CN115975864A - Qishu endogenous Bacillus belgii, screening method and application thereof - Google Patents

Abstract

The invention relates to Qishu endogenous Bacillus belgii, a screening method and application thereof. Qi Shuna Bacillus belgii has been deposited at 26/10 of 2022 in China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC M20221664. Qi Shuna Bacillus belezii HSU-3 has wide antibacterial property, the bacterial strain has obvious antibacterial effect on Curvularia leaf spot disease, corn stalk rot disease, cotton wilt disease and wheat scab disease, and the antibacterial rate reaches more than 45%; wherein the bacteriostatic rate of the bacterial strain on curvularia leaf spot is the highest, and the bacteriostatic rate reaches 63.72%; meanwhile, the fermentation product of the strain has very obvious biocontrol effect on curvularia leaf spot germ, and the bacteriostatic rate is further remarkably improved to 75.61 percent.

Description

Qishu endogenous Bacillus belgii, screening method and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to Qishu endogenous Bacillus belgii, a screening method and application thereof.

Background

Qi Shu (Atractylodes macrocepha Koidz., japan), which is known as Kotech, winter Qi Shu, etc., belongs to Atractylodes (Atractylodes DC., japan) of the family Compositae (Compositae) and the family Cynara (Cynareae), and is a perennial herbaceous plant. Qi Shu is used as a medicine with root-like stem, and is an important bulk Chinese medicinal material; originally recorded in Shen nong Ben Cao Jing, it is sweet and bitter in taste, warm in nature, entering spleen and stomach meridians, listed as the superior, and centrally distributed in Anhui, zhejiang and Jiangxi, etc., wherein Qin Qi Shu has the best quality and efficacy. Qi Qishu (Qishu for short) is a famous species in Huangshan mountain in Anhui province, and is a famous genuine medicinal material in Anhui province.

The existing research shows that Qi Shu mainly contains medicinal components such as volatile oil, lactones, polysaccharide and the like, and has multiple medicinal functions such as diabetes treatment, aging resistance, tumor resistance, inflammation resistance, bacteria resistance and the like. Therefore, qi Shu has good medicinal value and development prospect. However, qi Shu mainly uses wild resources and has harsh growth environment; meanwhile, with the massive excavation and long-term lack of protection of human beings on wild keemun operation, qi Shuye living species are in an extremely endangered state, and protection is urgently needed. Therefore, the development of medicinal value of the Chinese medicinal composition while protecting the Chinese medicinal resources is imminent.

The endophyte is planted in a host body, has rich varieties, can generate metabolites which are the same as or similar to the host plant, and becomes an important resource for screening secondary metabolites with bacteriostatic activity. In recent years, much attention is paid to the research of disease control by utilizing plant endophytes, for example, bi Jiangtao and the like research on the biocontrol potential of endophytes in ammopiptanthus mongolicus plants, and the separated endophytes AMER007 and AMER008 are found to have obvious antagonistic action on lycium barbarum nigrum and wheat take-all pathogen (Bi Jiangtao, yang Wei, li Ping, he Da Han. Separation of endangered medicinal plants ammopiptanthus mongolicus endophytes and the primary analysis of the antibacterial activity thereof [ J ]. Natural product research and development, 2013,25 (12): 1621-1626.); ding Ting and the like are used for separating and purifying endophytic fungi in eucommia ulmoides plants to obtain 32 strains, and an in vitro antagonistic screening test finds that 22 strains of endophytic fungi have an antibacterial effect on corn sheath blight bacteria (Ding Ting, sun Weiwei, wang Shuai and Jiang Haiyang. Screening of the strain with corn sheath blight activity in the eucommia ulmoides endophytic fungi [ J ] plant protection, 2014,40 (6): 29-35.). However, the research on keemun art is mainly focused on nutrient components, growth and reproduction, active components and the like, and no report is made on the research on Qi Shu endophytes.

Aiming at the situation, the strain with the biocontrol effect is separated and screened from wild Qi Shu of Qimen in Anhui, identified by using a morphological and molecular biological method and researched for the bacteriostatic activity of a secondary metabolite of the strain.

