Strain with growth promoting and biocontrol functions and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to Rahnella and Burkholderia with growth promotion and biological control functions and a composition thereof, and the Rahnella comprises a 16S rDNA sequence shown in SEQ ID NO: 1 or a complementary sequence thereof or a sequence which is at least 85% identical with the sequence shown in SEQ ID NO: 1; the burkholderia comprises a 16S rDNA sequence as shown in SEQ ID NO: 2 or a complementary sequence of the 16S rDNA sequence or a sequence which is at least 85% identical with the sequence as shown in SEQ ID NO: 2. The new strains Rahnella victoriana Rahn.R5, Burkholderia ubonensis Burk.R2 and the application of the composition of the Rahnella victoriana Rahn.R5 and the Burkholderia ubonensis Burk.R2, which are provided by the invention, in promoting crop growth and / or preventing and treating plant diseases, have important significance on improving the crop yield and promoting agricultural green production.

Description

Technical Field

[0001] The present application relates to the field of microorganisms, and specifically relates to Rahnella and Burkholderia with growth-promoting and biocontrol functions and their applications. Background Art

[0002] In the past more than 40 years, the stable increase in grain production has been achieved in agricultural production by applying chemical fertilizers and pesticides. However, the excessive application of chemical fertilizers and pesticides has brought a series of environmental impacts. The excessive application of chemical fertilizers easily leads to the destruction of soil structure, the loss of soil nutrients, soil compaction and secondary salinization of the soil environment; the unreasonable application of pesticides will bring problems of excessive pesticide residues in agricultural products and environmental pollution. Plant growth-promoting rhizobacteria refer to beneficial microorganisms that can colonize the rhizosphere of plants and have promoting effects on plant growth, development and stress resistance. The development and utilization of plant rhizosphere beneficial bacteria resources can reduce the application amount of chemical fertilizers, and some strains can replace the application of pesticides, thereby alleviating the environmental impacts brought by chemical fertilizers and pesticides.

[0003] As a beneficial bacterium, in the related research of Rahnella sp., Patent CN 110643531 B discloses a Rahnella victor that has a good degradation effect on cellulose, but this strain does not have the functions of promoting crop growth and biocontrol; Patent CN 108504595 B discloses a plant growth-promoting rhizobacterium Rahnella aquatilis Gro, which can produce high yields of indole acetic acid and can solve the conversion of insoluble potassium silicate into soluble potassium salts, and has growth-promoting characteristics such as decomposing organic phosphorus and nitrogen fixation. However, this strain does not have the biocontrol function against pathogenic bacteria.

[0004] Therefore, there is an urgent need to develop new bacterial strains with both plant growth-promoting and biocontrol functions to reduce the use of chemical fertilizers and pesticides, thereby reducing environmental pollution. Summary of the Invention

[0005] To this end, the embodiments of the present application provide a new strain of Rahnella with both growth-promoting and biocontrol functions, a compound preparation containing the same, and their applications.

[0006] The first aspect of the embodiments of the present application provides a Rahnella 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 with SEQ ID NO: 1.

[0007] In some embodiments, the Rahnella bacterium is of the genus Rahnella; preferably, the Rahnella bacterium has the 16S rDNA sequence shown in SEQ ID NO: 1; more preferably, the Rahnella bacterium is Rahnella victoriana Rahn.R5 with the preservation number GDMCC No: 63461.

[0008] An embodiment of the second aspect of the present application provides an agricultural preparation, which contains the Raoultella as described in any embodiment of the first aspect above and optionally Burkholderia.

[0009] In some embodiments, the Burkholderia contains 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; optionally, the Burkholderia is of the genus Burkholderia; preferably, the Burkholderia has the 16S rDNA sequence shown in SEQ ID NO: 2; more preferably, the Raoultella is Burkholderia ubonensis Burk.R2 with the preservation number GDMCC No: 63569.

[0010] 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.

[0011] An embodiment of the third aspect of the present application proposes the application of the Raoultella as described in any embodiment of the first aspect above or the agricultural preparation as described in any embodiment of the second aspect above in promoting plant growth.

[0012] In some embodiments, the Raoultella or the agricultural preparation is specifically used for one or more of the following: nitrogen fixation, phosphorus solubilization, siderophore production, hormone production, and ACC deaminase production. Optionally, the phosphorus solubilization includes dissolving organic phosphorus and dissolving inorganic phosphorus. Optionally, the siderophore includes siderophilin. Optionally, the hormone includes indoleacetic acid, gibberellin or a combination thereof.

[0013] An embodiment of the fourth aspect of the present application proposes the application of the Raoultella as described in any embodiment of the first aspect above or the agricultural preparation as described in any embodiment of the second aspect above in plant disease control.

[0014] In some embodiments, the plant disease control includes one or more of the following: inhibiting Rhizoctonia solani, inhibiting Fusarium oxysporum, and inhibiting Fusarium graminearum, preferably inhibiting Fusarium graminearum.

[0015] In some embodiments, the plant diseases include one or more of the following: black spot, anthracnose, root rot, and lawn mottle caused by Rhizoctonia solani; scab, root rot, stem rot, ear rot, basal stem rot, flower rot, rice blast, smut, black spot, brown spot, and mosaic disease caused by Fusarium oxysporum; and scab, root rot, stem rot, ear rot, basal stem rot, flower rot, rice blast, smut, black spot, and brown spot caused by Fusarium graminearum.

[0016] In some embodiments, the plant is selected from crops, vegetables, fruit trees, flowers and turfgrasses. Optionally, the crop is selected from rice, corn, wheat, barley, oats, millet, soybeans, peanuts and cotton, and the vegetable is selected from Chinese cabbage, carrots, ginger, string beans, beets, cucumbers, peppers, tomatoes, onions, bracken and alfalfa.

[0017] In some embodiments, the plant is selected from Gramineae plants and Leguminosae plants. Preferably, the Gramineae plant is selected from corn, millet and upland rice, and the Leguminosae plant is selected from soybeans and alfalfa.

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

[0019] The present application first proposes the application of the new strain of Rahnella victoriana Rahn.R5 and its compound preparation with Burkholderia ubonensis Burk.R2 in promoting plant growth and / or controlling plant diseases caused by Fusarium oxysporum, especially in effectively fixing nitrogen, inhibiting the fungus Fusarium oxysporum and promoting plant growth, thereby reducing the application amount of chemical fertilizers and pesticides and alleviating the environmental impact brought by chemical fertilizers and pesticides. The new strain of Rahnella with growth-promoting and biocontrol functions proposed in the present application is of great significance for improving the yield of agricultural crops. At the same time, by making the microbial fertilizer suitable for agricultural production with Rahnella victoriana Rahn.R5, the application amount of chemical fertilizers can be reduced and green agricultural production can be promoted. 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 the embodiments. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is the phylogenetic tree of Rahn.R5 according to Embodiment 2 of the present application.

