Bacillus amyloliquefaciens strain, biocontrol inoculant, fermentation broth, and preparation method and application of bacillus amyloliquefaciens strain and biocontrol inoculant

Abstract

The invention discloses a bacillus amyloliquefaciens strain, a biocontrol inoculant, a fermentation broth, a preparation method and an application, the bacillus amyloliquefaciens strain is bacillus amyloliquefaciens Ba-D3, and is preserved in the China Center for Type Culture Collection, the preservation date is December 3, 2024, and the preservation number is CCTCC NO: M 20242712. The bacillus strain is subjected to activation, seed culture and production culture to prepare the biocontrol inoculant. The biocontrol inoculant is subjected to fermentation culture and separation, and supernate is taken to prepare fermentation liquor. The bacillus amyloliquefaciens Ba-D3 antagonizes the growth of various fusarium germs, and the biological control effect on root rot caused by fusarium is remarkably improved. The biocontrol microbial inoculum or fermentation broth prepared by the method is applied to root irrigation, and has the advantages of low production cost, good application effect, no toxicity, environmental protection and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticide technology, and particularly relates to a strain of Bacillus amyloliquefaciens, a biocontrol agent, a fermentation broth, a preparation method, and an application. Background Technology

[0002] Root rot, a serious plant disease caused by soil-borne fungal pathogens, primarily infects the root system and rhizomes of plants. By damaging the vascular tissue of the root system, it hinders the transport of water and nutrients, leading to the rotting and necrosis of the underground rhizomes and wilting, yellowing and withering of the leaves in the above-ground parts, ultimately causing plant death. It poses a significant threat to crop production and agricultural development. The main pathogens causing root rot include those of the genus *Fusarium* (…). Fusarium spp. Fusarium solani (soil fungus) Fusarium solani Fusarium oxysporum ( Fusarium oxysporum ), Verticillium ( Fusarium whorl-shaped ), Rhizoctonia spp. Rhizoctonia spp. Rhizoctonia solani ( ) Rhizoctonia solani ) and Pythium ( Pythiumspp. The ultimate Pythium ( The last Pythium ), Pythium spp. ( Pythium aphaniderm Among these, Fusarium pathogens are highly pathogenic and cause particularly serious damage. This disease widely infects many important economic crops, including grain crops such as wheat, corn, and rice, cash crops such as soybeans, peanuts, and cotton, as well as fruit trees, vegetables, and other agricultural crops.

[0003] Currently, methods for controlling crop root rot include chemical control, physical control, and biological control. Among these, chemical control is the most widely used, such as pesticide spraying and environmental fumigation. However, chemical control methods can easily cause environmental pollution, pesticide residues, and alterations to the soil microbial community structure during application. While physical control methods are environmentally friendly, they suffer from technical problems such as being time-consuming and labor-intensive, having a limited range of control, and incomplete eradication of pathogens. Biological control, by introducing antagonistic or endogenous bacteria, effectively inhibits pathogens and combines high efficiency, environmental friendliness, and economy, making it a key measure and means for modern green disease control. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a method for preparing Bacillus amyloliquefaciens strains, biocontrol agents, and fermentation broth, as well as their application in root rot control. Addressing the deficiencies of existing technologies, this invention significantly improves the biocontrol effect against Fusarium-induced root rot by utilizing Bacillus amyloliquefaciens Ba-D3 to antagonize the growth of various Fusarium pathogens.

[0005] To address the aforementioned technical problems, this invention provides a Bacillus strain, wherein the Bacillus is Bacillus amyloliquefaciens Ba-D3 (… Bacillus amyloliquefaciens (), deposited at the China Center for Type Culture Collection, on December 3, 2024, with accession number CCTCC NO: M 20242712.

[0006] Based on a general technical concept, the present invention provides a biocontrol agent, which is prepared by the Bacillus strain through activation, seed culture, and production culture.

[0007] Based on a general technical concept, the present invention provides a method for preparing the biocontrol agent, the method comprising the following steps: S1. Spread the preserved strain of Bacillus amyloliquefaciens Ba-D3 onto a solid culture medium and culture it using the plate culture method until yellow single colonies appear. S2. The yellow single colony is inoculated into a sterile liquid culture medium and cultured to the logarithmic growth phase to obtain the desired bacterial solution; S3. The cultured bacterial solution is inoculated into the production culture medium for production culture until the bacterial cells reach the logarithmic growth phase, and the biocontrol agent is prepared.

