Ternobacter rhizosphere, microbial inoculum containing same and application of ternobacter rhizosphere and microbial inoculum

Abstract

The invention relates to the technical field of microorganisms, and discloses rhizosphere agrobacterium, a fungicide containing the rhizosphere agrobacterium and application of the rhizosphere agrobacterium and the fungicide. The preservation number of the Pedobacter rhizosphaerae is CGMCC (China General Microbiological Culture Collection Center) No.28364, and the preservation number of the Pedobacter rhizosphaerae is CGMCC No.28364. The invention further provides a method for preventing and treating plant diseases and / or promoting plant growth and a method for promoting plant seed germination. The agrobacterium rhizosphere has a remarkable growth promoting effect on plants, not only can promote germination of plant seeds, but also can promote plant growth of the plants, so that physiological indexes such as plant height, leaf length, leaf width and root length are remarkably improved; in addition, the compound has relatively good inhibitory activity to lauraceae solanaceae, and has a high control effect on bacterial wilt.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically, to a Pedobacter rhizosphaerae, a microbial agent containing the bacterium, and their applications. Background Art

[0002] Under the background of population growth and the improvement of living standards, people's demand for high-quality agricultural products has been increasing continuously. The traditional farming methods that use a large amount of chemical fertilizers and pesticides seriously affect the yield and quality of agricultural products. Excessive use of chemical fertilizers will cause soil compaction, acidification, and a decrease in microbial diversity, ultimately affecting the growth of crops. Excessive use and abuse of chemical pesticides will cause many problems such as drug resistance, environmental pollution, and pesticide residues. With the increasing demand for a high-quality life, while reducing the use of traditional chemical fertilizers and pesticides, using microbial resources to improve soil fertility and control plant diseases has become an optimal solution for the green development of agriculture.

[0003] Bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum, which generally harms solanaceous crops including tomatoes, peppers, potatoes, tobacco, eggplants, etc. Once crops are infected, it is difficult to cure them with drugs. At present, a variety of microbial agents have been found to be able to effectively inhibit the growth of Ralstonia solanacearum and reduce the population of Ralstonia solanacearum in the soil, thereby reducing the incidence of solanaceous crops. However, affected by survival conditions, most microbial agents have poor colonization ability in the rhizosphere, showing the drawback of unstable control efficacy. Plant commensal microorganisms are a class of microorganisms attached to the surface of plants, which have the characteristics of rapid colonization and propagation, and can make up for the defect of unstable control efficacy in the field of biocontrol bacteria from the soil. They are the most potential biocontrol strains for development. Previous studies have sporadically carried out the screening of biocontrol bacteria and growth-promoting bacteria for solanaceous crops, and so far, there has been no report on Pedobacter that can effectively promote the growth of solanaceous plants and simultaneously control Ralstonia solanacearum pathogens. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art, and provide a Pedobacter rhizosphaerae, a microbial agent containing the bacterium, and their applications. The Pedobacter rhizosphaerae has significant growth-promoting ability, can promote the germination of plant seeds and the growth of plants, and has good inhibitory activity against Ralstonia solanacearum.

[0005] To achieve the above purpose, in the first aspect of the present invention, a Pedobacter rhizosphaerae is provided, and the preservation number of the Pedobacter rhizosphaerae is CGMCC No. 28364.

[0006] In the second aspect of the present invention, a microbial agent is provided, and the microbial agent contains the above-mentioned Pedobacter rhizosphaerae.

[0007] Preferably, the microbial agent contains the cells and / or metabolites of the Geobacter radicis.

[0008] Preferably, the microbial agent is a liquid microbial agent and / or a solid microbial agent, preferably a liquid microbial agent.

[0009] Preferably, the content of Geobacter radicis in the microbial agent is not less than 10 8 cfu / mL.

[0010] The third aspect of the present invention provides a method for preparing a microbial agent, which includes: fermenting and culturing the above-mentioned Geobacter radicis.

[0011] Preferably, the conditions for the fermentation culture include: the inoculum amount is 5×10 5 -5×10 6 cfu / mL, the temperature is 20-35 °C, and the rotation speed is 150-200 rpm.

[0012] Preferably, the process of the preparation method further includes: performing solid-liquid separation on the fermentation broth obtained by the fermentation culture to obtain cells, and mixing the cells with water.

[0013] Preferably, the OD of the cell suspension formed by mixing the cells with water 600 is 0.2-0.6.

