A strain of Pseudomonas capable of enhancing peanut immunity and promoting growth and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a strain of Pseudomonas having the effects of enhancing peanut immunity and promoting growth and its application. The present invention provides a strain of Pseudomonas ( Pseudomonas sp.), which can effectively antagonize pathogens, significantly enhance plant immunity and play a significant growth-promoting role.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Pseudomonas capable of enhancing plant immunity and promoting growth and application thereof. Background Art

[0002] With peanuts ( Arachis hypogaea L.) production, it is susceptible to various diseases such as white rot, root rot and fruit rot during the planting process, among which the Sclerotium rolfsii Sacc. ) seriously restricts the yield and quality of peanuts and is one of the most serious soil-borne fungal diseases of peanuts. In order to improve the yield and quality of peanuts, farmers spray a large amount of chemical pesticides. As the number of consecutive cropping years increases, the resistance of pathogens increases, and traditional prevention and control methods are ineffective. In addition, the large-scale use of chemical pesticides has destroyed the ecological balance and biodiversity of soil microbial communities on the one hand, and also caused food safety problems such as pesticide residues on the other hand. It is urgent to seek effective alternative products.

[0003] Biological control measures are one of the effective strategies to replace traditional fertilizers and pesticides. Researchers visualized the cultivation of plant rhizosphere microorganisms, screened out strains that promote plant growth and prevent diseases, and then inoculated them back into the roots of plants after large-scale fermentation and cultivation. They promote plant growth and prevent plant diseases by secreting growth-promoting substances, antibacterial substances and inducing plant immunity.

[0004] Pseudomonas is widely present in soil, water, plants, and the intestines of humans and animals, occupying a large number of ecological niches. In recent years, studies have shown that Pseudomonas with good biocontrol effects have been isolated and identified from different plants. These Pseudomonas can secrete a variety of secondary metabolites to antagonize plant pathogens (such as siderophores, phenazine-1-carboxylic acid, and pyocyanin), and can prevent and control a variety of plant fungal and bacterial diseases. Although these secondary metabolites can directly or indirectly prevent and control plant diseases, plants mainly rely on their own immune system when resisting pathogen infection. Studies have shown that in addition to secreting metabolites to directly antagonize pathogens, some root microorganisms can also induce systemic acquired resistance (SAR) to protect plants from damage. It is mainly manifested in defense responses such as reactive oxygen species (ROS) burst, callose deposition, and lignin deposition.

[0005] There are various biocontrol products available, but in actual use, the effects are greatly reduced. Although this is related to the application method and environment, it is more caused by the genetic diversity and complexity of soil pathogens. For example, some genotypes of pathogens may develop tolerance to antagonistic bacteria, making it difficult for strains in biological agents to resist the invasion of pathogens through antagonism. Therefore, it is of great significance to isolate and screen multifunctional strain resources that have the functions of antagonizing pathogens, enhancing plant immunity and promoting growth from specific hosts. Summary of the invention

[0006] In view of this, the present invention provides a Pseudomonas strain having the effects of enhancing plant immunity and promoting growth and its application. The strain has multiple functions, can effectively antagonize pathogens, significantly enhance plant immunity and exert a significant growth-promoting effect, and well copes with the above-mentioned challenges.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a Pseudomonas sp. ( Pseudomonas sp.).

[0009] The present invention also provides the above-mentioned Pseudomonas ( Pseudomonas sp.) fermentation products.

[0010] The present invention also provides the above-mentioned Pseudomonas ( Pseudomonas sp.) or the use of the fermentation product thereof in at least one of the following:

[0011] (A1), siderophore production;

[0012] (A2), decomposition of organic phosphorus;

[0013] (A3), decomposition of inorganic phosphorus;

[0014] (A4), amylase production;

[0015] (A5), catalase;

[0016] (A6), bacterial biofilm production;

[0017] (A7), inhibit or antagonize pathogens.

[0018] In some specific embodiments of the present invention, the pathogenic bacteria used in the above application include Sclerotium sclerotiorum ( Sclerotium rolfsii Sacc. ).

[0019] The present invention also provides the above-mentioned Pseudomonas ( Pseudomonas sp.) or the use of the fermentation product thereof in at least one of the following:

[0020] (B1), promoting the production of ROS in plant leaves;

[0021] (B2) Promote callose deposition in plant roots;

[0022] (B3) Increase plant growth rate and aboveground biomass.