[0008] Based on a general technical concept, the present invention provides a fermentation broth, which is prepared by fermenting and culturing the biocontrol agent and separating the supernatant.

[0009] Based on a general technical concept, the present invention provides a method for preparing the fermentation broth, the method comprising the following steps: S1. Spread the preserved strain of Bacillus amyloliquefaciens Ba-D3 onto a solid culture medium and culture it until yellow single colonies appear. S2. The yellow single colony is inoculated into a sterile liquid culture medium and cultured to the logarithmic growth phase to obtain the desired bacterial solution; S3. The cultured bacterial solution is inoculated into the production culture medium for production culture until the bacterial cells reach the logarithmic growth phase, and the biocontrol agent is prepared. S4. The biocontrol agent is fermented to obtain a fermentation mixture; S5. Centrifuge the fermentation mixture, collect the supernatant, and obtain the fermentation broth.

[0010] In the above preparation method, the temperature of the plate culture in S1 is 28℃~32℃, and the culture time is 48 h~72 h; And / or, in S2, the seed culture temperature is 28℃~32℃, the rotation speed is 160 rpm~200 rpm, and the culture time is 48 h~72 h; And / or, in S3, the bacterial solution is inoculated at 2% to 5% of the total volume of the Bacillus amyloliquefaciens production culture medium.

[0011] In the above preparation method, further, the concentration of the biocontrol agent in step S4 is 1×10⁻⁶. 8 cfu / mL.

[0012] Further, in the above preparation method, the solid culture medium in step 7 comprises: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, and pH adjusted to 7.2-7.4; the liquid culture medium and the production culture medium both comprise: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, and pH adjusted to 7.2-7.4.

[0013] In the above preparation method, the fermentation culture is specifically carried out at a temperature of 28℃~32℃ and a rotation speed of 160 rpm~200 rpm for 48 h~72 h; And / or, the centrifugation conditions are: centrifugation at 6000 rpm to 10000 rpm for 5 min to 10 min.

[0014] Based on a general technical concept, the present invention provides the application of the Bacillus strain, the Bacillus amyloliquefaciens biocontrol agent, and the Bacillus amyloliquefaciens fermentation broth in the prevention and control of root rot in cowpea.

[0015] Based on a general technical concept, this invention provides the application of the aforementioned Bacillus strain, the aforementioned Bacillus amyloliquefaciens biocontrol agent, and the aforementioned Bacillus amyloliquefaciens fermentation broth in the preparation of a Fusarium antagonist. The Fusarium is one or more of Fusarium oxysporum, Fusarium rosenbergii, and Fusarium fusiforme.

[0016] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides a Bacillus amyloliquefaciens strain Ba-D3, which is effective against Fusarium oxysporum, a bacterium that causes root rot. Fusarium oxysporum Fusarium rosenbergii ( ), Fusarium incarnate It has a significant antagonistic effect on various pathogenic bacteria of the Fusarium genus, such as ( ).

[0017] (2) This invention provides a biocontrol agent and fermentation liquid, which has the characteristics of simple, fast and efficient preparation process, green and environmentally friendly, biosafe and pollution-free. This technology provides an effective biological control solution for the prevention and control of root rot.

[0018] A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The depositary institution is the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20242712. The address of the depositary institution is located at Wuhan University, China, and the deposit date is December 3, 2024.

[0019] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the accompanying drawings and tables. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] Figure 1 To investigate the inhibitory effect of Bacillus amyloliquefaciens fermentation broth on the mycelial growth of root rot pathogens.

[0022] Figure 2 The fermentation broth of Example 3 shows the inhibition of Fusarium oxysporum spore germination.

[0023] Figure 3 The fermentation broth of Example 3 shows the inhibition of Fusarium rosenbergii spore germination.

[0024] Figure 4 The fermentation broth of Example 3 shows the inhibition of Fusarium spore germination.

[0025] Figure 5 To investigate the effects of Bacillus amyloliquefaciens on the hyphal morphology of Fusarium oxysporum, Fusarium rosenbergii, and Fusarium fusiforme.

[0026] Figure 6 The effects of water treatment (left) and Bacillus amyloliquefaciens biocontrol agent treatment (right) on the control of root rot in chili peppers in the field. Detailed Implementation

[0027] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention. The materials, reagents, and instruments used in the following embodiments can all be purchased commercially. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art.

