Pseudomonas and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to pseudomonas and application thereof.The pseudomonas comprises a 16S rDNA sequence shown in SEQ ID NO: 1 or a complementary sequence of the 16S rDNA sequence or a sequence which is at least 85% identical with the sequence shown in SEQ ID NO: 1, and particularly relates to Pseudomonas sp.Pseu.R1 with the preservation number of GDMCC No.63566. The invention further relates to the application of the pseudomonas. The Pseu.R1 provided by the embodiment of the invention has multiple functions of fixing nitrogen, dissolving organophosphorus, promoting plant growth, increasing yield and the like, discovers and enriches available microbial resources, and has important significance on increasing the crop yield.

Description

Technical Field

[0001] The present application relates to the field of microorganisms, and in particular to a Pseudomonas sp. and its application. Background Art

[0002] Fertilizers and pesticides play a major role in increasing agricultural production. However, their large-scale application has brought about a series of environmental impacts. For example, excessive application of fertilizers can easily lead to soil structure destruction, soil nutrient loss, soil compaction and secondary salinization of the soil environment. Irrational application of pesticides can lead to problems such as excessive pesticide residues in agricultural products and environmental pollution.

[0003] In recent years, the emergence of biofertilizers and biocontrol has alleviated the environmental impacts of chemical fertilizers and pesticides, and these beneficial microorganisms, such as plant rhizosphere growth-promoting bacteria, have been increasingly used in agricultural production. The genus Pseudomonas was established by Migula in 1894. Pseudomonas sp. is a Gram-negative bacterium that has the functions of producing antibiotics, iron carriers, and utilizing nutrients in the matrix. Therefore, it has great potential to promote crop growth and inhibit soil pathogens. In the related art, Pseudomonas has been shown to be useful for the prevention and control of plant pathogens (CN 114990009 A), and has a growth-promoting and yield-increasing effect on plants such as beans, peppers, and soybeans (CN 109880778 B, CN 114015629 B).

[0004] The development of new Pseudomonas strains with growth-promoting, yield-increasing and disease-resistant functions is of great significance for reducing the application of chemical fertilizers and promoting green agricultural development. Summary of the invention

[0005] To this end, the present application proposes a new strain of Pseudomonas and related applications thereof.

[0006] The first embodiment of the present application provides a Pseudomonas sp., which comprises the 16SrDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO: 1.

[0007] In some embodiments, the Pseudomonas is of the genus Pseudomonas, and the Pseudomonas has a 16S rDNA sequence as shown in SEQ ID NO:1.

[0008] In some embodiments, the Pseudomonas is Pseudomonas sp. Pseu.R1 having a deposit number of GDMCC No.63566.

[0009] The second aspect of the present application provides a method for culturing Pseudomonas as described in any embodiment of the first aspect, comprising inoculating the Pseudomonas into a culture medium for culturing, wherein the culture temperature is 20 to 40°C, preferably 25 to 37°C, more preferably 26 to 30°C, and most preferably 28°C.

[0010] In some embodiments, the culture medium is beef extract peptone medium.

[0011] In some embodiments, the pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.6.

[0012] In some embodiments, the culture medium includes peptone 8g / L to 12g / L, preferably 10g / L, beef extract 2g / L to 4g / L, preferably 3g / L, and sodium chloride 4g / L to 6g / L, preferably 5g / L.

[0013] The third aspect of the present application provides an agricultural preparation, comprising the Pseudomonas described in any embodiment of the first aspect or a fermentation broth thereof.

[0014] In some embodiments, the agricultural preparation is a liquid preparation or a freeze-dried preparation, and the dosage form of the agricultural preparation is selected from: wettable powder, water dispersible granules, suspension, aqueous emulsion, granules, seed coating agent or a combination thereof.

[0015] In some embodiments, the fermentation medium of the fermentation broth is beef extract peptone medium.

[0016] The fourth aspect of the present application proposes an application of the Pseudomonas described in any embodiment of the first aspect or the agricultural preparation described in any embodiment of the third aspect in plant nitrogen fixation.

[0017] The fifth aspect of the present application proposes the use of the Pseudomonas described in any embodiment of the first aspect or the agricultural preparation described in any embodiment of the third aspect in dissolving organic phosphorus.