[0023] In some specific embodiments of the present invention, the plant used above includes peanut;

[0024] The peanuts include colorful peanuts.

[0025] The present invention also provides the above-mentioned Pseudomonas ( Pseudomonas sp.) or the use of the fermentation product thereof in at least one of the following:

[0026] (C1) Prevent, reduce or eliminate plant diseases;

[0027] (C2), promote plant growth and development;

[0028] (C3), enhance plant immunity or disease resistance;

[0029] (C4) Preparation of products for planting.

[0030] In some specific embodiments of the present invention, the plant used above includes peanut;

[0031] The peanuts include colorful peanuts.

[0032] In some specific embodiments of the present invention, the disease in the above application includes white rot.

[0033] In some specific embodiments of the present invention, the above-mentioned application of white rot includes peanut white rot.

[0034] In some specific embodiments of the present invention, the peanut white rot mentioned in the application is Sclerotium solani ( Sclerotium rolfsii Sacc. ) caused by peanut white rot.

[0035] The present invention also provides a preparation comprising:

[0036] (D1), the above-mentioned Pseudomonas ( Pseudomonas sp.); or

[0037] (D2) the above fermentation products.

[0038] In some specific embodiments of the present invention, the above-mentioned formulation further comprises MgCl2.

[0039] In some specific embodiments of the present invention, the preparation method of the above-mentioned preparation comprises resuspending the above-mentioned Pseudomonas ( Pseudomonas sp.) steps.

[0040] The present invention also provides a product for planting, comprising:

[0041] (E1), the above-mentioned Pseudomonas ( Pseudomonas sp.); or

[0042] (E2), the above fermentation products; or

[0043] (E3) the above preparation.

[0044] In some specific embodiments of the present invention, the above-mentioned product comprises a microbial fertilizer.

[0045] The present invention also provides the above-mentioned Pseudomonas ( Pseudomonas sp.) culture method.

[0046] In some specific embodiments of the present invention, the above-mentioned culturing method comprises the above-mentioned Pseudomonas ( Pseudomonas sp.) steps of inoculation and culture medium.

[0047] The present invention also provides a method for planting crops, based on any of the following planting:

[0048] (F1), the above-mentioned Pseudomonas ( Pseudomonas sp.); or

[0049] (F2), the above fermentation products; or

[0050] (F3) the above preparations.

[0051] In some specific embodiments of the present invention, the crops described in the above planting method include peanuts;

[0052] The peanuts include colorful peanuts.

[0053] The strain and application of the present invention have the following effects:

[0054] On the one hand, affected by plant species, varieties and regional environment, plants are selective for strains that colonize in tissues. Most strains can only colonize in the soil around the rhizosphere. During the re-inoculation process, they are easily affected by the environment and have a low survival rate. The strains isolated from the host tissues are highly specific and have advantages in the re-inoculation and colonization process. They can more easily enter plant tissues, establish a symbiotic relationship with plants, and exert their functions. The strains of the present application are isolated from the root tissues of the colorful peanuts. When the strains are re-inoculated back into the host roots in the form of bacterial liquid or biological agents, they are more likely to colonize in plant tissues and establish a symbiotic relationship with plants.

[0055] On the other hand, due to the diversity and complexity of plant pathogen genes, single-function strains are difficult to compete, so mining strains with multiple functions (such as antagonizing pathogens, enhancing plant immunity and promoting growth) can better cope with the impact of pathogen diversity and other aspects. The strains of this application happen to have multiple functions, which can effectively antagonize pathogens, significantly enhance plant immunity and play a significant role in promoting growth, thus effectively coping with the challenges brought by various diversities. Specifically:

[0056] (1) The strain of the present application is an endophytic bacterium isolated from the root system of the seven-color peanut, a native variety unique to Yunnan. The strain can better establish a symbiotic relationship with the plant and reduce rejection reactions.

[0057] (2) The roots of colorful peanut plants were exposed to the bacterial solution OD 600 Soaking roots in 0.2 medium for 6 hours has a good effective prevention and control rate against white rot of colorful peanut (Sclerotium rufin).

[0058] (3) Inoculation of bacterial solution into the roots of colorful peanut plants (OD 600 0.5) After 7 days, under the stimulation of 500 μg / mL chitin, the living tissue of the plant leaves was able to produce a large amount of ROS, and a large amount of callose was deposited in the root system, which effectively enhanced the immunity of the plant.

