Burkholderia parakholderia with plant growth promoting effect and application of burkholderia parakholderia

Abstract

The invention relates to the field of microorganisms, in particular to burkholderia parakholderia with a plant growth promoting effect and application of the burkholderia parakholderia, and the burkholderia parakholderia 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. The application of the burkholderia paraburkholderia strydomiana Raa.R5 and the composition of the burkholderia paraburkholderia strydomiana Raa.R5 provided by the invention in promoting the growth of the crops has an important significance in improving the yield of the crops and promoting the green production of the agriculture.

Description

Technical Field

[0001] This application relates to the field of microorganisms, and particularly to a Paraburkholderia and its application with the function of promoting plant growth. Background Art

[0002] Although the application of chemical fertilizers helps to increase grain production steadily, excessive application thereof is likely to cause environmental problems such as damage to soil structure, loss of soil nutrients, soil compaction, and secondary salinization of soil environment. Plant growth-promoting rhizobacteria 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 growth-promoting rhizobacteria resources can reduce the application amount of chemical fertilizers and lower the utilization rate of chemical fertilizers, and thus have received extensive attention in recent years.

[0003] Therefore, the development of new bacterial strains that can be used for plant growth promotion to reduce the use of chemical fertilizers is of great significance for reducing environmental pollution and achieving green production. Summary of the Invention

[0004] To this end, the embodiments of this application provide a new strain of Paraburkholderia with growth-promoting function, a composite preparation containing the same, and their applications.

[0005] The first aspect of the embodiments of this application provides a Paraburkholderia, which is characterized by containing 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.

[0006] In some embodiments, the Paraburkholderia is of the genus Paraburkholderia sp.; preferably, the systematic classification of the Paraburkholderia is Paraburkholderia strydomiana; more preferably, the Paraburkholderia has the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence; most preferably, the Paraburkholderia is Paraburkholderia strydomiana Rara.R5 with the preservation number GDMCC No. 63462.

[0007] The second aspect of the embodiments of this application provides an application of the Paraburkholderia according to any one of the above embodiments in promoting plant growth.

[0008] In some embodiments, the Paraburkholderia is used to promote plant nitrogen fixation and / or nodulation.

[0009] In some embodiments, the plants are selected from gramineous plants and leguminous plants. Preferably, the gramineous plants are selected from corn, millet, and upland rice, and the leguminous plants are selected from soybean and alfalfa.

[0010] An embodiment of the third aspect of the present application provides a composition, which comprises Burkholderia parabrevis as described in any embodiment of the first aspect above and Rhizobium sp.

[0011] In some embodiments, the Rhizobium sp. comprises the 16S rDNA sequence shown in SEQ ID NO: 2 or its complementary sequence or a sequence with at least 85% identity compared to SEQ ID NO: 2.

[0012] In some embodiments, the systematic classification of the Rhizobium is Rhizobium grahamii; preferably, the Rhizobium has the 16S rDNA sequence shown in SEQ ID NO: 2 or its complementary sequence; more preferably, the Rhizobium is Rhizobium grahamii Rhiz.R2 with the preservation number GDMCC No. 63570.

[0013] An embodiment of the fourth aspect of the present application provides an agricultural preparation, which comprises Burkholderia parabrevis as described in any embodiment of the first aspect above or the composition as described in any embodiment of the third aspect above.

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

[0015] An embodiment of the fifth aspect of the present application provides an application of the composition as described in any embodiment of the third aspect above or the agricultural preparation as described in any embodiment of the fourth aspect above in promoting plant growth.

[0016] In some embodiments, the composition or the agricultural preparation is used to promote plant nitrogen fixation and / or nodulation.

[0017] In some embodiments, the plants are selected from gramineous plants and leguminous plants. Preferably, the gramineous plants are selected from corn, millet and upland rice, and the leguminous plants are selected from soybean and alfalfa.

[0018] The present application achieves the following beneficial effects:

[0019] It was found in this application that strains of Paraburkholderia sp., especially those under the species Paraburkholderia strydomiana, have plant growth-promoting effects, and a new strain of Paraburkholderia, Rara.R5, under this genus was isolated. This strain showed good growth-promoting and yield-increasing effects when applied alone or in combination with strains of Rhizobium sp., especially those under the species Rhizobium grahamii. In particular, the combination of the two bacteria achieved synergistic effects in nitrogen fixation and nodulation. Therefore, the combination of Paraburkholderia and Rhizobium proposed in the examples of this application can be effectively used for plant growth promotion, especially for promoting root nodulation and nitrogen fixation, which is of great significance for increasing crop yields and achieving green agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 Phylogenetic tree of Rara.R5 according to Example 2 of the present application.

[0022] Figure 2 Phylogenetic tree of Rhiz.R2 according to Example 2 of the present application.

[0023] Figure 3 Plate growth diagram of Rara.R5 according to Example 3 of the present application.

[0024] Figure 4 Shows the growth of Rara.R5 on Ashby nitrogen-free medium according to Example 4 of the present application.

