Bacterium Bacillus timonensis and application thereof
By screening and identifying the bacteria Bacillus timonensis 2212CXJCT3-11, this strain has the function of producing IAA, which solves the problem of insufficient research on promoting bacteria in cabbage, achieves a significant improvement in cabbage growth, provides the application of new microbial fertilizers, and promotes cabbage Growth of plants such as hearts.
Patent Information
- Application Number
- CN202411205365.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, there are few researches on the probiotics of cabbage, and agriculture tends to reduce the use of inorganic fertilizers and PGP microorganisms, and there is a lack of sustainable probiotic development.
The bacteria Bacillus timonensis 2212CXJCT3-11 was screened and identified. This strain has the function of producing IAA, which can promote the growth of cabbage hearts, including improving the bud, root, and germination rate of seedlings, and is used to develop new microbial fertilizers.
By significantly increasing the plant height, fresh ground weight, maximum leaf width, root length and root weight of cabbage hearts by using Bacillus timonensis 2212CXJCT3-11, it provides a sustainable bacterial solution suitable for the growth promotion of cabbage hearts and other plants .
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Figure CN119120272A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a bacterium Bacillus timonensis and applications thereof. Background Art
[0002] Chinese cabbage (Brassica parachinensis L.) is a plant in the genus Brassica of the family Cruciferae, which produces scapes. It is also known as Guangdong cauliflower, cauliflower, etc. The root system of Chinese cabbage is shallow, with many fibrous roots and strong regeneration ability. The stem is shortened before bolting, and the scape is round, yellow-green or green. The leaves are broadly oval or elliptical, green or yellow-green, with obvious veins. The petiole is narrow and has shallow grooves. Chinese cabbage has a racemose inflorescence, with a yellow, cross-shaped corolla. The fruit is a long silique, which is yellow-brown when mature. The seeds are nearly round, brown or dark brown.
[0003] Chinese cabbage originated in southern my country and is one of the main plants in southern China. Chinese cabbage is rich in calcium, iron, crude fiber, vitamins, carotene and other nutrients, and has the effects of preventing anemia, enhancing immunity, preventing iron deficiency anemia, lowering cholesterol, clearing away heat and detoxifying, relieving restlessness and thirst, and diuresis and laxative.
[0004] At present, research on Chinese cabbage has found that Bacillus polymyxa and Bacillus amyloliquefaciens can antagonize anthracnose; disease-resistant varieties react quickly to anthracnose infection, and their ability to self-regulate and restore to normal state and resist membrane lipid peroxidation is stronger than that of susceptible varieties; mineral nutrients such as nitrogen, potassium, and silicon can improve the disease resistance of Chinese cabbage. However, there are few reports on growth-promoting bacteria in Chinese cabbage. The current trend in agriculture is to reduce the input of inorganic fertilizers and the use of PGP microorganisms to achieve sustainability, so it is necessary to develop and research growth-promoting bacteria.
[0005] Bacillus timonensis was screened from cauliflower basic soil. Currently, there are few reports on the functions of Bacillus timonensis, and there is no report on its growth-promoting function on plants. Summary of the invention
[0006] The purpose of the present application is to overcome the shortcomings of the above-mentioned prior art and provide a bacterium Bacillustimonensis and its application. The bacterium Bacillus timonensis of the present application has the function of producing IAA, which can promote the growth of Chinese cabbage (increase the sprout length, root length and germination rate of Chinese cabbage seedlings). The bacterium Bacillustimonensis with growth-promoting function is expected to become a new type of microbial fertilizer or preparation.
[0007] The present application provides a bacterium Bacillus timonensis, wherein the bacterium Bacillus timonensis is Bacillus timonensis 2212CXJCT3-11, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with a classification name of Bacillus timonensis, a deposit number of GDMCC No: 64309, and a deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0008] The 16S rDNA sequence of Bacillus timonensis 2212CXJCT3-11 screened in the present application is shown as SEQ ID NO:1.
[0009] The Bacillus timonensis 2212CXJCT3-11 with growth-promoting function screened in the present application has the function of producing IAA, which can promote the growth of plants, specifically promote the growth of buds and roots and improve the germination rate, and further improve the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight of the plant. Therefore, the Bacillus timonensis2212CXJCT3-11 screened in the present application is expected to become a new type of microbial fertilizer or strain to promote plant growth.
[0010] The Bacillus timonensis 2212CXJCT3-11 of the present application was obtained by separation and screening from the Chinese cabbage basic soil collected from the Chinese cabbage planting base in Futian, Huizhou City, Guangdong Province.