[0059] (4) The roots of colorful peanut plants were exposed to the bacterial solution OD 600 Root immersion in 0.2 medium for 6 hours promoted the growth and development of colorful peanuts to a certain extent under the condition of no pathogen stress.

[0060] (5) Pseudomonas B1207 has the functions of producing siderophores, solubilizing organic and inorganic phosphorus, producing amylase, producing H2O2 enzyme, and producing biofilm, which play an important role in colonization, disease resistance and growth promotion.

[0061] (6) The strain described in this application has multiple functions such as disease resistance, growth promotion and enhancement of plant immunity, providing strain resources for the development of plant "live vaccines".

[0062] Biological Deposit Description

[0063] Culture name: Pseudomonas sp. B1207 ( Pseudomonas sp. B1207), was deposited in the China Center for Type Culture Collection on April 1, 2024. The address of the collection is: Wuhan University, Wuhan, China; the collection number is CCTCCNO: M 2024598.

[0064] The B1207 mentioned in this application is the strain with the deposit number of CCTCC NO: M 2024598. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0066] Figure 1 Pseudomonas Pseudomonas sp.) B1207 morphology;

[0067] Figure 2 Shows the results of homologous sequence comparison;

[0068] Figure 3 The pathogenicity test of Sclerotium sclerotium is shown;

[0069] Figure 4 The results of antagonistic strain screening are shown;

[0070] Figure 5 The morphological effect of antagonistic strains in controlling white june disease plants is shown;

[0071] Figure 6 The morphological effect of antagonistic strains on the root and stem of white jujube disease was shown;

[0072] Figure 7 Shows the results of ROS burst test of antagonistic strains;

[0073] Figure 8 Shows callose deposition in the roots of the antagonistic strain B1207; a shows CK roots at 50 times magnification, with a scale bar of 250 μm, b shows CK roots at 100 times magnification, with a scale bar of 100 μm, c shows CK roots at 200 times magnification, with a scale bar of 50 μm, d shows B1207 roots at 50 times magnification, with a scale bar of 250 μm, e shows B1207 roots at 100 times magnification, with a scale bar of 100 μm, and f shows B1207 roots at 200 times magnification, with a scale bar of 50 μm;

[0074] Fig. 9 Shown is the gel detection diagram of B1207 16s rDNA extraction and amplification;

[0075] Fig.10 Shows the test results of the growth-promoting effect of B1207;

[0076] Fig.11 Shows the siderophore production capacity test results;

[0077] Fig.12 Shows the test results of the ability to dissolve organic phosphorus;

[0078] Fig.13 Shows the test results of inorganic phosphorus solubility;

[0079] Fig.14 Shows the test results of amylase production capacity;

[0080] Fig.15 Shows the results of the H2O2 production enzyme ability test;

[0081] Fig.16 Shows the biofilm test results. DETAILED DESCRIPTION

[0082] The present invention discloses a strain of Pseudomonas with plant immunity enhancement and growth promotion effects and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0083] The present invention screened out a strain of Pseudomonas that has the effects of preventing and controlling white rot of seven-color peanuts and promoting growth, and the identification results are as follows:

[0084] 1. Morphological identification:

[0085] The strain was cultured on TSA solid medium [formula: 17 g / L casein (tryptic hydrolysate), 3 g / L soy peptone, 5 g / L NaCl, 2.5 g / L K2HPO4, 2.5 g / L glucose (Dextrose), 20 g / L agar powder, adjusted to pH 7.2 with NaOH, then fixed to 1 L, sterilized at 121°C for 15 min] at 30°C for 24 h. The colony diameter was 2.0 mm~3.0 mm, the colony morphology was nearly round, slightly raised, grayish white, opaque, and an obligate aerobe. Figure 1 As shown. After the strain was cultured in TSB liquid medium [formula: 17 g / L casein (tryptic hydrolysate), 3 g / L soy peptone, 5 g / L NaCl, 2.5 g / L K2HPO4, 2.5 g / L glucose (Dextrose), 20 g / L agar powder, adjusted to pH 7.2 with NaOH, then fixed to 1 L, sterilized at 121℃ for 15 min] for several days, the color of the medium gradually turned dark green, a large amount of biofilm was produced, and the bacterial liquid was viscous. Under the microscope, the cells were rod-shaped, the size of the bacteria was (1.5~3.0) μm × (0.5~0.8) μm, arranged singly or in pairs, with 1~3 flagella, active movement, no spores, able to form capsules, easily stained by common dyes, and Gram-negative.

