CN108865925B - A strain of Bacillus amyloliquefaciens, fermentation product and preparation method and application thereof - Google Patents

Abstract

The invention discloses bacillus amyloliquefaciens, a fermentation product, a preparation method and application thereof. The strain is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) E3, is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan university in Wuhan, China in 2017 and 12 and 7 months, and has the preservation number of CCTCC NO: m2017768; the strain can effectively inhibit the infection of the rice basic rot fungi on rice seeds, improves the germination rate, and can be used as a biocontrol strain to be applied to the prevention and control of the rice basic rot. The invention also provides a preparation method of the fermentation stock solution of the strain, which is simple and easy to operate. The invention also provides the antibacterial lipopeptide of the strain and an extraction method thereof, the antibacterial lipopeptide shows an inhibiting effect on various plant pathogenic bacteria, has a wider antibacterial spectrum, and can be used for developing efficient, broad-spectrum, low-toxicity and environment-friendly microbial agents.

Description

A strain of Bacillus amyloliquefaciens, fermentation product and preparation method and application thereof

technical field

The invention belongs to the high-tech field of biology, and particularly relates to a strain of Bacillus amyloliquefaciens, a fermentation product and a preparation method and application thereof.

Background technique

In the process of agricultural production, plant diseases caused by plant pathogens seriously affect the growth, development and yield of plants, and even lead to plant death. At present, the control of plant diseases mainly relies on chemical pesticides, which have made great contributions to the safe production of agricultural products, but the widespread use of chemical pesticides and the increase in the amount of pesticides have caused a series of problems, increasing the resistance of certain pathogenic bacteria, and at the same time It will cause environmental pollution, so the development of biological pesticides is imminent, and the use of microorganisms to inhibit plant pathogens provides the possibility of biological control of diseases. Among them, Bacillus is a non-pathogenic bacteria widely existing in nature, which can produce a variety of antibacterial substances, and is a research hotspot in the biological control of plant diseases, because the bacteria itself and the antibacterial substances produced are in the control of disease occurrence, They have good performance in promoting plant growth and other aspects, and they are easy to reproduce and have strong ability to adapt to the environment, showing broad application prospects in the biological control of plant diseases.

Biological control is the use of biocontrol bacteria itself or secondary metabolites in the growth process to inhibit the growth and reproduction of pathogenic microorganisms through antagonism, parasitism, predation, and competition with pathogenic bacteria for living space and nutrients. Plant diseases have the advantages of green, safe and pollution-free. The secretion of Bacillus subtilis strains isolated from soil by Liu Yongfeng et al. had an inhibition rate of 74% against watermelon Fusarium wilt, 95.4% and 100% against rice sheath blight and rice smut, respectively. The area control of rice sheath blight obtained better disease control effect. Kaewklom et al. discovered a new bacteriocin PPB from Bacillus amyloliquefaciens SP113LM, which can strongly inhibit the growth of microorganisms such as Listeria monocytogenes, and found that PPB also has a broad-spectrum antibacterial activity active.

Bacillus strains that have been commercialized include B.subtilis GB03, B.subtilis MB1600, B.subtilis QST713 and B.amyloliquefaciens FZB24 in the United States, which can be used to control beans, wheat, cotton and other diseases caused by Fusarium and Rizoctonia, respectively. Root diseases of peanuts, etc.; Liming Wu et al. found that FZB42 has a good control effect on rice bacterial blight (Xanthomonas oryzae pv.oryzae) and rice stripe spot (Xanthomonas oryzaepv.oryzicola). There are also B.subtilis RB14 and B.subtilisNB22GB03 developed in Japan, B.subtilis A-13 developed in Australia and other biocontrol strains.

Rice basal rot fungus (Dickya zeae), short rod-shaped, solitary, blunt at both ends, with pericyte flagella, no spores and capsules, Gram-negative, bacteria on beef extract protein gland agar ( The color of the colony cultivated on the NA) medium was colostrum white and then turned to khaki, and the colony was amoeba-like, dull and anaerobic.

Rice bacterial basal rot can occur in the entire growth period of rice. It invades when the seeds germinate. The invasion of the bacteria will cause the seeds or buds to rot. It can occur from the tillering stage to the grain-filling stage of rice. Rice disease that occurs in the tillering stage At first, the heart leaves were green, and then the heart leaves turned yellow, and the base began to turn brown and rot; after the rice booting stage, the disease occurred, and the plants lost water and died. Half-withered or withered ears, the rotten part of the base of the diseased plant increases, accompanied by a small amount of inverted roots.

