Bacillus safensis hnxj-b6 and application thereof

Abstract

The application discloses Bacillus safensis HNXJ-B6 and application thereof, is preserved in China Microorganism Strain Preservation Management Committee general microorganism center, and its preservation number is: CGMCC NO: 32528, and the preservation date is November 7, 2024.The application is identified by 16S rDNA strain, and it is confirmed that HNXJ-B6 is Bacillus safensis.The inhibition to four pathogenic bacteria of Ralstonia solanacearum, Cryphonectria parasitica, Cytospora chrysosperma and Gymnosporangium yamadae is obvious, and the application provides a new way and new biological pesticide selection for the biological control of the four pathogenic bacteria, and has good application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial application, in particular to a Bacillus safensis HNXJ-B6 and application thereof. BACKGROUND

[0002] Bacillus can achieve the effect of preventing and treating plant diseases through competition, antagonism, growth promotion, induced resistance and multi-mechanism synergy. Bacillus safensis is a gram-positive bacterium of Bacillus genus, which was first isolated from the spacecraft assembly facilities of the American Jet Propulsion Laboratory. The research on the bacterium mainly focuses on its growth promotion, for example, Sharma et al. isolated Bacillus safensis with growth promotion from saffron, Breedt et al. applied Bacillus safensis in field trials, which effectively improved the yield of corn in different soil types. Xu Tongwei screened Bacillus safensis from the root soil of black shank tobacco, which can effectively prevent and treat tobacco black shank disease. Wang Ruiqi isolated 75 strains of rhizosphere bacteria from the rhizosphere soil of rape in Tibet, among which the antagonistic effect of Bacillus safensis on wheat scab reached more than 50%, which shows that Bacillus safensis has great application potential in promoting plant growth. Bacillus safensis has rich diversity, and there are many strains with potential development value. Based on the current situation, it is of great application value and practical significance to develop Bacillus safensis with new functions. SUMMARY

[0003] In view of the above defects of the prior art, the present application provides a Bacillus safensis HNXJ-B6 and application thereof to solve the problems in the background art.

[0004] To achieve the above purpose, the present application provides the following technical solutions:

[0005] A Bacillus safensis HNXJ-B6, the preservation number of which is CGMCC NO: 32528, the preservation date of which is November 7, 2024, and which is preserved in the China General Microbiological Culture Collection Center.

[0006] Preferably, the present application further includes a fermentation bacteria agent containing Bacillus safensis HNXJ-B6.

[0007] Preferably, the preparation method of the fermentation bacteria agent includes the following steps:

[0008] (1) The frozen Bacillus safensis HNXJ-B6 is streak inoculated on NA culture medium and placed in a 28℃ incubator for culture for 48h, and then a single colony is picked to NB liquid culture medium and placed in a 30℃, 170rpm shaking culture for 12h to complete the activation;

[0009] (2) Take the activated Bacillus sabinatus HNXJ-B6 and inoculate it into LB medium at an inoculation ratio of 1-3% v / v. After culturing in a shaker at 28-37℃ and pH 7.0-9.0 for 48-96 hours, the fermentation agent is obtained.

[0010] Preferably, the NA culture medium is prepared by adding 5.0g of bacterial peptone, 1.0g of yeast powder, 10.0g of glucose, 3.0g of beef powder, and 20.0g of agar powder to every 1000mL of distilled water, adjusting the pH to 6.8-7.2, and then sterilizing at 121℃ for 20min.

[0011] Preferably, the NB liquid culture medium is prepared by adding 5.0g of bacterial peptone, 1.0g of yeast powder, 10.0g of glucose, and 3.0g of beef powder to every 1000mL of distilled water, adjusting the pH to 6.8-7.2, and then sterilizing at 121℃ for 20min.

[0012] Preferably, the LB medium is prepared by adding 5.0g of yeast extract, 10.0g of sodium chloride, and 10.0g of tryptone to every 1000mL of distilled water, and then sterilizing at 121℃ for 20min.

