Bacterium Rhodobacter lindaniclasticus and application thereof
By screening and applying bacteria with IAA-producing phosphorus relieving functions, Rhodanobacter lindaniclasticus 2212CXJCT1-12, the problem of insufficient research on promoting bacteria in cabbage is solved, and a significant improvement in the growth of cabbage is achieved. It provides a new microbial fertilizer, which is better than other soil probiotics. The effect of bacteria.
Patent Information
- Application Number
- CN202411205224.6
- 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
There are few researches on the proliferation of cabbage in the prior art. Rhodanobacter lindaniclasticus is mainly focused on pesticide degradation, lacking reports on the proliferation function of plants. In addition, agriculture tends to reduce the use of inorganic fertilizers, which is of great significance to promote the development of microbial agents. .
The bacteria Rhodanobacter lindaniclasticus 2212CXJCT1-12 has the function of producing IAA and phosphorus soluble. By preparing microbial preparations, it promotes the growth of vegetable hearts, which specifically includes screening, purification, culture and application in vegetable heart base soil to improve buds. , root growth and germination rate, further improving the plant height, fresh weight on the ground, maximum leaf width and root length of the vegetable heart.
Rhodanobacter lindaniclasticus 2212CXJCT1-12 significantly promotes the growth of vegetable hearts, improves the plant height, fresh weight on the ground, maximum leaf width, root length and root weight, and provides the theoretical basis of new microbial fertilizers, which is better than other soil probiotics. Effect.
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Figure CN119120269A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of microbial technology, and in particular to a bacterium Rhodanobacterlindaniclasticus and an application thereof. Background Art
[0002] Chinese cabbage (Brassica parachinensis L.) is a vegetable in the genus Brassica of the family Cruciferae, with flower stalks as its product. 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 vegetables in South China. Current research on Chinese cabbage has found that Bacillus polymyxa and Bacillus amyloliquefaciens can antagonize anthrax; 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 promote the use of microbial agents with growth-promoting functions to achieve sustainability. Therefore, it is necessary to develop and research growth-promoting bacteria.
[0003] Rhodanobacter lindaniclasticus was screened from the soil where Chinese cabbage was grown. Current research is mostly focused on in vitro experiments, and its research mainly focuses on pesticide degradation, but there are few reports on its plant growth-promoting function. 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 bacterium Rhodanobacterlindaniclasticus and its application. The bacterium Rhodanobacterlindaniclasticus of the present application has the functions of producing IAA and solubilizing phosphate, which helps to promote the growth of Chinese cabbage and lays a theoretical foundation for microbial fertilizer.
[0005] To achieve the above purpose, the technical solution adopted by this application is:
[0006] The present application provides a bacterium Rhodanobacter lindaniclasticus, wherein the bacterium Rhodanobacter lindaniclasticus is Rhodanobacter lindaniclasticus 2212CXJCT1-12, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with a classification name of Rhodanobacterlindaniclasticus, a collection number of GDMCC No: 64308, and a collection address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0007] The colony morphology of Rhodanobacter lindaniclasticus 2212CXJCT1-12 of the present application is round, raised, light yellow and opaque, with a smooth surface, and is a Gram-negative bacterium.
[0008] The 16S rDNA sequence of Rhodanobacter lindaniclasticus 2212CXJCT1-12 is shown in SEQ ID NO:1.
[0009] The Rhodanobacter lindaniclasticus2212CXJCT1-12 with growth-promoting function screened out in the present application has the functions of producing IAA and solubilizing phosphate, and can promote the growth of vegetables, 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 of vegetables. Therefore, the Rhodanobacter lindaniclasticus2212CXJCT1-12 screened out in the present application is expected to become a new type of microbial fertilizer or preparation to promote the growth of vegetables.
[0010] The screening process of Rhodanobacter lindaniclasticus 2212CXJCT1-12 comprises the following steps:
[0011] 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, picked and streaked onto a new NA solid medium plate for purification, and purified continuously for more than 3 generations until a pure strain was isolated, and the Rhodanobacterlindaniclasticus 2212CXJCT1-12 of the present application was screened.
[0012] The present application also provides a microbial preparation, wherein the bacterium Rhodanobacterlindaniclasticus is described in the microbial preparation.
