AU2020418980A1

AU2020418980A1 - Endophytic bacillus from Pu'er tea tree leaves and application thereof

Info

Publication number: AU2020418980A1
Application number: AU2020418980A
Authority: AU
Inventors: Fangzhou LI; Wenyong LOU; Jiguo YANG; Yingjie ZENG; Minhua ZONG
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-12-30
Filing date: 2020-10-23
Publication date: 2022-08-25
Also published as: WO2021135544A1; CN111019866B; CN111019866A

Abstract

An endophytic bacillus from Pu'er tea tree leaves and an application thereof. The endophytic bacillus from Pu'er tea tree leaves is named Bacillus velezensis FZ06, and was deposited at the China Center for Type Culture Collection in Wuhan University, Wuhan, China on 25 October 2019, with deposit number CCTCC NO: M 2019854. The bacterium can produce lipopeptide substances, including 27 species in three categories, with a higher richness than other previously reported Bacillus velezensis. The lipopeptides produced have obvious inhibitory activity on pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus, and Aspergillus parasiticus, and can be used to inhibit pathogenic bacteria.

Description

ENDOPHYTIC BACILLUS FROM PU'ER TEA TREE LEAVES AND APPLICATION THEREOF

Field of the Invention

The present disclosure belongs to the technical field of microorganisms, and particularly relates to an endophytic bacillus from Pu'er tea tree leaves and an application thereof.

Background of the Invention

Lipopeptides are an important class of non-ionic biosurfactants, mainly derived from secondary metabolic pathways of some bacteria and fungi. They are diverse in species and have complicated structures. Lipopeptides are generally constituted of a fatty acid chain and a peptide chain which form a ring by a lactone or an amido bond. As a molecular weapon of microorganisms, the lipopeptides in the natural environment often have natural functions of anti-virus, antagonizing bacteria and even antagonizing fungi. Therefore, They are widely used in the development of plant disease and insect pest resistance reagents. As a class of the nonionic surfactants, the antibacterial mechanism of the lipopeptides may be similar to that of conventional antifungal surface-active molecules: the nonionic biosurfactants bind and separate essential proteins related to survival on a target fungal cell membrane, so that target fungi die. Due to the differences in structure and properties, the lipopeptides generated by different microorganisms theoretically have different antibacterial spectra, and a single compound often may not achieve broad bacteriostasis; and the adaptive evolution of the microorganisms is considered, a long-term application of a single antibiotic is likely to make some fungi generate drug resistance. However, if the lipopeptides with more diverse structures and richer properties are used in combination, the above problem may be solved. Therefore, looking for the microorganisms that may generate more abundant lipopeptide substances and using lipopeptide compounds thereof in combination are of great significance in aspects of expanding the antibacterial spectra of the lipopeptide compounds, further reducing the adaptive evolution of the antibiotics in the target fungi, and even reducing the bacteriostasis concentration.

