Biocontrol bacillus amyloliquefaciens WYP1-4 for degrading feathers and application of biocontrol bacillus amyloliquefaciens WYP1-4

Abstract

The invention discloses a biocontrol bacillus amyloliquefaciens WYP1-4 for degrading feathers and application thereof, and belongs to the technical field of microorganisms. The bacterial strain is classified and named as bacillus amyloliquefaciens WYP1-4, and is preserved in the Guangdong Microbiological Culture Collection Center, the preservation number is GDMCC No.65114, and the preservation address is the 5th floor, No.59 floor, Institute of Microbiology, Academy of Sciences, Guangdong Province. The feather degradation rate of the strain reaches 93% (28 h), and the strain has high industrial utilization value and has a good antagonistic effect on tea tree pathogenic bacteria pestalotiopsis pseudomonas CLBB1 and aspergillus niger YC01.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a biocontrol agent of feather-degrading Bacillus amyloliquefaciens WYP1-4 and its applications. Background Technology

[0002] my country is a major poultry farming and consumption country, generating a large amount of feather waste annually from poultry slaughter. While a small portion of this waste is used to produce high-value-added products such as down products and decorations, or converted into feather powder through steam pressure and / or chemical treatment for limited use as a dietary protein supplement in animal feed, the vast majority is discarded without proper utilization. Feathers have a very stable structure; direct disposal into the environment without proper treatment causes serious environmental pollution. Feathers contain over 90% protein and are a good source of peptides, amino acids, and minerals. However, the most abundant protein in feathers is keratin, which is difficult to digest and naturally degrade.

[0003] Keratin is an insoluble protein, chemically stable and difficult to degrade due to its numerous disulfide bonds, hydrogen bonds, and hydrophobic interactions. Traditional keratin processing mainly involves high-temperature, high-pressure hydrolysis of keratin to convert feathers into feed (feather meal), or acid-base hydrolysis to break down keratin into soluble peptides and amino acids. These methods consume large amounts of energy, placing a significant burden on the environment. Furthermore, high-temperature treatment often leads to the degradation of heat-sensitive amino acids such as methionine, lysine, and tryptophan, as well as the significant loss of essential amino acids, reducing the nutritional value of the hydrolysates. In recent years, microorganisms have shown great potential in keratin waste treatment (such as converting feathers into soluble proteins, peptides, and amino acids). Microorganisms can hydrolyze feather keratin by producing keratinase, avoiding the destruction of amino acids such as methionine, tryptophan, and lysine, thus achieving the biotransformation of feather waste into nutritionally balanced, digestible feather pyrolysates.

[0004] There are a large number of microorganisms in nature that can produce keratinase to degrade feathers, including bacteria, fungi and actinomycetes. However, the microorganisms currently used for feather degradation generally have problems such as low keratinase activity, poor thermal stability and low feather degradation efficiency, making them unsuitable for industrial-scale applications. Summary of the Invention

[0005] One technical problem solved by this invention is to provide a *Bacillus amyloliquefaciens* WYP1-4. Another technical problem solved by this invention is to provide an application of *Bacillus amyloliquefaciens* WYP1-4 in feather degradation. A further technical problem solved by this invention is to provide an application of *Bacillus amyloliquefaciens* WYP1-4 in antagonizing the tea tree pathogens *Pseudomonas pseudochaetus* CLBB1 and *Aspergillus niger* YC01. A third technical problem solved by this invention is to provide an application of the fermentation broth and / or volatile metabolites of *Bacillus amyloliquefaciens* WYP1-4 in antagonizing the tea tree pathogen *Pseudomonas pseudochaetus* CLBB1.

[0006] Technical Solution: To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] A strain of Bacillus amyloliquefaciens, classified as Bacillus amyloliquefaciens WYP1-4, has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 65114, located on the 5th floor of Building 59, Institute of Microbiology, Guangdong Academy of Sciences.

[0008] This strain was isolated and screened from distiller's grains.

[0009] Biological characteristics of the strain: The optimal growth temperature is 30-40℃, it can tolerate 50℃, and the optimal pH is 5.0-8.0.

[0010] Colony and cell morphology characteristics: Strain WYP1-4 grew well on LB solid medium. The colonies were milky white, raised, moist and smooth. When picked up with an inoculation needle, the colonies felt sticky and were not easy to pick up. As the culture time increased, the colony surface wrinkled. It is a Gram-positive strain with short rod-shaped cells.