[0018] The sixth aspect of the present application proposes a use of the Pseudomonas as described in any embodiment of the first aspect or the agricultural preparation as described in any embodiment of the third aspect in promoting plant growth.

[0019] In some embodiments, the application includes applying the Pseudomonas as described in any embodiment of the first aspect or the agricultural formulation as described in any embodiment of the third aspect to the plant. Optionally, the application includes: root irrigation, injection into the plant body and / or spraying on the plant surface. The plant surface optionally includes the surface of roots, stems, leaves, flowers, fruits and / or seeds.

[0020] In some embodiments, the plant is selected from the group consisting of Leguminosae, Poaceae and Solanaceae plants. Preferably, the Leguminous plant is selected from soybeans and beans, the Poaceae plant is millet, and the Solanaceae plant is pepper.

[0021] The embodiments of the present application achieve the following beneficial effects:

[0022] This application is the first to isolate and identify a new strain of Pseudomonas Pseudomonas sp. Pseu.R1 under the genus Pseudomonas, which exhibits many functions such as nitrogen fixation, dissolution of organic phosphorus, promotion of plant growth and increase in yield. The discovery and use of the new species of Pseudomonas proposed in this application enriches the available microbial resources and is of great significance to increasing crop yields; at the same time, by making it into a microbial fertilizer suitable for agricultural production, it can reduce the use of chemical fertilizers and promote green agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is a phylogenetic tree of Pseudomonas sp. Pseu.R1 according to Example 2 of the present application;

[0025] Figure 2 This is a plate growth diagram of Pseudomonas sp. Pseu. R1 according to Example 3 of the present application;

[0026] Figure 3 shows the microscopic morphology of Pseudomonas sp. Pseu. R1 according to Example 3 of the present application;

[0027] Figure 4 The growth of Pseudomonas sp. Pseu. R1 according to Example 4 of the present application on Ashby nitrogen-free medium is shown;

[0028] Figure 5 The phosphate dissolving effect of Pseudomonas sp. Pseu. R1 according to Example 4 of the present application is shown;

[0029] Figure 6 The growth-promoting effect of Pseudomonas sp. Pseu. R1 on soybean according to Example 5 of the present application is shown;

[0030] Figure 7The results show the growth-promoting and yield-increasing effect of Pseudomonas sp. Pseu. R1 on millet according to Example 6 of the present application.

[0031] Instructions for strain preservation:

[0032] Pseudomonas sp.Pseu.R1: The deposit registration number is GDMCC No.63566, and the deposit institution is: Guangdong Microbiological Culture Collection Center (GDMCC for short); the address of the deposit unit is: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the deposit time is June 16, 2023. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0034] This application is made based on the following knowledge of the inventor:

[0035] With the growth of the world's population and the reduction of arable land, food security has become a hot topic of concern. Over the past 40 years, my country has achieved a stable increase in national grain production through the application of chemical fertilizers and pesticides. However, the excessive application of chemical fertilizers and pesticides has brought about a series of environmental impacts. Excessive application of chemical fertilizers can easily lead to soil structure destruction, soil nutrient loss, soil compaction and secondary salinization of the soil environment; irrational application of pesticides will lead to excessive pesticide residues in agricultural products and environmental pollution. Plant rhizosphere growth-promoting bacteria refer to beneficial microorganisms that can colonize in the rhizosphere of plants and promote plant growth, development and stress resistance. The development and utilization of plant rhizosphere growth-promoting bacteria resources can reduce the amount of chemical fertilizers used, improve the utilization rate of chemical fertilizers, partially replace the application of pesticides, and alleviate the environmental impact of chemical fertilizers and pesticides.

[0036] The genus Pseudomonas was established by Migula in 1894. Pseudomonas is a Gram-negative bacterium that has the ability to produce antibiotics, iron carriers, and utilize nutrients in the matrix, so it has great potential to promote crop growth and inhibit soil pathogens. In addition, because Pseudomonas can grow rapidly, can show good colonization ability in the rhizosphere, and has strong adaptability to the environment, the application of Pseudomonas in agriculture has shown broad development prospects in recent years.