Summary of the Invention

A primary purpose of the present disclosure is to overcome the shortcomings and deficiencies of the prior art, and to provide an endophytic bacillus from Pu'er tea tree leaves. Another purpose of the present disclosure is to provide an application of the above endophytic bacillus from the Pu'er tea tree leaves. Another purpose of the present disclosure is to provide a lipopeptide substance prepared by the above endophytic bacillus from the Pu'er tea tree leaves and an application thereof. In order to achieve the above purposes, the present disclosure is achieved by the following technical schemes: An endophytic bacillus from Pu'er tea tree leaves, named Bacillus velezensis FZ06, is separated and purified from fresh Pu'er tea tree leaves in Xishuangbanna. The endophytic bacillus from the Pu'er tea tree leaves is Bacillus velezensis FZ06, and its deposit information is as follows: deposit unit: China Center for Type Culture Collection (CCTCC), deposit date: October 25, 2019, deposit address: Wuhan University, Wuhan, China, and deposit number: CCTCC NO: M 2019854. The application of the endophytic bacillus from the Pu'er tea tree leaves in preparation of the lipopeptide substance preferably includes the following steps: (1) fermentation culture: inoculating seed solution of the endophytic bacillus from the Pu'er tea tree leaves into a fermentation medium for fermentation culture, to obtain fermentation solution; (2) acid precipitate separation and washing: performing first centrifugation on the fermentation solution obtained in the step (1) to remove bacterial cells, adding an acid to an obtained supernatant to adjust pH to 2.0, performing second centrifugation after standing to remove the supernatant, washing the obtained precipitate, and then performing third centrifugation, to obtain an acid precipitate; (3) acid precipitate extraction: adding the acid precipitate obtained in the step (2) to a solvent for extraction, then performing solid-liquid separation, drying the obtained liquid, and then adding water to redissolve and obtain an aqueous phase extract solution; (4) separation and purification of lipopeptide substance: performing first drying on the aqueous phase extract solution obtained in the step (3), adding the solvent to an obtained dry substance to redissolve, then performing chromatographic purification, performing second drying on an obtained eluate containing a lipopeptide substance, adding the water to redissolve, and finally performing third drying, to obtain the lipopeptide substance. The seed solution in the step (1) is preferably prepared by the following steps: activating the preserved endophytic bacillus from the Pu'er tea tree leaves, and expanding culture, to obtain the seed solution. The activating step is preferably as follows: inoculating the preserved endophytic bacillus from the Pu'er tea tree leaves into an activation medium, and culturing under a condition of 35-39°C for 24~48 hours, to obtain the activated endophytic bacillus from the Pu'er tea tree leaves. The cultural condition is preferably that it is cultured at 36-38°C for 24 hours. The activation medium is preferably a potato dextrose agar (PDA) medium. The step of expanding the culture is preferably as follows: inoculating the activated endophytic bacillus from the Pu'er tea tree leaves into a seed medium, and culturing under conditions of 35-39°C and 150~170 rpm for 24~48 hours, to obtain the seed solution. The inoculation amount of the activated endophytic bacillus from the Pu'er tea tree leaves is preferably 1~5% of the seed medium by a volume ratio; and more preferably 1% of the seed medium. The seed medium is preferably a liquid Luria-Bertani (LB) medium. The fermentation culture condition in the step (1) is preferably that it is cultured at 36-39°C and 150~170 rpm for 48-72 hours; and more preferably it is cultured at 36-38°C and 160 rpm for 72 hours. The first centrifugation condition in the step (2) is preferably that it is centrifuged at 4°C and 10,000 rpm for 10 min. The acid in the step (2) is preferably a hydrochloric acid; and more preferably the hydrochloric acid with a concentration of 6 mol/L. The standing condition in the step (2) is preferably that it is standing at 4°C for 12-24 hours; and more preferably it is standing at 4°C for 12 hours. The second centrifugation condition in the step (2) is preferably that it is centrifuged at 4°C and 10,000 rpm for 5 min. The washing in the step (2) is preferably that it is washed with a dilute hydrochloric acid; and more preferably it is washed with the dilute hydrochloric acid with pH=2.0. The washing times in the step (2) are preferably 2 times. The third centrifugation condition in the step (2) is preferably that it is centrifuged at 10,000 rpm for 10 min. The solvent in the step (3) is preferably an anhydrous methanol. The solid-liquid separation mode in the step (3) is preferably suction filtration; and more preferably vacuum suction filtration. The drying in the step (3) is preferably that it is hang-dried by concentration; more preferably it is hang-dried by vacuum concentration; and most preferably it is dried at 40°C by a rotary evaporator. The water in the step (3) is preferably deionized water. The first drying and the third drying in the step (4) are preferably freeze-drying. The solvent in the step (4) is preferably the anhydrous methanol. The chromatographic purification in the step (4) is preferably that the purification is performed by using a Sephadex gel chromatographic column; and more preferably a Sephadex LH-20 gel column is used, the eluent is 100% methanol, and the flow rate is 0.5 mL/min. The second drying in the step (4) is preferably that it is hang-dried by concentration; more preferably it is hang-dried by vacuum concentration; and most preferably it is rotated and evaporated to dryness at 40°C by the rotary evaporator. A lipopeptide substance is obtained by separation and purification of the fermentation broth by the above endophytic bacillus from the Pu'er tea tree leaves. The lipopeptide substance includes a Surfactin-like homologue, an Iturin-like homologue and a Fengycin-like homologue. The Surfactin-like homologue is preferably C 12-Leu -Surfactin, C 13-Leu 7 -Surfactin, C 14 -Leu7 -Surfactin, C 15 -Leu 7 -Surfactin, Ci 6 -Leu 7 -Surfactin, C 12-Val 7 -Surfactin, C13-Val 7 -Surfactin, C 14-Val 7-Surfactin, C 1 5 -Val7 -Surfactin and

C 14-Val 7 -Surfactin. The Iturin-like homologue is preferably C 14-Bacillomycin F, C1 5 -Bacillomycin F,

C 16-Bacillomycin F, C 17-Bacillomycin F, C 14-Bacillomycin D, C1 5 -Bacillomycin D, C 16-Bacillomycin D and C 17-Bacillomycin D.

The Fengycin-like homologue is preferably C 15-Ala -Fengycin, C 1 6-Ala -Fengycin, C 17-Ala -Fengycin, C 15-Abu6 -Fengycin, C 1 6-Abu6-Fengycin, C 17-Abu 6 -Fengycin, C 15-Val-Fengycin, C 1 6-Val-Fengycin and C1 7 -Val-Fengycin. The lipopeptide substance is applied in the inhibition of pathogenic microorganisms. The pathogenic microorganisms include but are not limited to Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillusfavus and Aspergillus parasiticus. The Escherichiacoli are preferably Escherichiacoli GIMI.708. The Staphylococcus aureus is preferably Staphylococcus aureus subsp. aureus GIM1.441. The Salmonella is preferably Salmonella choleraesuis subsp. choleraesuis GIM1.163. The Aspergillusfiavus is preferably Aspergillusfavus GIM 3.493. The Aspergillus parasiticus is preferably Aspergillus parasiticus Speare GIM 3.395. Compared with the prior art, the present disclosure has the following advantages and effects: (1) The present disclosure separates Bacillus velezensis FZ06 from fresh Pu'er tea tree leaves for the first time, the lipopeptide substances that the bacteria produce include 27 species of 3 categories, and its richness is more than that of Bacillus velezensis which is already reported at present. Herein, a polypeptide chain amino acid sequence of the Fengycin-like homologue containing an amino fatty acid chain with 15-17 carbon atoms is observed in the Bacillus velezensis for the first time. (2) The method for preparing the lipopeptide substance provided by the present disclosure is simple and easy to implement, mild in conditions, and easy to control, and provides a new path for large-scale production of the lipopeptide substance. (3) The lipopeptide substance produced by the Bacillus velezensis FZ06 in the present disclosure has the apparent inhibitory activity effect on the pathogenic microorganisms such as Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus and Aspergillus parasiticus, and may be well applied to inhibit the pathogenic microorganisms. A new idea is provided for improving the postharvest preservation performance or disease resistance of a host plant abscission substance by improving the interaction between the endophytic bacteria and a host plant.

