CN108913622B

CN108913622B - Bacillus megaterium BM22 and preparation and application of spore powder thereof

Info

Publication number: CN108913622B
Application number: CN201810800529.XA
Authority: CN
Inventors: 朱涵明月; 朱天辉
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2018-07-06
Filing date: 2018-07-06
Publication date: 2020-08-21
Anticipated expiration: 2038-07-06
Also published as: CN108913622A

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

Bacillus megaterium BM22 and preparation and application of spore powder thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus megaterium BM22 strain, spore powder thereof and application thereof in preventing and treating photinia fraseri leaf spot.

Background

Photinia fraseri (a scientific name: Photinia x fraserdes) is a hybrid of Photinia in Rosaceae, and is evergreen small arbor or shrub, the height of the arbor can reach 5 meters, and the height of the shrub can reach 2 meters. The tree crown is spherical, the leaf is leathery, is long round to inverted egg-shaped, is in a needle shape, the leaf end is gradually sharp, the leaf base is wedge-shaped, the leaf edge is provided with sawteeth with glands, the flower is more and dense, the inflorescence of the umbrella house is repeated, the flower is white, the pear is yellowish red, the pear is opened in 5-7 months, and the 9-10 months. Mainly distributed in the southeast and eastern parts of asia and in subtropical and temperate regions of north america, and has been widely cultivated in many provinces of china. The photinia fraseri is used as a street tree, and the stem of the photinia fraseri is like a torch; making a hedge which is like a dragon; the landscape can be made in various shapes by trimming, and the landscape effect is beautiful. The photinia fraseri is named because its new tips and tender leaves are bright red. The red Robin and the red lip are common, wherein the leaf color of the red Robin is bright and attractive, and the ornamental value is better. In spring and autumn, the new tips and tender leaves of the photinia fraseri are scarlet, the color is gorgeous and durable, and the photinia fraseri is extremely vivid. In summer, the leaves turn bright green at high temperature, giving people a fresh and cool feeling. Photinia fraseri is named for its bright red new shoots and young leaves.

Photinia fraseri leaf spot [ Cercospora eriomotryae (Enjojii) Saw ] mainly causes the leaf damage. The lesion spots on the leaves are round to irregular, dark red with the size of 2-15 mm, gray in the center and dark red at the edges. In the later period, a plurality of black specks, namely pathogenic bacteria fruiting bodies, grow on the front surface of the leaves. The pathogenic bacteria mostly live through the winter on the dead leaves, and the conidia are primarily infected and re-infected by airflow propagation in the next spring. The disease enters the full period of the disease in 7-9 months each year. Generally, the disease is easy to occur in rainy season or in high-temperature and humid conditions. The disease is mainly chemical prevention, but because the chemical pesticide is applied for a long time, the environment is easily polluted, the ecology is damaged, and the research on the comprehensive prevention and treatment measures of the disease is still needed.

Disclosure of Invention

The invention aims to solve the technical problem of the prior art in preventing and treating the photinia fraseri leaf spot disease, and provides the application of bacillus megaterium BM22 and spore powder thereof in preventing and treating the photinia fraseri leaf spot disease.

The invention is realized by the following technical scheme:

the Bacillus megaterium BM22 strain is separated from healthy eucommia leaves in Sichuan Mali invertebral fossa medical field by adopting a plate dilution coating method in 2017, 6 and 15 days, and is preserved in China general microbiological culture Collection center (CGMCC) of institute of microbiology, China academy of sciences, microbiological research institute, China general microbiological culture Collection center (CGMCC) in 2017, 12 and 15 days, wherein the address is No. 3 of North Chen West Lu No.1 of the sunward region in Beijing, and the preservation number is CGMCC No. 15070.

The single colony of the bacillus megaterium BM22 cultured on the NA plate culture medium for 2d is milky white, round, rough in surface, wavy in edge, opaque and non-adhesive.

The 16S rDNA sequence of the bacillus megatherium BM22 is shown in SEQ ID NO. 1.

The invention relates to spore powder taking bacillus megaterium as an active ingredient, which is prepared by taking bacillus megaterium BM22 as a strain through seed culture, fermentation culture and diatomite adsorption.

The spore powder taking the bacillus megaterium BM22 as a strain contains live spores with the number not less than 1 × 10¹⁰cfu/g。

The spore powder taking the bacillus megaterium BM22 as a strain is prepared by the following method:

1) first-order inclined plane seeds: preparing a beef extract peptone slant culture medium, inoculating bacillus megaterium, and culturing to prepare a first-class seed;

2) secondary liquid seeds: sterilizing the nutrient meat culture solution under high pressure, placing in a triangular flask under the control of oxygen, inoculating bacillus megaterium slant seeds in an aseptic state, and performing shaking culture to obtain bacillus megaterium liquid seeds;

3) the culture solution is sterilized under high pressure, placed in a triangular flask under the control of oxygen, inoculated with Bacillus megaterium liquid seeds in a sterile state, the inoculum size is 15 percent of the total volume of the liquid, after shaking culture for 4 days, centrifuged for 10min, the supernatant is discarded, spore spores at the bottom of the centrifugation cup are adsorbed by diatomite, and the culture solution is stored in a refrigerator at 4 ℃ for standby.

