Bacterium Sutcliffiella rhizosphaerae and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a bacterium Sutcliffia rhizosphaerae and an application of the bacterium Sutcliffia rhizosphaerae. The bacterium Sutcliffia rhizosphaerae is Sutcliffia rhizosphaerae 2212CXJCT4-14, is preserved in the Guangdong Microbial Culture Collection Center on January 24, 2024, and has the preservation number of GDMCC (China General Microbiological Culture Collection Center) No: 64311, and the preservation number of the bacterium Sutcliffia rhizosphaerae is CGMCC No: 64311. The Sutcliffia rhizosphaerae 2212CXJCT4-14 obtained by screening has a growth promoting function, and the strain has the functions of producing IAA (indoleacetic acid), dissolving phosphorus and the like, can remarkably promote the growth of the Futian flowering cabbage, and is expected to become a microbial fertilizer or preparation.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a bacterium Sutcliffiellarhizosphaerae and an application 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] Sutcliffiella rhizosphaerae was screened from the basal soil of Chinese flowering cabbage. Current research is mostly focused on in vitro experiments. There are few reports on the functions of Sutcliffiella rhizosphaerae, and no reports on its growth-promoting function for 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 Sutcliffiella rhizosphaerae and its application. The bacterium Sutcliffiella rhizosphaerae of the present application has the functions of producing IAA and solubilizing phosphate, and can significantly promote the growth of Chinese cabbage, which is specifically reflected in increasing the sprout length, root length and germination rate of Chinese cabbage seedlings. The bacterium Sutcliffiella rhizosphaerae with growth-promoting function is expected to become a microbial fertilizer or preparation.

[0007] The present application provides a bacterium Sutcliffiella rhizosphaerae, wherein the bacterium Sutcliffiella rhizosphaerae is Sutcliffiella rhizosphaerae 2212CXJCT4-14, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the classification name Sutcliffiella rhizosphaerae, the collection number GDMCC No: 64311, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] As a preferred embodiment of the bacterium Sutcliffiella rhizosphaerae described in the present application, the 16S rDNA sequence of Sutcliffiella rhizosphaerae 2212CXJCT4-14 is shown in SEQ ID NO: 1.

[0009] The Sutcliffiella rhizosphaerae 2212CXJCT4-14 with growth-promoting function screened out in the present application has the functions of producing IAA and solubilizing phosphate, and can significantly promote the growth of plants, specifically promoting the growth of buds and roots and improving the germination rate, and further improving the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight. Therefore, the Sutcliffiella rhizosphaerae 2212CXJCT4-14 screened out in the present application is expected to become a microbial fertilizer or strain, significantly promoting the growth of plants.

[0010] The Sutcliffiella rhizosphaerae 2212CXJCT4-14 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 Sutcliffiella rhizosphaerae 2212CXJCT4-14 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 -5 100 μl of the diluted bacterial solution was coated 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 Sutcliffiellarhizosphaerae 2212CXJCT4-14 of the present application was obtained by screening.

[0013] The present application also provides a fermentation broth of the above-mentioned bacterium Sutcliffiella rhizosphaerae.

[0014] As a preferred embodiment of the fermentation broth of the present application, the total concentration of live bacteria of Sutcliffiellarhizosphaerae 2212CXJCT4-14 in the fermentation broth is 10 7 cfu / g cfu / g.

[0015] In the technical solution of the present application, when the above total concentration of live bacteria is used in the fermentation liquid, Sutcliffiellarhizosphaerae2212CXJCT4-14, 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. The strain is more manifested in significantly promoting the growth of Chinese cabbage.

[0016] The present application also provides a method for preparing the above fermentation broth, comprising the following steps:

[0017] A single colony of the Sutcliffiella rhizosphaerae 2212CXJCT4-14 was picked and inoculated into a liquid culture medium for cultivation to obtain a fermentation broth of Sutcliffiella rhizosphaerae 2212CXJCT4-14.

[0018] As a preferred embodiment of the method for preparing the fermentation broth 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 method for preparing the fermentation broth 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 Sutcliffiella rhizosphaerae or the fermented bacterial liquid in promoting plant growth.

[0021] As a preferred embodiment of the application described in the present application, the plant includes Chinese cabbage.

[0022] 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 them can use Sutcliffiella rhizosphaerae2212CXJCT4-14 screened by this application to significantly promote the growth of Chinese cabbage.

[0023] As a preferred embodiment of the application described in the present application, promoting plant growth includes any one or two or more of the following (1) to (4):

[0024] (1) Promote the growth of buds;

[0025] (2) Promote root elongation;

[0026] (3) Improve the germination rate of plants;

[0027] (4) Increase plant height, aboveground fresh weight, leaf width, root length and root weight.

[0028] The present application also provides the use of the above-mentioned bacteria Sutcliffiella rhizosphaerae in the preparation of a microbial preparation or microbial fertilizer for promoting the growth of Chinese cabbage.

[0029] The present application also provides a method for promoting plant growth, which comprises adding the above-mentioned bacteria Sutcliffiella rhizosphaerae or the fermentation bacterial liquid.