[0025] Figure 5 Shows the legume growth-promoting effect of Rara.R5 according to Example 5 of the present application.

[0026] Figure 6 Shows the grass growth-promoting effect of Rara.R5 according to Example 5 of the present application, where a, upland rice; b, millet; c, corn.

[0027] Figure 7 Shows the nodulation-promoting effect of Rhiz.R2 according to Example 6 of the present application.

[0028] Figure 8It shows the synergistic effect of Rara.R5 and Rhiz.R2 in promoting plant growth according to Embodiment 7 of the present application.

[0029] Description of strain preservation:

[0030] Paraburkholderia strydomiana Rara.R5: The preservation registration number is GDMCC No: 63462, and the preservation institution is: Guangdong Provincial Microbial Culture Collection Center; The address of the preservation unit is: 5th Floor, Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; The preservation time is May 14, 2023.

[0031] Rhizobium grahamii Rhiz.R2: The preservation registration number is GDMCC No: 63570, and the preservation institution is: Guangdong Provincial Microbial Culture Collection Center; The address of the preservation unit is: 5th Floor, Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; The preservation time is June 16, 2023. Specific embodiments

[0032] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention and do not limit the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0033] This application is based on the following understanding of the inventors:

[0034] In the related art, some strains of the genus Paraburkholderia sp. have been reported to have the functions of degrading petroleum hydrocarbons and transforming organically bound cadmium in soil, and can be used for the treatment of contaminated soil (CN 113430138 A, CN 110317741B).

[0035] Through a large number of experimental analyses, the inventors of this application found that Paraburkholderia sp. has the function of promoting plant growth. A strain of Paraburkholderia strydomiana Rara.R5 (hereinafter referred to as Rara.R5) was isolated from soybean rhizosphere soil. This strain showed good effects of promoting growth and increasing yield when applied alone or in combination with rhizobia (Rhizobium sp.), especially the strains under Rhizobium grahamii. In particular, the combined application of the two bacteria achieved synergistic effects in promoting nitrogen fixation and nodulation of plants. Therefore, the combination of Paraburkholderia and rhizobia proposed in the embodiments of this application can be effectively used for promoting plant growth, especially promoting root nodulation and nitrogen fixation, which is of great significance for increasing crop yields and realizing green agricultural production.

[0036] Furthermore, the embodiments of this application also propose a rhizobium Rhiz.R2, which shows synergistic effects when applied in combination with Rara.R5, providing a new resource for the green production of plants.

[0037] The embodiments of the first aspect of this application propose a Paraburkholderia bacterium, which contains the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity compared to SEQ ID NO: 1.

[0038] In the embodiments of this application, the percentage of identity generally describes the degree of similarity between two sequences, that is, it generally describes the percentage of nucleotides that are the same as the reference sequence at their sequence positions. In the embodiments of this application, the "sequence with at least 85% identity" refers to a sequence with an identity between 85% and 100% (including the endpoint values) compared to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. For example, it can have a sequence identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, as well as the sequence identity shown by countless decimals between two adjacent integers. For example, compared to the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, it has a sequence identity of at least 98.57%, 99.64%, 99.7%, 99.8% or 99.9%.

[0039] In some embodiments, the Paraburkholderia is Paraburkholderia sp.; preferably, the systematic classification of the Paraburkholderia is Paraburkholderia strydomiana; more preferably, the Paraburkholderia has a 16S rDNA sequence as shown in SEQ ID NO: 1 or its complementary sequence; most preferably, the Paraburkholderia is Paraburkholderia strydomiana Rara.R5 (hereinafter referred to as Rara.R5) with the deposit number GDMCC No. 63462.

[0040] The Latin scientific name of the genus Paraburkholderia (Paraburkholderia sp.) is a short rod-shaped cell belonging to Gram-negative bacilli. Through phylogenetic analysis, Rara.R5 proposed in the embodiments of the present application is a strain under the species strydomiana of the genus Paraburkholderia, and it is named Paraburkholderia strydomiana Rara.R5 and deposited in the Guangdong Microbial Culture Collection Center; the deposit registration number is GDMCC No: 63462; the address of the depositary institution: 5th Floor, Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; the deposit date is May 14, 2023.

[0041] In the embodiments of the present application, the strain Rara.R5 with the 16S rDNA sequence shown in SEQ ID NO: 1 can be understood by those skilled in the art as the original strain. A strain with a genomic sequence having 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 Rara.R5 with the sequence shown in SEQ ID NO: 1. It can be understood that a strain such as strain Rara.R5 can undergo spontaneous mutation or be artificially cultured to form a variant strain, for example, a deletion of nucleotides, an addition of nucleotides, or a substitution of nucleotides. The "variant strain" has a highly identical gene sequence and extremely similar biological functions to the "strain Rara.R5", and the mutated genes basically do not affect the conserved sequence of the strain Rara.R5, thus not affecting the genetic stability of the strain Rara.R5. More specifically, the "variant strain" is also a strain of the Rara.R5 species and exhibits the physiological activity characteristics of the Rara.R5 species. The specific species corresponding to Rara.R5 and all bacterial strains under the species also fall within the protection scope of the present application.

