Pogostemon cablin growth-promoting rhizobacteria variovorax strain and application thereof

Abstract

According to the pogostemon cablin rhizosphere growth-promoting bacterium variovorax guangxiensis strain and the application thereof, the variovorax guangxiensis strain is separated from rhizosphere soil of healthy pogostemon cablin, the variovorax guangxiensis strain YFJ1LB8 has the effects of generating ammonium ions and indoleacetic acid and has the biofilm forming ability, and the variovorax guangxiensis strain has the effects of promoting plant growth, promoting growth of plants and promoting growth of the pogostemon cablin. Particularly, the growth of the pogostemon cablin is promoted excellently. The fresh weight, the dry weight, the stem fresh weight, the stem dry weight, the leaf fresh weight and the leaf dry weight of the pogostemon cablin plant can be obviously improved; the biological fertilizer can replace chemical fertilizers and pesticides and is used for promoting growth of field crops; the use of chemical fertilizers and pesticides is effectively reduced.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and specifically relates to a strain of the plant growth-promoting rhizobacterium Variovorax in Pogostemon cablin roots and its application. Background Art

[0002] During the cultivation of medicinal plants, the application of chemical fertilizers and pesticides plays a crucial role in increasing yields and combating pests and diseases. However, if these chemicals are used in an unscientific manner for a long time, a series of serious problems will occur. These problems include a significant reduction in resource utilization efficiency, a gradual degradation of soil quality, and a continuous decline in the quality of medicinal plants; not only will it affect the efficacy of the medicinal materials, but it will also cause irreversible damage to the environment.

[0003] Therefore, there is an urgent need to explore and develop a new type of green and efficient method to replace the use of traditional chemical fertilizers and pesticides. It can promote the healthy growth of medicinal plants while ensuring environmental sustainability. By adopting eco-friendly planting techniques such as organic agriculture, biological control, and ecological agriculture, the negative impact on the environment can be effectively reduced, while the quality and efficacy of the medicinal materials can be improved. In addition, through scientific soil management and plant nutrition regulation, the utilization efficiency of resources can be further optimized to achieve the sustainable production of medicinal plants. This can ensure human health while protecting and maintaining the natural environment on which we depend for survival.

[0004] Plant growth-promoting rhizobacteria (PGPR) are a class of beneficial microorganisms that can effectively colonize plant roots and promote the growth and development of host plants, defend against pathogenic bacteria, etc. through the production of a series of complex molecular signal substances. They have both the dual functions of disease prevention and growth promotion and have received extensive attention from researchers in multiple disciplines as a hot topic in the research of microbial fertilizers. Rhizosphere-promoting bacteria have high adaptability, fast growth rates, and biochemical diversity in various environments, can metabolize a wide range of natural and exogenous compounds, and are an indispensable part of the rhizosphere biota; when rhizosphere-promoting bacteria grow in association with host plants, they can stimulate the growth of the host. Therefore, plant growth-promoting rhizobacteria are of great agricultural significance for improving crop productivity, and it is crucial to explore new rhizosphere-promoting bacteria in agricultural production.

[0005] Previous studies have confirmed that PGPR has a direct growth-promoting effect, which can improve the availability of soil nutrients through nitrogen fixation, phosphorus solubilization, potassium solubilization and other processes. For example, strains such as Bacillus velezensis can secrete organic acids and enzymes to promote the release of elements such as phosphorus and potassium. Some PGPR can secrete hormones such as indole acetic acid (IAA) and gibberellin (GA) to regulate plant growth. For example, Bacillus velezensis JB0319 can efficiently synthesize IAA and significantly promote the root development of potted crops. Some PGPR help plants cope with stresses such as salinity and drought by synthesizing osmoregulatory substances (such as proline and betaine) or regulating the plant antioxidant system. On the other hand, PGPR can also play an indirect growth-promoting role. For example, PGPR can inhibit pathogenic bacteria by competing for nutrients and secreting antibacterial substances (such as antibiotics and siderophores). For example, some strains can control diseases such as tobacco black shank and potato late blight; or enhance the broad-spectrum resistance to pathogenic bacteria by activating the expression of plant defense genes.