[0022] Figure 2 It is the phylogenetic tree of Burk.R2 according to Embodiment 2 of the present application.

[0023] Figure 3 It is the plate growth diagram of Rahn.R5 according to Embodiment 3 of the present application.

[0024] Figure 4 It is the plate growth diagram of Burk.R2 according to Embodiment 3 of the present application.

[0025] Figure 5The growth of Rahn.R5 according to Embodiment 4 of the present application on Ashby nitrogen-free medium is shown.

[0026] Figure 6 The growth of Burk.R2 according to Embodiment 4 of the present application on Ashby nitrogen-free medium is shown.

[0027] Figure 7 The phosphorus-solubilizing effect of Rahn.R5 according to Embodiment 5 of the present application is shown.

[0028] Figure 8 The phosphorus-solubilizing effect of Burk.R2 according to Embodiment 5 of the present application is shown.

[0029] Figure 9 The siderophore-producing effect of Burk.R2 according to Embodiment 6 of the present application is shown.

[0030] Figure 10 The inhibitory effect of Rahn.R5 against Rhizoctonia solani according to Embodiment 7 of the present application is shown.

[0031] Figure 11 The inhibitory effect of Rahn.R5 against Fusarium oxysporum according to Embodiment 7 of the present application is shown.

[0032] Figure 12 The inhibitory effect of Rahn.R5 against Fusarium graminearum according to Embodiment 7 of the present application is shown.

[0033] Figure 13 The inhibitory effect of Burk.R2 against Rhizoctonia solani according to Embodiment 7 of the present application is shown.

[0034] Figure 14 The inhibitory effect of Burk.R2 against Fusarium oxysporum according to Embodiment 7 of the present application is shown.

[0035] Figure 15 The inhibitory effect of Burk.R2 against Fusarium graminearum according to Embodiment 7 of the present application is shown.

[0036] Figure 16 The growth-promoting effect of Rahn.R5 according to Embodiment 8 of the present application is shown, where upland rice is on the left and millet is on the right.

[0037] Figure 17 The growth-promoting effect of Burk.R2 according to Embodiment 8 of the present application is shown, where a, maize; b, upland rice; c, peanut; d, alfalfa.

[0038] Description of strain preservation:

[0039] Rahnella victoriana Rahn.R5: The preservation registration number is GDMCC No: 63461, 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 date is May 14, 2023.

[0040] Burkholderia ubonensis Burk.R2: The preservation registration number is GDMCC No: 63569, 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 date is June 16, 2023. Specific implementation manners

[0041] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments 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 constitute any limitation to the present invention in any way.

[0042] This application is based on the following understanding of the inventors:

[0043] The excessive application of chemical fertilizers and pesticides in agricultural production has brought a series of environmental problems, such as the destruction of soil structure, soil nutrient loss, soil compaction and secondary salinization of the soil environment, as well as the problems of excessive pesticide residues in agricultural products and environmental pollution.

[0044] Soil, as a treasure house of microbial resources, has a complex composition and a wide variety of microorganisms. There are approximately 2,000 - 18,000 species of microorganisms in 1 g of soil. Rhizosphere microorganisms are extremely susceptible to plant hosts, soil types, nutritional status, and climate factors. The selection of microorganisms by plant hosts does not seem to be completely random. It seems that by secreting specific small-molecule nutrients, some potentially beneficial plant probiotics for plant growth are attracted to colonize around them, while modifying the soil pH and improving the soil quality, etc. Screening out potentially beneficial bacteria with application value purposefully under the guidance of big data analysis results can provide possibilities for agricultural applications.

[0045] In the embodiments of the present application, through a large number of experiments and data analyses, a new strain of Rahnella, Rahnella victoriana Rahn.R5 (hereinafter referred to as Rahn.R5), is isolated from soybean rhizosphere soil samples. It is capable of nitrogen fixation, phosphorus solubilization, siderophore production, hormone production, etc., and it is first discovered that, compared with other strains in its genus, it particularly has the function of inhibiting Fusarium graminearum. Thus, while significantly promoting the growth of crops, it has an antibacterial effect and can effectively reduce the application amounts of pesticides and chemical fertilizers.

[0046] Furthermore, in the embodiments of the present application, a Burkholderia is also isolated, which has more excellent growth promotion and antibacterial functions compared with other strains in its species.

[0047] The Rahnella proposed in the first aspect embodiments of the present application includes 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.

[0048] In the embodiments of the present application, the percentage of identity generally describes the degree of identity 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 the present application, the "sequence with at least 85% identity" refers to a sequence with an identity of any value between 85% and 100% (including the end values) compared with 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 sequence identities indicated by countless decimals between two adjacent integers. For example, it has a sequence identity of at least 98.57%, 99.64%, 99.7%, 99.8% or 99.9% compared with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0049] In some embodiments, the Rahnella is of the genus Rahnella; preferably, the Rahnella has the 16S rDNA sequence shown in SEQ ID NO: 1; more preferably, the strain is Rahnella victoriana Rahn.R5 (hereinafter referred to as Rahn.R5) with the preservation number GDMCC No: 63461.

[0050] The Latin scientific name of the genus Rahnella (Izard et al., 1981) consists of small rod-shaped cells, 0.5 - 0.7 μm × 2 - 3 μm, and belongs to the family Enterobacteriaceae under aerobic or facultative anaerobic fermentative Gram-negative bacilli. Through the analysis of the evolutionary status, Rahn. R5 proposed in the embodiments of the present application is a new species under the genus Rahnella, named Rahnella victoriana Rahn.R5, and is deposited in the Guangdong Microbial Culture Collection Center; the deposit registration number is GDMCC No: 63461; the address of the deposit unit is the 5th floor of the Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the deposit date is May 14, 2023.

[0051] In the embodiments of the present application, the strain Rahn.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 Rahn.R5 with the sequence shown in SEQ ID NO: 1. It can be understood that a strain such as Rahn.R5 can undergo spontaneous mutations or be artificially cultured to form variant strains, such as nucleotide deletions, nucleotide additions, or nucleotide substitutions. The "variant strain" has a highly identical gene sequence and extremely similar biological functions to the "strain Rahn.R5", and the mutated genes basically do not affect the conserved sequence of the strain Rahn.R5, thus not affecting the genetic stability of the strain Rahn.R5. More specifically, this "variant strain" is also a strain of the species Rahn.R5, showing the physiological activity characteristics of the species Rahn.R5. All bacterial strains of the specific species corresponding to Rahn.R5 and under the species also fall within the protection scope of the present application.

