A bacterium, Bosea spartocytisi, and its applications

Abstract

The application relates to the technical field of microorganisms, and specifically discloses a bacterium Bosea spartocytisi and application thereof. The bacterium Bosea spartocytisi is Bosea spartocytisi 2212CXJCT2-15, which has been preserved in the Guangdong Microbial Culture Collection Center on January 24, 2024, and the preservation number is GDMCC No: 64313. Bosea spartocytisi 2212CXJCT2-15 screened in the application has a growth promoting function, the strain can produce IAA and has a function of dephosphorization, and the strain can significantly promote the growth of Fokienia Hodginsii, and is expected to become a microbial fertilizer or preparation.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a bacterium, Bosea spartocytisi, and its applications. Background Technology

[0002] Chinese choy (Brassica parachinensis L.) is a plant in the Brassicaceae family, genus Brassica, whose main product is its flower stalks. It is also known as Guangdong choy, cauliflower, etc. Chinese choy has a shallow root system with many fibrous roots and strong regenerative ability. Before bolting, the stem is short and the flower stalks are round, yellowish-green or green. The leaves are broadly ovate or elliptical, green or yellowish-green, with prominent veins; the petioles are narrow and long with shallow grooves. Chinese choy has racemose inflorescences with yellow, cruciform corollas; the fruit is a silique, turning yellowish-brown when ripe; 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 the region. It is rich in calcium, iron, dietary fiber, vitamins, carotene, and other nutrients, and has various health benefits including preventing anemia, boosting immunity, preventing iron-deficiency anemia, lowering cholesterol, clearing heat and detoxifying, relieving thirst, and promoting urination and bowel movements.

[0004] Current research on Chinese cabbage has found that *Bacillus polymyxa* and *Bacillus amyloliquefaciens* can antagonize anthracnose; resistant varieties respond quickly to anthracnose infection, exhibit stronger self-regulation and recovery capabilities, and greater resistance to membrane lipid peroxidation than susceptible varieties; adequate mineral nutrition, such as nitrogen, potassium, and silicon, can enhance the disease resistance of Chinese cabbage. However, there are few reports on growth-promoting bacteria in Chinese cabbage. Current agricultural trends focus on reducing the input of inorganic fertilizers and the use of PGP microorganisms to achieve sustainability; therefore, research and development of growth-promoting bacteria is necessary.

[0005] Bosea spartocytisi was screened from the basic soil of Chinese cabbage. According to literature reports, current research mainly focuses on in vitro experiments. It is a root-nodulating endophytic bacterium, Gram-negative, non-spore-forming, aerobic, and rod-shaped. Currently, there are few reports on the functions of Bosea spartocytisi, and its growth-promoting effects on plants have not been reported. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a bacterium, Bosea spartocytisi, and its application. The bacterium Bosea spartocytisi of this application has functions such as producing IAA and solubilizing phosphorus, which can significantly promote the growth of Chinese cabbage. This growth-promoting bacterium, Bosea spartocytisi, is expected to become a microbial fertilizer or preparation.

[0007] This application provides a bacterium, *Bosea spartocytisi*, specifically *Bosea spartocytisi* 2212CXJCT2-15, which was deposited on January 24, 2024, at the Guangdong Provincial Center for Microbial Culture Collection. Its classification name is *Bosea spartocytisi*, its accession number is GDMCC No.: 64313, and its deposit address is Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0008] As a preferred embodiment of the bacterium Bosea spartocytisi described in this application, the 16S rDNA sequence of Boseaspartocytisi 2212CXJCT2-15 is shown in SEQ ID NO: 1.

[0009] As a preferred embodiment of the bacteria Bosea spartocytisi described in this application, primer pairs such as SEQ ID NO: 2-3 are used to detect Bosea spartocytisi 2212CXJCT2-15.

[0010] Using the above primer pair can better detect Bosea spartocytisi 2212CXJCT2-15.

[0011] The *Bosea spartocytisi* 2212CXJCT2-15 strain obtained in this application has growth-promoting functions such as IAA production and phosphorus solubilization, which can significantly promote plant growth, specifically by promoting bud and root growth and increasing germination rate, further increasing plant height, above-ground fresh weight, maximum leaf width, root length, and root weight. Therefore, the *Bosea spartocytisi* 2212CXJCT2-15 strain obtained in this application has the potential to become a microbial fertilizer or strain that significantly promotes plant growth.