[0042] When the newly isolated strain Rara.R5 in the embodiments of the present application is used alone, it can effectively improve the agronomic traits of plants, especially can be used for plant growth promotion, such as promoting nitrogen fixation and root growth of gramineous plants and leguminous plants. The newly isolated strain Rara.R5 in the embodiments of the present application provides new biological resources for green agricultural production.

[0043] The second aspect of the embodiments of the present application proposes the application of Burkholderia paraperfectomarina as described in any of the above embodiments in promoting plant growth.

[0044] In some embodiments, the Burkholderia paraperfectomarina can be used to promote the growth of the above-ground parts of plants, such as plant height, stem diameter, leaves, flowers and / or fruits, to promote plant growth, thereby increasing the above-ground biomass and improving plant yield. In some embodiments, the Burkholderia paraperfectomarina can also be used to promote the growth of the underground parts of plants, such as root length, root diameter, root nodulation, etc., to promote plant growth, thereby increasing the underground biomass and improving plant yield.

[0045] In some embodiments, the Burkholderia paraperfectomarina can be used to promote plant nitrogen fixation and / or nodulation.

[0046] In some embodiments, the plants include but are not limited to crops, vegetables, fruit trees, flowers and turfgrasses. Optionally, the crops are selected from rice, corn, wheat, barley, oats, millet, soybeans, peanuts, cotton and potatoes, and the vegetables are selected from Chinese cabbage, carrots, ginger, green beans, beets, cucumbers, peppers, tomatoes, onions, ferns and alfalfa.

[0047] In some embodiments, the plants are selected from gramineous plants and leguminous plants. Preferably, the gramineous plants are selected from corn, millet and upland rice, and the leguminous plants are selected from soybeans and alfalfa.

[0048] The third aspect of the embodiments of the present application proposes a Rhizobium sp. which contains the 16S rDNA sequence shown in SEQ ID NO: 2 or its complementary sequence or a sequence with at least 85% identity compared with SEQ ID NO: 2.

[0049] In some embodiments, the systematic classification of the Rhizobium sp. is Rhizobium grahamii; preferably, the Rhizobium sp. has the 16S rDNA sequence shown in SEQ ID NO: 2 or its complementary sequence; more preferably, the Rhizobium sp. is Rhizobium grahamii Rhiz.R2 with the preservation number GDMCC No. 63570 (hereinafter referred to as Rhiz.R2).

[0050] The rhizobia proposed in the embodiments of the present application can effectively promote plant growth. Specifically, it can promote plant growth by promoting nodulation in plant roots to enhance the nitrogen fixation function of plants.

[0051] The fourth aspect of the embodiments of the present application proposes a composition, which contains Burkholderia paraphytica and rhizobia as described in any of the above embodiments.

[0052] When the rhizobia proposed in the embodiments of the present application are co-applied with the above-mentioned Burkholderia paraphytica, they show synergistic effects in aspects such as promoting plant growth, such as promoting nodulation. This indicates that the bacteria can be used in the form of a composition with Burkholderia paraphytica for promoting plant growth, which is of great significance for increasing crop yields and promoting green agricultural production.

[0053] The fifth aspect of the embodiments of the present application proposes an agricultural preparation, which includes Burkholderia paraphytica or the composition as described in any of the above embodiments. Since the above-mentioned strain also includes mutant strains of the same genus as strains Rara.R5 and Rhiz.R2, the specific genera of strains Rara.R5 and Rhiz.R2 and all bacterial strains under these genera also fall within the protection scope of the agricultural preparation of the present application.

[0054] In some embodiments, the agricultural preparation can be or include a fermentation broth containing Burkholderia paraphytica as described in any of the above embodiments and optional Rhiz.R2 bacteria, especially strains Rara.R5 and optional Rhiz.R2. The fermentation broth contains strains Rara.R5 and optional Rhiz.R2 and their metabolites, and these metabolites can also be further used as biological fertilizers or biocontrol drugs. In some embodiments, the fermentation medium of the fermentation broth can be a beef extract peptone medium. It can be understood that as long as the fermentation medium of the fermentation broth of any of the above strains in the embodiments of the present application can provide the normal growth and metabolic fermentation of these strains, the present application does not limit this.

[0055] In some embodiments, the agricultural preparation may further comprise adjuvants, where the adjuvants may be organic matter and / or inorganic matter. In the embodiments of the present application, the organic matter may be additional bacterial fertilizers other than strain Rara.R5 and optionally Rhiz.R2 that can promote plant growth. For example: bacterial fertilizers that increase soil nitrogen and crop nitrogen nutrition, such as rhizobium fertilizer, azotobacter fertilizer, nitrogen-fixing blue-green algae fertilizer, etc.; bacterial fertilizers that decompose soil organic matter, such as organic phosphorus bacteria fertilizer, comprehensive bacterial fertilizer; bacterial fertilizers that decompose insoluble minerals in the soil, such as phosphorus bacteria fertilizer, potassium bacteria fertilizer, mycorrhizal fungi fertilizer; bacterial fertilizers that stimulate plant growth, such as growth-promoting bacterial fertilizer; bacterial fertilizers that increase the stress resistance of crop roots, such as antibiotic bacterial fertilizer, stress-resistant bacterial fertilizer. In the embodiments of the present application, the organic matter may also be organic fertilizers required for plant growth, such as manure, etc. It can be understood that the organic matter in the embodiments of the present application, alone or in combination, as long as it can ensure a promoting effect on plant growth, is not limited in this application.