Chemical agents are currently one of the effective ways to prevent and control rice basal rot. The occurrence and prevalence of bacterial basal rot disease of rice is fast. Generally, pesticides are applied after the disease occurs, and it is often easy to miss the best control time.

For the chemical control of bacterial base rot, the residues of chemical agents will pollute the environment and also bring some hidden dangers to food safety. The use of biological control methods shows its advantages in production. An antagonistic bacteria isolated by Polish scientists Sylwia Jafra and others on bulb crops such as onions and lilies can inhibit the corrosion of hyacinth roots by Dickya zeae. Wang Yinhan et al screened out a strain that has a good antagonistic effect on pineapple bacterial soft rot, and the antagonistic bacteria can secrete active substances with antibacterial properties.

The production of antibacterial active substances through secondary metabolism, inhibiting the growth of harmful microorganisms or directly killing pathogenic microorganisms is one of the important mechanisms for the control of plant diseases by biocontrol Bacillus. Bacillus amyloliquefaciens has a broad antibacterial spectrum, has strong secondary metabolite production ability, and can produce a variety of antibacterial substances. The antibacterial substances are mainly antibacterial proteins, lipopeptides and polyketones.

In actual production, the control of rice basal rot should be assisted by control agents in addition to the disease-resistant varieties. Since the disease was first reported in the late 1970s, the disease is currently aggravating and spreading year by year. However, there is still a lack of more in-depth and systematic research on the pathogenesis of this disease at home and abroad.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a strain of Bacillus amyloliquefaciens, which was screened by the inventor from the rhizosphere soil by the plate inhibition zone method. The strain has strong resistance to rice basal rot. The antagonism can effectively inhibit the rice basal rot pathogen and improve the germination rate of rice seeds.

Another object of the present invention is to provide a method for preparing the fermentation product of Bacillus amyloliquefaciens.

Another object of the present invention is to provide the antibacterial lipopeptide of Bacillus amyloliquefaciens.

Another object of the present invention is to provide applications of the Bacillus amyloliquefaciens, its fermentation products and/or antimicrobial lipopeptides.

The object of the present invention is achieved through the following technical solutions:

A strain of Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens E3, was deposited on December 7, 2017 in the China Collection of Type Cultures (CCTCC) located in Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M2017768.

The morphological and physiological characteristics of the Bacillus amyloliquefaciens are as follows: the colonies are white, round, with irregular edges, protruding around, depression in the middle, smooth membrane coating on the initial surface, and folds on the later surface.

The physiological and biochemical characteristics of the described Bacillus amyloliquefaciens are:

The 16S rDNA gene characteristics of the Bacillus amyloliquefaciens: its 16S rDNA sequence is shown in the nucleotide sequence of SEQ ID NO. 3, and the sequence length is 1456 bp.

The preparation method of the fermentation product of described Bacillus amyloliquefaciens, comprises the steps:

The Bacillus amyloliquefaciens were inoculated into LB liquid medium, and cultured at 160-200 rpm for 48-72 hours at 28°C.

The inoculum of the Bacillus amyloliquefaciens is preferably 1%.

A fermentation product is prepared by the method for preparing the fermentation product of Bacillus amyloliquefaciens.

A preparation method of the antibacterial lipopeptide of Bacillus amyloliquefaciens, comprising the steps:

(1) HCl precipitation: the fermentation product of Bacillus amyloliquefaciens is centrifuged, the precipitation is discarded, HCl is slowly added to the supernatant liquid while stirring, the pH value is adjusted to 2 to 3, and left to stand overnight at 2 to 6 °C ;

(2) Organic solvent extraction: centrifuge the solution obtained in step (1), take the precipitate, add acetone for extraction, take the supernatant, and dry to obtain the antimicrobial lipopeptide.

The pH value described in step (1) is further preferably 2.

The temperature for standing still described in the step (1) is preferably 4°C.

The conditions for centrifugation described in step (1) are preferably centrifugation at 8000 rpm for 15 min.

The conditions for centrifugation described in step (2) are preferably centrifugation at 8000 rpm for 15 min.

The amount of acetone described in step (2) is preferably 5 times that of the precipitation.

After adding acetone for extraction as described in step (2), the precipitate can also be subjected to at least one acetone extraction.

The drying described in step (2) is preferably air drying.

The antimicrobial lipopeptide described in step (2) is preferably stored at -80°C.

An antibacterial lipopeptide is prepared by the method for preparing the antibacterial lipopeptide of Bacillus amyloliquefaciens.