[0012] The Bosea spartocytisi 2212CXJCT2-15 used in this application was obtained by separation and screening from basic soil samples collected from the Chinese cabbage planting base in Futian, Huizhou City, Guangdong Province.

[0013] Specifically, the screening process for the Bosea spartocytisi 2212CXJCT2-15 includes the following steps:

[0014] Basic soil samples of Chinese cabbage were collected from the Futian Chinese cabbage planting base in Huizhou City, Guangdong Province. The samples were mixed thoroughly and stored at 4℃. A sterilized conical flask containing glass beads was used; 45ml of sterile water was added, along with 5g of the collected rhizosphere soil sample. The mixture was shaken at 160rpm for 30min. 0.5ml of the supernatant was then gradually diluted into test tubes containing 4.5ml of sterile water to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 Soil suspension, take 10 -4 and 10 -5 100 μl of diluted bacterial suspension was spread onto NA solid medium plates and then incubated in a constant temperature incubator for 2 days. Single colonies of different morphologies were selected and streaked onto fresh NA solid medium plates for purification. The purification process was repeated for at least 3 generations until a pure strain was isolated. The strain was then screened to obtain the Boseaspartocytisi2212CXJCT2-15 of this application.

[0015] This application also provides a bacterial suspension of the aforementioned bacterium, *Bosea spartocytisi*.

[0016] In the technical solution of this application, the bacteria Bosea spartocytisi screened in this application has the characteristic of significantly promoting plant growth, specifically by enhancing the bud growth, root elongation and germination rate of plants, and further increasing the plant height, above-ground fresh weight, maximum leaf width, root length and root weight. This lays a theoretical foundation for the microbial promotion of plant growth, and this strain is particularly effective in significantly promoting the growth of Chinese cabbage.

[0017] This application also provides a method for preparing the above-mentioned bacterial solution, including the following steps:

[0018] Single colonies of *Bosea spartocytisi* 2212CXJCT2-15 were picked and inoculated into liquid culture medium to obtain a bacterial suspension of *Bosea spartocytisi* 2212CXJCT2-15.

[0019] In a preferred embodiment of the method for preparing the bacterial culture described in this application, the culture conditions are: a culture temperature of 28°C and a culture time of 24 hours.

[0020] As a preferred embodiment of the method for preparing bacterial culture described in this application, the liquid culture medium used in this application includes LB liquid culture medium, but is not limited to LB liquid culture medium. The liquid culture medium may also include liquid culture media commonly used in the art, as long as it can achieve the effect of culturing bacteria.

[0021] This application also provides the application of the aforementioned bacteria, Bosea spartocytisi, or the bacterial solution thereof, in promoting plant growth.

[0022] In a preferred embodiment of the application described in this application, the plant includes Chinese cabbage.

[0023] The choy sum mentioned includes Fukuda choy sum, and the types of choy sum are not limited to the above-mentioned types, but also include common types of choy sum in the field. All of them can achieve a significant growth-promoting effect on choy sum by using Bosea spartocytisi 2212CXJCT2-15 obtained by screening in this application.

[0024] As a preferred embodiment of the application described in this application, promoting plant growth includes any one or more of the following (1) to (4):

[0025] (1) Promotes bud growth;

[0026] (2) Promotes root elongation;

[0027] (3) Improve the germination rate of plants;

[0028] (4) Increase plant height, above-ground fresh weight, leaf width, root length and root weight.

[0029] This application also provides the application of the above-mentioned bacteria Bosea spartocytisi or the bacterial solution thereof in the preparation of microbial preparations or microbial fertilizers that promote the growth of Chinese cabbage.

[0030] This application also provides a method for promoting plant growth, the method comprising adding the aforementioned bacteria Boseaspartocytisi or the bacterial solution described above.

[0031] This application obtained a bacterial strain from Chinese cabbage soil that exhibits IAA production, phosphorus solubilization, and significant plant growth promotion. The strain was identified as *Bosea spartocytisi* and named *Bosea spartocytisi2212CXJCT2-15*. The strain's IAA production capacity reached 16.57 mg / L, and its phosphorus solubilization content was quantitatively determined to be 1.95 ± 0.19 mg / L.