The nutrient meat culture solution comprises the following components: 7g of beef extract, 10g of peptone, 5g of NaCl, and 1000mL of photinia fraseri leaf impregnation liquid.

The culture solution is as follows: 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 flour, KH₂PO₄0.005～0.006％，(NH₄)₂SO₄0.006～0.008％，MnSO₄0.004～0.005％，CaCO₃0.0005～0.002％，MgSO₄·7H₂0.005-0.006% of O, and the balance of Photinia fraseri leaf impregnation liquid, wherein the pH value is 7.0-7.5.

The spore powder which is prepared by the preparation method and takes the bacillus megaterium BM22 as an active ingredient is also within the protection scope of the invention.

The spore powder is applied to treating photinia fraseri red spot, and the powder is prepared into the powder with the concentration of 1 multiplied by 109cfu/mL, and then diluted and applied.

The bacillus megatherium BM22 disclosed by the invention is applied to prevention and treatment of photinia fraseri leaf spot.

The bacillus megatherium BM22 spore powder is applied to preventing and treating photinia fraseri leaf spot and is sprayed during the seedling stage or the plant growth process.

The method for preventing and treating the photinia fraseri leaf spot disease by using the bacillus megaterium BM22 also belongs to the protection scope of the invention.

The invention has the beneficial effects that:

the bacillus megatherium BM22 provided by the invention has a special effect on photinia fraseri leaf spot, tests show that the inhibition rate of photinia fraseri leaf erythema germs reaches 98%, the bacillus megatherium BM22 is prepared into spore powder, has a good prevention and treatment effect, is suitable for commercial production, can avoid the serious consequence of environmental pollution caused by chemical drugs, has the characteristics of stress resistance, good storability, high temperature resistance, low temperature resistance and drying resistance, conforms to the strategy of national sustainable development, has the characteristic of low cost compared with the chemical drugs, and has a wide application prospect.

Drawings

FIG. 1 is an electrophoretogram of PCR amplification product of BM22rDNA-ITS sequence of Bacillus megaterium of the present invention;

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

Detailed Description

The technical solutions of the present invention are further described with reference to the following embodiments, which are merely preferred embodiments of the present invention, and not limiting the present invention in other forms, and any person skilled in the art may change or modify the technical contents disclosed above into equivalent embodiments with equivalent changes. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the scope of the present invention, unless they depart from the technical spirit of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation and characterization of Bacillus megaterium BM22

1.1 isolation, purification and preservation of endophytic bacteria

Taking healthy eucommia leaves in the generation area of eucommia black spot in the Sichuan Mali hupehensis Uygur nest medicine field. The collected sample is washed cleanly by sterile water, 1.0g of tissue is weighed, the tissue is soaked in 70% alcohol for 1-2min, then sterilized by 1-3% sodium hypochlorite solution for 3-5min, after being washed for a plurality of times by sterile water, 100 mu L of the last washing liquid is absorbed to smear an NA flat plate (3 g of beef extract, 10g of peptone, 5g of sodium chloride, 15-20g of agar powder, 1000mL of distilled water and pH 7.0, the mixture is evenly mixed and subpackaged and then is sterilized at 121 ℃ for 30min under high pressure), and the mixture is cultured in the dark at 27 ℃ for 24h for comparison of surface sterilization and checking whether the surface sterilization is complete or not.

Cutting the surface-sterilized bark tissue, placing into a sterile mortar, adding sterilized quartz sand and 10mL sterile water, grinding, standing for 30min, and diluting for 10min^-1，10^-2，10^-3，10^-4，10^-5Total 5 gradients, 100. mu.L 10 aspirated^-3，10^-4，10^-5The 3 gradient grinding liquids are respectively coated on NA plates (beef extract 3g, peptone 10g, sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7.0, and after uniform mixing and subpackaging, autoclaving at 121 ℃ for 30min), each treatment is repeated for 3 times, culturing is carried out at 27 ℃ for 48h, and single colonies with obvious colony morphology difference are selected and enter a primary screen.

According to the difference of colony size, color, protrusion, edge characteristic, smooth surface, transparency, etc., selecting single colony, streaking and purifying on NA plate, and transferring the NA slant to 4 deg.c for storage. A total of 36 endophytic bacteria were obtained.