[0056] In the embodiments of the present application, the inorganic matter may be chemical components that can be used in agriculture, such as agriculturally acceptable carriers, excipients, diluents, adjuvants, vehicles or combinations thereof, and / or inorganic fertilizers that do not affect the microbial activity concentration in the agricultural preparation, and this application is not limited thereto.

[0057] In the embodiments of the present application, the dosage form of the agricultural preparation may be selected from: wettable powder, water dispersible granule, suspension, emulsion in water, granule, seed coating agent or combinations thereof. It can be understood that the dosage form of the agricultural preparation in the embodiments of the present application, as long as it can ensure application to plants in a certain form, is not limited in this application.

[0058] In the embodiments of the present application, "application" may include: root irrigation, injection into the plant body, and / or spraying on the plant surface, and the plant surface may optionally include the surfaces of roots, stems, leaves, flowers, fruits, and / or seeds.

[0059] The agricultural preparation proposed in the embodiments of the present application, by using Rara.R5, can be effectively used to improve the agronomic traits of plants, which can effectively fix nitrogen, promote nodulation, etc., and thus shows a significant effect of promoting plant growth and increasing plant yield. In addition, by further adding Rhiz.R2 to the agricultural preparation, its synergistic effect with Rara.R5 can further significantly enhance the growth-promoting effect, thereby greatly reducing the application amount of chemical fertilizers and alleviating the environmental impact caused by chemical fertilizers.

[0060] The embodiments of the sixth aspect of the present application propose the application of the Burkholderia paraperuensis as described in any of the above embodiments or the agricultural preparation as described in any of the above embodiments in promoting plant growth.

[0061] In some embodiments, the Burkholderia paraperuensis or a composition thereof with rhizobium can be used for nitrogen fixation and / or nodulation promotion.

[0062] In some embodiments, the composition of Burkholderia and Rhizobium can be used to promote the growth of the above-ground parts of plants, such as plant height, stem diameter, leaves, flowers, and / or fruits, so as to promote the growth of plants, thereby increasing the above-ground biomass and enhancing the plant yield. In some embodiments, the composition of Burkholderia and Rhizobium can also be used to promote the growth of the underground parts of plants, such as root length, root diameter, root nodulation, etc., to promote the growth of plants, thereby increasing the underground biomass and increasing the plant yield. The Burkholderia Rara.R5 or the agricultural preparation containing it proposed in the embodiments of the present application shows a significant effect of promoting plant growth through nitrogen fixation, tumor promotion, etc. In addition, by further combining it with Rhizobium, compared with the separate application of the two, it shows a better comprehensive synergistic growth promotion and yield increase effect, thereby being able to greatly reduce the application amount of chemical fertilizers and alleviate the environmental impact brought by chemical fertilizers and pesticides.

[0063] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out 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 can be obtained from commercial channels unless otherwise specified.

[0064] Unless otherwise specified, in the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0065] Example 1: Isolation of new strains Rara.R5 and Rhiz.R2

[0066] Collect soybean rhizosphere soil samples, pour the rhizosphere soil samples into a mortar, add 2 - 3 ml of sterile water, and directly grind them into a powder to obtain a grinding solution. Dilute the grinding solution with sterile water to gradient concentrations of 10 2 to 10 5 . Coat the gradient dilutions onto the nutrient agar solid medium respectively, and at the same time coat sterile water without bacterial solution as a blank control. After coating, incubate in an inverted position at 28°C for 48 - 72 hours. Pick single colonies into the liquid nutrient agar medium for amplification culture respectively, and then streak and amplify repeatedly for multiple times to obtain monoclonal strains numbered Rara.R5 and Rhiz.R2. Sequence the 16S rDNA genes of the monoclonal strains Rara.R5 and Rhiz.R2, and the specific sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

[0067] Rara.R5 16S rDNA sequence (SEQ ID NO: 1):

[0068] CGCATGCTTAATATGCAGTCGACGGCAGCACGGGGGCAACCCTGGTGGCGAGTGGCG

[0069] AACGGGTGAGTAATACATCGGAACGTGTCCTGTAGTGGGGGATAGCCCGGCGAAAGC

[0070] CGGATTAATACCGCATACGCTCTGCGGAGGAAAGCGGGGGATCCTTCGGGACCTCGCG

[0071] CTACAGGGGCGGCCGATGGCAGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGC

[0072] GACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCC

[0073] CAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATC

[0074] CAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAA

[0075] AGAAAACCTCGTGGTTAATACCCGTGGGGGATGACGGTACCGGAAGAATAAGCACCG

[0076] GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTA

[0077] CTGGGCGTAAAGCGTGCGCAGGCGGTTCGCTAAGACAGATGTGAAATCCCCGGGCTT

[0078] AACCTGGGAACTGCATTTGTGACTGGCGGGCTAGAGTATGGCAGAGGGGGGTAGAAT

[0079] TCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAG

[0080] CCCCCTGGGCCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTA

[0081] GATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTCGGGTCTTCATTGACT

[0082] TGGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAA

[0083] AACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGAT

[0084] GCAACGCGAAAAACCTTACCTACCCTTGACATGTATGGAACCCTGCTGAGAGGTGGGG

[0085] GTGCCCGAAAGGGAGCCATAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGT

[0086] GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCTAGTTGCTACGCAAG

[0087] AGCACTCCAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAG

[0088] TCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGT

[0089] CGCCAACCCGCGAGGGGGAGCCAATCCCAGAAAACCGATCGTAGTCCGGATCGCACT

[0090] CTGCAACTCGAGTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGG

[0091] TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTCACCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTACCACGGTGATCATGCTC(SEQ ID NO:1)

[0092] Rhiz.R2 16S rDNA sequence (SEQ ID NO:2):

[0093] GACGTCGGTCTACACATGCAGTCGAGCGGCAGCGGGAAGTAGCTTGCTACTTTGCCGG

[0094] CGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTA

[0095] CTGGAAACGGTAGCTAATACCGCATGACCTCGCAAGAGCAAAGTGGGGGACCTTCGG

[0096] GCCTCACGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCA

[0097] CCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGA

[0098] CACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAG

[0099] CCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTAGGGTTGTAAAGCACTTTCAG

[0100] CGAGGAGGAAGGGTTCAGTGTTAATAGCACTGTGCATTGACGTTACTCGCAGAAGAA

[0101] GCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATC

[0102] GGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCC

[0103] GAGCTTAACTTGGGAACTGCATTTGAAACTGGCAAGCTAGAGTCTTGTAGAGGGGGGT

[0104] AGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAA

[0105] GGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAG

[0106] GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTGCCCTTG

[0107] AGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAA

[0108] GGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA

[0109] ATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGAT

[0110] AGCTTAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT

[0111] TGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCG

[0112] AGTCATGTCGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGAT

[0113] GACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATA

[0114] CAAAGAGAAGCGAACTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGG

[0115] ATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAAT

[0116] GCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGG

[0117] GTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTACCACTTGGATCAT(SEQ ID NO:2)

[0118] Example 2: Identification and determination of the evolutionary status of the new strain Rara.R5

[0119] The approximately 1.4 Kb sequence of Rara.R5 that is nearly the full-length 16S rDNA obtained by sequencing (i.e., SEQ ID NO: 1) was aligned in the 16S rDNA gene database of EzBioCloud (https: / / www.ezbiocloud.net / ). The alignment results showed that the strain with the highest homology to the 16S rDNA gene of Rara.R5 in the database is Paraburkholderia strydomiana WK1.1f(T) (accession number: HF674688), with a similarity of 100%. Further, the sequences of the related strains of Rara.R5 were used to construct a phylogenetic tree using MAGA with the neighbor-joining method (1000 replicates), and the results are as Figure 1 shown. It can be seen from Figure 1 that Rara.R5 and Paraburkholderia strydomiana WK1.1f(T) (accession number: HF674688) are clustered together, indicating that Rara.R5 isolated in Example 1 belongs to the species strydomiana of the genus Paraburkholderia.

[0120] Similarly, the approximately 1.4 Kb sequence of Rhiz.R2 obtained by sequencing, which is nearly the full length of 16S rDNA, was aligned in the 16S rDNA gene database of EzBioCloud. The alignment results showed that the strain with the highest homology to the 16S rDNA gene of Rhiz.R2 in the database is Rhizobium grahamii | CCGE 502 (accession number: AEYE01000061), with a similarity of 98.49%. Further, the sequences of the related strains of Rhiz.R2 were used to construct a phylogenetic tree by neighbor-joining bootstrap analysis (1000 replicates) using MAGA, and the results are as Figure 1 shown. It can be Figure 2 seen that Rhiz.R2 and Rhizobium grahamii | CCGE 502 (accession number: AEYE01000061) form a single branch together. Therefore, it can be determined that Rhiz.R2 belongs to the genus Rhizobium grahamii and is named Rhiz.R2.

[0121] Therefore, combining the 16S rDNA sequence alignment and the MAGA tree construction results, it can be determined that Rara.R5 is a new strain under Paraburkholderia strydomiana. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 14, 2023, with the deposit number GDMCC No. 63462 and named Paraburkholderia strydomiana Rara.R5.

[0122] Similarly, Rhiz.R2 was deposited in the Guangdong Provincial Microbial Culture Collection Center on June 16, 2023, with the deposit number GDMCC No. 63570 and named Rhizobium grahamii Rhiz.R2.

