Pseudomonas chlororaphis and application thereof

Abstract

The invention relates to pseudomonas chlororaphis and application thereof, and belongs to the technical field of microorganisms. The method comprises the following steps: firstly, screening and separating a strain which has an antagonistic effect on a plurality of pathogenic bacteria of corns and soybeans from crop rhizosphere soil samples collected from a strip-shaped composite planting field of the soybeans and the corns, and preserving the strain in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.31744; through verification, the pseudomonas chlororaphis provided by the invention has a very good antagonistic effect on various pathogenic bacteria of corn and soybeans, and has a very good growth promoting effect on plants such as corn and soybeans; the strain provided by the invention can be used as a biocontrol strain for preventing and treating corn and soybean diseases, and provides guarantee for improving the yield and quality of corn and soybeans.

Description

Technical Field

[0001] The invention relates to Pseudomonas chlororaphis and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] The strip-shaped composite planting of soybeans and corn is an important technology for stabilizing grain production and increasing oil production. It can effectively ensure the maximization of the effectiveness, efficiency and benefits of grain and oil planting, guarantee stable and high yields of grain crops, and expand the planting and supply of oil crops. It is an important way to alleviate the contradiction of land competition for grain and oil in my country and achieve the unity of tapping potential and ensuring supply with sustainable development.

[0003] However, the occurrence of soybean and corn diseases is one of the important factors that restrict their harvest yield and quality. At present, soybean and corn diseases are mainly controlled by chemical agents, and drug residues and environmental pollution are the main risks. Excessive use of chemical agents will not only make pathogens resistant to drugs, but also directly pollute water and soil, thus posing risks to human health.

[0004] Using biocontrol strains or fungicides to control diseases is one of the technical means to reduce the use of pesticides. In recent years, biological control has attracted attention due to its environmental friendliness and high safety. Among them, microbial fungicides have become increasingly valued due to their relative safety, no residue, and environmental friendliness, and have become the focus of international green pesticide development. However, at present, there are reports on biocontrol bacteria that can control soybean or corn diseases alone, but there are no reports on biocontrol strains that can be used on both soybean and corn crops at the same time. Summary of the invention

[0005] In view of some problems existing in the prior art, the present invention provides a Pseudomonas chlororaphis and its application. The strain provided by the present invention also has a good antagonistic effect on corn and soybean pathogens. The present invention provides a possible biocontrol strain for reducing the use of pesticides in soybean and corn strip planting fields.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] The present invention first provides Pseudomonas chlororaphis, which is classified and named as Pseudomonas chlororaphis DY8, and is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on August 26, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 31744.

[0008] Wherein, the Pseudomonas chlororaphis DY8 is derived from the crop rhizosphere soil of the soybean-corn strip composite planting field.

[0009] Further, according to the embodiments of the present invention, in experience, the strains screened and isolated by the present invention have the following morphological characteristics: forming orange-yellow colonies on the LB plate, with raised surfaces, neat edges, and gloss.

[0010] The present invention also provides a bacterial suspension or fermentation liquid containing the Pseudomonas chlororaphis.

[0011] The present invention also provides the metabolites of the Pseudomonas chlororaphis.

[0012] The present invention also provides a biocontrol agent containing the Pseudomonas chlororaphis, or the bacterial suspension or fermentation liquid, or the metabolite.

[0013] The concentration of Pseudomonas chlororaphis DY8 in the biocontrol agent is 1.8×10 9 CFU / mL.

[0014] The present invention also provides the use of the Pseudomonas chlororaphis, or the bacterial suspension or fermentation liquid in promoting plant growth, wherein the plants include corn and soybean.

[0015] The present invention also provides the use of the Pseudomonas chlororaphis, or the bacterial suspension or fermentation liquid, or the metabolite, or the biocontrol agent in antagonizing plant pathogens, wherein the plant pathogens include: corn sheath blight, corn leaf blight, soybean phomopsis, soybean root rot verticillium and soybean root rot Fusarium.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The chlororaphis Pseudomonas DY8 provided by the present invention has good antagonistic activity against common corn and soybean pathogens; among them, the width of the inhibition zone against corn sheath blight is 6 mm, the width of the inhibition zone against corn leaf blight is 12 mm, the width of the inhibition zone against soybean phomopsis is 8 mm, the width of the inhibition zone against soybean root rot verticillium is 10 mm, and the width of the inhibition zone against soybean root rot Fusarium is 11 mm, all of which have obvious inhibition zones.