Disclosure of Invention

The endophytic strain HSU-3 is obtained by separating from a traditional Chinese medicinal material Qi Shu, identified as Bacillus subtilis (Bacillus velezensis) by morphological and molecular biological methods, and found to exist in fermentation products through bacteriostatic activity experiments; the antibacterial substance in the fermentation product has strong stability to proteinase K and trypsin, good thermal stability and ultraviolet stability, and good antibacterial ability in alkaline environment.

The first aspect of the present invention is: providing an internal generation Bacillus belief in Qishu, which is separated from Qishu collected in Qimen county of Huangshan city, anhui province; is named as Qi Shuna raw Bacillus belgii HSU-3, and the Qi Shuna raw Bacillus belgii is preserved in China Center for Type Culture Collection (CCTCC) at 26/10.2022 with the preservation number of CCTCC M20221664.

After separation and purification, the Qishu endogenous B.baileyi strain is a round single colony, the edge is irregular, the surface is rough and opaque, and the color of the center of the colony is darker and light yellow; the thalli is rod-shaped and round-ended and has the size of 1.5-2.0 multiplied by 0.8-1 μm through further observation by a scanning electron microscope.

Further, the Qi Shuna bacillus belezii has antagonistic effect on plant pathogenic fungi.

Further, the Qi Shu endophytic bacillus beijerinckii fermentation product has antagonistic effect on plant pathogenic fungi.

Further, the Qi Shu endophytic Bacillus belgii fermentation product has antagonistic effect on Curvularia lunata.

Further, the method for extracting the Qi Shu endophytic bacillus beiLeisi fermentation product comprises the following steps: inoculating Qi Shuna Bacillus belius HSU-3 into a culture medium for culture, inoculating 80-120 mu L of the culture medium for continuous culture, centrifuging the fermentation liquid, and taking the supernatant for later use.

Furthermore, it can be known through plate confrontation experiments that Qi Shuna live Bacillus belgii HSU-3 has antagonistic activity against Curvularia leaf spot disease, gibberella zeae, corn stalk rot, cotton wilt, apple anthracnose, tomato wilt, rice blast, anthracnose, phytophthora capsici, sclerotinia sclerotiorum.

Further, the Qi Shuna method for isolating B.belgii comprises: cleaning fresh rhizome with sterile water, pretreating, and cutting into small pieces of about 2-5 mm; planting the tissue segments on a PDA culture medium in a sterile environment, after bacteria grow out from the cut of the rhizome cultured at a constant temperature, transferring the tissue segments to a fresh PDA culture medium for purification; transferring the purified strain to PDA slant culture medium, and storing for use

Wherein, the PDA culture medium comprises: 200g of peeled potatoes, 20g of glucose and 15g of agar, and double distilled water is added to the mixture to reach the constant volume of 1000mL.

The second aspect of the present invention is:

the invention adopts a plate marking method to obtain Qi Shuna single colonies of Bacillus belezii HSU-3, utilizes a universal primer to carry out 16S rRNA amplification, constructs a phylogenetic tree and defines the classification status of the strain.

Further, a phylogenetic tree was constructed using the 16S rRNA sequence, and it was found that the strain was located in the same branch as Bacillus velezensis strain FZB42 and the self-expansion support rate was 89%. Therefore, the strain was further determined to be Bacillus belgii, named B.velezensis strain HSU-3.

Wherein, the sequence of Qi Shuna 16S rRNA gene of Bacillus belezensis is shown in SEQ ID NO. 1.

The third aspect of the present invention is: provides the application of Qi Shuna Bacillus beleisis, including the following applications:

the application of the strain as an antagonistic bacterium of curvularia leaf spot;

the application of the strain as an antagonistic bacterium of wheat scab germs;

the application of the strain as antagonistic bacteria of the corn stalk base rot germs;

the application of the antagonistic bacteria of cotton fusarium wilt germs;

the application of the antagonistic bacteria of apple anthracnose bacteria;

the application of the compound as an antagonistic bacterium of tomato blight disease bacteria;

the application of the antagonistic bacteria of rice blast germs;

the application of the antibacterial agent as the antagonistic bacteria of the germs of the anthracnose of the jujube;

the application of the strain as an antagonistic bacterium of phytophthora capsici;

can be used as antagonistic bacteria of sclerotinia sclerotiorum.