[0086] 2. Molecular identification

[0087] Based on the 16S rDNA sequence, the phylogenetic tree was constructed using MEGA 7.0 software. Figure 2 As shown, it can be seen that the strain Pseudomonas aeruginosa The closest relative.

[0088] The results of morphological characteristics and homologous sequence analysis showed that the above strains belonged to the bacterial kingdom, Pseudomonas genus ( Pseudomonas sp.), Pseudomonas aeruginosa species ( Pseudomonas aeruginosa ). It was deposited in the China Center for Type Culture Collection (CCTCC) of Wuhan University, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; the deposit date is April 1, 2024; the deposit number is CCTCC NO: M 2024598; named Pseudomonas ( Pseudomonas sp.) B1207.

[0089] The present invention provides a strain of Pseudomonas ( Pseudomonas sp.) B1207 and its applications.

[0090] The Pseudomonas B1207 provided by the present invention has a strong colonization ability on colorful peanuts, can effectively prevent and treat white rot caused by Sclerotium sclerotiorum, and has the effect of promoting plant growth and development.

[0091] The invention comprises a method for isolating and obtaining the Pseudomonas B1207, which is a strain isolated from the root system of seven-color peanuts collected in Menglian County, Yunnan Province, and is obtained by adopting a plate dilution method.

[0092] The invention comprises application of the Pseudomonas B1207 in preventing and treating the white rot of colorful peanut caused by Sclerotium sclerotiorum and a method thereof.

[0093] The invention includes the application of the Pseudomonas B1207 in promoting the growth of colorful peanut plants and a method thereof.

[0094] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0095] The present invention will be further described below in conjunction with the embodiments.

[0096] Example 1: Isolation and disease resistance screening of Pseudomonas B1207

[0097] (1) Sample collection: Root samples of colorful peanuts were collected in Menglian County, Yunnan Province on August 17, 2023. After being rinsed with sterile water, they were placed in a 50 mL centrifuge tube containing 25% glycerol and stored in a dry ice bucket for later use.

[0098] (2) Isolation and purification: In a clean bench, rinse the sample with sterile water, sterilize with 75% alcohol for 30 seconds, rinse with sterile water three times, sterilize with 3% sodium hypochlorite for 2 minutes, rinse with sterile water three times, and finally dry the water with sterilized filter paper, and quickly bake it back and forth over the flame of an alcohol lamp 2 to 3 times. Thoroughly disinfect the surface of the sample. Put it in a sterile mortar, add a small amount of sterile water for grinding, dilute the slurry into suspensions of different gradients, take 100 μL and spread it on TSA solid culture medium, and culture it at 30℃ in a constant temperature incubator for 24 to 72 hours. Select single colonies with different morphology, color, and size, streak them on TSA plates, and culture them at 30℃ for 24 to 48 hours. Repeat the above operation until purified colonies are obtained, number them, and store them in 25% glycerol after liquid culture. Store them in a -80℃ refrigerator for the next step of screening antagonistic strains.

[0099] (3) Cultivation of sterile seedlings of colorful peanut potted plants: The potting medium was a mixture of vermiculite and organic matrix in a ratio of 4:1, which was sterilized at 121°C for 90 min. Selected colorful peanut seeds with full morphology were sterilized by soaking in 75% anhydrous ethanol for 30 s, rinsed with sterile water three times, and then soaked in 2.5% hydrogen peroxide silver ion disinfectant + 0.01% Tween-20 for 48 h, rinsed with sterile water four times, and sown in sterile matrix. The seeds were kept in a day-night mode of 16 h / 8 h, temperature of 30°C / 25°C, and light quantum flux of 200 μmol·m -2 ·s -1 Grow it in a greenhouse and you will get sterile peanut seedlings after about a week.

[0100] (4) Pathogenicity test of the pathogen (Sclerotium truncatum): This strain was isolated from a seven-color peanut continuous cropping test field with severe white peanut disease in the laboratory. It was determined to be Sclerotium truncatum through morphological characteristics and molecular identification and was stored in a -80℃ refrigerator in the laboratory.

[0101] Preparation for activation of Sclerotium uniformis: The preserved Sclerotium uniformis strain was activated on PDA medium (formula: 6 g / L potato flour, 20 g / L glucose, 20 g / L agar, adjusted to pH 5.6 with NaOH, then made up to 1 L, and sterilized at 121°C for 15 min). When the mycelium covered the plate, it was placed in the dark and cultured for more than 45 days to obtain Sclerotium uniformis covered with sclerotia.