[0011] Specifically, the screening process of Bacillus timonensis 2212CXJCT3-11 comprises the following steps:
[0012] The cauliflower soil collected from the cauliflower planting base in Futian, Huizhou City, Guangdong Province was mixed and stored in a 4°C refrigerator. A sterilized conical flask with glass beads was added with 45 ml of sterile water and 5 g of the rhizosphere soil sample. The mixture was shaken at 160 rpm for 30 min. 0.5 ml of the supernatant was gradually diluted into a test tube containing 4.5 ml of sterile water to prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 of soil suspension, take 10 -4 and 10 -5100 μl of the diluted bacterial solution was spread on a NA solid medium plate, and then placed in a constant temperature incubator for 2 days. Single colonies with different morphologies were selected and streaked onto a new NA solid medium plate for purification, and the purification was continued for more than 3 generations until a pure strain was isolated, and the Bacillustimonensis 2212CXJCT3-11 of the present application was obtained by screening.
[0013] The present application also provides a novel microbial preparation, which comprises the above-mentioned Bacillustimonensis 2212CXJCT3-11.
[0014] As a preferred embodiment of the novel microbial preparation described in the present application, the total concentration of live bacteria of Bacillus timonensis 2212CXJCT3-11 in the novel microbial preparation is 10 7 cfu / g.
[0015] In the technical solution of the present application, when Bacillus timonensis2212CXJCT3-11 in the novel microbial preparation adopts the above total concentration of live bacteria, it helps to promote plant growth, specifically by improving the bud growth and root growth of plants and increasing the germination rate, further increasing the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight of plants, which is conducive to the theoretical foundation of microorganisms promoting plant growth. This strain is more reflected in promoting the growth of Chinese cabbage.
[0016] As a preferred embodiment of the novel microbial preparation described in the present application, the preparation method of the novel microbial preparation comprises the following steps:
[0017] A single colony of the Bacillus timonensis 2212CXJCT3-11 is picked and inoculated into a liquid culture medium for cultivation to obtain a bacterial liquid of Bacillus timonensis 2212CXJCT3-11, i.e., a novel microbial preparation.
[0018] As a preferred embodiment of the novel microbial preparation described in the present application, the culture conditions are: the culture temperature is 28° C. and the culture time is 24 hours.
[0019] As a preferred embodiment of the novel microbial preparation described in the present application, the liquid culture medium used in the present application includes LB liquid culture medium, but is not limited to LB liquid culture medium. The liquid culture medium may also include liquid culture medium commonly used in the art, as long as it can achieve the effect of culturing bacteria.
[0020] The present application also provides the use of the above-mentioned bacteria Bacillus timonensis or the novel microbial preparation in promoting plant growth.
[0021] As a preferred embodiment of the application described in the present application, the bacterium Bacillus timonensis has the function of producing IAA.
[0022] As a preferred embodiment of the application described in the present application, the bacterium Bacillus timonensis increases the shoot length, root length and germination rate of plants.
[0023] The present application also provides the use of the above-mentioned bacteria Bacillus timonensis in preparing a new microbial preparation or microbial fertilizer for promoting the growth of Chinese cabbage.
[0024] The present application also provides a method for promoting the growth of flowering cabbage, the method comprising adding the above-mentioned bacteria Bacillustimonensis or the new microbial preparation.
[0025] The Chinese cabbage includes Futian Chinese cabbage. The types of Chinese cabbage are not limited to the above types, but also include common types of Chinese cabbage in the art, all of which can use Bacillus timonensis 2212CXJCT3-11 screened by the present application to promote the growth of Chinese cabbage.
[0026] In the technical scheme of the present application, compared with other soil probiotics (Microbacterium laevaniformans), the Bacillus timonensis2212CXJCT3-11 with growth-promoting function obtained by screening in the present application helps to promote the growth of Chinese cabbage, which is specifically manifested in that the plant height, aboveground fresh weight, maximum leaf width, root length and root weight of the Chinese cabbage are better, indicating that Bacillustimonensis 2212CXJCT3-11 is expected to become a new type of microbial fertilizer or preparation.
[0027] Compared with the prior art, this application has the following beneficial effects:
[0028] The present application provides a bacterium Bacillus timonensis and an application thereof. The present application screened out Bacillus timonensis 2212CXJCT3-11 with a growth-promoting function, which has the effects of high IAA production and the like, and helps to promote the growth of Chinese cabbage, specifically by promoting the growth of buds and roots and improving the germination rate, further improving the plant height, above-ground fresh weight, maximum leaf width, root length, and root weight of Chinese cabbage, and laying a theoretical foundation for microbial fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1This is the phylogenetic tree of Bacillustimonensis 2212CXJCT3-11 strain constructed based on the 16SrRNA gene sequence using the Neighbor-joining method. DETAILED DESCRIPTION
[0030] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.
[0031] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.