[0032] In the technical solution of this application, compared with other soil probiotics (Bacillus zanthoxyli), the Bosea spartocytisi 2212CXJCT2-15 with growth-promoting function obtained by screening in this application helps to promote the growth of Chinese cabbage (promoting bud growth, root elongation and improving germination rate). Specifically, the plant height, above-ground fresh weight, maximum leaf width, root length and root weight of the Chinese cabbage are better, indicating that Bosea spartocytisi 2212CXJCT2-15 has the potential to become a microbial fertilizer or preparation.

[0033] Compared with the prior art, this application has the following beneficial effects:

[0034] This application provides a bacterium, Bosea spartocytisi, and its application. The Bosea spartocytisi 2212CXJCT2-15 with growth-promoting function was screened and has functions such as high IAA production and phosphorus solubilization. It can significantly promote the growth of Chinese cabbage, specifically by promoting bud growth, root elongation and increasing germination rate. It further increases the plant height, above-ground fresh weight, maximum leaf width, root length and root weight of Chinese cabbage, and further increases the resources of growth-promoting bacteria for Chinese cabbage in Futian, laying a theoretical foundation for microbial fertilizer. Attached Figure Description

[0035] Figure 1 Morphological diagram of Bosea spartocytisi 2212CXJCT2-15 strain;

[0036] Figure 2 Electrophoresis image of the 16S rDNA amplified fragment of the Bosea spartocytisi 2212CXJCT2-15 strain;

[0037] Figure 3 Phylogenetic tree of the Boseaspartocytisi 2212CXJCT2-15 strain constructed using the Neighbor-joining method based on the 16S rRNA gene sequence. Detailed Implementation

[0038] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0039] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0040] In the following examples, Bacillus zanthoxyli (GDAAS 02416) was obtained from the National Germplasm Bank of Agricultural Environmental Microorganisms (Guangdong);

[0041] NA solid medium: 10.0g peptone, 3.0g beef extract powder, 5.0g NaCl, 18.0g agar, 1000mL distilled water, pH 7.3±0.2, sterilized at 121℃ for 15min.

[0042] LB liquid medium: 10.0g peptone, 10.0g NaCl, 5g yeast extract, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 30min.

[0043] PKO Inorganic Phosphorus Medium: Glucose 10.0g, Ca3(PO4)2 5.0g, (NH4)2SO4 0.50g, NaCl 0.20g, KCl 0.20g, MgSO4·7H2O 0.30g, MnSO4 0.03g, FeSO4·7H2O 0.03g, Yeast Extract 0.50g, Distilled Water 1000mL, pH 6.8-7.0, Sterilize at 121℃ for 30min.

[0044] King's liquid medium: 20.0g tryptone, 2.0g dipotassium hydrogen phosphate, 1.5g magnesium sulfate, 0.1g tryptophan, 15mL glycerol, pH 7.0, sterilized at 121℃ for 30min.

[0045] Example 1: Screening of the Boseaspartocytisi 2212CXJCT2-15 strain

[0046] Basic soil samples of Chinese cabbage were collected from the Futian Chinese cabbage planting base in Huizhou City, Guangdong Province. The samples were mixed thoroughly and stored at 4℃. A sterilized conical flask containing glass beads was used; 45ml of sterile water was added, along with 5g of the collected rhizosphere soil sample. The mixture was shaken at 160rpm for 30min. 0.5ml of the supernatant was then gradually diluted into test tubes containing 4.5ml of sterile water to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 Soil suspension, take 10 -4 and 10 -5 100 μL of diluted bacterial suspension was spread onto NA solid medium plates and incubated for 2 days. Single colonies of different morphologies were selected and streaked onto fresh NA solid medium plates for purification. This purification process was repeated for at least 3 generations until a pure strain was isolated. The purified strain was then classified, labeled, and cryopreserved in glycerol tubes at -80°C for later use.

[0047] The purified strain was subjected to IAA and phosphate solubility tests, and the strain with good growth-promoting effect was obtained through pot experiments.

[0048] Single colonies were picked from the purified strain and inoculated into 5 mL of LB liquid medium. The culture was then incubated at 28°C for 24 h to obtain the bacterial culture of the strain.