A microbial inoculum contains at least one of the Bacillus amyloliquefaciens, its fermentation product and antibacterial lipopeptide.

Application of the Bacillus amyloliquefaciens, its fermentation products and/or antibacterial lipopeptides in preventing and treating rice diseases.

The rice disease is rice basal rot.

Compared with the prior art, the present invention has the following advantages and effects:

1. The research that utilizes Bacillus amyloliquefaciens to prevent and control rice base rot is rarely reported in China, and the present invention provides a new Bacillus amyloliquefaciens E3, which is an antagonistic antibacterial with stronger control effect to rice base rot, and this bacterial strain can effectively Inhibit the infection of rice seeds by rice basal rot fungus and improve the germination rate of rice seeds.

2. The present invention also provides a method for preparing the Bacillus amyloliquefaciens E3 fermentation stock solution, which is simple and easy to operate.

3. The present invention also provides the antibacterial lipopeptide of Bacillus amyloliquefaciens E3 and an extraction method thereof, and the production of lipopeptide bacteriostatic active substances by Bacillus amyloliquefaciens E3 can effectively inhibit rice basal rot, and has a strong biological It has a certain inhibitory effect on a variety of plant pathogens including bacteria and fungi, and has a broad antibacterial spectrum, which can be used to develop high-efficiency, broad-spectrum, low-toxicity, and environmentally friendly microbial inoculants.

Description of drawings

Fig. 1 is a photograph of the inhibition zone produced by Bacillus amyloliquefaciens E3 in rescreening.

Fig. 2 is a morphological photograph of Bacillus amyloliquefaciens E3.

Figure 3 is a phylogenetic tree diagram constructed based on the 16S rDNA gene sequence.

Figure 4 is the result of PCR electrophoresis of the β-mannanase gene of strain E3; wherein, lane M is DNAMarker (DS5000), lane 1 is 16S rDNA, and lane 2 is using primers Bsu-man-1F and Bsu-man-1R The amplification results of ; Lane 3 is the amplification results of Bam-man-1F and Bam-man-1R.

Figure 5 is a result analysis diagram of the effect of Bacillus amyloliquefaciens E3 on the germination rate of rice seeds.

Figure 6 is a graph showing the results of biocontrol effects of Bacillus amyloliquefaciens E3 crude lipopeptide on different strains; wherein, A is Fusarium solanacearum, B is Aspergillus flavus, C is Aspergillus niger, D is Fusarium nivalis, and E is Escherichia coli, F is citrus canker, G is rice basal rot, H is Laurella solanaceae, I is Burkholderia cepacia, and J is banana basal rot.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1 Screening and identification of Bacillus amyloliquefaciens E3 strain

1. Screening of strains

(1) Preliminary screening of Bacillus amyloliquefaciens: 1 g of soil from a citrus orchard in Hunan Province was added to 10 mL of sterile water, incubated at 200 rpm for 30 min, and then diluted with water. The dilution gradients were 10×, 100×, and 1000×. The dilution solution was coated on the bacteria containing rice basal rot (hereinafter referred to as EC1, the full-length genome of this strain has been published on NCBI GenBank, the serial number is NCBI Reference Sequence: NZ_CP006929, the website is https://www.ncbi.nlm.nih .gov/nuccore/NZ_CP006929.1) on LB solid medium. Preparation of LB solid medium containing EC1: Cultivate EC1 bacteria with LB liquid medium at 28°C and 200rpm until the OD ₆₀₀ is about 1, and then at 40-50°C, 10μL of bacterial suspension is added per ml of LB medium. The ratio of the liquid was mixed, and about 10 mL of medium was poured into each dish to obtain a plate containing EC1, which was incubated at 28 °C for 48 h, and observed whether there was an inhibition zone.

(2) Rescreening: streak and purify the bacteria obtained from the primary screening, pick a single colony and cultivate it in a 96-well plate containing 200 μL of LB liquid medium until the bacteria liquid is turbid (about 12h) to obtain a biocontrol bacteria liquid. The LB solid medium containing EC1 was punched with a hole punch with a diameter of 5 mm, 10 μL of biocontrol bacteria solution was added into the hole, and cultured at 28° C. for 1 day to observe whether there was a bacteriostatic zone. A total of 19 strains with bacteriostatic zone were screened, and one of the strains had a larger bacteriostatic zone with a diameter of about 20 mm, as shown in Figure 1, named E3. This strain is preserved and the strain is subsequently classified and identified.

