Rhizosphere growth-promoting bacteria and applications thereof

Abstract

The application discloses rhizosphere growth promoting bacteria and application thereof, and provides bacterial strains for promoting plant growth and application thereof, wherein the bacterial strains are selected from at least one of the following: a strain 1 comprising a 16S rDNA sequence shown in SEQ ID NO: 1 or a complementary sequence thereof or a sequence with at least 85% identity compared with the SEQ ID NO: 1; a strain 2 comprising a 16S rDNA sequence shown in SEQ ID NO: 2 or comprising a sequence with at least 85% identity compared with the SEQ ID NO: 2; a strain 3 comprising a 16S rDNA sequence shown in SEQ ID NO: 3 or comprising a sequence with at least 85% identity compared with the SEQ ID NO: 3; and a strain 4 comprising a 16S rDNA sequence shown in SEQ ID NO: 4 or comprising a sequence with at least 85% identity compared with the SEQ ID NO: 4.

Description

Technical Field

[0001] This application relates to the field of microbiology, specifically to rhizosphere growth-promoting bacteria and their applications. Background Technology

[0002] Microorganisms are a large group of organisms including bacteria, viruses, and fungi. They are tiny individuals that have a close relationship with plants, and microorganisms can be found in the roots, stems, leaves, flowers, and seeds of plants. Various physiological functions of plant growth are affected by microorganisms. For example, microorganisms can promote plant growth and development, reduce soil heavy metal toxicity, enhance plant resistance to extreme environments (drought, flood, heat), and control pests and diseases.

[0003] Microorganisms and plant hosts have undergone millions of years of co-evolution. Root microorganisms are the hotspot for energy and material exchange between plants and microorganisms. 40% of the carbon source fixed by plant roots through photosynthesis is used as a source of nutrition, signal transduction and antimicrobial active substances for root soil microorganisms.

[0004] Therefore, there is an urgent need to develop plant rhizosphere growth-promoting bacteria to promote the growth and development of a wide range of plants. Summary of the Invention

[0005] Therefore, embodiments of this application provide rhizosphere growth-promoting bacteria and their applications.

[0006] The first aspect of this application provides a bacterial strain for promoting plant growth, wherein the bacterial strain is selected from at least one of the following:

[0007] Strain 1 contains the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO: 1;

[0008] Strain 2, comprising the 16S rDNA sequence shown in SEQ ID NO: 2 or comprising a sequence with at least 85% identity to SEQ ID NO: 2;

[0009] Strain 3, comprising the 16S rDNA sequence shown in SEQ ID NO: 3 or comprising a sequence with at least 85% identity to SEQ ID NO: 3; and

[0010] Strain 4 contains the 16S rDNA sequence shown in SEQ ID NO: 4 or contains a sequence with at least 85% identity to SEQ ID NO: 4.

[0011] In some embodiments, strain 1 is a member of the genus *Arthrobacter*. In some embodiments, strain 1 has a 16S rDNA sequence as shown in SEQ ID NO: 1. In some embodiments, strain 1 is *Arthrobacter* sp. *Arth4* with accession number GDMCC No: 62566.

[0012] In some embodiments, strain 2 is a species of Bacillus. In some embodiments, strain 2 is a species of Bacillus paralicheniformis. In some embodiments, strain 2 has a 16S rDNA sequence as shown in SEQ ID NO: 2. In some embodiments, strain 2 is Bacillus paralicheniformis Bacil9 with accession number GDMCCNo: 62569.

[0013] In some embodiments, strain 3 is a species of *Priestia*. In some embodiments, strain 3 is a species of *Priestia megaterium*. In some embodiments, strain 3 has a 16S rDNA sequence as shown in SEQ ID NO: 3. In some embodiments, strain 3 is *Priestia megaterium* Bacif4 with accession number GDMCC No: 62567.

[0014] In some embodiments, strain 4 is a species of Bacillus. In some embodiments, strain 4 is a species of Bacillus velezensis. In some embodiments, strain 4 has a 16S rDNA sequence as shown in SEQ ID NO: 4. In some embodiments, strain 4 is Bacillus velezensis Baci5 with accession number GDMCC No: 62568.

[0015] The second aspect of this application provides an agricultural formulation comprising bacterial strains as described in any embodiment of the first aspect of this application.

[0016] In some embodiments, the agricultural formulation further includes excipients.

[0017] In some embodiments, the formulation of the agricultural preparation is selected from: wettable powder, water-dispersible granules, suspension concentrate, emulsion, granules, seed coating agent, or a combination thereof.

[0018] The third aspect of this application provides for the use of the bacterial strains described in any embodiment of the first aspect of this application in the preparation of agricultural formulations.

[0019] The fourth aspect of this application provides a method for promoting plant growth, the method comprising applying to the plant a bacterial strain as described in any embodiment of the first aspect of this application or an agricultural preparation as described in any embodiment of the second aspect of this application.

