Bacillus cereus and application thereof

Abstract

The invention relates to bacillus cereus and application thereof, and belongs to the technical field of microorganisms. The bacillus cereus TT5-2 is separated from fresh soil for tobacco planting, and detection shows that the bacillus cereus TT5-2 can secrete urease, effectively decompose urea and effectively antagonize meloidogyne incognita. Tobacco growth promotion experiments prove that the screened bacillus cereus TT5-2 can effectively promote the growth of tobacco seedlings. The bacillus cereus TT5-2 screened by the invention not only can effectively antagonize meloidogyne incognita, but also can remarkably promote the growth of tobacco, can secrete urease, is a multifunctional bacillus cereus integrating biocontrol and growth promotion, and has important significance for enriching a bacillus strain library.

Description

Technical Field

[0001] The invention relates to Bacillus cereus and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Plant-parasitic nematodes are obligate root parasites, living on the roots, stems, leaves, flowers, buds, and fruits of various plants. Compared to other major pathogens (such as fungi, bacteria, and viruses), they actively invade their hosts and migrate to cause damage. They cause significant losses to agricultural production in my country each year and have become the second largest category of agricultural diseases. Surveys across the country have shown that nearly all greenhouse and field vegetable plots, including watermelon, bitter melon, cantaloupe, cucumber, tomato, loofah, and celery, suffer varying degrees of nematode infestation. The incidence is particularly severe in protected plots where continuous cropping is practiced, and in severe cases, the disease can lead to complete harvest loss.

[0003] There are numerous species of plant parasitic nematodes, including Meloidogyne, Anguina, Ditylenchus, Heterodera, Aphelenchoides, and ecto-radical nematodes. Root-knot nematodes and cyst nematodes are two of the most devastating plant parasitic nematodes in agricultural production, causing significant economic losses when they infect vegetables and grain crops. Root-knot nematode disease is a global disease, causing $170 billion in economic losses annually.

[0004] Tobacco, an annual or limited perennial herb in the Solanaceae family (Solanaceae), is a widely cultivated and important cash crop in my country. Currently, due to the long-term afforestation of most tobacco fields in my country and the use of pesticides and chemical fertilizers, the soil structure of tobacco-growing areas has deteriorated, and the number of root-knot nematodes in the soil has increased annually. Root-knot nematodes primarily infect tobacco roots, disrupting the differentiation and physiological activity of root tissue, causing leaf tips and margins to dry out and curl inward. Furthermore, the wounds caused by root-knot nematodes infecting tobacco roots promote the infection of other fungal and bacterial diseases, seriously affecting tobacco leaf yield and quality. Root-knot nematodes have become a major disease in my country's tobacco-producing areas.

[0005] Currently, methods for controlling root-knot nematodes fall into four main categories: agricultural control, physical control, chemical control, and biological control. Crop rotation and breeding nematode-resistant varieties are two effective agricultural methods for nematode control. However, crop rotation is significantly limited due to the wide host range of nematodes, limited arable land resources, and limited farming conditions. Currently, there is a lack of nematode-resistant varieties with good agronomic traits. Heat treatment is an effective nematode control method, but it is costly, indiscriminately kills beneficial microorganisms in the soil, and damages the soil microecological environment, hindering its widespread application. Chemical nematicides are highly effective and easy to use, making them the primary method for nematode control. However, with the increasing severity of long-term use, chemical nematicides have become increasingly toxic, have high residue levels, and pose serious environmental risks. They are no longer suitable for environmentally friendly and effective nematode control. Biological control, due to its long-lasting effects, ecological safety, and friendliness to humans and animals, has attracted considerable attention in the control of root-knot nematodes and plays a crucial role in sustainable agricultural development.