[0018] (2) The Pseudomonas chlororaphis DY8 provided by the present invention has a significant promoting effect on the rooting and germination of corn and soybean seeds. Experimental verification shows that the root length of corn seeds treated with DY8 is 12.46% longer than that of the control group, and the sprout height is 21.71% higher than that of the control group; the weight of soybean seeds treated with DY8 is 50.82% heavier than that of the control group; and the sprout height is 26.64% higher than that of the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1It is the morphological characteristics and phylogenetic tree diagram of Pseudomonas chlororaphis DY8; Figure A is the morphological characteristics diagram of Pseudomonas chlororaphis DY8; Figure B is the phylogenetic tree diagram of Pseudomonas chlororaphis DY8 constructed based on the 16S rRNA gene.

[0020] Figure 2 This is a graph showing the verification results of the antagonistic ability of Pseudomonas chlororaphis DY8 against soybean and corn pathogens.

[0021] Figure 3 This is the result of high-resolution mass spectrometry HRMS detection of active metabolites of Pseudomonas chlororaphis DY8.

[0022] Figure 4 It is the active metabolite of Pseudomonas chlororaphis DY8. 1 H NMR spectra.

[0023] Figure 5 It is the active metabolite of Pseudomonas chlororaphis DY8. 13 C NMR spectrum.

[0024] Figure 6 It is the result of liquid chromatography detection of active metabolites of Pseudomonas chlororaphis DY8.

[0025] Figure 7 These are the results of an experiment in which Pseudomonas chlororaphis DY8 promotes the rooting of corn and soybean seeds.

[0026] Figure 8 These are the results of an experiment in which Pseudomonas chlororaphis DY8 promoted the germination of corn and soybean seeds. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0028] In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods, and those processes involved in the embodiments that are not described in detail are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail again.

[0029] The preparation methods of the LB liquid culture medium, LB culture medium plate (LB plate), and PDA plate described in the examples are all conventional methods in the art.

[0030] Example 1: Strain screening and identification

[0031] Soybean-maize rhizosphere soil samples were collected from soybean-maize strip-composite planting fields in Liuhe District, Nanjing City, Jiangsu Province.

[0032] Place 1g of soil sample in a 50mL sterile centrifuge tube, add sterile water and glass beads, and shake at 180rpm / min for 30min; take the suspension after shaking and use sterile water to make gradient dilutions from 10 3 , 10 4 100 μL of each of the two-fold diluted bacterial suspensions was evenly spread on LB medium plates and cultured at 28°C for 48 h; single colonies with different morphologies were transferred to new LB plates for purification; the purified colonies were subjected to antagonism experiments with the test strains (corn sheath blight pathogen and soybean phomopsis pathogen, both of which were independently isolated and preserved by the applicant's laboratory from susceptible crops in soybean-corn strip composite planting fields in Liuhe District, Nanjing City, Jiangsu Province, and the isolation and preservation methods were conventional methods in the art).

[0033] The specific operation of the antagonism experiment in this embodiment is as follows:

[0034] The test strains of corn sheath blight and soybean phomopsis were inoculated on a PDA plate and incubated in a 28°C incubator for 3 days before use. The single colony screened and purified in this example was inoculated into LB liquid culture medium and incubated overnight in a horizontal shaker at 28°C. Use a 5mm diameter punch to punch a bacterial cake at the edge of the test bacterial colony, and inoculate the taken bacterial cake in the center of a new PDA plate. The bacterial suspension of the strain screened and purified in this example was spotted in the center of the bacterial cake and the edge of the plate, and then the plate was placed in a 28°C incubator for static incubation. After 4 days, the antibacterial effect was observed. The selected strains with good antagonistic effects were further protected with 20% glycerol as a protective agent, and then suspended and frozen in a -70°C ultra-low temperature refrigerator.

[0035] like Figure 1 As shown in A, the strain isolated and screened in the present invention forms orange-yellow colonies on the LB plate, and the colony surface is raised, the edges are neat and shiny.

[0036] The isolated strains were subjected to the following molecular biological identification:

[0037] The purified single colony on the LB medium was picked up, cultured with LB liquid medium, shaken at 180 rpm and 28 °C for 12 h, the bacteria were collected by centrifugation, and the genomic DNA of the strain was extracted using a DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd., catalog number: DP302-02).