[0006] Pogostemon cablin is an important medicinal plant, and the research on its rhizosphere growth-promoting bacteria has important value in agriculture and the cultivation of medicinal plants. Exploring excellent endophytic bacteria in the roots of Pogostemon cablin for the development of special bio-fertilizers for Pogostemon cablin to replace chemical fertilizers and pesticides, thereby reducing the damage to the environment while increasing crop yields and resisting pests and diseases, is a new type of green and effective method. Summary of the Invention

[0007] The embodiments of the present application provide a Variovorax strain of Pogostemon cablin rhizosphere growth-promoting bacteria and its application to solve the problems existing in the related technologies. The technical solutions are as follows:

[0008] In the first aspect, the embodiments of the present application provide a Variovorax strain of Pogostemon cablin rhizosphere growth-promoting bacteria, and the strain is Variovorax guangxiensis YFJ1LB8, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 25, 2025, and the deposit number is GDMCC No. 65897.

[0009] This strain was isolated from the rhizosphere soil of healthy Pogostemon cablin in the Pogostemon cablin planting base in Luoding City, Yunfu City, Guangdong Province, China, and has the growth-promoting characteristics of producing ammonium ions, producing indole acetic acid, and forming biofilms. The rhizosphere growth-promoting bacteria Variovorax YFJ1LB8 can promote the growth of Pogostemon cablin plants. It was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 25, 2025. The deposit address is the 5th floor of Building 59, Institute of Microbiology, Guangdong Academy of Sciences, 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 65897.

[0010] In one embodiment, the Variovorax guangxiensis strain YFJ1LB8 is light yellow on the plate, with a smooth surface, convex, neat edges, and circular shape; the morphological characteristics of the bacteria: Gram-negative bacteria, rod-shaped bacteria.

[0011] In one embodiment, the sequence of the Variovorax guangxiensis strain YFJ1LB8 is as shown in SEQ ID No.1.

[0012] In a second aspect, an embodiment of the present application provides a composition, which includes the above-mentioned plant growth-promoting bacterium Variovorax guangxiensis strain in the rhizosphere of Pogostemon cablin.

[0013] In a third aspect, an embodiment of the present application provides a microbial inoculant, which uses the above-mentioned Variovorax guangxiensis strain YFJ1LB8 as an active ingredient.

[0014] In one embodiment, the inoculant is a liquid inoculant; in the liquid inoculant, the OD of the viable bacteria of the Variovorax guangxiensis strain YFJ1LB8 600 = 0.6 - 1.0.

[0015] In a fourth aspect, an embodiment of the present application provides the application of the above-mentioned plant growth-promoting bacterium Variovorax guangxiensis strain in the rhizosphere of Pogostemon cablin, the composition, and the microbial inoculant in producing ammonium ions, indole acetic acid, and forming biofilms.

[0016] In a fifth aspect, an embodiment of the present application provides the application of the above-mentioned plant growth-promoting bacterium Variovorax guangxiensis strain in the rhizosphere of Pogostemon cablin, the composition, and the microbial inoculant in promoting the growth of Pogostemon cablin.

[0017] In one embodiment, the above-mentioned plant growth-promoting bacterium Variovorax guangxiensis strain in the rhizosphere of Pogostemon cablin, the composition, and the microbial inoculant are prepared into a bacterial suspension and used for root irrigation treatment of Pogostemon cablin.

[0018] In one embodiment, in the bacterial suspension, the OD of the viable bacteria of the Variovorax guangxiensis strain YFJ1LB8 600 = 0.6 - 1.0.

[0019] The advantages or beneficial effects in the above technical solutions at least include:

[0020] The strain YFJ1LB8 of the present invention is isolated from the rhizosphere soil of healthy Pogostemon cablin, has the functions of producing ammonium ions and indole acetic acid, and has the ability to form biofilms, which has an excellent promoting effect on promoting plant growth, especially the growth of Pogostemon cablin. It can significantly increase the fresh weight, dry weight of the Pogostemon cablin plant, fresh weight of the stem, dry weight of the stem, fresh weight of the leaf, and dry weight of the leaf; it can replace chemical fertilizers and pesticides and be used as a biological fertilizer for promoting the growth of field crops; it can effectively reduce the use of chemical fertilizers and pesticides.

[0021] The above - mentioned summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In the drawings, unless otherwise specified, the same reference numerals throughout the several views represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.

