Bacillus bacterium and application thereof

Abstract

The invention belongs to the technical field of plant growth promoting and yield increasing methods, and particularly relates to a bacillus bacterium and application thereof. The bacillus bacterium is bacillus velezensis FB007 and is preserved in the China Center for Type Culture Collection on December 28, 2023, and the preservation number of the bacillus bacterium is CCTCC NO: M 20232709. Experiments prove that when the bacillus velezensis FB007 is applied to root systems of pepper seedlings, lettuce seedlings and arabidopsis thaliana, the growth of the plants can be promoted, the yield of the plants can be increased, and the main effects are that the lateral roots of the plants are increased, the growth of the plants is promoted, and the dry weight and fresh weight of the plants are increased. The bacillus velezensis FB007 provided by the invention has an important application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant growth promotion and yield increase methods, and particularly relates to a bacterium of the genus Bacillus and its application. Background Art

[0002] Growth promotion is an important indicator for determining the success of plant yield. Plants must accurately combine internal signals and environmental signals and then initiate the growth process. The growth of plants is affected by the combined action of the natural environment and genetic mechanisms, showing rich diversity among and within species. At present, extensive research has been carried out on the mechanism of regulating plant growth from aspects such as physiology, biochemistry, metabolism, ecology, genetics, and evolution. Especially with the development of molecular biology, people can understand the mechanism of regulating plant growth at the molecular level of gene composition, expression regulation, and signal transduction, providing new ways to regulate plant growth by means of genetic engineering and other methods. Due to the complexity of the plant growth mechanism, it is very difficult to promote plant growth by traditional breeding means. Although genetic engineering and other means have opened up new ways to promote plant growth, the isolation of genes that can efficiently regulate plant growth has become the main limiting factor. Therefore, it is necessary to start from other aspects to find new ways to promote plant growth.

[0003] Plant growth-promoting rhizobacteria can colonize the rhizosphere of plants and promote plant growth through various mechanisms, such as nitrogen fixation, phosphorus solubilization, and potassium solubilization to increase the available nutrient elements for plants, produce plant growth hormones such as indole acetic acid and gibberellin to stimulate plant growth, and inhibit the growth of soil-borne pathogens by producing antibacterial substances or competing for sites, thereby enhancing the disease resistance of plants. In addition, they can also help plants resist stress. For example, in adverse environments such as drought and salinity, by regulating the physiological metabolism of plants, the stress resistance of plants is improved, and thus the yield and quality of plants are increased.

[0004] At present, microorganisms that can promote plant growth include plant growth-promoting rhizobacteria such as nitrogen-fixing bacteria, phosphorus bacteria, silicate bacteria, mycorrhizal fungi, photosynthetic bacteria, and Bacillus. Although these existing plant growth-promoting rhizobacteria can promote plant growth to a certain extent, they still have the defect of unstable effects in complex soil environments. Summary of the Invention

[0005] To solve the defect that the existing plant growth-promoting rhizobacteria in the prior art have unstable effects in complex soil environments, the present invention provides a bacterium of the genus Bacillus and its application. To achieve the above object, the present invention adopts the following technical solutions.

[0006] The present invention provides a bacterium of the genus Bacillus, which is Bacillus velezensis FB007. It was deposited at the China Center for Type Culture Collection on December 28, 2023, with the deposit number CCTCC NO: M 20232709.

[0007] The Bacillus velezensis FB007 has the following characteristics: on nutrient agar medium, the colonies are round, with a smooth surface, regular edges, milky white, opaque, viscous, shiny, and the colony diameter is 2.5 mm ± 0.5 mm; Gram-positive, rod-shaped, with spores, non-motile. This strain has strong nitrogen fixation, phosphorus solubilization and potassium solubilization abilities, can produce various plant growth hormones such as indole acetic acid and gibberellin, and has good stress resistance characteristics such as salt and alkali tolerance and drought tolerance. It can grow well in the range of pH 5.5 - 9.5 and temperature 20°C - 40°C, has an inhibitory effect on a variety of plant pathogens, has strong environmental adaptability, can be applied under different soil types and climatic conditions, can significantly promote plant root development, improve the absorption and utilization rate of plant nutrients, enhance the stress resistance and disease resistance of plants, and thus can promote plant growth and increase yield.