[0028] Example 1 An embodiment of the present invention: Bacillus amyloliquefaciens Ba-D3 ( Bacillus starch liquefier Ba-D3, isolated and purified from soil, is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20242712, located at 299 Bayi Road, Wuchang District, Wuhan, Hunan Province, China (China Center for Type Culture Collection, Wuhan University), on December 3, 2024. The preparation process of the Bacillus amyloliquefaciens biocontrol agent and biocontrol fermentation broth of this invention is simple, rapid, efficient, green, environmentally friendly, and biosafe, providing an effective biological control solution for root rot.

[0029] In this embodiment, Bacillus amyloliquefaciens Ba-D3 was mainly obtained through screening using the following methods: 10 g of soil sample collected from the field was added to an Erlenmeyer flask containing 90 ml of sterile water and incubated for 30 min to obtain a bacterial suspension. The suspension was then diluted and spread onto LB agar plates and incubated at 28℃~32℃ for 48 h~72 h until pale yellow single colonies appeared. Single colonies were selected and streaked onto agar plates for purification. The plates were incubated at 28℃~32℃ for 48 h~72 h until single colonies appeared. Single colonies with larger growth and regular shape were selected.

[0030] Based on our testing, the Bacillus amyloliquefaciens Ba-D3 of this embodiment has the following main characteristics: The optimal growth temperature is 28℃~32℃. When cultured on plate medium for 48 h~72 h, pale yellow round colonies are formed with regular edges, smooth surfaces, and a colony diameter of 0.20 mm~0.60 mm. Bacillus amyloliquefaciens strains turn yellow when cultured in liquid medium for 48 h~72 h.

[0031] Example 2 A biocontrol agent of the present invention is prepared from the Bacillus amyloliquefaciens strain of Example 1 through isolation, purification, seed culture, and production culture. Furthermore, the Bacillus amyloliquefaciens biocontrol agent and fermentation broth of the present invention possess characteristics such as environmental friendliness, safety for humans and animals, and no harm to crops, and are unlikely to induce drug resistance in pests and diseases.

[0032] The preparation method of the biocontrol agent in this embodiment includes: (1) Isolation: The Bacillus amyloliquefaciens strain of Example 1 was inoculated into a 100 ml Erlenmeyer flask for enrichment culture. The cultured bacterial suspension was diluted and spread to obtain single colonies.

[0033] (2) Purification: Single colonies were purified by streaking on LB agar plates until yellow single colonies appeared. The components of the LB agar plate were: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, and pH adjusted to 7.2-7.4.

[0034] (3) Seed culture: Yellow single colonies were inoculated into liquid culture medium for cultivation. The seed culture temperature was 28℃~32℃, and the rotation speed was 180 rpm (160 rpm~200 rpm is also acceptable). The pH value of the liquid culture medium was 7.2~7.4 to obtain bacterial suspension in the logarithmic growth phase. The components of the liquid culture medium were: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, and the pH was adjusted to 7.2~7.4.

[0035] (4) Production Cultivation: Inoculate the bacterial solution at 2% to 5% of the total volume of the Bacillus amyloliquefaciens production culture medium and carry out production cultivation at a temperature of 28℃ to 32℃ and a rotation speed of 180 rpm (160 rpm to 200 rpm is also acceptable) until the logarithmic growth phase of the cells, thus obtaining the Bacillus amyloliquefaciens biocontrol agent. The production culture medium formula is the same as that of the liquid culture medium, and the pH is adjusted to 7.2 to 7.4. The bacterial concentration of the biocontrol agent is 1×10⁻⁶. 6 cfu / mL ~ 1×10 8 cfu / mL.

[0036] Example 3 A fermentation broth of the present invention is prepared from the Bacillus amyloliquefaciens strain of Example 1 through isolation, purification, seed culture, production culture, fermentation culture, and centrifugation to collect the supernatant.

[0037] The method for preparing the fermentation broth in this embodiment includes: (1) Isolation: The Bacillus amyloliquefaciens strain of Example 1 was inoculated into a 100 ml Erlenmeyer flask for enrichment culture. The cultured bacterial suspension was diluted and spread to obtain single colonies.

[0038] (2) Purification: Single colonies were purified by streaking on LB agar plates until yellow single colonies appeared. The components of the LB agar plate were: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, and pH adjusted to 7.2-7.4.

[0039] (3) Seed culture: Yellow single colonies were inoculated into liquid culture medium for cultivation. The seed culture temperature was 28℃~32℃, and the rotation speed was 180 rpm (160 rpm~200 rpm is also acceptable). The pH value of the liquid culture medium was 7.2~7.4 to obtain bacterial suspension in the logarithmic growth phase. The components of the liquid culture medium were: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, and the pH was adjusted to 7.2~7.4.