[0023] Figure 1 Colony morphology of bacterium YFJ1LB8

[0024] Figure 2 Phylogenetic tree of bacterium YFJ1LB8 constructed based on 16S sequence

[0025] Figure 3 Detection result of ammonium - ion production by bacterium YFJ1LB8

[0026] Figure 4 Detection result of biofilm formation by bacterium YFJ1LB8

[0027] Figure 5 Effect diagram of promoting the growth of Pogostemon cablin potted plants by bacterium YFJ1LB8

[0028] Figure 6 Comparison result diagram of plant height and stem diameter of Pogostemon cablin potted plants in the experimental group and control group of bacterium YFJ1LB8

[0029] Figure 7 Comparison result diagram of fresh weight and dry weight of plants, stems, and leaves of Pogostemon cablin potted plants in the experimental group and control group of bacterium YFJ1LB8 DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In the following, only some exemplary embodiments are briefly described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0031] Patchouli (Pogostemon cablin) is an important medicinal plant, and the study of its plant growth-promoting rhizobacteria has important value in agriculture and the cultivation of medicinal plants. As a medicinal plant, it is more sensitive to the use of fertilizers and pesticides. Therefore, how to increase crop yields, resist pests and diseases while reducing the residual amount of chemical substances in patchouli, and reducing the damage to the environment; and exploring excellent endophytic bacteria in patchouli roots for the development of special bio-fertilizers for patchouli may be a new type of green and effective method.

[0032] Example 1

[0033] Isolation, preservation and identification of strain YFJ1LB8

[0034] 1. Isolation and preservation of strain YFJ1LB8

[0035] Collect rhizosphere soil samples of healthy patchouli from the patchouli planting base in Luoding City, Yunfu City, Guangdong Province. Weigh 1.0 g of soil samples and add them to 9 mL of sterile water. Shake them thoroughly at 28 °C and 220 r / min for 30 min. Pipette 100 μL of the soil suspension and add it to 900 μL of sterile water, and dilute it serially to 10 -3 、10 -4 、10 -5 、10 -6 . Pipette 100 μL of each concentration of diluted suspension and spread it on LB solid medium, and incubate it in a constant temperature incubator at 28 °C. Pick single colonies according to colony color, shape, size, elevation state, edge state, surface state, transparency, etc. Purify it repeatedly 3 - 5 times until a pure strain is obtained.

[0036] Inoculate the strain into LB liquid medium, shake it at 28 °C and 220 r / min for 24 - 48 h. Mix the bacterial liquid and 30% glycerol in a cryotube at a volume ratio of 1:1. After quick-freezing in liquid nitrogen, store it frozen at -80 °C.

[0037] 2. Identification of strain YFJ1LB8

[0038] Observation of morphological characteristics

[0039] Colony morphological characteristics: As Figure 1 shown, the colonies of strain YFJ1LB8 are light yellow on the plate, with a smooth surface, convex, neat edges, and circular; Bacterial morphological characteristics: Gram-negative bacteria, rod-shaped bacteria.

[0040] 16S sequence analysis

[0041] Primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’, SEQ ID No.2) and 1492R (5’-GGTTACCTTGTTACGACTT-3’, SEQ ID No.3) were selected for PCR amplification, and 16S rDNA sequence determination was carried out;

[0042] PCR system (40 μL): 2×Taq M-aster Mix PlusⅡ 20 μL, 27F 1.5 μL, 1492R 1.5 μL, template 1 μL, made up to 40 μL with water;

[0043] Amplification conditions: 98℃ for 3 min; 98℃ for 15 s, 56℃ for 30 s, 72℃ for 2 min, 35 cycles; 72℃ for 7 min;

[0044] The PCR amplification products were detected by 1% agarose gel electrophoresis and sequenced by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.; The sequencing sequence of the strain is shown in SEQ ID NO.1:

[0045] SEQ ID NO.1:

[0046]

[0047] The sequencing results were submitted to the GenBank database for BLAST analysis and comparison. It was found that the strain with a high homology to strain YFJ1LB8 was Variovorax guangxiensis. An NJ phylogenetic tree was constructed using the 16S sequence, as Figure 2 shown, strain YFJ1LB8 and Variovorax guangxiensis were in the same branch.