[0020] The embodiments of this application achieve the following beneficial effects:

[0021] This application, for the first time, proposes four bacterial strains—Arthrobacter sp. Arth4, Bacillus paralicheniformis Bacil9, Priestia megaterium Bacif4, and Bacillus velezensis Baci5—and their applications in improving plant agronomic traits. In particular, the genera and species to which these four strains belong can effectively promote root elongation and stem growth, thereby enhancing nutrient absorption and accumulation, and ultimately increasing crop yield. The rhizosphere growth-promoting bacteria proposed in this application are of great significance for increasing crop yield. Furthermore, by preparing Arth4, Bacil9, Bacif4, and Baci5 into microbial fertilizers suitable for agricultural production, the use of chemical fertilizers can be reduced, promoting green agricultural production. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a clustering diagram of Arth4 according to Embodiment 2 of this application;

[0024] Figure 2 This is a clustering diagram of Bacil9 according to Embodiment 2 of this application;

[0025] Figure 3 This is a clustering diagram of Bacif4 according to Embodiment 2 of this application;

[0026] Figure 4 This is a clustering diagram of Baci5 according to Embodiment 2 of this application;

[0027] Figure 5 This is a plate growth diagram according to Embodiment 3 of this application;

[0028] Figure 6 This is a diagram showing the aseptic soil growth of seedlings according to Example 4 of this application;

[0029] Figure 7 This is a field growth diagram of Huagu 12 according to Example 5 of this application;

[0030] Figure 8 This is another field growth diagram of Huagu 12 according to Example 5 of this application.

[0031] Instructions for the preservation of microbial strains:

[0032] Arthrobacter sp.Arth4: The deposit registration number is GDMCC No:62566, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, the address of the depositary institution is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit date is June 28, 2022.

[0033] Bacillus paralicheniformis Bacil9: Depository number GDMCC No:62569, depository institution: Guangdong Provincial Center for Microbial Culture Collection; address of depository institution: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; deposit date: June 28, 2022.

[0034] Priestia megaterium Bacif4: The deposit registration number is GDMCC No:62567, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, the address of the depositary institution is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit date is June 28, 2022.

[0035] Bacillus velezensis Baci5: The deposit registration number is GDMCC No:62568, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, the address of the depositary institution is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit date is June 28, 2022. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] This application is based on the inventor's following understanding:

[0038] Soil, a treasure trove of microbial resources, contains a complex and diverse array of microorganisms; approximately 2,000-18,000 species of microorganisms are found in 1 gram of soil. Root microorganisms are highly susceptible to influences from plant hosts, soil type, nutrient status, and climatic factors. The selection of microorganisms by plant hosts does not appear to be entirely random; it seems to attract beneficial plant growth bacteria, potentially probiotics, by secreting specific small-molecule nutrients, while simultaneously modifying soil pH and improving soil quality. However, how microorganisms actually help plant growth and increase crop yields remains unknown.

[0039] Studies have shown that, compared with non-plant-related microorganisms, plant-related microorganisms (rhizosphere microorganisms, root surface microorganisms, etc.) have evolved a large number of functional gene elements to adapt to the plant environment, such as plant root nodules, nitrogen fixation, plant hormone synthesis, T3SS and T6SS secretion systems, and flagellar movement.

[0040] Understanding the microbial composition surrounding plants, analyzing the interrelationships between microorganisms, plant growth, and environmental conditions, identifying the optimal microbial composition required for plant growth, and establishing controllable models for microbial support of crop health, efficient growth, and high yield can maximize food production. This requires establishing large-scale correlation analyses between plant-related microbial composition and functional gene information and crop growth and yield phenotypes to identify potential plant probiotics and functional characteristics. Guided by big data analysis results, targeted screening of potential probiotics with application value can provide possibilities for agricultural applications.

[0041] This application embodiment studies the composition of rhizosphere microorganisms in different varieties of millet through sequencing, while simultaneously collecting phenotypic data on millet growth and yield. By constructing a linear model, key microbial groups (marker bacteria) associated with millet growth and yield phenotypes were identified. Single bacterial strains were isolated from the millet rhizosphere soil, and 16S rRNA sequence alignment revealed four marker strains significantly associated with millet growth and yield. Furthermore, this application embodiment validates the growth-promoting function of these strains through a series of experiments, including plate growth-promoting experiments, sterile soil experiments, and field experiments.

[0042] The first aspect of this application provides a bacterial strain for promoting plant growth, wherein the bacterial strain is selected from at least one of the following:

[0043] Strain 1 contains the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO: 1;

[0044] Strain 2, comprising the 16S rDNA sequence shown in SEQ ID NO: 2 or comprising a sequence with at least 85% identity to SEQ ID NO: 2;

[0045] Strain 3, comprising the 16S rDNA sequence shown in SEQ ID NO: 3 or comprising a sequence with at least 85% identity to SEQ ID NO: 3; and

[0046] Strain 4 contains the 16S rDNA sequence shown in SEQ ID NO: 4 or contains a sequence with at least 85% identity to SEQ ID NO: 4.

