Bacillus BAF143, fermentation liquor crude extract thereof and application of bacillus BAF143 in corn storage and phytopathogen inhibition
By screening and identifying the rough bosomon of BAF143 and its fermentation solution, the growth and germination of pathogenic bacteria such as yellow mold in corn storage have achieved significant inhibitory effects and show the potential of broad -spectrum biophythy.
Patent Information
- Application Number
- CN202411826502.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-01-24
AI Technical Summary
During the storage process, corn is susceptible to pathogens such as Aspergillus aflatoxin, resulting in contamination and health risks. It is difficult for the existing technology to effectively inhibit the growth and germination of these pathogens.
Screening and identifying a rough bosomic bosopiablyblyblybacter BaF143 and its fermentation liquid, to prepare rough rats through steps such as shock cultivation, expansion of cultivation and extraction, significantly inhibiting the growth and germination of yellow mold.
The BAF143 strain and its fermentation broth crude extract can significantly reduce the germination rate of Aspergillus flavus spores from 97.44% to 9.76%. They have inhibitory effects on a variety of plant pathogenic bacteria and have broad-spectrum biocontrol effects.
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Figure CN119351273A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Bacillus BAF143 and a crude extract of fermentation liquid thereof and applications thereof in corn storage and inhibition of plant pathogens. Background Art
[0002] Corn, a plant of the Poaceae family and the genus Zea, is a major food crop and also one of the indispensable raw materials in the fields of medical care, light industry, and chemical industry. During its growth, corn is often infected by pathogens such as fungi, bacteria, and viruses. These pathogens are difficult to completely eliminate, and their spores can easily remain on the surface of crops and germinate again under suitable conditions. For example, during long-term storage, because the embryo of corn contains more than 30% protein and more soluble sugars, it has a certain hygroscopicity, and the breathing process will cause fever and condensation. At this time, if the environmental conditions are not strictly controlled, then these spores remaining in the crops will easily germinate and cause pollution, produce toxins, and harm human health.
[0003] The present application conducts screening in contaminated corn, hoping to obtain a strain that can inhibit the germination of Aspergillus flavus spores and is beneficial to corn storage, and further explore the effects of the strain on different pathogens. Summary of the invention
[0004] The purpose of the present invention is to provide a strain which is beneficial to corn storage and inhibits the growth and germination of Aspergillus flavus.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a Bacillus sp. BAF143, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of September 23, 2024 and a deposit number of CGMCC No.32046.
[0007] The present invention provides a crude extract of fermentation broth of Bacillus BAF143, and the preparation method thereof is as follows:
[0008] (1) inoculating the Bacillus sp. BAF143 into a culture medium, shaking and culturing the culture, and then expanding the culture to obtain a BAF143 fermentation liquid;
[0009] (2) After centrifuging the BAF143 fermentation broth, taking the supernatant and placing it in an organic solvent 1 for extraction, taking the organic phase and drying it to obtain a crude extract of the Bacillus subtilis BAF143.
[0010] Preferably, the culture medium in step (1) is LB culture medium;
[0011] The shaking culture conditions are 28-32° C., 150-250 r / min, 20-28 h;
[0012] The conditions for the expanded culture are 28-32° C. for 2-4 days.
[0013] Preferably, the centrifugation conditions in step (2) are: 7000-9000 rpm, 5-15 min;
[0014] The organic solvent 1 is ethyl acetate, and the volume ratio of the supernatant to the organic solvent 1 is 1:0.8-1.2.
[0015] Preferably, the crude extract of fermentation broth is used in the form of a liquid preparation, the solvent is methanol, and the effective concentration when used is 25-50 mg / mL.
[0016] The invention provides application of Bacillus BAF143 and crude extract of fermentation broth thereof in inhibiting the growth of Aspergillus flavus hyphae or spore germination.
[0017] The invention provides application of Bacillus BAF143 and crude extract of fermentation broth thereof in corn storage.
[0018] The present invention also provides the use of Bacillus BAF143 in inhibiting plant pathogenic bacteria.
[0019] Preferably, the plant pathogens include Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani anthracnose pathogen, mango anthracnose pathogen and banana anthracnose pathogen.
[0020] Preferably, the effective bacterial suspension concentration of Bacillus subtilis BAF143 is (0.8-1.2)×10 8 CFU / mL.
[0021] The invention screened and identified a strain of Bacillus BAF143, which can significantly inhibit the growth of Aspergillus flavus. The crude extract of fermentation broth prepared by the strain can significantly inhibit the germination of Aspergillus flavus spores, and can reduce the germination rate of Aspergillus flavus spores from 97.44±4.44% to 9.76±0.21%, effectively preventing the growth and germination of Aspergillus flavus in corn, and has good application prospects in corn storage.