[0040] (4) Production Cultivation: Inoculate the bacterial solution at 2% to 5% of the total volume of the Bacillus amyloliquefaciens production culture medium and carry out production cultivation at a temperature of 28℃ to 32℃ and a rotation speed of 180 rpm (160 rpm to 200 rpm is also acceptable) until the logarithmic growth phase of the cells, thus obtaining the Bacillus amyloliquefaciens biocontrol agent. The production culture medium formula is the same as that of the liquid culture medium, and the pH is adjusted to 7.2 to 7.4. The bacterial concentration of the biocontrol agent is 1×10⁻⁶. 8 cfu / mL (1×10) 6 cfu / mL ~ 1×10 8 (cfu / mL is acceptable).

[0041] (5) Fermentation culture: The biocontrol agent is fermented in a fermentation medium at a temperature of 28℃~32℃ and a rotation speed of 180 rpm (160 rpm~200 rpm is acceptable) for 60 h (48 h~72 h is acceptable) to obtain a fermentation mixture.

[0042] (6) Centrifugation: Centrifuge the fermentation mixture at 8000 rpm (6000 rpm ~ 10000 rpm is acceptable) for 10 min (5 min ~ 10 min is acceptable), collect the supernatant to obtain the fermentation broth, and store it at 4℃ for later use. The fermentation medium formula is the same as that of the liquid medium, and the pH is adjusted to 7.2~7.4.

[0043] Example 4 The application of the fermentation broth of Example 3 in the control of root rot pathogens, the application method includes: Diseased tissue samples and diseased areas were collected from cowpeas (large-scale root rot was found in cowpea growing areas in Hainan; samples showing blackish-brown rot symptoms on the roots and necks were selected) for pathogen isolation. Three pathogenic strains were isolated, purified, and identified, namely *Fusarium oxysporum*. Fusarium oxysporum (Fo), Fusarium rosenbergii Fusarium incarnate (Fi), Fusarium Fusarium falcate (Ff). Use a 6 mm punch to make holes in the mycelium cake and transfer it to a new growth medium for growth until the medium is fully colonized.

[0044] Take the prepared Bacillus amyloliquefaciens Ba-D3 biocontrol fermentation broth and growth medium, and evenly spread 100 µl and 200 µl of each onto LB solid medium plates used for culturing root rot pathogens Fo, Fi, and Ff, respectively. After the spread solution is absorbed by the medium and the plate surface is dry, use a 6 mm aseptic punch to take mycelial cakes of different root rot pathogens and place them in the center of the solid medium surface. Use unspread blank solid medium as a control. Set up three replicates for each treatment. Incubate at 28℃. After the control plate has reached full mycelial growth, measure the colony diameter of each treatment group using the cross-cross method and calculate the mycelial growth inhibition rate R. The formula for calculating the mycelial growth inhibition rate R is: R(%)=(R1-R2) / R1×100%.

[0045] Where R is the mycelial growth inhibition rate, R1 is the colony growth diameter of the blank control group, and R2 is the colony growth diameter of the treatment group.

[0046] Figure 1 The fermentation broth is used to treat Fusarium oxysporum, the pathogen of root rot. Fusarium oxysporum Fusarium rosenbergii Fusarium incarnate Fusarium Fusarium falcate The control effect diagram is shown below. a is the blank control with no inoculated pathogen, b is the treatment with 100 µl of growth medium, c is the treatment with 200 µl of growth medium, d is the treatment with 100 µl of fermentation broth from Example 3, and e is the treatment with 200 µl of fermentation broth from Example 3.

[0047] Table 1: Inhibitory effects of fermentation broth and growth medium from Example 3 on different pathogens of root rot.

[0048] from Figure 1 As shown in Table 1, compared with the control culture medium, the pathogens (Fi, Ff, Fo) grown under the 100 µl and 200 µl growth medium plating conditions did not achieve significant inhibition rates. However, after treatment with 100 µl of fermentation broth, compared with the control group, the inhibition rates against pathogens Fi, Ff, and Fo were 20.7%, 33.3%, and 46.1%, respectively; when the treatment concentration was increased to 200 µl, the inhibition rates reached 26.8%, 60.2%, and 53.8%, respectively, indicating that the inhibitory effect changed with increasing concentration. Especially at the treatment concentration of 200 µl, the inhibition rates against pathogens Ff and Fo were both greater than 50%, showing a significant inhibitory effect, indicating that the Bacillus amyloliquefaciens fermentation broth has significant antagonistic activity against pathogens in root rot.