[0013] Preferably, the method for preparing the microbial preparation comprises the following steps:
[0014] A single colony of the Rhodanobacter lindaniclasticus 2212CXJCT1-12 was picked and inoculated into LB liquid culture medium, and cultured at 28° C. for 24 hours to obtain a 2212CXJCT1-12 bacterial liquid, i.e., a microbial preparation.
[0015] As a preferred embodiment of the microbial preparation of the present application, the total concentration of live bacteria of Rhodanobacter lindaniclasticus in the microbial preparation is 10 7 cfu / g.
[0016] The use of the above total live bacteria concentration of Rhodanobacter lindaniclasticus in microbial preparations can better promote the growth of vegetables, enhance the sprout growth and root growth of vegetables, and increase the germination rate, and further increase the plant height, aboveground fresh weight, maximum leaf width, root length, and root weight of vegetables.
[0017] As a preferred embodiment of the microbial preparation described in the present application, the microbial preparation further includes an auxiliary material, and the auxiliary material includes vermiculite.
[0018] The present application also provides primers for detecting the bacterium Rhodanobacter lindaniclasticus, and the sequences of the primers are shown in SEQ ID NOs: 2-3.
[0019] The present application also provides the use of the above-mentioned bacterium Rhodanobacter lindaniclasticus in the preparation of a microbial preparation or microbial fertilizer for promoting vegetable growth.
[0020] As a preferred embodiment of the application described in the present application, the vegetable includes Chinese cabbage heart.
[0021] As a preferred embodiment of the application described in the present application, the total concentration of live bacteria of Rhodanobacter lindaniclasticus in the microbial preparation or microbial fertilizer is 10 7 cfu / g.
[0022] The present application also provides a method for promoting vegetable growth, which comprises adding the bacterium Rhodanobacter lindaniclasticus or the microbial preparation.
[0023] As a preferred embodiment of the method for promoting vegetable growth described in the present application, the vegetables include Chinese cabbage hearts.
[0024] In the technical scheme of the present application, compared with other soil probiotics (Bacillus zanthoxyli), the present application adopts the screened Rhodanobacter lindaniclasticus 2212CXJCT1-12 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 the Chinese cabbage are better.
[0025] Compared with the prior art, this application has the following beneficial effects:
[0026] The present application provides a bacterium Rhodanobacter lindaniclasticus and an application thereof. The bacterium Rhodanobacter lindaniclasticus of the present application has the functions of producing IAA and solubilizing phosphate, and can promote the growth of Chinese cabbage, specifically promote the growth of buds and roots and improve the germination rate, further improve the plant height, aboveground 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
[0027] Figure 1 This is the colony morphology of Rhodanobacter lindaniclasticus;
[0028] Figure 2 The gel electrophoresis diagram of the 16S rDNA amplified fragment of Rhodanobacter lindaniclasticus;
[0029] Figure 3 This is the phylogenetic tree of Rhodanobacterlindaniclasticus 2212CXJCT1-12 strain constructed based on the 16S rRNA 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, Bacillus zanthoxyli (GDAAS 02416) 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] 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.
[0036] Example 1: Screening of Rhodanobacter lindaniclasticus 2212CXJCT1-12 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 -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.
[0038] The purified Chinese cabbage rhizosphere bacteria were subjected to IAA and phosphorus solubility test, and the Chinese cabbage rhizosphere bacteria 2212CXJCT1-12 with better growth-promoting effect was obtained through potted plant verification.
[0039] A single colony was picked from the purified Chinese cabbage rhizospheric bacteria 2212CXJCT1-12 and inoculated into 5 mL of LB liquid culture medium, and cultured at 28° C. for 24 h to obtain a 2212CXJCT1-12 bacterial solution.
[0040] The DNA of the rhizosphere bacteria 2212CXJCT1-12 of Chinese cabbage was extracted using a bacterial DNA extraction kit (purchased from Ark Biosafety Technology Co., Ltd.), and PCR amplification was performed using bacterial 16S rRNA universal primers. The DNA of the rhizosphere bacteria 2212CXJCT1-12 of Chinese cabbage extracted with universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3) 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 2212CXJCT1-12; the PCR amplification product of the rhizosphere bacteria 2212CXJCT1-12 of Chinese cabbage 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. The sequenced nucleic acid sequence was compared in the EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the alignment result was identified as Rhodanobacter lindaniclasticus.