[0022] In addition, strain BAF143 has an inhibitory effect on different plant pathogens, among which the inhibition rate of Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani anthracnose pathogen, Fusarium solani anthracnose pathogen and Fusarium solani anthracnose pathogen is higher than 50%, and the inhibition rate of Fusarium solani is the highest, which is 69.70%. It can be used as a broad-spectrum biocontrol bacteria. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The morphological characteristics of BAF143, where A is the colony morphology on LB medium, B is the unstained bacterial morphology under a microscope, and C is the Gram-stained bacterial morphology under a microscope.
[0024] Figure 2 This is a phylogenetic tree constructed based on 16S sequences.
[0025] Figure 3 Schematic diagram of the plate design for the cross-seeding method in Example 2.
[0026] Figure 4 This is the effect of strain BAF143 in Example 2 on the growth of Aspergillus flavus hyphae.
[0027] Figure 5 Schematic diagram of the flat plate confrontation in the flat plate punching method in Example 3.
[0028] Figure 6 This is the effect of the crude extract of the fermentation broth of strain BAF143 in Example 3 on the growth of Aspergillus flavus hyphae.
[0029] Figure 7 The effect of the crude extract of the fermentation broth of strain BAF143 in Example 3 on the germination of Aspergillus flavus spores.
[0030] Figure 8 This is the effect of the crude extract of the fermentation broth of strain BAF143 in Example 4 on the infection of Aspergillus flavus in corn.
[0031] Fig. 9 The results of plate confrontation of strain BAF143 in Example 5 against 10 plant pathogens.
[0032] Fig.10 It is the inhibition rate of strain BAF143 in Example 5 against 10 plant pathogens.
[0033] Collection Instructions
[0034] Bacillus sp. BAF143, the strain is deposited in the General Microbiology Center of China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 23, 2024, and the deposit number is CGMCC No.32046. DETAILED DESCRIPTION
[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0036] Example 1 Screening and identification of strains
[0037] 1. Morphological identification: In the culture of Aspergillus flavus, a contaminated bacterial strain was found to be antagonistic to Aspergillus flavus. The bacteria was picked on the LB plate and purified by the plate streak method. The strain BAF143 was further cultured on LB medium at 30°C for 24 hours, and the colony characteristics were observed and recorded.
[0038] like Figure 1 As shown in A, the colonies on the LB medium are milky white, dry and dull on the surface, with rough edges, wrinkles, opaque, difficult to pick up with an inoculation needle, and sticky; when observed under a microscope, the bacterial cells ( Figure 1 B and Figure 1 C) It is rod-shaped, has no spores, and is a Gram-positive bacterium.
[0039] 2. Classification and identification:
[0040] Total bacterial DNA was obtained by chelex-100 method and amplified with 16S primers (SEQ ID NO.1: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and SEQ ID NO.2: 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the PCR products were sent to Guangzhou BGI for sequencing. The obtained sequences were aligned on the EzBiocloud.net website and NCBI (National Center for Biotechnology Information) and the sequences of related strains were downloaded. All tree-building sequences were aligned in Clustal X 1.83, and ML trees were constructed in MEGA v6.06, with maximum likelihood bootstrap proportions (MLBP) higher than 50%.
[0041] The sequence of strain BAF143 is shown in SEQ ID NO.3:
[0042] SEQ ID NO.3:
[0043]
[0044] Depend on Figure 2 It can be seen that strain BAF143 and MK618595 Bacillus amyloliquefaciens CC12 are clustered in a small branch, the maximum likelihood support rate of this branch is not high (MLBP=70%), and the similarity with this species is 99.20%. Therefore, strain BAF143 is tentatively identified as Bacillus sp.
[0045] Example 2 Effect of strain BAF143 on the growth of Aspergillus flavus hyphae
[0046] The strain was selected and placed in liquid LB medium, cultured in a shaking incubator at 30°C for 24 h, and plated against Aspergillus flavus. The cross-inoculation method (cross-inoculation method as Figure 3 First, holes were punched at about 1 cm from the edge and in the center of the plate using a 0.4 cm diameter puncher. The agar block was taken out and 10 μL of the bacterial BAF143 suspension (10 8 CFU / mL), 5 μL of Aspergillus flavus spore suspension (10 8 CFU / mL). After culturing in a 30°C incubator for 5 days, the diameter of the Aspergillus flavus colonies was observed and measured, photographed and recorded, and the inhibition rate was calculated according to formula (1).