[0013] Preferably, the application of Bacillus saffron HNXJ-B6 or fermentation agent of the present invention in the prevention and control of plant fungal diseases caused by Cryphonectria parasitica.

[0014] Preferably, the application of the Bacillus saffron HNXJ-B6 or fermentation agent of the present invention in the prevention and control of plant fungal diseases caused by Cytospora chrysosperma.

[0015] Preferably, the application of Bacillus sabensis HNXJ-B6 or fermentation agent of the present invention in the prevention and control of plant fungal diseases caused by Yamada rust fungus.

[0016] Preferably, the application of the Bacillus saefolius HNXJ-B6 or fermentation agent of the present invention in the prevention and control of bacterial diseases in tobacco plants caused by Ralstonia solanacearum.

[0017] Compared with existing technologies, the beneficial effects of this invention are: the *Bacillus saeformis* HNXJ-B6 or its agent of this invention exhibits significant inhibitory effects on *Ralstonia solanacearum*, *Cryphonectria parasitica*, *Cytospora chrysosperma*, and *Gymnosporangium yamadae*, with high safety. The *Bacillus saeformis* HNXJ-B6 or its agent of this application is readily available, inexpensive, and provides good control effects. This invention provides a new approach and new biological agent selection for the biological control of the above four pathogens, and has significant application value. Attached Figure Description

[0018] Figure 1 To isolate bacterial strains from the rhizosphere soil of healthy plants and diseased plants, the left side is the JT primary screening and the right side is the BT primary screening.

[0019] Figure 2 Purification of strains for screening rhizosphere soil of healthy plants and rhizosphere soil of diseased plants, with the left side showing JT purification and the right side showing BT purification;

[0020] Figure 3 The antagonistic effect of HNXJ-B6 on Ralstonia solanacearum was initially screened.

[0021] Figure 4 The antagonistic effect of HNXJ-B6 secondary screening on Ralstonia solanacearum;

[0022] Figure 5 The colony morphology of strain HNXJ-B6;

[0023] Figure 6 The electrophoretic bands were amplified by PCR of the 16S rRNA sequence of strain HNXJ-B6.

[0024] Figure 7 The antibacterial effect of B6 at different temperatures;

[0025] Figure 8 The antibacterial effect of B6 at different pH values;

[0026] Figure 9 The antibacterial effect of B6 on LB medium was shown, with CK serving as the blank control group. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0028] Experiment 1: Isolation and Purification of Soil Strains

[0029] 1.1 The test bacteria were isolated using the dilution plate method.

[0030] (1) Prepare a sterilized 200mL Erlenmeyer flask in advance, weigh 10g of soil sample (collected from soil near where cigar tobacco wilt disease occurs), add 90mL of sterile water, shake gently to fully dissolve the soil sample in sterile water; place it on a constant temperature shaker at 220rpm for 30min, then remove it and place it on a clean bench for 10min.

[0031] (2) Use a pipette to take the supernatant of the soil bacterial suspension after it has been allowed to stand, and use a serial dilution method to prepare 10 μL of each solution. -2 10 -3 10 -4 and 10 -5 Diluents; take 100 μL of each concentration gradient diluent and add it to the prepared NA medium. Shake evenly back and forth to spread the bacterial suspension evenly. Perform 3 replicates for each concentration gradient. Incubate upside down in a 28℃ constant temperature incubator.

[0032] (3) After 2 days, the NA medium was removed and the growth of the colonies was observed in a clean bench. Based on the colony morphology and color, different single colonies were picked out and placed in NB liquid medium and cultured in a constant temperature shaker (30℃, 170rpm). After the NB liquid medium was inoculated with single colonies, it was shaken and cultured for 12 hours. Then, it was strewn on a plate and cultured in a constant temperature incubator at 28℃ for 48 hours. Each strewn plate was replicated in 3 times to purify the isolated bacteria.

[0033] (4) Repeat step (3) once until all colonies on the NA medium are uniform, and the purification is complete. After culturing the purified bacteria in NB liquid medium overnight, mix with 80% glycerol at a ratio of 1:3 and store at -80°C.

