A kind of white bacillus and its application

Abstract

The present invention discloses a white bacillus and its application, belonging to the field of microbial technology. In order to provide an effective method for inhibiting Rhizoctonia solani, a method for promoting the growth of sugar beets, and a microorganism and method for producing cellulase. The present invention provides a white bacillus ( Bacillus albus ), the white Bacillus is named Sb‑3, and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2024875, and a deposit date of May 8, 2024. The present invention provides a good strain resource for the future development of plant-specific microbial agents and fertilizers.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a white bacillus. Background Art

[0002] beet( Beta vulgaris L.) belongs to the genus Beta in the family Amaranthaceae, and is a perennial or biennial herb. As an important sugar crop, beet occupies an important position in agricultural production. Beets were first domesticated from sea beets and are now grown in more than 40 countries around the world, second only to the first sugar crop, sugarcane, and provide about 20% of the world's edible sugar. Beets have a high economic volume in the market and can provide ingredients for many industries such as medicine, food processing, and chemical synthesis.

[0003] Beet blight is an important disease in beet production. Blight is caused by a variety of pathogenic fungi, among which Rhizoctonia solani ( Rhizoctonia solani ) is relatively common. Rhizoctonia solani is a widely distributed soil-borne plant pathogenic fungus belonging to the phylum Ascomycetes and the genus Rhizoctonia. The mycelium is white in the early stage and light brown in the later stage. The optimal growth temperature is 25°C and the optimal pH is 6.0-7.0. Rhizoctonia solani has a heavy infection rate and a low seedling survival rate. Rhizoctonia solani occurs frequently in all sugar beet producing areas in China, with a general incidence rate of 20% to 40%, and severe cases can reach 60% to 80%, and even destroy the seeds. Single or combined infection of the pathogen can lead to the occurrence of Rhizoctonia solani, resulting in the loss of nutrients and water in the plant. At the same time, damage to the stem fibers causes the plant to fall, ultimately resulting in a reduction in crop yield.

[0004] Endophytic bacteria are defined as bacteria that colonize inside plants without causing disease. They adapt to ecological niches and can improve plant growth and disease resistance under different conditions. Currently, there are few discoveries about biological control of beet damping-off disease, and there are no reports on the use of Bacillus albus to control beet damping-off disease. Due to the easy mutation of biocontrol bacteria, the effect of a biocontrol bacterium often loses its effect over time. Therefore, the development of other highly effective biocontrol agents is particularly urgent for sugar beet production. Summary of the invention

[0005] The purpose of the present invention is to provide an effective method for inhibiting Rhizoctonia solani, a method for promoting the growth of sugar beets, and a microorganism and method for producing cellulase.

[0006] The present invention provides a white Bacillus ( Bacillus albus ), the white Bacillus was deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 2024875 and the deposit time of May 8, 2024.

[0007] The present invention provides a microbial preparation containing the above-mentioned albicans bacillus.

[0008] The present invention provides an application of the white bacillus or the microbial preparation in preparing pesticides for preventing or treating damping-off disease.

[0009] The present invention provides an application of the white bacillus or the microbial preparation in promoting the growth of sugar beet.

[0010] The present invention provides the use of the above-mentioned Bacillus albicans or the above-mentioned microbial preparation in producing extracellular enzymes.

[0011] It is further defined that the extracellular enzyme is cellulase.

[0012] The present invention provides a method for producing cellulase, which comprises culturing the white bacillus at 36°C for 3 days.

[0013] It is further defined that the culture medium for culturing Bacillus albicans contains: CMC-Na 10 g·L -1 , K2HPO41g·L -1 , NH4NO31.0 g·L -1 , CaCl20.02 g·L -1 , MgSO4·7H2O 0.2 g·L -1 , FeCl3·6H2O0.05 g·L -1 and agar 15 g·L -1 .

[0014] The present invention provides a method for preventing or treating beet damping-off disease, which is characterized in that the white bacillus or the biological agent is made into a bacterial suspension and then poured into the root of the beet.