[0037] CN 114990009 A proposes the application of plant rhizosphere growth-promoting strain F13 in the preparation of disease-resistant growth-promoting and yield-increasing microbial agents, which are classified and named Pseudomonas aeruginosa, have antagonistic effects on plant pathogens, have drug-sensitive effects on antibiotics, can be used for field prevention and control of powdery mildew of summer black grapes or beans, and have growth-promoting and yield-increasing effects on beans. CN 109880778 B proposes a composite microbial agent with growth-promoting and yield-increasing effects on peppers and its application, the composite microbial agent includes fermentation liquid of Bacillus flexus HGD12, Pseudomonas putida HGD3 and Bacillus velezensis HP9 strains, which can achieve fertilizer reduction and pepper yield increase. In addition, CN 114015629 B proposes a composite bacterial agent that has a growth-promoting effect on soybeans. The composite bacterial agent is composed of a mixture of Pseudomonas sp. H1 fermentation liquid and Bacillus altitudinis Y1 fermentation liquid, and can be applied to soybean planting to promote the growth of soybean plants.

[0038] It can be seen that strengthening the screening and identification of new strains of Pseudomonas bacteria and further screening out new strains of Pseudomonas with growth-promoting functions are of great significance for reducing the application of chemical fertilizers and promoting green development of agriculture.

[0039] The present application proposes a new strain of Pseudomonas sp. Pseu.R1 (hereinafter referred to as "Pseu.R1") under the genus Pseudomonas, which has the functions of nitrogen fixation, dissolving organic phosphorus and promoting plant growth. The application of Pseu.R1 proposed in the present application can promote the absorption and utilization of nutrients such as nitrogen and phosphorus during plant growth, promote plant growth, and provide a new microbial strain resource for reducing the amount of chemical fertilizers and increasing their efficiency in agricultural production.

[0040] In a first aspect, an embodiment of the present application provides a Pseudomonas sp. comprising a 16S rDNA sequence shown in SEQ ID NO: 1 or a complementary sequence thereof or a sequence having at least 85% identity to SEQ ID NO: 1.

[0041] In the present application examples, the percentage of identity generally describes the degree to which two sequences are identical, that is, it generally describes the percentage of nucleotides corresponding to the same nucleotides of the reference sequence at their sequence positions. In the present application examples, the "sequence with at least 85% identity" refers to a sequence with an identity shown by any numerical value (including endpoint values) between 85% and 100% compared to the sequence shown in SEQ ID NO: 1, for example, it can have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and sequence identity shown by countless decimals between two adjacent integers, for example, it has at least 98.57%, 99.64%, 99.7%, 99.8% or 99.9% sequence identity compared to the sequence shown in SEQ ID NO: 1.

[0042] In the embodiments of the present application, Pseu.R1 is of the genus Pseudomonas. In some embodiments, Pseu.R1 has a 16S rDNA sequence as shown in SEQ ID NO:1.

[0043] Pseudomonas is an obligately aerobic, Gram-negative, non-spore-bearing, encapsulated bacillus that is rod-shaped or slightly curved. The body size is (0.5-1)×(1.5-4) microns. It has terminal flagella and can move. Some strains produce fluorescent pigments or (and) water-soluble pigments such as red, blue, yellow, and green, and do not ferment sugars. Pseudomonas exists in soil, fresh water, and sea water. After identification, Pseu.R1 proposed in the examples of this application is a new species under the genus Pseudomonas, which is named Pseudomonas sp.Pseu.R1 and is preserved in the Guangdong Provincial Microbiological Culture Collection Center. The address of the preservation unit is: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the preservation time is June 16, 2023; the preservation number is GDMCC No.63566.

[0044] In the embodiments of the present application, the strain Pseu.R1 having the sequence shown in SEQ ID NO:1 can be understood by those skilled in the art as the original strain. The strain having a genomic sequence with at least 85% identity to the sequence shown in SEQ ID NO:1 can be understood by those skilled in the art as a variant strain of the strain Pseu.R1 having the sequence shown in SEQ ID NO:1. It is understood that a strain (such as strain Pseu.R1) can undergo spontaneous mutation or be artificially cultured to form a variant strain, such as a nucleotide deletion, nucleotide addition, or nucleotide replacement. The "variant strain" has a highly identical gene sequence and extremely similar biological functions to the "strain Pseu.R1", and the mutated gene will basically not affect the conserved sequence of the strain Pseu.R1, and thus will not affect the genetic stability of the strain Pseu.R1. More specifically, the "variant strain" is also a strain of the species Pseu.R1, showing the physiological activity characteristics of the species Pseu.R1. The specific species corresponding to Pseu.R1 and all bacterial strains under it also fall within the scope of protection of the present application.