[0052] When the newly isolated strain Rahn.R5 in the embodiments of the present application is used alone, it can effectively improve the agronomic traits of plants, especially effectively fix nitrogen, dissolve phosphorus, produce siderophores, and can inhibit Fusarium oxysporum, Rhizoctonia solani, especially Fusarium graminearum, thereby significantly promoting plant growth and preventing plant diseases, thus reducing the application amount of chemical fertilizers and pesticides and alleviating the environmental impact brought by chemical fertilizers and pesticides. The new strain of Rahnella with growth-promoting and biocontrol functions proposed in the present application is of great significance for improving crop yields.

[0053] An embodiment of the present application further provides a Burkholderia bacterium, 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 to SEQ ID NO: 2. The Burkholderia bacterium belongs to the genus Burkholderia; preferably, it has the 16S rDNA sequence shown in SEQ ID NO: 2. Through phylogenetic analysis, a new strain of Burkholderia bacterium was isolated in the embodiment of the present application, named Burkholderia ubonensis Burk.R2 and deposited in the Guangdong Provincial Culture Collection Center of Microorganisms, with the deposit registration number GDMCC No: 63569; the address of the deposit unit is the 5th floor of the Experimental Building, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the deposit date is June 16, 2023.

[0054] Burkholderia sp. is a Gram-negative bacterium widely present in water, soil, plants, and the human body. It is a straight or slightly curved bacillus, arranged singly or in pairs; it has one or several polar flagella, is motile, and has no spores. Some bacteria of the genus Burkholderia have functions such as biological control, plant growth promotion, and bioremediation. Compared with other strains of the same species and genus as Burkholderia ubonensis Burk.R2 (Burkholderia ubonensis Burk.R2, hereinafter simply referred to as "Burk.R2") proposed in the embodiment of the present application, it has stronger plant growth promotion and biocontrol effects, especially for growth promotion effects such as nitrogen fixation and inhibition performance against pathogenic fungi such as Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, and Aspergillus niger, and can be more effectively used for plant disease control and growth promotion.

[0055] An embodiment of the second aspect of the present application provides an agricultural preparation, including the Rahnella bacterium described in any embodiment of the first aspect above and an optional Burkholderia bacterium.

[0056] Since the above-mentioned strain also includes mutant strains of the same species as strains Rahn.R5 and Burk.R2, the specific species of strains Burk.R2 and Burk.R2 and all bacterial strains under their species also fall within the protection scope of the agricultural preparation of the present application.

[0057] In some embodiments, the agricultural preparation can be or include a fermentation broth containing any one of the above-mentioned Rahnella and optionally any one of Burkholderia, especially the fermentation broth of strains Rahn.R5 and optionally Burk.R2. This fermentation broth contains strains Rahn.R5 and optionally Burk.R2 and their metabolites, and these metabolites can also be further used as biofertilizers or biocontrol drugs. In some embodiments, the fermentation medium of the fermentation broth can be a beef extract peptone medium. It can be understood that the fermentation medium of the fermentation broth of any one of the above-mentioned Rahnella and optionally any one of Burkholderia strains in the embodiments of the present application only needs to be able to provide the normal growth and metabolic fermentation of these strains, and the present application does not limit this.

[0058] In some embodiments, the agricultural preparation may further contain excipients, where the excipients can be organic matter and / or inorganic matter. In the embodiments of the present application, the organic matter can be additional bacterial fertilizers other than strains Rahn.R5 and optionally Burk.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 soil-insoluble minerals, such as phosphorus bacteria fertilizer, potassium bacteria fertilizer, mycorrhizal fungal 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 can 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 only needs to ensure that it can promote plant growth, and the present application does not limit this.

[0059] In the embodiments of the present application, the inorganic matter can 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 the present application does not limit this.

[0060] In the embodiments of the present application, the dosage form of the agricultural preparation can 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 only needs to ensure that it can be applied to plants in a certain form, and the present application does not limit this.

[0061] In the embodiments of the present application, "application" can include: root irrigation, in-plant injection and / or plant surface spraying, and the plant surface optionally includes the surfaces of roots, stems, leaves, flowers, fruits and / or seeds.

[0062] The agricultural preparation proposed in the embodiments of the present application can effectively improve the agronomic traits of plants by using Rahn.R5. It can effectively fix nitrogen, dissolve phosphorus, produce siderophores, inhibit Fusarium oxysporum, Rhizoctonia solani, and Fusarium graminearum, especially inhibit Fusarium graminearum, thereby showing a significant dual effect of promoting growth and preventing diseases. In addition, by further adding Burk.R2 to the agricultural preparation, its synergy with Rahn.R5 can further significantly improve the growth promotion and disease prevention effects, thereby greatly reducing the application amounts of chemical fertilizers and pesticides and alleviating the environmental impacts brought by chemical fertilizers and pesticides.

[0063] An embodiment of the third aspect of the present application provides an application of the Rahnella described in any embodiment of the first aspect above or the agricultural preparation described in any embodiment of the second aspect above in promoting plant growth.

[0064] In some embodiments, the Rahnella or the agricultural preparation is specifically used for one or more of the following: nitrogen fixation, phosphorus dissolution, siderophore production, hormone production, and ACC deaminase production. In some embodiments, the Rahnella or the agricultural preparation containing it can be used for effectively fixing nitrogen, dissolving phosphorus, producing siderophores, producing hormones, and producing ACC deaminase, etc. to promote plant growth. Embodiments of the present application thus provide an application of the Rahnella or the agricultural preparation in nitrogen fixation, phosphorus dissolution, siderophore production, hormone production, and / or ACC deaminase production to promote plant growth.

[0065] In the embodiments of the present application, "phosphorus dissolution" refers to the process in which under the action of microorganisms, organic phosphorus compounds in the soil are converted into phosphates (POT) or insoluble phosphorus in the soil is converted into soluble phosphorus, and the forms of phosphorus existence can be organic phosphorus and inorganic phosphorus. It can be understood that the Rahnella or the agricultural preparation containing it in the embodiments of the present application can increase the content of available phosphorus in the soil, which is beneficial to promoting plant growth.

[0066] In the embodiments of the present application, "siderophore" refers to low-molecular-weight organic compounds produced by microorganisms and plants that have specific binding ability to Fe3+ in the environment. These organic substances have a high affinity for Fe3+ and can coordinate with it to form stable octahedral chelates. These chelates are absorbed through the cell membrane to supplement iron nutrition for cells. In some embodiments, the siderophore includes siderophilin. It can be understood that the Rahnella, Burkholderia, or the agricultural preparation containing it in the embodiments of the present application has a good stimulating effect on plant growth and development through siderophore production and can prevent certain plant root diseases.