The invention has the advantages that:

(1) Qi Shuna Bacillus belezii HSU-3 in the invention has wide antibacterial property, the bacterial strain has obvious antibacterial effect on Curvularia leaf spot of rice, basal rot of corn, blight of cotton and gibberellic disease of wheat, and the antibacterial rate reaches more than 45%;

(2) The invention discovers Qi Shuna Bacillus belezii HSU-3 for the first time that the antagonistic effect of the Bacillus belezii on curvularia leaf spot germ is most obvious, and the bacteriostasis rate reaches 63.72%; meanwhile, the fermentation product of the strain has very obvious biocontrol effect on curvularia leaf spot bacteria, and the antibacterial rate is further remarkably improved to 75.61 percent. The Qi Shu endophyte fermentation product can obviously improve the antagonism to curvularia leaf spot germ, which also provides an important novel antagonistic bacterium resource for preventing curvularia leaf spot disease of paddy rice;

(3) The Qi Shuna raw Bacillus belgii HSU-3 fermentation liquor has strong thermal stability and ultraviolet resistance, has good bacteriostatic activity in a slightly alkaline environment, and has good development prospect.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 shows the morphology of Bacillus belgii HSU-3 colonies;

FIG. 2 shows the morphology of Bacillus beilesiensis HSU-3 cells under a scanning electron microscope;

FIG. 3 is a graph showing the antagonistic action of Bacillus belgii HSU-3 on Curvularia lunata Bl.C;

FIG. 4 is a graph showing the antagonistic effect of Bacillus beiLeisi HSU-3 on Gibberella zeae;

FIG. 5 is a graph of the antagonistic effect of Bacillus belgii HSU-3 on Colletotrichum malaccensis;

FIG. 6 is a graph showing the antagonistic effect of Bacillus belgii HSU-3 on Fusarium oxysporum F.sp.sp.gossypii;

FIG. 7 is a phylogenetic tree constructed based on Bacillus 16S rRNA;

FIG. 8 is a graph showing the antagonistic action of Bacillus beiLeisi HSU-3 fermentation products on Curvularia lunata;

FIG. 9 is a graph showing the antagonistic effect of Bacillus beiLeisi HSU-3 fermentation product on Gibberella zeae;

FIG. 10 is a graph of the antagonistic effect of Bacillus belgii HSU-3 fermentation products on apple anthracnose pathogen;

FIG. 11 is a graph showing the antagonistic effect of Bacillus beiLeisi HSU-3 fermentation products on Fusarium oxysporum F.sp.sp.gossypii;

FIG. 12 is a graph comparing the effect of two different proteases on B.beiLeisi HSU-3 fermentation broth antagonistic activity;

FIG. 13 is a graph comparing the effect of different pH on the antagonistic activity of Bacillus beiLensis HSU-3 fermentation broth;

FIG. 14 is a graph comparing the effect of different temperatures on the antagonistic activity of Bacillus beiLensis HSU-3 fermentation broth;

FIG. 15 is a graph comparing the effect of UV light on the antagonistic activity of Bacillus beijerinckii HSU-3 fermentation broth at different times.

Detailed Description

The invention is further illustrated by the following examples, which are intended to illustrate, but not to limit the invention further.

The invention provides Qizhu endogenous belie bacillus which is separated from Qizhu collected in Qimen county of Huangshan City in Anhui province, and samples are collected and stored in a subject group laboratory for later use in a refrigerator at 4 ℃.

The main instruments used in the present invention include: agar, glucose (national drug group), ZGP-2050 type constant temperature incubator (shanghai zhicheng analytical instruments manufacturing ltd.), SW-CJ-ICU type super clean bench (shanghai new seedling ltd.), DSX-280B type autoclave (shanghai Shen An medical instruments ltd.), MQD-B2R type shaking incubator (shanghai Min spring instruments ltd.), pH meter (shanghai instruments electroscience instruments ltd.).