[0014] The fourth aspect of the present invention provides the application of the above-mentioned Geobacter radicis, the above-mentioned microbial agent or the microbial agent prepared by the above-mentioned preparation method in at least one of preventing and controlling plant diseases, promoting plant seed germination and promoting plant growth.

[0015] Preferably, the prevention and control of plant diseases is to inhibit plant pathogenic bacteria and / or inhibit plant diseases.

[0016] Preferably, the plant pathogenic bacteria is Ralstonia solanacearum; the plant disease is a plant disease mediated by Ralstonia solanacearum, more preferably bacterial wilt.

[0017] Preferably, the promotion of plant growth is to increase at least one of the plant height, leaf length and leaf width.

[0018] Preferably, the plant is tobacco and / or solanaceous crops.

[0019] The fifth aspect of the present invention provides a method for preventing and controlling plant diseases and / or promoting plant growth, the method includes: spraying and / or drenching the plants with the above-mentioned Geobacter radicis, the above-mentioned microbial agent or the microbial agent prepared by the above-mentioned preparation method.

[0020] Preferably, the plant is tobacco.

[0021] Preferably, the process of root irrigation includes: irrigating the plants of each plant with 15 - 25 mL of the microbial agent with an OD 600 of 0.2 - 0.4 on the 12th - 16th day and the 18th - 24th day after transplantation. 600

[0022] The sixth aspect of the present invention provides a method for promoting the germination of plant seeds, the method includes: soaking the seeds of plants with the above-mentioned Rhizobacter terricola, the above-mentioned microbial agent or the microbial agent prepared by the above-mentioned preparation method.

[0023] Preferably, the plant is tobacco and / or solanaceous crops.

[0024] Preferably, the process of soaking treatment includes: after soaking the seeds of the plant with hydrogen peroxide, soaking with ethanol, and cleaning, mixing and soaking with the microbial agent with an OD 600 of 0.2 - 0.4; 600

[0025] Preferably, the conditions for soaking with hydrogen peroxide include: the concentration of hydrogen peroxide is 10 - 20% by weight, and the time is 8 - 12 min;

[0026] The conditions for soaking with ethanol include: the concentration of ethanol is 70 - 80% by volume, and the time is 20 - 40 s;

[0027] The time for the mixed soaking condition is 25 - 35 min.

[0028] Through the above technical solutions, the beneficial effects of the present invention are as follows:

[0029] The Rhizobacter terricola provided by the present invention is an endophytic bacterium isolated from the root surface of healthy tobacco plants. This strain has a significant growth-promoting effect on plants, can not only promote the germination of plant seeds (especially solanaceous crops), but also promote the growth of plant plants, significantly improving physiological indexes such as plant height, leaf length, leaf width, and root length; in addition, this strain has good inhibitory activity against Ralstonia solanacearum and has a high control effect on bacterial wilt, and has the potential to be further developed into a biological preparation for plants.

[0030] The Rhizobacter terricola provided by the present invention is highly efficient and safe, does not cause environmental pollution problems, has simple culture conditions and is easy to preserve, and is suitable for industrial production, so it has good practical application value.

[0031] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation part.

[0032] Biological preservation

[0033] The strain provided by the present invention is Pedobacter rhizosphaerae, which was deposited on September 5, 2023, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101; the abbreviation of the depositary institution is CGMCC), and the deposit number is CGMCC No. 28364. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a colony morphology diagram of the strain Cas204 on NA medium in Example 1;

[0035] Figure 2 It is a phylogenetic tree of the 16S rDNA sequence of the strain Cas204 in Example 1;

[0036] Figure 3 It is a diagram showing the differences in physiological indexes of the plants treated with the strain Cas204 in Example 2 and the control group;

[0037] Figure 4 It is a diagram showing the effect of the strain Cas204 on seed germination in Example 3;

[0038] Figure 5 It is an effect diagram showing the inhibition of the incidence of tobacco seedlings by the strain Cas204 under greenhouse conditions in Example 4, where the left figure is the Cas204 treatment group and the right figure is the water treatment group. DETAILED DESCRIPTION OF THE INVENTION

[0039] The following provides a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0040] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0041] In the first aspect, the present invention provides a strain of Pedobacter rhizosphaerae, wherein the deposit number of the Pedobacter rhizosphaerae is CGMCC No. 28364.

[0042] The Pedobacter rhizosphaerae provided by the present invention is isolated from the root tissue samples of healthy tobacco plants. The isolation of the Pedobacter rhizosphaerae can adopt the conventional methods for isolating new strains in the art, such as the gradient dilution isolation method.