[0067] In the embodiments of the present application, the Rahnella or Burkholderia can also secrete hormones during metabolism to promote plant growth. In some embodiments, the hormones include but are not limited to indoleacetic acid, gibberellin, or a combination thereof.

[0068] In the embodiments of the present application, "ACC deaminase (ACCD)" refers to an active substance that can decompose ACC into ammonia and α-ketobutyric acid, thereby reducing ethylene synthesis. It can be understood that the Rahnella or Burkholderia in the embodiments of the present application can inhibit ethylene synthesis in plants, reduce the sensitivity of plants to adversity, and improve the stress resistance of plants by producing ACC deaminase.

[0069] The Rahnella Rahn.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, phosphorus solubilization, siderophore production, hormone production, ACC deaminase production, etc. In addition, by further adding Burkholderia Burk.R2 (which shows better comprehensive growth promotion effect compared to other strains in its genus) to the agricultural preparation, its synergy with Rahn.R5 can further enhance the growth promotion effect, thereby greatly reducing the application amount of chemical fertilizers and pesticides and alleviating the environmental impact brought by chemical fertilizers and pesticides.

[0070] The embodiments of the fourth aspect of the present application propose the application of the Rahnella as described in any one of the above first aspect embodiments or the agricultural preparation as described in any one of the above second aspect embodiments in the prevention and control of plant diseases.

[0071] In some embodiments, the prevention and control of plant diseases includes one or more of the following: inhibiting Rhizoctonia solani, inhibiting Fusarium oxysporum, and inhibiting Fusarium graminearum, especially inhibiting Fusarium graminearum.

[0072] In the embodiments of the present application, "Fusarium oxysporum" belongs to the family Tuberculariaceae and the genus Fusarium. It is a soil-borne pathogenic fungus distributed worldwide with a wide host range and can cause the occurrence of wilt diseases in more than 100 plants such as cucurbits, solanaceae, bananas, cotton, legumes, and flowers. The Rahnella Rahn.R5, Burkholderia Burk.R2 or the agricultural preparation containing it proposed in the embodiments of the present application can effectively inhibit Fusarium oxysporum, thereby effectively preventing and controlling the wilt diseases of plants such as cucurbits, solanaceae, bananas, cotton, legumes, and flowers caused by Fusarium oxysporum.

[0073] In the embodiments of the present application, "Rhizoctonia solani" is a soil-borne fungus with a wide host range, which can infect at least more than 260 plants in 43 families, including Gramineae (rice, wheat, corn, etc.), Solanaceae (tomato, potato, tobacco, etc.), Leguminosae (peanut, soybean, alfalfa, etc.), etc. It is an important pathogenic bacterium affecting agricultural production. This pathogenic bacterium mainly directly penetrates the epidermis of plant leaves, leaf sheaths and stems by producing infection pads, hyphal coils and other infection structures, absorbs the nutrients of the host, destroys the original tissue cell structure, causes leaf rot, sheath rot and stem base rot, appears oval or irregular yellow-brown leaf spots, and finally leads to plant chlorosis, wilting and even death. The Rahnella Rahn.R5, Burkholderia Burk.R2 or the agricultural preparation containing them proposed in the embodiments of the present application can effectively inhibit Rhizoctonia solani, and thus can effectively prevent and control rice sheath blight, potato black scurf and soybean root rot caused by Rhizoctonia solani, etc.

[0074] In the embodiments of the present application, "Fusarium graminearum" is a highly infectious pathogenic bacterium that can infect parts of plants such as spikes, stems, stem bases and roots of cereal crops such as wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oryza sativa), oats (Arena sativa), etc., causing diseases such as ear rot, stem rot, stem base rot and root rot. The present application first discovers that Rahnella Rahn.R5 has a good inhibitory effect on Fusarium graminearum, thus providing a richer strain resource for the prevention and control of plant diseases. In addition, Burkholderia Burk.R2 proposed in the embodiments of the present application shows better inhibitory ability against the above-mentioned pathogenic bacteria compared with other strains in its genus.

[0075] In some embodiments, the plant diseases include one or more of the following: black spot, anthracnose, root rot and lawn leaf spot caused by Rhizoctonia solani; scab, root rot, stem rot, ear rot, stem base rot, flower rot, rice blast, smut, black spot, brown spot and mosaic disease caused by Fusarium oxysporum; and scab, root rot, stem rot, ear rot, stem base rot, flower rot, rice blast, smut, black spot and brown spot caused by Fusarium graminearum.

[0076] In some embodiments, the plants are selected from crops, vegetables, fruit trees, flowers and lawn grasses. Optionally, the crops are selected from rice, corn, wheat, barley, oats, millet, soybean, peanut and cotton, and the vegetables are selected from Chinese cabbage, carrot, ginger, kidney bean, beet, cucumber, pepper, tomato, onion, bracken and alfalfa.

[0077] In other embodiments, the plant is selected from Gramineae plants and Leguminosae plants. Preferably, the Gramineae plants are selected from corn, millet and upland rice, and the Leguminosae plants are selected from soybean and alfalfa.

[0078] In the following examples, the experimental methods are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0079] Unless otherwise specified, in the following examples, the quantitative tests are all set with three repeated experiments, and the results are averaged.

[0080] Example 1: Isolation of new strains Rahn.R5 and Burk.R2

[0081] Collect the rhizosphere soil sample of soybean. Pour the rhizosphere soil sample into a mortar, add 2 - 3 ml of sterile water, and directly grind it into powder to obtain a grinding liquid. Dilute the grinding liquid with sterile water to gradient concentrations of 10 2 to 10 5 . Coat the gradient dilution liquids onto the nutrient agar solid medium respectively, and at the same time coat the sterile water without bacterial liquid as a blank control. After coating, incubate it in an inverted position at 28 °C for 48 - 72 hours. Pick the single colonies into the liquid nutrient agar medium for amplification culture respectively, and then repeatedly streak for amplification to purify the monoclonal strains numbered Rahn.R5 and Burk.R2. Sequence the 16S rDNA genes of the Rahn.R5 and Burk.R2 monoclonal strains, and the specific sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

[0082] Rahn.R5 16S rDNA sequence (SEQ ID NO: 1):

[0083] GGCAGCGGGAAGTAGCTTGCTACTTTGCCGGCGAGCGGCGGACGGGTGAGTAATGTC

[0084] TGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATGA

[0085] CCTCGCAAGAGCAAAGTGGGGGACCTTCGGGCCTCACGCCATCGGATGTGCCCAGAT

[0086] GGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTG

[0087] AGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCA

[0088] GCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGA

[0089] AGAAGGCCTTAGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGGTTCAGTGTTAATA

[0090] GCACTGTGCATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGC

[0091] CGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCA

[0092] GGCGGTTTGTTAAGTCAGATGTGAAATCCCCGAGCTTAACTTGGGAACTGCATTTGAA

[0093] ACTGGCAAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATG

[0094] CGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACG

[0095] CTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTG

[0096] TAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGT

[0097] TAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGG

[0098] GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACC

[0099] TACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAACTCTGAG

[0100] ACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCA

[0101] ACGAGCGCAACCCTTATCCTTTGTTGCCAGCGAGTCATGTCGGGAACTCAAAGGAGAC

[0102] TGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACG

[0103] AGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGAACTCGCGAGAG

[0104] CAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCAT

[0105] GAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGG(SEQ ID NO:1)

[0106] Burk.R2 16S rDNA sequence (SEQ ID NO:2):

[0107] GGATCTGGCGGCATGCCTTAACATGCAGTCGAACGGCAGCACGGGTGCTTGCACCTGG

[0108] TGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGC

[0109] CCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGACCTTCG

[0110] GGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCT

[0111] ACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAG

[0112] ACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAA

[0113] GCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTG

[0114] TCCGGAAAGAAATCCTTGGTTCTAATATAGCCGGGGGATGACGGTACCGGAAGAATAA

[0115] GCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCG

[0116] GAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGCTAAGACCGATGTGAAATCCCCG

[0117] GGCTCAACCTGGGAACTGCATTGGTGACTGGCAGGCTAGAGTATGGCAGAGGGGGGT

[0118] AGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAA

[0119] GGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGG

[0120] ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATT

[0121] TCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGAT

[0122] TAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTC

[0123] GATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCTGCTGAGAGGCG

[0124] GGAGTGCTCGAAAGAGAACCGGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT

[0125] CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGC

[0126] AAGAGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC

[0127] AAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAG

[0128] GGTTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGC

[0129] ACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCG

[0130] CGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTT

[0131] TACCAGAAGTGGCTAGTCTAACCGCAAGGAGGACGGTCACCACGTAGATCAGTTGCCT(SEQ ID NO:2)

[0132] Example 2: Identification and determination of the evolutionary status of the new strains Rahn.R5 and Burk.R2

[0133] The approximately 1.4 Kb sequence of Rahn.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 Rahn.R5 in the database is Rahnella victoriana FRB 225(T) (accession number: KF308403), with a similarity of 99.63%. Further, the sequences of the related strains of Rahn.R5 were used to construct a phylogenetic tree by the neighbor-joining method (1000 replicates) using MAGA, and the results are as Figure 1 shown. It can be Figure 1 seen that Rahn.R5 and Rahnella victoriana FRB 225(T) (accession number: KF308403) are clustered in one branch, indicating that after 16S rDNA alignment, Rahn.R5 isolated in Example 1 can be temporarily classified as Rahnella victoriana (i.e., Rahnella victoriana).

[0134] Further, the whole genome of the monoclonal strain obtained in Example 1 was sequenced, and the average nucleotide identity (ANI) analysis was performed between the Rahn.R5 sequence obtained by sequencing and its related bacteria. As shown in the following table, the ANI value of strain Rahn.R5 and Rahnella woolbedingensis DSM 27399 is the highest, at 90.96%. The ANI value with Rahnella victoriana FRB 225 is only 88.44%, both of which are less than the new bacteria determination standard of 95%. Therefore, based on the sequence of the whole genome of Rahn.R5, it can be determined that Rahn.R5 is a new bacterial species belonging to the genus Rahnella.

[0135] ANI value analysis between Rahn.R5 and other related bacteria of the genus Rahnella

[0136] Related species ANI (%) Rahnella victoriana FRB225 88.44 Rahnella woolbedingensis DSM 27399 90.96 Ewingella americana ATCC 33852 88.48 Rahnella aceris SAP-19 88.48 Rouxiella chamberiensis 130333 79.81 Serratia liquefaciens ATCC 27592 79.12 Serratia fonticola LMG 7882 79.03 Yersinia ruckeri ATCC 29473 78.95 Yersinia proxima P37424 78.87

[0137] Therefore, combining the 16S rDNA sequence alignment, whole genome sequence alignment, and MAGA tree construction results, it can be determined that Rahn.R5 is a new bacterial species under the genus Rahnella. It was deposited in the Guangdong Provincial Culture Collection of Microorganisms on May 14, 2023, with the deposit number GDMCC No. 63461 and named Rahnella victoriana Rahn.R5.

[0138] Similarly, the approximately 1.4 Kb sequence of Burk.R2 obtained by sequencing, which is nearly the full length of 16S rDNA (i.e., SEQ ID NO: 2), 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 Burk.R2 in the database is Burkholderia ubonensis CIP 107078(T) (accession number: EU024179), with a similarity of 99.39%. Further, the sequences of the related strains of Burk.R2 were used to construct a phylogenetic tree by neighbor-joining bootstrap analysis (1000 replicates) using MAGA, and the results are as Figure 2 shown. It can be Figure 2 seen that Burk.R2 and Burkholderia ubonensis CIP 107078(T) (accession number: EU024179) form a single branch together. Therefore, it can be determined that Burk.R2 belongs to the genus Burkholderia ubonensis, and it was named Burkholderia ubonensis Burk.R2. It was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms on June 16, 2023, with the deposit number GDMCC No. 63569.

[0139] Example 3: Colony characteristics of the new strains Rahn.R5 and Burk.R2

[0140] The new strain Rahn.R5 provided in this application appears milky white and opaque on the beef extract peptone solid medium plate. The colonies are round and smooth, and the cells are viscous (as Figure 3 shown).

[0141] The strain Burk.R2 provided in this application appears yellow on the beef extract peptone solid medium plate, with a neat edge and opaque. The colonies are round and smooth, and the cells are viscous ( Figure 4 ).

[0142] Example 4: The new strains Rahn.R5 and Burk.R2 have nitrogen fixation characteristics

[0143] Using a pipette, 5 μL of the bacterial suspensions of strains Rahn.R5 and Burk.R2 were respectively spotted on the Ashby nitrogen-free medium and then cultured in a constant temperature biochemical incubator at 30 °C for 48 hours, and the results were observed. The results showed that Rahn.R5 and Burk.R2 could grow well on the Ashby nitrogen-free medium, confirming that both strains have good nitrogen fixation ability (as Figure 5 and Figure 6as shown). 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.