[0102] Sterile peanut seedlings were selected, and after removing the cotyledons, they were transplanted into a 18×35 cm round glass pot. The mouth of the pot was sealed with a 0.45 μm filter membrane. The day and night mode was 16 h / 8 h, the temperature was 30℃ / 25℃, the humidity was above 90%, and the light quantum flux was 200 μmol·m -2 ·s -1After growing in a greenhouse for about 3 days, the plants were inoculated with neat sclerotia (3-4 sclerotia or 3 pieces of 3 mm cakes) and the incidence of the plants was observed and counted. Figure 3 shown.

[0103] The results of the pathogenicity test showed that lesions of varying degrees appeared on the rhizomes of the plants 4 days after inoculation of the sclerotia (or cakes) of Sclerotium sclerotiorum. As time went on, the main symptoms of the plants were irregular lesions, constriction symptoms, yellowing and wilting of leaves on the rhizomes, which were consistent with the symptoms of the field disease, with an incidence rate of 100%, and strong infectivity and pathogenicity. According to Koch's postulates, the diseased rhizomes in the experiment were placed on PDA plates for strain isolation, and the resulting strains had the same morphology as the inoculated strains, proving that the inoculated strains and the diseased strains were the same strains (Sclerotium sclerotiorum).

[0104] (5) Initial screening of antagonistic bacteria: The antagonistic activity of the above-mentioned isolated strains against S. rolf-milk pathogen was evaluated, and the antagonistic strains were screened out. The antagonistic test measured the radius of the test strain and the inhibition zone formed between the test strain and the S. rolf-milk pathogen. The smaller the radius of the S. rolf-milk pathogen, the stronger the test strain's ability to resist the S. rolf-milk pathogen. Three replicates of antagonism tests were performed independently for each strain. The pathogen strain was inoculated in the middle of the PDA plate, and the test strain was inoculated approximately 2 cm on both sides of the pathogen. The PDA plate inoculated with sterile water was used as the CK treatment. When the CK just grew all over the plate, the antagonistic activity of each tested strain was evaluated. The inhibition efficiency percentage was calculated using the following formula:

[0105] Inhibition efficiency percentage = (radius of pathogenic bacteria of Saccharomyces rolfsii in CK - radius of pathogenic bacteria of Saccharomyces rolfsii in test strain) / radius of pathogenic bacteria of Saccharomyces rolfsii in CK × 100%;

[0106] A total of 8 strains with significant antagonistic effects against Sclerotium were obtained. Figure 4 , Table 1.

[0107] Table 1: Results of confrontation experiments between different strains and Sclerotium uniformis

[0108]

[0109] (6) Rescreening of antagonistic strains: Rescreen the strains that have an inhibitory effect on pathogens in the initial screening. First, inoculate 0.5% of the rescreened strains into 2 mL of TSB liquid culture medium for recovery, and then inoculate 2% into 50 mL of TAB culture medium. Culture at 30°C and 120 rpm until the logarithmic growth phase (OD 600 value 0.8), the cells were collected after centrifugation, and resuspended with 10 mmol / L sterile MgCl2 solution. The bacterial solution was diluted to OD 600The value was 0.2. Sterile peanut seedlings with uniform shape and size were selected, 20 for each treatment, and the roots were immersed in the re-screened bacterial solution for 12 h (the control was immersed in sterile MgCl2), then transplanted into a 18×35 cm round glass flower pot. The bottle mouth was sealed with a 0.45 μm filter membrane. The day and night mode was 16 h / 8 h, the temperature was 30℃ / 25℃, the humidity was above 90%, and the photon flux was 200 μmol·m -2 ·s -1 After growing in a greenhouse for about 3 days, the plants were inoculated with sclerotia or cakes of Sclerotium sclerotiorum, and the incidence of the plants was observed and counted. It was found that the three endophytic bacteria, strains B0861, B0927 and B1207, showed a good control rate on the plants. Compared with the control, there were no symptoms of infection of Sclerotium sclerotiorum hyphae in the rhizomes. Figure 5 and Figure 6 shown.