[0048] Determination of physiological and biochemical characteristics

[0049] The physiological and biochemical characteristics of strain YFJ1LB8 were detected according to the methods in Bergey's Manual of Determinative Bacteriology and Manual of Systematic Bacteriology of Common Bacteria. The results are shown in Table 1.

[0050] Table 1 Physiological and biochemical characteristics of strain YFJ1LB8

[0051]

[0052] Based on the above morphological characteristic analysis, 16S rDNA phylogenetic tree analysis, and physiological and biochemical characteristics, strain YFJ1LB8 was identified as Variovorax guangxiensis.

[0053] Example 2

[0054] Analysis of the growth-promoting characteristics of strain YFJ1LB8

[0055] 1. Determination of ammonium ion production activity

[0056] A pure colony of YFJ1LB8 was picked and inoculated into a test tube containing peptone (10 g / L) culture medium. It was cultured at 28 °C with shaking at 220 r / min for 72 h. 1 mL of Nessler's reagent was added to each test tube. If a yellow-brown precipitate appeared, it indicated the presence of ammonium ion production activity; if no yellow-brown precipitate appeared, it indicated the absence of ammonium ion production activity. The results are as Figure 3 and Table 2 shown, strain YFJ1LB8 had the activity of producing ammonium ions.

[0057] 2. Determination of IAA (indole-3-acetic acid) production ability

[0058] Pick the pure colonies of YFJ1LB8 and inoculate them into the LB medium containing 0.5 g / L tryptophan. Use the uninoculated medium as a control. Incubate at 28 °C and 220 r / min with shaking for 48 h. Take 1 mL of the culture solution, centrifuge at 12,000 r / min for 5 min. Take 500 μL of the supernatant and add an equal volume of Salkowski reagent. After color development in the dark at room temperature for 30 min, observe the color change. If the color turns red, it indicates that the strain has the ability to produce IAA. Measure the absorbance at 530 nm, using the blank medium as a control, and use the absorbance corresponding to pure IAA (set the concentration gradients of the IAA standard product as 1, 3, 5, 10, 20 μg / mL) to make a standard curve, and calculate the output of IAA (μg / mL). The obtained IAA standard curve equation is y = 0.0061x + 0.0053 (R 2 = 0.9991). The results are shown in Table 2. The strain YFJ1LB8 has the ability to produce IAA. Using the linear formula of the absorbance value, the amount of IAA secreted by the strain YFJ1LB8 can be calculated as 4.55 ± 0.74 μg / mL.

[0059] 3. Determination of the ability to form biofilms

[0060] Dilute the YFJ1LB8 bacterial suspension with LB culture solution to OD 600 to 0.2. Take 150 μL and inoculate it into a 96-well plate, and incubate at 28 °C for 72 h. Subsequently, carefully aspirate to remove the unbound cells in the plate, and wash with 150 μL of sterile water. After air-drying for 45 min, add 150 μL of 0.1% crystal violet solution to stain for 20 min, wash three times with sterile water, dry, add 200 μL of 95% ethanol to dissolve the dye for 20 min, measure the absorbance at 590 nm, and the blank control is the uninoculated LB culture solution. Repeat 5 times. The darker the purple color, the stronger the ability to form biofilms, and colorless transparency indicates no ability to form biofilms. The results are as Figure 4 shown in Table 2. The results show that the strain YFJ1LB8 has the ability to form biofilms.

[0061] Table 2 Analysis results of the growth-promoting characteristics of the strain YFJ1LB8

[0062] Strain number Ammonium-producing example IAA production (μg / mL) <![CDATA[Forming biofilm (OD 590 )]]> YFJ1LB8 + 4.55±0.74 0.32±0.09