[0049] DNA was extracted from the bacterial strain using a bacterial DNA extraction kit (purchased from Ark Biosafety Technology Co., Ltd.). PCR amplification was performed using universal primers for bacterial 16S rRNA. The universal primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:3). Using the extracted DNA as a template, PCR amplification was performed using 2×PCR Mix (PCR amplification program shown in Table 1). The PCR products of the strain were obtained. The PCR amplification products were detected by 1% agarose gel electrophoresis, purified, and recovered. Then, first-generation sequencing was performed to obtain the nucleic acid sequences of the PCR products.

[0050] The nucleic acid sequence obtained from sequencing was compared with the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the comparison result identified it as Bosea spartocytisi.

[0051] The strain was named *Bosea spartocytisi* 2212CXJCT2-15 and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 24, 2024. Its classification name is *Bosea spartocytisi*, its accession number is GDMCC No.: 64313, and its deposit address is Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0052] The colony morphology of Bosea spartocytisi 2212CXJCT2-15 is as follows: Figure 1 As shown.

[0053] The 16S rDNA sequence of Bosea spartocytisi 2212CXJCT2-15 is shown in SEQ ID NO:1. Its gel electrophoresis image is shown below. Figure 2 As shown.

[0054] SEQ ID NO:1:

[0055]

[0056] The phylogenetic tree of Boseaspartocytisi2212CXJCT2-15 constructed using the Neighbor-joining method based on the 16S rRNA gene sequence is as follows: Figure 3 As shown.

[0057] Table 1 PCR amplification program

[0058]

[0059] Example 2: Determination of soluble phosphorus and IAA content in Bosea spartocytisi 2212CXJCT2-15

[0060] The selected *Bosea spartocytisi* 2212CXJCT2-15 strain was inoculated onto NA agar plates and incubated at 28°C for 24 hours. A small amount of the bacterial suspension was picked and inoculated into 2 ml of sterile water, shaken well, and the OD value of the bacterial suspension was measured. 600 =1 (controlling the initial concentration), take 0.1 ml of bacterial suspension and inoculate it into 9.9 ml of PKO inorganic phosphorus medium. Use 0.1 ml of sterile water added to the medium as a control. Each treatment is repeated three times and cultured on a shaker for 5 days (28℃, 150 r / min). Then, centrifuge the culture medium at 4℃ (4000 r / min) for 15 min, collect the supernatant, and determine the soluble phosphorus content using the molybdenum antimony colorimetric method.

[0061] The selected *Bosea spartocytisi* 2212CXJCT2-15 strain was inoculated onto NA solid plates and incubated at 28°C for 24 h. Single colonies were picked and transferred to King's liquid medium containing 100 mg / L tryptophan, with King's liquid medium containing 100 mg / L tryptophan serving as a blank control. The plates were incubated in the dark for 35 min, and the absorbance at 530 nm was measured. The IAA content was calculated using the IAA standard curve.

[0062] The results are shown in Table 2.

[0063] Table 2

[0064] Group IAA mg / L Phosphate solubility value (mg / L) CK 6.18 5.01±0.14 Bosea spartocytisi 2212CXJCT2-15 16.57 19.5±0.19

[0065] As shown in Table 2, compared with the control group (CK group), the Bosea spartocytisi2212CXJCT2-15 strain screened in this application has growth-promoting function. This strain has functions such as IAA production and phosphorus solubilization, which can significantly promote the growth of Chinese cabbage.

[0066] Example 3: Determination of the culture of Fukuda Chinese cabbage using Boseaspartocytisi2212CXJCT2-15

[0067] The *Bosea spartocytisi* 2212CXJCT2-15 strain obtained from the above screening was inoculated onto NA solid plates and cultured at 28°C for 24 h. A single colony was picked and inoculated into 5 ml of LB medium, and cultured for 24 h. The resulting bacterial culture was diluted 1000 times for later use. 5 mL of the diluted bacterial culture was added to each petri dish, containing 10 *Chenopodium album* seeds. The petri dishes containing the seeds were cultured at 30°C in the dark for 3 days. The shoot length, root length, and germination rate of the *Chenopodium album* seeds in different treatment groups were recorded. Each treatment was repeated three times, with a 1000-fold dilution of LB medium as a control.

[0068] Data on sprout length, root length, and germination rate of Chinese cabbage were processed and statistically analyzed using SPSS 25.0 software. The results are shown in Table 3.