[0015] The present invention provides a method for promoting the growth of sugar beets, characterized in that the white bacillus or the microbial preparation is made into a bacterial suspension and poured into the roots of sugar beets in the seedling stage.

[0016] Beneficial effects: The white Bacillus Sb-3 obtained by the present invention is derived from sugar beet and can be well colonized in sugar beet; it can effectively inhibit Rhizoctonia solani, and the incidence is significantly different from that of the control, and can be used for biological control of sugar beet to reduce the occurrence of sugar beet damping-off in the seedling stage; it can avoid problems such as excessive pesticide residues, environmental pollution, and drug resistance of pathogens caused by chemical control methods, so it is more green and environmentally friendly, and can ensure the safety of sugar beet consumption. White Bacillus is made into a bacterial suspension and inoculated into plant seedlings through root irrigation, which can significantly increase the plant height, stem thickness and dry fresh weight of sugar beet, and the difference is significant compared with the control. Therefore, the present invention provides a good strain resource for the future development of plant-specific microbial agents and fertilizers.

[0017]

Biological Deposit Information

[0018] Figure 1 This is a single colony image of the white Bacillus Sb-3 in Example 1 of the present invention on LB medium;

[0019] Figure 2 Phylogenetic tree of strain Sb-3 in Example 1 of the present invention constructed based on 16S rRNA gene;

[0020] Figure 3 It is a blank control group diagram of Example 2 of the present invention and a confrontation effect diagram of Bacillus albicans Sb-3 against Rhizoctonia solani plate;

[0021] Figure 4 This is a diagram showing the growth-promoting effect of strain Sb-3 on beet potted plants in Example 3 of the present invention;

[0022] Figure 5 The changes of physiological indicators of sugar beet after being treated with Bacillus albus Sb-3 in Example 3;

[0023] Figure 6 The nitrogen fixation ability test results of strain Sb-3 in Example 3 of the present invention are as follows;

[0024] Figure 7 This is a diagram showing the effect of strain Sb-3 on beet damping-off in potted plants in Example 4 of the present invention;

[0025] Figure 8 The results of the assay of the protease production capacity of strain Sb-3 in Example 4 of the present invention;

[0026] Fig. 9 The results are shown in Table 1. These are the results of the cellulase production ability test of strain Sb-3 in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0028] Example 1: Isolation, screening and identification of endophytic white Bacillus Sb-3 in sugar beet

[0029] (1) LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, pH 7.2.

[0030] (2) Preparation of potato dextrose agar medium (PDA medium): 200 g potatoes, 20 g glucose, 15 g agar powder, 1 L deionized water, sterilized at 121°C for 20 min.

[0031] (3) Isolation of endogenous white Bacillus in sugar beet: Healthy sugar beet seedlings were collected from fields with severe damping-off disease. 2 g of roots, petioles and leaves were collected respectively. The tissues were first rinsed carefully with distilled water to ensure that the soil and impurities on the surface were removed. The surface of the tissue was gently wiped with sterile paper to remove excess water. On a sterile workbench, the tissues were cut into small pieces of 2 mm × 2 mm for subsequent disinfection and grinding. During the disinfection process, the small pieces were first disinfected with 75% alcohol for 30 s and rinsed with sterile water 3 to 4 times to remove residual alcohol. They were rinsed with 0.1% raw mercuric solution for 30 s to further kill possible microorganisms. They were rinsed with sterile water again 3 to 4 times to ensure that the disinfectant on the surface of the tissue was completely removed. The small pieces were cut into smaller pieces with a sterile blade and placed in a mortar. They were soaked in saline and ground with a mortar and pestle to break the tissue and release the microorganisms. Finally, use a sterilized inoculation loop to spread the grinding liquid on the streak to facilitate subsequent microbial isolation and screening.

[0032] (4) Identification of endophytic white Bacillus in sugar beet: Based on the physiological and biochemical characteristics and molecular biological test results of strain Sb-3, it was identified as white Bacillus ( Bacillus albus The specific identification results are as follows:

[0033] First, the strain with obvious antagonistic effect was purified several times, inoculated into LB medium, and cultured at 25℃ for 36 h before observing the colony morphology: the single colony of strain Sb-3 was large, waxy, white, opaque, shiny, and with uneven edges. Figure 1 shown.

