Bacillus tequilensis with lignin degradation and black shank antagonism functions and application of bacillus tequilensis
By isolating and identifying Bacillus tekirae JZF3, the problem of single-function microbial strains has been solved, achieving efficient degradation of lignin and prevention and control of tobacco black shank disease, and promoting plant growth.
Patent Information
- Application Number
- CN202511168083.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-20
- Publication Date
- 2025-12-12
AI Technical Summary
In existing technologies, microbial strains have limited functions and cannot simultaneously achieve efficient lignin degradation and antagonistic effects against tobacco black shank. Furthermore, traditional treatment methods suffer from environmental pollution and high energy consumption.
A strain of Bacillus tequilensis JZF3 was isolated and identified, which has dual functions of lignin degradation and antagonism against tobacco black shank disease. By optimizing culture conditions and specific culture media, its application in fermented distiller's grains fertilizer was realized.
It achieves efficient degradation of lignin and effective prevention and control of tobacco black shank disease. The degradation solution has a growth-promoting effect on plants and has high market prospects.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the prevention and treatment of black shank disease and straw degradation technology, and in particular to a multi-functional strain Bacillus tequilensis JZF3 with lignin degradation and tobacco black shank disease antagonistic functions and its application. BACKGROUND
[0002] Straw contains rich nutrients, among which lignin, as a natural physical barrier, wraps and reinforces cellulose and hemicellulose, and its complex aromatic polymer structure directly affects the efficiency of straw degradation, making the degradation of lignin a key problem for the utilization of straw resources. In the traditional disposal method, burning will produce various pollutants to threaten the atmospheric environment, and direct application to the field can improve soil organic matter, but high lignin content will also lead to a prolonged composting period. Physical and chemical treatment methods such as high temperature and high pressure, organic solvents, etc. can achieve lignin depolymerization, but there are problems such as high equipment requirements, high energy consumption and secondary pollution. Compared with the above methods, microbial degradation technology has the advantages of environmental friendliness and low cost, and has become the most promising solution. Therefore, it is crucial to tap high-efficiency lignin-degrading microbial resources.
[0003] Microbial degradation systems mainly include fungi and bacteria. White rot fungi can completely degrade lignin at room temperature, but have the defect of long reaction period. Bacterial groups such as Bacillus and Pseudomonas also have lignin-degrading properties, and have the advantages of fast growth and strong environmental adaptability. Han Yujie successfully screened several bacterial strains with high-efficiency lignin-degrading ability from straw application soil, and improved the lignin-degrading efficiency by optimizing the culture conditions. However, the existing strains have single function, which is different from the actual application needs, and the multi-functional strain resources need to be strengthened. For example, the cellulose-degrading bacteria screened by Qiang Zhenyu can degrade wheat straw and promote the growth of corn; the strains isolated from soil by Qi Yongzhi not only can accelerate straw degradation, but also can prevent and treat wheat sharp eyespot. These multi-functional strains can improve the soil micro-ecology through rhizosphere colonization, and reduce the use of chemical pesticides, which is of great importance to promote straw resource utilization and ecological protection.
[0004] Bacillus is one of the most common gram-positive bacteria, which can be isolated from various habitats. Bacillus tequilensis was first isolated from a 2000-year-old shaft tomb in Mexico by Gatson JW et al. in 2006, and was identified as a new species of Bacillus based on the results of sodA gene sequence, fatty acid profile and DNA hybridization. Previous studies have found that Bacillus tequilensis BT8 has the ability of fiber degradation, which increases the nutritional value of rice and corn straw silage feed. However, the research on Bacillus tequilensis mainly focuses on its taxonomic status and single functional characteristics, and the exploration in the fields of practical application such as biodegradation of agricultural waste and biological control of plant diseases is limited. SUMMARY
[0005] The technical problem to be solved by the present application is to provide a Bacillus tequilensis with lignin degradation and black leg disease antagonistic functions and its application, which can overcome the shortcomings of the prior art.
[0006] The technical solution of the present application is: a Bacillus tequilensis JZF3, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation date is June 9, 2025, and the preservation number is CGMCC No. 34784.
[0007] The Bacillus tequilensis JZF3 is used for lignin degradation.
[0008] The Bacillus tequilensis JZF3 is used for black leg disease antagonistic function.