[0123] Example 3: Colony characteristics of the new strain Rara.R5

[0124] The new strain Rara.R5 provided in this application is milky white on the beef extract peptone solid medium plate, with a neat edge, opaque, round and smooth colonies, and viscous cells (as Figure 3 shown).

[0125] Example 4: The new strain Rara.R5 has nitrogen fixation characteristics

[0126] After pipetting 5 μL of the bacterial liquid of strain Rara.R5 onto the Ashby nitrogen-free medium and incubating it in a constant temperature biochemical incubator at 30°C for 48 hours, the results were observed. The results showed that Rara.R5 could grow well on the Ashby nitrogen-free medium, confirming that the strain has good nitrogen fixation ability (asFigure 4 As shown in the figure). The formula of Ashby nitrogen-free medium is: 10 g / L mannitol, 0.2 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 0.2 g / L NaCl, 0.1 g / L CaSO4·2H2O, 5 g / L CaCO3.

[0127] Example 5: The new strain Rara.R5 has the function of promoting plant growth

[0128] 5.1 Growth-promoting function of the new strain Rara.R5 on leguminous plants

[0129] After activating the strain Rara.R5, it was inoculated into the beef extract peptone medium and fermented by shaking at 28 °C and 180 r / min for 5 days to obtain the Rara.R5 fermentation broth.

[0130] Soybeans (cultivar Zhonghuang 13) were planted in flower pots filled with field soil. After 5 days of seed germination, 5 ml of the prepared bacterial liquid was poured around the roots of the germinated plants in each pot, and 3 parallel groups were set for each treatment. Then, it was watered once every 5 days for a total of 3 times. A control group was set, and the control group was treated with an equal amount of sterile water.

[0131] After 25 days of growth, the plants were sampled and the biomass data were statistically analyzed. The specific measurement data are shown in Table 1, and the growth conditions of the treatment group and the control group are as Figure 5 shown.

[0132] Table 1 Effects of inoculating Rara.R5 on the growth of soybeans

[0133]

[0134]

[0135] Table 1 shows the measured values of each growth index of soybeans after applying the strain Rara.R5 provided in this application. As can be seen from Table 1, the significant difference analysis shows that compared with the control group, the plant height, root length, aboveground fresh weight, underground fresh weight and number of root nodules of soybeans after applying Rara.R5 are all higher than those of the control group, indicating that Rara.R5 can promote the rooting, nodulation and growth of the underground part of soybeans (the growth rates are 1.89%, 18.90% and 25.93% respectively), significantly increase the plant height of soybeans (p < 0.05), and significantly increase the aboveground fresh weight of soybeans (p < 0.05), and the growth rates are 18.69% and 25.75% respectively. It shows that the strain Rara.R5 has significant effects on promoting root growth and increasing plant biomass, especially the growth of the aboveground part, when applied to soybeans.

[0136] Figure 5 shows the growth promotion effect of Rara.R5 according to this embodiment. By Figure 5It can be seen that in the pot experiment, the soybeans after applying the strain Rara.R5 provided by the present application grew more vigorously than those in the control group. The plants were generally taller, stronger and more upright, and the leaves were dark green, indicating that the strain Rara.R5 provided by the present invention has a good promoting effect on the growth of the stems and leaves of soybeans, and it can be effectively used for promoting the growth and increasing the yield of leguminous plants such as soybeans.

[0137] 5.2 Growth-promoting function of the new strain Rara.R5 on gramineous plants

[0138] After activating the strain Rara.R5, it was inoculated into the beef extract peptone medium and fermented by shaking at 28 °C and 180 r / min for 5 days to obtain the Rara.R5 fermentation broth. When in use, the fermented broth was diluted with sterile water at a ratio of 1:100. Upland rice, millet and corn were respectively planted in the field plots, and the crop varieties were Luyin 46, Huagu 12 and Jinxiangyu respectively. Inoculation was carried out at the time of sowing and seedling stage of crop growth, and an equal amount of the fermented dilution was irrigated for each plant. After 35 days of growth, the crops were sampled and the biomass data were statistically analyzed. The specific measurement data are shown in Table 2, Table 3 and Table 4, and the growth conditions of the treatment group and the control group are as Figure 6 shown (a, upland rice; b, millet; c, corn).

[0139] Table 2 Effects of inoculating Rara.R5 on the field growth of upland rice

[0140]

[0141] Table 3 Effects of inoculating Rara.R5 on the field growth of millet

[0142]

[0143] Table 4 Effects of inoculating Rara.R5 on the field growth of corn

[0144]

[0145] Tables 2-4 are respectively the measured values of each growth index of upland rice, millet and corn after applying the new strain Rara.R5 provided by the present invention. As can be seen from Table 2, the significant difference analysis shows that applying the Rara.R5 strain can extremely significantly increase the root length and underground fresh weight of upland rice, and the growth rates are 43.43% and 109.74% respectively, and effectively promote the above-ground fresh weight and stem diameter of upland rice. It shows that the Rara.R5 strain has an obvious effect on promoting the root growth and the accumulation of biomass in the above-ground and underground parts of upland rice, thus effectively promoting the yield increase of upland rice.