[0008] As can be seen from the above, compared with the existing plant growth-promoting rhizobacteria, the Bacillus velezensis FB007 provided by the present invention has a better effect in promoting plant growth, will not show unstable conditions, and solves the defect that the existing plant growth-promoting rhizobacteria in the prior art have unstable growth-promoting effects in complex soil environments.

[0009] Preferably, the nucleotide sequence of the 16S rRNA of the Bacillus velezensis FB007 is as shown in SEQ ID NO.1:

[0010] Preferably, the Bacillus velezensis FB007 can promote the growth of at least one of plants such as pepper, lettuce and Arabidopsis thaliana.

[0011] Preferably, the Bacillus velezensis FB007 can increase the yield of at least one of plants such as pepper, lettuce and Arabidopsis thaliana.

[0012] The present invention also provides the use of the Bacillus bacterium or the culture of the Bacillus bacterium in promoting plant growth and increasing plant yield.

[0013] Preferably, the Bacillus velezensis FB007 or the culture of the Bacillus velezensis FB007 is used for preparing a microbial inoculant for promoting plant growth and increasing plant yield.

[0014] The above-mentioned microbial inoculant for preparing a microbial inoculant for promoting plant growth and increasing plant yield is simply referred to as a microbial inoculant. This microbial inoculant can improve soil structure and fertility, inhibit the reproduction of soil-borne pathogens, reduce the occurrence of soil diseases, recruit other microorganisms to form a microbial flora, play a synergistic role, enhance the promotion effect on plant growth, and increase the yield.

[0015] Preferably, the microbial inoculant uses the Bacillus velezensis FB007, the culture of the Bacillus velezensis FB007 or the lysate of the Bacillus velezensis FB007 as an active ingredient.

[0016] Preferably, the roots of plants are treated with the microbial inoculant, thereby promoting plant growth and increasing plant yield.

[0017] The plants include at least one of pepper, lettuce and Arabidopsis thaliana.

[0018] Preferably, the OD 600nm value of the microbial inoculant is 0.2.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a Bacillus bacterium. The Bacillus bacterium provided by the present invention is Bacillus velezensis FB007. The Bacillus velezensis FB007 has strong nitrogen fixation, phosphorus solubilization and potassium solubilization abilities, can produce various plant growth hormones such as indoleacetic acid and gibberellin, and has good stress resistance characteristics of salt tolerance and drought tolerance, has strong environmental adaptability, can be applied under different soil types and climate conditions, can significantly promote plant root development, improve the absorption and utilization rate of plant nutrients, enhance the stress resistance and disease resistance of plants, and thus can promote plant growth and increase plant yield.

[0020] The Bacillus velezensis FB007 provided by the present invention has a better effect in promoting plant growth compared to these existing plant growth-promoting rhizobacteria, and there will be no unstable situation, solving the defect that these existing plant growth-promoting rhizobacteria in the prior art have unstable growth-promoting effects in complex soil environments.

[0021] 2. It has been experimentally proven in the present invention that the Bacillus velezensis FB007 provided by the present invention can promote the growth of Arabidopsis thaliana and increase the number of roots, promote the growth of peppers and lettuce, and increase the yields of peppers and lettuce. The Bacillus velezensis FB007 provided by the present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the effect of Bacillus velezensis FB007 on the root growth of Arabidopsis thaliana seedlings in the present invention; among them, Figure 1 A in it is the growth diagram of Arabidopsis thaliana seedlings inoculated with Bacillus velezensis FB007 under tissue culture conditions, which are five parallel samples; Figure 1 B in it is the growth diagram of Arabidopsis thaliana seedlings not inoculated with Bacillus velezensis FB007 under tissue culture conditions, which are five parallel samples.