Brief Description of the Drawings

Fig. 1 is a graph showing an antibacterial phenomenon of endophytic bacteria in Pu'er tea tree leaves antagonizing black spore-generating endophytic fungi. Fig. 2 is a scanning electron microscope (SEM) image of Bacillus velezensis FZ06: herein, A is the SEM image of the Bacillus velezensis FZ06 under 1000x magnification times; and B is the SEM image of the Bacillus velezensis FZ06 under 5000x magnification times. Fig. 3 is the phylogenetic tree diagram of 16S rDNA of the Bacillus velezensis FZ06. Fig. 4 is a total ion current diagram of lipopeptide substance solution prepared by the Bacillus velezensis FZ06: herein, A is the total ion current diagram of a Surfactin-like homologue; B is the total ion current diagram of an Iturin-like homologue; and C is a total ion current diagram of a Fengycin-like homologue. Fig. 5 is the graph showing the inhibitory effect of the lipopeptide substance prepared by the Bacillus velezensis FZ06 and sterile water on bacterial pathogens: herein, A is a graph showing an inhibitory effect of the lipopeptide substance prepared by the Bacillus velezensis FZ06 and the sterile water on Escherichia coli GIMI.708; B is a graph showing an inhibitory effect of the lipopeptide substance prepared by the Bacillus velezensis FZ06 and the sterile water on Staphylococcus aureus subsp. aureus GIM1.441; and C is a graph showing an inhibitory effect of the lipopeptide substance prepared by the Bacillus velezensis FZ06 and the sterile water on Salmonella choleraesuissubsp. choleraesuis GIM1.163. Fig. 6 is the graph showing an inhibitory effect of the lipopeptide substance prepared by the FZ06 and the sterile water on fungal pathogens: herein, A is a graph showing an inhibitory effect of the lipopeptide substance prepared by the Bacillus velezensis FZ06 and the sterile water on Aspergillus flavus GIM 3.493; and B is a graph showing an inhibitory effect of the lipopeptide substance prepared by the Bacillus velezensis FZ06 and the sterile water on Aspergillus parasiticusSpeare GIM 3.395.

Detailed Description of the Embodiments

The present disclosure is further described in detail below with reference to embodiments and drawings, but implementation modes of the present disclosure are not limited to this. Test methods that do not specify specific experimental conditions in the following embodiments are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by manufacturers. Materials, reagents and the like used, unless otherwise specified, are reagents and materials obtained from commercial sources.

Embodiment 1: Separation and identification of antifungal endophytic bacteria (endophytic bacillus from Pu'er tea tree leaves) (1) Pretreatment of fresh Pu'er tea tree leaves: collected fresh and complete Pu'er tea tree leaves (from Menghai County, Xishuangbanna, Yunnan Province) with the same leaf age are transported to a laboratory within 24 hours, and a surface disinfection operation is performed, it is specifically as follows: in a sterile operation bench, the fresh and complete Pu'er tea tree leaves are firstly soaked in 75% (volume fraction) ethanol solution for 15 seconds, then rinsed with sterile water and wipe-dried with sterile cotton; then, it is soaked in 3.25% (available chlorine content) sodium hypochlorite solution for 15 seconds, rinsed with the sterile water and wipe-dried with the sterile cotton; and the above surface disinfection operation is repeated for three times. The last time of rinse water is spread in a PDA medium, and then cultured at a temperature of 37°C for 2 weeks. If a microorganism does not appear, the leaf surface is considered to be completely disinfected. The fresh Pu'er tea tree leaves with complete surface disinfection are placed in a ventilated sterile bag, and stored at 4°C for 30 days. After 30 days, the complete Pu'er tea tree leaves that still remain green and do not have the apparent endophytic fungal growth leading to a decomposition phenomenon of the plant leaves are selected. The above surface disinfection operation is repeated for three times. The last time of rinse water is spread in the PDA medium, and then cultured at the temperature of 37°C for 2 weeks. If the microorganism does not appear, the leaf surface is considered to be completely disinfected. (2) Culture and separation of antifungal endophytic bacteria: The Pu'er tea tree leaves obtained in the step (1), which remain green and have the secondary disinfection on the surface, are aseptically sliced, to obtain a plant slice with a diameter of about 0.5 cm, the plant slice is placed in the PDA medium, and cultured at 30°C for one week, and results are shown in Fig. 1: the phenomenon (an upper right part of the medium) of the endophytic bacteria antagonizing the black spore-generating endophytic fungi is apparent. A colony of the endophytic bacteria that inhibit the black spore-generating endophytic fungi is picked and streaked, and the separation and purification are repeated for three times, to obtain the antifungal endophytic bacteria. The characteristics of solid culture of the bacterial cells are as follows: a ring of the purified antifungal endophytic bacteria is taken and cultured on the PDA medium at 36-38°C for 3-5 days, and an initial colony is light white, and gradually turns yellow; and the edge of the colony is irregular, and is fluffy. The characteristics of liquid culture of the bacterial cells are as follows: a ring of the purified antifungal endophytic bacteria is taken and cultured in 50 mL of a potato dextrose liquid medium at 36-38°C for 3 days: on the first day, the medium is changed from clarification to turbidity, and there is an apparent bacterial liquid culture phenomenon; on the second day, it enters a stable period, and the turbidity is not changed; and on the third day, the surface of fermentation solution forms a wrinkle. The morphology of the bacterial cells under a scanning electron microscope is shown in Fig. 2: it is a rod-like structure, and a single cell is 0.7-0.8 x 2~3 m. (3) 16S identification of single colony: a separated strain is inoculated into 50 mL of an LB medium, shaken and cultured at 37°C and 160 rpm for 24 hours, and 10 mL of bacterial solution is taken to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. The 16S rDNA sequence of the strain is shown in SEQ ID NO: 1: (1483 bp) TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGA CAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGT GGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATAC CGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTA CCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTT CCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGT TTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATA GGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGC CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGC

GTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTC AACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAG AGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACAC CAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAG CGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGAT GAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCAT TAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAAT TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAAC GCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGA CGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTC GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTT AGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACC GGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCT ACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTA AGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGAC TGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAAT ACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAAC ACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGA CAGATGATTGGGGTGAAGTCGT Its 16S rDNA sequence is used for homology analysis in National Center of Biotechnology Information (NCBI) with Blast, several strains are selected for phylogenetic tree analysis, and results are shown in Fig. 3: it is indicated that the separated antifungal endophytic bacterial strain is Bacillus velezensis, and named as Bacillus velezensis FZ06.

Embodiment 2: Preparation of lipopeptide substance by Bacillus velezensis FZ06 (1) Activation of strain: under a sterile condition, 1 ring of Bacillus velezensis FZ06 is taken from a glycerol cryopreservation tube and inoculated into a PDA medium, and activated and cultured at 36-38°C for 24 hours; (2) Seed solution culture: under the sterile condition, 1 mL of the activated Bacillus velezensis FZ06 is taken and transferred into 100 mL of a liquid LB medium, and cultured at 36-38°C and 160 rpm in a shaker for 24 hours, to obtain seed solution; (3) fermentation culture: under the sterile condition, 20 mL of the seed solution obtained in the step (2) is taken and inoculated into 2 L of a liquid LB medium, and cultured at 36-38°C and 160 rpm in the shaker for 72 hours, to obtain fermentation solution; (4) acid precipitate separation and washing: the fermentation solution is centrifuged at 4°C and 10,000 rpm in a low-temperature high-speed centrifuge for 10 min to remove the bacterial cells, and then 6 mol/L HCl is added to a centrifuged supernatant to adjust pH to 2.0, it is standing for 12 hours in an environment of 4°C, to obtain acid-precipitated fermentation solution; the acid-precipitated fermentation solution is centrifuged at 4°C and 10,000 rpm in the low-temperature high-speed centrifuge for 5 min, the supernatant is discarded and a precipitate is collected, the precipitate is washed twice with 200 mL of dilute hydrochloric acid water with pH=2.0, and the precipitate is collected after being centrifuged at 10,000 rpm for 10 min, to obtain an acid precipitate; (5) acid precipitate extraction: the acid precipitate is extracted with 200 mL of an anhydrous methanol, extract solution is subjected to vacuum suction filtration, suction filtration solution obtained is rotated and evaporated to dryness by using a rotary evaporator at 40°C, and it is redissolved in deionized water, to obtain aqueous phase extract solution; and (6) separation and purification of lipopeptide substance: the aqueous phase extract solution is freeze-dried, to obtain a freeze-dried substance, 50 mL of the anhydrous methanol is added to redissolve, redissolved solution obtained is subjected to Sephadex LH-20 column chromatography purification (an eluent is 100% methanol, and the flow rate is 0.5 mL/min), the obtained eluent containing a lipopeptide substance is concentrated in vacuum to dryness, then water is added to redissolve, redissolved solution is freeze-dried, to obtain the lipopeptide substance generated by the Bacillus velezensis FZ06, and the yield is 1.03 g/L.

Embodiment 3: Structure identification of lipopeptide substance prepared by Bacillus velezensis FZ06 50 mg of lipopeptide powder obtained by extraction of Embodiment 2 is redissolved in 50 mL of an anhydrous methanol, and after being diluted by 1000 times with the anhydrous methanol, it is subjected to ultra-high performance liquid chromatography-electrospray-secondary mass spectrometry UPLC-ESI-MS/MS structure determination, and specific analysis conditions are as follows: Chromatographic separation condition: chromatographic column: Waters

