CN111349589B - Bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus roxburghii and application thereof - Google Patents

Abstract

The invention discloses bacillus amyloliquefaciens for preventing and treating stem rot of anoectochilus formosanus and application thereof, and belongs to the technical field of crop disease prevention and treatment. The strain is bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) GZ5, which has been registered and preserved in the China general microbiological culture Collection center on 21.11.2019 with the preservation number of CGMCC No. 18987. The strain GZ5 is obtained from healthy tomato rhizosphere soil in Guangxi county, Fujian province, is harmoniously compatible with soil ecology, and is non-toxic and non-pathogenic. The bacillus amyloliquefaciens GZ5 strain is obtained by screening through a plate confrontation test, can effectively inhibit hypha growth and spore germination of the stem rot pathogen of the anoectochilus roxburghii, has good compatibility with the environment, and has good development and application prospects.

Description

A kind of Bacillus amyloliquefaciens for controlling clematis stem rot and its application

technical field

The invention relates to Bacillus amyloliquefaciens for preventing and controlling stem rot of clematis and application thereof, and belongs to the technical field of crop disease control.

Background technique

Clematis is a rare Chinese medicinal material in my country, and the whole plant can be used as medicine either fresh or dried. Because wild clematis has strict requirements on the ecological environment, poor adaptability, low reproduction rate, slow growth, and due to its unique nutritional value and medicinal value, it has been artificially over-excavated and has become an endangered species, which has been listed in Fujian Province. It is a protected species of provincial wild medicinal materials. At present, clematis is mainly cultivated artificially, but the problem of disease has become increasingly prominent during the cultivation process. Among them, stem rot caused by Fusarium oxysporum is one of the main diseases in clematis cultivation, which can cause harm to clematis in different growth periods. At the time of the disease, yellow-brown water-soaked lesions appeared at the base of the stem of the plant, which rapidly developed to a circle around the stem, and the diseased part was dry and rotten. Stem rot disease develops rapidly, and seedlings quickly collapse and die. When the disease is serious, the damage rate is as high as 90%, which brings huge economic losses to growers.

There are few reports on the comprehensive prevention and control of clematis stem rot. Chemical control is the main control method for the disease in current production. The research by Shao Qingsong and others showed that the inhibitory effect of tebuconazole EC on clematis stem rot bacteria Better, its inhibitory virulence is 92.50 times that of carbendazim. In addition, commonly used fungicides include chlorothalonil, mancozeb and thiophanate-methyl. Chemical fungicides have played a certain role in the prevention and control of clematis stem rot, but with the large and unscientific use of chemical fungicides, many disadvantages have become increasingly prominent, such as pathogen resistance, environmental pollution and ecosystem damage. damage, pesticide residues, etc. In recent years, with the deepening of people's understanding of ecological agriculture, the exploration of effective and environmentally compatible control measures has attracted widespread attention from scholars at home and abroad. Biological control is an important development concept in current agricultural practice. It has the characteristics of being harmless to humans and animals, non-polluting, non-residue, renewable, difficult to make harmful organisms resistant to drugs, easy to industrialize, and low cost. The use of pesticides to reduce the environmental pollution of pesticides has very unique advantages, which are in line with the current trend of sustainable agricultural development in my country and have extremely broad development prospects.

Bacillus amyloliquefaciens is a gram-positive bacterium with high affinity with bacillus subtilis, widely distributed in nature, easy to isolate and culture, and has broad-spectrum antibacterial activity, does not pollute the environment, and is non-toxic to humans and animals , Strong resistance to stress and good stability. It can produce a variety of high-efficiency active metabolites, effectively inhibit the activity of fungi and other bacteria, and at the same time promote plant growth and induce organisms to produce resistance, and control the growth of plant pathogens and the damage to organisms in many ways. At present, the fungus has been widely used in the control of flowers, fruits and various crop diseases. Studies by Qiu Guang et al. have shown that Bacillus amyloliquefaciens can rapidly colonize the rhizosphere of strawberries, preventing and controlling soil-borne diseases, improving disease resistance of strawberries, improving strawberry quality and increasing yield; studies by He Bibo et al. The germination rate of seeds and the plant height, root length, fresh and dry weight of sesame seedlings were significantly promoted, and it had a control effect on sesame stem spot blight. Studies have shown that Bacillus amyloliquefaciens can produce the plant growth hormone IAA, stimulate crop growth and induce resistance in its defense system, thereby inhibiting plant pathogens and preventing plant diseases, while promoting the growth of plant roots and increasing crop yield.