[0030] The present application screened out a bacterial strain from Chinese cabbage soil that has the characteristics of producing IAA and solubilizing phosphate, and has a significant growth-promoting function for plants. The strain was identified as Sutcliffiella rhizosphaerae and named Sutcliffiellarhizosphaerae 2212CXJCT4-14. The strain has an IAA production capacity of up to 13.88 mg / L, and its phosphate solubilization content is quantitatively determined to be 35.88±1.88 mg / L.

[0031] In the technical scheme of the present application, compared with other soil probiotics (Bacillus zanthoxyli), the Sutcliffiella rhizosphaerae 2212CXJCT4-14 with growth-promoting function obtained by screening in the present application helps to promote the growth of Chinese cabbage (promote bud growth, root elongation and increase germination rate), which is specifically manifested in that the plant height, above-ground fresh weight, maximum leaf width, root length and root weight of the Chinese cabbage are better, indicating that Sutcliffiella rhizosphaerae2212CXJCT4-14 is expected to become a microbial fertilizer or preparation.

[0032] Compared with the prior art, this application has the following beneficial effects:

[0033] The present application provides a bacterium Sutcliffiella rhizosphaerae and an application thereof. The present application screened out Sutcliffiella rhizosphaerae 2212CXJCT4-14 with growth-promoting function, which has the effects of high IAA production and phosphorus solubilization, and is helpful to promote the growth of Chinese cabbage, specifically by significantly promoting bud growth, root elongation and improving germination rate, further improving plant height, aboveground 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

[0034] Figure 1 is a morphological diagram of the Sutcliffiella rhizosphaerae 2212CXJCT4-14 strain;

[0035] Figure 2 The electrophoresis diagram of the amplified fragment of 16s rDNA of Sutcliffiella rhizosphaerae 2212CXJCT4-14 strain;

[0036] Figure 3 This is the phylogenetic tree of Sutcliffiellarhizosphaerae 2212CXJCT4-14 strain constructed based on 16S rRNA gene sequence using the neighbor-joining method. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] In the following examples, Bacillus zanthoxyli (GDAAS 02416) was derived from the National Agricultural Environment Microbial Germplasm Resource Bank (Guangdong);

[0040] 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.

[0041] 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.

[0042] PKO inorganic phosphorus medium: glucose 10.0 g, Ca3(PO4)2 5.0 g, (NH4)2SO4 0.50 g, NaCl 0.20 g, KCl 0.20 g, MgSO4·7H20 0.30 g, MnSO4 0.03 g, FeSO4·7H20 0.03 g, yeast extract 0.50 g, distilled water 1000 mL, pH 6.8-7.0, sterilization at 121°C for 30 min.

[0043] 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.

[0044] Example 1: Screening of Sutcliffiellarhizosphaerae 2212CXJCT4-14 strain

[0045] 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 -5of soil suspension, take 10 -4 and 10 -5 100ul 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℃ refrigerator for later use.

[0046] The purified strains were subjected to IAA and phosphate solubilization value determination, and the strains with better growth-promoting effects were obtained through potted plant verification.

[0047] 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.

[0048] 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.

[0049] The sequenced nucleic acid sequence was compared in the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the alignment result was identified as Sutcliffiella rhizosphaerae.

[0050] The above strain was named Sutcliffiella rhizosphaerae 2212CXJCT4-14, and was deposited in Guangdong Provincial Microbiological Culture Collection on January 24, 2024. The classification name is Sutcliffiella rhizosphaerae, the collection number is GDMCC No: 64311, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0051] The 16S rDNA sequence of Sutcliffiella rhizosphaerae 2212CXJCT4-14 is shown in SEQ ID NO: 1. The colony morphology of Sutcliffiella rhizosphaerae 2212CXJCT4-14 is shown in Figure 1 As shown in the gel electrophoresis diagram Figure 2 shown.

[0052] SEQ ID NO: 1:

[0053]

[0054] The phylogenetic tree of Sutcliffiellarhizosphaerae 2212CXJCT4-14 constructed based on 16S rRNA gene sequences using the neighbor-joining method is shown in Figure 3 shown.

[0055] Table 1 PCR amplification program

[0056]

[0057] Example 2, determination of soluble phosphorus and IAA in Sutcliffiellarhizosphaerae2212CXJCT4-14 content

[0058] The Sutcliffiella rhizosphaerae 2212CXJCT4-14 obtained by the above screening was inoculated onto a NA solid plate and cultured at 28°C for 24 h. The selected bacteria were inoculated into 2 ml of sterile water, shaken, and the OD of the bacterial suspension was measured. 600 = 1 (control initial concentration), take 0.1 ml of bacterial suspension, inoculate into 9.9 ml PKO inorganic phosphorus medium, add 0.1 ml of sterile water to the medium as control, repeat each treatment three times, and culture on a shaker for 5 days (28°C, 150 r / min). Then centrifuge the culture solution at 4°C (4000 r / min) for 15 min, aspirate the supernatant, and determine the soluble phosphorus content by molybdenum antimony colorimetric method.

[0059] Sutcliffiella rhizosphaerae 2212CXJCT4-14 obtained by the above screening was inoculated onto 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.