[0047] In the embodiments of this application, the percentage of identity typically describes the degree to which two sequences are identical, that is, it typically describes the percentage of nucleotides that correspond to the same nucleotides in the reference sequence at their sequence positions. In the embodiments of this application, the "sequence with at least 85% identity" refers to a sequence that has an identity of any value (including endpoint values) between 85% and 100% compared to the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. For example, it can have sequence identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. It can also refer to sequence identity represented by an infinite number of decimals between two adjacent integers, such as having at least 98.57%, 99.64%, 99.7%, 99.8%, or 99.9% sequence identity compared to the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0048] In this embodiment, strain 1 is a member of the genus *Arthrobacter*. In some embodiments, strain 1 has the sequence shown in SEQ ID NO: 1. In some embodiments, strain 1 may be *Arthrobacter* sp. *Arth4*, which has accession number GDMCC No: 62566.

[0049] Arthrobacter is a Gram-positive bacterium that exhibits a distinct rod-to-spherical growth cycle on complex culture media, with spherical Arthrobacter being a typical species. Phylogenetic analysis revealed that Arth4, as described in this application, is a novel species within the genus Arthrobacter. Arth4 is an Arthrobacter species belonging to the genus Arthrobacter, named *Arthrobacter sp. Arth4*; its accession number is GDMCC No:62566; the depository is Guangdong Provincial Center for Microbial Culture Collection; the address of the depository is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province; the deposit date is June 28, 2022.

[0050] In this application embodiment, strain Arth4, having the sequence shown in SEQ ID NO:1, can be understood by those skilled in the art as the original strain. Strains with a genome sequence having at least 85% identity with the sequence shown in SEQ ID NO:1 can be understood by those skilled in the art as variant strains of strain Arth4, having the sequence shown in SEQ ID NO:1. It is understood that strains such as Arth4 can undergo spontaneous mutations or be artificially cultured to form variant strains, for example, by nucleotide deletions, additions, or substitutions. The "variant strain" and "strain Arth4" have highly identical gene sequences and extremely similar biological functions; the mutated genes do not substantially affect the conserved sequences of strain Arth4, and therefore do not affect the genetic stability of strain Arth4. More specifically, this "variant strain" is also a strain of species Arth4, exhibiting the physiological activity characteristics of species Arth4. The specific genus corresponding to strain 1 and all species within that genus also fall within the protection scope of this application.

[0051] In this embodiment, strain 2 is a species of Bacillus. In some embodiments, strain 2 is a species of Bacillus paralicheniformis. In some embodiments, strain 2 has the sequence shown in SEQ ID NO: 2. In some embodiments, strain 2 may be Bacillus paralicheniformis Bacil9 with accession number GDMCC No: 62569.

[0052] In this embodiment, strain 3 is a species of *Priestia*. In some embodiments, strain 3 is a species of *Priestia megaterium*. In some embodiments, strain 3 has the sequence shown in SEQ ID NO: 3. In some embodiments, strain 3 may be *Priestiamegaterium Bacif4* with accession number GDMCC No: 62567.

[0053] In this embodiment, strain 4 is a species of Bacillus. In some embodiments, strain 4 is a species of Bacillus velezensis. In some embodiments, strain 4 has the sequence shown in SEQ ID NO: 4. In some embodiments, strain 4 may be Bacillus velezensis Baci5 with accession number GDMCC No: 62568.

[0054] Bacil9 is a paralicheniformis bacillus belonging to the genus Bacillus. Its deposit registration number is GDMCC No:62569. The depository institution is Guangdong Provincial Center for Microbial Culture Collection. The address of the depository is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. The deposit date is June 28, 2022.

[0055] Bacif4 is a species of Priestiamegaterium, belonging to the genus Priestia. Its name is Priestiamegaterium Bacif4. The deposit registration number is GDMCC No:62567. The depository institution is Guangdong Provincial Center for Microbial Culture Collection. The address of the depository institution is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. The deposit date is June 28, 2022.

[0056] Baci5 is a species of Bacillus velezensis belonging to the genus Bacillus. Its name is Bacillus velezensis Baci5. The depositary number is GDMCC No:62568. The depositary institution is the Guangdong Provincial Center for Microbial Culture Collection. The address of the depositary institution is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province. The deposit date is June 28, 2022.