So far, there have been relevant reports on the chemical control of clematis stem rot, but there are few reports on the biological control of clematis stem rot. A strain of Bacillus amyloliquefaciens GZ5 was isolated and obtained in our laboratory in the early stage, and this strain was used as a biocontrol bacteria to study its inhibitory effect on mycelial growth and strain fermentation of Fusarium oxysporum, a pathogen of clematis stem rot. The inhibitory effect of the liquid and its sterile filtrate on the mycelial growth and spore germination of the clematis stem rot pathogen provides a practical basis for the biological control of clematis stem rot and the in-depth development and application of the biocontrol bacteria.

SUMMARY OF THE INVENTION

The object of the present invention is: for the current clematis stem rot serious occurrence, and the area expands year by year, the chemical control effect is not ideal, brings the problem of environmental pollution simultaneously, a kind of amyloliquefaciens spore for preventing and treating clematis stem rot is provided Bacilli and their applications.

The present invention is achieved through the following technical solutions:

A Bacillus amyloliquefaciens for the prevention and treatment of clematis stem rot, identified as Bacillus amyloliquefaciens GZ5 by morphological observation, observation of culture characteristics and molecular biology research, and has been preserved in China on November 21, 2019 The Ordinary Microbiology Center of the Microbial Culture Collection Management Committee, the address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the culture collection number is CGMCC No.18987.

The strain GZ5 was streaked in LB medium (10 g tryptone, 5 g yeast powder, 5 g sodium chloride and 15 g agar powder, distilled water was added to the volume to 1000 mL, dissolved and adjusted to pH 7.0-7.2, autoclaved. The colony characteristics and morphological characteristics were observed after culturing at 37 °C for 48 h. Colony characteristics: The colony of this strain is round, milky white, opaque, with neat edges and a raised center. The surface is moist and slightly sticky. Morphological characteristics: Strain GZ5 is Gram-positive, with short rod-shaped cells, obtuse ends at both ends, endospores, sporangia not inflated, oval-shaped spores, meso- to sub-terminal.

The biocontrol agent prepared by the described Bacillus amyloliquefaciens strain, its preparation process is as follows:

The strains were inoculated into LB liquid medium at 37°C, 180 r/min, and cultured for 24 h to prepare seed liquid, and 10 mL of seed liquid was inoculated into the fermentation medium. The optimized fermentation medium formula was as follows: 10 g tryptone, 5 g yeast powder, 5 g sodium chloride and 15 g agar powder were added to distilled water to make up to 1000 mL, dissolved and adjusted to pH 7.0-7.2. Through the exploration of the culture conditions, the optimal fermentation conditions were obtained: the inoculum volume was 5% (v/v), the bottling volume was 100mL/250mL, the pH value of the fermentation medium was 7.0-7.2, 37°C, 180 r/min, and the culture was 48 h, the fermentation broth of strain GZ5 with a concentration of about 10 ⁸ CFU/ml was prepared. The fermentation broth was centrifuged at 4 °C and 8000 r/min for 20 min, and then filtered with a 0.22 μm bacterial filter to obtain GZ5 sterile body fermentation broth, which was the biocontrol agent.

The application of described Bacillus amyloliquefaciens GZ5 in the prevention and treatment of clematis stem rot, specifically in the method for inhibiting clematis stem rot mycelial growth and spore germination as follows:

Inhibitory effect of strain GZ5 on the growth of mycelial mycelium of clematis stem rot pathogen

Take the spare PDA and LB medium, melt them in a microwave oven, and mix them in equal proportions. Inoculate the Fusarium oxysporum mycelial block (D=7 mm) in the center of the PDA and LB mixed culture dish in equal proportions, at 2.5 cm from the opposite sides of the mycelial block, dip the GZ5 bacterial solution on the plate with an inoculation ring and scratch it. Line, only received the same size of Fusarium oxysporum mycelial block as the control, repeated 3 times, placed in a 28 ℃ constant temperature incubator for cultivation, and the antibacterial rate was counted.

