Bacillus safensis HNXJ-B6 and application thereof

Abstract

The invention discloses bacillus safensis HNXJ-B6 and application thereof, the bacillus safensis HNXJ-B6 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number of the bacillus safensis HNXJ-B6 is CGMCCNO: 32528, and the preservation date is November 7, 2024. According to the invention, the HNXJ-B6 is confirmed to be bacillus safensis through 16S rDNA (ribosomal deoxyribonucleic acid) strain identification. According to the present invention, with the application of the compound, the inhibition effect on the four pathogenic bacteria such as Ralstonia solanacearum, Cryphonectria paraaspora, Cytosporospora chrysosporium and Gymnosporospora yamadae can be significant, the new approach and the new biological agent selection can be provided for the biological prevention and control of the four pathogenic bacteria, and the good application value can be provided.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to Bacillus sabdariffa HNXJ-B6 and application thereof. Background Art

[0002] Bacillus can achieve the effect of preventing and controlling plant diseases through competition, antagonism, growth promotion, induced resistance and multi-mechanism synergy. Bacillus sabfungi is a Gram-positive bacterium of the genus Bacillus. It was first discovered and isolated from the spacecraft assembly facility of the Jet Propulsion Laboratory in the United States. Research on this bacterium mainly focuses on its growth-promoting effect. For example, Sharma et al. isolated Bacillus sabfungi with growth-promoting effect from saffron, and Breedt et al. used Bacillus sabfungi in field experiments to effectively increase corn yields in different soil types. Xu Tongwei screened Bacillus sabfungi from the root soil of black shank disease tobacco plants, which can effectively prevent and control tobacco black shank disease. Wang Ruiqi isolated 75 rhizosphere bacteria from the rhizosphere soil of rapeseed in Linzhi, Tibet, among which Bacillus sabfungi had an antagonistic effect of more than 50% on wheat fusarium rust. It can be seen that Bacillus sabfungi has great application potential in promoting plant growth. Bacillus sabdariffa has rich diversity, among which there are many strains with potential development value. Based on the current situation, developing Bacillus sabdariffa with new functions has very important application value and practical significance. Summary of the invention

[0003] In view of the above-mentioned defects of the prior art, the present invention proposes a Bacillus safovici HNXJ-B6 and its application to solve the problems raised by the above-mentioned background technology.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] A Bacillus shaforniensis HNXJ-B6, whose preservation number is: CGMCCNO: 32528, the preservation date is November 7, 2024, and it is preserved in the General Microbiology Center of China Microbiological Culture Collection Administration.

[0006] Preferably, the present invention also includes a fermentation bacterial agent containing Bacillus safovici HNXJ-B6.

[0007] Preferably, the method for preparing the fermentation agent comprises the following steps:

[0008] (1) The frozen Bacillus sabogen HNXJ-B6 was streaked onto NA medium and cultured in a 28°C incubator for 48 h. A single colony was picked and transferred to NB liquid medium and cultured at 30°C and 170 rpm for 12 h to complete activation.

[0009] (2) The activated Bacillus sabdariffa HNXJ-B6 was inoculated into LB medium at a ratio of 1-3% v / v, and cultured on a shaking platform at 28-37° C. and pH 7.0-9.0 for 48-96 hours, and then taken out to obtain a fermentation agent.

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

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

[0012] Preferably, 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.

[0013] Preferably, the Bacillus sabdariffa HNXJ-B6 or fermented bacteria agent of the present invention can be used to prevent and control plant fungal diseases caused by Cryphonectria parasitica.

[0014] Preferably, the Bacillus sabdariffa HNXJ-B6 or fermented bacteria agent of the present invention can be used to prevent and control plant fungal diseases caused by Cytospora chrysosperma.

[0015] Preferably, the Bacillus sabdariffa HNXJ-B6 or fermented bacteria agent of the present invention can be used to prevent and control plant fungal diseases caused by Puccinia yamada.

[0016] Preferably, the Bacillus sabdariffa HNXJ-B6 or fermented bacterial agent of the present invention can be used to prevent and control bacterial diseases of tobacco plants caused by Ralstonia solanacearum.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the bacillus sabdariffa HNXJ-B6 or the bacterial agent of the present invention has obvious inhibitory effect on Ralstonia solanacearum, Cryphonectria parasitica, Cytospora chrysosperma and Gymnosporangium yamadae, and has high safety. The bacillus sabdariffa HNXJ-B6 or the bacterial agent of the present application is easy to obtain, has low cost, and has good control effect. The present invention provides a new approach and new biological agent selection for the biological control of the above four pathogens, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the isolation of bacterial strains in the rhizosphere soil of healthy plants and diseased plants, with the left side showing the JT primary screening and the right side showing the BT primary screening;

[0019] Figure 2 The purification of strains screened from the rhizosphere soil of healthy plants and diseased plants, with JT purification on the left and BT purification on the right;

[0020] Figure 3 This is the initial screening of the antagonistic effect of HNXJ-B6 against Ralstonia solanacearum;

[0021] Figure 4 To screen HNXJ-B6 for its antagonistic effect against Ralstonia solanacearum;

[0022] Figure 5 This is the colony morphology of the HNXJ-B6 strain;

[0023] Figure 6 This is the electrophoresis band of PCR amplification of 16S rRNA sequence of HNXJ-B6 strain;

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

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

[0026] Fig. 9 is the antibacterial effect of B6 on LB medium, where CK is the blank control group. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0028] Experimental Example 1 Isolation and purification of soil strains

[0029] 1.1 Isolation of the test bacteria was carried out by dilution plate spreading method.