2. Classification and identification of E3 strains:

(1) Observation of morphological characteristics

The growth morphology of E3 strain on LB solid plate (cultivated at 28°C) is shown in Figure 2: the colony is white, round, with irregular edges, protruding around, depression in the middle, smooth film coating on the initial surface, and surface formation in the later stage. folds.

(2) Physiological and biochemical characteristics

The E3 strain was subjected to physiological and biochemical detection of biocontrol bacteria E3 according to MS(i)/C005-C01 ("Common Bacterial System Identification Manual", "Berger Bacterial Identification Manual") according to standard methods, and the obtained results are shown in Table 1. :

Table 1 Physiological and biochemical characteristics of biocontrol strain E3

Description: "+" means positive; "-" means negative

(3) Classification of strains based on 16S rDNA sequences

Design primers that can specifically amplify 16SrDNA. The primer sequences are as follows:

(1) F27: 5'-AGAGTTTGATCATGGCTCAG-3;

(2) R1492: 5'-TACGGTTACCTTGTTACGACTT-3'.

Using the genomic DNA of E3 strain as the template, PCR amplification was carried out with F27 and R1492 as the primer pair, and the target band was recovered and sequenced to obtain a DNA fragment of about 1456 bp. The resulting 16S rDNA sequence is shown below:

TAGCATGCGCAGCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCT TCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGTAAGAAGTTATGACT

The obtained sequences were compared in GenBank by BLAST, and the results showed that the 16S rDNA gene sequence of strain E3 had a sequence similarity of 99% with that of Bacillus amyloliquefaciens. According to the comparison results, it was preliminarily determined to be Bacillus amyloliquefaciens.

A phylogenetic tree of strain E3 was constructed based on the 16S rDNA gene sequence, as shown in Figure 3.

(4) Amplification of β-mannanase gene sequence

Design primers that can amplify the β-mannanase gene sequence, wherein the primer pairs Bsu-man-1F and Bsu-man-1R can specifically amplify the Bacillus subtilis β-mannanase gene; the primer pair Bam-man -1F and Bam-man-1R can specifically amplify the β-mannanase gene of Bacillus amyloliquefaciens. The primer pair sequences are as follows:

Bsu-man-1F: 5'-CAGGCTCACACTTTGTCTTG-3';

Bsu-man-1R: 5'-TGAACACAGTCCTGGGTTAG-3';

Bam-man-1F: 5'-TCGGTTTCACATCCTTCATC-3';

Bam-man-1R: 5'-TTTGTCAGCGTGTCTTTCTG-3';.

The specific PCR amplification of the two primer pairs was 1287bp.

Using the amplified 16S rDNA and β-mannanase gene sequences as primers, the genomic DNA of E3 strain was used as the template for PCR amplification. The results are shown in Figure 4: Swimming lane 1 is the primer pair (F27 and R1492) PCR amplification The results of the 16S rDNA fragment, electrophoresis results showed a band with a size of about 1500bp; lane 2 is the primer pair (Bsu-man-1F and Bsu-man-1R) to specifically amplify the Bacillus subtilis β-mannanase gene fragment. As a result, electrophoresis showed no amplified band; lane 3 was the result of specific PCR amplification of Bacillus amyloliquefaciens β-mannanase gene fragment with primer pair (Bam-man-1F and Bam-man-1R), showing There is a band of size 1287bp. The E3 strain did not have the Bacillus subtilis-specific β-mannanase gene band amplification in lane 2, while the Bacillus amyloliquefaciens β-mannanase gene 1287bp specific amplification appeared in lane 3, indicating that the E3 strain belongs to amylolytica Bacillus. Therefore, the obtained E3 strain was finally determined to be Bacillus amyloliquefaciens.

The E3 strain is biologically preserved, and the preservation information is as follows: the name is Bacillus amyloliquefaciens E3, which was deposited in the China Collection of Typical Cultures (CCTCC) located in Wuhan University, Wuhan, China on December 7, 2017, and the preservation number is CCTCC NO: M 2017768.

Example 2

(1) Preparation of fermentation stock solution

Prepare 100 mL of LB liquid medium, inoculate the biocontrol bacteria (ie E3 bacteria) obtained in Example 1 according to the inoculation amount of 1% (volume ratio), cultivate at 28° C. and 200 rpm for three days to obtain a fermentation stock solution.