[0043] The tissue isolation method specifically may include: after removing the soil attached to the root surface of the tobacco plant, putting the root tissue sample into physiological saline, and using the ultrasonic oscillation method to collect the soil on the root surface; the remaining after taking out the fine roots is the soil stock solution; adopting the gradient dilution method to obtain soil dilutions with concentrations of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 ; respectively coating different soil dilutions on the isolation medium, and culturing for 40 - 60 h at a temperature of 20 - 35°C to obtain a plurality of colonies; according to the growth characteristics of the colonies such as color, size, degree of elevation, transparency, hardness, and whether the edge is regular, etc., picking single colonies, and performing three-zone streaking on the picked single colonies on the isolation medium, and purifying 2 - 4 times to obtain pure cultured strains.

[0044] According to the present invention, the isolation medium can adopt any medium capable of enriching strains. Exemplarily, the isolation medium contains beef extract, peptone, NaCl, and agar. Preferably, the isolation medium contains 3 - 5 g / L of beef extract, 8 - 12 g / L of peptone, 3 - 5 g / L of NaCl, and 15 - 20 g / L of agar.

[0045] The inventors of the present invention carried out morphological identification and molecular biological identification on the isolated pure cultured strains. The results showed that the strain is Pedobacter rhizosphaerae, and it was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 5, 2023, with the deposit number of CGMCC No. 28364.

[0046] The Pedobacter rhizosphaerae provided by the present invention can produce a large amount of cells and / or metabolites of Pedobacter rhizosphaerae after fermentation culture. The present invention has no particular limitation on the fermentation culture method, as long as the Pedobacter rhizosphaerae can be proliferated in large quantities by this fermentation culture method. For example, the Pedobacter rhizosphaerae can be inoculated into a liquid medium and cultured with shaking at a temperature of 20 - 35°C and a rotation speed of 150 - 200 rpm to obtain a fermentation broth. Among them, the liquid medium can be a medium commonly used in the art. For example, it can contain beef extract, peptone, and NaCl; preferably, the liquid medium contains 3 - 5 g / L of beef extract, 8 - 12 g / L of peptone, and 3 - 5 g / L of NaCl.

[0047] In a second aspect, the present invention provides a bacterial agent, wherein the bacterial agent contains the above-mentioned Geobacter radicis.

[0048] According to the present invention, the bacterial agent preferably contains the cells and / or metabolites of Geobacter radicis. In the present invention, the term "metabolite" refers to the metabolites produced by Geobacter radicis during fermentation or culture (including intracellular metabolites and / or extracellular metabolites).

[0049] According to the present invention, there is no particular limitation on the dosage form of the bacterial agent. Depending on different intended uses, it can be prepared into different dosage forms and corresponding excipients (formulating agents) and other components can be added. For example, the bacterial agent can be a liquid bacterial agent (such as a dilution of cells and / or metabolites) and / or a solid bacterial agent (such as the cells of Geobacter radicis), and preferably a liquid bacterial agent. Among them, those skilled in the art are familiar with what excipients are added to which dosage form of the bacterial agent, and will not be elaborated here in detail.

[0050] In the present invention, there is no particular limitation on the concentration of Geobacter radicis in the bacterial agent, and specific selection can be made according to specific circumstances. Preferably, the content of Geobacter radicis in the bacterial agent is not less than 10 8 cfu / mL. More preferably, the content of Geobacter radicis in the bacterial agent is 10 8 -10 9 cfu / mL.

[0051] The bacterial agent provided in the present invention can be obtained by any conventional method in the art.

[0052] In a third aspect of the present invention, a method for preparing a bacterial agent is provided, and the preparation method includes: fermenting and culturing the above-mentioned Geobacter radicis.

[0053] The Geobacter radicis provided by the present invention can produce a large number of viable Geobacter radicis cells through fermentation culture. The present invention has no particular limitation on the method of fermentation culture, as long as the Geobacter radicis can be proliferated in large quantities through this fermentation culture method.

[0054] According to the present invention, preferably, the Geobacter radicis is activated before the fermentation culture and then inoculated into the fermentation medium.

[0055] Among them, the activation can adopt any method of activating bacterial strains. Preferably, the activation process includes: inoculating Geobacter radicis (specifically, Geobacter radicis preserved in a glycerol tube, 0.5 - 2 mL) into the activation medium for activation culture, then inoculating into the seed medium for seed culture to obtain a seed solution, and inoculating the seed solution into the fermentation medium for the fermentation culture.