[0038] The 16S rDNA sequences of the strains were amplified using the 16S rDNA universal primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′.

[0039] The PCR reaction system (25 μL) was: 10.5 μL ddH2O, 12.5 μL 2×Es Taq MasterMix (Dye), 0.5 μL each of 27F and 1492R primers, and 1 μL of strain DNA.

[0040] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, renaturation at 72°C for 1 min, repeated 30 cycles, and extension at 72°C for 10 min.

[0041] The PCR product was sent to a sequencing company for sequencing after being confirmed by agarose gel electrophoresis. The 16SrDNA gene sequence of the strain obtained by sequencing is shown in SEQ ID No: 1. The sequence obtained by sequencing was subjected to BLASTA sequence comparison analysis in NCBI. The sequence of the strain separated and purified in this example was 98.41%-99.37% consistent with the 16S rDNA gene sequence of Pseudomonas chlororaphis. The sequences of the standard strains with high similarity in the comparison results were selected, and the phylogenetic tree was constructed using MEGA7.0.26 software, as shown in FIG. Figure 1 As shown in B, the strain separated and purified by the present invention is most closely related to Pseudomonas chlororaphis and they are clustered into one branch.

[0042] Based on the above molecular identification results, the strain isolated and purified in the present invention belongs to Pseudomonas chlororaphis, and is named Pseudomonas chlororaphis DY8.

[0043] The sequence information of the 16S rRNA fragment of Pseudomonas chlororaphis DY8 separated and purified by the present invention is shown in SEQ ID No: 1:

[0044]

[0045] Example 2: Preparation of Pseudomonas chlororaphis DY8 bacterial agent

[0046] The glycerol strain of Pseudomonas chlororaphis DY8 of the present invention was streaked onto an LB medium plate and cultured at 28° C. for 24 h; a single colony was picked and inoculated into LB liquid culture medium and cultured on a shaker at 28° C. for 18 h with a shaking frequency of 180 rpm.

[0047] The bacterial concentration of Pseudomonas chlororaphis DY8 inoculum obtained by dilution plate spreading method was 1.8×10 9 CFU / mL.

[0048] The Pseudomonas chlororaphis DY8 screened by the present invention can be used to prepare a bacterial agent for biological control.

[0049] Example 3: Detection of biocontrol ability of Pseudomonas chlororaphis DY8

[0050] The common pathogens of soybean and corn diseases were used as test strains, and the plate antagonism method was used to detect the biocontrol ability of DY8.

[0051] The plant pathogens tested include: corn sheath blight, corn leaf blight, soybean phomopsis, soybean root rot verticillium, and soybean root rot fusarium (the laboratory independently isolated and preserved them from susceptible crops in the soybean-corn strip-planting fields in Liuhe District, Nanjing City, Jiangsu Province, and the isolation and preservation methods are conventional methods in the field).

[0052] The above test strains were inoculated on PDA plates and cultured in a 28°C incubator for 3 days before use.

[0053] Pick a single colony of DY8, inoculate it into LB liquid medium, and culture it overnight on a horizontal shaker at 28°C. Use a 5mm diameter puncher to punch a bacterial cake at the edge of the pathogen colony, and inoculate the taken bacterial cake in the center of a new PDA plate. Dot the cultured DY8 bacterial suspension in the center of the bacterial cake and the edge of the plate, and then place the plate in a 28°C incubator for static culture. After 4 days, observe the antibacterial effect.

[0054] The final antibacterial effect of DY8 is as follows Figure 2 As shown, the width of the inhibition zone of DY8 against corn sheath blight is 6 mm, the width of the inhibition zone against corn leaf blight is 12 mm, the width of the inhibition zone against soybean phomopsis is 8 mm, the width of the inhibition zone against soybean root rot verticillium is 10 mm, and the width of the inhibition zone against soybean root rot Fusarium is 11 mm. In summary, Pseudomonas chlororaphis DY8 has good antagonistic activity against the five pathogenic fungi tested.