[0045] The new strain Pseu.R1 isolated in the examples of the present application can be effectively used to improve the agronomic traits of plants, especially to effectively fix nitrogen, dissolve phosphorus and promote plant growth, thereby reducing the amount of fertilizer applied and alleviating the environmental impact of fertilizers. The new strain of Pseudomonas with growth-promoting function proposed in the present application is of great significance to increasing crop yields.

[0046] A second aspect of an embodiment of the present application provides a method for culturing the Pseudomonas described in any embodiment of the first aspect, comprising inoculating the Pseudomonas into a culture medium for culturing, wherein the culture temperature is 20 to 40°C, preferably 25 to 37°C, more preferably 26 to 30°C, and most preferably 28°C.

[0047] In some embodiments, the culture medium is beef extract peptone medium.

[0048] In some embodiments, the pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.6,

[0049] In some embodiments, the culture medium includes peptone 8g / L to 12g / L, preferably 10g / L, beef extract 2g / L to 4g / L, preferably 3g / L, and sodium chloride 4g / L to 6g / L, preferably 5g / L.

[0050] It is understandable that the present application has no intention to limit the type of culture medium and specific parameters of the culture medium (such as pH, content of each component, etc.) involved in the culture method of Pseudomonas proposed in the examples, as long as the normal growth of Pseudomonas can be guaranteed.

[0051] An embodiment of the third aspect of the present application provides an agricultural preparation, comprising the Pseudomonas described in any embodiment of the first aspect or a fermentation broth containing the same.

[0052] In an embodiment of the present application, the agricultural preparation may contain any of the above-mentioned Pseudomonas strains, including: Pseudomonas comprising the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity compared with SEQ ID NO: 1, Pseudomonas having the 16S rDNA sequence shown in SEQ ID NO: 1 and / or Pseudomonas sp. Pseu. R1 having the deposit number GDMCC No. 63566; at the same time, since the above-mentioned strains also include mutant strains of the same species as Pseu.R1, the specific species of Pseu.R1 and all bacterial strains under it also fall within the protection scope of the agricultural preparation of the present application.

[0053] In an embodiment of the present application, the agricultural preparation further includes auxiliary materials, wherein the auxiliary materials may be organic matter and / or inorganic matter. In an embodiment of the present application, the organic matter may be, in addition to the strain Pseu.R1, other additional fertilizers that can promote plant growth, for example: fertilizers that increase soil nitrogen and crop nitrogen nutrition, such as rhizobium fertilizers, nitrogen-fixing fertilizers, nitrogen-fixing cyanobacteria fertilizers, etc.; fertilizers that decompose soil organic matter, such as organophosphorus bacterial fertilizers, comprehensive fertilizers; fertilizers that decompose soil insoluble minerals, such as phosphorus bacterial fertilizers, potassium bacterial fertilizers, mycorrhizal fungal fertilizers; fertilizers that stimulate plant growth, such as growth-promoting fertilizers; fertilizers that increase the resistance of crop roots to stress, such as antibiotic fertilizers, stress-resistant bacterial fertilizers. In an embodiment of the present application, the organic matter may also be organic fertilizers required for plant growth, such as manure, etc. It is understandable that the organic matter in the embodiment of the present application alone or in combination as long as it can ensure that it has a promoting effect on plant growth, the present application does not limit this.

[0054] In the embodiments of the present application, the inorganic substance may be a chemical component that can be used in agriculture, such as an agriculturally acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof, and / or an inorganic fertilizer that does not affect the concentration of microbial activity in the agricultural preparation, and the present application does not impose any restrictions on this.

[0055] In the embodiments of the present application, the agricultural preparation can be a liquid preparation or a freeze-dried preparation, and the specific dosage form can be selected from: wettable powder, water dispersible granules, suspension, aqueous emulsion, granules, seed coating, or a combination thereof. It is understood that the dosage form of the agricultural preparation in the embodiments of the present application only needs to ensure that it can be applied to plants in a certain form, and the present application does not limit this.