[0056] In the present invention, the activation medium and the seed medium can each independently be a medium conventionally used in the art. Further preferably, the components of the activation medium and the seed medium each contain beef extract, peptone, and NaCl, specifically, they can contain 3 - 5 g / L of beef extract, 8 - 12 g / L of peptone, and 3 - 5 g / L of NaCl.

[0057] The conditions such as temperature and time for the activation culture and the seed culture can be selected according to the growth requirements of Agrobacterium rhizogenes. Preferably, the conditions for the activation culture and the seed culture each independently include: the temperature is 20 - 35°C, the rotation speed is 150 - 200 rpm, and the time is 20 - 30 h.

[0058] In the present invention, the medium for the fermentation culture can be a fermentation medium conventionally used in the art. For example, it can contain beef extract, peptone, and NaCl, and more preferably, it contains 3 - 5 g / L of beef extract, 8 - 12 g / L of peptone, and 3 - 5 g / L of NaCl.

[0059] In the present invention, the conditions such as the inoculum amount, temperature, rotation speed, and time for the fermentation culture can be selected according to the growth requirements of Agrobacterium rhizogenes. Preferably, the conditions for the fermentation culture include: the inoculum amount is 5×10 5 -5×10 6 cfu / mL (generally about 1×10 6 cfu / mL); the temperature is 20 - 35°C, specifically, it can be 20°C, 23°C, 26°C, 29°C, 32°C, 35°C, or any value between the above two values; the rotation speed is 150 - 200 rpm, specifically, it can be 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, or any value between the above two values; the time is 10 - 30 h, specifically, it can be 10 h, 15 h, 20 h, 25 h, 30 h, or any value between the above two values. Under these preferred conditions, it is beneficial to promote the growth of Agrobacterium rhizogenes.

[0060] In addition, the cells and metabolites of Agrobacterium rhizogenes in the fermentation broth of the above fermentation culture can be further separated. There are no particular limitations on the separation method, as long as the cells and metabolites can be enriched from the culture broth. Preferably, the process of the preparation method further includes: performing solid-liquid separation on the fermentation broth obtained from the fermentation culture to obtain the cells, and mixing the cells with water to form a cell suspension as a microbial agent. Further preferably, the OD of the cell suspension formed by mixing the cells with water 600 is 0.2 - 0.6, specifically, it can be 0.2, 0.3, 0.4, 0.5, 0.6, or any value between the above two values.

[0061] In the present invention, the water in the bacterial suspension is sterile water; solid-liquid separation can be carried out by any separation method, specifically centrifugation or filtration, preferably centrifugation. Exemplarily, the conditions for centrifugation include: a rotation speed of 8000 - 9000 rpm and a time of 1 - 5 min.

[0062] The Rhizobacter terricola provided by the present invention has a significant plant growth-promoting effect on plants. It can not only promote the germination of plant seeds (especially tobacco seeds and Solanaceae crop seeds), but also promote the growth of plant plants, significantly improving physiological indexes such as plant height, leaf length, leaf width, and root length. In addition, this strain has good inhibitory activity against Ralstonia solanacearum and a high control effect on bacterial wilt. Based on this, in the fourth aspect, the present invention provides the application of the above-mentioned Rhizobacter terricola, the above-mentioned bacterial agent, or the bacterial agent prepared by the above-mentioned preparation method in at least one of preventing and controlling plant diseases, promoting plant seed germination, and promoting plant growth.

[0063] According to the present invention, preferably, the prevention and control of plant diseases is to inhibit plant pathogens and / or inhibit plant diseases. Further preferably, the plant pathogen is Ralstonia solanacearum; the plant disease is a plant disease mediated by Ralstonia solanacearum, more preferably bacterial wilt, such as tobacco bacterial wilt.

[0064] According to the present invention, preferably, the promotion of plant growth is to increase at least one of the plant height, leaf length, and leaf width.

[0065] According to the present invention, the plant can be any kind of crop. Preferably, the plant is a Solanaceae crop, more preferably tobacco (such as Yunyan) and / or tomato (such as Guifei Cherry).

[0066] In the fifth aspect, the present invention provides a method for preventing and controlling plant diseases and / or promoting plant growth, the method comprising: spraying and / or root irrigation treatment of the plants of the plant with the above-mentioned Rhizobacter terricola, the above-mentioned bacterial agent, or the bacterial agent prepared by the above-mentioned preparation method.