[0047]
[0048] Depend on Figure 4 It can be seen that compared with the control, the bacterial strain BAF143 has a significant inhibitory effect on Aspergillus flavus in LB medium, Martin medium and PDA medium, and its inhibition rate is higher than 40%. Among them, the inhibition rate on PDA medium is the highest, reaching 52.73±0.53%.
[0049] Example 3 Effect of crude extract of fermentation broth of strain BAF143 on the growth of Aspergillus flavus hyphae
[0050] 1. Preparation of Crude Extracts
[0051] The strain was picked from the plate and inoculated into a 250 mL conical flask containing 100 mL LB liquid medium. After shaking and culturing at 200 r / min for 24 h, the seed solution was used. The seed solution was inoculated into a new LB liquid medium at a ratio of 1:100 and cultured at 30°C for 3 days to obtain the fermentation liquid (OD 600=1.82). The fermentation liquid was centrifuged at 8000rpm for 10min, the supernatant was taken, an equal volume of ethyl acetate was added, and the mixture was gently shaken and mixed, and then allowed to stand. After the two liquids were separated, the upper layer (ethyl acetate layer) was taken out, and the extraction was repeated 3 times to obtain the organic phase, which was rotary evaporated on a rotary evaporator at 40°C and 50rpm to an extract, dried, and weighed; the crude extract after drying was dissolved in methanol to a concentration of 50mg / mL and stored in a refrigerator at 4°C for use.
[0052] 2. Effect of crude extract of fermentation broth of strain BAF143 on the growth of Aspergillus flavus hyphae
[0053] The effect of crude extract on the growth of Aspergillus flavus hyphae was determined by plate punch method (plate punch method as Figure 5 ), punch holes about 1 cm from the edge and in the center of the plate with a 0.4 cm diameter puncher, take out the agar block, inoculate 50 μL of 50 mg / mL crude extract in the holes on both sides, pure methanol solvent in the control group, and 5 μL of Aspergillus flavus spore suspension (10 8 CFU / mL), and after 5 days of incubation at 30℃, the diameter of Aspergillus flavus colonies was measured (the plate confrontation results were as follows Figure 6 A), observe and take photos, and calculate the inhibition rate according to formula (1). The results are as follows Figure 6 As shown in B.
[0054] Depend on Figure 6 It can be seen that the crude extract of strain BAF143 can significantly inhibit the growth of Aspergillus flavus, with an inhibition rate of 33.85±1.09%.
[0055] 3. Effect of crude extract of fermentation broth of strain BAF143 on spore germination of Aspergillus flavus
[0056] Pipette 100 μL of Aspergillus flavus spore suspension (10 8 CFU / mL), inoculated into 900 μL PDB medium containing crude extract of fermentation broth of strain BAF143, so that the final concentration of crude extract was 25 mg / mL, and the control group was not added with crude extract, and cultured at 30°C constant temperature shaker, 200 rpm for 24 h, and the spore germination was observed under an optical microscope (the results are shown in Figure 7 A, where a is the control group and b is the treatment group), and the spore germination rate was counted, and the statistical results are shown in Figure B.
[0057] Depend on Figure 7 It can be seen that the PDB control group ( Figure 7 Aa) and the bacterial strain BAF143 ( Figure 7There was a significant difference between the crude extract group and the control group (P<0.01), indicating that the crude extract of bacterial strain BAF143 has a very significant inhibitory effect on the germination of Aspergillus flavus spores. According to statistics, the germination rate of Aspergillus flavus spores in the control group reached 97.44±4.44%, while the germination rate of Aspergillus flavus spores in the BAF143 group was only 9.76±0.21% ( Figure 7 B).
[0058] Example 4 Effect of crude extract of fermentation broth of strain BAF143 on the growth of Aspergillus flavus hyphae in corn
[0059] Select corn with good integrity, uniform shape and no contamination for disinfection. Soak it in 0.05% sodium hypochlorite for 3 minutes, then wash it with sterile water for 30 seconds, soak it in 75% alcohol for 1 minute, and finally rinse it with sterile water for 3 times until there is no alcohol smell. Soak the disinfected corn in aflatoxin suspension (10 8 CFU / mL), 30℃, 150rpm, soak for 30min, then divide the corn into two groups, 10 corns in each group, put them in a sterile culture dish, with 3 replicates in each group, the surface of the corn in the treatment group was sprayed with 500μL (50μL / corn) of crude extract with a concentration of 50mg / mL, and the surface of the corn in the control group was sprayed with an equal amount of methanol solvent, all samples were sealed and cultured in a 30℃ incubator for 60d, and the moldy condition of the corn was observed and counted.