[0056] In the embodiments of the present application, the agricultural preparation can also be a fermentation broth comprising any of the above-mentioned Pseudomonas strains, the fermentation broth contains the Pseudomonas and its metabolites, and these metabolites can also be further used as biofertilizers or biocontrol drugs. In some embodiments, the fermentation medium of the fermentation broth can be a beef extract peptone medium. It is understood that the fermentation medium of the fermentation broth of the Pseudomonas strain in the embodiments of the present application can provide normal growth and metabolic fermentation of Pseudomonas, and the present application does not limit this.

[0057] The agricultural preparations proposed in the examples of this application can be effectively used to improve the agronomic traits of plants by using Pseu.R1, especially to effectively fix nitrogen, dissolve phosphorus and promote plant growth, thereby reducing the amount of fertilizer applied and alleviating the environmental impact of fertilizers. The new strain of Pseudomonas with growth-promoting function proposed in this application is of great significance to increasing crop yields.

[0058] The embodiments of the fourth aspect of the present application propose the use of the Pseudomonas as described in any embodiment of the first aspect or the agricultural preparation as described in any embodiment of the third aspect in plant nitrogen fixation, organic phosphorus dissolution and / or plant growth promotion.

[0059] In some embodiments, the use comprises applying the Pseudomonas described in any embodiment of the first aspect or the agricultural formulation described in any embodiment of the third aspect to the plant.

[0060] In some embodiments, the application may include: root irrigation, plant body injection and / or plant surface spraying, wherein the plant surface may include roots, stems, leaves, flowers, fruits and / or seed surfaces. In some embodiments, the plant may be subjected to root irrigation treatment during the sowing period and / or seedling stage of the plant.

[0061] In some embodiments, the plant is selected from the Leguminosae, Gramineae and Solanaceae plants. Preferably, the Leguminous plant is selected from soybeans and beans, more preferably soybeans; the Gramineae plant can be millet; the Solanaceae plant can be pepper.

[0062] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0063] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0064] Example 1: Isolation of strains

[0065] Collect the rhizosphere soil samples of soybeans, pour them into a mortar, add 2-3 ml of sterile water and grind them into powder. Use sterile water to dilute the grinding liquid to 10 2 Up to 10 5 Gradient concentrations. The gradient dilutions were spread on beef extract peptone solid culture medium, and sterile water without bacterial solution was spread as a blank control. After the coating was completed, it was inverted and cultured at 28°C for 48-72 hours. A single colony was picked and amplified in beef extract peptone liquid culture medium, and then repeatedly streaked and amplified, and purified to obtain a strain numbered Pseu.R1. The 16S rDNA gene of the Pseu.R1 monoclonal strain was sequenced, and the specific sequence is shown in SEQ ID NO: 1.

[0066] Pseu.R1 16S rDNA gene sequence (SEQ ID NO: 1):

[0067] CCACGGGGGGCGGCGGTCTACACATGCAAGTCGAGCGGATGAAGGGAGCTTGCTCTC

[0068] TGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGGATCTGCCTGGTAGTGGGGGACA

[0069] ACGTCTCGAAAGGGACGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGCTCTT

[0070] CGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGC

[0071] TCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTG

[0072] AGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGA

[0073] AAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACCTT

[0074] AAGTTGGGAGGAAAGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAAT

[0075] AAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGGTGCAAGCGTTA

[0076] ATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGC

[0077] CCCGGGCTCAACCCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTAGGGCAGAGG

[0078] GTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGG

[0079] CGAAGGCGACCACCTGGGCTCATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAA

[0080] ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAAT

[0081] CCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCC

[0082] GCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG

[0083] GTTAATTCGAAGCAACGCGAAGAACCTAACCAGGCCTTGACATCCAGTGAACTTACCA

[0084] GAGATGGTTTGGTGCCTTCGGGAACACTGAGACAGGTGCTGCATGGCTGTCGTCAGCT

[0085] CGTGTCGTGAGAATGTAGGGTTAAGTCCCGTAACGGAGCGCAACCCTTGTCCTTAGTT

[0086] ACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGG

[0087] TGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAAT

[0088] GGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGT

[0089] AGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGA

[0090] ATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT

[0091] GGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTACCACGGTTGATCTTCTGAG (SEQ ID NO: 1)