[0146] As can be seen from Table 3, the analysis shows that the application of strain Rara.R5 has a promoting effect on the root length, above-ground fresh weight, and above-ground fresh weight of foxtail millet, and the growth rates compared with the control group are 24.90%, 34.01%, and 87.10% respectively; at the same time, the significance difference analysis shows that the application of strain Rara.R5 can significantly increase the plant height of foxtail millet and extremely significantly increase the stem diameter, with growth rates of 9.90% and 28.06% respectively, indicating that strain Rara.R5 can promote the growth of the underground part of foxtail millet while effectively promoting the growth of the above-ground parts such as the plant height and stem diameter of foxtail millet, thus effectively promoting the yield increase of foxtail millet.

[0147] As can be seen from Table 4, compared with the control group, after the application of strain Rara.R5, the indexes such as the plant height, root length, above-ground fresh weight, underground fresh weight, and stem diameter of maize have all increased to a certain extent, and the growth rates are 8.13%, 4.98%, 40.81%, 46.70%, and 10.63% respectively. Among them, the significance difference analysis shows that the application of strain Rara.R5 can significantly increase the plant height and above-ground fresh weight of maize, proving that the application of strain Rara.R5 can promote the growth of the underground part of maize while also significantly promoting the growth of the above-ground part of maize, and it shows a promoting effect on the fruiting of the above-ground part, which can be effectively used for the yield increase of maize.

[0148] As Figure 6 can be seen, in the field experiment, the plants after applying the strain Rara.R5 provided by the present invention are more lush in the above-ground or underground parts compared with the control group in different crops. For example, the root system is more developed after applying Rara.R5 to upland rice; after applying Rara.R5 to foxtail millet and maize, the above-ground parts are generally taller, stronger, and the leaves are dark green, indicating that the strain Rara.R5 proposed in the embodiments of the present application can be effectively used for the growth promotion and yield increase of gramineous crops.

[0149] Example 6: Verification of the nodulation ability of strain Rhiz.R2

[0150] After activating strain Rhiz.R2, it was inoculated into the beef extract peptone medium and shaken and fermented at 28 °C and 180 r / min for 5 days. Then, the OD of the bacterial liquid 600Adjust to 0.5, take the bacterial liquid, centrifuge at 5000 rpm for 10 min, remove the supernatant, resuspend the cells with sterile water, and set aside. Another strain of rhizobia Rhiz.E1 isolated from soybean rhizosphere soil (Rhizobium esperanzae, Cordeiro AB, Ribeiro RA, Helene LCF, Hungria M (2017). Rhizobium esperanzae sp. nov., a N2-fixing root symbiont of Phaseolus vulgaris from Mexican soils. Int. J. Syst. Evol. Microbiol. 67, 3937-3945.) was operated in the same way as a control for measuring the nodulation ability of Rhiz.R2.

[0151] Surface-sterilize soybean seeds (Zhonghuang 13) with 1% sodium hypochlorite solution for 5 min, and rinse with sterile distilled water 5 times. Plant the sterilized seeds in sterile soil pots, where the sterile soil was obtained after autoclaving at 120 °C. Plant 5 plants in each pot. Five days after the seeds germinated, pour 5 ml of bacterial liquid Rhiz.R2 and Rhiz.E1 around the roots of the germinated plants in each pot respectively. Set 3 parallel groups for each treatment, and the control group was watered 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 the number of nodules. The number of nodules in the blank group was 0 because no rhizobia were inoculated. The specific measured values of nodules in the treatment group are shown in Table 5 below. The growth of nodules in the treatment group and the control group is as Figure 7 shown.

[0152] Table 5

[0153] Group Number of root nodules (mean ± standard deviation) p correction p correction symbol Rhiz.E1 46.77±23.44 / ns Rhiz.R2 73.63±38.25 0.00922 **

[0154] As can be seen from Table 5 and Figure 7 it can be seen that the number of nodules of soybeans inoculated with Rhiz.R2 is extremely significantly higher than that of soybeans inoculated with Rhiz.E1, indicating that Rhiz.R2 has good nodulation-promoting ability, suggesting that Rhiz.R2 proposed in the embodiments of the present application can be effectively used for plant growth promotion, especially nodulation and nitrogen fixation.

[0155] Example 7: Synergistic effect of Rara.R5 and Rhiz.R2 in plant growth promotion

[0156] After activating strains Rara.R5 and Rhiz.R2 respectively, they were inoculated into the beef extract peptone medium and fermented with shaking at 28°C and 180 r / min for 5 days to obtain the fermentation broths of Rara.R5 and Rhiz.R2. Alfalfa was planted in field plots respectively to verify the synergistic effect of Rara.R5 and Rhiz.R2 on leguminous crops. The fermentation broths were inoculated at the seedling stage and jointing stage of millet respectively. When in use, the fermented bacterial liquid was diluted with water at a ratio of 1:100, and an equal amount of the fermented dilution was watered for each plant. The control group was not inoculated with the fermentation broth, treatment group 1 was inoculated with the Rara.R5 fermentation broth alone, treatment group 2 was inoculated with the Rhiz.R2 fermentation broth alone, and treatment group 3 was inoculated with the mixed fermentation broth of Rara.R5 and Rhiz.R2. After 90 days of growth, the crops were sampled and the biomass data were statistically analyzed. The specific measurement data are shown in Table 6 respectively, and the growth conditions of the treatment groups and the control group are as Figure 8 shown.

