Bacillus safensis hnxj-b1 and application thereof

Abstract

The application discloses Bacillus safensis HNXJ-B1 and application thereof, is preserved in China Microorganism Strain Preservation Management Committee general microorganism center, and its preservation number is: CGMCC NO: 32527, and the preservation date is November 7, 2024.The application is identified by 16S rDNA strain, and it is confirmed that HNXJ-B1 is Bacillus safensis.The inhibition effect on Ralstonia solanacearum, Diaporthe sclerotioides, Trichophyton mentagrophytes and Malassezia is obvious, can simultaneously inhibit plant disease pathogenic bacteria and animal pathogenic fungi, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial application, in particular to a Bacillus safensis HNXJ-B1 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-B1 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-B1, the preservation number of which is CGMCC NO: 32527, 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-B1.

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

[0008] (1) inoculate the frozen Bacillus safensis HNXJ-B1 on NA culture medium, and place it in a 28℃ incubator for 48h, then pick single colonies to NB liquid medium, and place it in a 30℃, 170rpm shaking incubator for 12h to complete the activation;

[0009] (2) Take the activated Bacillus safensis HNXJ-B1, inoculate into LB medium according to 1-3% v / v inoculation ratio, take out after 48-96h culture at 28-37℃, pH 7.0-9.0 on a shaking table, and obtain the fermentation inoculant.

[0010] Preferably, the configuration method of the NA medium is to add bacterial protein peptone 5.0g, yeast powder 1.0g, glucose 10.0g, beef powder 3.0g, and agar powder 20.0g in 1000mL distilled water, adjust pH to 6.8-7.2, and then sterilize at 121℃ for 20min.

[0011] Preferably, the configuration method of the NB liquid medium is to add bacterial protein peptone 5.0g, yeast powder 1.0g, glucose 10.0g, and beef powder 3.0g in 1000mL distilled water, adjust pH to 6.8-7.2, and then sterilize at 121℃ for 20min.

[0012] Preferably, the configuration method of the LB medium is to add yeast paste 5.0g, sodium chloride 10.0g, and trypsin 10.0g in 1000mL distilled water, and then sterilize at 121℃ for 20min.

[0013] Preferably, the Bacillus safensis HNXJ-B1 or fermentation inoculant of the application can be applied to prevent and control plant fungal diseases caused by Diaporthe sclerotioides.

[0014] Preferably, the Bacillus safensis HNXJ-B1 or fermentation inoculant of the application can be applied to prevent and control fungal disease drugs caused by Trichophyton mentagrophytes.

[0015] Preferably, the Bacillus safensis HNXJ-B1 or fermentation inoculant of the application can be applied to prevent and control fungal disease drugs caused by Malassezia.

[0016] Preferably, the Bacillus safensis HNXJ-B1 or fermentation inoculant of the application can be applied to prevent and control tobacco plant bacterial diseases caused by Ralstonia solanacearum.

[0017] Compared with the prior art, the beneficial effects of the present application are that the bacillus safensis HNXJ-B1 or microbial inoculant of the present application has obvious inhibitory effect on Ralstonia solanacearum, Diaporthe sclerotioides, Trichophyton mentagrophytes and Malassezia, has high safety, can simultaneously inhibit plant disease pathogens and animal pathogenic fungi, has fast growth and reproduction speed of HNXJ-B1, low production cost, simple process and wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The left side is JT primary screening, and the right side is BT primary screening for isolation of bacterial strains in the rhizosphere of healthy plants and the rhizosphere of diseased plants;

[0019] Figure 2 The left side is JT purification, and the right side is BT purification for purification of strains screened from the rhizosphere of healthy plants and the rhizosphere of diseased plants;

[0020] Figure 3 The antagonistic effect of HNXJ-B1 on Ralstonia solanacearum in primary screening;

[0021] Figure 4 The antagonistic effect of HNXJ-B1 on Ralstonia solanacearum in secondary screening;