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

[0031] (2) Use a pipette to take the supernatant of the soil bacterial suspension after standing, and use the gradient dilution method to prepare 10 -2 , 10 -3 , 10 -4 and 10 -5 Take 100 μL of dilution of each concentration gradient, add it to the NA medium prepared in advance, shake it back and forth evenly to spread the bacterial suspension evenly, and repeat 3 times for each concentration gradient; invert and culture in a constant temperature incubator at 28°C;

[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 selected and placed in NB liquid medium and cultured in a constant temperature oscillator (30°C, 170 rpm). After the NB liquid medium inoculated with a single colony was shaken for 12 hours, it was placed in a 28°C constant temperature incubator and cultured for 48 hours using the plate streaking method. Each streaked plate was replicated three times, and the isolated bacteria were purified.

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

[0034] 1.2 Preliminary screening of test bacteria

[0035] The plate confrontation method was used to screen out bacteria that have antagonistic effects on the pathogen Ralstonia solanacearum.

[0036] (1) Activate the pathogen in advance and prepare it to a concentration of 10 6 cfu / mL bacterial suspension;

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

[0038] (3) Take 5 μL of the purified test bacteria and inoculate them on the NA medium containing pathogens. Repeat 4 times for each treatment, and set up a blank control. Incubate the culture inverted at 28°C for 48 hours, and observe whether an inhibition zone is formed.

[0039] 1.3 Rescreening of test bacteria

[0040] The bacterial suspension (10 6 cfu / mL), take 100 μL of the bacterial suspension on the NA medium and spread it evenly. The bacteria (HNXJ-B6) that have antagonistic effects on pathogens in the initial screening results were transferred to LB medium and cultured to a concentration of 10 9 cfu / mL (OD = 0.8-1), take 5 μL of the bacteria to be tested and inoculate it in the center of the plate. Each treatment is repeated 3 times, and a blank control is set at the same time. After inverted culture in a 28°C constant temperature incubator for 48 hours, observe whether there is an inhibition zone formed, and use the cross method to measure the size of the inhibition zone.

[0041] Test Example 2 Identification of HNXJ-B6 strain

[0042] (1) Morphological observation: The HNXJ-B6 strain with antagonistic effects was transferred to LB medium and cultured for 10 9 cfu / mL (OD=0.8-1), and inoculated into NA medium again. Observe the colony morphology after 48 hours.

[0043] (2) Molecular Biological Identification:

[0044] The 16S rRNA fragment was amplified and sequenced using bacterial universal primers, and the sequencing results were 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] PCR reaction conditions: 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 is completed, a 1.2% agarose gel is prepared for electrophoresis to detect whether there is a band of the target length, and the PCR stock solution with a band is sent for sequencing. After obtaining the sequencing results, the sequence is compared in the NCBI database to obtain the strain information.

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

[0054] The fermentation conditions of Bacillus sabdariffa HNXJ-B6 were optimized, and the optimal culture medium, temperature, pH value and culture time of the strain were explored through gradient experiments. The antibacterial conditions were observed and recorded every 48 hours.

[0055] The experimental conditions are as follows:

[0056] Table 2 Fermentation conditions settings

[0057]

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

[0059] Test Example 4 Test results and analysis of HNXJ-B6 strain against Ralstonia solanacearum

[0060] 4.1 Isolation and purification of rhizosphere soil microorganisms

[0061] The prepared soil suspension was diluted into different concentration gradients of 10 -2 , 10 -3 , 10 -4 and 10 -5 The suspension was then spread on solid NA medium and cultured at 28°C for two days for preliminary screening (see Figure 1 ), observed the colony morphology on the plate, selected 9 strains from the culture medium of the rhizosphere soil of the diseased plants, and selected 11 bacterial colonies with different colony morphology from the culture medium of the rhizosphere soil of the healthy plants in the diseased field. These colonies were purified and 20 purified bacterial strains were successfully obtained (see Figure 2 ), and the colony morphology of 20 bacterial strains 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] Using the plate confrontation method, 20 strains of the tested bacteria were 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 hours to observe the antibacterial effect. HNXJ-B6 (see Figure 3 ) produced an inhibition zone.

[0066] 4.3 Results of rescreening of HNXJ-B6 strain

[0067] The HNXJ-B6 strain was rescreened and after 2 days of culture, an obvious inhibition zone was observed for B6. The size of the B6 inhibition zone was about 20 mm (see Figure 4 ).

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

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

[0070] The HNXJ-B6 strain sequence fragment was successfully amplified using the bacterial universal primers 27F / 1492R. The PCR stock solution was sent for sequencing, and a B6 fragment of about 1415 bp was obtained (see Figure 6 ), the sequence of HNXJ-B6 strain was compared with the NCBI database by Blast, and based on the results of morphological and molecular biological identification, B6 was identified as Bacillus sabdariffa.