[0067] According to the present invention, the plant can be any kind of crop. Preferably, the plant is a Solanaceae crop, more preferably tobacco (such as Yunyan) and / or tomato (such as Guifei Cherry).

[0068] According to the present invention, the root irrigation can be carried out by a conventional operation method. Preferably, the process of root irrigation for preventing and controlling plant diseases includes: performing at least two root irrigation treatments on each plant after transplantation, with an interval of 4 - 6 d between adjacent root irrigation treatments, and each root irrigation treatment using 15 - 25 mL OD 600A bacterial agent with an OD of 0.5 - 0.6; when promoting plant growth, the process of root irrigation includes: root-irrigating each plant of the plants on the 12th - 16th day and the 18th - 24th day after transplantation with 600 a bacterial agent with an OD of 0.2 - 0.4, 15 - 25 mL. In this preferred embodiment, it is beneficial to improve the effect of Agrobacterium terregens in controlling plant diseases and better promote plant growth.

[0069] Sixthly, the present invention provides a method for promoting the germination of plant seeds, and the method includes: soaking the seeds of plants with the above-mentioned Agrobacterium terregens, the above-mentioned bacterial agent or the bacterial agent prepared by the above-mentioned preparation method.

[0070] According to the present invention, the plant can be any kind of crop. Preferably, the plant is a solanaceous crop, and more preferably it is tobacco (such as Yunyan) and / or tomato (such as Guifei Cherry).

[0071] According to the present invention, preferably, the process of the soaking treatment includes: after soaking the seeds of the plants with hydrogen peroxide, soaking with ethanol, and cleaning, mixing and soaking with 600 a bacterial agent with an OD of 0.2 - 0.4.

[0072] According to the present invention, preferably, the conditions for soaking with hydrogen peroxide include: the concentration of hydrogen peroxide is 10 - 20% by weight, specifically it can be 10% by weight, 12% by weight, 14% by weight, 16% by weight, 18% by weight, 20% by weight, or any value between the above two values; the time is 8 - 12 min, specifically it can be 8 min, 9 min, 10 min, 11 min, 12 min, or any value between the above two values. The conditions for soaking with ethanol include: the concentration of ethanol is 70 - 80% by volume, specifically it can be 70% by volume, 72% by volume, 74% by volume, 76% by volume, 78% by volume, 80% by volume, or any value between the above two values; the time is 20 - 40 s, specifically it can be 20 s, 25 s, 30 s, 35 s, 40 s, or any value between the above two values. The time for the conditions of the mixed soaking is 25 - 35 min, specifically it can be 25 min, 27 min, 29 min, 31 min, 33 min, 35 min, or any value between the above two values. In this preferred embodiment, it is beneficial to better utilize Agrobacterium terregens to promote the germination of plant seeds.

[0073] In the present invention, the cleaning can be carried out 2 - 4 times with sterile water.

[0074] The present invention will be described in detail below through examples.

[0075] In the following examples, the components of NB medium are: 3 g / L of beef extract, 10 g / L of peptone, and 5 g / L of NaCl;

[0076] The components of NA medium are: 3 g / L of beef extract, 10 g / L of peptone, 5 g / L of NaCl, and 15 g / L of agar.

[0077] The seeds of tobacco K326 were purchased from Yuxi Cigarette Seed Co., Ltd., the seeds of Yunyan 87 were purchased from Yuxi Cigarette Seed Co., Ltd., the seeds of Guifei Cherry were purchased from Qingxian Xingyun Seed Industry Co., Ltd., and Ralstonia solanacearum was donated by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences; without special instructions, other raw materials and reagents are commercially available products.

[0078] Example 1 Isolation, culture and species identification of strains

[0079] Collect root tissue samples of healthy tobacco plants (collected from Tongren area, Guizhou). After brushing off the soil samples attached to the root surface with a brush, put the root tissue samples into a centrifuge tube containing normal saline (NaCl concentration is 0.85% by weight), and use the ultrasonic oscillation method to collect the root surface soil (specifically: add pre-cooled deionized water at 4°C to the ultrasonic cleaner, place the centrifuge tube in the water, perform ultrasonic treatment 2 times, 20 s each time, with a 5 s interval between the two ultrasonic treatments); take out the fine roots in the centrifuge tube with sterile forceps, and the remaining is the soil stock solution. Use the gradient dilution method to obtain soil dilutions with concentrations of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 . Spread different soil dilutions on NA medium respectively and culture at 28°C for 48 h to obtain multiple colonies. According to the growth characteristics of the colonies such as color, size, elevation degree, transparency, hardness, and whether the edge is regular, pick single colonies, and perform three-zone streaking on the picked single colonies on NA medium and purify 3 times to obtain the pure cultured strain Cas204.