[0057] Bacillus belongs to the family Bacillusaceae and the genus Bacillus. It is a group of Gram-positive bacteria capable of producing resistant endospores. The cells are rod-shaped and covered with a large amount of calcium pyridine dicarboxylate. Its cortex, located between the core and the spore shell, is rich in peptidoglycan; the core is a highly concentrated, inert chromosome; the outermost wall is a peptidoglycan wall, and there are one or more layers of a protein-rich spore coat. Bacillus also possesses broad-spectrum bacterial activity, producing bacteriocins to inhibit pathogens. An important characteristic of this genus is its ability to produce spores with specific resistance to adverse conditions. Currently, commonly used types of Bacillus include Bacillus subtilis, Bacillus licheniformis, and Bacillus cereus. In the embodiments of this application, strains 2-4 all belong to the genus Bacillus. Through further phylogenetic analysis, it was found that Bacil9 isolated in the embodiments of this application is a new strain of Bacillus paralicheniformis; Bacif4 isolated in the embodiments of this application is a new strain of Priestia megaterium; and Baci5 isolated in the embodiments of this application is a new strain of Bacillus velezensis.

[0058] It is understood that, as discussed above regarding strain 1 variants, strain variants with sequences that are at least 85% identical to those shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 also belong to the genus Bacillus and its corresponding species, and also possess physiological activity characteristics similar to strains 2-4. The specific genera corresponding to strains 2-4 and all species under those genera also fall within the scope of protection of this application.

[0059] The four novel strains Arth4, Bacil9, Bacif4, and Baci5 isolated in this application embodiment can effectively improve the agronomic traits of plants when used alone or in combination. In particular, the bacterial genera and species to which these four strains belong can effectively promote root elongation and stem growth, thereby improving nutrient absorption and accumulation and ultimately increasing crop yield. The rhizosphere growth-promoting bacteria proposed in this application embodiment are of great significance for improving crop yield.

[0060] The second aspect of this application also provides an agricultural formulation comprising the bacterial strains described in any embodiment of the first aspect of this application.

[0061] In the embodiments of this application, the agricultural formulation may contain any of the above-mentioned bacterial strains, wherein the bacterial strains include: strain 1, containing the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO: 1; strain 2, containing the 16S rDNA sequence shown in SEQ ID NO: 2 or containing a sequence with at least 85% identity to SEQ ID NO: 2; strain 3, containing the 16S rDNA sequence shown in SEQ ID NO: 3 or containing a sequence with at least 85% identity to SEQ ID NO: 3; and strain 4, containing the 16S rDNA sequence shown in SEQ ID NO: 4 or containing a sequence with at least 85% identity to SEQ ID NO: 4. Therefore, the agricultural formulation in the embodiments of this application may be a single strain 1-4 or any combination of strains 1-4; simultaneously, since the above-mentioned strains also contain mutant strains of the same genus as strains 1-4, the specific genera of strains 1-4 and all species under those genera also fall within the protection scope of the agricultural formulation of this application.

[0062] In this embodiment, strain 2 is a species of Bacillus. In some embodiments, strain 2 is a species of Bacillus paralicheniformis. In some embodiments, strain 2 has the sequence shown in SEQ ID NO: 2. In some embodiments, strain 2 may be Bacillus paralicheniformis Bacil9 with accession number GDMCC No: 62569.

[0063] In this embodiment, strain 3 is a species of *Priestia*. In some embodiments, strain 3 is *Priestia megaterium*. In some embodiments, strain 3 has the sequence shown in SEQ ID NO: 3. In some embodiments, strain 3 may be *Priestiamegaterium Bacif4* with accession number GDMCC No: 62567.

[0064] In this embodiment, strain 4 is a species of Bacillus. In some embodiments, strain 4 is a species of Bacillus velezensis. In some embodiments, strain 4 has the sequence shown in SEQ ID NO: 4. In some embodiments, strain 4 may be Bacillus velezensis Baci5 with accession number GDMCC No: 62568.

[0065] In this application embodiment, the agricultural formulation further includes excipients, which can be organic matter and / or inorganic matter. In this application embodiment, the organic matter can be other microbial fertilizers that promote plant growth, besides strains 1-4, such as: microbial fertilizers that increase soil nitrogen and crop nitrogen nutrition, such as rhizobium fertilizer, nitrogen-fixing bacterial fertilizer, nitrogen-fixing cyanobacteria fertilizer, etc.; microbial fertilizers that decompose soil organic matter, such as organophosphate bacterial fertilizers, comprehensive microbial fertilizers; microbial fertilizers that decompose insoluble minerals in the soil, such as phosphorus bacterial fertilizers, potassium bacterial fertilizers, mycorrhizal fungal fertilizers; microbial fertilizers that stimulate plant growth, such as growth-promoting microbial fertilizers; and microbial fertilizers that increase the stress resistance of crop roots, such as antibiotic fertilizers, stress-resistant microbial fertilizers. In this application embodiment, the organic matter can also be organic fertilizers required for plant growth, such as manure. It is understood that the organic matter in this application embodiment, alone or in combination, is sufficient as long as it can ensure a promoting effect on plant growth; this application does not impose any limitations on this.