Inhibitory effect of fermentation broth of strain GZ5 on mycelial growth of clematis stem rot pathogen

Place the Fusarium oxysporum mycelium block (D=7 mm) in the center of the PDA plate, place sterilized filter paper sheets with a diameter of 5 mm at 2.5 cm away from the upper and lower relative positions of the mycelial block, and add 10 mL dropwise to each of the filter paper sheets For the fermentation broth of strain GZ5, the same size of Fusarium oxysporum mycelial block was used as the control, repeated three times, and cultured at a constant temperature of 28 °C, and the antibacterial rate was counted.

Inhibitory effect of fermentation broth of strain GZ5 on spore germination of clematis stem rot pathogen

The germination rate of spores was determined by the concave glass slide spore germination method, that is, the fermentation broth of strain GZ5 was added dropwise to the concave glass slide in a volume ratio of 1:1:1, and the spore suspension of Fusarium oxysporum at a concentration of 1×10 ⁵ cells/mL was used. liquid, 1 wt.% aqueous glucose solution, and LB liquid medium was added to the control group to replace the fermentation broth. The concave glass slides were placed in a large petri dish covered with wet filter paper and sealed with plastic wrap. After 24 hours of treatment, the spore germination of Fusarium oxysporum was observed under an optical microscope. The germination standard was that the germ tube length reached the spore. If the diameter is half, if there is no germination after 24 hours or the number of germination is small, observe it every 2 hours, and observe at least 5 fields of view, and observe the germination of 30-40 spores in each field of view.

Inhibitory effect of aseptic fermentation broth of strain GZ5 on the growth of mycelium of clematis stem rot pathogen

Add 2 mL of the aseptic fermentation broth of strain GZ5 into the melted 20 mL of PDA medium cooled to about 45 °C, mix and pour it into a plate to prepare a bacteriostatic plate of the aseptic fermentation broth of GZ5. Fusarium oxysporum hyphae with a diameter of 7 mm were inoculated in the center of the plate. Add 2 mL of LB liquid medium to the melted 20 mL of PDA medium cooled to about 45 °C, mix and pour the plate, and also inoculate a 7 mm diameter Fusarium oxysporum mycelial block in the center of the plate as a control, repeat 3 times , placed in a 28 ℃ incubator, and the growth diameter of the colony was measured by the cross method, and the antibacterial rate was calculated.

Inhibitory effect of aseptic fermentation broth of strain GZ5 on spore germination of clematis stem rot pathogen

The germination inhibition rate of spores was determined by the concave glass slide spore germination method, and the sterile body fermentation broth of strain GZ5 was sequentially dropped on the concave glass slide in a volume ratio of 1:1:1, with a concentration of 1×10 ⁵ spores/mL of cuspidium. Fusarium spore suspension, 1% aqueous dextrose solution, and control were added LB liquid medium instead of sterile body fermentation broth, and each treatment was repeated 3 times. The spore germination of Fusarium oxysporum was observed under a light microscope after 24 h of treatment. The germination standard was that the length of the germ tube reached half the diameter of the spore. If the germ tube did not germinate or the number of germination was small after 24 h, observation was performed every 2 h. Once, observe at least 5 fields of view, and observe the germination of 30-40 spores in each field of view.

Beneficial effects of the present invention:

1. The Bacillus amyloliquefaciens GZ5 screened by the present invention has a strong inhibitory effect, and can obviously inhibit the mycelial growth and spore germination of the clematis stem rot fungus. Since the strain is isolated from the tomato rhizosphere soil, it is harmonious and compatible with the soil ecology, and it is a biological agent, so it does not have a series of problems caused by the use of chemical pesticides, can reduce agricultural pollution, and can effectively prevent clematis stem rot, Has a good development and application prospects.

2. The Bacillus amyloliquefaciens strain of the present invention has simple culture conditions, is easy to preserve, and is easy to industrialize production, and is an ideal biocontrol bacteria.

Description of drawings

Figure 1 Inhibitory effect of strain GZ5 on the growth of mycelium of clematis stem rot pathogen.

Fig. 2 Inhibitory effect of the fermentation broth of strain GZ5 on the growth of mycelium of the pathogenic fungus of clematis stem rot.

Fig. 3 The inhibitory effect of the fermentation broth of strain GZ5 on the spore germination of clematis stem rot pathogen.

Fig. 4 Inhibitory effect of sterile body fermentation broth of strain GZ5 on mycelial growth of clematis stem rot pathogen.