ACQUITY UPLC BEHC18 column (2.1 mmx100 mm, 1.7 m); column temperature: 30°C; flow rate: 0.4 mL/min; mobile phase: A--ultrapure water containing 0.1% formic acid, B--acetonitrile containing 0.1% formic acid; gradient elution condition: 0-0.5 min, 60% A; 0.5-3.5 min, 60-20% A; 3.5-4 min, 20% A; 4-6 min, 20~5% A; 6-7 min, 5~2% A; 7~10 min, 2% A; 10~10.5 min, 2~60% A; 10.5~15 min, 60% A; and loading amount: 1 L. Mass spectrometry analysis condition: an Agilent G6545A QTOF mass spectrometer is selected, in a positive ion scanning mode, the mass scanning range of the primary mass spectrometry is set to 110-1600 amu, and the mass scanning range of the secondary mass spectrometry is set to 501600 amu; the ion source temperature is 150°C, and the voltage of a capillary tube is 3.26 kV; the flow rate of a N 2 flow is 600 L/h, and the flow rate of an Ar 2 flow is 50 L/h; and mass spectrometry data acquisition and processing software is Agilent MassHunter. UPLC-MS results are shown in Fig. 4: the lipopeptide substance generated by the Bacillus velezensis FZ06 contains Surfactin-like homologues with molecular weights of 994, 1008, 1022, 1036, and 1050 and the like that differ by one methylene (-CH2) (Fig. 4-A), Iturin-like homologues with molecular weights of 1043, 1057, 1071, and 1085 and the like that differ by one methylene (-CH2) (Fig. 4-B) and Fengycin-like homologues with molecular weights of 1435, 1449, 1463, 1477, 1491, and 1505 and the like that differ by one methylene (-CH2) (Fig. 4-C). By the determination of UPLC-ESI-MS/MS, the Surfactin-like homologue is a cyclic structure formed by a hydroxy fatty acid chain containing 12-16 carbon atoms and a polypeptide chain containing 7 amino acids, there are two types of the polypeptide chains, and its amino acid sequence is: glutamic acid (Glu) - leucine (Leu) - leucine (Leu) - valine (Val) - aspartic acid (Asp) - leucine (Leu) - leucine ( Leu) or glutamic acid (Glu) - leucine (Leu) - leucine (Leu) - leucine (Leu) - aspartic acid (Asp) - leucine (Leu) - valine (Val); the Iturin-like homologue is a cyclic structure formed by an amino fatty acid chain containing 14-17 carbon atoms and a polypeptide chain containing 7 amino acids, there are two types of the polypeptide chains, and its amino acid sequence is: aspartic acid (Asn) - tyrosine (Tyr) - aspartic acid (Asn) - glutamine (Gln) - proline (Pro) - aspartic acid (Asn) - threonine (Thr) or aspartic acid (Asn) tyrosine (Tyr) - glutamine (Gln) - aspartic acid (Asn) - proline (Pro) - aspartic acid (Asn) - threonine (Thr); and the Fengycin-like homologue is a cyclic structure formed by an amino fatty acid chain containing 15-17 carbon atoms and a polypeptide chain containing 10 amino acids, there are three types of the polypeptide chains, and its amino acid sequence is: glutamic acid (Glu)- ornithine (Orn) - tyrosine (Tyr) threonine (Thr) - glutamic acid (Glu) - valine (Val) - proline (Pro) - glutamine (Gln) tyrosine (Tyr) - isoleucine (Ile) - glutamic acid (Glu) - omithine (Om) - tyrosine (Tyr) - threonine (Thr)- glutamic acid (Glu) - aminobutyric acid (Abu) - proline (Pro) glutamine (Gln) - tyrosine (Tyr) - isoleucine (Ile) or glutamic acid (Glu) - ornithine (Om) - tyrosine (Tyr) - threonine (Thr) - glutamic acid (Glu) - alanine (Ala) - proline (Pro) - glutamine (Gln) - tyrosine (Tyr) - isoleucine (Ile). Specific molecular formulas and structural formulas of the lipopeptide substances are shown in Table 1: Table 1: Chemical name, molecular formula and structural formula of lipopeptide substance obtained in Embodiment 2 Substance Chemical name Molecular formula Structural formula category Surfactin-like such as Surfactin A, C 1 2 -Leu 7-Surfactin CsoHs 7N70 13 homologue n=6

C 5 1H 8 9N 70 1 3 such as Surfactin A, C 13 -Leu 7 -Surfactin n=7

Cs2H91N7013 such as Surfactin A, C 14 -Leu 7 -Surfactin n=8

Cs3H93N7013 such as Surfactin A, Cis-Leu 7- Surfactin n=9

C 5 4 H 8 5N 7 0 13 such as Surfactin A, C 16-Leu 7 -Surfactin n=10

C 5 oH8 7N 70 13 such as Surfactin B, C 12 -Val 7 -Surfactin n=6

C 5 1H 8 9N 70 13 such as Surfactin B, C 13 -Val 7 -Surfactin n=7

Cs2H91N7013 such as Surfactin B, C14-Val7_-Surfactin n=8

Cs3H93N7013 such as Surfactin B, Cis-Val7_-Surfactin n=9

C 5 4 H 8 5N 7 0 1 3 such as Surfactin B, C 16-Val 7 -Surfactin n=10 Iturin-like such as Bacillomycin C 14 -Bacillomycin F C 4 8H 7 4 N 1 2 0 1 4 homologue F, n=7

such as Bacillomycin F, n=8 Cis-Bacillomycin F C49H78N12Oi4 Fsuch =as Bacillomycin such-ascillomycinFi C 1 -Bacillomycin F CsoH 7 sN 1 2 0 1 4 F, n=9

such as Bacillomycin C 1 7-Bacillomycin F C 5 1Hs8 N 1 2 0 1 4 F, n=10 such as Bacillomycin C 14-Bacillomycin D C 4 sH 7 4 N 1 2 0 1 4 D, n=7

such as Bacilomycin C 1 -BacillomycinD C49H 12I4 D,n=8

such as Bacilomycin C 1 -BacillomycinD CsoH 7 sN 1 2 i0 4 D,n=9

such as Bacilomycin C 17 -BacillomycinD C 5 1 HIoN 1 I0 4 2 D, n=10

Fengycin-like such as Fengycin A, C 1 -Ala 6 -Fengycin C 7 1HI1 4N 1 2 0 20 homologue n=11