[0080] Streak the strain Cas204 onto a fresh NA medium and culture at 28°C for 24 h. The single colony of the strain Cas204 is shown in Figure 1 , showing orange-red, opaque, and smooth edges. Use a 10 μL pipette tip to pick the purified bacterial lawn into a 2 mL centrifuge tube, and extract the strain DNA using a bacterial genomic DNA extraction kit (produced by Nanjing Novozymes Biotech Co., Ltd.).

[0081] Using genomic DNA as a template, the 16S rRNA gene of the strain was amplified using bacterial primers (nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2); the PCR system was 3 μL of 10× buffer; 3 μL of dNTP; 0.3 μL of primer; 0.15 μL of rTaq; 1 μL of DNA template; 22.25 μL of ddH2O; the PCR reaction conditions were pre-denaturation at 94 °C for 5 min; 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min, with 30 cycles; extension at 72 °C for 10 min.

[0082] 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO:1),

[0083] 1492R: 5’-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO:2);

[0084] The PCR products were analyzed by agarose gel electrophoresis, and the obtained fragments were sequenced. The sequenced sequences were aligned on the EzBioCloud website (http: / / www.ezbiocloud.net / ), and a phylogenetic tree was constructed using MEGA 11 software. The taxonomic status of the strain was finally determined based on the sequence similarity and the position in the phylogenetic tree.

[0085] By 16S rDNA sequencing, a gene sequence with a length of 1402 bp was obtained, and the nucleotide sequence is shown in SEQ ID NO:3; this gene sequence was submitted to the NCBI database with the accession number OR506097. The obtained sequence was compared with the sequences of formally named strains in the EzBioCloud database, and it was found that the strain Cas204 had the highest similarity of 99.79% with Pedobacter rhizosphaerae 01-09T. A phylogenetic tree was constructed using the 16S rDNA sequences of Cas204 and other standard strains of the genus Pedobacter, and the results are shown in Figure 2 It was found that the strain Cas204 clustered with Pedobacter rhizosphaerae, and finally Cas204 was determined to be Pedobacter rhizosphaerae.

[0086] The strain Cas204 was streaked on a slant in a test tube, 5 mL of a sterile glycerol preservation solution (glycerol concentration: 20 wt%) was added, vortexed, the bacterial suspension was dispensed into 2-mL cryotubes, stored at -80 °C, and sent to the China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 28364.

[0087] Example 2

[0088] The strain Cas204 was inoculated into NB liquid medium (inoculation amount: about 1×10 6 cfu / mL), cultured overnight at 28 °C with a rotation speed of 180 rpm, the cells were collected by centrifugation at 8000 rpm for 5 min, and the supernatant was discarded; the cells were resuspended with sterile water, and the OD 600 of the bacterial suspension was adjusted to 0.3. After transplanting tobacco plants (K326), they were divided into two groups with 20 plants in each group. At 14 d and 21 d after transplanting, 20 mL of the bacterial suspension was irrigated to the roots of each plant in the treatment group, and 20 mL of sterile water was irrigated to the roots of each plant in the control group. At 28 d after transplanting, the differences in plant height, leaf length, leaf width, root length, etc. between the treatment group and the control group were measured. The results are shown in Table 1 and Figure 3 .

[0089] As can be seen from Table 1 and Figure 3 , after irrigating the roots with the strain Cas204, the physiological indexes such as plant height, leaf length, and leaf width of the plants were significantly increased compared with the control group (p < 0.05). The plant height increased by about 0.651 cm, the leaf length increased by about 2.10 cm, the leaf width increased by about 1.42 cm, and the root length increased by 0.71 cm. It shows that irrigating the roots with the bacterial suspension of the strain Cas204 can promote plant growth and has a good growth-promoting effect.

[0090] Table 1 Growth-promoting effect of the strain Cas204 on plants

[0091] Processing Plant height cm Leaf length cm Leaf width cm Root length cm Control 0.77±0.06 4.03±0.12 3.25±0.05 2.97±0.06 Cas204 1.38±0.08*** 6.13±0.35** 4.67±0.14*** 3.68±0.13*

[0092] Note: In the table, "*" represents p < 0.05; "**" represents p < 0.01; "***" represents p < 0.001.