(2) Preparation of EC1 bacterial solution:

Inoculate EC1 single colony LB liquid medium, cultivate at 28°C, 200rpm, grow to about _OD600 =0.6, centrifuge at 5000rpm for 10 minutes to collect bacteria, suspend with sterile water, and use colony plate counting method. EC1 bacterial liquid, respectively 1.4×10 ⁷ cfu/mL, 1.4×10 ⁶ cfu/mL, 1.4×10 ⁵ cfu/mL, 1.4×10 ⁴ cfu/mL, 1.4×10 ³ cfu/mL;

(3) Disease prevention and growth promotion characteristics of Bacillus amyloliquefaciens E3 on rice seeds

In order to test the bacteriostatic effect of biocontrol bacteria E3 on rice basal rot, the germination experiments of rice seeds were carried out.

Choose healthy and plump rice seeds, rinse with double-distilled water several times before use. The experiment was divided into two groups, 30 seeds were selected in each group, 1.4×10 ⁷ cfu/mL, 1.4×10 ⁶ cfu/mL, 1.4×10 ⁵ cfu/mL, 1.4×10 ⁴ cfu/mL mL, 1.4×10 ³ cfu/mL EC1 bacterial solution was soaked at 28°C for 5 hours, the bacterial solution was discarded, and the seeds were rinsed three times with sterile water. They were placed in petri dishes lined with 3 to 4 pieces of filter paper (wet with sterile water). After 12 hours of moisturizing cultivation at 30°C, one group was sprayed with sterile fermentation broth (10 ¹¹ cfu/mL) of biocontrol bacteria E3 to make the fermentation broth evenly cover the seeds. The other group was not inoculated with biocontrol bacteria E3. The control was soaked in sterile fermentation broth (10 ¹¹ cfu/mL) of biocontrol bacteria E3 and sterile water for 5 hours, and then rinsed the seeds three times with sterile water, and also placed 3 to 4 pieces of filter paper (sterile water) on the pad. wet) in a petri dish. The germination situation was observed every day at 30°C moisturizing culture, and sterile water was appropriately added for moisturizing, and the seed germination situation was counted after 4 days.

The rice basal rot fungus can invade when the seeds germinate, and the invasion of the fungus will cause the seeds or buds to rot. It can be seen from the results in Figure 5 that the negative control sterile water and biocontrol bacteria E3, the rice seeds germinate normally, and only the positive control inoculated with EC1 bacteria, when the concentration is 1.4×10 ⁷ cfu/mL, 1.4×10 ⁶ cfu/mL , 1.4×10 ⁵ cfu/mL, 1.4×10 ⁴ cfu/mL significantly inhibited the germination of rice seeds; in the experimental group inoculated with biocontrol bacteria E3, the EC1 concentration was 1.4×10 ⁷ cfu/mL, 1.4×10 ⁶ cfu/mL, the control effect is not good, but the germination rate of rice seeds at the concentration of 1.4×10 ⁵ cfu/mL, 1.4×10 ⁴ cfu/mL and 1.4×10 ³ cfu/mL was significantly higher than that of the uninoculated biocontrol bacteria. test group. These results indicate that Bacillus amyloliquefaciens E3 strain can effectively inhibit the infection of rice basal rot to a certain extent.

Example 3 Bacteriostatic spectrum test of crude lipopeptide of Bacillus amyloliquefaciens E3

1. Antibacterial lipopeptide extraction

Lipopeptides were extracted by HCl precipitation combined with organic solvent extraction. Take 100 mL of E3 fermentation stock solution, centrifuge at 8000 rpm for 15 min, discard the precipitate, slowly add HCl to the fermentation supernatant while stirring, adjust the pH value to 2.0, and let stand at 4 °C overnight. The precipitated protein was centrifuged at 8000 r/min for 15 min, the supernatant and the precipitate after salting out the protein were collected, and the precipitate was extracted with 5 times the volume of acetone. After extraction, centrifuge at 8000 r/min for 15 minutes, collect the supernatant, extract the precipitate again with the same organic solvent, centrifuge, and collect the supernatant extract. The two extracts were air-dried in a fume hood to obtain crude extracts, and the air-dried crude extracts were collected and stored at -80°C.