1.2 plate antagonism of endophytic bacteria against Photinia fraseri erythematodes (Enjojii) Saw.)

Plate confrontation method: inoculating 7d of photinia fraseri erythematodes (Cercospora obotyae (Enjojii) Saw.) cake (diameter 6mm) in the center of the culture dish, dipping 2d of endophytes from the cake about 3cm away from the cake by using inoculating rings, respectively drawing a thin line on two symmetrical sides of each germ, culturing at 28 ℃, taking a flat plate only inoculated with the germ as a control, measuring the colony growth diameter of the germ when the control is full of the dish, and repeating the treatment 3 times each time. The colony growth inhibition was calculated as follows:

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

Table 1 shows that 32 endophytic bacteria have a good antagonistic effect on Photinia fraseri erythematodes (Enjojii) Saw), and the BM22 inhibition rate reaches 98%.

TABLE 1 antagonistic action of endophytic bacteria against Photinia fraseri Erythrophyta (7d)

1.3 identification of the species of endogenous antagonistic bacteria

The endophyte BM22 was identified as B.megaterium by morphological observation and combination of physiological and biochemical indicators (Table 2) and molecular biology.

TABLE 2 physiological and biochemical indexes of strain BM22

A single colony of 2d BM22 strain cultured on NA plate medium was milky white, round, rough-surfaced, wavy-edged, opaque, and non-adherent.

Molecular biological identification (16S rDNA): extracting bacterial DNA to perform PCR amplification and electrophoresis, and recycling a product to a biotechnology company for sequencing; the determined sequences were subjected to homology BLAST analysis with sequences already reported in GenBank databases and multiple sequence comparisons with Clustalx (1.83) software, and phylogenetic trees were constructed using the adjacency method in Mega4.0 software to determine the phylogenetic status of the BM22 strain in microorganisms. The molecular biology identification obtains a DNA fragment with the length of 949bp (figure 1), the 16S rDNA sequence of the BM22 strain is submitted to a GenBank database for BLAST analysis, the bacterial 16S rDNA sequence with higher homology is selected and is subjected to multiple matching arrangement analysis by Clustalx software, and a phylogenetic tree is constructed by Mega analysis software (figure 2). It can be seen that the BM22 strain supports a poly-1 branch with 99% nucleotide sequence similarity of the 16SrRNA gene and a higher abduction value with JX286698 and is more distant from other Bacillus, indicating that BM22 has a closest relationship to Bacillus megaterium.

Based on the characteristics, the BM22 strain is classified and named as Bacillus megaterium (BM 22), and has been stored in the general microbiological culture Collection center of the institute of microbiology, China Committee for culture Collection of microorganisms (CGMCC) No.15070 in 2017, 12 months and 15 days.

Example 2 preparation of Bacillus megaterium BM22 spore powder

The bacillus megaterium BM22 obtained in example 1 was cultured by seed culture and fermentation to prepare a spore powder preparation, which was prepared by the following method:

a beef extract peptone slant culture medium (5 g of beef extract, 10g of peptone, 3.5g of NaCl, 18g of agar and 1000mL of water) is prepared by a conventional method, inoculated with bacillus megaterium and cultured to prepare a first-class seed.

The nutrient meat culture solution (7 g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of magnolia bark root bark maceration extract) is sterilized under high pressure, bottled in a 300mL triangular flask according to the proportion of 100mL of liquid under the control of oxygen introduction, inoculated with 2 inclined plane seeds in an aseptic state, and subjected to shaking culture to prepare the liquid bacillus megaterium seeds. Wherein 10g of Photinia fraseri leaves are boiled in 1000mL of water for 10 minutes and filtered, and the obtained filtrate is Photinia fraseri leaf impregnation liquid.

Liquid fermentation and diatomite adsorption into powder: 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 flour, KH₂PO₄0.005～0.006％，(NH₄)₂SO₄0.006～0.008％，MnSO₄0.004～0.005％，CaCO₃0.0005～0.002％，MgSO₄·7H₂0.005-0.006% of O, and the balance of water (Photinia fraseri leaf impregnation liquid), and the pH value is 7.0-7.5. The culture solution is sterilized under high pressure, placed in a triangular flask under the control of oxygen, inoculated with Bacillus megaterium liquid seeds in a sterile state, the inoculum size is 15 percent of the total volume of the liquid, after shaking culture for 4 days, centrifuged for 10min, the supernatant is discarded, spore spores at the bottom of the centrifugation cup are adsorbed by diatomite, and the culture solution is stored in a refrigerator at 4 ℃ for standby.

And (3) preparation preservation: the bottled preparation can be stored at normal temperature for 1 year or at low temperature (4 deg.C) for 2 years without affecting the preventing and treating effects.