[0069] Table 3

[0070]

[0071] As shown in Table 3, compared with the control group (CK group), the Bosea spartocytisi2212CXJCT2-15 of this application has growth-promoting function, specifically, it can promote the growth of Chinese cabbage buds (bud length is 1.51±0.26cm), root elongation (root length is 1.00±0.18cm), and increase the germination rate (reaching 80%). It can significantly promote the growth of Chinese cabbage and further increase the resources of growth-promoting bacteria for Chinese cabbage.

[0072] Example 4: Pot Experiment

[0073] The bacterial culture obtained in Example 3 and the existing control strain (Bacillus zanthoxyli) were diluted 1000 times for later use; the germinated Chinese cabbage seeds obtained from the germination experiment were used for pot experiments.

[0074] The roots of germinated *Brassica rapa* seeds were soaked in a 1000-fold diluted bacterial culture for 15 minutes, then transplanted into planting cups and cultured at 25-30℃ under 12h / 12h light / dark conditions. In the second and third weeks after transplanting, the roots were drenched with a 10-fold diluted bacterial culture. Seedlings were harvested 26 days after transplanting, and the fresh weight of the above-ground parts, plant height, maximum leaf width, root length, and root weight were recorded. The culture medium served as a control group.

[0075] The seedling harvest data were statistically analyzed using SPSS 25 software. One-way ANOVA was used to compare differences among multiple groups, and Duncan's method was used for multiple comparisons. The results are shown in Table 4.

[0076] Table 4

[0077]

[0078] As shown in Table 4, compared with other soil probiotics (Bacillus zanthoxyli) and the control group (CK), the plant growth-promoting Bosea spartocytisi 2212CXJCT2-15 screened in this application can be applied to the growth of Futian cabbage, showing a significant effect on promoting its growth. Specifically, the plant height was 15.90±0.40cm, the above-ground fresh weight was 4.68±0.35g, the maximum leaf width was 5.15±0.35cm, the root length was 26.10±0.10cm, and the root weight was 0.84±0.04g, indicating a better effect on promoting cabbage growth. This demonstrates that the Bosea spartocytisi 2212CXJCT2-15 screened in this application has a better growth-promoting function, which is beneficial to the growth of plants such as Futian cabbage.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A type of bacteria Bosea spartocytisi Its characteristics are, The bacteria Bosea spartocytisi for Bosea spartocytisi 2212CXJCT2-15 was deposited on January 24, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 64313), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The bacteria as described in claim 1 Bosea spartocytisi Its characteristics are, The Bosea spartocytisi The 16S rDNA sequence of 2212CXJCT2-15 is shown in SEQ ID NO:

1.

3. The bacteria as described in claim 1 Bosea spartocytisi Its characteristics are, Detection was performed using primer pairs such as SEQ ID NO: 2-3. Bosea spartocytisi 2212CXJCT2-15.

4. The bacteria as described in any one of claims 1 to 3 Bosea spartocytisi The bacterial solution.

5. The method for preparing bacterial solution as described in claim 4, characterized in that, Includes the following steps: Pick the Bosea spartocytisi A single colony of 2212CXJCT2-15 was obtained and inoculated into liquid culture medium for culture, yielding... Bosea spartocytisi Bacterial solution of 2212CXJCT2-15.

6. The bacteria as described in any one of claims 1 to 3 Bosea spartocytisi Alternatively, the application of the bacterial solution as described in claim 4 in promoting plant growth, wherein the plant is Chinese cabbage.

7. The application as described in claim 6, characterized in that, Promoting plant growth includes any one or more of the following (1) to (4): (1) Promotes bud growth; (2) Promotes root elongation; (3) Improve the germination rate of plants; (4) Increase plant height, above-ground fresh weight, leaf width, root length and root weight.

8. The bacteria as described in any one of claims 1 to 3 Bosea spartocytisi Alternatively, the bacterial solution as described in claim 4 can be used in the preparation of microbial preparations or microbial fertilizers that promote the growth of Chinese cabbage.

9. A method for promoting plant growth, characterized in that, The method includes adding bacteria as described in any one of claims 1 to 3. Bosea spartocytisi Or the bacterial solution as described in claim 4; the plant is Chinese cabbage.

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