Fig. 5 Inhibitory effect of the sterile body fermentation broth of strain GZ5 on spore germination of clematis stem rot pathogen.

Fig. 6 Electron microscope observation of strain GZ5 after in vivo treatment of clematis stem rot pathogen.

Detailed ways

In order to better understand the present invention, the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified.

Example 1 Isolation and identification of antagonistic bacteria

l. Bacteria isolation

Gently shovel the topsoil around the rhizosphere of healthy tomato plants, use a soil sampler to take soil samples at a depth of 5-10 cm, pour them into sterilized ziplock bags, make real-time records, and bring them back to the laboratory for separation . Randomly weigh 10 g of each soil into a 250 mL conical flask containing 90 mL of sterile water and a small amount of glass beads, fix the bottle with sealing film, place it on a 200 r/min shaker for 30 min, and let it stand for 5 min Then, use a pipette to suck up 1 ml of the soil sample suspension, add it into a test tube containing 9 ml of sterile water, and mix it well, that is, ^10-2 times the soil sample dilution. Using the same method, prepare 10-2 times in turn ^{. 3} , ^10-4 , ^10-5 , ^10-6 times soil sample dilution. Take 100 ul of the prepared soil sample dilutions of each gradient multiple and spread them evenly on LB plates, seal them with parafilm, and place the plates upside down in a 37°C biochemical incubator for 2-4 d. Observe the color, shape, transparency and other characteristics of the grown bacterial colonies, and pick out different types of single colonies for repurification.

The antagonistic strains of Fusarium oxysporum were screened by plate confrontation method. Take the spare PDA and LB medium, melt them in a microwave oven and mix them in equal proportions. Use a hole punch with a diameter of 7 mm to punch out the bacterial cake with the same diameter at the edge of the activated Fusarium oxysporum colony, and then inoculate the bacterial cake in the center of the PDA and LB mixed petri dish in equal proportions, and at the same time 2.5 from the opposite sides of the bacterial cake. At cm, dip the activated antagonistic solution to be tested on the LB medium with an inoculating loop and draw a line, and inoculate only the same size of Fusarium oxysporum cake in the center of the plate as a control, each treatment is repeated 3 times, and placed Incubate in a constant temperature incubator at 28 °C, and measure the size of the inhibition zone when the control group grows to the edge of the plate.

, morphological observation

The antagonistic strain GZ5 with the most obvious inhibition zone was streak-inoculated in LB medium (10 g tryptone, 5 g yeast powder, 5 g sodium chloride and 15 g agar powder plus distilled water to make up to 1000 mL, dissolve and adjust pH. 7.0-7.2, autoclaved for later use), and observed colony characteristics and morphological characteristics after culturing at 37 °C for 48 h. Colony characteristics: The colony of this strain is round, milky white, opaque, with neat edges and a raised center. The surface is moist and slightly sticky. Morphological characteristics: Strain GZ5 is Gram-positive, with rod-shaped cells, obtuse ends at both ends, endospores, sporangia not inflated, spores oval, mesogenic to subterminal. According to the morphological characteristics, it is preliminarily determined that the strain GZ5 belongs to the genus Bacillus sp .

, 16S rDNA sequence analysis

After the genomic DNA of the strain GZ5 was extracted by the genome extraction kit, the universal primers L1: 5-AGTCGTAACAACGTAGCCGT-3' and L2: 5-GTGCCAAGGCATCCACC-3' were used for PCR amplification of 16S rDNA. The PCR amplification products were recovered, and after ligation, transformation and identification, the positive clones were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the full-length sequence was 1000 bp (see Sequence). The obtained sequence was submitted to the GenBank database for BLAST analysis and alignment, and it was found that it had more than 99% sequence homology with Bacillus amyloliquefaciens.

sequence:

GTGCAAGGGCGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGATTTATTTCGAAGCAACGCGAAGATCTTACCAGTTTTGACATCCTCTGATATCCTAGAGATAGACGTCCCT .

Combining morphological and cultural characteristics, strain GZ5 was identified as Bacillus amyloliquefaciens .