[0092] Example 2: Identification and evolutionary status determination of the new strain Pseu.R1

[0093] The sequence of Pseu.R1, about 1.4Kb, which is almost the full length of 16S rDNA, obtained by sequencing (i.e., SEQ ID NO: 1), was compared in the 16S rDNA gene database of EzBioCloud (https: / / www.ezbiocloud.net / ). The comparison results showed that the two strains with the highest homology to the 16S rDNA gene of Pseu.R1 in the database were Pseudomonashunanensis LV (T) (sequence number: JX545210) and Pseudomonas brassicae MAFF 212427 (T) (sequence number: LC514379), with similarities of 97.67% and 97.39%, respectively (both less than the new bacteria judgment standard of 98.65%). Based on the information of 16SrDNA, it can be determined that Pseu.R1 is a new species belonging to the genus Pseudomonas.

[0094] Furthermore, the sequences of closely related strains of Pseu.R1 were used to construct a phylogenetic tree using the neighbor-merging method using MAGA. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that Pseu.R1 is not clustered with other species of bacteria, which also proves that strain 1 isolated in Example 1 is a new species under the genus Pseudomonas.

[0095] Therefore, combined with the 16S rDNA sequence alignment and MAGA tree construction results, it can be determined that Pseu.R1 is a new species of the genus Pseudomonas, named Pseudomonas sp.Pseu.R1, and preserved in Guangdong Provincial Microbiological Culture Collection with the accession number GDMCC No.63566.

[0096] Example 3: Morphological observation of the new strain Pseu.R1

[0097] After cultivation, it was observed that the new strain Pseu.R1 provided in the present application was milky white, opaque, with round and smooth colonies and viscous ( Figure 2 ).

[0098] Figure 3 This is a microscopic observation image of Pseudomonas sp. Pseu. R1 in the embodiment of the present application. Figure 3 As shown, Pseudomonas sp.Pseu.R1 is a Gram-negative bacterium with a rod-shaped body.

[0099] Example 4: The new strain Pseu.R1 has nitrogen fixation and phosphorus solubilization properties

[0100] 5uL of the bacterial solution of strain Pseu.R1 was pipetted onto the Ashby nitrogen-free medium and then placed in a 30°C constant temperature biochemical incubator for 48 hours to observe the growth results. The results showed that Pseu.R1 could grow well on the Ashby nitrogen-free medium, confirming that the Pseu.R1 strain had good nitrogen fixation ability ( Figure 4 ), indicating that Pseu.R1 proposed in the embodiment of the present application is a nitrogen-fixing bacterium that can improve soil fertility. The formula of Ashby nitrogen-free medium is: 10g / L mannitol, 0.2g / L KH2PO4, 0.2g / L MgSO4·7H2O, 0.2g / L NaCl, 0.1g / L CaSO4·2H2O, 5g / L CaCO3.

[0101] 5uL of the bacterial solution of strain Pseu.R1 was pipetted on the solid medium of organophosphate in Montana and then placed in a 30℃ constant temperature biochemical incubator for 48 hours to observe the growth results. The results showed that Pseu.R1 could form an obvious phosphorus dissolving circle on the liquid medium of Montana, confirming that the strain Pseu.R1 had a good ability to dissolve organophosphate ( Figure 5), indicating that Pseu.R1 proposed in the embodiment of the present application is a phosphate-solubilizing bacterium, which can dissolve insoluble phosphorus and release microorganisms of effective phosphorus that can be absorbed by plants, and therefore has the potential to improve soil quality and structure, increase the effective utilization rate of phosphorus in the soil, save fertilizer and increase production. The formula of the Mengjina organic phosphorus solid culture medium is: MnSO4·4H2O 0.03g; FeSO4·7H2O 0.03g; CaCO35.0g; Glucose 10.0g; (NH4)2SO4 0.5g; Lecithin 0.2g; NaCl 0.3g; KCl 0.3g; Yeastextract paste 0.4g; Agar 20.0g, distilled water is used to make up to 1000mL, pH 7.0-7.5.

[0102] Example 5: The new strain Pseu.R1 has a soybean growth effect

[0103] 5.1 After activating the strain Pseu.R1, inoculate it into beef extract peptone medium and ferment it at 28°C and 180r / min for 5 days. Then, adjust the OD600 of the bacterial solution to 0.5, take the bacterial solution, centrifuge it at 5000rpm for 10min, remove the supernatant, and resuspend the bacteria in sterile water for later use.