[0055] Example 4: Detection of active metabolites of Pseudomonas chlororaphis DY8

[0056] A single colony of DY8 was picked and inoculated into 200 mL of LB liquid culture medium, and cultured on a horizontal shaker at 28°C for 48 h. The metabolites in the fermentation broth were extracted with 200 mL of ethyl acetate, and the organic phase was evaporated to dryness using a rotary evaporator, and the crude extract was dissolved with 1 mL of methanol. High performance liquid chromatography was used to separate and purify the substances in the crude extract using ddH2O and acetonitrile as mobile phases. The purified samples were detected by nuclear magnetic resonance and mass spectrometry, and it was finally determined that the active metabolites of Pseudomonas chlororaphis were phenazine-1-carboxylic acid, 2-hydroxy-phenazine-1-carboxylic acid, 2-hydroxyphenazine ( Figure 3-Figure 6 ), these three substances have been reported to have antibacterial activity against pathogenic fungi, bacteria, etc.; it further shows that the green needle Pseudomonas DY8 screened and isolated by the present invention has antibacterial activity against pathogenic fungi, bacteria, etc., and can be used as a biocontrol agent for disease prevention and control.

[0057] Example 5: Detection of the growth-promoting ability of Pseudomonas chlororaphis DY8

[0058] Filter paper was placed in 4 glass culture dishes and sterile water was added to keep them moist. In 2 of the culture dishes, 50 untreated corn and soybean seeds (commercially available, corn variety is Lianhuanuo No. 2, soybean variety is Zhongjiang 202) were placed as the control group (CK), and in the other 2 culture dishes, 50 corn and soybean seeds soaked in DY8 bacterial suspension were placed as the experimental group (DY8). The 4 culture dishes were placed in a 28°C incubator for rooting experiments. After 4 days, it was observed that the roots of corn seeds treated with DY8 increased significantly, while the roots of soybean seeds treated with DY8 were significantly thicker. After statistical analysis of root weight / length, it was found that DY8 had a significant growth-promoting effect on the rooting of soybeans and corn. Among them, the root length of corn seeds increased significantly after DY8 treatment, with a growth rate of 12.46%, and the root and stem weight of soybean seeds treated with DY8 increased significantly, with a weight gain rate of 50.82% ( Figure 7 ).

[0059] Sterilized nutrient soil (purchased from Huaian Wolong Agricultural Technology Co., Ltd.) with a thickness of about 8 cm was spread in two flower pots. 36 untreated corn and soybean seeds were planted in the left half of the two flower pots, and 36 corn and soybean seeds soaked in DY8 bacterial suspension were planted in the right half. The flower pots were placed in a 28°C incubator for germination experiments. After 7 days, it was observed that the sprout lengths of corn and soybean treated with DY8 were both longer than those of the control group. The sprout lengths of corn treated with DY8 were 21.71% and 26.64% higher than those of the control group ( Figure 8 ), revealing that Pseudomonas chlororaphis DY8 has a significant growth-promoting effect on soybean and corn germination.

[0060] The above embodiments are detailed descriptions of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. For those skilled in the art, any modifications made without departing from the content, spirit and scope of the present invention are within the protection scope of the present invention.

Claims

1. A Pseudomonas chlororaphis, characterized in that The classification name of the Pseudomonas chlororaphis is Pseudomonas chlororaphis DY8, and the preservation number is CGMCC No.31744.

2. The Pseudomonas chlororaphis according to claim 1, characterized in that The Pseudomonas chlororaphis DY8 is derived from the crop rhizosphere soil of a soybean-corn strip composite planting field.

3. A bacterial suspension or fermentation broth comprising the Pseudomonas chlororaphis according to claim 1.

4. The metabolite of Pseudomonas chlororaphis according to claim 1.

5. A biocontrol agent comprising the Pseudomonas chlororaphis according to claim 1, or the bacterial suspension or fermentation broth according to claim 3, or the metabolite according to claim 4.

6. The biocontrol agent according to claim 5, characterized in that In the biocontrol agent, the concentration of Pseudomonas chlororaphis DY8 is 1.8×10 9 CFU / mL.

7. Use of the Pseudomonas chlororaphis according to claim 1 or 2, or the bacterial suspension or fermentation liquid according to claim 3 in promoting plant growth or seed germination.

8. The use according to claim 7, characterized in that: The plants include corn and soybean.

9. Use of the Pseudomonas chlororaphis according to claim 1 or 2, or the bacterial suspension or fermentation broth according to claim 3, or the metabolite according to claim 4, or the biocontrol agent according to claim 5 in preventing or antagonizing plant pathogens.

10. The use according to claim 9, characterized in that: The plant pathogens include: corn sheath blight pathogen, corn leaf blight pathogen, soybean phomopsis pathogen, soybean root rot verticillium pathogen and soybean root rot fusarium pathogen.

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