[0060] The results are shown in Table 2.

[0061] Table 2

[0062] Group IAAmg / L Phosphorus solubility mg / L CK 6.18 5.01±0.14 Sutcliffiella rhizosphaerae 2212CXJCT4-14 13.88 35.88±1.88

[0063] It can be seen from Table 2 that compared with the control group (CK group), the Sutcliffiellarhizosphaerae2212CXJCT4-14 screened in this application has a growth-promoting function. This strain has the functions of producing IAA and solubilizing phosphate, and can significantly promote the growth of Chinese cabbage.

[0064] Example 3: Experiment on the determination of Sutcliffiellarhizosphaerae2212CXJCT4-14 in the culture of Futian Chinese cabbage

[0065] Sutcliffiella rhizosphaerae 2212CXJCT4-14 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 5 ml LB and cultured for 24 hours. The obtained bacterial culture was diluted 1000 times for standby use. 5 mL of the bacterial culture dilution was added to a culture dish. Each culture dish contained 10 seeds of Futian Chinese cabbage. The culture dish containing the Chinese cabbage seeds was 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.

[0066] 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.

[0067] Table 3

[0068]

[0069] It can be seen from Table 3 that compared with the control group (CK group), the Sutcliffiellarhizosphaerae2212CXJCT4-14 of the present application has a growth-promoting function, which is specifically manifested in that it can promote the growth of Chinese cabbage sprouts (sprout length is 1.58±0.24a cm), root elongation (root length is 1.22±0.23a cm) and increase the germination rate (reaching 85%), which can significantly promote the growth of Futian Chinese cabbage and further increase the resources of Futian Chinese cabbage growth-promoting bacteria.

[0070] Example 4, potted plant experiment

[0071] The bacterial culture obtained in Example 3 and the existing control strain (Bacillus zanthoxyli) were diluted 1000 times for later use; the sprouted Chinese cabbage seeds obtained in the germination experiment were used for potted plant experiments.

[0072] 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.

[0073] 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.

[0074] Table 4

[0075]

[0076] It can be seen from the data in Table 4 that compared with other soil probiotics (Bacillus zanthoxyli) and the control group (CK), the Sutcliffiella rhizosphaerae 2212CXJCT4-14 with growth-promoting function obtained by screening in this application can be applied to the growth of Chinese cabbage, and has a significant effect on promoting the growth of Chinese cabbage, which is specifically manifested in that the plant height of the Chinese cabbage is 12.10±1.10cm, the aboveground fresh weight is 4.19±0.19g, the maximum leaf width is 3.43±0.12cm, the root length is 23.90±4.10cm, and the root weight is 0.37±0.13g, which promotes the growth of Chinese cabbage better. It shows that the Sutcliffiella rhizosphaerae 2212CXJCT4-14 obtained by screening in this application has a better growth-promoting function, which is beneficial to the growth of plants such as Futian Chinese cabbage.

[0077] 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 Sutcliffiella rhizosphaerae, characterized in that The bacterium Sutcliffiellarhizosphaerae is Sutcliffiella rhizosphaerae 2212CXJCT4-14, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the deposit number GDMCC No: 64311, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The bacterium Sutcliffiella rhizosphaerae according to claim 1, characterized in that The 16S rDNA sequence of Sutcliffiella rhizosphaerae 2212CXJCT4-14 is shown in SEQ ID NO:

1.

3. The fermentation broth of the bacterium Sutcliffiella rhizosphaerae as claimed in claim 1 or 2.

4. The fermentation liquid according to claim 3, characterized in that The total concentration of live bacteria of Sutcliffiellarhizosphaerae 2212CXJCT4-14 in the fermentation broth is 10 7 cfu / g.

5. The method for preparing a fermentation liquid according to claim 3 or 4, characterized in that: The following steps are involved: A single colony of the Sutcliffiella rhizosphaerae 2212CXJCT4-14 was picked and inoculated into a liquid culture medium for cultivation to obtain a fermentation broth of Sutcliffiella rhizosphaerae 2212CXJCT4-14.

6. The method for preparing a fermentation solution according to claim 5, characterized in that: The culture conditions are as follows: the culture temperature is 28° C. and the culture time is 24 h.

7. Use of the bacterium Sutcliffiella rhizosphaerae as claimed in claim 1 or 2 or the fermented bacterial liquid as claimed in claim 3 or 4 in promoting plant growth.

8. The use according to claim 7, characterized in that Promoting plant growth includes any one or more of the following (1) to (4): (1) Promote the growth of buds; (2) Promote root elongation; (3) Improve the germination rate of plants; (4) Increase plant height, aboveground fresh weight, leaf width, root length and root weight.

9. Use of the bacterium Sutcliffiella rhizosphaerae as claimed in claim 1 or 2 in preparing a microbial preparation or microbial fertilizer for promoting the growth of flowering cabbage.

10. A method for promoting plant growth, characterized in that: The method comprises adding the bacterium Sutcliffiella rhizosphaerae according to claim 1 or 2 or the fermentation broth according to claim 3 or 4.

Images