[0110] Example 2: Test on the enhancement of plant immunity by Pseudomonas B1207

[0111] (1) Plant pretreatment

[0112] The strains with better control rate against Sclerotium in the rescreening (B0861, B0927, B1207) were selected and inoculated into 2 mL TSB liquid medium at a ratio of 0.5% for recovery, and then inoculated into 50 mL TAB medium at a ratio of 2%, and cultured at 30°C and 120 rpm until the logarithmic growth phase (OD 600 value 0.8), the cells were collected after centrifugation, and the bacterial solution was resuspended in 10 mmol / L sterile MgCl2 solution and diluted to OD 600 The value was 0.5; sterile seedlings of uniform shape and size were selected and transplanted into a 18×35 cm round glass flower pot. The bottle mouth was sealed with a 0.45 μm filter membrane. The day and night mode was 16 h / 8 h, the temperature was 30℃ / 25℃, the humidity was above 90%, and the light quantum flux was 200 μmol·m -2 ·s -1 After growing in a greenhouse for 3 days, the bacterial solution was inoculated into the roots, with 10 replicates for each treatment.

[0113] (2) Plant leaf ROS test

[0114] Seven days after inoculation, the leaf with the best growth was selected, and the living tissue was cut out with a 5 mm borer. The leaf was placed face up in a 96-well plate containing 100 μL of sterile water and incubated overnight at 22°C under continuous light to reduce stress response and measure the oxidative stress response of peanut leaves.

[0115] Remove the sterile water with a pipette, add 100 μL of immune test solution, which contains 200 μM luminol (3-amino-benzenedicarboxylic acid hydrazide) and 100 μg / mL Horseradish Peroxidase and 500 μg / mL chitin, then immediately place the plate in the microplate reader, measure the luminescence intensity once a minute at 470 nm (chemiluminescence measurement mode), and continue to measure for more than 80 minutes until the luminescence intensity drops to the control level. A blank control is set for each test, including a negative blank in which no leaves are added and physiological saline is used instead of bacterial solution. The integral data of the ROS kinetic curve is calculated using the Sintegral command of the R software Bolstad2 package, and the results are shown in the figure. Figure 7 As shown, it can be seen that the living tissue of the leaves can produce a large amount of ROS, which effectively enhances the immunity of the plant.

[0116] (3) Detection of callose deposition in plant roots

[0117] 7 days after inoculation, the plant was removed from the flowerpot, the root system was kept intact as much as possible, the root system was rinsed with sterile water, the main root segment about 2 cm to 5 cm away from the root tip was cut, and it was directly immersed in Carnoy's fixative (V acetic acid: V ethanol = 1:3) for 24 hours; it was immersed in anhydrous ethanol twice, 1 minute each time, and then transferred to at least 10 times the volume of 100% ethanol for preservation; before staining, the tissue was removed, immersed in 50% ethanol for 30 minutes, and slightly drained; then immersed in 1× PBS for 30 minutes, and slightly drained; the staining working solution was prepared before use, and the root tissue was immersed in callose staining working solution and stained for 1 hour at room temperature in the dark. 25 μL of aqueous gelatin was added to the slide to seal the slide, and the stained tissue was transferred to the slide. The images were taken using a fluorescence microscope (Leica DM6B) (excitation light 370 nm, emission light 590 nm) at 5x, 10x, and 20x objectives, with a resolution of 1,300 × 1,030; the exposure times were 700 ms, 350 ms, and 150 ms, respectively. Figure 8 As shown, a large amount of callus is deposited in the root system, which effectively enhances the immunity of the plant.

[0118] Example 3: Identification of Pseudomonas B1207

[0119] The bacterial genomic DNA of B1207 was extracted using a bacterial fungal genome extraction kit. 16S rDNA sequence amplification primers: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1), 1492R 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2). PCR amplification conditions: 96℃ 5 min; 96℃ 30 s; 56℃ 30 s; 72℃ 1 min; 30 cycles in total; 72℃ 10 min; 4℃ storage, PCR products were detected by 1% agarose gel electrophoresis, marked with 100~5000 bp marker, and the results were as follows Fig. 9 As shown, the DNA sequences were sent to Shenzhen BGI Co., Ltd. for purification and sequence determination. Basic BLAST on the NCBI website was used to compare the homology of the DNA sequences obtained by strain sequencing, and the phylogenetic tree was constructed using MEGA 7.0 software using the NJ method, as shown in Figure 2 As shown. Pseudomonas sp. were in the same branch of the evolutionary tree and were identified as Pseudomonas and named Pseudomonas ( Pseudomonas sp.) B1207.