[0060] like Figure 8 As shown, it can be clearly seen that 4 corn kernels in the control group have grown white hyphae ( Figure 8 The red arrow in the figure points to the right), while no white mycelium appeared in the 10 corn kernels sprayed with the crude extract, indicating that the crude extract of the bacteria can inhibit the growth of Aspergillus flavus in corn.
[0061] Example 5 Effects of strain BAF143 on different plant pathogenic fungi
[0062] Using the cross method, 10 μL of bacterial suspension (10 8 CFU / mL), the central hole was inoculated with a plant pathogenic fungus cake punched with a hole punch of the same diameter. After culturing at 30°C for 5 days, the diameter of the plant pathogenic fungus colony was observed and measured and photographed (such as Fig. 9 ), the inhibition rate was calculated according to formula (1) (e.g. Fig.10 ).
[0063] The 10 pathogens are Fusarium oxysporum (FO), Fusarium fujikuroi (FF), Fusarium solani (FS), banana wilt pathogen 1 (FOC1), banana wilt pathogen 4 (FOC4), Fusarium oxysporum (JJZ), Fusarium fujikuroi (ZZZ), Fusarium solani (ZZT), mango anthracnose pathogen (MGT), and banana anthracnose pathogen (XJT).
[0064] Depend on Fig. 9 and Fig.10 It can be seen that compared with the control, in Martin medium, the bacterial strain BAF143 has a significant inhibitory effect on these 10 plant pathogenic fungi, and the inhibition rate is higher than 30%, among which the inhibition rate of Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani and Fusarium solani is higher than 50%, and the inhibition rate of Fusarium solani is the highest, which is 69.70%. It can be seen that the Bacillus bacterial strain BAF143 is a broad-spectrum biocontrol bacterium.
[0065] It can be seen from the above examples that the present invention screened and identified a Bacillus sp. BAF143, which can significantly inhibit the growth of Aspergillus flavus. The crude extract prepared from it can significantly inhibit the germination of Aspergillus flavus spores, and has good application prospects in corn storage.
[0066] In addition, strain BAF143 has an inhibitory effect on different plant pathogens, among which the inhibition rate of Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani anthracnose pathogen, Fusarium solani anthracnose pathogen and Fusarium solani anthracnose pathogen is higher than 50%, and the inhibition rate of Fusarium solani is the highest, which is 69.70%. It can be used as a broad-spectrum biocontrol bacteria.
[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A strain of Bacillus sp. BAF143, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 23, 2024, and the deposit number is CGMCC No.32046.
2. The crude extract of fermentation broth of Bacillus BAF143 according to claim 1, characterized in that The preparation method of the fermentation broth crude extract is as follows: (1) inoculating the Bacillus sp. BAF143 into a culture medium, shaking and culturing the culture, and then expanding the culture to obtain a BAF143 fermentation liquid; (2) After centrifuging the BAF143 fermentation broth, taking the supernatant and placing it in an organic solvent 1 for extraction, taking the organic phase and drying it to obtain a crude extract of the fermentation broth of Bacillus subtilis BAF143.
3. The crude extract of fermentation broth as claimed in claim 2, characterized in that The culture medium in step (1) is LB culture medium; The shaking culture conditions are 28-32° C., 150-250 r / min, 20-28 h; The conditions for the expanded culture are 28-32° C. for 2-4 days.
4. The crude extract of fermentation broth as claimed in claim 3, characterized in that The centrifugal conditions in step (2) are: 7000-9000 rpm, 5-15 min; The organic solvent 1 is ethyl acetate, and the volume ratio of the supernatant to the organic solvent 1 is 1:0.8-1.
2.
5. The crude extract of fermentation broth as claimed in claim 4, characterized in that The fermentation broth crude extract is used in the form of a liquid preparation, the solvent is methanol, and the effective concentration when used is 25-50 mg / mL.
6. Use of the crude extract of fermentation broth of Bacillus BAF143 according to claim 1 or Bacillus BAF143 according to any one of claims 2 to 5 in inhibiting the hyphae growth or spore germination of Aspergillus flavus.
7. Use of a crude extract of fermentation broth of Bacillus BAF143 according to claim 1 or Bacillus BAF143 according to any one of claims 2 to 5 in corn storage.
8. Use of the Bacillus BAF143 according to claim 1 in inhibiting plant pathogens.
9. The use according to claim 8, characterized in that The plant pathogens include Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani, Fusarium solani anthracnose pathogen, mango anthracnose pathogen and banana anthracnose pathogen.
10. The use according to claim 9, characterized in that The effective bacterial suspension concentration of Bacillus subtilis BAF143 is (0.8-1.2)×10 8 CFU / mL.