[0034] 1.2 Initial screening of the bacteria to be tested

[0035] Bacteria with antagonistic activity against Ralstonia solanacearum pathogen were screened using the plate confrontation method.

[0036] (1) Activate the pathogen in advance and prepare a solution with a concentration of 10. 6 CFU / mL bacterial suspension;

[0037] (2) Use a pipette to measure 100 μL of bacterial suspension and spread it evenly on the sterilized NA medium.

[0038] (3) Take 5 μL of the purified test bacteria and inoculate it onto NA medium containing the pathogen. Each treatment has 4 replicates, and a blank control is also set up. After incubating upside down in a 28℃ incubator for 48 h, observe whether an inhibition zone forms.

[0039] 1.3 Secondary screening of test bacteria

[0040] Bacterial suspension (10) 6 (cfu / mL), take 100 μL of bacterial suspension and spread it evenly on NA medium. Transfer the bacteria (HNXJ-B6) that showed antagonistic activity against the pathogen in the initial screening results to LB medium and culture until the concentration reaches 10. 9 CFU / mL (OD = 0.8-1), 5 μL of the test bacteria was inoculated into the center of the plate. Each treatment was repeated in triplicate, with a blank control included. After incubation at 28℃ for 48 hours, the presence and size of inhibition zones were observed and measured using the cross-sectional method.

[0041] Identification of strain HNXJ-B6 in Experimental Example 2

[0042] (1) Morphological observation: The HNXJ-B6 strain, which was screened to have antagonistic effects, was transferred to LB medium and cultured for 10 hours. 9 The culture was inoculated again with cfu / mL (OD = 0.8-1) onto NA medium. Colony morphology was observed after 48 hours.

[0043] (2) Molecular biological identification:

[0044] The 16S rRNA fragment was amplified using universal bacterial primer pairs and sequenced. The sequencing results were then compared with the NCBI database.

[0045] Universal primers for bacteria:

[0046] 27F(5'-AGAGTTTGATCCTGGCTCAG-3');

[0047] 1492R(5'-GGTTACCTTGTTACGACTT-3');

[0048] The PCR reaction system is as follows:

[0049] Table 1 PCR reaction system

[0050]

[0051] PCR reaction conditions: pre-denaturation 95℃ for 5 min, denaturation 95℃ for 30 s, annealing 55℃ for 30 s, extension 72℃ for 30 s, 30 cycles, final extension 72℃ for 5 min.

[0052] After PCR, a 1.2% agarose gel was prepared for electrophoresis to check for bands of the target length. The PCR stock solution containing the bands was then sent for sequencing. The sequencing results were then compared with the NCBI database to obtain the bacterial strain information.

[0053] Experimental Example 3: Optimization of fermentation conditions for strain HNXJ-B6

[0054] Fermentation conditions for Bacillus sabovella HNXJ-B6 were optimized using gradient experiments to explore the optimal culture medium, temperature, pH, and culture time for the strain. Antibacterial activity was observed and recorded every 48 hours.

[0055] The experimental conditions are as follows:

[0056] Table 2 Fermentation Conditions

[0057]

[0058] (Three replicates were set up for each bacterium and each condition, and one Ralstonia solanacearum plate was set up as a control for each condition)

[0059] Test results and analysis of Ralstonia solanacearum strain HNXJ-B6 in Experiment 4

[0060] 4.1 Isolation and purification of rhizosphere soil microorganisms

[0061] The dilution plate coating method was used. The prepared soil suspension was diluted to different concentration gradients of 10. -2 10 -3 10 -4 and 10 -5 The suspension was then spread onto solid NA medium and incubated at 28°C for two days before preliminary screening (see details). Figure 1 Colony morphology was observed on plates. Nine bacterial colonies were selected from the rhizosphere soil of diseased plants, and 11 bacterial colonies with different morphologies were selected from the rhizosphere soil of healthy plants in the diseased field. These colonies were purified, and 20 purified bacterial strains were successfully obtained (see details). Figure 2 The colony morphology of 20 bacterial strains was recorded, as shown in Table 3.