[0120] Example 4: Evaluation of the Growth-Promoting Effect of Pseudomonas B1207

[0121] The preserved strain of Pseudomonas B1207 was inoculated into 2 mL of TSB liquid medium at a ratio of 0.5% for recovery, and then inoculated into 50 mL of TAB medium at a ratio of 2%, and cultured at 30°C and 120 rpm until the logarithmic growth phase (OD 600 value 0.8), the cells were collected after centrifugation, and the bacterial solution was resuspended in 10 mmol / L sterile MgCl2 solution and diluted to OD 600 The value was 0.2. Sterile peanut seedlings of uniform shape and size were selected. Ten seeds were used for each treatment. The roots were immersed in the rescreened bacterial solution for 12 h (the control was immersed in 10 mmol / L sterile MgCl2). Then, the seeds were transplanted into a 10×15 cm round pot and kept in a day / night mode of 16 h / 8 h, temperature of 30℃ / 25℃, and light quantum flux of 200 μmol·m -2 ·s -1 The plants were grown in a greenhouse at about 30 s and potted, and the growth of the plants was evaluated 30 days after transplanting.

[0122] Fig.10The results showed that under the condition of no pathogen stress, the strain could effectively promote plant growth and development. After 30 days of inoculation, the average plant height of the control was 7.5 cm, and the average plant height of B1207 was 10.57 cm, which was 40.9% higher than that of the control.

[0123] Example 5: Study on the Growth-Promoting Function of Pseudomonas B1207

[0124] 1. Siderophore production ability test: The activated single colony of Pseudomonas B1207 was inoculated on a universal CAS medium for detecting siderophores (chrome azurol 60.5 mg, cetyltrimethylammonium bromide 72.9 mg, FeCl3·6H2O 2.645 mg, peptone 4.5 g, glucose 9 g, beef extract powder 2.7 g, NaCl 4.5 g, agar 20 g, distilled water 1000 mL) and cultured at 30°C for 5 days. If a transparent zone appeared around the colony, it was positive. The diameter of the transparent zone D (cm) and the colony diameter d (cm) were measured, and the D / d value was calculated. The larger the ratio, the greater the siderophore production activity. Three repeated parallel experiments were performed.

[0125] Fig.11 The results showed that there was an obvious transparent zone around the Pseudomonas B1207 strain, and the D / d value was 1.80, indicating that the strain had the ability to produce siderophores.

[0126] 2. Test of organophosphorus solubility: Pick a single colony of activated Pseudomonas B1207 and spot-inoculate it on a plate of Mengina organophosphorus medium (formula: (NH4)2SO40.5 g, NaCl 0.3 g, KCl 0.3 g, glucose 10 g, FeSO4·7H2O 0.03 g, MnSO40.03 g, CaCO35 g, MgSO4·7H2O 0.3 g, lecithin 0.2 g, agar 20 g, distilled water 1000 mL), and culture it at 30℃ for 5 d. The appearance of a transparent zone around the colony is positive. The diameter of the transparent zone D (cm) and the diameter of the colony d (cm) are measured, and the D / d value is calculated. The larger the ratio, the stronger the organophosphorus solubility ability. Set up 3 repeated parallel experiments.

[0127] Fig.12 The results showed that there was an obvious transparent zone around the Pseudomonas B1207 strain, and the D / d value was 2.20, indicating that the strain had the ability to dissolve organic phosphorus.

[0128] 3. Test of inorganic phosphorus solubilization ability: Pick a single colony of activated Pseudomonas B1207 and inoculate it on a plate of Pikovasky's phosphate solubilization medium (NaCl 0.3 g, MgSO4·7H2O 0.3 g, MnSO40.03 g, KCl 0.3 g, (NH4)2SO40.5 g, FeSO4·7H2O 0.03 g, Ca3(PO4)25 g, glucose 10 g, agar 20 g, distilled water 1000 mL), and culture it at 30℃ for 5 days. If a transparent circle appears around the colony, it means that the functional strain has the ability to solubilize inorganic phosphorus. Determine the diameter D (cm) of the transparent circle and the colony diameter d (cm), and calculate the D / d value. The larger the ratio, the stronger the ability to solubilize inorganic phosphorus. Each experiment is repeated 3 times.

[0129] Fig.13 The results showed that there was an obvious transparent zone around the Pseudomonas B1207 strain, and the D / d value was 2.33, indicating that the strain had the ability to solubilize inorganic phosphorus.