[0011] The 16S rDNA nucleotide sequence of strain Bacillus amyloliquefaciens WYP1-4 is as follows:

[0012] CCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACA

[0013] GATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTG

[0014] CCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACC

[0015] GCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCA

[0016] TTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAG

[0017] GGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA

[0018] GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG

[0019] TTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGC

[0020] ACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT

[0021] ACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCT

[0022] TAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAAC

[0023] TTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGT

[0024] GGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAA

[0025] AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGC

[0026] TAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTG

[0027] GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG

[0028] TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTC

[0029] TGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTT

[0030] GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGAT

[0031] CTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGA

[0032] AGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAA

[0033] TGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCT

[0034] CAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGG

[0035] ATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACG

[0036] AGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTA

[0037] The gyrA nucleotide sequence of Bacillus amyloliquefaciens strain WYP1-4 is as follows:

[0038]

[0039] The 16S rDNA and gyrA sequences were BLAST-aligned on NCBI to construct a phylogenetic tree. This strain was found to be in the same branch as Bacillus amyloliquefaciens, and was identified as Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens WYP1-4.

[0040] The above-mentioned application of Bacillus amyloliquefaciens WYP1-4 in antagonizing plant pathogens.

[0041] The plant pathogens used in this application are *Pseudomonas pseudobulb* CLBB1 and *Aspergillus niger* YC01.

[0042] Application of the fermentation broth and / or volatile metabolites of Bacillus amyloliquefaciens WYP1-4 in antagonizing the plant pathogen Pseudomonas polychaete CLBB1.

[0043] The application, preparation of fermentation broth: Strain WYP1-4 was inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 24 h. The culture was then centrifuged at 10000 rpm and 4°C for 10 min. The resulting supernatant was collected and filtered using a 0.22 μm syringe filter.

[0044] The application, preparation of volatile metabolites: strain WYP1-4 is inoculated into LB solid medium and cultured at 37°C for 1-5 days to obtain volatile metabolites produced by strain WYP1-4.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0046] (1) The Bacillus amyloliquefaciens WYP1-4 provided by the present invention can directly utilize feather waste as a carbon and nitrogen source to produce keratinase with strong activity and rapidly decompose feathers. This strain can decompose feathers to only feather residue in 28 hours, with a feather degradation rate of 93%, which has high industrial utilization value.

[0047] (2) The Bacillus amyloliquefaciens WYP1-4 provided by the present invention has a good antagonistic effect on the tea tree pathogens Pseudomonas polychaete and Aspergillus niger.

[0048] (3) The fermentation broth and volatile metabolites of Bacillus amyloliquefaciens WYP1-4 provided by the present invention have a good antagonistic effect on the tea tree pathogen Pseudomonas polychaete. Attached Figure Description

[0049] Figure 1This is a graph showing the results of secondary screening of protease-producing strains;

[0050] Figure 2 These are photographs of the colony morphology of strains 1-4;

[0051] Figure 3 Microscopic morphological photographs of strains 1-4;

[0052] Figure 4 To construct a phylogenetic tree of Bacillus amyloliquefaciens based on the 16S rDNA sequence;

[0053] Figure 5 To construct a phylogenetic tree of Bacillus amyloliquefaciens based on the gyrA sequence;

[0054] Figure 6 Figure showing the effect of temperature on the growth of strain WYP1-4;

[0055] Figure 7 The graph shows the effect of pH on the growth of strain WYP1-4;

[0056] Figure 8 The figure shows the effect of NaCl content in the culture medium on the growth of strain WYP1-4.

[0057] Figure 9 The images show the appearance of the feather degradation medium and the feather degradation medium after 28 hours of degradation by strain WYP1-4.

[0058] Figure 10 The graph shows the changes in feather degradation rate at different fermentation times when strain WYP1-4 degrades feathers.

[0059] Figure 11 Figure showing the results of the antagonistic assay for Bacillus amyloliquefaciens strains and pathogens;

[0060] Figure 12 The figure shows the inhibition results of sterile filtrate of strain WYP1-4 culture medium on Polychaete pseudodiscoids.

[0061] Figure 13 The figure shows the inhibition results of volatile metabolites of strain WYP1-4 on Polychaete pseudodiscoids. Detailed Implementation

[0062] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0063] Example 1

[0064] strain screening

[0065] (1) Preliminary screening of bacterial strains

[0066] Dissolve 1g of distiller's grains in 9mL of sterile water, and spread 0.2mL evenly on the screening medium. Incubate at 37℃ for 48h. By measuring the diameter of the hydrolysis zone (D) and the diameter of the colony (d), and using the D / d value as the initial screening criterion, strains with a diameter-to-zone ratio >4.00 (Table 1) were screened and purified five times. All obtained strains were stored in glycerol cryovials (containing 20% ​​sterile glycerol) and stored at -20℃.

