A rhizosphere growth-promoting bacterium Kushnera HH-2 for promoting plant growth and its application

Abstract

The present invention discloses a rhizosphere growth-promoting bacterium Kushnera HH-2 for promoting plant growth and its application, belonging to the field of microbial technology. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30312. The strain has good tolerance to adversity and can produce NH 3 , produce indoleacetic acid, produce siderophores, and promote plant growth, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a rhizosphere growth-promoting bacterium Kushnera HH-2 for promoting plant growth and an application thereof. Background Art

[0002] At present, salt stress is one of the main factors limiting agricultural productivity, and soil salinization has become a huge obstacle to world food production. Soil salinization is caused by reasons such as saline irrigation, water shortage and sea level rise caused by global warming, which not only affects crop productivity but also threatens the sustainability of global agricultural ecosystems. High salinity (100-200mM) reduces the osmotic potential of soil solution and causes osmotic stress. The reduction in soil solution osmotic potential leads to a decrease in water conductivity, which in turn leads to a decrease in plant absorption of water and solutes, forming physiological drought. Salt stress can also affect various physiological and biochemical mechanisms and pathways of plants, leading to imbalanced nutrient absorption, changes in growth-inducing regulatory factors, and inhibition of protein synthesis and photosynthesis. These factors lead to decreased plant growth and yield.

[0003] Plant growth-promoting rhizobacteria (PGPR) can form a symbiotic relationship with plants and may play an important role in salt stress. The rhizosphere is an important soil chemical and ecological zone close to the plant roots, where a large number of interactions between plants and microorganisms occur. Some bacteria in the rhizosphere that can promote plant growth and protect plants from biotic and abiotic stresses are called plant growth-promoting bacteria. The close connection between PGPR and plants and soil exists throughout the life cycle of plants, promoting plant growth through direct and indirect mechanisms, such as nutrient acquisition under salt stress, siderophore chelation, synthesis of plant hormones, and induction of systemic resistance of host plants to pathogens. Therefore, PGPR has important agricultural significance for improving crop productivity under saline-alkali conditions, and the discovery of new PGPR is crucial in agricultural production. Summary of the invention

[0004] The purpose of the present invention is to provide a rhizosphere growth-promoting bacterium Kushnera HH-2 for promoting plant growth and its application, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Kushneria avicenniae strain HH-2, which was deposited in the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30312.

[0007] Furthermore, the strain was isolated from the roots of Suaeda salsa in the tidal flats of Huanghua City, Cangzhou City, Hebei Province, China, and has certain salt tolerance, acid and alkali tolerance and acid-base regulation capabilities, as well as the ability to produce NH 3 , producing indoleacetic acid and siderophore-producing growth-promoting properties. The rhizosphere growth-promoting bacteria HH-2 can promote seed germination and growth of wheat under salt stress.

[0008] The present invention also provides a microbial agent, comprising the Kushnerella HH-2 and / or its metabolites.

[0009] The present invention also provides the use of the Kushnerella HH-2 or the microbial agent in promoting plant growth.

[0010] Preferably, the plant comprises wheat.

[0011] The present invention also provides the use of the Kushnerella HH-2 or the microbial agent in producing indoleacetic acid.

[0012] The present invention also provides the Kushnerella HH-2 or the microbial agent in producing NH 3 Application in.

[0013] The present invention also provides the use of the Kushnerella HH-2 or the microbial agent in producing siderophores.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention provides a Kushnerella strain HH-2, which has good stress tolerance and can produce NH 3 , produce indoleacetic acid, produce siderophores, and promote plant growth, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the 7-day growth curve of HH-2 under different NaCl concentration conditions;

[0018] Figure 2 This is the growth curve of HH-2 under different pH conditions;

[0019] Figure 3 This is the acid-base regulating ability curve of HH-2;

[0020] Figure 4 Producing NH for HH-2 3 Test results;

[0021] Figure 5 This is the result of the siderophore production test of HH-2;

[0022] Figure 6 This is a picture showing the effect of HH-2 in promoting wheat germination;

[0023] Figure 7 This is the picture showing the potted plant growth promotion effect of HH-2. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0030] In the following examples, the culture medium information is as follows:

[0031] Culture medium: CAS culture medium: Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; LB liquid culture medium, LB solid culture medium: Jinclone (Beijing) Biotechnology Co., Ltd.