[0009] The lignin degradation liquid metabolites of the Bacillus tequilensis JZF3 are used for promoting plant growth.
[0010] The obtaining method of Bacillus tequilensis JZF3 is that the distiller's spent grains matured fertilizer is taken into a sterile 50 ml centrifuge tube, an appropriate amount of sterile water is added, and the mixture is shaken for 1 min and mixed uniformly, and then is left to stand. 1 ml of supernatant is taken as a stock solution by using a pipette gun, and then is diluted with sterile water in stages to 10-3. 100 μL of the stock solution and the diluted sample are uniformly coated on TSA plate culture medium, and different gradients are repeated for 3 times. The plate is placed at 28℃ and left to stand for 2-3 days until single colonies are grown. Colonies with different shapes, sizes and colors are picked, numbered and purified, and the obtained Bacillus tequilensis JZF3 has irregular colony morphology on NA medium, is light yellow and opaque, has a darker middle and a lighter edge, a smooth surface, and an irregular edge with a slightly raised center.
[0011] The preparation method of the Bacillus toyoi is that the NA solid culture medium is composed of the following raw materials in a mass ratio: tryptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g, agar powder 20.0 g, and distilled water 1 L.
[0012] The obtaining method of the lignin degradation liquid metabolite of the aforementioned Bacillus tequilensis JZF3 is that the Bacillus tequilensis JZF3 is inoculated into NB liquid medium, and is cultured overnight at 28℃ and 180 rpm. When the absorbance value of the bacterial liquid at 600 nm wavelength reaches 1.0, 10 mL of the bacterial liquid is taken, and the bacterial body is collected by centrifugation at 6000 rpm for 10 min at 4℃. After being washed with sterile water, the bacterial body is all inoculated into 200 mL of liquid medium with rice straw as the only carbon source, and is cultured for 7 days at 28℃ and 180 rpm.
[0013] The obtaining method of the lignin degradation liquid metabolite of the aforementioned Bacillus tequilensis JZF3 is that the liquid medium with rice straw as the only carbon source is prepared by mixing the following raw materials in a mass ratio: rice straw powder 25 parts, KH2PO4 1 part, MgSO4 0.2 part, K2HPO4 1 part, (NH4)2SO4 2 parts, CaCl2 0.1 g, peptone 0.1 g, and distilled water 1000 parts.
[0014] Compared with the prior art, the present application separates a Bacillus tequilensis JZF3 from distiller's spent grain compost, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation date is June 9, 2025, and the preservation number is CGMCC No. 34784. The Bacillus tequilensis JZF3 has lignin degradation and legume disease antagonistic functions, and the lignin degradation liquid metabolites of the Bacillus tequilensis JZF3 have a plant promoting effect, so that the Bacillus tequilensis JZF3 has a high market prospect. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 The colony morphological characteristics of the degradation bacteria JZF3 on the NA culture medium; Figure 2 The 16S rDNA phylogenetic tree of JZF3; Figure 3 The effect picture of JZF3 producing decolorization circles on aniline blue-PDA culture medium; Figure 4 The effect picture of JZF3 inhibiting Phytophthora parasitica; Figure 5 The promoting effect of the metabolites of JZF3 degrading rice straw on tobacco. DETAILED DESCRIPTION
[0016] Example 1: Isolation and identification of strain JZF3 (1) Strain isolation The strain JZF3 is isolated from distiller's spent grain compost. The specific isolation method is as follows: take the distiller's spent grain compost into a sterile 50 ml centrifuge tube, add an appropriate amount of sterile water, shake for 1 min, and mix well, and then stand still. Use a pipette to take 1 ml of supernatant as a stock solution, and then dilute it with sterile water to 10-3 step by step, and take 100 μL of the stock solution and the diluted sample to evenly coat the TSA plate (10.0 g of tryptone, 3.0 g of beef extract, 5.0 g of sodium chloride, 20.0 g of agar powder, and 1 L of distilled water), repeat 3 times at different gradients, and place it at 28°C for 2-3 days until single colonies are formed. Pick colonies with different shapes, sizes, and colors, number them, and purify and culture them.
[0017] (2) Morphological observation Inoculate the strain numbered JZF3 into NA solid culture medium (10.0 g of tryptone, 3.0 g of beef extract, 5.0 g of sodium chloride, 20.0 g of agar powder, and 1 L of distilled water), and culture at 28°C for 2 days, as shown in FIG. 1. Figure 1As shown, JZF3 colonies on NA medium are irregular in shape, pale yellow, opaque, darker in the center and lighter at the edges; the surface is smooth, the edges are irregular, and the center is slightly raised.