[0006] Organisms found to be effective in nematode control include fungi, bacteria, viruses, nematodes, insects, mites, and some invertebrates. Fungi and bacteria are the most studied of these. While most biocontrol fungi demonstrate good control efficacy in indoor and potted experiments, they often struggle to achieve optimal results in field trials due to limitations such as colonization ability, temperature, and humidity. Bacteria are generally easy to culture, reproduce rapidly, and have strong colonization abilities, making them important biocontrol agents for nematodes. Bacillus species are a common biocontrol agent, producing numerous secondary metabolites with nematicidal activity that damage the nematode's body wall or intestinal tract, achieving nematicidal effects. Some Bacillus species can also induce systemic resistance in their hosts, promoting plant growth while suppressing disease. Although numerous biocontrol agents for root-knot nematodes have been identified, very few have been used in production. Therefore, to improve tobacco yield and quality in my country, identifying biocontrol agents that possess both root-knot nematode antagonism and other desirable properties remains crucial. Summary of the Invention

[0007] The first object of the present invention is to provide a Bacillus cereus that can antagonize root-knot nematodes and significantly promote plant growth.

[0008] The second purpose of the present invention is to use Bacillus cereus in plant cultivation, and to provide a Bacillus cereus that can antagonize root-knot nematodes and significantly promote plant growth for use in plant cultivation.

[0009] In order to achieve the above object, a technical solution of Bacillus cereus in the present invention is:

[0010] A Bacillus cereus, wherein the preservation number of the Bacillus cereus is CGMCC NO.27097.

[0011] The beneficial effect of the above technical solution is that the Bacillus cereus of the present invention is a pioneering invention. The present invention isolated a strain of Bacillus cereus TT5-2 from fresh soil of tobacco planting. Testing found that the bacteria can secrete urease, effectively decompose urea, and can effectively antagonize southern root-knot nematodes. The tobacco growth promotion experiment proved that the Bacillus cereus TT5-2 screened out by the present invention can effectively promote the growth of tobacco seedlings. The Bacillus cereus TT5-2 screened out by the present invention can not only effectively antagonize southern root-knot nematodes, but also significantly promote the growth of tobacco, and can secrete urease. It is a multifunctional Bacillus cereus that integrates biocontrol and growth promotion, and is of great significance for enriching the Bacillus strain library.

[0012] Specifically, urease-producing microorganisms have the potential to replace or surpass traditional pure urease preparations through the synergistic effect of their urease active center and auxiliary proteins. Its application prospects are broad, mainly reflected in the following aspects: (1) Promoting plant growth: Urease-producing bacteria can be used in agriculture and forestry to decompose nitrogen fertilizers and urea to produce NH + , providing nitrogen sources for plants; (2) MICP (Microbial Induced Calcite Precipitation) technology: Microorganisms induce crystallization through urea decomposition or photosynthesis, and are widely used in the preparation of engineering materials, soil reinforcement and removal of heavy metal ions. (3) Detoxification of alcoholic beverages: Urease-producing microorganisms can effectively remove urea from alcoholic beverages, reduce the production of carcinogen ethyl carbamate, and improve the quality of alcoholic beverages. (4) Wastewater treatment: Compared with traditional pure urease, the application of urease-producing microorganisms in the purification of urea wastewater is more economical, efficient, and the process is simple. (5) Treatment of uremia: Introducing urease-producing microorganisms into the human body can decompose urea in the body, which is expected to alleviate the symptoms of uremia.

[0013] In order to achieve the above-mentioned object, the technical solution of the application of Bacillus cereus in plant cultivation in the present invention is:

[0014] The invention discloses an application of Bacillus cereus in plant cultivation, wherein the application is for preventing and controlling plant root-knot nematode diseases and promoting plant growth.

[0015] The beneficial effects of the above technical solution are as follows: the present invention isolates a strain of Bacillus cereus TT5-2 from fresh tobacco soil, and uses its bacterial solution to analyze the lethality of second-instar larvae of the southern root-knot nematode, finding that the strain has a high mortality rate against second-instar larvae of the southern root-knot nematode. This invention fully demonstrates that Bacillus cereus TT5-2 can effectively antagonize southern root-knot nematodes, providing a new microbial resource for controlling root-knot nematode diseases, expanding the variety of biocontrol bacteria, and providing technical guidance for the efficient microbial control of southern root-knot nematode diseases.