[0144] Example 5: The new strains Rahn.R5 and Burk.R2 have the characteristic of dissolving phosphorus

[0145] After respectively pipetting 5 μL of the bacterial solutions of strains Rahn.R5 and Burk.R2 with a pipette gun and spotting them on the Mengjinna organic phosphorus solid medium, they were placed in a constant temperature biochemical incubator at 30 °C and cultured for 48 hours, and the growth results were observed. The results showed that Rahn.R5 could form an obvious phosphorus-dissolving circle on the Mengjinna liquid medium, and the ratio of the diameter of the phosphorus-dissolving circle to the diameter of the colony was 1.42 ± 0.22, confirming that the strain Rahn.R5 has good ability to dissolve organic phosphorus ( Figure 7 ). Burk.R2 also could form an obvious phosphorus-dissolving circle on the Mengjinna liquid medium, and the ratio of the diameter of the phosphorus-dissolving circle to the diameter of the colony was 1.82 ± 0.12, confirming that the strain Burk.R2 also has good ability to dissolve organic phosphorus ( Figure 8 ). The formula of the Mengjinna organic phosphorus solid medium is: 0.03 g of MnSO4·4H2O; 0.03 g of FeSO4·7H2O; 5.0 g of CaCO3; 10.0 g of Glucose; 0.5 g of (NH4)2SO4; 0.2 g of Lecithin; 0.3 g of NaCl; 0.3 g of KCl; 0.4 g of Yeast extract paste; 20.0 g of Agar, made up to 1000 mL with distilled water, pH 7.0 - 7.5.

[0146] The above results suggest that Rahn.R5 and Burk.R2 proposed in the examples of this application are phosphate-solubilizing bacteria, which can dissolve organic phosphorus or insoluble phosphorus, release available phosphorus that can be absorbed by plants, and thus have the potential to improve soil quality and structure, increase the effective utilization rate of phosphorus in the soil, save fertilizers and increase yields.

[0147] Example 6: The strain Burk.R2 has the characteristic of producing siderophores

[0148] Using the chrome azurol plate method, pick the colony of Burk.R2 with an inoculation loop and inoculate it in the center of the plate, and culture it upside down at 28 °C for 2 days, and observe the growth of the strain. The strain producing siderophores shows an orange-yellow halo formed around the bacterial lawn, and if no orange-yellow halo is produced, it means that the strain has no ability to produce siderophores. The formula of the chrome azurol medium is: containing 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein per 100 mL, 1 mmol·L -1100 μL of CaCl2, 1 mmol·L -1 2 mL of MgSO4, 1.8 g of agar. Slowly add 5 mL each of phosphate buffer solution and CAS staining solution at about 60 °C to obtain chrome azurol medium.

[0149] The results showed that Burk.R2 could form an obvious orange-yellow halo on the chrome azurol medium. The ratio of the diameter (D) of the orange-yellow halo to the diameter (d) of the colony was 2.44 ± 0.03. Therefore, it could be judged that Burk.R2 had a strong ability to produce siderophores ( Figure 9 ), indicating that the Burk.R2 strain in the embodiment of this application could produce a low-molecular-weight organic compound with specific binding ability to Fe 3+ to supplement iron nutrition for plant cells.

[0150] Example 7: The new strains Rahn.R5 and Burk.R2 have antibacterial properties

[0151] The antibacterial abilities of Rahn.R5 and Burk.R2 were tested by the plate confrontation method. The pathogenic fungus Rhizoctonia solani was activated from the slant to the center of the PDA plate at 25 °C and cultured for 5 - 7 d. When the colony grew to half of the plate diameter, it was reserved after determining the target strain according to the colony characteristics and microscopic characteristics. Rhizoctonia solani, Fusarium oxysporum, and Fusarium graminearum were respectively made into 5-mm-diameter agar discs and placed in the center of the PDA plate. 5 μL of the bacterial solutions of Rahn.R5 and Burk.R2 were respectively pipetted and spotted at the edge of the plate. When the pathogenic bacteria covered the plate, the radius of the pathogenic bacteria from the proximal end of the bacteria was measured. The results showed that Rahn.R5 had inhibitory effects on Rhizoctonia solani, Fusarium oxysporum, and Fusarium graminearum (as shown in 10, 11, and 12 respectively). Burk.R2 also had inhibitory effects on Rhizoctonia solani, Fusarium oxysporum, and Fusarium graminearum (as shown in 13, 14, and 15 respectively). The widths of the antibacterial zones were 42.86 mm, 28.64 mm, and 31.38 mm respectively.

[0152] The above experimental results indicated that Rahn.R5 and Burk.R2 proposed in the embodiment of this application had strong antibacterial properties and inhibitory functions on a variety of soil-borne pathogenic bacteria, indicating that Rahn.R5 and Burk.R2 proposed in the embodiment of this application and their combination could be effectively used for the prevention and control of plant diseases caused by Rhizoctonia solani, Fusarium oxysporum, Fusarium graminearum, etc., and had a comprehensive inhibitory ability on soil-borne pathogenic bacteria and could be used as a multi-faceted and potent biocontrol agent.

[0153] Example 8: The new strains Rahn.R5 and Burk.R2 have the function of promoting plant growth

[0154] 8.1 Verification of the plant growth promotion function of the new strain Rahn.R5

[0155] After activating the strain Rahn.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 Rahn.R5 fermentation broth. Upland rice and millet were planted in the field plots, and the crop varieties were Luyin 46 and Huagu 12 respectively. The strain Rahn.R5 was inoculated at the time of sowing and seedling stage of crop growth, and an equal amount of fermented dilution (the fermentation broth was diluted at a ratio of 1:100 with sterile water) was irrigated for each plant; at the same time, a control group was set up, and the control group was treated with an equal amount of sterile water.

[0156] After 35 days of growth, the crops were sampled and the biomass data were statistically analyzed. The specific measurement data are shown in Table 1 and Table 2, and the growth conditions of the treatment group and the control group are as Figure 16 shown (left, upland rice; right, millet).

[0157] Table 1 Effects of inoculating Rahn.R5 on the field growth of upland rice

[0158]

[0159] Tables 1-2 are the measured values of each growth index of upland rice and millet after applying the strain Rahn.R5 provided by this application. As can be seen from Table 1, the significance difference analysis shows that compared with the control group, applying Rahn.R5 can significantly increase the root length of upland rice (p<0.05), and extremely significantly increase the aboveground fresh weight, underground fresh weight and stem diameter of upland rice (p<0.01), and the growth rates are 20.00%, 102.05%, 111.66% and 86.87% respectively. It shows that the application of the Rahn.R5 strain to upland rice has significant growth-promoting effects such as promoting root growth, strengthening stem stalks, and promoting plant biomass growth.