[0022] Figure 5 The colony morphology of HNXJ-B1 strain;

[0023] Figure 6 The 16S rRNA sequence PCR amplification electrophoresis band of HNXJ-B1 strain;

[0024] Figure 7 The antibacterial effect of B1 at different temperatures;

[0025] Figure 8 The antibacterial effect of B1 at different pH values;

[0026] Figure 9 The antibacterial effect of B1 on LB medium, wherein CK is a blank control group. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0028] Test Example 1: Isolation and purification of soil strains

[0029] 1.1 Isolation of the tested bacteria, using dilution plate coating 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] The bacterial suspension (10 6 cfu / m L), and 100 μL of the bacterial suspension was evenly smeared on the NA medium. The bacteria HNXJ-B1 with antagonistic effect on the pathogenic bacteria in the preliminary screening result were transferred to the LB medium for culture until the concentration was 10 9 cfu / m L (OD = 0.8-1), and 5 μL of the bacteria to be tested was inoculated in the center of the plate. Three repetitions were set for each treatment, and a blank control was simultaneously set. After being placed in a constant temperature incubator at 28°C and cultured for 48 h, it was observed whether there was a bacteriostatic ring, and the size of the bacteriostatic ring was measured by the cross method.

[0041] Test Example 2 Identification of HNXJ-B1 strain

[0042] (1) Morphological observation: the HNXJ-B1 strain with antagonistic effect screened was transferred to the LB medium for culture until the concentration was 10 9 cfu / mL (OD = 0.8-1), and then inoculated on the NA medium. After 48 h, the colony morphology was observed.

[0043] (2) Molecular biological identification:

[0044] The 16S rRNA fragment was amplified by using bacterial universal primers and sequenced, the sequencing result was compared with the NCBI database,

[0045] Bacterial universal primers:

[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] The PCR reaction conditions are as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and final extension at 72°C for 5 min.

[0052] After the PCR, a 1.2% agarose gel was prepared, and electrophoresis was performed to detect whether there was a band of the target length (1500 bp). The PCR original solution with the band was sent for sequencing. After obtaining the sequencing result, the sequence was compared in the NCBI database to obtain the strain information.

[0053] Test Example 3 Optimization of fermentation conditions of HNXJ-B1 strain

[0054] The fermentation conditions of Bacillus safensis HNXJ-B1 were optimized, and the optimal culture medium, temperature, pH value and culture time of the strain were explored through gradient experiments. The antibacterial effect was observed and recorded every 48 h.

[0055] The experimental conditions were as follows:

[0056] Table 2 Setting of fermentation conditions

[0057]

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

[0059] Test Example 4 Test results and analysis of HNXJ-B1 strain on Ralstonia solanacearum

[0060] 4.1 Isolation and purification of rhizosphere soil microorganisms

[0061] Dilution plate coating method was used. The prepared soil suspension was diluted into different concentration gradients of 10 -2 , 10 -3 , 10 -4 and 10 -5 , and then coated on solid NA medium, and cultured at 28°C for two days, then preliminary screening was carried out (see Figure 1 ), and the colony morphology was observed on the plate. 9 strains were selected from the culture medium of the rhizosphere soil of diseased plants, and 11 strains of bacteria with different colony morphologies were selected from the culture medium of the rhizosphere soil of healthy plants in the diseased field. These colonies were purified, and 20 purified bacteria were successfully obtained (see Figure 2 ), and the colony morphology of the 20 bacteria was recorded, as shown in Table 3.

[0062] Table 3 Colony morphology observation table of 20 screened strains

[0063]

[0064] 4.2 Preliminary screening results of the tested bacteria

[0065] Plate confrontation method was used, and the 20 tested bacteria were respectively preliminarily screened on the prepared NA medium containing Ralstonia solanacearum (4 replicates for each strain). The NA medium was cultured in a 28°C incubator for 48 h, and then the antibacterial effect was observed. It was observed that HNXJ-B1 (see Figure 3 ) produced an antibacterial circle.