[0018] (3) Molecular identification The 16S rDNA sequence of strain JZF3 was analyzed. A single colony of JZF3 was picked, and its 16S rDNA sequence was amplified using the following primers: 27 F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1) 1492 R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).
[0019] Sequencing revealed that the 16S rDNA sequence of JZF3 was 1419 bp in length, as shown in SEQ ID NO.3.
[0020] Fourteen standard strain sequences were obtained from the NCBI (GenBank) database. The 16S rDNA sequences of the isolated strains and reference strains were analyzed using MEGA 11, and a Neighbor-joining phylogenetic tree was constructed. Figure 2 As shown, the JZF3 strain provided by this invention is most closely related to Bacillus tequilensis. Based on the colony characteristics of this strain on petri dishes, the isolated strain JZF3 of this invention was identified as Bacillus tequilensis, named Bacillus tequilensis JZF3, and deposited on June 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34784.
[0021]
[0022] Example 2: Lignin degradation ability of strain JZF3 0.10 g of aniline blue was dissolved in 1 L of PDA medium (6.0 g potato starch, 20.0 g glucose, 20.0 g agar, 1 L distilled water) to prepare PDA-aniline blue medium. Bacillus equilensis JZF3 obtained in Example 1 was inoculated into TSB liquid medium (17.0 g tryptone, 3.0 g soybean papain hydrolysate, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 1 L distilled water). After incubation at 28℃ and 180 rpm for 24 h with shaking, the culture was transferred to PDA-aniline blue plates using the spot inoculation method. Each strain was replicated three times. After incubation at 28℃ for 3 days, the presence of a decolorization zone was observed. The colony diameter d (cm) and the diameter D (cm) of the colony and its surrounding transparent zone were measured using the cross-cross method. The ratio (D / d) represented the strain's ability to degrade lignin. The experimental results are as follows: Figure 3 As shown, strain JZF3 can secrete extracellular enzymes to decolorize and degrade aniline blue, with a ratio D / d of 1.74.
[0023] Example 3: Antagonistic ability of strain JZF3 against Tobacco Black Shank Bacterium. Phytophthora tobaccoii was inoculated onto oat medium and cultured for 3 days. Strain JZF3 was inoculated onto NB medium and cultured on a shaker at 28℃ and 180 rpm for 24 h. A 5 mm mycelial cake was collected from the edge of a Phytophthora tobaccoii colony and inoculated into the center of an oat solid medium. 3 μL of JZF3 bacterial suspension was added 2.5 cm from the edge of the plate. Three replicates were set up, with no bacterial suspension added to the control group. After culturing at 28℃ for 3 days, antagonistic activity was observed and the inhibitory effect was recorded. The inhibition rate was calculated using the formula: (Coronavirus colony radius in control group - Coronavirus colony radius in treatment group) / Coronavirus colony radius in control group × 100%. The experimental results are shown below. Figure 4 As shown, strain JZF3 achieved an inhibition rate of 67% against black shank bacillus.
[0024] Example 4: Degradation of straw by strain JZF3 Strain JZF3 was inoculated into NB liquid medium and cultured overnight at 28℃ and 180 rpm. When the absorbance of the bacterial suspension at 600 nm reached approximately 1.0, 10 mL of bacterial suspension was collected and centrifuged at 6000 rpm for 10 minutes at 4℃ to collect the cells. After washing with sterile water, the entire sample was inoculated into 200 mL of liquid medium with rice straw as the sole carbon source (25 g sieved rice straw powder, 1 part KH2PO4, 0.2 parts MgSO4, 1 part K2HPO4, 2 parts (NH4)2SO4, 0.1 g CaCl2, 0.1 g peptone, 1 L distilled water). The medium was then cultured with shaking at 28℃ and 180 rpm for 7 days. The uninoculated medium served as a blank control group, and three independent replicates were set up for both the control and treatment groups. The degradation rate of straw was determined using the loss-of-gravity method. The fermentation broth was centrifuged at 6000 r / min for 10 min after cultivation. The precipitate was repeatedly washed with sterile water to remove residual culture medium components, and then dried in an 80℃ oven to constant weight. The straw degradation rate (%) was calculated using the formula (W0-W1) / W0×100%, where W0 represents the dried mass (g) of the remaining straw in the control group after cultivation, and W1 represents the dried mass (g) of the remaining straw in the treatment group. The experimental results showed that *Bacillus tekiria* JZF3 can directly degrade rice straw without any chemical pretreatment. After 7 days of cultivation, strain JZF3 achieved a degradation rate of 22.5% for rice straw.