[0093] Example 3

[0094] Tobacco seeds (K326) and tomato seeds (Guifei Cherry variety) were soaked in an aqueous H2O2 solution with a concentration of 15 wt% for 10 min, and then soaked and disinfected with ethanol with a concentration of 75 vol% for 30 s, and washed three times with sterile water to obtain pretreated seeds; the strain Cas204 was inoculated into NB liquid medium (inoculation amount: about 1×10 6(cfu / mL), the culture solution was obtained by overnight culture at 28 °C and 180 rpm. The culture solution was centrifuged at 8000 rpm for 5 min to collect the bacteria, which were resuspended with sterile water, and the OD of the bacterial suspension was adjusted 600 = 0.3; The above pretreated seeds were divided into a treatment group and a control group. The seeds in the treatment group were soaked in the bacterial suspension for 30 min, and the seeds in the control group were soaked in sterile water for 30 min; Double-layer sterilized filter paper was laid in a petri dish and moistened with 2 mL of sterile water; 100 tobacco seeds / tomato seeds were evenly placed on the filter paper and cultured at 28 °C, and the seed germination was recorded. The results are shown in Figure 4 ("*" represents p < 0.05, with significant difference; "***" represents p < 0.001, with extremely significant difference).

[0095] From Figure 4 it can be seen that after treatment with the Cas204 bacterial suspension (OD 600 = 0.3), the germination rate of tobacco seeds reached 96.29 ± 3.21%, while the germination rate of tobacco seeds treated with sterile water (control) was only 86.11 ± 2.78%. The Cas204 treatment group was significantly higher than the control group; After treatment with the Cas204 bacterial suspension (OD 600 = 0.3), the germination rate of tomato seeds reached 71.33 ± 0.91%, while the germination rate of tobacco seeds treated with sterile water (control) was only 50.10 ± 1.77%. The Cas204 treatment group was significantly higher than the control group. Based on the above results, it shows that treating seeds with the Cas204 bacterial suspension can significantly promote the germination of crop seeds.

[0096] Example 4

[0097] Tobacco seeds (Yunyan 87) were soaked in an aqueous solution of H2O2 with a concentration of 15% by weight for 10 min, and then disinfected by soaking in ethanol with a concentration of 75% by volume for 30 s, and washed three times with sterile water to obtain pretreated seeds; The pretreated seeds were sown into a seedling-raising substrate (organic matter content is 23%, purchased from Shouguang Ward Agricultural Science and Technology Co., Ltd.). When the tobacco seedlings grew 4 leaves, they were transplanted into flower pots with a diameter of 10 cm, and divided into an experimental group and a control group, with 15 pots of tobacco seedlings in each group for replication.

[0098] The test strain Cas204 and Ralstonia solanacearum were respectively inoculated into NB medium (inoculation amount was about 1×10 6 cfu / mL), and the culture solution was obtained by culturing at 28 °C and 200 rpm for 24 h. The culture solution was centrifuged at 900 rpm for 1 min, resuspended with sterilized water, and the OD was adjusted 600 to 0.6 to obtain the Cas204 bacterial suspension and the Ralstonia solanacearum bacterial suspension respectively.

[0099] In the experimental group, 20 mL of the Cas204 bacterial suspension was used for root irrigation of tobacco seedlings, and the root irrigation was carried out twice with an interval of 5 days (the first root irrigation was on the 14th day after transplanting). The control group was irrigated with an equal amount of clear water. 29 days after transplanting, 20 mL of the Ralstonia solanacearum bacterial suspension was used for root irrigation of tobacco plants. After applying Ralstonia solanacearum, the incidence of bacterial wilt was observed in the plants. When the first diseased plant appeared, the disease incidence of each treatment was observed every 2 days for a total of 20 days. The results are shown in Figure 5 .

[0100] The disease severity was classified as follows:

[0101] Grade 0: Healthy plants,

[0102] Grade 1: 1% - 25% of the leaves were diseased,

[0103] Grade 3: 26% - 50% of the leaves were diseased,

[0104] Grade 5: More than 50% of the leaves were diseased,

[0105] Grade 7: More than two-thirds of the leaves were diseased,

[0106] Grade 9: The whole plant died;

[0107] The incidence rate, disease index and control effect of each treatment group were calculated by the following formulas;

[0108] Incidence rate = number of diseased plants / total number of investigated plants × 100%,

[0109] Disease index = Σ (number of plants at each disease grade × number of diseased plants) / (number of investigated plants × 9) × 100%,

[0110] Control effect = (control disease index - test disease index) / control disease index × 100%.