[0160] Table 2 Effects of inoculating Rahn.R5 on the field growth of millet

[0161]

[0162] As can be seen from Table 2, the data show that applying the Rahn.R5 strain can significantly increase the stem diameter of millet, and the growth rate is 24.87%. In addition, the application of Rahn.R5 also has obvious promoting effects on the plant height, root length, aboveground fresh weight and underground fresh weight of millet, and the growth rates are 9.87%, 1.98%, 40.76% and 129.03% respectively. It shows that the application of the Rahn.R5 strain to gramineous plants such as millet also plays a significant role in promoting root growth, strengthening stem stalks, and promoting plant biomass growth.

[0163] From Figure 16It can be seen that in the field experiment, compared with the control group, the plants treated with the strain Rahn.R5 provided in this application showed a more vigorous growth trend in the above-ground or underground parts in different crops. For example, after applying Rahn.R5 to upland rice, the plant height was higher, the leaves were wider, the stems were thicker, and the roots were more developed; after applying Rahn.R5 to foxtail millet, the above-ground part was higher and the stems were also thicker.

[0164] Therefore, the plant growth-promoting rhizosphere strain Rahn.R5 isolated and identified in the embodiments of this application has a good promoting effect on the growth of plant stems, leaves, and roots, and is an effective growth-promoting microorganism.

[0165] 8.2 Verification of the plant growth promotion function of the strain Burk.R2

[0166] Use Burk.R2 to treat corn, upland rice, peanut, and alfalfa respectively, and the varieties are Jinxiangyu, Luyin 46, Yuhua 22, and alfalfa. The specific experimental procedures such as bacterial liquid fermentation and irrigation treatment are the same as those in 8.1.

[0167] After 35 days of growth, samples of the plants were taken and the biomass data were statistically analyzed. The specific measurement data are shown in Tables 3, 4, 5, and 6, and the growth conditions of the treatment group and the control group are as Figure 17 shown (a, corn; b, upland rice; c, peanut; d, alfalfa).

[0168] Tables 3-6 are the measured values of various growth indexes of corn, upland rice, peanut, and alfalfa after applying Burk.R2 provided by the present invention respectively. As can be seen from Table 3, the data analysis shows that applying the Burk.R2 strain can significantly increase the plant height, above-ground fresh weight, underground fresh weight, and stem diameter of corn. The growth rates compared with the control group are 3.69%, 36.64%, 83.02%, and 12.90% respectively, proving that the Burk.R2 strain has obvious growth-promoting effects such as promoting the biomass growth of the above-ground and underground parts when acting on corn.

[0169] As can be seen from Table 4, compared with the control group, applying the Burk.R2 strain can increase the plant height, root length, above-ground fresh weight, and stem diameter of upland rice, especially the root length and above-ground fresh weight, with growth rates of 26.86% and 35.31% respectively, indicating that the Burk.R2 strain can promote the growth of the above-ground part and rooting of upland rice to a certain extent, and has a promoting effect on the fruiting of the above-ground part, and can be effectively used for increasing the yield of upland rice.

[0170] As can be seen from Table 5, the data analysis shows that applying the Burk.R2 strain has a certain promoting effect on the plant height, root length, and underground fresh weight of peanuts, especially on the promotion of root length and underground fresh weight. It shows that the Burk.R2 strain can be used for the growth promotion of peanuts, and has a promoting effect on the fruiting of the underground part, and can be effectively used for increasing the yield of peanuts.

[0171] As can be seen from Table 6, data analysis shows that the application of strain Burk.R2 significantly promotes the plant height, above-ground fresh weight, and underground fresh weight of alfalfa. The growth rates compared with the control group are 32.50%, 191.97%, and 161.86% respectively. At the same time, it also promotes the growth of roots, proving that the application of strain Burk.R2 can significantly promote the growth of above-ground stems and leaves and underground roots of alfalfa.

[0172] Table 3 Effects of inoculating Burk.R2 on the field growth of maize

[0173] Different treatments Plant height (mm) Root length (mm) Aboveground fresh weight (g) Underground fresh weight (g) Stem diameter (mm) Control group (mean ± standard deviation) 101.50±10.08 25.13±1.55 118.93±30.36 19.59±5.46 21.65±4.48 Burk.R2 (mean ± standard deviation) 105.25±8.05 24±3.34 162.5±46.99 35.85±13.93 24.44±4.1 Growth rate 3.69％ -4.48％ 36.64％ 83.02％ 12.90％ p value 0.509 0.678 0.0727 0.0291 0.286 p value symbol - - - * -

[0174] Table 4 Effects of inoculating Burk.R2 on the field growth of upland rice

[0175]

[0176] Table 5 Effects of inoculating Burk.R2 on the field growth of peanut

[0177] Different treatments Plant height (mm) Root length (mm) Aboveground fresh weight (g) Underground fresh weight (g) Control group (mean ± standard deviation) 36.63±1.14 17±1.54 83.86±31.99 3.73±1.31 Burk.R2 (mean ± standard deviation) 38.33±0.24 23.67±2.62 75.49±30.01 4.25±1.64 Growth rate 4.66％ 39.22％ -9.97％ 14.11％ p value 1 0.0158 1 1 p value symbol - * - -

[0178] Table 6 Effects of inoculating Burk.R2 on the field growth of alfalfa

[0179] Different treatments Plant height (mm) Root length (mm) Aboveground fresh weight (g) Underground fresh weight (g) Control group (mean ± standard deviation) 49±6.69 25.7±2.52 20.65±5.82 2.99±0.43 Burk.R2 (mean ± standard deviation) 64.93±7.56 27.9±2.18 60.29±13.93 7.84±2.66 Growth rate 32.50％ 8.56％ 191.97％ 161.86％ p value 0.0155 0.418 0.0132 0.0491 p value symbol * - * *

[0180] From Figure 17 it can be seen that in the field experiment, the plants after applying the strain Burk.R2 provided by the present invention showed a more lush growth trend in the underground part compared with the control group in different crops. For example, the roots of the plants were more developed after applying Burk.R2 in maize, upland rice, peanut, and alfalfa. In addition, the plant height of upland rice and alfalfa was higher and the above-ground biomass was larger after applying Burk.R2, indicating that the Burk.R2 strain proposed in the embodiments of the present application will have beneficial effects on promoting plant growth and increasing crop yield.

[0181] Example 9: The new strains Rahn.R5 and Burk.R2 have disease resistance functions

[0182] After activating strains Rahn.R5 and Burk.R2, they were respectively inoculated into beef extract peptone medium and fermented by shaking at 28 °C and 180 r / min for 5 days to obtain the fermentation broths of Rahn.R5 and Burk.R2. The disinfected soybean Heilong 35 seeds were planted in sterile soil pots, and 5 - 10 plants were planted in each pot. The growth temperature of the plants was 18 - 21 °C, and the light condition was set as 16 h of light and 8 h of dark environment.