Cis-Ala 6-Fengycin C7Ho100such asFengycin A, n-i2

C 1 7-Ala 6-Fengycin C 73 H 1i 2 N 12 0 20 such as Fengycin A, n=13

Cis-Abu 6 -Fengycin C7Ho100such asFengycin B, n=11

Cis-Abu 6 -Fengycin C 7 3 Hii 2 N 12 0 2 0 such as Fengycin B, n=12

C 1 7 -Abu 6 -Fengycin C7H4100such asFengycin B, n=13

Cis-Val-Fengycin C73 Hii 2 N 12 0 2 0 such as Fengycin C, n=11

C 7 4HI 1 4N 1 2 0 20 such as Fengycin C, C 16-Val 6-Fengycin n=12

C 7 5H 116N 12 02 0 such as Fengycin C, C 17-Val 6-Fengycin n=13 Herein, the structural formulas of Surfactin A, Surfactin B, Bacillomycin F, Bacillomycin D, Fengycin A, Fengycin B, and Fengycin C are as follows:

HOOC NH

140

(C NH NH COOH

Surfactin A NH NHH

HOOC 0 QH NH 10 H 1

HN

NH 0

100

HO

0 NH 2

H2N 0 N H

O 00 NH NH

CH NH

NH2

O N HN N HO NH NH,

Bacillomycin F HO

NH, ° O ' HN 0 HO NCOO Ot 0 NH 0 ( CCH2NH NHNH 0 NH,

N 0 3 CHN

COOH4N

NHN HN

SBacillomycin D OH O

HOONH HN HO COON

H N o HN N N N 0 0 NH OH 0 0

0 0

0 NH H

0 NH

0 HO

Fengycin A

H 2N HO COOH

H 0 HN

(CH2 ) NH N NH O 0N: 0 ON NH OH O

CDOH 0

0 0 0 NH H

5N

Fengycin B

0 H0

H2N HO COOH

H COHN

HO Fengycin H (CHO N NHN

H 0

HO' 0

Embodiment 4: Bacterial inhibitory effect test of lipopeptide substance prepared by Bacillus velezensis FZ06

(1) Three common bacterial pathogens are selected: Escherichiacoli GIM1.708 (purchased from Guangdong Microbial Culture Collection Center), Staphylococcus aureus subsp. aureus GIM1.441 (purchased from Guangdong Microbial Culture Collection Center) and Salmonella choleraesuis subsp. Choleraesuis GIM1.163 (purchased from Guangdong Microbial Culture Collection Center), under a sterile condition, a small amount of Escherichia coli GIM1.708, Staphylococcus aureus subsp. aureus GIM1.441 and Salmonella choleraesuis subsp. Choleraesuis GIM 1. 163 stored at -80°C are picked with an inoculating needle and respectively inoculated into a solid LB medium test tube, and activated at 36-38°C for 24 hours, to obtain the activated Escherichiacoli GIM1.708, Staphylococcus aureus subsp. aureus GIM1.441 and Salmonella choleraesuissubsp. Choleraesuis GIM1.163; (2) under the sterile condition, a ring of the activated Escherichiacoli GIMI.708, Staphylococcus aureus subsp. aureus GIMI.441 and Salmonella choleraesuis subsp. Choleraesuis GIM1.163 are taken and respectively transferred to 50 mL of a liquid LB seed medium, cultured at 36-38°C and 160 rpm in a shaker for 24 hours, to obtain Escherichia coli GIMI.708 seed solution, Staphylococcus aureus subsp. aureus GIMI.441 seed solution and Salmonella choleraesuis subsp. Choleraesuis GIM1.163 seed solution; (3) under the sterile condition, the obtained seed solution is respectively diluted with sterile water to about 1 x 108 cfu/mL, and then spread in an LB agar solid medium, to obtain a culture dish containing the Escherichiacoli GIMI.708, a culture dish containing the Staphylococcus aureus subsp. aureus GIMI.441 and a culture dish containing the Salmonella choleraesuissubsp. Choleraesuis GIM1.163; (4) 10 mg of the lipopeptide substance freeze-dried powder prepared in Embodiment 2 is taken, and 2 mL of deionized water is added, to obtain 5 mg/mL of lipopeptide solution; and (5) a Oxford cup (the inner diameter is 6 mm, and it is sterilized) is respectively placed in the culture dishes containing the bacterial pathogens obtained in the step (3); in an experimental group, 200 L of 5 mg/mL lipopeptide solution is added to the Oxford cup, and in a blank control group, 200 L of the sterile water is added to the sterile Oxford cup, and then placed in a 37°C incubator and cultured for 24 hours at a constant temperature, to observe its bacteriostatic effect, results are shown in Fig. 5, and at the same time, the size of its inhibition zone is measured, and results are shown in Table 2; and it is indicated that the lipopeptide substance prepared by the Bacillus velezensis FZ06 has the apparent inhibitory effect on the bacterial pathogens.