[0023] Figure 2 It is a growth comparison diagram of Arabidopsis thaliana seedlings treated with the Bacillus velezensis FB007 agent of the present invention and Arabidopsis thaliana seedlings in the untreated control group; among them, Figure 2 A in it is the growth diagram of Arabidopsis thaliana seedlings inoculated with the Bacillus velezensis FB007 agent under potting conditions, which are three parallel samples; Figure 2 B in it is the growth diagram of Arabidopsis thaliana seedlings not inoculated with the Bacillus velezensis FB007 agent under potting conditions, which are three parallel samples.

[0024] Figure 3 It is a growth comparison diagram of pepper seedlings treated with the Bacillus velezensis FB007 agent of the present invention and pepper seedlings in the untreated control group; among them, Figure 3 A in it is the growth diagram of pepper seedlings inoculated with the Bacillus velezensis FB007 agent under potting conditions; Figure 3 B in it is the growth diagram of pepper seedlings not inoculated with the Bacillus velezensis FB007 agent under potting conditions.

[0025] Figure 4 It is a growth comparison diagram of lettuce seedlings treated with the Bacillus velezensis FB007 agent of the present invention and lettuce seedlings in the untreated control group; among them, Figure 4 A in it is the growth diagram of lettuce seedlings inoculated with the Bacillus velezensis FB007 agent under potting conditions; Figure 4Figure B in [reference] shows the growth diagram of lettuce seedlings without inoculating with Bacillus velezensis FB007 inoculant under potted conditions.

[0026] Figure 5 This is the effect of Bacillus velezensis FB007 inoculant treatment on the stem diameter, fresh weight and dry weight of lettuce seedlings in the present invention; among them, Figure 5 Figure A in [reference] is the analysis diagram of the stem diameter of lettuce seedlings; Figure 5 Figure B in [reference] is the analysis diagram of the fresh weight of lettuce seedlings; Figure 5 Figure C in [reference] is the analysis diagram of the dry weight of lettuce seedlings. Specific Embodiments

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0028] The culture media and materials used in the embodiments are as follows: (1) Culture Media LB liquid medium: Dissolve 10 g of tryptone, 5 g of yeast extract and 5 g of sodium chloride in 1 L of distilled water, then sterilize at 121 °C for 15 min and use after cooling.

[0029] LB solid medium: Add agar to the LB liquid medium to make its concentration 15 g / L; then sterilize at 121 °C for 15 min. Pour the liquid medium cooled to about 55 °C into a petri dish and let it cool naturally.

[0030] (2) Materials Wild-type Arabidopsis thaliana ( Arabidopsis thaliana ) Columbia-0 subtype is described in the following literature: Lundberg DS, Lebeis SL, Paredes SH, et al. Defining the core Arabidopsis thaliana root microbiome. Nature. 2012;488(7409):86-90.

[0031] The wild-type Arabidopsis thaliana Columbia-0 subtype is hereinafter referred to as Arabidopsis thaliana for short.

[0032] The seeds of Arabidopsis thaliana are from China Agricultural University.

[0033] The seeds of pepper are from China Agricultural University.

[0034] The seeds of lettuce are from China Agricultural University.

[0035] Example 1: Isolation, Identification and Preservation of Bacillus velezensis FB007 I. Isolation of Bacillus velezensis FB007 1. Add 5 g of rhizosphere soil sample to 45 mL of phosphate buffer, stir for 15 min, and let stand for 10 min. Then, take 1 mL of the supernatant and add it to a sterile test tube containing 9 mL of phosphate buffer, and mix well. At this time, the dilution factor is recorded as 10 -1 . Then, pipette 1 mL from the sterile test tube and add it to another sterile test tube containing 9 mL of phosphate buffer, and mix well. And so on, prepare the above dilutions into 10 -2 、10 -3 、10 -4 and 10 -5 bacterial suspensions with different dilutions.

[0036] After taking 0.1 mL of each of the above bacterial suspensions with different dilution gradients, spread them evenly on the LB solid medium and incubate at 28 °C for 2 days with static culture.

[0037] Among them, the rhizosphere soil sample was collected from the soil of Tomato Commune in Yingcheng City, Xiaogan.