Example 2: Inhibitory effect of strain GZ5 in vivo on the growth of clematis stem rot pathogenic mycelium

Take the spare PDA and LB medium, melt them in a microwave oven, and mix them in equal proportions. Inoculate the Fusarium oxysporum mycelial block (D=7 mm) in the center of the PDA and LB mixed culture dish in equal proportions, at 2.5 cm from the opposite sides of the mycelial block, dip the GZ5 bacterial solution on the plate with an inoculation ring and scratch it. Line, only received the same size of Fusarium oxysporum mycelial block as the control, repeated 3 times, placed in a 28 ℃ constant temperature incubator for cultivation, and the antibacterial rate was counted. The results are shown in Figure 1. The hyphae of Fusarium oxysporum grew vigorously on the medium without GZ5 in vivo, while the growth of hyphae was significantly inhibited on the medium containing GZ5 in vivo. After 6 days of culture, the inhibition rate of mycelial growth was 63.75%.

Embodiment 3: the inhibitory effect of bacterial strain GZ5 fermentation broth on the growth of clematis stem rot pathogenic fungus mycelium

Place the Fusarium oxysporum mycelium block (D=7 mm) in the center of the PDA plate, place sterilized filter paper sheets with a diameter of 5 mm at 2.5 cm away from the upper and lower relative positions of the mycelial block, and add 10 mL dropwise to each of the filter paper sheets For the fermentation broth of strain GZ5, only the same size of Fusarium oxysporum mycelial block was used as a control, repeated three times, placed at 28 °C for constant temperature cultivation, and the antibacterial rate was counted. The results are shown in Figure 2, the fermentation broth of strain GZ5 has obvious inhibitory effect on the growth of Fusarium oxysporum mycelium, and the inhibition rate of mycelium growth after 6 days of culture is 62.58%.

Embodiment 4: Inhibitory effect of bacterial strain GZ5 fermentation broth on spore germination of clematis stem rot pathogen

The germination rate of spores was determined by the concave glass slide spore germination method, that is, the fermentation broth of strain GZ5 was added dropwise to the concave glass slide in a volume ratio of 1:1:1, and the spore suspension of Fusarium oxysporum at a concentration of 1×10 ⁵ cells/mL was used. liquid, 1 wt.% aqueous glucose solution, and LB liquid medium was added to the control group to replace the fermentation broth. The 28 ℃ moisturizing culture is to place the concave glass slide in a large petri dish covered with wet filter paper and seal it with plastic wrap. After 24 hours of treatment, the spore germination of Fusarium oxysporum is observed under an optical microscope. The germination standard is that the germ tube length reaches The diameter of the spores is half. If the spores have not germinated after 24 h or the number of germinations is small, observe them every 2 h, and observe at least 5 fields of view for each treatment, and observe the germination of 30-40 spores in each field of view. The results are shown in Figure 3. After the GZ5 fermentation broth was treated for 24 h, the spore germination rate was 11.1%, and the spore germination inhibition rate was 78.83%, indicating that the GZ5 fermentation broth significantly inhibited the spore germination of Fusarium oxysporum.

Example 5: Inhibitory effect of bacterial strain GZ5 aseptic fermentation broth on mycelial growth of clematis stem rot pathogenic bacteria

Add 2 mL of the sterile body fermentation broth of strain GZ5 into the melted 20 mL PDA medium cooled to about 45 °C, mix and pour it into a plate to prepare a bacteriostatic plate of the sterile body fermentation broth of GZ5. Fusarium oxysporum hyphae with a diameter of 7 mm were inoculated in the center of the plate. Add 2 mL of LB liquid medium to the melted 20 mL of PDA medium cooled to about 45°C, mix and pour the plate, and inoculate a 7 mm diameter Fusarium oxysporum mycelial block in the center of the plate as a control, repeat 3 times , placed in a 28 ℃ incubator, and the growth diameter of the colony was measured by the cross method, and the antibacterial rate was calculated. The results are shown in Figure 4. In the medium added with GZ5 sterile body fermentation broth, the growth of Fusarium oxysporum was slow and sparse, and the amount of mycelium was significantly reduced. After 6 days of culture, the growth diameter of the colony was 22 mm. Mycelial growth inhibition rate was 63.33%.

