the following methods:

Beef extract peptone slant medium (beef extract 5g, peptone 10g, NaCl 3.5g, agar 18g, water 1000mL) was prepared by a conventional method, and after inoculation with Bacillus megaterium, the first-class seeds were cultivated.

Nutritious meat culture liquid (beef extract 7g, peptone 10g, NaCl 5g, Magnolia bark dipping solution 1000mL) after autoclaving, under the control of oxygen supply, bottled according to the proportion of 100mL liquid in 300mL conical flask, in sterile State inoculated with slanted seeds of Bacillus megaterium, inoculated with 2 slanted seeds per bottle, and shaken cultured to make liquid seeds of Bacillus megaterium. Wherein, 10 g of heather leaves are boiled in 1000 mL of water for 10 minutes, and filtered, and the obtained filtrate is heather leaf dipping solution.

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 flour 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 remainder The water (Panthera japonica leaf dipping solution), pH 7.0 ~ 7.5. After the culture medium was autoclaved, it was placed in a triangular flask under the control of oxygen supply, and the liquid seeds of Bacillus megaterium were inoculated in a sterile state, and the inoculation amount was 15% of the total volume of the liquid. The spores at the bottom of the centrifuge cup were adsorbed with diatomaceous earth, and stored in a 4°C refrigerator for later use.

Preservation of preparations: Preservation of bottled preparations at room temperature for 1 year or at low temperature (4°C) for 2 years will not affect the preventive and therapeutic effects.

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

When the inoculum is used, the spore powder preparation diluent is sprayed (powder) on the seedlings.

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

In the red-leaf heather cultivation field, according to the occurrence degree of erythema, it was divided into 4 occurrence areas (I: 5-10%; II: 11-30%; III: 31-60%; IV: >60%). In April-May, the microbial inoculum prepared in Example 2 was divided into concentrations by spray method: diluted 50 times, diluted 100 times, diluted 200 times, diluted 400 times, diluted 800 times, diluted 1600 times, and diluted 2400 times. Heather leaves were treated, sprayed evenly along the leaf surface, 100 mL per plant, and used sterile water as a control, repeated 3 times, and the incidence of red-leaf heather was counted. 20 plants per treatment.

15 days after treatment, disease investigation and statistics were carried out according to the disease classification standards in Table 3, and the control effect was calculated.

Table 3 Red leaf heather erythema grading standard

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 spray test, and the biocontrol bacteria showed a good control effect on P. 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. In general, when the disease occurs below 10%, 800 times the solution is the economic concentration; when the disease occurs 10-30%, the 400-fold solution is the economic concentration; when the disease occurs 30-60%, the 200-fold solution is the economic concentration. When it is more than 60%, the economic concentration is 100 times.

Table 4 The effect of BM22 spraying on the control of red leaf heather 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

On the whole, the Bacillus megaterium BM22 strain isolated by the present invention has special effects on erythema red leaf heather, 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 embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present 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

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 949

<212> DNA

<213> Bacillus megaterium

<400> 1

caggctgcgg ggctatacat gcagtcgagc gaactgatta gaagcttgct tctatgacgt 60

tagcggcgga cgggtgagta acacgtgggc aacctgcctg taagactggg ataacttcgg 120

gaaaccgaag ctaataccgg ataggatctt ctccttcatg ggagatgatt gaaagatggt 180

ttcggctatc acttacagat gggcccgcgg tgcattagct agttggtgag gtaacggctc 240

accaaggcaa cgatgcatag ccgacctgag agggtgatcg gccacactgg 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 (7)

1. a preparation method of the spore powder of active ingredient with Bacillus megaterium (Bacillus megaterium) BM22, is characterized in that, comprises the following steps: 1) First-grade slanted seeds: Make beef extract peptone slanted medium, inoculate with Bacillus megaterium BM22, and cultivate to make first-grade slanted seeds; 2) Secondary liquid seeds: After autoclaving the nutrient succulent culture medium, it is placed in a triangular flask under the control of oxygen, inoculated with primary slanted seeds in a sterile state, and shaken to cultivate to make secondary liquid seeds; 3) After the fermentation medium is autoclaved, it is placed in a triangular flask under the control of oxygen supply, and the secondary liquid seeds are inoculated in a sterile state. The inoculation amount is 15% of the total volume of the liquid. The clear liquid was absorbed with diatomaceous earth and the spores at the bottom of the centrifuge cup were stored in a 4°C refrigerator for later use; The Bacillus megaterium BM22 has been deposited in the China General Microorganism Culture Collection and Management Center on December 15, 2017, and the deposit number is CGMCC No.15070; The components of the nutritious succulent culture solution are: 7 g of beef extract, 10 g of peptone, 5 g of NaCl, and 1000 mL of a dipping solution of rhododendron leaves. 2. The preparation method according to claim 1, wherein the fermentation broth is: corn flour 0.5-0.6%, corn starch 0.08-0.2%, bran 0.9-1.2%, glucose 0.18-0.54%, Soybean cake flour 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 of water, pH 7.0-7.5. 3. preparation method according to claim 1 is characterized in that, the composition of described beef extract peptone slant medium is: beef extract 5 g, peptone 10 g, NaCl 3.5 g, agar 18 g, water 1000 mL. 4. The spore powder with Bacillus megaterium BM22 as an active ingredient prepared by the preparation method of claim 1. 5 . The spore powder according to claim 4 , wherein the number of viable spores contained in the spore powder is not less than 1×10 ¹⁰ cfu/g. 6 . 6. The application of Bacillus megaterium BM22 with the deposit number of CGMCC No. 15070 in the prevention and treatment of erythema erythraea caused by Cercospora eriobotryae. 7. the application of the spore powder according to claim 4 in preventing and treating the red leaf heather erythema caused by Cercospora eriobotryae, it is characterized in that, carry out dusting or spray application in seedling stage or plant growth process.

Images