[0063] Example 3

[0064] Growth-promoting effect of the strain YFJ1LB8 on Pogostemon cablin

[0065] After culturing the strain in LB culture solution at 28 °C and 220 r / min for 24 h, centrifuge at 8,000 r / min for 10 min to collect the bacteria, resuspend with sterile water, and adjust OD 600It was 0.6 - 1.0. Pogostemon cablin seedlings with consistent growth were selected and transplanted into flowerpots with a diameter of 13.00 cm and filled with the same culture medium, one plant per pot. The experimental group (bacterial suspension) and the control group (sterile water) were set up, with 6 pots for each treatment. 7 days after transplantation, the root irrigation treatment was carried out with the bacterial suspension, and 25 mL of the bacterial suspension was poured into each pot of Pogostemon cablin seedlings. The second bacterial suspension was poured 28 days later, and the treatment was carried out 2 times in total. The agronomic traits were measured on the 28th, 42nd, and 56th days after treatment, and the fresh weight and dry weight of each part were weighed on the 56th day. The growth effect of Pogostemon cablin plants was as Figure 5 shown; the comparison chart of the changes in plant height and stem diameter was as Figure 6 shown; the comparison of the fresh weight and dry weight of plants, stems, and leaves was as Figure 7 shown.

[0066] It can be seen from Figures 5 - 7 that after the application of the bacterial suspension prepared from strain YFJ1LB8, the growth promotion effect on Pogostemon cablin was obvious, and the fresh weight of Pogostemon cablin plants, the dry weight of plants, the fresh weight of stems, the dry weight of stems, the fresh weight of leaves, and the dry weight of leaves were significantly increased. It shows that this strain has a good plant growth promotion effect, and this bacterium can be used to develop microbial inoculants and biological fertilizers for promoting the growth of field crops.

[0067] In summary, the strain YFJ1LB8 of the present application has the functions of producing ammonium ions and indole acetic acid, and has the ability to form biofilms, which has an excellent promoting effect on promoting plant growth, especially the growth of Pogostemon cablin. It can significantly increase the fresh weight of Pogostemon cablin plants, the dry weight of plants, the fresh weight of stems, the dry weight of stems, the fresh weight of leaves, and the dry weight of leaves; it can replace chemical fertilizers and pesticides and be used as a biological fertilizer for promoting the growth of field crops; it can effectively reduce the use of chemical fertilizers and pesticides.

[0068] In the description of this specification, the descriptions with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" 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 application. 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.

[0069] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" can explicitly or implicitly include at least one of these features. In the description of the present application, "a plurality" means two or more unless otherwise specifically defined.

[0070] As described above, it is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application can easily think of various changes or substitutions thereof, and these should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A plant growth-promoting rhizobacterium Variovorax strain of Pogostemon cablin, characterized in that, The strain is Variovorax guangxiensis YFJ1LB8, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on February 25, 2025, with the deposit number GDMCC No. 65897.

2. A composition, characterized in that, The composition comprises the plant growth-promoting rhizobacteria Variovorax guangxiensis described in claim 1.

3. A microbial inoculant, characterized in that, The microbial inoculum uses the Variovorax guangxiensis strain YFJ1LB8 described in claim 1 as the active ingredient.

4. The microbial inoculant according to claim 3, wherein The microbial agent is a liquid microbial agent; the viable bacteria concentration of the Variovorax sp. strain YFJ1LB8 in the liquid microbial agent is OD 600 = 0.6 - 1.

0.

5. Use of the plant growth-promoting rhizobacteria Variovorax guangxiensis described in claim 1, the composition described in claim 2, and the microbial inoculum described in claim 3 or 4 in ammonium ion production.

6. Use of the plant growth-promoting rhizobacteria Variovorax guangxiensis described in claim 1, the composition described in claim 2, and the microbial inoculum described in claim 3 or 4 in indoleacetic acid production.

7. Use of the plant growth-promoting rhizobacteria Variovorax guangxiensis described in claim 1, the composition described in claim 2, and the microbial inoculum described in claim 3 or 4 in biofilm formation.

8. Use of the plant growth-promoting rhizobacteria Variovorax guangxiensis described in claim 1, the composition described in claim 2, and the microbial inoculum described in claim 3 or 4 in promoting the growth of Pogostemon cablin.

9. The application according to claim 8, wherein Prepare the plant growth-promoting rhizobacteria Variovorax guangxiensis described in claim 1, the composition described in claim 2, and the microbial inoculum described in claim 3 or 4 into a bacterial suspension, and perform root irrigation treatment on Pogostemon cablin.

10. The application according to claim 9, characterized in that, In the bacterial suspension, the OD of viable cells of the bacterium Variovorax sp. strain YFJ1LB8 600 is 0.6 - 1.0.

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