Rhizosphere bacterium Solitalea sp. And application thereof
The microbial agent of the rhizosphere bacteria Solitalea sp.2212CXGJT6-5 obtained through screening, using its IAA-producing phosphorus and relieving function, solved the problem of insufficient research on promoting bacteria in cabbage, significantly improved the growth performance of cabbage, and provided more Excellent growth-promoting effect.
Patent Information
- Application Number
- CN202411205373.2
- 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 probiotics of cabbage, and agriculture tends to reduce the use of inorganic fertilizers and PGP microorganisms, resulting in an urgent need for the development of sustainable probiotics.
The screened rhizosphere bacteria Solitalea sp.2212CXGJT6-5 has the function of producing IAA and phosphorus soluble. It promotes the growth of cabbage heart through its microbial agent, improves bud, root growth and germination rate, and enhances plant height, fresh weight on the ground, and maximum The leaves are wide and the roots are long.
It significantly promotes the growth of cabbage heart, provides new microbial fertilizers, enhances the growth performance of cabbage heart, and has better effect than other soil probiotics.
Smart Images
Figure CN119120274A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a rhizosphere bacterium Solidea sp. 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 flower stalks. It is also known as Guangdong Chinese cabbage, Chinese cabbage, cauliflower, etc. Chinese cabbage originated in southern my country and is one of the main plants in South China. Current research on Chinese cabbage has found that Bacillus polymyxa and Bacillus amyloliquefaciens can antagonize anthracnose; disease-resistant varieties react quickly to anthracnose infection, and have stronger self-regulation and ability to restore normal state and resist membrane lipid peroxidation than 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 study growth-promoting bacteria.
[0003] Solitalea sp. was screened from the rhizosphere soil of Chinese cabbage. Currently, most studies focus on in vitro experiments, and it was found that it has certain biological control functions. However, there are few reports on its growth-promoting function in plants. Summary of the invention
[0004] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide a rhizospheric bacterium Solidea sp. and its application. The rhizospheric bacterium Solidea sp. of the present application has the functions of producing IAA and solubilizing phosphate, which helps to promote the growth of Chinese cabbage.
[0005] The present application provides a rhizospheric bacterium Solidalea sp., wherein the rhizospheric bacterium Solidalea sp. is Solidalea sp.2212CXGJT6-5, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the classification name Solidalea sp., the collection number is GDMCC No: 64312, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0006] The colony morphology of Solidea sp. 2212CXGJT6-5 of the present application is round, raised, milky white and opaque, with a smooth surface, and is a Gram-negative bacterium.
[0007] The 16S rDNA sequence of Solitalea sp.2212CXGJT6-5 is shown in SEQ ID NO:1.
[0008] The Solidea sp.2212CXGJT6-5 with growth-promoting function screened out in this application has the functions of producing IAA and solubilizing phosphorus, which can promote the growth of plants, specifically promote the growth of buds and roots and improve the germination rate, and further increase the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight. Therefore, the Solidea sp.2212CXGJT6-5 screened out in this application is expected to become a new type of microbial fertilizer or strain to promote plant growth.
[0009] The screening process of Solidea sp.2212CXGJT6-5 comprises the following steps:
[0010] 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 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. The purification was continued for more than 3 generations until a pure strain was isolated, and the Solidale asp.2212CXGJT6-5 of the present application was obtained by screening.
[0011] The present application provides a microbial agent, which includes the above-mentioned rhizospheric bacteria Solidale asp.
[0012] As a preferred embodiment of the microbial agent of the present application, the total concentration of live bacteria of the rhizosphere bacteria Solidalea sp. in the microbial agent is 10 7 cfu / g.
[0013] The rhizospheric bacteria Solidalea sp. screened in the present application have a growth-promoting effect. The use of the above total concentration of live bacteria of the rhizospheric bacteria Solidalea sp. in the microbial agent can better promote plant growth, enhance plant bud growth, root growth and germination rate, and further increase plant height, aboveground fresh weight, maximum leaf width, root length and root weight.
[0014] As a preferred embodiment of the microbial agent described in the present application, the preparation method of the microbial agent comprises the following steps:
[0015] A single colony of the Solidea sp. 2212CXGJT6-5 was picked and inoculated into LB liquid culture medium, and cultured at 28° C. for 24 hours to obtain a bacterial solution of Solidea sp. 2212CXGJT6-5, i.e., a microbial agent.
[0016] The present application also provides a reagent for detecting the above-mentioned rhizospheric bacteria Solidea sp., wherein the reagent comprises primers whose sequences are shown in SEQ ID NOs: 2-3.