[0157] Table 6

[0158]

[0159] As can be seen from Table 6, compared with the control group, all treatment groups increased the plant height, root length, above-ground fresh weight and underground fresh weight of alfalfa to varying degrees, indicating that both Rara.R5 and Rhiz.R2 proposed in the embodiments of the present application have plant growth-promoting effects. Among them, the growth rates of the plant height, root length, above-ground fresh weight and underground fresh weight of alfalfa in treatment group 3 were 13.78%, 55.88%, 158.59% and 197.28% respectively, which were significantly higher than those in treatment group 1 and treatment group 2. From Figure 8 this, it can be seen that the growth of the plants in the treatment groups was better than that in the control group. Among them, the plants in treatment group 3 were taller and more lush, showing a more vigorous growth trend, indicating that Rara.R5 and Rhiz.R2 can synergistically promote the growth and yield increase of plants such as leguminous crops.

[0160] Therefore, the newly isolated and identified plant growth-promoting rhizobacteria strain Rara.R5 and its composition with Rhiz.R2 in the embodiments of the present application have good promoting effects on the growth of various plants. Specifically, it is mainly reflected in the promoting effects on the growth of plant stems, leaves and roots. For example, in leguminous plants and gramineous plants, it can effectively promote the growth of the above-ground part to promote fruiting, indicating that the newly isolated and identified plant growth-promoting rhizobacteria strain Rara.R5 and Rhiz.R2 and their combined preparations in the embodiments of the present application can be effectively used for the growth promotion of various plants including gramineous crops and leguminous plants, thereby increasing crop yields. Further, compared with the single application of Rara.R5 and Rhiz.R2, the combined application of the two achieves synergistic effects in growth promotion. This can effectively reduce the application amount of chemical fertilizers and alleviate the environmental impact brought by chemical fertilizers and pesticides. The newly discovered Paenibacillus strains Rara.R5 and Rhiz.R2 with growth-promoting and biocontrol functions proposed in the present application are of great significance for improving crop yields and environmental protection.

[0161] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection 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 can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0162] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Burkholderia paraphytica, characterized in that, Comprising the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence having at least 85% identity compared to SEQ ID NO:

1.

2. The Burkholderia parabrevis according to claim 1, characterized in that, The Paraburkholderia is of the genus Paraburkholderia sp.; preferably, the systematic classification of the Paraburkholderia is Paraburkholderia strydomiana; more preferably, the Paraburkholderia has the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence; most preferably, the Paraburkholderia is Paraburkholderia strydomiana Rara.R5 with the deposit number GDMCC No. 63462.

3. Use of the Paraburkholderia according to claim 1 or 2 in promoting plant growth.

4. The application according to claim 3, characterized in that The Paraburkholderia is used to promote plant nitrogen fixation and / or nodulation.

5. The application according to claim 3 or 4, characterized in that, The plant is selected from Gramineae plants and Leguminosae plants. Preferably, the Gramineae plants are selected from maize, millet and upland rice, and the Leguminosae plants are selected from soybean and alfalfa.

6. A composition, characterized in that, Comprising the Paraburkholderia according to claim 1 or 2 and Rhizobium sp., Optionally, the Rhizobium comprises the 16S rDNA sequence shown in SEQ ID NO: 2 or its complementary sequence or a sequence having at least 85% identity compared to SEQ ID NO: 2; Preferably, the systematic classification of the Rhizobium is Rhizobium grahamii; more preferably, the Rhizobium has the 16S rDNA sequence shown in SEQ ID NO: 2 or its complementary sequence; most preferably, the Rhizobium is Rhizobium grahamii Rhiz.R2 with the deposit number GDMCC No. 63570.

7. An agricultural preparation, characterized in that, Comprising the composition according to claim 1 or 2 or the composition according to claim 6, Optionally, the agricultural preparation is a liquid preparation and / or a freeze-dried preparation, and the dosage form of the agricultural preparation is selected from: wettable powder, water dispersible granule, suspension, emulsion in water, granule, seed coating agent or a combination thereof.

8. Use of the composition according to claim 6 or the agricultural preparation according to claim 7 in promoting plant growth.

9. The application according to claim 8, wherein The composition or the agricultural preparation is used to promote plant nitrogen fixation and / or nodulation.

10. The application according to claim 8 or 9, characterized in that, The plant is selected from Gramineae plants and Leguminosae plants. Preferably, the Gramineae plants are selected from maize, millet and upland rice, and the Leguminosae plants are selected from soybean and alfalfa.

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