[0104] 5.2 Use 1% sodium hypochlorite solution to disinfect the surface of soybean seeds (Zhonghuang 13) for 5 minutes, and rinse with sterile distilled water 5 times. Plant the disinfected seeds in sterile soil pots, where the sterile soil is obtained after high-temperature sterilization at 120°C. Plant 5 plants in each pot. After 5 days of seed germination, pour 5 ml of bacterial solution Pseu.R1 around the roots of the germinated plants in each pot. Set up 3 parallel groups for each treatment, and irrigate the control group with sterile water. Water once every 7 days, for a total of 2 times. After 30 days, take out the plants in the pots and measure their growth indicators. The specific measurement data are shown in Table 1. The growth conditions of the treatment group and the control group are shown in Table 1. Figure 6 shown.

[0105] Table 1

[0106] Different treatment Plant height(cm) Root length (cm) Fresh weight above ground (g) Underground fresh weight (g) Control group (mean ± SD) 54.60±8.87 24.70±6.42 7.56±3.31 2.77±1.67 Pseu.R1 (mean ± standard deviation) 77.8±11.25 27.4±7.12 9.98±1.91 2.80±1.63 growth rate 42.49％ 10.93％ 31.99％ 1.16％ p-value 0.0195 0.54 0.266 0.985 p-value sign * - - -

[0107] As can be seen from Table 1, compared with the control group, the application of the Pseu.R1 strain effectively increased the plant height, root length, aboveground fresh weight and underground fresh weight of soybeans, with growth rates of 42.49%, 10.93%, 31.99% and 1.16% respectively, that is, the effect of promoting plant growth was reflected in all indicators. The significant difference analysis showed that the application of the Pseu.R1 strain significantly increased the plant height of soybeans, indicating that the Pseu.R1 strain proposed in the examples of the present application has a beneficial effect on promoting the growth of the main stem.

[0108] Depend on Figure 6 It can be seen that in the planting experiment, the growth trend of the plants after applying the strain Pseu.R1 provided by the present invention was good, and compared with the control group, the plant height and underground root system were more luxuriant, indicating that the application of the Pseu.R1 strain can significantly promote the growth of leguminous plants such as soybeans; at the same time, the more luxuriant underground part is also conducive to fully exerting the nitrogen fixation function of the plant, improving the nutrient absorption of the plant rhizosphere, etc., thereby increasing the yield of the crop.

[0109] Example 6: The new strain Pseu.R1 has the effect of promoting millet growth and increasing yield

[0110] After the strain Pseu.R1 was activated, it was inoculated into beef extract peptone medium and fermented at 28°C and 180r / min for 5 days to obtain Pseu.R1 fermentation liquid. Millet (Huagu 12) was planted in field plots to verify the growth-promoting effect of Pseu.R1 on gramineous crops. Pseu.R1 fermentation liquid was inoculated at the seedling stage and jointing stage of millet respectively. When used, the fermentation liquid was diluted with sterile water at a ratio of 1:100, and each crop was irrigated with an equal amount of fermentation dilution liquid; at the same time, the plants were treated with an equal amount of sterile water as a control. After 35 days of growth, the crops were sampled and the biomass data were counted. The specific measurement data are shown in Table 2. The growth conditions of the treatment group and the control group are shown in Table 2. Figure 7 shown.

[0111] Table 2

[0112] Different treatment Plant height(cm) Root length (cm) Fresh weight above ground (g) Underground fresh weight (g) Ear weight (g) Control group (mean ± SD) 79.84±8.93 14.90±2.62 15.55±4.19 3.06±1.41 1.35±0.46 Pseu.R1 (mean ± standard deviation) 97.42±6.80 9.62±3.97 23.44±3.59 5.68±1.52 2.00±0.4 growth rate 22.02％ -35.44％ 50.74％ 85.62％ 48.15％ p-value 0.0058 0.0280 0.0164 0.0120 0.0316 p-value sign ** * * * *