[0111] It can be seen from Figure 5 that under greenhouse conditions, the strain Cas204 has an obvious inhibitory effect on the incidence of bacterial wilt in tobacco seedlings; after treatment with 20 mL of the Cas204 bacterial suspension (OD 600 = 0.6), the incidence rate of tobacco plants was 27%, which was significantly lower than the incidence rate of 53% in the control group; the disease index of the treatment group was 8.74, which was significantly lower than the disease index of 29.78 in the control group, and the control effect of Cas204 on bacterial wilt reached 70.65%.

[0112] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A rhizobacterium, characterized in that The deposit number of the Pedobacter rhizosphaerae is CGMCC No.28364.

2. A bacterial agent, characterized in that The bacterial agent contains the rhizospheric soil bacterium according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent contains the bacterial body and / or metabolites of the rhizospheric soil bacterium.

4. The bacterial agent according to claim 2 or 3, characterized in that The bacterial agent is a liquid bacterial agent and / or a solid bacterial agent, preferably a liquid bacterial agent; Preferably, the content of Rhizobacter in the liquid bacterial agent is not less than 10 8 cfu / mL.

5. A method for preparing a bacterial agent, characterized in that: The preparation method comprises: fermenting and culturing the rhizosphere soil bacterium described in claim 1.

6. The preparation method according to claim 5, characterized in that: The fermentation culture conditions at least meet the following requirements: the inoculation volume is 5×10 5 -5×10 6 cfu / mL, temperature 20-35°C, speed 150-200rpm, time 10-30h; Preferably, the preparation method further comprises: performing solid-liquid separation on the fermentation liquid obtained by the fermentation culture to obtain bacterial cells, and mixing the bacterial cells with water; Preferably, the bacterial suspension OD formed by mixing the bacterial cells with water is 600 It is 0.2-0.

6.

7. Use of the rhizospheric soil bacterium according to claim 1, the bacterial agent according to any one of claims 2 to 4, or the bacterial agent prepared by the preparation method according to claim 5 or 6 in at least one of preventing and controlling plant diseases, promoting plant seed germination, and promoting plant growth.

8. The use according to claim 7, characterized in that: The plant disease prevention and control is to inhibit plant pathogens and / or inhibit plant diseases; Preferably, the plant pathogen is Ralstonia solanacearum; the plant disease is a plant disease caused by Ralstonia solanacearum, more preferably bacterial wilt; Preferably, the promoting plant growth is to increase at least one of the plant height, leaf length and leaf width; Preferably, the plant is a crop of the Solanaceae family, more preferably tobacco and / or tomato.

9. A method for preventing and controlling plant diseases and / or promoting plant growth, characterized in that: The method comprises: spraying and / or root irrigation treatment on plants using the rhizobacterium described in claim 1, the bacterial agent described in any one of claims 2 to 4, or the bacterial agent prepared by the preparation method described in claim 5 or 6; Preferably, the plant is a Solanaceae crop, more preferably tobacco and / or tomato; Preferably, the root irrigation process for preventing and controlling plant diseases comprises: performing at least two root irrigation treatments on each plant after transplanting, with an interval of 4-6 days between two adjacent root irrigation treatments, and each root irrigation treatment using 15-25 mL OD 600 The bacterial agent is 0.5-0.6; The root irrigation process for promoting plant growth includes: irrigating the roots of each plant at OD 12-16 days and 18-24 days after transplanting. 600 15-25mL of 0.2-0.4 bacterial agent.

10. A method for promoting plant seed germination, characterized in that: The method comprises: soaking plant seeds with the rhizobacterium described in claim 1, the bacterial agent described in any one of claims 2 to 4, or the bacterial agent prepared by the preparation method described in claim 5 or 6; Preferably, the plant is a Solanaceae crop, more preferably tobacco and / or tomato; Preferably, the soaking treatment process comprises: soaking the seeds of the plant in hydrogen peroxide, ethanol, washing, and then mixing with OD 600 Soak in 0.2-0.4% bacterial agent mixture; Preferably, the conditions for soaking in hydrogen peroxide include: a concentration of hydrogen peroxide of 10-20% by weight and a soaking time of 8-12 minutes; The conditions for ethanol soaking include: ethanol concentration of 70-80% by volume and time of 20-40s; The time of the mixed soaking condition is 25-35 minutes.

Images