[0066] In the embodiments of this application, the inorganic matter can be an agriculturally usable chemical component, such as an agriculturally acceptable carrier, excipient, diluent, adjuvant, medium, excipient, carrier or combination thereof, and / or an inorganic fertilizer that does not affect the concentration of microbial activity in the agricultural formulation. This application does not limit this.

[0067] In the embodiments of this application, the formulation of the agricultural preparation can be selected from: wettable powder, water-dispersible granules, suspension concentrate, emulsion, granules, seed coating agents, or combinations thereof. It is understood that the formulation of the agricultural preparation in the embodiments of this application only needs to ensure that it can be applied to plants in a certain form, and this application does not impose any restrictions on this.

[0068] The agricultural formulations proposed in this application, using Arth4, Bacil9, Bacif4, and Baci5 alone or in combination, can effectively improve the agronomic traits of plants. In particular, the genera and species of these four strains effectively promote root elongation and stem growth, thereby enhancing nutrient absorption and accumulation, and ultimately increasing crop yield. The rhizosphere growth-promoting bacteria proposed in this application are of great significance for increasing crop yield. Furthermore, by preparing Arth4, Bacil9, Bacif4, and Baci5 into a microbial fertilizer suitable for agricultural production, the use of chemical fertilizers can be reduced, promoting green agricultural production.

[0069] The third aspect of this application also proposes the use of a bacterial strain as described in any embodiment of the first aspect of this application in the preparation of agricultural formulations.

[0070] The fourth aspect of this application also proposes a method for promoting plant growth, comprising introducing a bacterial strain as described in any embodiment of the first aspect of this application or an agricultural preparation as described in any embodiment of the second aspect of this application.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0072] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0073] Example 1: Isolation of strains

[0074] Rhizosphere soil samples were collected from millet plants grown in Yangling, Shaanxi Province. The samples were diluted with sterile PBS-S buffer to form soil solutions, and then centrifuged at 1200 rpm for 5 minutes. The supernatant was collected. The supernatant was then diluted to 10⁻⁶ ppm. -1 Up to 10 -7 The gradient concentrations were taken as 10. -4 and 10 -6 Two μl of the concentrated dilution was inoculated into 96-well microtiter plates containing LB medium and incubated at 28°C for 48–72 hours. The culture was then passaged three times on LB solid medium to obtain single clones of the strain. Sequencing of the 16S rRNA gene of the single clones revealed microbial strains 1–4 with growth-promoting functions, as detailed below.

[0075] Strain 1: Arth4, whose 16S rDNA gene sequence is shown below as SEQ ID NO: 1.

[0076]

[0077] Strain 2: Bacil9, whose 16S rDNA gene sequence is shown below as SEQ ID NO: 2.

[0078]

[0079] Strain 3: Bacif4, whose 16S rDNA gene sequence is shown below as SEQ ID NO: 3.

[0080]

[0081] Strain 4: Baci5, whose 16S rDNA gene sequence is shown below as SEQ ID NO: 4.

[0082]

[0083] Example 2: Identification of the isolated strain

[0084] The inventors further identified strains 1-4 isolated in Example 1 by comparing the 16S rRNA sequences of strains 1-4 with databases.

[0085] 2.1 Arth4 (strain 1)

[0086] The nearly full-length 16S rDNA sequence of Arth4 (approximately 1.4 kb, SEQ ID NO: 1) obtained through sequencing was compared with the 16S rRNA gene database of EzBioCloud to preliminarily obtain the species classification information of strain 1. According to the comparison results, the two strains with the highest homology to the 16S rRNA gene of Arth4 in the database are *Arthrobacter bambusae* GM18 (serial number: KF150696) and *Paenarthrobacter aurescens* NBRC12136 (serial number: BJMD01000050), with similarities of 98.57% and 98.50%, respectively. Based on the 16S rRNA information, Arth4 can be preliminarily identified as a new species belonging to the genus *Arthrobacter*.

[0087] Furthermore, a phylogenetic tree was constructed using MAGA with neighbor-to-neighbor merging for closely related strains of Arth4, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that Arth4 does not cluster with other species of bacteria, which also proves that strain 1 isolated in Example 1 is a new species of Arthrobacter.

[0088] Simultaneously, the whole genome of Arth4 was sequenced, and its genome sequence was analyzed using FastANI software to determine the average nucleotide identity (ANI) of the genomes of Arthrobacter_bambusae (Genebank sequence number: GCA_022606295.1) and Paenarthrobacter aurescens NBRC 12136 (Genebank sequence number: GCA_006538985.1), which showed the highest similarity. ANI is an indicator for comparing the phylogenetic relationship between two genomes at the nucleotide level. ANI is defined as the average base similarity between homologous segments of two microbial genomes, and its characteristic is a high degree of distinguishability between closely related species. Analysis showed that the genome ANI of Arth4 was only 80.5% and 84.4% of that of Arthrobacter_bambusae and Paenarthrobacter aurescens NBRC 12136, respectively, which is far lower than the 95% ANI value of the same species (see Jain C, Rodriguez-R LM, Phillippy AM, et al. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries[J]. Nature Communications, 2018, 9(1): 5114, 95% is considered to be the threshold for determining whether they are the same species). Therefore, Arth4 was further determined to be a new strain of Arthrobacter, named Arthrobacter sp. Arth4, and deposited in Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No: 62566.