[0066] 4.3 Rescreening results of HNXJ-B1 strain

[0067] HNXJ-B1 strain was rescreened, and after 2 days of culture, B1 was observed to have a clear inhibition zone. The size of the inhibition zone of B1 was about 20 mm (see Figure 4 ).

[0068] 4.4 Morphological observation and molecular biological identification of HNXJ-B1 strain

[0069] The B1 colony was milky white, medium-sized, dry, round, no halo, translucent, petal-shaped edge, and rough surface (see Figure 5 ).

[0070] The sequence fragment of HNXJ-B1 strain was successfully amplified using bacterial universal primers 27F / 1492R. The PCR original solution was sent for sequencing, and a B1 fragment of about 1500 bp was obtained (see Figure 6 ), and the sequence of HNXJ-B1 strain was compared with the sequences in the NCBI database by Blast, and it was found that the sequence coverage reached 99% with Bacillus safensis strain AHB11 chromosome (accession number: CP097374.1). Based on the results of morphological and molecular biological identification, B1 was Bacillus safensis.

[0071] 4.5 Optimization of fermentation conditions of HNXJ-B1 strain

[0072] The HNXJ-B1 strain with good antagonistic effect was screened for fermentation condition optimization, and the most suitable culture medium, temperature, pH value, and culture time were explored through gradient experiments.

[0073] After treatment at different temperatures (25°C, 28°C, and 37°C), the results showed that 28°C was the most suitable temperature, and after 48 h of culture, HNXJ-B1 strain had a clear inhibitory effect on Ralstonia solanacearum (see Figure 7 ).

[0074] Under different pH values (pH 9.0, pH 7.0, and pH 5.2) at 28°C, after 48 h of culture, HNXJ-B1 was observed to have an inhibition zone at pH 9 and pH 7.0 (see Figure 8 ).

[0075] After screening at pH 7.0 and 28°C, the results showed that after 48 h of culture, HNXJ-B1 had a better inhibitory effect on Ralstonia solanacearum in LB medium than in NA medium, and the size of the inhibition zone of HNXJ-B1 could reach 23 mm (see Figure 9 ).

[0076] Through the comparison of the above fermentation conditions, the optimal antibacterial fermentation conditions of HNXJ-B1 are as follows: using LB medium, pH 7, incubating at 28°C, and culturing for 48-96h.

[0077] Test Example 5: Inhibition test of HNXJ-B1 fermentation agent on Diaporthe sclerotioides, Trichophyton mentagrophytes and Malassezia

[0078] 5.1 Source of pathogenic bacteria: Diaporthe sclerotioides was purchased from Nuogen Gene Technology (Wuhan) Co., Ltd., Trichophyton mentagrophytes was purchased from Shanghai Xiaoya Biological Technology Co., Ltd., and Malassezia (ATCC44344) was purchased from Jiangxi Jianglan Pure Biological Reagent Co., Ltd.

[0079] 5.2 Inhibition test

[0080] Plate confrontation method was used

[0081] (1) The three pathogenic bacteria, Diaporthe sclerotioides, Trichophyton mentagrophytes and Malassezia, were activated in advance, and a bacterial suspension with a concentration of 10 6 cfu / mL was prepared;

[0082] (2) 100 μL of the bacterial suspension was taken with a pipette gun and evenly spread on the NA medium to which sterilized NA medium had been added;

[0083] (3) The frozen Bacillus safensis HNXJ-B1 was streak-inoculated on the NA medium and placed in a 28°C incubator for 48h. A single colony was picked and inoculated in NB liquid medium and placed in a 30°C, 170rpm shaking incubator for 12h to complete the activation. The activated Bacillus safensis HNXJ-B1 was inoculated in LB medium at a ratio of 3%, and after 48h of shaking culture at 37°C and pH 9.0, the fermentation agent was obtained.