Example 6: Inhibitory effect of bacterial strain GZ5 aseptic fermentation broth on spore germination of clematis stem rot pathogen

The germination inhibition rate of spores was determined by the concave glass slide spore germination method, and the sterile body fermentation broth of strain GZ5 was added dropwise to the concave glass slide in a volume ratio of 1:1:1, with a concentration of 1×10 ⁵ cells/mL of Fusarium oxysporum. Bacterial spore suspension, 1 wt.% aqueous glucose solution, and control, LB liquid medium was added to replace the sterile body fermentation broth, and each treatment was repeated 3 times. The spore germination of Fusarium oxysporum was observed under a light microscope after 24 h of treatment. The germination standard was that the length of the germ tube reached half the diameter of the spore. If the germ tube did not germinate or the number of germination was small after 24 h, observation was performed every 2 h. once. At least 5 visual fields were observed for each treatment, and 30-40 spore germinations were observed in each visual field. The results are shown in Figure 5. After 24 hours of treatment with the aseptic body fermentation broth, the spore germination rate was 26.73%, and the spore germination inhibition rate was 54.59%, indicating that the aseptic body fermentation broth of strain GZ5 had a certain effect on the spore germination of Fusarium oxysporum. inhibition.

Embodiment 7: Electron microscope observation of strain GZ5 in vivo treatment of clematis stem rot pathogen

After the treatment of Fusarium oxysporum according to the method of Example 2, a small amount of mycelium at the edge of the inhibited colony was picked in the experimental group, and the mycelium at the edge of the colony was picked in the control group, and the mycelia were observed under an electron microscope respectively. Shape. The results are shown in Figure 6. The control mycelium grew vigorously, the mycelia were long, uniform, and plump, while the experimental group had deformed mycelia, shriveled and shrunken. It can be seen that GZ5 in vivo treatment can cause obvious changes in the hyphal morphology of Fusarium oxysporum.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Institute of Plant Protection, Fujian Academy of Agricultural Sciences

<120> A kind of Bacillus amyloliquefaciens for preventing and treating clematis stem rot and its application

<130> 3

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1000

<212> DNA

<213> Artificial sequences

<400> 1

gtgcaagggc ggcgtgctat acatgcaagt cgagcggaca gatgggagct tgctccctga 60

tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 120

cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt 180

ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240

ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300

acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360

tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420

gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg 480

gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540

gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600

gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660

tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720

gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780

cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840

acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900

gggggcccgc acaagcggtg gagcatgtga tttatttcga agcaacgcga agatcttacc 960

agttttgaca tcctctgata tcctagagat agacgtccct 1000

<210> 2

<211> 20

<212> DNA

<213> Artificial sequences

<400> 2

agtcgtaaca acgtagccgt 20

<210> 3

<211> 17

<212> DNA

<213> Artificial sequences

<400> 3

gtgccaaggc atccacc 17

Claims (5)

1. a Bacillus amyloliquefaciens bacterial strain for the prevention and treatment of clematis stem rot, is characterized in that, described bacterial strain is amyloliquefaciens Bacillus amyloliquefaciens GZ5 has been deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee on November 21, 2019, with the preservation number of CGMCC No. 18987. 2. the biocontrol agent prepared by the Bacillus amyloliquefaciens strain as claimed in claim 1. 3 . The biocontrol agent according to claim 2 , wherein the biocontrol agent is obtained by fermentation of the Bacillus amyloliquefaciens strain GZ5. 4 . 4. The biocontrol agent of claim 3, wherein the bacterial strain is inoculated in LB liquid medium at 37°C, 180 r/min, and cultivated for 24 h to prepare seed liquid, and 10 mL of seeds are taken. The liquid was inoculated into the fermentation medium, and the fermentation conditions were as follows: the inoculum volume was 5% (v/v), the bottling volume was 100mL/250mL, the pH value of the fermentation medium was 7.0-7.2, 37°C, 180 r/min, and cultured for 48h. The fermentation broth of strain GZ5 with a concentration of 10 ⁸ CFU/ml was obtained; the fermentation broth was centrifuged at 4°C, 8000 r/min for 20 min, and filtered with a 0.22 μm bacterial filter to obtain a sterile fermentation broth, which was the biocontrol The formula of the fermentation medium is: 10 g of tryptone, 5 g of yeast powder, 5 g of sodium chloride and 15 g of agar powder and distilled water to make up to 1000 mL, dissolve and adjust the pH to 7.0-7.2. 5. the application of Bacillus amyloliquefaciens strain as claimed in claim 1 in preventing and treating clematis stem rot.

Images