Example 1

1. 8978 isolation and purification of zxft 8978 Bacillus belezii HSU-3:

separating and purifying internal Bacillus belezii HSU-3 by tissue block separation method, which comprises the following steps: cleaning fresh rhizome with sterile water, pretreating with 5% NaClO solution and 75% ethanol solution, and cutting into small pieces of about 3 mm; the tissue segments were plated on PDA medium in sterile conditions and a control group was set. Culturing at the constant temperature of 28 ℃ for 3-5 days, and transferring the rhizomes of the experimental group to a fresh PDA culture medium for purification after the rhizomes grow out of the rhizomes; the purified strain is transferred to a PDA slant culture medium and stored in a refrigerator at 4 ℃ for later use.

Wherein, PDA culture medium: 200g of peeled potatoes, 20g of glucose and 15g of agar, and double distilled water is added to the mixture to reach the constant volume of 1000mL.

2. And (3) morphological observation: the Qi Shuna Bacillus beleisi HSU-3 strain is obtained by separation and purification, and the growth condition is shown in figure 1. The bacterial strain is a circular single bacterial colony, the edge is irregular, the surface is rough and opaque, and the color of the center of the bacterial colony is dark and light yellow; further observation by a scanning electron microscope revealed that the cells had rod-like and round ends and the cell size was 1.5 to 2.0X 0.8 to 1 μm, as shown in FIG. 2.

3. Qi Shuna antagonistic action of B.beiLensis HSU-3 against pathogenic fungi:

the method for measuring the antagonism of internal B.leiensis HSU-3 in keemun by adopting a flat plate opposing method comprises the following steps: transferring the pathogenic fungi cakes (5 mm) of each test plant to the center of a PDA culture medium respectively, inoculating 4 blocks of Keemum endophyte cakes (5 mm) with the same size at a position 30mm away from the pathogenic fungi by a cross symmetry method after the pathogenic fungi grow out, and carrying out the confrontation experiment. Each group was repeated 5 times while using a plate inoculated with only the pathogenic fungus to be tested as a blank, and cultured in a constant temperature incubator at 28 ℃. And observing the growth state of the pathogenic fungi every 24h, and measuring the colony diameter of the pathogenic fungi to calculate the bacteriostasis rate.

The plate confrontation method is adopted to determine the bacteriostasis of Qi Shuna Bacillus belgii HSU-3 to 10 plant pathogenic fungi such as curvularia leaf spot pathogen of rice, fusarium head blight pathogen of wheat, corn stalk rot pathogen and the like, and the results are shown in Table 1:

TABLE 1 bacterial strain HUS-3 bacteriostat

It should be noted that the error bars in the table are shown as standard deviations and the different letters indicate significant differences (P < 0.05) as tested by Duncan's repolarization method.

In addition, the strain has the antagonistic effect on 4 plant pathogenic fungi of curvularia leaf spot, fusarium graminearum, colletotrichum malorum and fusarium oxysporum, and the results are shown in fig. 3-6.

The result shows that Qi Shuna Bacillus belezii HSU-3 has obvious bacteriostatic effect on curvularia leaf spot disease of rice, basal rot disease of corn, fusarium wilt disease of cotton and fusarium head blight disease of wheat, and the bacteriostatic rate reaches over 45%.

It is emphasized that Qi Shuna Bacillus beiLensis HSU-3 has the highest bacteriostatic effect on Curvularia lunata, the bacteriostatic rate is as high as 63.72%, and the antagonistic effect is excellent.

Therefore, the Qishu endogenous Bacillus belezii HSU-3 has wide antibacterial property and application and development potential.

4. The strain of the Qishu endogenous Bacillus belgii HSU-3 is identified:

the Qi Shuna single colony of Bacillus belezensis HSU-3 is obtained by plate marking method, the morphological characteristics of the colony are observed, and the thallus is observed by an electron microscope.