[0089] 2.2 Baci9 (strain 2)

[0090] Similarly, the nearly full-length 16S rDNA sequence of Bacil9 (Bacil9_16S_rRNA_gene, SEQ ID NO: 2) obtained from sequencing was compared with the 16S rRNA gene database of EzBioCloud to preliminarily obtain the species classification information of strain 2. According to the comparison results, the strain with the highest homology to Bacil9's 16S rRNA gene in the database is Bacillus paralicheniformis (sequence number: KY694465), with a sequence similarity of 98.98%. Based on the 16S rRNA information, it can be preliminarily determined that Bacil9 belongs to the species Bacillus paralicheniformis.

[0091] Furthermore, a phylogenetic tree was constructed using MAGA with neighbor-to-neighbor merging for closely related strains of Bacil9, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that Bacil9 clusters together with Bacillus paralicheniformis KJ-16 (Genebank sequence number: GCA_001042485.2), which also verifies that strain 2 isolated in Example 1 belongs to the species Bacillus paralicheniformis.

[0092] Simultaneously, the whole genome of Bacil9 was sequenced, and its genome sequence was analyzed using FastANI software with the genome of Bacillus paralicheniformis KJ-16 (Genebank sequence number: GCA_001042485.2), which had the highest similarity. The analysis results showed that the ANI of the Bacil9 genome with that of Bacillus paralicheniformis KJ-16 (Genebank sequence number: GCA_001042485.2) reached 99.24% (greater than 95%). Therefore, Bacil9 was further identified as a new strain of Bacillus paralicheniformis, named Bacillus paralicheniformis Bacil9, and deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:62569.

[0093] 2.3 Bacif4 (strain 3)

[0094] Similarly, the nearly full-length 16S rDNA sequence of Bacif4 (Bacif4_16S_rRNA_gene, i.e., SEQ ID NO: 3) obtained from sequencing was compared with the 16S rRNA gene database of EzBioCloud to preliminarily obtain the species classification information of strain 3. According to the comparison results, the strain with the highest homology to the 16S rRNA gene of Bacif4 in the database is Priestia megaterium NBRC 15308 (serial number: JJMH01000057), with a similarity of 99.93%. Based on the 16S rRNA information, it can be preliminarily determined that Bacif4 belongs to the species Priestia megaterium.

[0095] Furthermore, a phylogenetic tree was constructed using MAGA with neighbor-to-neighbor merging for the sequences of closely related strains of Bacif4, and the results are as follows: Figure 3 As shown. By Figure 3 It can be seen that Bacif4 is closely related to Priestia megaterium NBRC 15308 (Genebank sequence number: GCA_009935415.1), which also verifies that strain 3 isolated in Example 1 belongs to the Priestia megaterium species.

[0096] Simultaneously, the whole genome of Bacif4 was sequenced, and its genome sequence was analyzed using FastANI software with the genome of Priestia megaterium NBRC 15308 (Genebank sequence number: GCA_009935415.1), which had the highest similarity. The analysis results showed that the ANI of the Bacif4 genome with that of Priestia megaterium NBRC15308 (Genebank sequence number: GCA_009935415.1) reached 99.18% (greater than 95%). Therefore, Bacif4 was further identified as a novel strain of Priestia megaterium, named PriestiamegateriumBacif4, and deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:62567.

[0097] 2.4Baci5 (strain 4)

[0098] Similarly, the nearly full-length 16S rDNA sequence of Baci5 (Baci5_16S_rRNA_gene, SEQ ID NO: 4) obtained from sequencing was compared with the 16S rRNA gene database of EzBioCloud to preliminarily obtain the species classification information of strain 4. According to the comparison results, the strains with the highest homology to Baci5's 16S rRNA gene in the database are Bacillus siamensis KCTC 13613 (serial number: AJVF01000043) and Bacillus velezensis CR-502 (serial number: AY603658), with a similarity of 99.93%. Based on the 16S rRNA information, Baci5 can be preliminarily determined to belong to the genus Bacillus.

[0099] Furthermore, a phylogenetic tree was constructed using MAGA with neighbor-to-neighbor merging for closely related strains of Baci5, and the results are as follows: Figure 4 As shown. By Figure 4 It can be seen that Baci5 clusters together with Bacillus siamensis KCTC 13613 (Genebank sequence number: GCA_000262045.1) and Bacillus velezensis CR-502 (Genebank sequence number: GCA_001461825.1), which also verifies that strain 4 isolated in Example 1 belongs to the genus Bacillus.