[0016] Furthermore, the present invention demonstrated through urea decomposition experiments that the bacteria can effectively decompose urea, indicating that it can secrete urease, and the results show that its secretion ability is relatively strong. Furthermore, through tobacco growth promotion experiments, it was demonstrated that the bacteria can promote the growth of tobacco seedlings by increasing the length and width of tobacco leaves. The Bacillus cereus TT5-2 screened by the present invention can both secrete urease and effectively promote tobacco growth, providing a new tobacco growth-promoting bacterium and laying a foundation for improving tobacco quality and yield.

[0017] As a further improvement, the promoting plant growth is to increase the length and width of leaves of plant seedlings.

[0018] As a further improvement, the plant is tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology diagram of Bacillus cereus strain TT5-2 in Example 1 of the present invention;

[0020] Figure 2 This is the phylogenetic tree constructed based on the 16S rRNA sequences of different strains of Bacillus cereus in Example 1 of the present invention;

[0021] Figure 3 The results of the culture of Bacillus cereus TT5-2 strain in Christensen's Urean Agar in Example 2 of the present invention are as follows;

[0022] Figure 4 The lethal effect of the Bacillus cereus TT5-2 strain solution in Example 3 of the present invention on the second-instar larvae of the root-knot nematode (wherein, A represents the morphological effect of different treatments on the second-instar larvae of the southern root-knot nematode, and B represents the corrected mortality rate (%) of the second-instar larvae of the southern root-knot nematode treated with different strains);

[0023] Figure 5 Figure 4 shows the growth-promoting effect of the Bacillus cereus TT5-2 strain on tobacco in Example 4 of the present invention (wherein, A represents the effect of different treatments on the morphology of tobacco seedlings, and B represents the effect of different treatments on the leaves of tobacco seedlings. Different letters in the figure represent significant differences). DETAILED DESCRIPTION

[0024] At present, most of my country's tobacco fields have been under long-term cultivation and use of pesticides and chemical fertilizers, which has caused the soil structure of tobacco planting to deteriorate, and the situation of pests and diseases (such as root knot nematode disease) has become more serious year by year. In addition, the residue of pesticides will also affect human health and cause environmental pollution, which seriously restricts the sustainable development of tobacco. In order to increase the yield and output of tobacco, it is necessary to develop resources for tobacco biocontrol bacteria and growth-promoting bacteria in the soil environment of tobacco planting, screen good microbial strains that promote tobacco growth, and be able to better adapt to the environment of tobacco growth and give full play to the functions of microorganisms. It is extremely important to develop high-quality growth-promoting bacteria products to increase tobacco yield. This is not only in line with the development concept of green environmental protection, but also can promote the sustainable development of tobacco. Based on this, the present invention provides a Bacillus cereus TT5-2.

[0025] The present invention will be further described below in conjunction with specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0026] The formula and preparation process of the culture medium and some solutions used in the embodiments of the present invention are as follows:

[0027] LB agar medium: Prepare according to the ratio of 10 g / L tryptone, 5 g / L yeast powder, 5 g / L sodium chloride, 1 g / L glucose, and 15 g / L agar, and finally place it in an autoclave for sterilization at 121°C for 15 min.

[0028] Christensen's Urean Agar medium: 1g / L peptone, 5g / L sodium chloride, 1g / L glucose, 2g / L potassium dihydrogen phosphate, 0.012g / L phenol red, 20g / L agar, pH 6.8±0.2. Combine all ingredients except urea and agar, boil in 950mL of sterile water, dissolve, adjust pH to 6.8±0.2, add agar, and autoclave at 121°C for 15 minutes. Cool to 50-55°C, then add 50mL of sterile-filtered 40% urea solution for a final urea concentration of 2%.

[0029] Preparation of MS solid culture medium: MS culture medium basal salt 4.43 g / L, sucrose 30 g / L, plant gel 3 g / L, pH 5.8, high pressure sterilization at 121℃ for 15 min.

[0030] Preparation of 10% sodium hypochlorite solution: add 9 mL of sterile water to 1 mL of sodium hypochlorite solution.

[0031] The tobacco variety used in the following examples of the present invention is the cultivated tobacco variety K326 (Nicotiana tabacum L.) planted in an artificial climate chamber of the National Tobacco Gene Research Center at a temperature of 26-28° C., a relative humidity of 60%, and a photoperiod of 16 h light / 8 h dark.