Qi Shuna Bacillus belgii HSU-3 whole genome DNA was extracted with a bacterial genome DNA kit, 16S rRNA was amplified with the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3')/1492R (5'-TACGGTTACCTTGTTACGACTT-3'), and sent to Genetian Biotechnology (Anhui) Ltd for sequencing. Obtaining a homologous sequence of the strain sequence by using BLAST alignment in a GenBank database of NCBI, carrying out sequence alignment on the sequence obtained by using ClustalX software and the searched homologous sequence, constructing a phylogenetic tree by using MEGA software according to a Maximum Likelihood method (Maximum Likeliood), and carrying out detection by using a self-expanding method (Bootstrap) for 1000 times.

By utilizing molecular phylogenetic analysis, 16S rRNA sequence of Qi Shuna live Bacillus belgii HSU-3 is obtained, a phylogenetic tree is constructed as shown in figure 7, and the strain and Bacillus belgii velezensis strain FZB42 are found to be positioned in the same branch, and the self-expansion support rate is 89%.

Therefore, the strain was further determined to be Bacillus belgii, named B.velezensis strain HSU-3.

Qi Shuna the sequence of the 16S rRNA gene of B.belief is shown in SEQ ID No. 1.

Example 2

1. 3242 extraction of fermentation product of Qi Shuna Bacillus beleisi HSU-3:

qi Shuna Bacillus belgii HSU-3 was inoculated in PD liquid medium, cultured at 28 deg.C and 180rpm for 24h, and 100. Mu.L was inoculated in 100mL PD liquid medium and cultured for 3d. And centrifuging the endophyte fermentation liquor at 9500rpm for 25min, and taking the supernatant for later use.

Wherein the PD culture medium is a corresponding liquid culture medium of PDA culture medium (peeling potato 200g, glucose 20g, agar 15g, adding double distilled water to reach a constant volume of 1000 mL), and no agar is added.

2. Qi Shuna Bacillus beleisis HSU-3 fermentation product antagonistic action against phytopathogenic fungi test:

qi Shuna Bacillus belgii HSU-3 fermentation liquid and culture medium are fully mixed according to 1:9 volume ratio to prepare culture medium containing endophyte sterile fermentation liquid, and sterile water is used as blank control. 4 test plant pathogenic fungi, namely curvularia leaf spot pathogen of rice, fusarium graminearum, colletotrichum gloeosporioides and fusarium oxysporum of cotton are respectively inoculated into the center of the plate, the thickness of a fungus cake is 5mm, and each treatment is repeated for 5 times. And each test fungus was incubated at 28 ℃. And measuring the growth diameters of the colonies of the control group and the treatment group when the colony growth conditions of the control group are proper, and calculating the bacteriostasis rate of the endophyte fermentation liquor.

Sterile water is used as a blank control, fermentation liquor is used as an experimental group, 4 test bacteria of curvularia lunata, fusarium graminearum, colletotrichum malorum and fusarium oxysporum f.sp.gossypii are respectively inoculated into the sterile water and a fermentation liquor flat plate, the relative bacteriostatic activity of the Qi Shuna live bacillus belgii HSU-3 fermentation liquor on 4 common pathogenic fungi is measured, and specific antagonistic action graphs are shown in figures 8-11.

The results show that the Qi Shuna raw Bacillus belgii HSU-3 fermentation liquor has good bacteriostatic effect on curvularia leaf spot pathogen of rice and gibberellic disease of wheat, the relative bacteriostatic rate on curvularia leaf spot pathogen of rice can reach 75.61%, the relative bacteriostatic rate on gibberellic disease of wheat can reach 42.42%, and the bacteriostatic effect on fusarium oxysporum and anthracnose of apple is poor.