[0032] In the following examples, Microbacterium laevaniformans (GDAAS 02931) was derived from the National Agricultural Environment Microbial Germplasm Resource Bank (Guangdong);
[0033] NA solid medium: peptone 10.0 g, beef extract powder 3.0 g, NaCl 5.0 g, agar 18.0 g, distilled water 1000 mL, pH 7.3±0.2, sterilization at 121℃ for 15 min.
[0034] LB liquid medium: peptone 10.0 g, NaCl 10.0 g, yeast extract powder 5 g, distilled water 1000 mL, pH 7.0, sterilization at 121 °C for 30 min.
[0035] King's liquid medium: 20.0 g of tryptone, 2.0 g of dipotassium hydrogen phosphate, 1.5 g of magnesium sulfate, 0.1 g of tryptophan, 15 mL of glycerol, pH 7.0, sterilization at 121°C for 30 min.
[0036] Example 1. Screening of Bacillus timonensis 2212CXJCT3-11 strain
[0037] The cauliflower soil collected from the cauliflower planting base in Futian, Huizhou City, Guangdong Province was mixed and stored in a 4°C refrigerator. A sterilized conical flask with glass beads was added with 45 ml of sterile water and 5 g of the rhizosphere soil sample. The mixture was shaken at 160 rpm for 30 min. 0.5 ml of the supernatant was gradually diluted into a test tube containing 4.5 ml of sterile water to prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 of soil suspension, take 10 -4 and 10 -5100 μl of the diluted bacterial solution was spread on a NA solid medium plate and then placed in a constant temperature incubator for 2 days. Single colonies with different morphologies were selected and streaked on a new NA solid medium plate for purification. Purification was continued for more than 3 generations until a pure strain was isolated. The purified strains were classified and labeled, frozen in glycerol tubes, and stored in a -80°C refrigerator for later use.
[0038] The purified strains were subjected to IAA assay, and strains with better growth-promoting effects were obtained through potted plant verification.
[0039] A single colony was picked from the purified strain, inoculated into 5 mL of LB liquid culture medium, and cultured at 28° C. for 24 h to obtain a bacterial liquid of the strain.
[0040] The DNA of the strain was extracted using a bacterial DNA extraction kit (purchased from Ark Biosafety Technology Co., Ltd.), and bacterial 16S rRNA universal primers were used for PCR amplification. The universal primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3). The DNA of the strain was extracted as a template and PCR amplification was performed under the action of 2×PCR Mix (the PCR amplification program is shown in Table 1) to obtain the PCR product of the strain; the PCR amplification product of the strain was detected by 1% agarose gel electrophoresis, purified and recovered, and then subjected to first-generation sequencing to obtain the nucleic acid sequence of the PCR product.
[0041] The nucleic acid sequences obtained by sequencing were compared in the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the comparison results were identified as Bacillus timonensis.
[0042] The above strain was named Bacillus timonensis 2212CXJCT3-11, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024. The classification name is Bacillus timonensis, the collection number is GDMCCNo: 64309, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0043] The 16S rDNA sequence of Bacillus timonensis 2212CXJCT3-11 is shown in SEQ ID NO:1.
[0044] SEQ ID NO: 1:
[0045]
[0046] The phylogenetic tree of Bacillustimonensis 2212CXJCT3-11 constructed based on 16S rRNA gene sequences using the neighbor-joining method is shown in Figure 1 shown.
[0047] Table 1 PCR amplification program
[0048]
[0049] Example 2: Determination of IAA content in Bacillus timonensis 2212CXJCT3-11
[0050] The Bacillus timonensis 2212CXJCT3-11 obtained by the above screening was inoculated on a NA solid plate and cultured at 28°C for 24 hours. A single colony was picked and added to King's liquid medium containing 100 mg / L tryptophan, and King's liquid medium containing 100 mg / L tryptophan was used as a blank control. The plate was placed in the dark for 35 minutes, and the absorbance at 530 nm was measured. The IAA content was calculated using the IAA standard curve.
[0051] The results are shown in Table 2.
[0052] Table 2
[0053] Group IAA(mg / L) CK 6.18 Bacillus timonensis 2212CXJCT3-11 36.95
[0054] As can be seen from Table 2, compared with the control group (CK group), the Bacillus timonensis 2212CXJCT3-11 screened in the present application has a growth-promoting function, has functions such as high IAA yield, and can significantly promote the growth of Chinese cabbage.