[0038] Phosphate buffer, 1×: NaCl 136 mM, KCl 2.7 mM, Na2HPO4 10 mM, NaH2PO4 1.8 mM, pH 7.3, purchased from Sinopharm Group.

[0039] 2. After the cultivation is completed, pick the single colonies on the LB solid medium, purify them repeatedly 3 times, and screen out a strain of bacteria. The screened bacteria were named bacteria FB007.

[0040] II. Identification of Bacteria FB007 The result of comparing the 16S rRNA sequence of bacteria FB007 with the sequences in NCBI shows that bacteria FB007 has the highest homology with the strains of the genus Bacillus ( Bacillus velezensis ), reaching 99.93%.

[0041] Among them, the nucleotide sequence of the 16S rRNA sequence of bacteria FB007 is as shown in SEQ ID NO.1:

[0042] III. Preservation Based on the above 16S rRNA sequence homology analysis results, the bacterium FB007 obtained by the above separation and purification was identified as Bacillus velezensis FB007.

[0043] Strain preservation: A single colony of Bacillus velezensis FB007 was inoculated into LB liquid medium and cultured on a shaker at 28 °C and 200 r / min for 8 h. After the culture was completed, a bacterial culture solution was obtained.

[0044] One volume of the bacterial culture solution was mixed evenly with one volume of glycerol with a volume ratio of 60% to obtain a bacterial solution, which was stored at -80 °C.

[0045] Bacillus velezensis FB007 is currently preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20232709.

[0046] Example 2: Preparation of microbial inoculum 1. The bacterial solution stored at -80 °C in Example 1 was streaked and activated on LB solid medium.

[0047] 2. A single colony on the LB solid medium was picked and inoculated into a sterilized conical flask containing 100 mL of LB liquid medium, and cultured at 28 °C and 200 r / min for 8 h. After the culture was completed, the OD 600nm value of the obtained bacterial solution was adjusted to 0.2, and this bacterial solution was the prepared microbial inoculum. This microbial inoculum is also called Bacillus velezensis FB007 inoculum.

[0048] Among them, the specification of the conical flask is 250 mL.

[0049] Example 3: Role of Bacillus velezensis FB007 in promoting plant growth I. Preparation of culture medium Specification of culture dish: 10 cm × 10 cm.

[0050] 1 / 2MS solid medium: 2.23 g of 1 / 2MS basic medium powder, 15 g of sucrose and 20 g of agar were dissolved in distilled water, and then made up to 1 L with distilled water, the pH value was adjusted to 5.8, and then sterilized at 121 °C for 15 minutes. The liquid medium cooled to about 55 °C was poured into the culture dish and used after natural cooling.

[0051] Among them, the 1 / 2MS basic medium powder was purchased from Coolaber Company.

[0052] II. Influence of Bacillus velezensis FB007 inoculum on plant growth and yield increase This part of the experiment studies the effect of the secretions of Bacillus velezensis FB007 inoculant on the root growth of Arabidopsis thaliana. The specific operations are as follows: 1. Take Arabidopsis thaliana seeds, treat them with an alcohol solution with a volume ratio of 75% for 30 seconds, then wash them three times with sterile water, and then treat them with a sodium hypochlorite aqueous solution with a volume ratio of 2.6% for 6 minutes, and finally wash them three times with sterile water.

[0053] 2. After completing step 1, sow the Arabidopsis thaliana seeds on 1 / 2 MS solid medium. After vernalization at 4°C for two days, then place them at 22°C; 14 h light / 10 h dark; and cultivate them in a constant temperature incubator with a light intensity of 5000 Lx for 7 days to harvest Arabidopsis thaliana seedlings.

[0054] 3. Pick the Arabidopsis thaliana seedlings completed in step 2 and transplant them into 1 / 2 MS solid medium, with 5 seedlings placed in each medium. Divide them into a control group and a treatment group.