3. Qi Shuna stability of Bacillus beleisis HSU-3 fermentation products

The influence of Qi Shuna Bacillus beiLensis HSU-3 fermentation products on the stability of the inhibition effect of curvularia leaf spot of rice is verified by changing the treatment conditions, and the specific test is as follows:

(1) Enzyme stability

Mixing 5 mu g/mL trypsin and proteinase K with endophyte fermentation liquor respectively, carrying out shaking table reaction for 2.5h at 28 ℃ and 180rpm, taking sterile water without proteinase treatment as a blank control, taking curvularia leaf spot pathogen of rice as a test bacterium, and inoculating the test bacterium into a fermentation liquor plate after proteinase treatment. Each treatment was repeated 5 times. And calculating the relative inhibition rate of Qi Shu endophyte fermentation liquor.

The result of the influence of protease treatment on the antibacterial activity of the Qishu endogenous Bacillus belgii HSU-3 fermentation liquor is as follows: qi Shuna B.beiLeisi HSU-3 fermentation broth was treated with proteinase K and trypsin, and the enzyme stability of the broth was determined, the results are shown in FIG. 12. The result shows that the bacteriostatic activity of the fermentation product of the strain HSU-3 has no obvious influence, which indicates that the strain has stronger tolerance to protease.

(2) Acid-base stability

The pH value of Qi Shu endophyte fermentation liquor is respectively adjusted to 2, 4, 6, 8, 10 and 12 by a pH meter and an acid-base buffer solution, and after standing for 24 hours, the pH value is adjusted to be 7.3. The untreated sterile water is used as a blank control, the fermentation liquor which is not subjected to acid-base treatment is used as a control, the curvularia leaf spot germ of the rice is used as a test bacterium, and the experimental treatment method and the relative bacteriostasis rate calculation method are the same as the above.

The influence of acid and alkali treatment on the antibacterial activity of Qishu endogenous Bacillus beleisi HSU-3 fermentation liquor is as follows: the fermentation broth was treated with different pH gradients, and the acid-base stability of the fermentation broth was determined, with the results shown in fig. 13.

The results show that the relative inhibition rate is maximum at 105.3% when the pH is 8.0. But the bacteriostatic activity is in a descending trend under the treatment of strong acid and strong alkali, and when the pH value is 2.0, the bacteriostatic activity is the lowest, and the relative bacteriostatic rate is 45 percent; the antibacterial activity of the antibacterial substance of the fermentation product is obviously reduced under the condition of strong acid or strong alkali, and the antibacterial substance has better stability under the condition of partial alkalinity.

(3) Thermal stability

Treating endophyte fermentation liquor in water bath kettle at 40 deg.C, 60 deg.C, 80 deg.C and 100 deg.C for 30min, cooling to room temperature, using untreated fermentation liquor as control, using non-temperature treated sterile water as blank control, using curvularia leaf spot of rice as test bacteria, and performing experimental treatment and relative antibacterial rate calculation.

Effect of heat treatment on the bacteriostatic activity of Qi Shuna raw bacillus beleisi HSU-3 fermentation broth: the fermentation broth was treated at different temperatures of 40-100 ℃ for 30min, and the thermal stability of the fermentation broth was determined, with the results shown in FIG. 14.

The experimental result shows that the relative bacteriostasis rate of the fermentation liquid can reach more than 100% after being treated at different temperatures, and particularly, after the fermentation supernatant of Qi Shuna live Bacillus beleisi HSU-3 is treated at 100 ℃, the relative bacteriostasis rate still reaches more than 100%, which indicates that the bacteriostatic active substances in the fermentation product have stronger tolerance to different temperatures.

(4) UV stability

Placing 1 ultraviolet lamp (UV-C, 30W) right above the endophyte fermentation liquor, wherein the vertical distance between the lamp and the liquid level in the culture dish is 15cm, and the irradiation time is 1h, 2h and 3h. The untreated fermentation liquor is used as a control, the sterile water without temperature treatment is used as a blank control, the curvularia leaf spot germ is used as a test bacterium, and the experimental treatment method and the relative bacteriostasis rate calculation method are the same as the above.

Influence of ultraviolet rays on the bacteriostatic activity of Qi Shuna live Bacillus belezii HSU-3 fermentation broth: the fermentation broth was irradiated with ultraviolet light for 1-3 hours, and the effect of different ultraviolet irradiation times on the stability of the fermentation broth was determined, with the results shown in fig. 15.