[0067] Table 1. Initial screening results of protease-producing strains

[0068] serial number Hydrolysis ring diameter Colony diameter Pivot diameter ratio D / d 1-1 12.67±0.89 3.61±0.83 3.63±0.76 1-2 13.26±0.03 2.70±0.26 4.95±0.51 1-3 11.20±0.70 3.18±0.54 3.57±0.44 1-4 12.90±1.58 2.18±0.47 6.00±0.52 1-5 8.62±0.41 1.76±0.27 4.95±0.58 1-6 4.53±1.07 1.44±0.14 3.15±0.74

[0069] (2) Secondary screening of strains

[0070] The strains (1-2, 1-4, and 1-5) obtained from the initial screening were activated and inoculated into LB medium. The cultures were then incubated at 37°C and 180 rpm for 12 h to obtain seed culture. The seed culture was inoculated into a secondary screening medium at a 2% inoculation rate and incubated at 37°C and 180 rpm for 24 h. The fermentation broth was then centrifuged at 12000 rpm for 10 min to obtain crude enzyme solution. The secondary screening medium (g / L) consisted of: glucose 20 g, soybean meal 40 g, wheat bran 30 g, potassium dihydrogen phosphate 0.3 g, and sodium hydrogen phosphate dodecahydrate 0.4 g.

[0071] By detecting the acidic, neutral, and alkaline protease activities of each strain, and following the protease activity assay method according to GB / T23527-2009 "Protein Preparations", strains 1-4 with relatively high activity were finally obtained. Figure 1 ).

[0072] Example 2

[0073] strain identification

[0074] (1) Observation of colony and cell morphology

[0075] Streaking of target strains 1-4 onto LB agar plates and incubation at 37℃ for 24 h showed good growth on LB solid medium. The colonies were milky white, raised, moist, and smooth. When picked up with an inoculation needle, the colonies felt sticky and were difficult to pick up. With prolonged incubation, the colony surface showed signs of shrinkage. Figure 2 Fresh bacterial cells were selected, Gram-stained, and examined under a microscope (10×100) to observe the morphology. This strain was Gram-positive, and the bacterial cells were short rod-shaped. Figure 3 ).

[0076] (2) Physiological and biochemical identification of the strain

[0077] The carbon source utilization, acid production, and enzyme activity of strains 1-4 were detected using HBI Bacillus biochemical identification strips (Qingdao Haibo Biotechnology Co., Ltd.). The specific procedures were performed according to the manufacturer's instructions. Table 2 shows that the strain was positive for VP test, gelatin liquefaction, nitrate reduction, and starch hydrolysis. It can grow under 7% NaCl and pH 5.7 conditions. Reactions for citrate, propionate, D-xylose, L-arabinose, and D-mannitol utilization were negative.

[0078] Table 2. Physiological and biochemical characteristics of strain WYP1-4

[0079]

[0080] (3) Molecular biological identification of the strain

[0081] The target strain was inoculated into LB medium and cultured at 37°C and 180 r / min for 24 h. DNA was extracted and 16S rDNA and gyrA were sequenced. The 16S rDNA nucleotide sequence is as follows:

[0082] CCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACA

[0083] GATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTG

[0084] CCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACC

[0085] GCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCA

[0086] TTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAG

[0087] GGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA

[0088] GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG

[0089] TTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGC

[0090] ACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT

[0091] ACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCT

[0092] TAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAAC

[0093] TTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGT

[0094] GGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAA

[0095] AGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGC

[0096] TAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTG

[0097] GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG

[0098] TGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTC

[0099] TGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTT

[0100] GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGAT

[0101] CTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGA

[0102] AGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAA

[0103] TGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCT

[0104] CAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGG

[0105] ATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACG

[0106] AGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTA

[0107] The gyrA nucleotide sequence is as follows:

[0108]

[0109] The obtained 16S rDNA and gyrA sequences were BLAST-aligned on NCBI, and a phylogenetic tree was constructed. The results are as follows: Figure 4 and Figure 5 As shown, this strain belongs to the same branch as Bacillus amyloliquefaciens, and it was identified as Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens WYP1-4.

[0110] The strain was deposited on September 9, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC, 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences, No. 100 Xianlie Middle Road, Guangzhou, China), with accession number GDMCC No. 65114.