[0062] Table 3 shows the colony morphology of the 20 selected strains.

[0063]

[0064] 4.2 Initial screening results of the bacteria to be tested

[0065] The plate confrontation method was used to initially screen 20 bacterial strains on prepared NA medium containing Ralstonia solanacearum (four replicates for each strain). After incubation at 28°C for 48 hours, the antibacterial effect was observed. HNXJ-B6 (see details) was observed to be effective. Figure 3 It produces an inhibition zone.

[0066] 4.3 Results of secondary screening of strain HNXJ-B6

[0067] After secondary screening of strain HNXJ-B6 and incubation for 2 days, a clear inhibition zone was observed in B6. The inhibition zone of B6 was approximately 20 mm in size (see details). Figure 4 ).

[0068] 4.4 Morphological observation and molecular biological identification of strain HNXJ-B6

[0069] B6 colonies are milky white, medium-sized, dry, round, without a halo, translucent, with petal-like edges and a rough, patterned surface (see details). Figure 5 ).

[0070] The HNXJ-B6 strain sequence fragment was successfully amplified using the universal bacterial primer 27F / 1492R. The PCR stock solution was sequenced, yielding a fragment of approximately 1415 bp in size (see details). Figure 6 The sequence of strain HNXJ-B6 was compared with the NCBI database using BLAST. Based on the combined morphological and molecular biological identification results, B6 was identified as Bacillus sabolicii.

[0071] Optimization of fermentation conditions for strain 4.5HNXJ-B6

[0072] The fermentation conditions of the HNXJ-B6 strain, which showed good antagonistic activity, were optimized through gradient experiments to explore the optimal culture medium, temperature, pH, and culture time.

[0073] Treatment at different temperatures (25℃, 28℃, and 37℃) showed that 28℃ was the optimal temperature. After 48 hours of cultivation, strain HNXJ-B6 exhibited significant inhibitory activity against Ralstonia solanacearum (see details). Figure 7 ).

[0074] Under different pH values ​​(pH 9.0, pH 7.0, and pH 5.2), after culturing at 28℃ for 48 h, HNXJ-B6 showed inhibition zones at both pH 9 and pH 7.0 (see details). Figure 8 ).

[0075] Screening was performed on NA and LB media at pH 7.0 and 28℃. The results showed that after 48 h of culture, HNXJ-B6 exhibited better inhibitory activity against Ralstonia solanacearum in LB medium than in NA medium, with the inhibition zone of HNXJ-B6 reaching 50 mm (see details). Figure 9 ).

[0076] Based on the comparison of the above fermentation conditions, the optimal antibacterial fermentation conditions for HNXJ-B6 are LB medium, pH 7, 28℃, and culture for 48-96 hours.

[0077] Experimental Example 5: Inhibition Test of HNXJ-B6 Fermentation Agent on *Cryptocoryne parasiticus*, *Cryptocoryne aurea*, and *Gnaphalium affine*.

[0078] 5.1 Sources of pathogens: *Cryphonectria parasitica* was purchased from Shanghai Fusheng Industrial Co., Ltd., *Cytospora chrysosperma* was purchased from Shanghai Yanzun Biotechnology Co., Ltd., and *Gy mnosporangium yamadae* was extracted from apples showing symptoms of apple rust.

[0079] 5.2 Inhibition Test

[0080] Using the flat plate confrontation method

[0081] (1) Activate the three pathogenic fungi in advance: *Cryphonectria parasitica*, *Cytospora chrysosperma*, and *Gymnosporangium yamadae*, and prepare a solution with a concentration of 10. 8 CFU / mL bacterial suspension;

[0082] (2) Use a pipette to measure 100 μL of bacterial suspension and spread it evenly on the sterilized NA medium.

[0083] (3) The frozen Bacillus sabolicii HNXJ-B6 was streaked onto NA medium and incubated in an incubator at 28°C for 48 hours. Single colonies were picked and transferred to NB liquid medium and incubated at 30°C and 170 rpm for 12 hours to complete the activation. The activated Bacillus sabolicii HNXJ-B6 was then inoculated into LB medium at an inoculation ratio of 3% and incubated in a shaker at 37°C and pH 9.0 for 48 hours to obtain the fermentation agent.