[0113] As shown in Table 2, compared with the control group, the application of Pseu.R1 strain effectively increased the plant height, aboveground fresh weight, underground fresh weight and ear weight of millet, with growth rates of 22.02%, 50.74%, 85.62% and 48.15%, respectively. The significant difference analysis showed that the application of Pseu.R1 strain significantly increased the plant height of millet and significantly increased the aboveground fresh weight, underground fresh weight and ear weight of millet. Figure 7 It can be seen that compared with the control group, the plants after applying the strain Pseu.R1 provided by the present invention have a good growth trend, the plants are taller and the ears are larger, indicating that the application of the Pseu.R1 strain can significantly promote the growth of grass plants such as millet and has the function of increasing production. In addition, although the root length of millet is reduced after applying the Pseu.R1 strain compared with the control group, the underground fresh weight is significantly increased (Table 2). Figure 7 It can be seen that the root system of the treatment group is thicker, indicating that the application of Pseu.R1 strain can promote root development by promoting the thickness of roots and the increase in the number of roots, thereby achieving the beneficial effect of promoting plant growth.

[0114] Therefore, the rhizosphere growth-promoting bacteria Pseu.R1 newly isolated and identified in the examples of the present application has a good promoting effect on plant growth, indicating that the rhizosphere growth-promoting bacteria Pseu.R1 newly isolated and identified in the examples of the present application and the agricultural preparations containing it can be effectively used to promote the growth of various plants including Solanaceae plants and Leguminosae plants, which can effectively increase agricultural production, reduce the amount of fertilizers used, and alleviate the environmental impact of fertilizers. The new Pseudomonas strain Pseu.R1 with growth-promoting function proposed in this application is of great significance to increasing crop yields, preventing and controlling plant diseases, and protecting the environment.

[0115] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0116] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Pseudomonas, characterized in that The invention comprises the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence having at least 85% identity with SEQ ID NO:

1.

2. The Pseudomonas according to claim 1, characterized in that The Pseudomonas is of the genus Pseudomonas and has a 16S rDNA sequence as shown in SEQ ID NO:

1.

3. The Pseudomonas according to claim 1 or 2, characterized in that The Pseudomonas is Pseudomonas sp. Pseu. R1 with a deposit number of GDMCC No.63566.

4. A method for culturing the Pseudomonas according to any one of claims 1 to 3, characterized in that: The method comprises inoculating the Pseudomonas into a culture medium and culturing the culture at a temperature of 20 to 40° C., preferably 25 to 37° C., more preferably 26 to 30° C., and most preferably 28° C. Optionally, the culture medium is beef extract peptone medium, Optionally, the pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.6, Optionally, the culture medium comprises peptone 8g / L to 12g / L, preferably 10g / L, beef extract 2g / L to 4g / L, preferably 3g / L, and sodium chloride 4g / L to 6g / L, preferably 5g / L.

5. An agricultural preparation, characterized in that: A method comprising the Pseudomonas according to any one of claims 1 to 3 or a fermentation broth comprising the same, Optionally, the agricultural preparation is a liquid preparation or a freeze-dried preparation, and the dosage form of the agricultural preparation is selected from: wettable powder, water dispersible granules, suspension, aqueous emulsion, granules, seed coating agent or a combination thereof, Optionally, the fermentation medium of the fermentation broth is beef extract peptone medium.

6. Use of the Pseudomonas according to any one of claims 1 to 3 or the agricultural preparation according to claim 5 in nitrogen fixation in plants.

7. Use of the Pseudomonas according to any one of claims 1 to 3 or the agricultural preparation according to claim 5 in dissolving organic phosphorus.

8. Use of the Pseudomonas according to any one of claims 1 to 3 or the agricultural preparation according to claim 5 in promoting plant growth.

9. The use according to claim 8, characterized in that: The use comprises applying the Pseudomonas according to any one of claims 1 to 3 or the agricultural preparation according to claim 5 to plants, Optionally, the application comprises: root irrigation, injection into the plant body and / or spraying on the plant surface, and the plant surface optionally comprises the surface of roots, stems, leaves, flowers, fruits and / or seeds.

10. The use according to claim 8 or 9, characterized in that: The plant is selected from the group consisting of Leguminosae, Gramineae and Solanaceae plants. Preferably, the Leguminous plant is selected from soybeans and beans, the Gramineae plant is millet, and the Solanaceae plant is pepper.

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