[0183] On the sowing day, Rahn.R5 and Burk.R2 were inoculated respectively, and a sterile liquid inoculation group was set as the control group. Each treatment had 3 parallel groups, and the inoculation amount was 2 mL of the fermentation broth poured into the roots of each plant. After growing for 7 days, Rahn.R5 and Burk.R2 were inoculated for the second time. And 2 mL of the Fusarium oxysporum suspension (OD600 = 0.3) was inoculated after growing for 14 days. The disease severity of soybean growth after 30 days of planting was statistically analyzed. The disease index was divided into 0 - 4, where 0 means no wilting; 1 means 1 - 25% of the leaves withered; 2 means 25 - 50% of the leaves withered; 3 means 51 - 75% of the leaves withered; 4 means 76 - 100% of the leaves withered. The statistical methods for the disease index and the disease prevention effect are as follows, and the specific statistical results are shown in Table 7:

[0184]

[0185]

[0186] Table 7

[0187] Control group Rahn.R5 Burk.R2 Number of plants (disease level 0) 0 1 2 Number of plants (disease level 1) 2 5 4 Number of plants (disease level 2) 3 1 1 Number of plants (disease level 3) 2 0 0 Number of plants (disease level 4) 0 0 0 Total number of plants 7 7 7 Highest level 3 2 2 Disease index 66.67％ 50.00％ 42.86％ Biocontrol effect / 25.00％ 35.71％

[0188] Table 7 is the statistical result of the disease prevention effect of Rahn.R5 and Burk.R2 according to the embodiments of the present application. It can be seen from Table 7 that the highest disease level of the control group was level 3, and the number of plants infected at disease levels 1, 2, and 3 was 2 plants, 3 plants, and 2 plants respectively, and the disease index was 66.67%. After inoculating Rahn.R5, the highest disease level dropped to level 2, and 1 plant was not infected. The number of plants infected at disease levels 1 and 2 was 5 plants and 1 plant respectively, and the disease index dropped to 50%, achieving a control effect of 25% for the biocontrol effect. After inoculating Burk.R2, the highest disease level also dropped to level 2, and 2 plants were not infected. The number of plants infected at disease levels 1 and 2 was 4 plants and 1 plant respectively, and the disease index dropped to 42.86%, achieving a control effect of 35.71% for the biocontrol effect. Thus, it is proved that the strains Rahn.R5 and Burk.R2 of the present invention both have strong biocontrol functions for inhibiting Fusarium oxysporum and can be effectively used for biological control of field crops and the like.

[0189] Therefore, the newly isolated and identified plant growth-promoting rhizobacteria strains Rahn.R5 and Burk.R2 in the embodiments of the present application have good promoting effects on the growth of various crops. Specifically, it is mainly reflected in the promotion of the growth of crop stems, leaves and roots, indicating that the newly isolated and identified plant growth-promoting rhizobacteria strains Rahn.R5 and Burk.R2 and their combined preparations in the embodiments of the present application can not only achieve plant disease control, but also be effectively used for the growth promotion of various plants including gramineous crops and leguminous plants, which can effectively reduce the application amount of chemical fertilizers and pesticides and alleviate the environmental impact brought by chemical fertilizers and pesticides. The newly discovered Bacillus - like strains Rahn.R5 and Burk.R2 with growth-promoting and biocontrol functions proposed in the present application are of great significance for improving crop yields, controlling plant diseases and environmental protection.

[0190] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. mean 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.

[0191] 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 limiting 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 Rahnella sp., 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 Rahnella bacterium according to claim 1, characterized in that, The Rahnella bacterium is of the genus Rahnella; preferably, the Rahnella bacterium has the 16S rDNA sequence shown in SEQ ID NO: 1; more preferably, the Rahnella bacterium is Rahnella victoriana Rahn.R5 with the deposit number GDMCC No: 63461.

3. An agricultural preparation, characterized in that, Comprising the Rahnella bacterium as described in claim 1 or 2 and optionally a Burkholderia bacterium, Optionally, the Burkholderia bacterium 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; Optionally, the Burkholderia bacterium is of the genus Burkholderia; preferably, the Burkholderia bacterium has the 16S rDNA sequence shown in SEQ ID NO: 2; more preferably, the Rahnella bacterium is Burkholderia ubonensis Burk.R2 with the deposit number GDMCC No: 63569.

4. The agricultural preparation according to claim 3, wherein 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.

5. Use of the Rahnella bacterium as described in claim 1 or 2 or the agricultural preparation as described in claim 3 or 4 in promoting plant growth.

6. The application according to claim 5, characterized in that, The Rahnella bacterium or the agricultural preparation is specifically used for one or more of the following: nitrogen fixation, phosphorus solubilization, siderophore production, hormone production and ACC deaminase production, Optionally, the phosphorus solubilization includes dissolving organic phosphorus and dissolving inorganic phosphorus, Optionally, the siderophore includes siderophilin, Optionally, the hormone includes indoleacetic acid, gibberellin or a combination thereof.

7. Use of the Rahnella bacterium as described in claim 1 or 2 or the agricultural preparation as described in claim 3 or 4 in the control of plant diseases.

8. The application according to claim 7, characterized in that, The control of plant diseases includes one or more of the following: inhibiting Rhizoctonia solani, inhibiting Fusarium oxysporum and inhibiting Fusarium graminearum, preferably inhibiting Fusarium graminearum.

9. The application according to claim 8, characterized in that, The plant diseases include one or more of the following: black spot, anthracnose, root rot and lawn leaf spot caused by Rhizoctonia solani; scab, root rot, stem rot, ear rot, basal stem rot, flower rot, rice blast, smut, black spot, brown spot and mosaic disease caused by Fusarium oxysporum; and scab, root rot, stem rot, ear rot, basal stem rot, flower rot, rice blast, smut, black spot and brown spot caused by Fusarium graminearum.

10. The use as described in claim 5 or 6 or the use as described in any one of claims 7 to 9, characterized in that The plant is selected from crops, vegetables, fruit trees, flowers and turfgrasses. Optionally, the crop is selected from rice, corn, wheat, barley, oats, millet, soybean, peanut and cotton, and the vegetable is selected from Chinese cabbage, carrot, ginger, kidney bean, beet, cucumber, pepper, tomato, onion, bracken and alfalfa; or The plant is selected from gramineous plants and leguminous plants. Preferably, the gramineous plant is selected from corn, millet and upland rice, and the leguminous plant is selected from soybean and alfalfa.

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