[0055] In the control group, 5 μL of LB liquid medium was inoculated at the lower quarter of the 1 / 2 MS solid medium corresponding to each Arabidopsis thaliana seedling; in the treatment group, 5 μL of Bacillus velezensis FB007 inoculant was inoculated at the lower quarter of the 1 / 2 MS solid medium corresponding to each Arabidopsis thaliana seedling. The treatment method is shown in Figure 1 . Among them, Bacillus velezensis FB007 inoculant is the microbial inoculant.

[0056] After the Arabidopsis thaliana seedlings were vertically cultured subsequently, the root growth of the Arabidopsis thaliana seedlings was inhibited and could not reach the position inoculated with Bacillus velezensis FB007 inoculant.

[0057] 4. Vertically culture the 1 / 2 MS solid medium completed in step 3 for 18 days, and observe the growth status of Arabidopsis thaliana seedlings.

[0058] The growth status of Arabidopsis thaliana seedlings is as shown in Figure 1 and Figure 2 .

[0059] Among them, Figure 1 Figure A in shows the growth diagram of Arabidopsis thaliana seedlings under tissue culture conditions after treatment with Bacillus velezensis FB007 inoculant, Figure 1 and Figure B in is the control group. Figure 2 Figure A in shows the growth diagram of Arabidopsis thaliana seedlings under potted conditions after treatment with Bacillus velezensis FB007 inoculant, Figure 2 and Figure B in is the control group.

[0060] Figure 1 The result of is that under the condition of inoculating Bacillus velezensis FB007 inoculant, the growth status of Arabidopsis thaliana seedlings growing on the medium is good, and the number of lateral roots increases significantly.

[0061] Figure 2 The result is as follows: Under the condition of inoculating the Bacillus velezensis FB007 bacterial agent, it can promote the growth of Arabidopsis thaliana seedlings under the condition of potting substrate.

[0062] The above results show that under the condition of inoculating the Bacillus velezensis FB007 bacterial agent, the growth of Arabidopsis thaliana seedlings grown on the culture medium is good, and the number of lateral roots increases significantly. It can be found that the Bacillus velezensis FB007 bacterial agent can also promote the growth of plants under the condition of potting substrate. Thus, Bacillus velezensis FB007 plays an important role in promoting plant growth.

[0063] Example 4: Application of Bacillus velezensis FB007 in promoting the growth and increasing the yield of peppers and lettuces I. Preparation of culture medium Specification of the culture dish: 10 cm × 10 cm.

[0064] MS solid culture medium: Dissolve 4.46 g of 1 / 2 MS basal medium powder, 20 g of sucrose and 3 g of phytagel in distilled water, then make up the volume to 1 L with distilled water, adjust the pH value to 5.8, and then sterilize at 121 °C for 15 minutes and use after cooling.

[0065] Among them, the 1 / 2 MS basal medium powder is purchased from Coolaber Company.

[0066] II. Effects of Bacillus velezensis FB007 bacterial agent on the growth and yield increase of peppers and lettuces 1. Take pepper seeds and lettuce seeds, treat them with an alcohol solution with a volume ratio of 75% for 30 seconds respectively, then wash them three times with sterilized water, then treat them with a sodium hypochlorite aqueous solution with a volume ratio of 2.6% for 6 min, and finally wash them three times with sterilized water.

[0067] 2. After completing step 1, sow the pepper seeds and lettuce seeds on the MS solid culture medium respectively. After vernalization at 4 °C for two days, place them at 22 °C respectively; 14 h light / 10 h dark; in a constant temperature incubator with a light intensity of 5000 Lx for cultivation. Until the pepper seedlings and lettuce seedlings grow two cotyledons respectively, transplant them respectively.

[0068] 3. Transplant the germinated pepper seedlings and germinated lettuce seedlings completed in step 2 into pots filled with substrate soil, one plant per pot, and obtain 20 pots of pepper seedlings and 20 pots of lettuce seedlings respectively.

[0069] Among them, the germinated pepper seedlings are the pepper seedlings after growing two cotyledons; the germinated lettuce seedlings are the lettuce seedlings after growing two cotyledons.