[0017] The present application also provides the use of the above-mentioned rhizospheric bacteria Solidea sp. or the above-mentioned microbial agent in promoting plant growth.
[0018] The present application also provides the use of the above-mentioned rhizospheric bacteria Solidea sp. in the preparation of a microbial agent or microbial fertilizer for promoting plant growth.
[0019] As a preferred embodiment of the application described in the present application, the plant includes Chinese cabbage.
[0020] The present application also provides a method for promoting plant growth, which comprises adding the above-mentioned rhizospheric bacteria Solidea sp. or the above-mentioned microbial agent.
[0021] As a preferred embodiment of the method for promoting plant growth described in the present application, the plant includes Chinese flowering cabbage.
[0022] In the technical scheme of the present application, compared with other soil probiotics (Bacillus zanthoxyli), the present application adopts the screened Solidea sp.2212CXGJT6-5 with growth-promoting function and applies it to the growth of Chinese cabbage, which has a better effect of promoting the growth of Chinese cabbage, and the plant height, aboveground fresh weight, maximum leaf width, root length and root weight of Chinese cabbage are better.
[0023] Compared with the prior art, this application has the following beneficial effects:
[0024] The present application provides a rhizospheric bacterium Solidea sp. and its application. The rhizospheric bacterium Solidea sp. screened in the present application has the effects of high IAA production and phosphorus solubilization, can promote the growth of Chinese cabbage, can promote the growth of buds and roots and improve the germination rate, further improve the plant height, above-ground fresh weight, maximum leaf width, root length and root weight of Chinese cabbage, and lay a theoretical foundation for microbial fertilizer. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the colony morphology of Solidea sp.2212CXGJT6-5;
[0026] Figure 2 It is the gel electrophoresis image of the 16S rDNA amplified fragment of Solidea sp.2212CXGJT6-5;
[0027] Figure 3 This is the phylogenetic tree of Solidale asp. 2212CXGJT6-5 strain constructed based on the 16S rRNA gene sequence using the neighbor-joining method. DETAILED DESCRIPTION
[0028] 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.
[0029] 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.
[0030] In the following examples, Bacillus zanthoxyli (GDAAS 02416) was derived from the National Agricultural Environment Microbial Germplasm Resource Bank (Guangdong);
[0031] 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.
[0032] 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.
[0033] 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.
[0034] Example 1: Screening of Solidea sp. 2212CXGJT6-5 strain
[0035] 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 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.
[0036] The purified Chinese cabbage rhizosphere bacteria were used to measure IAA and phosphorus solubilization values, and the rhizosphere bacteria with better growth-promoting effect were obtained through potted plant verification.
[0037] A single colony was picked from the purified rhizosphere bacteria and inoculated into 5 mL of LB liquid culture medium, and cultured at 28° C. for 24 h to obtain a bacterial solution of the rhizosphere bacteria.
[0038] The DNA of rhizosphere bacteria was extracted by a bacterial DNA extraction kit (purchased from Ark Biosafety Technology Co., Ltd.), and PCR amplification was performed using bacterial 16S rRNA universal primers. The universal primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3). The DNA of rhizosphere bacteria extracted was used 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 rhizosphere bacteria; the PCR amplification product of rhizosphere bacteria was detected by 1% agarose gel electrophoresis, purified and recovered, and then sequenced by the first generation to obtain the nucleic acid sequence of the PCR product. The nucleic acid sequence obtained by sequencing was compared in the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the comparison result was identified as Solidea sp.
[0039] The above strain was named Solidalea sp.2212CXGJT6-5, and the strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024. The classification name is Solidalea sp., the collection number is GDMCC No: 64308, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0040] The colony morphology of Solidalea sp.2212CXGJT6-5 in the present application is round, raised, milky white and opaque, with a smooth surface, and is a Gram-negative bacterium. Figure 1 shown.
[0041] The 16S rDNA sequence of Solitalea sp.2212CXGJT6-5 is shown in SEQ ID NO: 1. The gel electrophoresis diagram is shown in Figure 2 shown.
[0042] SEQ ID NO: 1:
[0043]
[0044] The phylogenetic tree of Solidale asp.2212CXGJT6-5 constructed based on 16S rRNA gene sequence using the neighbor-joining method is shown in Figure 3 shown.