[0025] Example 5: Growth-promoting effects of JZF3's metabolites after degrading rice straw on plants. A pot experiment was conducted to test plant growth promotion using tobacco as the material. Tobacco seeds were sown in seedling trays, and after emergence, seedlings with uniform growth were selected for transplanting. The experiment included a treatment group and a control group. The treatment group was watered with rice straw fermentation liquid fermented for 7 days using strain JZF3 (the supernatant was obtained after centrifugation and diluted 7 times), while the control group was watered with an equal volume of untreated rice straw supernatant. Each treatment was replicated in triplicate, with 10 pots of tobacco seedlings per replicate. Each pot was watered with 20 mL of the treatment liquid for 2 consecutive days. After 10 days of cultivation, the growth indicators of the tobacco seedlings were measured, with the leaf area calculated using the formula 0.6345 × (leaf length × leaf width).
[0026] The results are as follows Figure 5 As shown, the metabolites of JZF3 after degrading rice straw can promote the growth of tobacco. Compared with the control group, the leaf area of the treatment group increased by 28% (P<0.05) and the fresh weight increased by 63% (P<0.01).
[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A strain of Bacillus tequilensis JZF3, deposited at the China General Microbiological Culture Collection Center (CGMCC), deposited on June 9, 2025, accession number: CGMCCNo.34784.
2. The application of Bacillus tequilensis JZF3 as described in claim 1 for lignin degradation.
3. The application of the Bacillus tequilensis JZF3 strain as described in claim 1 for tibial antagonistic function.
4. The application of the metabolites of Bacillus tequilensis JZF3 lignin degradation solution according to claim 1 for promoting plant growth.
5. The method for obtaining Bacillus tequilensis JZF3 as described in claim 1, characterized in that: Take the fermented distiller's grains into a sterile 50ml centrifuge tube, add an appropriate amount of sterile water, shake for 1 minute to mix, and let stand. Use a pipette to draw 1 ml of supernatant as the stock solution, then serially dilute it with sterile water to 10⁻³. Take 100 μL of the stock solution and the diluted sample and spread them evenly on TSA agar plates. Repeat the process three times for each gradient. Incubate at 28°C for 2-3 days until single colonies grow. Pick colonies with different shapes, sizes, and colors, number them, and purify them. The colonies of Bacillus tequilensis JZF3 on NA agar are irregular in morphology, pale yellow, opaque, darker in the center and lighter at the edges; the surface is smooth, the edges are irregular, and the center is slightly raised.
6. The method for preparing Bacillus orientalis according to claim 1, characterized in that: The NA solid culture medium is composed of the following raw materials in the specified mass ratio: 10.0 g tryptone, 3.0 g beef extract, 5.0 g sodium chloride, 20.0 g agar powder, and 1 L distilled water.
7. The method for obtaining the metabolites of Bacillus tequilensis JZF3 lignin degradation solution as described in claim 5, characterized in that: Bacillus tequilensis JZF3 was inoculated into NB liquid medium and cultured overnight at 28°C and 180 rpm. When the absorbance of the bacterial suspension at 600 nm reached 1.0, 10 mL of the bacterial suspension was collected by centrifugation at 6000 rpm for 10 minutes at 4°C. After washing with sterile water, the entire suspension was inoculated into 200 mL of liquid medium with rice straw as the sole carbon source and cultured with shaking at 28°C and 180 rpm for 7 days.
8. The method for obtaining the metabolites of Bacillus tequilensis JZF3 lignin degradation solution as described in claim 7, characterized in that, The liquid culture medium using rice straw as the sole carbon source is prepared by mixing the following raw materials in the following mass ratio: 25 parts rice straw powder, 1 part KH2PO4, 0.2 parts MgSO4, 1 part K2HPO4, 2 parts (NH4)2SO4, 0.1 g CaCl2, 0.1 g peptone, and 1000 parts distilled water.