2. Antibacterial lipopeptide antibacterial spectrum test

In order to test whether the crude lipopeptide has a broad spectrum, some pathogenic bacteria were selected for the antibacterial spectrum test of crude lipopeptide. Aspergillus niger (strain number GIM3.576), Aspergillus flavus (strain number GIM3.493), Fusarium solani (strain number GIM 3.501), Fusarium nival (strain number GIM 3.503) (the described Aspergillus niger, Aspergillus flavus, Fusarium solanacearum, Fusarium nivalensis were purchased from Guangdong Provincial Institute of Microbiology) vacuum freeze-dried strains were activated and cultivated on PDA plates, growing out Mycelium preservation is performed after the mycelium. The fungal antibacterial activity test adopts the plate confrontation method. Select an appropriate position on the PDA plate to place two bacterial cakes, punch a hole with a 5 mm punch in the middle of the bacterial cake, add 10 μL of crude lipopeptide, incubate at 28 °C, and observe. Fungal growth. The bacteriostatic activity was tested by agar diffusion method, and the crude lipopeptide was tested against Escherichia coli DH5α (NCBI code: GCA_000755445.1), citrus canker Xanthomonas citri pv.citri strain jx-6 (NCBI code: NZ_CP011827.2) , Dickya zeae EC1 (NCBI number GCA_000816045.1), Ralstonia solanacearum GMI1000, NCBI number GCA_000009125.1), Burkholderia cepacia (strain Burkholderia cenocepacia H111, NCBI number GCF_000236215. 2), the antibacterial activity of banana base rot pathogen (Dickya zeae MS1, NCBI number is PRJNA194072) (described Laurella solanaceae, rice base rot bacteria, Burkholderia cepacia, banana base rot bacteria and Citrus canker is a preserved strain in the laboratory, and the relevant information of the strain has been published on NCBI), add the corresponding overnight cultured pathogen in the LB medium at a ratio of 1:100, pour the plate to air dry, and punch it with a 5mm punch. 10 μL of crude lipopeptide was added to the well, and incubated at 28°C.

Test crude lipopeptide against Aspergillus niger, Aspergillus flavus, Fusarium solanacearum, Fusarium nivale, Escherichia coli, Citrus canker, S. The bacteriostatic effect of pathogenic bacteria, the results are shown in Figure 6: the crude lipopeptide of E3 strain not only has biocontrol effect on rice basal rot fungus, but also on Fusarium solanacearum, citrus canker fungus, banana basal rot fungus, R. solanacearum, Escherichia coli also showed the function of biological control. However, it has no control effect on Aspergillus niger, Aspergillus flavus, Fusarium nivalis and Burkholderia cepacia. It can be seen that the crude lipopeptide of Bacillus amyloliquefaciens E3 has a certain inhibitory effect on bacteria and fungi, and has a broad antibacterial spectrum.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

<120> A strain of Bacillus amyloliquefaciens, fermentation product and its preparation method and application

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<223> Primer F27

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agagtttgat catggctcag 20

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<223> Primer R1492

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gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120

aaccggggct aataccggat ggttgtctga accgcatggt tcagacataa aaggtggctt 180

cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 240

caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300

gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360

gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga 420

acaagtgccg ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa 480

ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540

taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 600

ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660

gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720

tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780

cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840

ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg 900

cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960

cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt 1020

ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080

aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa 1140

accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200

gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat 1260

ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa 1320

tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380

ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct ttatggagcc agccgccgaa 1440

gtaagaagtt atgact 1456

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<400> 4

caggctcaca ctttgtcttg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer Bsu-man-1R

<400> 5

tgaacacagt cctgggttag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer Bam-man-1F

<400> 6

tcggtttcac atccttcatc 20

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer Bam-man-1R

<400> 7

tttgtcagcg tgtcttctg 19

Claims (7)

1. A bacillus amyloliquefaciens is characterized in that:

is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) E3, deposited in the China center for type culture Collection at Wuhan university in Wuhan, China in 2017, 12 months and 7 days, with the deposition number of CCTCC NO: m2017768.

2. The method for producing a fermentation product of Bacillus amyloliquefaciens according to claim 1, comprising the steps of:

inoculating the bacillus amyloliquefaciens into an LB liquid culture medium, and culturing at the temperature of 28 ℃ and the rpm of 160-200 for 48-72 hours.

3. The method for producing a fermentation product of bacillus amyloliquefaciens according to claim 2, wherein:

the inoculation amount of the bacillus amyloliquefaciens is 1 percent.

4. A fermentation product characterized by:

the method for producing a fermentation product of Bacillus amyloliquefaciens according to claim 2 or 3.

5. A microbial inoculant characterized by:

comprising at least one of the Bacillus amyloliquefaciens of claim 1 and the fermentation product of claim 4.

6. Use of the bacillus amyloliquefaciens according to claim 1 and/or the fermentation product according to claim 4 for controlling rice diseases.

7. Use according to claim 6, characterized in that:

the rice disease is rice basal rot.

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