[0111] Example 3

[0112] Biological characteristics study of strains

[0113] (1) Growth temperature experiment: The bacterial culture was inoculated into LB liquid medium at a 1% inoculum and cultured at 30℃, 40℃, 50℃ and 60℃, and 180 r / min. Samples were taken at 0h, 12h, 24h and 36h, and the OD value was measured at 600nm. The results of the growth temperature experiment are shown in […]. Figure 6 It can be seen that the strain grows well at 30-40℃, with the optimal growth temperature being 40℃, and can tolerate high temperatures of 50℃.

[0114] (2) Growth pH experiment: The bacterial culture was inoculated at a rate of 1% into LB liquid medium with initial pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively, and cultured at 37℃ and 180 r / min. OD was measured at 0h, 12h, 24h, and 36h. 600 The results of the growth pH experiment are shown below. Figure 7 It can be seen that the strain grows well at pH 5.0 to 8.0, with the optimal growth pH range being pH 5.0 to 7.0.

[0115] (3) Salt tolerance test: The bacterial culture was inoculated at a rate of 1% into culture media with NaCl concentrations of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, respectively, and cultured at 37℃ and 180 r / min. OD was measured at 0h, 12h, 24h, and 36h. 600 The experimental results are shown in [the table]. Figure 8It can be seen that the strain grows well in NaCl concentrations of 0.0-4.0%, with an optimal salinity of 2.0% and a maximum salt tolerance of 7.0%.

[0116] Example 4

[0117] Feather degradation test

[0118] The strain WYP1-4 was inoculated into the feather degradation medium at an inoculum of 7%, and cultured at 37℃ and 180r / min for 28h with shaking. The feather degradation phenomenon was observed, and the keratinase activity was measured.

[0119] Determination of keratinase activity: Take 200 μL of crude enzyme solution, preheat at 55℃ for 2 min, add 500 μL of 20% TCA (trichloroacetic acid) to the control group, and add 300 μL of the same preheated 20 g / L casein solution (pH 8.0) to the sample group. React in a water bath at 55℃ for 10 min. After the reaction, immediately add 20% TCA to the sample group to stop the reaction, and add 300 μL of casein to the control group. Let stand for 10 min, then centrifuge at 12000 rpm for 2 min. Take 400 μL of the supernatant, add 2 mL of 0.5 mol / L Na2CO3 and 400 μL of Folin-Ciocalteu reagent respectively, and measure the absorbance at 680 nm. Each experiment is repeated three times and the average value is taken.

[0120] Enzyme activity definition: Under conditions of pH 5.0 and 55℃, the production of 1 μg of tyrosine per minute is defined as one enzyme activity unit (U).

[0121] The feather degradation medium uses feathers as the sole carbon and nitrogen source. The medium is initially clear and transparent, but gradually becomes turbid as fermentation time increases and microorganisms grow. After 16 hours of culture, the shedding of down from the main shaft of the feathers is clearly visible. Figure 9 After 28 hours of fermentation, the culture medium became turbid, and most of the feathers had been degraded. Figure 10 As shown, after 28 hours of fermentation, the feather degradation rate reached 93%, and the keratinase activity reached 45.13 U / mL.

[0122] Feather degradation medium (g / L): Feather 2, Magnesium sulfate 0.1, Sodium chloride 0.5, Dipotassium hydrogen phosphate 1.4, Potassium dihydrogen phosphate 0.7, natural pH, sterilized at 121℃ for 30 min.

[0123] Example 5

[0124] Strain-pathogen antagonism assay

[0125] The agar plate confrontation experiment was used, inoculating strain WYP1-4 and the pathogen onto the same plate using a cross-inoculation method, and incubating at 30℃ for 4 days to observe the growth of the strain. *Pseudomonas pseudotrichous* CLBB1 and *Aspergillus niger* YC01 were selected as the test pathogens.

[0126] The fungus *Pseudomonas pseudochaetes* CLBB1 enters a dormant state, escaping the cold winter by attaching conidia or mycelium to diseased leaves. When temperature and humidity conditions are suitable the following year, it produces conidia to reproduce and begin a new round of infection on tea plants. *Pseudomonas pseudochaetes* CLBB1 typically infects tea plants from the leaf tips or edges, or through wounds. After a 1-2 week incubation period, it causes leaf disease, forming lesions. The fruiting bodies are spread by wind and rain, allowing for multiple reinfections. Infection by this pathogen leads to branch dieback, leaf decay, and weakened plant vigor, resulting in a severe decline in yield and quality, making it one of the major diseases affecting tea plants.

[0127] Aspergillus niger YC01 is an important plant pathogen that is widely distributed in grains, plant products and soil. It can easily multiply and cause grains to become moldy.