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

[0072] The fermentation conditions of the screened HNXJ-B6 strain with better antagonistic effect were optimized, and the optimal fermentation 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. After 48 hours of culture, HNXJ-B6 strain had a significant inhibitory effect on Ralstonia solanacearum (see Figure 7 ).

[0074] Under different pH values ​​(pH9.0, pH7.0 and pH5.2), after culturing for 48 h at 28°C, inhibition zones of HNXJ-B6 were observed at both pH9 and pH7.0 (see Figure 8 ).

[0075] The results showed that after 48 h of culture, HNXJ-B6 had a better antibacterial effect on Ralstonia solanacearum in LB medium than in NA medium, and the size of the inhibition zone of HNXJ-B6 could reach 50 mm (see Fig. 9 ).

[0076] Through the comparison of the above fermentation conditions, the best antibacterial fermentation condition of HNXJ-B6 is to use LB medium, pH 7, 28℃ cultivation conditions, and culture for 48-96h.

[0077] Test Example 5: Inhibition test of HNXJ-B6 fermentation agent on parasitic red shell fungus, golden shell cystsporum and Yamada glue rust fungus

[0078] 5.1 Source 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] Flat plate standoff method

[0081] (1) Three pathogens, Cryphonectria parasitica, Cytospora chrysosperma, and Gymnosporangium yamadae, were activated in advance and prepared at a concentration of 10 8 cfu / mL bacterial suspension;

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

[0083] (3) The frozen Bacillus sabogen HNXJ-B6 was streaked onto NA medium, and cultured in a 28°C incubator for 48 hours. A single colony was picked and placed in NB liquid medium, and cultured at 30°C and 170 rpm for 12 hours to complete activation; the activated Bacillus sabogen HNXJ-B6 was taken and inoculated into LB medium at a ratio of 3%, and cultured in a shaking incubator at 37°C and pH 9.0 for 48 hours, and then taken out to obtain a fermentation agent.

[0084] (4) Take 2 μL of the fermentation agent and inoculate it on the NA medium containing pathogens. Repeat 3 times for each treatment and take the average value. At the same time, set up a blank control (normal saline) and a positive control. Penthiopyrad was used as a positive control for Cystosporium chrysosporium and Glechoma yamadaensis, and Cycloheximide was used as a positive control for Cryptidella parasitica. After inverted culture in a 28°C constant temperature incubator for 48 hours, observe the formation of the inhibition zone.

[0085] 5.3 Test Results

[0086] As shown in Table 4:

[0087] Table 4 Inhibition effect

[0088]

[0089] As can be seen from Table 4, the HNXJ-B6 fermentation agent has a good inhibitory effect on parasitic Cryptocaryon, Chrysopora and Glechoma yamada, and is significantly better than the bactericidal effect of existing conventional fungicides. The subsequent HNXJ-B6 fermentation agent can be used as an inhibitor to prevent the above-mentioned pathogens. The present invention provides a new approach and new biological agent selection for the biological control of the above-mentioned pathogens, and has a good 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus safovici HNXJ-B6, characterized in that It is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCCNO: 32528 and the deposit date being November 7, 2024.

2. A fermentation agent containing the Bacillus safovici HNXJ-B6 according to claim 1.

3. The fermentation agent according to claim 2, characterized in that: The preparation method of the fermentation agent comprises the following steps: (1) Streaking frozen Bacillus sabogensis HNXJ-B6 onto NA medium, culturing in an incubator, and picking a single colony to activate in NB liquid medium; (2) The activated Bacillus sabdariffa HNXJ-B6 was inoculated into LB medium at a ratio of 1-3% v / v, and cultured on a shaking platform at 28-37° C. and pH 7.0-9.0 for 48-96 hours, and then taken out to obtain a fermentation agent.

4. The fermentation agent according to claim 3, characterized in that: The NA medium is prepared by adding 5.0 g of bacterial 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 to every 1000 mL of distilled water, adjusting the pH to 6.8-7.2, and then sterilizing at 121° C. for 20 min.

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

6. The fermentation agent according to claim 3, characterized in that: The LB medium was 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 sterilized at 121° C. for 20 min.

7. Use of the Bacillus sabdariffa HNXJ-B6 according to claim 1 or the fermentation agent according to claim 2 in preventing and controlling plant fungal diseases caused by Cryphonectria parasitica.

8. Use of the Bacillus sabdariffa HNXJ-B6 according to claim 1 or the fermentation agent according to claim 2 in preventing and controlling plant fungal diseases caused by Cytospora chrysosperma.

9. Use of the Bacillus saffron HNXJ-B6 according to claim 1 or the fermentation agent according to claim 2 in preventing and controlling plant fungal diseases caused by Puccinia yamada.

10. Use of the Bacillus sabdariffa HNXJ-B6 of claim 1 or the fermentation agent of claim 2 in preventing and controlling bacterial diseases of tobacco plants caused by Ralstonia solanacearum.

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