[0055] Example 3: Experiment on the determination of Bacillus timonensis 2212CXJCT3-11 in the cultivation of Futian Chinese cabbage
[0056] The Bacillus timonensis 2212CXJCT3-11 obtained by the above screening was inoculated on a NA solid plate and cultured at 28°C for 24 hours. A single colony was picked and inoculated into 5ml LB and cultured for 24 hours. The obtained bacterial culture was diluted 1000 times for standby use. 5mL of the bacterial culture dilution was added to a culture dish. Each culture dish contained 10 seeds of Futian Chinese cabbage. The culture dishes containing Chinese cabbage seeds were cultured at 30°C in the dark for 3 days. The sprout length, root length and germination rate of the Chinese cabbage seeds in different treatment groups were counted. Each treatment was repeated 3 times, and the LB culture medium was diluted 1000 times as a control.
[0057] The data processing of Chinese cabbage sprout length, root length and germination rate was statistically performed using SPSS25.0 software, and the results are shown in Table 3.
[0058] Table 3
[0059]
[0060] It can be seen from Table 3 that compared with the control group (CK group), the Bacillus timonensis2212CXJCT3-11 of the present application has a growth-promoting function, which is specifically manifested in that it can promote the growth of Chinese cabbage sprouts and roots and increase the germination rate. It can significantly promote the growth of Futian Chinese cabbage and further increase the resources of Futian Chinese cabbage growth-promoting bacteria.
[0061] Example 4, potted plant experiment
[0062] The bacterial culture obtained in Example 3 and the existing control bacteria (Microbacterium laevaniformans) were diluted 1000 times for later use; the sprouted Chinese cabbage seeds obtained in the germination experiment were used for potted plant experiments.
[0063] The roots of the germinated seeds of Futian Chinese cabbage were soaked in a 1000-fold diluted bacterial culture for 15 minutes, transplanted to a planting cup, and cultured at 25-30°C with a light / dark cycle of 12h / 12h. The roots were irrigated with a 10-fold diluted bacterial culture in the second and third weeks after transplantation. The seedlings were harvested 26 days after transplantation, and the fresh weight of the aboveground part of the Chinese cabbage, plant height, maximum leaf width, root length and root weight were counted. The culture medium was used as the control group.
[0064] The data of seedling harvest were statistically analyzed using SPSS25 software, and the differences among multiple groups were compared using one-way analysis of variance, and multiple comparisons were performed using the Duncan method. The results are shown in Table 4.
[0065] Table 4
[0066]
[0067] As shown in Table 4, compared with other soil probiotics (Microbacterium laevaniformans) and the control group (CK), the Bacillus timonensis 2212CXJCT3-11 with growth-promoting function obtained by the present application can be applied to the growth of Chinese cabbage, and has a better effect of promoting the growth of Chinese cabbage, which is specifically manifested in the plant height (18.7±2.1acm), aboveground fresh weight (4.87±0.04ag), maximum leaf width (7.2±0.4acm), root length (31.15±3.85a cm), and root weight (1.64±0.28ag) of the Chinese cabbage. This shows that the Bacillus timonensis 2212CXJCT3-11 obtained by the present application has a better growth-promoting function, which is beneficial to the growth of plants such as Futian Chinese cabbage.
[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.
Claims
1. A bacterium Bacillus timonensis, characterized in that: The bacterium Bacillus timonensis is Bacillus timonensis 2212CXJCT3-11, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the deposit number GDMCC No: 64309, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A novel microbial preparation, characterized in that: The novel microbial preparation comprises Bacillus timonensis 2212CXJCT3-11 as claimed in claim 1.
3. The novel microbial preparation according to claim 2, characterized in that: The total concentration of live bacteria of Bacillustimonensis 2212CXJCT3-11 in the novel microbial preparation is 10 7 cfu / g.
4. The novel microbial preparation according to claim 2, characterized in that: The preparation method of the novel microbial preparation comprises the following steps: A single colony of the Bacillus timonensis 2212CXJCT3-11 is picked and inoculated into a liquid culture medium for cultivation to obtain a bacterial liquid of Bacillus timonensis 2212CXJCT3-11, i.e., a novel microbial preparation.
5. The novel microbial preparation according to claim 4, characterized in that: The culture conditions are as follows: the culture temperature is 28° C. and the culture time is 24 h.
6. Use of the bacterium Bacillus timonensis according to claim 1 or the novel microbial preparation according to any one of claims 2 to 5 in promoting plant growth.
7. The use according to claim 6, characterized in that The bacterium Bacillus timonensis has the function of producing IAA.
8. The use according to claim 6, characterized in that The bacterium Bacillus timonensis increases the shoot length, root length and germination rate of plants.
9. Use of the bacterium Bacillus timonensis according to claim 1 in preparing a novel microbial preparation or microbial fertilizer for promoting the growth of flowering cabbage.
10. A method for promoting the growth of Chinese cabbage, characterized in that: The method comprises adding the bacterium Bacillus timonensis according to claim 1 or the novel microbial preparation according to any one of claims 2 to 5.