[0041] The above strain was named Rhodanobacter lindaniclasticus 2212CXJCT1-12, and the strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024. The classification name is Rhodanobacterlindaniclasticus, the collection number is GDMCC No: 64308, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0042] The colony morphology of Rhodanobacter lindaniclasticus 2212CXJCT1-12 is round, raised, opaque yellow, and smooth. It is a Gram-negative bacterium. Figure 1 shown.
[0043] The 16S rDNA sequence of Rhodanobacter lindaniclasticus 2212CXJCT1-12 is shown in SEQ ID NO: 1. The gel electrophoresis diagram thereof is shown in Figure 2 shown.
[0044] SEQ ID NO: 1:
[0045]
[0046] The phylogenetic tree of Rhodanobacterlindaniclasticus 2212CXJCT1-12 constructed based on 16S rRNA gene sequences using the neighbor-joining method is shown in Figure 3 shown.
[0047] Table 1 PCR amplification program
[0048]
[0049] Example 2: Determination of soluble phosphorus content of Rhodanobacter lindaniclasticus 2212CXJCT1-12 Amount and IAA content
[0050] The Rhodanobacter lindaniclasticus 2212CXJCT1-12 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 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.
[0051] The Rhodanobacter lindaniclasticus 2212CXJCT1-12 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.
[0052] The results are shown in Table 2.
[0053] Table 2
[0054]
[0055] As can be seen from Table 2, the Rhodanobacter lindaniclasticus 2212CXJCT1-12 of the present application has the functions of producing IAA and solubilizing phosphate, and can promote the growth of Chinese cabbage.
[0056] Example 3: Determination of the activity of Rhodanobacter lindaniclasticus 2212CXJCT1-12 in the culture of Chinese cabbage experiment
[0057] The Rhodanobacter lindaniclasticus 2212CXJCT1-12 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 and 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.
[0058] 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.
[0059] Table 3
[0060]
[0061] Note: Different lowercase letters represent differences in physiological parameters among different treatments (P<0.05).
[0062] As can be seen from Table 3, the Rhodanobacter lindaniclasticus 2212CXJCT1-12 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.
[0063] Example 4, potted plant experiment
[0064] The bacterial culture obtained in Example 3 and the existing control bacteria (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.
[0065] 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.
[0066] 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.
[0067] Table 4
[0068]
[0069] Note: Different lowercase letters represent differences in physiological parameters among different treatments (P<0.05).
[0070] It can be seen from Table 4 that compared with other soil probiotics (Bacillus zanthoxyli) and the control group (CK), the Rhodanobacter lindaniclasticus2212CXJCT1-12 with growth-promoting function obtained by screening 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.
[0071] 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 Rhodanobacter lindaniclasticus, characterized in that The bacterium Rhodanobacterlindaniclasticus is Rhodanobacter lindaniclasticus 2212CXJCT1-12, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 24, 2024, with the collection number GDMCCNo: 64308, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A microbial preparation, characterized in that: The microbial preparation comprises the bacterium Rhodanobacter lindaniclasticus as claimed in claim 1.
3. The microbial preparation according to claim 2, characterized in that The total concentration of live bacteria of Rhodanobacter lindaniclasticus in the microbial preparation is 10 7 cfu / g.
4. The microbial preparation according to claim 2, characterized in that The microbial preparation further comprises auxiliary materials, and the auxiliary materials comprise vermiculite.
5. A primer for detecting the bacterium Rhodanobacter lindaniclasticus as claimed in claim 1, characterized in that The sequences of the primers are shown in SEQ ID NOs: 2-3.
6. Use of the bacterium Rhodanobacter lindaniclasticus as claimed in claim 1 in preparing a microbial preparation or microbial fertilizer for promoting vegetable growth.
7. The use according to claim 6, characterized in that The vegetables include choy sum.
8. The use according to claim 6, characterized in that The total concentration of live bacteria of Rhodanobacter lindaniclasticus in the microbial preparation or microbial fertilizer is 10 7 cfu / g.
9. A method for promoting vegetable growth, characterized in that: The method comprises adding the bacterium Rhodanobacter lindaniclasticus according to claim 1 or the microbial preparation according to claim 2.
10. The method for promoting vegetable growth according to claim 9, characterized in that: The vegetables include choy sum.