[0084] (4) 2 μL of the fermentation agent was inoculated on the NA medium containing the pathogenic bacteria, with 3 replicates for each treatment, and the mean value was taken. Blank control (saline) and positive control were set up at the same time. 1 g / L ketoconazole was used as the positive control for Malassezia and Trichophyton mentagrophytes, and 0.5 g / L of mesnigmycin was used as the positive control for Diaporthe sclerotioides. After 48h of inverted culture in a 28°C constant temperature incubator, the formation of inhibition zones was observed.

[0085] 5.3 Test results

[0086] As shown in Table 4:

[0087] Table 4 inhibition effect

[0088]

[0089] From table 4, it can be seen that the HNXJ-B1 fermentation microbial agent has good inhibition effect on diaporthe phaseolorum, trichophyton mentagrophytes and malassezia, and is obviously superior to the treatment effect of the existing conventional drugs, the HNXJ-B1 fermentation microbial agent can be used as an inhibitor for preventing, relieving and treating the above pathogenic bacteria, and it has never been found that serratia fonticola has inhibition effect on diaporthe phaseolorum, trichophyton mentagrophytes and malassezia, and the application range of serratia fonticola is further expanded.

[0090] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A type of Bacillus safranin ( Bacillus safensis HNXJ-B1, characterized in that, The HNXJ-B1 is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO: 32527, and the preservation date is November 7, 2024.

2. A fermentation inoculant containing the Bacillus safensis HNXJ-B1 of claim 1.

3. The fermentation inoculant of claim 2, wherein, The preparation method of the fermentation inoculant comprises the following steps: (1) The frozen Bacillus safensis HNXJ-B1 is streak inoculated on NA medium and cultured in a culture box, and a single colony is picked to NB liquid medium for activation; (2) The activated Bacillus safensis HNXJ-B1 is inoculated into LB medium at a inoculation ratio of 1-3% v / v, and is taken out after being cultured at 28-37℃ and pH 7.0-9.0 for 48-96 h, to obtain the fermentation inoculant.

4. The fermentation inoculant of claim 3, wherein, The configuration method of the NA medium is that 5.0 g of bacterial protein peptone, 1.0 g of yeast powder, 10.0 g of glucose, 3.0 g of beef powder and 20.0 g of agar powder are added into 1000 mL of distilled water, the pH is adjusted to 6.8-7.2, and then sterilized at 121℃ for 20 min.

5. The fermentation inoculant of claim 3, wherein, The configuration method of the NB liquid medium is that 5.0 g of bacterial protein peptone, 1.0 g of yeast powder, 10.0 g of glucose and 3.0 g of beef powder are added into 1000 mL of distilled water, the pH is adjusted to 6.8-7.2, and then sterilized at 121℃ for 20 min.

6. The fermentation inoculant of claim 3, wherein, The configuration method of the LB medium is that 5.0 g of yeast paste, 10.0 g of sodium chloride and 10.0 g of pancreatic protein peptone are added into 1000 mL of distilled water, and then sterilized at 121℃ for 20 min.

7. The use of Bacillus safensis HNXJ-B1 of claim 1 or the use of the fermentation microbial agent of claim 2 in the prevention and treatment of plant fungal diseases caused by Diaporthe phaseolorum (D. phaseoli). Diaporthe sclerotioides ).

8. The *Bacillus saboides* HNXJ-B1 of claim 1 or the fermentation agent of claim 2 in the preparation of a treatment for the prevention and control of *Trichophyton mentagrophytes* (… Trichophyton mentagrophyte Application in drugs for fungal diseases caused by s).

9. Use of Bacillus safensis HNXJ-B1 of claim 1 or the fermentation microbial agent of claim 2 in the preparation of a drug for preventing and treating fungal diseases caused by Malassezia (Pityrosporum) spp. Malassezia ).

10. The use of Bacillus safensis HNXJ-B1 of claim 1 or the use of the fermentation microbial agent of claim 2 in the prevention and treatment of tobacco plant bacterial disease caused by Ralstonia solanacearum (Xuj 1). Ralstonia solanacearum )

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