[0034] Secondly, the physiological and biochemical characteristics of Sb-3 were tested. The results showed that the strain Sb-3 was Gram-positive, could hydrolyze starch, could hydrolyze cellulose, was positive in the VP test, and could use glucose and sucrose as the only carbon source for growth.

[0035] Finally, the strain Sb-3 was identified by molecular biology. The 16S rRNA gene sequence was amplified using primers 27F: 5′-AGAGTTTGATCCTGGTCAGAACGAACGCT-3′ (SEQ ID NO.1) and 1492R: 5′-TACGGCTACCTTGTTACGACTTCACCCC-3′ (SEQ ID NO.2). The sequencing results were used to construct a phylogenetic tree. The sequence was analyzed and compared with the 16S rDNA sequence in the database using the BLAST program of NCBI. The results showed that the Sb-3 strain had 100% homology with Bacillus albicans, confirming that its classification was named Bacillus albicans ( Bacillus albus ), named Bacillus albicans ( Bacillus albus ) Sb-3. The results are as follows Figure 2 shown.

[0036] 16S rRNA sequence (SEQ ID NO.3):

[0037]

[0038] Example 2: Determination of the antibacterial activity of endophytic white Bacillus Sb-3 in sugar beet by confrontation culture method

[0039] The plate confrontation method was used to detect the antagonistic properties of sugar beet endophyte Sb-3. Use a hole puncher (5 mm) to take bacterial blocks from the pathogen culture medium and place them in the center of the PDA. Then, the endophytic bacteria are connected to four symmetrical squares 15 mm away from the center of the culture medium. A control group must be set up and repeated three times. After culturing at 25°C for 3-5 days, the growth of the colonies in the culture medium was observed regularly. Pay special attention to whether there are inhibition zones between bacterial colonies, and whether the hyphae at the edge of the colony become sparse, dry or deformed. When the number of pathogens no longer increases, measure the radius and calculate the inhibition rate according to the formula: Inhibition rate = (colony radius of the control group − colony radius of the treatment group) / colony radius of the control group × 100%. The results are as follows. Figure 3 As shown, the antibacterial effect is obvious, and the inhibition rate is 37.53%.

[0040] Example 3: Determination of the growth-promoting effect of Bacillus albus Sb-3 on sugar beet based on sugar beet pot experiment

[0041] (1) Preparation of bacterial suspension: The isolated and purified Sb-3 was inoculated into LB liquid culture medium and cultured at 36℃ and 140 r / min for 16 h. After the culture was completed, the bacterial fermentation broth was subjected to refrigerated centrifugation at 4℃ and 10,000 r / min. The supernatant was discarded, the bacterial precipitate was separated, and a small amount of sterile water was added to make a suspension. The bacterial concentration in the suspension was measured using a Neubauer hemocytometer and adjusted to 10 by dilution. 8 CFU / mL.

[0042] (2) Treatment of beet seedlings with endophytes: The beet seeds KWS7748 were washed and coated, and then sown in pots of uniform size and pore size, with 16 seeds sown in each pot, and each treatment was repeated 4 times. After the beets emerged, 5 mL of bacterial suspension was poured into the roots, and the roots were irrigated once every 5 days. The control group was irrigated with distilled water. 25 days after the application of the bacteria, the growth of the beet plants under the two treatments was observed. The results are shown in Figure 2. Figure 4 shown.

[0043] (3) Measurement of sugar beet physiological indicators: plant height, stem diameter, dry weight and fresh weight of sugar beets under the two treatments were measured respectively. Figure 5 As shown in Table 1, inoculation with endophyte Sb-3 can significantly promote the growth of sugar beet, with the fresh weight, dry weight and leaf area being 65%, 53% and 52.38% higher than those of the control group, respectively.