[0032] 1. A specific embodiment of a Bacillus cereus of the present invention:

[0033] The Bacillus cereus of the present invention is Bacillus cereus TT5-2, with a deposit number of CGMCCNO.27097; the deposit date is April 12, 2023; the depositary is the General Microbiology Center of the China Culture Collection Administration; the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. The specific implementation operations are as follows:

[0034] Example 1 Screening and Identification of Bacillus cereus TT5-2

[0035] In this example, a strain of Bacillus cereus TT5-2 was isolated from fresh soil of tobacco plants. The specific implementation steps are as follows:

[0036] 1. Isolation of TT5-2 strain

[0037] Weigh 5 g of fresh soil after sieving, add it to 45 mL of cooled sterile water, shake at 30°C and 180 rpm for 30 min to obtain 10 -1 dilution, and then dilute 10 times to get 10 -2 , 10 -3 , 10 -4 , 10 -5 Dilution. Pipette 60μL 10 -3 , 10 -4 , 10 -5 Three dilutions of soil suspension were smeared on LB medium plates, with 3 plates for each dilution. The plates were cultured in a 37°C incubator overnight. Plates with clearly identifiable and evenly growing single colonies were selected. The single colonies were picked and purified using the parallel streak method. Once confirmed as single colonies, they were stored for later use.

[0038] 2. Cultivation of TT5-2 strain

[0039] (1) Activation of bacterial strains: Take 200 μL of bacterial solution stored in glycerol at -80°C and transfer it to LB liquid medium. Incubate at 30°C and 200 rpm for 24 h.

[0040] (2) Plate culture: Take a small amount of bacterial solution from step (1) and spread it on LB medium plate, culture at 30°C for 48 hours.

[0041] (3) Expansion culture: Select a single colony with good growth from step (2) and transfer it to LB liquid culture medium. The shaking medium expansion culture conditions are 200 r / min, temperature 30 ° C, and the strain OD 600=1, collect the bacterial liquid.

[0042] 3. Identification of TT5-2 strain

[0043] 1. Morphological identification

[0044] The colony morphology of Bacillus cereus TT5-2 is as follows Figure 1 As shown in the figure, the colonies are milky white and have a smooth surface.

[0045] 2. Molecular Biology Identification

[0046] DNA of Bacillus cereus TT5-2 was extracted and used as a template for PCR amplification using bacterial 16S rRNA universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3', shown in SEQ ID NO. 1) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3', shown in SEQ ID NO. 2) as amplification primers.

[0047] The PCR amplification reaction system and reaction conditions are shown in Tables 1 and 2.

[0048] Table 1 PCR amplification reaction system

[0049]

[0050] Table 2 PCR reaction conditions

[0051]

[0052]

[0053] 1% TAE solution was used as the buffer solution, and the PCR product was detected by 1% agarose gel electrophoresis. The size of the PCR product was observed by a gel imager. The sample produced a single band, indicating that the amplification was qualified. The PCR product was sequenced by Beijing Novogene Sequencing Co., Ltd.

[0054] The sequencing results and analysis of the 16S rRNA gene sequence of the TT5-2 strain showed that the 16S rRNA gene sequence of the TT5-2 strain (shown in SEQ ID NO.3) was compared with the 16S rRNA gene sequence of Bacillus cereus in GeneBank (see Figure 2 The results showed that the TT5-2 strain belonged to Bacillus cereus.

[0055] Based on the results of morphological and molecular biological identification, the strain was identified as Bacillus cereus, named TT5-2, and the preservation number was CGMCC NO.27097; the preservation date was April 12, 2023; the preservation unit was the General Microbiology Center of the China Culture Collection Administration; the preservation address was No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101.

[0056] Example 2 Urease assay of Bacillus cereus TT5-2

[0057] This example demonstrates that the bacteria can secrete urease through urea decomposition experiments. The specific implementation steps are as follows:

[0058] A small amount of bacterial solution was spread on Christensen's Urean Agar plates and cultured at 30°C for 48 hours. Three replicates were set up. Control group 1 (no bacterial solution), control group 2 (P. umsongensis ZT17-18) and control group 3 (P. baetica ZT17-19) were also prepared.