Embodiment 5: Fungal inhibitory effect test of lipopeptide substance prepared by Bacillus velezensis FZ06 (1) Two common fungal pathogens are selected: Aspergillus flavus GIM 3.493 (purchased from Guangdong Microbial Culture Collection Center) and Aspergillus parasiticus Speare GIM 3.395 (purchased from Guangdong Microbial Culture Collection Center), under a sterile condition, a small amount of Aspergillus flavus

GIM 3.493 and Aspergillus parasiticusSpeare GIM 3.395 stored at -80°C are picked with an inoculating needle and respectively inoculated into a PDA medium test tube, and activated at 36-38°C for 24 hours, to obtain the activated Aspergillusfavus GIM 3.493 and AspergillusparasiticusSpeare GIM 3.395; (2) the activated Aspergillus flavus GIM 3.493 and Aspergillus parasiticus Speare GIM 3.395 are inoculated into the sterilized PDA medium, and cultured in a 36-38°C incubator for 96 hours, to obtain the Aspergillus flavus GIM 3.493 with spores and the AspergillusparasiticusSpeare GIM 3.395 with spores; (3) the Aspergillus flavus GIM 3.493 spores and the Aspergillus parasiticus Speare GIM 3.395 spores are scraped and placed in sterile saline respectively, shaken by a vortex shaker for 3 min, and filtered with sterile filter paper, to obtain uniform Aspergillus flavus GIM 3.493 spore suspension and Aspergillus parasiticus Speare GIM 3.395 spore suspension; (4) the obtained Aspergillusflavus GIM 3.493 spore suspension and Aspergillus parasiticus Speare GIM 3.395 spore suspension obtained in the step (3) are respectively diluted with the sterile saline to 1 x 106 cfu/mL, and then spread in a PDA agar solid medium respectively, to obtain a culture dish containing the Aspergillusfavus GIM 3.493 and a culture dish containing the Aspergillus parasiticus Speare GIM 3.395; (5) 10 mg of the lipopeptide substance freeze-dried powder prepared in Embodiment 2 is taken, and 2 mL of deionized water is added, to obtain 5 mg/mL of lipopeptide solution; and (6) a Oxford cup (the inner diameter is 6 mm, and it is sterilized) is respectively placed in the culture dishes containing the fungal pathogens obtained in the step (4); in an experimental group, 200 L of 5 mg/mL lipopeptide solution is added to the Oxford cup, and in a blank control group, 200 L of the sterile water is added to the sterile Oxford cup, and then placed in a 37°C incubator and cultured for 5 days at a constant temperature, to observe its bacteriostatic effect, results are shown in Fig. 6, and at the same time, the size of its inhibition zone is measured, and results are shown in Table 2; and it is indicated that the lipopeptide substance prepared by the Bacillus velezensis FZ06 has the apparent inhibitory effect on the fungal pathogens. Table 2: Antibacterial activity of lipopeptide substances generated by strains extracted in Embodiment 2

Sample/Control Radius of inhibition zone (unit: mm) Escherichia Staphylococcus Salmonella Aspergillus Aspergillus

coli aureus subsp. choleraesuis flavus parasiticus

GIM 1.708 aureus subsp. GIM 3.493 Speare GIM 1.441 Choleraesuis GIM 3.395 GIM1.163 Lipopeptide 6.04±0.12 10.23±0.10 6.16±0.15 15.86±0.34 16.65±0.24 substance

Blank control 0 0 0 0 0

The above embodiments are preferred embodiments of the present disclosure, but implementation modes of the present disclosure are not limited by the above embodiments, and any other changes, modifications, replacements, combinations and simplifications made without departing the spirit essence and principle of the present disclosure should be equivalent substitution modes, and are all included in a scope of protection of the present disclosure.

Claims

1. An endophytic bacillus from Pu'er tea tree leaves, characterized in that: the name is Bacillus velezensis FZ06, it is deposited at the China Center for Type Culture Collection in Wuhan University, Wuhan, China on 25 October 2019, with the deposit number CCTCC NO: M 2019854.

2. An application of the endophyte bacillus from the Pu'er tea tree leaves according to claim 1 in preparation of a lipopeptide substance, characterized by comprising the following steps: (1) fermentation culture: inoculating seed solution of the endophytic bacillus from the Pu'er tea tree leaves according to claim 1 into a fermentation medium for fermentation culture, to obtain fermentation solution; (2) acid precipitate separation and washing: performing first centrifugation on the fermentation solution obtained in the step (1) to remove bacterial cells, adding an acid to an obtained supernatant to adjust pH to 2.0, performing second centrifugation after standing to remove the supernatant, washing an obtained precipitate, and then performing third centrifugation, to obtain an acid precipitate; (3) acid precipitate extraction: adding the acid precipitate obtained in the step (2) to a solvent for extraction, then performing solid-liquid separation, drying obtained liquid, and then adding water to redissolve, to obtain aqueous phase extract solution; and (4) separation and purification of lipopeptide substance: performing first drying on the aqueous phase extract solution obtained in the step (3), adding the solvent to an obtained dry substance to redissolve, then performing chromatographic purification, performing second drying on an obtained eluate containing a lipopeptide substance, adding the water to redissolve, and finally performing third drying, to obtain the lipopeptide substance.