[0045] Table 1 PCR amplification program
[0046]
[0047] Example 2: Determination of Soluble Phosphorus and IAA Content of Solidea sp. 2212CXGJT6-5
[0048] The Solidalea sp. 2212CXGJT6-5 obtained from 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 well, and the OD of the bacterial suspension was measured. 600 =1 (control initial concentration), take 0.1ml of bacterial suspension, inoculate into 9.9ml PKO inorganic phosphorus medium, add 0.1ml sterile water to the medium as control (CK), repeat three times for each treatment, and culture on a shaker for 5 days (28°C, 150r / min). Then centrifuge the culture solution at 4°C (4000r / min) for 15min, aspirate the supernatant, and determine the soluble phosphorus content by molybdenum antimony colorimetric method.
[0049] The Solidea sp. 2212CXGJT6-5 obtained by the above screening was inoculated into NA solid culture medium and cultured at 28°C for 24 h. A single colony was picked and added to King's liquid culture medium containing 100 mg / L tryptophan. King's liquid culture medium containing 100 mg / L tryptophan was used as a blank control (CK). The culture was allowed to stand in the dark for 35 min, the absorbance at 530 nm was measured, and the IAA content was calculated using the IAA standard curve.
[0050] The results are shown in Table 2.
[0051] Table 2
[0052] Group IAA(mg / L) Phosphorus solubility value (mg / L) CK 6.18 5.01±0.14 Solitalea sp.2212CXGJT6-5 12.76 48.74±2.34
[0053] As can be seen from Table 2, the Solidea sp. 2212CXGJT6-5 of the present application has the function of promoting growth, high IAA production, phosphorus solubilization and the like, and can significantly promote the growth of Chinese cabbage.
[0054] Example 3: Experiment on the determination of Solidale asp. 2212CXGJT6-5 in the cultivation of Chinese cabbage
[0055] The Solidalea sp.2212CXGJT6-5 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.
[0056] 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.
[0057] Table 3
[0058]
[0059] Note: Different lowercase letters represent differences in physiological parameters among different treatments (P<0.05).
[0060] As can be seen from Table 3, the Solidea sp. 2212CXGJT6-5 of the present application can promote the growth of buds and roots of Chinese cabbage and increase the germination rate, 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 (Bacillus zanthoxyli) were diluted 10 times and 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] Note: Different lowercase letters represent differences in physiological parameters among different treatments (P<0.05).
[0068] As shown in Table 4, compared with other soil probiotics (Bacillus zanthoxyli) and the control group (CK), the Solidalea sp.2212CXGJT6-5 with growth-promoting function screened out in this application is applied to the growth of Chinese cabbage, which has a better effect of promoting the growth of Chinese cabbage, and the plant height, aboveground fresh weight, maximum leaf width, root length and root weight of Chinese cabbage are better. This shows that the Solidalea sp.2212CXGJT6-5 screened out in this application has a better growth-promoting function, which is beneficial to the growth of plants such as Chinese cabbage.
[0069] 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 rhizobacterium Solidea sp., characterized in that The rhizospheric bacteria Solidea sp. is Solidea sp.2212CXGJT6-5, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 24, 2024, with the collection number GDMCC No: 64312, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A microbial agent, characterized in that: The microbial agent includes the rhizospheric bacteria Solidea sp. as claimed in claim 1.
3. The microbial agent according to claim 2, characterized in that The total concentration of live bacteria of the rhizosphere bacteria Solidalea sp. in the microbial agent is 10 7 cfu / g.
4. The microbial agent according to claim 2, characterized in that The preparation method of the microbial agent comprises the following steps: A single colony of the Solidea sp. 2212CXGJT6-5 was picked and inoculated into LB liquid culture medium, and cultured at 28° C.-30° C. for 24 h-48 h to obtain a bacterial solution of Solidea sp. 2212CXGJT6-5, i.e., a microbial agent.
5. A reagent for detecting the rhizobacterium Solidea sp. according to claim 1, characterized in that: The reagents include primers whose sequences are shown in SEQ ID NOs: 2-3.
6. Use of the rhizospheric bacteria Solidea sp. according to claim 1 or the microbial agent according to any one of claims 2 to 4 in promoting plant growth.
7. Use of the rhizospheric bacteria Solidea sp. according to claim 1 in the preparation of a microbial agent or microbial fertilizer for promoting plant growth.
8. The use according to claim 7, characterized in that The plants include Chinese cabbage.
9. A method for promoting plant growth, characterized in that: The method comprises adding the rhizospheric bacteria Solidea sp. according to claim 1 or the microbial agent according to claim 2.
10. The method for promoting plant growth according to claim 9, characterized in that: The plants include Chinese cabbage.