[0084] (4) Take 2 μL of fermentation inoculum and inoculate it onto NA medium containing pathogens. Each treatment has 3 replicates, and the mean value is taken. A blank control (physiological saline) and a positive control are set up. Pyrimethanil is used as a positive control for *Cryptococcus aureus* and *Gnaphalium affine*, and actinomycete ketone is used as a positive control for *Cryptococcus parasiticus*. After incubating in an inverted incubator at 28℃ for 48 h, the formation of inhibition zones is observed.

[0085] 5.3 Test Results

[0086] As shown in Table 4:

[0087] Table 4 Inhibition effect

[0088]

[0089] As shown in Table 4, the HNXJ-B6 fermentation agent has a good inhibitory effect on *Cryptospira spp.*, *Cryptospira chrysophagus*, and *Gnaphalium affine*, and is significantly better than the bactericidal effect of existing conventional bactericides. The HNXJ-B6 fermentation agent can be used as an inhibitor to prevent the above-mentioned pathogens. This invention provides a new approach and a new selection of biological agents for the biological control of these pathogens, and has great application value.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus safranin ( Bacillus safensis HNXJ-B6, characterized in that, The HNXJ-B6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 32528 and deposit date of November 7, 2024.

2. A fermentation agent containing Bacillus sabinatus HNXJ-B6 as described in claim 1.

3. The fermentation agent according to claim 2, characterized in that, The preparation method of the fermentation agent includes the following steps: (1) The frozen Bacillus sabolicus HNXJ-B6 was streaked onto NA medium, placed in an incubator for culture, and a single colony was picked and activated in NB liquid medium; (2) Take the activated Bacillus sabinatus HNXJ-B6 and inoculate it into LB medium at an inoculation ratio of 1-3% v / v. After culturing in a shaker at 28-37℃ and pH 7.0-9.0 for 48-96 h, the fermentation agent is obtained.

4. The fermentation agent according to claim 3, characterized in that, The NA culture medium is prepared by adding 5.0g of bacterial peptone, 1.0g of yeast powder, 10.0g of glucose, 3.0g of beef powder, and 20.0g of agar powder to every 1000mL of distilled water, adjusting the pH to 6.8-7.2, and then sterilizing at 121℃ for 20min.

5. The fermentation agent according to claim 3, characterized in that, The preparation method for NB liquid culture medium is as follows: add 5.0g of bacterial peptone, 1.0g of yeast powder, 10.0g of glucose, and 3.0g of beef powder to every 1000mL of distilled water, adjust the pH to 6.8-7.2, and then sterilize at 121℃ for 20min.

6. The fermentation agent according to claim 3, characterized in that, The LB medium is prepared by adding 5.0 g of yeast extract, 10.0 g of sodium chloride, and 10.0 g of tryptone to every 1000 mL of distilled water, and then sterilizing at 121°C for 20 min.

7. The *Bacillus sabinatus* HNXJ-B6 of claim 1 or the fermentation agent of claim 2 in the prevention and control of *Cryptococcus faecium* (… Cryphonectria parasitic a) Application in plant fungal diseases caused by fungi.

8. The *Bacillus sabensis* HNXJ-B6 of claim 1 or the fermentation agent of claim 2 in the prevention and control of *Bacillus aureus* (… Cytospora chrysosperma Applications in plant fungal diseases caused by fungi.

9. The *Bacillus sabensis* HNXJ-B6 of claim 1 or the fermentation agent of claim 2 in controlling *Bacillus sabensis* (Yamada rust fungus) Gymnosporangium yamadae Applications in plant fungal diseases caused by fungi.

10. The *Bacillus sabrio* HNXJ-B6 of claim 1 or the fermentation agent of claim 2 in the prevention and control of *Ralstonia solanacearum* (… Ralstonia solanacearum Application in bacterial diseases of tobacco plants caused by ).

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