[0100] Meanwhile, the whole genome of Baci5 was sequenced, and its genome sequence was analyzed using FastANI software with the genomes of Bacillus siamensis KCTC 13613 (Genebank sequence number: GCA_000262045.1) and Bacillus velezensis (Genebank sequence number: GCA_001461825.1), which had the highest similarity. Analysis showed that the genome ANI of Baci5 was 93.88% and 97.97% of that of Bacillus siamensis KCTC 13613 (Genebank sequence number: GCA_000262045.1) and Bacillus velezensis (Genebank sequence number: GCA_001461825.1), respectively. Therefore, it was further determined that Bacif5 does not belong to Bacillus siamensis, but is a new strain of Bacillus velezensis. It was named Bacillus velezensis Baci5 and deposited at Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:62568.

[0101] Example 3: Plate Growth Promotion Experiment

[0102] 3.1 After activating strains 1-4, they were inoculated into NY medium and fermented at 28°C with shaking at 180 rpm. Then, the OD of the bacterial culture was measured. 600 Adjust the concentration to 0.5, take the bacterial culture, centrifuge at 5000 rpm for 10 min, remove the supernatant, resuspend the bacterial cells in sterile water, and set aside for use.

[0103] 3.2 Huagu 12 seeds were surface-sterilized with 1% sodium hypochlorite for 5 minutes, followed by rinsing five times with sterile water. In sterile petri dishes, 15-20 seeds were evenly placed on each layer of sterile double-layered filter paper. 2 ml of the bacterial suspension 1-4 obtained in step 3.1 was sprayed onto the seeds, and they were removed after 7 days. The number of germinated seeds was counted, and the root length and plant height of the seedlings were recorded. Three parallel experimental groups were set up for each treatment. The control group was sprayed with sterile water.

[0104] 3.3 After 25 days of cultivation, the root length and plant height of the seedlings were measured. The specific measurements (average values) are shown in Tables 1 and 2, respectively. The growth of the treatment group and the control group are as follows: Figure 5 As shown.

[0105] Table 1

[0106] control group 3.63 / Bacif4 3.41 4.07E-01 Bacil9 3.95 2.12E-01 Baci5 3.96 2.12E-01 Arth4 4.30 2.40E-02

[0107] Table 2

[0108] control group 2.13 / Bacif4 2.83 1.10E-01 Bacil9 3.40 1.02E-08 Baci5 2.23 2.34E-03 Arth4 2.82 4.34E-06

[0109] Tables 1 and 2 show the root length and plant height of seedlings treated with microbial agents in the plate experiment. Table 1 shows that, under plate culture, compared with the control group, the root length of seedlings treated with agent 1 (Arth4) was significantly longer at the 5% level than the control group (*, p<0.05). Table 2 shows that, compared with the control group, the average root length of seedlings treated with agents 1-4 was longer than that of the control group, with highly significant differences between agents 1 (Arth4) and 2 (Bacil9) (***, p<0.001), and a significant difference at the 0.1% level for agent 4 (Baci5) (**, p<0.01). Therefore, this demonstrates that strains 1-4 have a good promoting effect on plant root length, especially plant height.

[0110] Example 4: Growth-promoting experiment in sterile soil

[0111] Millet seeds were disinfected with a 1% sodium hypochlorite solution for 5 minutes and rinsed 5 times with sterile distilled water. The disinfected seeds were then planted in sterile soil pots (the sterile soil was obtained by sterilizing field-grown millet soil at 120℃). 5-10 plants were planted in each pot. Five days after germination, 5 ml of bacterial solution (1-4) was poured around the roots of each germinated plant in each pot. Each treatment had three parallel groups, with the control group irrigated with sterile water. Irrigation was performed every 5 days for a total of 3 times. After 25 days, the plants were removed from the pots, and their root length and plant height were measured. The specific measurements (average values) are shown in Tables 3 and 4 below. The growth of the treatment and control groups is as follows: Figure 6 As shown.

[0112] Table 3

[0113] control group 3.39 / Bacif4 4.73 5.06E-04 Bacil9 4.43 1.33E-03 Baci5 4.13 3.29E-02 Arth4 3.95 6.86E-02

[0114] Table 4

[0115] control group 7.56 / Bacif4 9.20 2.71E-02 Bacil9 9.12 1.09E-02 Baci5 8.83 2.35E-02 Arth4 10.81 9.60E-07

[0116] Tables 3 and 4 show the root length and plant height of seedlings treated with bacterial solutions in the sterile soil experiment. Table 3 shows that, under sterile soil substrate cultivation, compared with the control group, the average root length of seedlings treated with bacterial agents 1-4 was longer, indicating that bacterial agents 1-4 all promoted plant root growth. Furthermore, bacterial agent 4 (i.e., Baci5) and bacterial agent 3 (i.e., Bacif4) showed extremely significant differences (***, p<0.001), while bacterial agent 2 (i.e., Bacil9) showed a significant difference at the 0.1% level (**, p<0.01), thus indicating that strains 2-4 can significantly promote plant root growth.