According to experimental results, after the fermentation liquor is irradiated by ultraviolet rays with different time gradients, the bacteriostatic activity of the fermentation liquor is not obviously changed; although the antibacterial activity is slightly reduced after 3 hours, the relative antibacterial rate still reaches 98.06 percent, which shows that the antibacterial substance in the fermentation liquor still has stronger antibacterial action and higher stability under the ultraviolet irradiation.

Finally, it should be noted that: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; it will be understood by those skilled in the art that the present invention may be modified and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.

Claims (10)

1. The Qishu endogenous Bacillus belgii is characterized in that Qi Shuna Bacillus belgii has been preserved in China Center for Type Culture Collection (CCTCC) at 26/10/2022 with the preservation number of CCTCC M20221664.

2. The Qi Shuna bacillus beiLevensis of claim 1, wherein the 16S rRNA gene sequence of bacillus beilesis as set forth in SEQ ID No. 1.

3. The bacillus beijerinckii Qi Shuna as in claim 1 wherein Qi Shuna beijerinckii is antagonistic to a phytopathogenic fungus.

4. The Qi Shuna Bacillus belgii producer of claim 1, wherein the Qi Shu Bacillus belgii fermentation product is antagonistic to a phytopathogenic fungus.

5. The Qi Shuna Bacillus belgii producer of claim 1, wherein the Qi Shu Bacillus belgii producer fermentation product is antagonistic to Curvularia lunata.

6. The Qi Shuna Bacillus belgii producer of claim 4, wherein the extraction process of the Qi Shu Bacillus belgii fermentation product comprises: inoculating Qi Shuna Bacillus beleisi HSU-3 into culture medium for culture, inoculating 80-120 μ L into culture medium for continuous culture, centrifuging fermentation liquid, and collecting supernatant.

7. The Qi Shuna bacillus belmentioned of claim 2, wherein said phytopathogenic fungi comprise curvularia leaf spot, fusarium graminearum, fusarium oxysporum, colletotrichum, fusarium solani, fusarium oxysporum, phytophthora capsici, and sclerotinia sclerotiorum.

8. The Qi Shuna bacillus belgii producer of claim 1, wherein the method of isolation of Qi Shuna bacillus belgii producer comprises: cleaning fresh rhizome with sterile water, pretreating, and cutting into small pieces of about 2-5 mm; planting the tissue segments on a PDA culture medium in a sterile environment, after bacteria grow out from the cut of the rhizome cultured at a constant temperature, transferring the tissue segments to a fresh PDA culture medium for purification; transferring the purified strain to PDA slant culture medium, and storing for use

Wherein the PDA culture medium comprises: 200g of peeled potatoes, 20g of glucose and 15g of agar, and double distilled water is added to the mixture to reach the constant volume of 1000mL.

9. The method of screening for Qi Shuna bacillus belgii sp as in claim 1, comprising the steps of: adopting a plate scribing method to obtain Qi Shuna single colonies of the Bacillus beleisis HSU-3, utilizing a universal primer to carry out 16S rRNA amplification, constructing a phylogenetic tree and determining the classification status of the strain.

10. The use of Qi Shuna Bacillus belgii producing bacteria according to claim 1,

the method comprises the following applications:

the application of the strain as an antagonistic bacterium of curvularia leaf spot;

the application of the antagonistic bacterium of wheat scab germs;

the application of the strain as antagonistic bacteria of the corn stalk base rot germs;

the application of the strain as antagonistic bacteria of cotton fusarium wilt pathogens;

the application of the antagonistic bacteria of apple anthracnose bacteria;

the application of the compound as an antagonistic bacterium of tomato blight disease bacteria;

the application of the antagonistic bacteria of rice blast germs;

the application of the antibacterial agent as the antagonistic bacteria of the germs of the anthracnose of the jujube;

the application of the strain as an antagonistic bacterium of phytophthora capsici;

can be used as antagonistic bacteria of sclerotinia sclerotiorum.

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