3. The application of the endophyte bacillus from the Pu'er tea tree leaves according to claim 2 in preparation of the lipopeptide substance, characterized in that: the inoculation amount of the seed solution in the step (1) is 1~5% of the fermentation medium by a volume ratio; and further 1% of the fermentation medium; the fermentation medium is a liquid Luria-Bertani (LB) medium; and the fermentation culture condition in the step (1) is that it is cultured at 35-39°C and 150~170 rpm for 48-72 hours; and further it is cultured at 36-38°C and 160 rpm for 72 hours.

4. The application of the endophyte bacillus from the Pu'er tea tree leaves according to claim 3 in preparation of the lipopeptide substance, characterized in that: the seed solution is prepared by the following steps: activating the preserved endophytic bacillus from the Pu'er tea tree leaves, and expanding culture, to obtain the seed solution; the activating step is as follows: inoculating the preserved endophytic bacillus from the Pu'er tea tree leaves into an activation medium, and culturing under a condition of 35-39°C for 24~48 hours, to obtain the activated endophytic bacillus from the Pu'er tea tree leaves; the activation medium is a potato dextrose agar (PDA) medium; the step of expanding the culture is as follows: inoculating the activated endophytic bacillus from the Pu'er tea tree leaves into a seed medium, and culturing under conditions of 35-39°C and 150~170 rpm for 24~48 hours, to obtain the seed solution; the inoculation amount of the activated endophytic bacillus from the Pu'er tea tree leaves is 1-5% of the seed medium by a volume ratio; and further 1% of the seed medium; and the seed medium is a liquid LB medium.

5. The application of the endophyte bacillus from the Pu'er tea tree leaves according to claim 2 in preparation of the lipopeptide substance, characterized in that: the first centrifugation condition in the step (2) is that it is centrifuged at 4°C and ,000 rpm for 10 min; the acid in the step (2) is a hydrochloric acid; and further the hydrochloric acid with a concentration of 6 mol/L; the standing condition in the step (2) is that it is standing at 4°C for 12-24 hours; and further it is standing at 4°C for 12 hours; the second centrifugation condition in the step (2) is that it is centrifuged at 4°C and 10,000 rpm for 5 min; the washing in the step (2) is that it is washed with a dilute hydrochloric acid; and further it is washed with the dilute hydrochloric acid with pH=2.0; the washing times in the step (2) are 2 times; the third centrifugation condition in the step (2) is that it is centrifuged at 10,000 rpm for 10 min; the solvent in the step (3) is an anhydrous methanol; the solid-liquid separation mode in the step (3) is suction filtration; and further vacuum suction filtration; the drying in the step (3) is that it is hang-dried by concentration; further it is hang-dried by vacuum concentration; and furthermore it is rotated and evaporated to dryness at 40°C by a rotary evaporator; and the water in the step (3) is deionized water.

6. The application of the endophyte bacillus from the Pu'er tea tree leaves according to claim 2 in preparation of the lipopeptide substance, characterized in that: the first drying and the third drying in the step (4) are freeze-drying; the solvent in the step (4) is the anhydrous methanol; the chromatographic purification in the step (4) is that the purification is performed by using a Sephadex gel chromatographic column; and further a Sephadex LH-20 gel column is used, the eluent is 100% methanol, and the flow rate is 0.5 mL/min; and the second drying in the step (4) is that it is hang-dried by concentration; further it is hang-dried by vacuum concentration; and furthermore it is rotated and evaporated to dryness at 40°C by the rotary evaporator.

7. A lipopeptide substance, characterized in that: it is obtained by fermenting, separating and purifying the endophytic bacillus from the Pu'er tea tree leaves according to claim 1.

8. The lipopeptide substance according to claim 7, characterized in that: the lipopeptide substance comprises a Surfactin-like homologue, an Iturin-like homologue and a Fengycin-like homologue.

9. The lipopeptide substance according to claim 8, characterized in that: the Surfactin-like homologue is C 12 -Leu7 -Surfactin, C 13-Leu 7-Surfactin,

C 14-Leu 7-Surfactin, C 1 5 -Leu7 -Surfactin, C 16 -Leu7 -Surfactin, C1 2-Val 7-Surfactin,

C13-Val 7 -Surfactin, C 14-Val 7 -Surfactin, C1 5 -Val 7 -Surfactin and C 14 -Val7 -Surfactin; the Iturin-like homologue is C1 4-Bacillomycin F, Ci 5-Bacillomycin F, Ci 6-Bacillomycin F, C1 7-Bacillomycin F, C1 4-Bacillomycin D, Ci 5 -Bacillomycin D, C 16-Bacillomycin D and C 17-Bacillomycin D; and the Fengycin-like homologue is C 1 5-Ala -Fengycin, C 16-Ala -Fengycin, C 17-Ala -Fengycin, C 1 5-Abu 6 -Fengycin, C 16-Abu 6 -Fengycin, C1 7-Abu6 -Fengycin, C 15-Val-Fengycin, C 16-Val-Fengycin and C 17 -Val-Fengycin.

10. An application of the lipopeptide substance according to any one of claims 7-9 in inhibiting pathogenic bacteria, characterized in that: the pathogenic bacteria comprise but not limited to Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillusflavus and Aspergillus parasiticus.

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