[0059] Specific observations such as Figure 3 As shown in the figure, it can be seen that after Bacillus cereus TT5-2 was cultured in urea agar medium for 48 hours, the culture medium turned pink compared with CK, indicating that Bacillus cereus TT5-2 contained urease that decomposes urea; compared with Pseudomonas sumsongensis ZT17-18 and Pseudomonas baetica ZT17-19, B. cereus TT5-2 has stronger urea decomposition ability.

[0060] 2. Specific examples of the application of Bacillus cereus in plant cultivation according to the present invention:

[0061] Example 3: Lethality of Bacillus cereus TT5-2 to Second-Instar Larvae of Southern Root-Knot Nematodes This example verifies the control effect of Bacillus cereus TT5-2 on plant root-knot nematodes. The specific implementation procedures are as follows:

[0062] In a 1.5 mL sterile centrifuge tube, add 100 μL of the second-instar larvae suspension of southern root-knot nematodes and 100 μL of sterile water to each well, and add OD 600 =1(2×10 8100 μL of a Bacillus cereus TT5-2 bacterial solution (100 μL) containing 100 μL of WT (100 μL) of Bacillus cereus TT5-2 (100 μL) of WT ...

[0063] Mortality rate = number of dead nematodes / number of tested nematodes × 100%;

[0064] Corrected mortality rate = (nematode mortality rate in the treatment group - nematode mortality rate in the control group) / (1 - nematode mortality rate in the control group) × 100%.

[0065] Specific results such as Figure 4 As shown by Figure 4 As shown in Figure A, after 24 hours of treatment with the bacterial solution of Bacillus cereus TT5-2, P. umsongensis ZT17-18, and P. baetica ZT17-19, microscopic observation revealed that some second-instar larvae of the root-knot nematode were in a state of rigidity and showed no reaction when the worm body was touched. Figure 4 As shown in Figure B, Bacillus cereus TT5-2 had the highest mortality rate against the second-instar larvae of the southern root-knot nematode, with the corrected mortality rate reaching 84.7%.

[0066] Example 4 Growth-promoting effect of Bacillus cereus TT5-2 on tobacco

[0067] This example uses K326 as the subject to verify the growth-promoting effect of Bacillus cereus TT5-2. The specific implementation procedures are as follows:

[0068] In a clean bench, take an appropriate amount of tobacco K326 seeds and place them in a 2mL centrifuge tube. Add 1mL of 10% sodium hypochlorite solution and wash them 5 times, each time for 2-3 minutes; then wash them 3 times with sterile water. Use sterile toothpicks to evenly sow the cleaned seeds on MS solid culture medium and then place them in an artificial climate chamber for cultivation. When the tobacco seedlings grow two true leaves, select tobacco seedlings of the same size and transplant them to new MS solid culture medium, transplanting 4 tobacco seedlings on each culture medium. Add 20μLOD 600 =0.6(4×10 7cfu / mL) was applied to the 1 / 5th depth of the MS culture medium (equivalent to applying the culture medium to the bottom of the medium, which effectively prevents direct contact between the culture medium and excessive plant tissue, facilitating observation of its effects on plant development and preventing excessive competition between microorganisms and plant tissue, which could inhibit plant growth). A culture medium without the culture medium served as a control. Three replicates were set up for each treatment. After 15 days of culture, the length and width of the largest leaf of each tobacco seedling were recorded.

[0069] Specific results such as Figure 5 As shown in the figure, compared with P. umsongensis ZT17-18 and P. baetica ZT17-19, Bacillus cereus TT5-2 significantly increased the leaf length and leaf width, and promoted the growth of tobacco seedlings.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Bacillus cereus, characterized in that: The deposit number of the Bacillus cereus is CGMCC NO.27097.

2. A use of the Bacillus cereus according to claim 1 in plant cultivation, characterized in that: The application is to prevent and control plant root knot nematode diseases and promote plant growth.

3. The use of Bacillus cereus in plant cultivation according to claim 2, characterized in that: The promoting of plant growth is to increase the length and width of leaves of plant seedlings.

4. The use of Bacillus cereus in plant cultivation according to claim 2 or 3, characterized in that: The plant is tobacco.

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