The powder prepared by the method and using the bacillus megaterium BM22 as a strain contains live spores with the number not less than 1 × 10¹⁰cfu/g。

When the microbial inoculum is used, the spore powder preparation diluent sprays (powder) to the nursery stock.

Example 3 Bacillus megaterium BM22 spore powder spray method control test

In the photinia fraseri cultivation land, the occurrence degree of the photinia fraseri is divided into 4 occurrence regions (I: 5-10%; II: 11-30%; III: 31-60%; IV: > 60%). In 4-5 months, the microbial inoculum prepared in the example 2 is subjected to concentration division by a spraying method: and (3) treating the leaves of the photinia fraseri by diluting 50 times, 100 times, 200 times, 400 times, 800 times, 1600 times and 2400 times, uniformly spraying 100mL of each leaf along the leaf surface, repeating for 3 times by using sterile water as a control, and counting the incidence condition of the photinia fraseri. Each 20 strains were treated.

And (5) carrying out disease investigation statistics according to the disease classification standard in the table 3 after 15d of treatment, and calculating the prevention and treatment effect.

TABLE 3 Classification Standard of Photinia fraseri Red Spot

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

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

the preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index x 100.

Table 4 shows the control effect of the endophytic bacteria BM22 after the spray test for 15d, and the biocontrol bacteria show good control effect on photinia fraseri anthrax. The control effect of the 50-time diluent in the four types of occurrence areas exceeds 80 percent, the disease spreading can be basically controlled, the control effect is reduced along with the increase of the dilution times, and the control effect difference is larger when the diluent is diluted 1600 times and 2400 times. In general, the disease occurs below 10%, the 800-time solution is economic concentration, when the disease occurs 10-30%, the 400-time solution is economic concentration, and when the disease occurs 30-60%, the 200-time solution is economic concentration. Above 60%, the economic concentration is 100 times.

TABLE 4 BM22 Effect (%) after spray-controlling Photinia fraseri leaf spot disease 15d

Concentration of	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 summary, the separated bacillus megaterium BM22 strain has special effect on the photinia fraseri leaf spot, has strong environmental suitability and good affinity, is suitable for commercial production, meets the strategic requirements of sustainable development, and has wide market prospect.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> Sichuan university of agriculture

<120> preparation and application of bacillus megaterium BM22 and spore powder thereof

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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

1. A method for preparing spore powder with Bacillus megaterium (BM 22) as active ingredient comprises the following steps:

1) first-order inclined plane seeds: preparing a beef extract peptone slant culture medium, inoculating bacillus megaterium BM22, and culturing to prepare a first-grade slant seed;

2) secondary liquid seeds: sterilizing the nutrient meat culture solution under high pressure, placing in a triangular flask under oxygen control, inoculating the first-stage slant seeds in an aseptic state, and performing shaking culture to obtain second-stage liquid seeds;

3) sterilizing the fermentation culture solution under high pressure, placing in a triangular flask under oxygen control, inoculating secondary liquid seeds in an aseptic state, wherein the inoculation amount is 15% of the total volume of the liquid, centrifuging for 10min after shaking culture days, removing supernatant, adsorbing spore spores at the bottom of the centrifugation cup with diatomite, and storing in a refrigerator at 4 deg.C for later use;

the bacillus megaterium BM22 has been preserved in China general microbiological culture collection center in 2017, 12 and 15 months, and the preservation number is CGMCC No. 15070;

the nutrient meat culture solution comprises the following components: 7g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of photinia fraseri leaf impregnation liquid.

2. The method according to claim 1, wherein the fermentation broth 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 flour, KH₂PO₄0.005～0.006%，(NH₄)₂SO₄0.006～0.008%，MnSO₄0.004～0.005%，CaCO₃0.0005～0.002%，MgSO₄·7H₂0.005-0.006% of O, and the balance of water, and pH 7.0-7.5.

3. The method according to claim 1, wherein the beef extract peptone slant medium comprises the following components: 5g of beef extract, 10g of peptone, 3.5g of NaCl, 18g of agar and 1000mL of water.

4. A spore powder comprising Bacillus megaterium BM22 as an active ingredient prepared by the method according to claim 1.

5. The spore powder of claim 4, wherein the spore powder has a viable spore number of not less than 1 × 10¹⁰cfu/g。

6. Application of bacillus megaterium BM22 with preservation number of CGMCC No.15070 in preventing and treating photinia fraseri leaf spot disease caused by Cercospora eriobotrya eriomotryae.

7. The use of the spore powders according to claim 4 for controlling photinia fraseri maculopathy caused by Cercospora erythraea, characterized in that the spore powders are applied by powder spraying or mist spraying at the seedling stage or during the growth of plants.