[0130] 4. Amylase production ability test: Pick a single colony of activated Pseudomonas B1207 and inoculate it on a screening medium [screening medium formula (g / L): beef extract 0.5, yeast powder 10.0, KH2PO4 20.0, MgSO4·7H2O 0.5, soluble starch 10.0, agar 20.0, distilled water 1000 mL] plate, and culture at 30℃ for 2 days. After adding Lugol's iodine solution to the screening medium, rinse with deionized water, and the appearance of a transparent circle on the plate indicates that the strain has the ability to produce amylase. Determine the diameter of the transparent circle (D), and repeat each experiment 3 times.

[0131] Fig.14 The results showed that there was an obvious transparent zone around the Pseudomonas B1207 strain, and the D value was 0.7, indicating that the strain had the ability to produce starch.

[0132] The preparation method of the above-mentioned Lugol's iodine solution is as follows: add 5 g of potassium iodide to 20 mL of pure water and dissolve it with a stirring rod, add 2.5 g of iodine and stir for a period of time to completely dissolve it, and then make the volume to 50 mL.

[0133] 5. Catalase production test: Pick up the colonies on the solid culture medium with a toothpick and place them in a clean test tube. Add 2 mL of 3% hydrogen peroxide solution and observe the results. If bubbles occur within half a minute, it is positive, and if no bubbles occur, it is negative.

[0134] Fig.15 The results showed that after adding 3% hydrogen peroxide to the test tube, a large number of bubbles were immediately produced, indicating that the strain has the ability to produce H2O2 enzyme.

[0135] 6. Bacterial biofilm test: (1) Bacterial culture: Pick a single colony on the solid culture medium and inoculate it into TSB liquid culture medium. Incubate it in a shaker at 30°C until the logarithmic growth phase. (2) Biofilm formation: Add 4 μL of bacterial solution (1:500 dilution) to a borosilicate test tube containing 2 mL of TSB culture medium and place it in a 30°C constant temperature incubator for static culture for 96 h. (3) Biofilm visualization: Collect biofilm samples after 72 h, gently aspirate the suspended bacteria with a pipette, wash with sterile water three times (the operation should be gentle to avoid damaging the biofilm), stain the formed biofilm with 2.5 mL of 0.1% crystal violet for 20 min without shaking, aspirate the dye with a pipette, and wash the unbound dye with sterile water. Repeat three times. After cooling and drying, add 2 mL of 100% ethanol to the test tube. The biofilm formation ability can be judged according to the color depth.

[0136] Fig.16 The results showed that after 72 h of static culture in the test tube, a thin film was visible on the liquid surface. After staining and re-dissolution, the solution was dark purple, indicating that the strain has the ability to form a biofilm.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Pseudomonas ( Pseudomonas sp.), characterized in that The deposit number is CCTCC NO: M 2024598.

2. The Pseudomonas according to claim 1 ( Pseudomonas sp.) in at least one of the following: (A1), siderophore production; (A2), decomposition of organic phosphorus; (A3), decomposition of inorganic phosphorus; (A4), amylase production; (A5), catalase; (A6), bacterial biofilm production; (A7), inhibit or antagonize Sclerotium sclerotiorum ( Sclerotium rolfsii Sacc. ).

3. The Pseudomonas as claimed in claim 1 ( Pseudomonas sp.) in at least one of the following: (B1), promoting the production of ROS in peanut leaves; (B2) Promote callus deposition in peanut roots; (B3) Increase the growth rate and aboveground biomass of peanut.

4. The Pseudomonas as claimed in claim 1 ( Pseudomonas sp.) in at least one of the following: (C1), reduce peanut white rot; (C2), promote the growth and development of peanuts; (C3) Enhance peanut immunity.

5. A preparation, characterized in that comprising the Pseudomonas according to claim 1 ( Pseudomonas sp.).

6. The preparation according to claim 5, characterized in that Also included is MgCl2.

7. A product for planting, characterized in that include: (D1), the Pseudomonas according to claim 1 ( Pseudomonas sp.); or (D2) The preparation according to claim 5 or 6.

8. A culture method, characterized in that comprising the Pseudomonas according to claim 1 ( Pseudomonas sp.) into the culture medium.

9. A method for planting peanuts, characterized in that: Planting based on any of the following: (E1), the Pseudomonas according to claim 1 ( Pseudomonas sp.); or (E2) The preparation according to claim 5 or 6.

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