[0049] Figure 2The fermentation broth of Example 3 shows the inhibition of Fusarium oxysporum spore germination. Figure 3 The fermentation broth of Example 3 shows the inhibition of Fusarium rosenbergii spore germination. Figure 4 The fermentation broth of Example 3 shows the inhibition of Fusarium spore germination. Figure 5 The effects of *Bacillus amyloliquefaciens* on the hyphal morphology of *Fusarium oxysporum*, *Fusarium rosenbergii*, and *Fusarium oxysporum* were investigated. The figures show that *Bacillus amyloliquefaciens* Ba-D3, its biocontrol agent, and fermentation broth significantly inhibited the germination of *Fusarium oxysporum*, *Fusarium rosenbergii*, and *Fusarium oxysporum* spores, as well as the morphology of their hyphae. This indicates that the fermentation broth of *Bacillus amyloliquefaciens* has significant antagonistic activity against pathogens causing root rot.

[0050] Example 5 Application of a biocontrol agent of Example 2 in the control of root rot of peppers in the field.

[0051] The biocontrol agent from Example 2 and water were applied to the root rot areas in the chili pepper growing area for root irrigation to investigate the control effect of root rot in the chili pepper growing area.

[0052] Figure 6 The study compared the actual control effects of water treatment (left) and the biocontrol agent treatment (right) in Example 2 on pepper root rot in the field. The results showed that the root rot in peppers was significantly alleviated in the area where the biocontrol agent was applied in Example 2, while the root rot was not alleviated in the area where water was applied, and even led to leaf wilting and death.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens, characterized in that, The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens Ba-D3 ( Bacillus amyloliquefaciens (), deposited at the China Center for Type Culture Collection, on December 3, 2024, with accession number CCTCC NO: M 20242712.

2. A biocontrol agent, characterized in that, The biocontrol agent is prepared from the Bacillus strain described in claim 1 through activation, seed culture, and production culture.

3. A method for preparing the biocontrol agent according to claim 2, characterized in that, The preparation method includes the following steps: S1. Spread the preserved strain of Bacillus amyloliquefaciens Ba-D3 onto a solid culture medium and culture it until yellow single colonies appear. S2. The yellow single colony is inoculated into a sterile liquid culture medium and cultured to the logarithmic growth phase to obtain the desired bacterial solution; S3. The bacterial solution is inoculated into the production culture medium for production culture until the bacterial cells reach the logarithmic growth phase, and the biocontrol agent is prepared.

4. A fermentation broth, characterized in that, The fermentation broth is prepared by fermenting and culturing the biocontrol agent described in claim 2 and then separating and taking the supernatant.

5. A method for preparing the fermentation broth according to claim 4, characterized in that, The preparation method includes the following steps: S4. The biocontrol agent is fermented to obtain a fermentation mixture; S5. Centrifuge the fermentation mixture, collect the supernatant, and obtain the fermentation broth.

6. The preparation method according to claim 3, characterized in that, The temperature for plate culture in S1 is 28℃~32℃, and the culture time is 48 h~72 h; And / or, in S2, the seed culture temperature is 28℃~32℃, the rotation speed is 160 rpm~200 rpm, and the culture time is 48 h~72 h; And / or, in S3, the bacterial solution is inoculated at 2% to 5% of the total volume of the Bacillus amyloliquefaciens production culture medium.

7. The preparation method according to claim 3, characterized in that, The solid culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder, and pH adjusted to 7.2-7.4; the liquid culture medium and the production culture medium both comprise: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, and pH adjusted to 7.2-7.

4.

8. The preparation method according to claim 5, characterized in that, The fermentation culture is specifically carried out at a temperature of 28℃~32℃ and a rotation speed of 160 rpm~200 rpm for 48 h~72 h; And / or, the centrifugation conditions are: centrifugation at 6000 rpm to 10000 rpm for 5 min to 10 min.

9. The application of the Bacillus amyloliquefaciens strain of claim 1, the biocontrol agent of claim 2, and the fermentation broth of claim 4 in the prevention and control of root rot in crops (peppers).

10. The use of the Bacillus amyloliquefaciens strain of claim 1, the biocontrol agent of claim 2, and the fermentation broth of claim 4 in the preparation of Fusarium oxysporum, Fusarium incarnatum, and Fusarium falciforme antagonists.

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