[0117] Regarding plant height, Table 4 shows that, under sterile soil substrate cultivation, the average plant height of seedlings treated with inoculants 1-4 was greater than that of the control group, indicating that strains 1-4 all promoted plant height growth. Furthermore, the plant height of seedlings treated with inoculants 2-4 was significantly higher than that of the control group at the 5% level (*, p<0.05), and inoculant 1 (i.e., Arth4) showed a highly significant difference compared to the control group (***, p<0.001), thus proving that strains 1-4 significantly promoted plant height growth.

[0118] In summary, strains 1-4 proposed in the embodiments of this application can be effectively used to promote plant growth.

[0119] Example 5: Field Experiment

[0120] Huagu 12 was planted in large pots filled with field soil and irrigated with bacterial solutions 1-4 respectively. After the plants grew, their growth traits were measured. Each treatment was performed in triplicate, while the control group was irrigated with sterile water. Specific measurement data (average values) are shown in Tables 5-7. The growth of the treatment and control groups is as follows: Figure 7 and Figure 8 As shown.

[0121] Table 5

[0122]

[0123]

[0124] Table 6

[0125] control group 14.36 / Arth4 17.64 0.0145 Bacil9 18.74 0.0116 Bacif4 15.45 0.3889 Baci5 14.77 0.7299

[0126] Table 7

[0127] control group 86 / Arth4 92.18 0.0852 Bacil9 90 0.3464 Bacif4 85.54 0.9036 Baci5 78.97 0.0637

[0128] Depend on Figure 7 and 8 As can be seen, in the field trial, the plants treated with bacterial agents 1-4 grew more vigorously than the control group, with generally taller, stronger, and more upright plants and dark green leaves, indicating that strains 1-4 in this application have a good promoting effect on the growth of plant stems and leaves. Meanwhile, from Figure 7 The results showed that, compared with the control group, the millet plants treated with inoculants 1-4 produced more ears of grain that were larger and fuller. This indicates that strains 1-4 can enable plants to absorb and accumulate more nutrients, and improve the grain setting of plants after promoting the overall growth of the plants, thereby increasing crop yield.

[0129] Tables 5-7 show the specific measurements of the main stem and plant height of the treatment and control groups, representing the quantitative values ​​of plant growth. As shown in Table 5, the average main stem diameter of plants treated with Bacif4 strain was comparable to that of the control group, while the main stem diameter of plants treated with other inoculants (Arth4, Bacil9, and Baci5) was greater than that of the control group. This demonstrates that strains 1-4 can promote plant stem growth, with Bacil9 (i.e., strain 2) exhibiting the thickest main stem, indicating that Bacil9 effectively promotes plant stem growth. Regarding plant height, Table 7 shows that in the field trial, the average plant height of the treatment group treated with Baci5 strain was lower than that of the control group, the average plant height of plants treated with Bacif4 strain was comparable to that of the control group, and the average plant height of plants treated with Arth4 and Bacil9 was higher than that of the control group. Regarding the diameter of the main stem spike, the average diameter of the main stem spikes applied with fungal agents 1-4 was greater than that of the control group. Since the fruit setting condition can directly reflect the nutrient absorption and accumulation status of plants, the data in Table 6 proves that fungal agents 1-4 can effectively promote the nutrient absorption status of plants.

[0130] Therefore, the rhizosphere growth-promoting strains 1-4 newly isolated and identified in this application have a good promoting effect on plant growth. Furthermore, the promotion of plant root growth by strains 1-4 can effectively improve the nutrient absorption of the plant rhizosphere, thereby increasing crop yield.

[0131] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0132] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bacterial strain for promoting plant growth, wherein the bacterial strain is strain 1, strain 1 is of the genus *Arthrobacter*, has a 16S rDNA sequence as shown in SEQ ID NO: 1, strain 1 is named *Arthrobacter sp.Arth4*, and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 28, 2022, with accession number: GDMCC No: 62566.

2. An agricultural formulation for promoting plant growth, wherein the agricultural formulation comprises the bacterial strain as described in claim 1.

3. The agricultural formulation according to claim 2, wherein the agricultural formulation further comprises excipients.

4. The agricultural formulation according to claim 2 or 3, wherein the formulation is selected from: wettable powder, water-dispersible granules, suspension concentrate, emulsion, granules, seed coating agent or a combination thereof.

5. Use of the strain according to claim 1 in the preparation of an agricultural formulation for promoting plant growth, wherein the plant is millet.

6. A method for promoting plant growth, the method comprising applying to the plant the bacterial strain as described in claim 1 or the agricultural agent as described in any one of claims 2 to 4, wherein the plant is millet.

Images