[0128] Results of strain-pathogen antagonism assay as follows Figure 11 As shown, Figure 11 The left image shows the experimental strain WYP1-4. This strain's growth is unaffected by the pathogen and exhibits good antagonistic ability, limiting the pathogen's growth. The right image shows LB medium used instead of the experimental strain; the pathogen grows well, covering the entire petri dish. The experimental results indicate that strain WYP1-4 has a good inhibitory effect on both *Pseudomonas pseudotrichous* CLBB1 and *Aspergillus niger* YC01.

[0129] Example 6

[0130] Inhibition test of fermentation supernatant of strain WYP1-4 against *Pseudomonas pseudochaetes* CLBB1

[0131] Strain strain WYP1-4 was inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 24 h. The culture was then centrifuged at 10,000 rpm and 4°C for 10 min. The supernatant was collected and filtered using a 0.22 μm syringe filter. The filtrate (CF) of this culture was used in an antifungal test against *Pseudomonas pseudodiscoids* CLBB1.

[0132] The CF of strain WYP1-4 was mixed with sterile, cooled PDA medium in an Erlenmeyer flask to achieve a final concentration of 30% (30% CF). A 6 mm diameter hyphal plug was taken from a 3-day-old colony of *Pseudomonas pseudotrichous* CLBB1 and placed in the center of the medium. The culture was incubated at 30°C for 5 days, and the antibacterial effect was observed. PDA medium without culture filtrate served as a control. Results are as follows: Figure 12 As shown, the growth of strain CLBB1 in the experimental group was inhibited, indicating that the sterile culture filtrate of strain WYP1-4 can inhibit the growth of *Pseudomonas pseudochaetes* CLBB1.

[0133] Example 7

[0134] Inhibition test of volatile metabolites (VOCs) of strain WYP1-4 against Polychaete pseudodiscoids

[0135] Strains WYP1-4 were inoculated into LB liquid medium and cultured at 37℃ and 180 r / min for 24 h. 100 μL of the culture was spread onto LB plates. A 6 mm diameter hyphal plug was taken from a 3-day-old colony of *Pseudomonas pseudochaetes* CLBB1 and placed in the center of a PDA medium. The bacterial and fungal plates were sealed together with a paraffin film. At the same time, a control LB plate coated with 100 μL of sterile distilled water was sealed together with the fungal plate in a similar manner. The plates were cultured at 30℃ for 5 days to observe the antibacterial effect.

[0136] The results are as follows Figure 13 As shown, the growth of strain CLBB1 in the experimental group was inhibited, indicating that strain WYP1-4 can produce volatile substances that inhibit the growth of *Pseudomonas pseudochaetes* CLBB1.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A species of Bacillus amyloliquefaciens, classified as Bacillus amyloliquefaciens WYP1-4, has been deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 65114, located at the 5th floor of Building 59, Institute of Microbiology, Guangdong Academy of Sciences.

2. The application of Bacillus amyloliquefaciens as described in claim 1 in the degradation of feathers.

3. The application according to claim 2, characterized in that, The amyloliquefaciens was inoculated into feather degradation medium and cultured by shaking.

4. The application according to claim 3, characterized in that, The inoculum size was 7%, and the culture conditions were: 37℃, 180r / min shaking culture for 28h.

5. The application according to claim 3, characterized in that, The formula for the feather degradation medium is as follows: feather 2 g / L, magnesium sulfate 0.1 g / L, sodium chloride 0.5 g / L, dipotassium hydrogen phosphate 1.4 g / L, potassium dihydrogen phosphate 0.7 g / L, natural pH, sterilized at 121℃ for 30 min.

6. The application of Bacillus amyloliquefaciens as described in claim 1 in antagonizing plant pathogens.

7. The application according to claim 6, characterized in that, The plant pathogens are *Pseudomonas pseudochaetes* CLBB1 and *Aspergillus niger* YC01.

8. The use of the fermentation broth and / or volatile metabolites of Bacillus amyloliquefaciens according to claim 1 in antagonizing the plant pathogen Pseudomonas polychaete CLBB1.

9. The application according to claim 8, characterized in that, Preparation of the fermentation broth: Strain strain WYP1-4 was inoculated into LB liquid medium and cultured at 37°C and 180 r / min for 24 h. The culture was then centrifuged at 10000 rpm and 4°C for 10 min. The resulting supernatant was collected and filtered using a 0.22 μm syringe filter.

10. The application according to claim 8, characterized in that, Preparation of the volatile metabolites: strain WYP1-4 was inoculated into LB solid medium and cultured at 37°C for 1-5 days to obtain the volatile metabolites produced by strain WYP1-4.

Images