[0044] Table 1

[0045]

[0046] (4) Determination of nitrogen fixation ability: The selected endophyte Sb-3 was activated in LB liquid medium and then inoculated into Axubei nitrogen-free solid medium and cultured at 26°C for 3 days. The results showed that an obvious transparent zone could be observed, indicating that the endophyte had the ability to fix nitrogen. Figure 6 shown.

[0047] Example 4: Determination of the ability of Bacillus albus Sb-3 to control sugar beet damping-off based on sugar beet pot plant experiment

[0048] Endophyte Sb-3 biocontrol potted plant test: Use a mixed solution of 5% sodium hypochlorite and 95% ethanol to disinfect the surface of beet seeds for 30 s. After disinfection, rinse the seeds repeatedly with tap water. Sow the seeds in pots filled with sterilized soil (autoclaved at 121℃ for 2 h). Before sowing, mix 400 g of sterilized soil with 0.067 g of the test Rhizoctonia solani inoculum, place it at the bottom of the pot, and then cover it with 300 g of sterilized soil and compact it. Sow 16 seeds in each pot, arrange them evenly, and finally cover them with 100 g of soil. After the beets emerge, take 5 mL of the bacterial suspension to irrigate the roots once every 5 days, and the control group is watered with distilled water. During the experiment, water needs to be added regularly to adjust the soil moisture, and the ambient temperature is controlled at 20-32℃ for 15 days. The growth and disease of beet plants under the two treatments are observed. The results are as follows Figure 7 As shown, the control effect of endophyte Sb-3 on sugar beet damping-off disease was 63.36%, indicating that endophyte Sb-3 can significantly inhibit the occurrence of sugar beet damping-off disease.

[0049] Example 5. Production of extracellular enzymes by Bacillus albus

[0050] Determination of extracellular enzyme activity of sugar beet endophyte Sb-3: 5 μL of activated bacterial solution was inoculated into the center of a skim milk powder solid culture medium plate and cultured at 28°C for 3 days. Figure 8 As shown in the figure, it was observed that the degradation zone was obvious in the protease detection medium, indicating that Sb-3 has the ability to produce protease. 5 μL of activated bacterial solution was taken and inoculated into the center of the cellulase solid medium plate and cultured at 28℃ for 3 days. After 3 days, it was stained with 0.07% Congo red solution for 30 minutes and then decolorized with 1 mol / L sodium chloride solution for 30 minutes. The results are shown in Fig. 9 As shown, strain Sb-3 produced degradation circles in cellulase medium, indicating that it has the ability to produce cellulase.

[0051] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention and be protected by the patent law.

Claims

1. A white Bacillus ( Bacillus albus ), characterized in that, The white Bacillus is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2024875 and the deposit date is May 8, 2024.

2. A microbial preparation containing the albicans bacillus according to claim 1.

3. Use of the white Bacillus according to claim 1 or the microbial preparation according to claim 2 in the preparation of pesticides for preventing or treating sugar beet damping-off disease, wherein the pathogenic bacteria of sugar beet damping-off disease is Rhizoctonia solani.

4. Use of the white Bacillus according to claim 1 or the microbial preparation according to claim 2 in promoting the growth of sugar beets.

5. Use of the white Bacillus according to claim 1 or the microbial preparation according to claim 2 in the production of protease and cellulase.

6. A method for producing cellulase, characterized in that: The white Bacillus according to claim 1 is cultured at 36° C. for 3 days.

7. The method according to claim 6, characterized in that The culture medium for culturing Bacillus albicans contains: CMC-Na 10 g·L -1 , K2HPO4 1 g·L -1 , NH4NO3 1.0 g·L -1 , CaCl2 0.02 g·L -1 , MgSO4·7H2O 0.2g·L -1 , FeCl3·6H2O 0.05 g·L -1 and agar 15 g·L -1 .

8. A method for preventing or treating beet damping-off disease, characterized in that: The white bacillus described in claim 1 or the biological preparation described in claim 2 is made into a bacterial suspension and poured into the roots of beets.

9. A method for promoting the growth of beets, characterized in that: The white Bacillus described in claim 1 or the microbial preparation described in claim 2 is made into a bacterial suspension and injected into the roots of sugar beets in the seedling stage.

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