[0070] 4. Take 10 pots of pepper seedlings and 10 pots of lettuce seedlings respectively. Use the root irrigation method to inoculate the Bacillus velezensis FB007 bacterial agent with an OD 600nm value of 0.2. Inoculate 5 mL of the Bacillus velezensis FB007 bacterial agent for each seedling, and inoculate once every two weeks. This serves as the treatment group. Take another 10 pots of pepper seedlings and 10 pots of lettuce seedlings respectively, and inoculate 5 mL of liquid LB medium every two weeks. Inoculate a total of 4 times, and the crops serve as the control group.

[0071] 5. Observe the growth and development phenotypes of the pepper seedlings and lettuce seedlings in the treatment group and the control group, as shown in Figure 3 and Figure 4 .

[0072] Figure 3 It shows that the plants of the pepper seedlings inoculated with the Bacillus velezensis FB007 bacterial agent are significantly larger, with increased height and more leaves. Figure 4 It shows that the leaves of the lettuce seedlings inoculated with the Bacillus velezensis FB007 bacterial agent are larger and the plants are taller.

[0073] 6. Measure and statistically analyze the stem diameter, fresh weight, and dry weight of the lettuce seedlings in the treatment group and the control group respectively. The statistical data is as shown in Figure 5 .

[0074] Figure 5 The results of show that under the treatment of the Bacillus velezensis FB007 bacterial agent, compared with the control group, the stem diameter, fresh weight, and dry weight of the lettuce seedlings all increased significantly.

[0075] Thus, it can be seen that Bacillus velezensis FB007 can promote plant growth and the accumulation of dry matter content, thereby increasing the yield.

[0076] From Figure 3 and Figure 4 , it can be seen that compared with the control group, Bacillus velezensis FB007 can promote the growth of pepper and lettuce plants.

[0077] In summary, the Bacillus velezensis FB007 provided by the present invention can promote plant growth and increase the yield.

[0078] Compared with the existing plant growth-promoting rhizobacteria, the Bacillus velezensis FB007 provided by the present invention has a better effect in promoting plant growth, will not show unstable conditions, and solves the defect that the existing plant growth-promoting rhizobacteria in the prior art have unstable growth-promoting effects in complex soil environments.

[0079] It should be noted that when the present invention involves numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent repetition, preferred embodiments of the present invention are described.

[0080] Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the basic creative concept, additional changes and modifications can be made to these embodiments, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A bacterium of the genus Bacillus, characterized in that, The Bacillus bacterium is Bacillus velezensis ( Bacillus velezensis ), FB007, which was deposited at the China Center for Type Culture Collection on December 28, 2023, with the deposit number CCTCC NO: M 20232709.

2. A Bacillus bacterium according to claim 1, characterized in that, The nucleotide sequence of the 16S rRNA of the Bacillus velezensis FB007 is as shown in SEQ ID NO.

1.

3. A Bacillus bacterium according to claim 1, characterized in that, The Bacillus velezensis FB007 can promote the growth of at least one plant among pepper, lettuce and Arabidopsis thaliana.

4. A Bacillus bacterium according to claim 1, characterized in that, The Bacillus velezensis FB007 can increase the yield of at least one plant among pepper, lettuce and Arabidopsis thaliana.

5. Use of the Bacillus bacterium according to claim 1 or a culture of the Bacillus bacterium according to claim 1 in promoting plant growth and increasing plant yield.

6. The application according to claim 5, wherein The Bacillus bacterium or the culture of the Bacillus bacterium is used for preparing a microbial inoculant for promoting plant growth and increasing plant yield.

7. The application according to claim 6, wherein The microbial inoculant uses the Bacillus velezensis FB007 or the culture of the Bacillus velezensis FB007 as an active ingredient.

8. The application according to claim 7, wherein Treat the root system of a plant with the microbial inoculant, thereby promoting plant growth and increasing plant yield; The plant includes at least one of pepper, lettuce and Arabidopsis thaliana.

9. The application according to claim 7, wherein The OD of the microbial inoculum 600nm value is 0.2.

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