[0032] Example 1

[0033] Isolation, preservation, screening, identification and storage of strains

[0034] Root samples of Suaeda salsa were collected from the tidal flats in Huanghua City, Cangzhou City, Hebei Province. 10 g of roots were weighed and placed in a conical flask containing 90 ml of sterile water. The flask was shaken at 180 r / min for 30 min. After standing for 10 min, 1 ml of the supernatant was drawn and diluted with sterile water to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Different dilution gradients of bacterial solution. Take 100 μL of each dilution and evenly spread it on the LB solid plate, and culture it at a constant temperature of 30℃ for 2-5 days. Select single colonies with different morphological characteristics from the dilution plates with low concentration to high concentration, streak them on a new LB solid plate, and purify them repeatedly 2-3 times until a pure strain is obtained. Inoculate the pure strain into LB liquid culture medium, shake and culture at 30℃, 180r / minn for 1-2 days, mix the bacterial solution with 50% glycerol 1:1 (v / v), freeze it with liquid nitrogen, and store it at -80℃.

[0035] 1. Identification of strains

[0036] The 16SrDNA of the strain was extracted and the 16SrDNA sequence was determined. The strain sequencing sequence is as follows:

[0037]

[0038] The sequencing results were compared in the NCBI database, and the NCBI taxonomy database was searched to obtain the best matching species classification information. The strain was determined to be Kushneria avicenniae strain in Latin, and was named Kushneria avicenniae strain HH-2.

[0039] 2. Preservation of strains

[0040] The Kushnerella strain HH-2 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 30312.

[0041] Example 2

[0042] Stress tolerance of strain HH-2

[0043] 1. Salt resistance

[0044] Pure colonies of HH-2 were selected and inoculated into 100 mL LB liquid medium containing 0, 1, 2, or 3 M NaCl, and cultured at 180 rpm and 30 °C for 7 days. The corresponding uninoculated LB liquid medium was used as a control, and the light absorption value (OD 600 ), draw a growth curve ( Figure 1 ). The result is Figure 1 As shown, under 0, 1, 2, and 3 M NaCl conditions, HH-2 can grow normally, accumulate to a higher concentration within 24 h, and enter an exponential growth period on the 4th day.

[0045] 2. Acid and alkali tolerance

[0046] Pure colonies of HH-2 were selected and cultured in LB liquid medium with pH values ​​of 4, 5, 6, 7, 8, 9, and 10 at 180 r / min and 30°C for 36 h. The corresponding uninoculated LB liquid medium was used as a control to measure the light absorption value (OD 600 ), draw a growth curve ( Figure 2 ). According to OD 600 The growth of the strains was divided into two values, OD 600 When the value is less than 0.2, the strain is considered to be unable to grow normally. 600 When the value is greater than 0.2, the strain is considered to be able to grow normally. The results showed that the strain HH-2 can grow normally in the pH range of 5 to 10.

[0047] 4. Acid-base regulation ability

[0048] Pick pure colonies of HH-2 and culture them in LB liquid medium with pH values ​​of 5, 6, 7, 8, 9, and 10 at 180 r / min and 30°C for 36 h. Observe the growth of the strain and measure the pH value of the fermentation solution. Calculate as follows:

[0049] p=|pH 1 -pH 2 | / pH 1 *100%,

[0050] Where: p is the acid-base regulation ability of the strain, pH 1 is the pH value of the culture medium before the strain fermentation; pH 2 is the pH value of the culture medium after the strain fermentation.

[0051] The pH change rate curve of the strain is plotted with percentage as the ordinate, such as Figure 3 As shown, when the pH value is 5-7, the strain HH-2 secretes alkaline substances to adjust the pH, and when the pH value is 8-10, the strain HH-2 secretes acidic substances to adjust the pH.

[0052] Example 3

[0053] Analysis of growth-promoting properties of strain HH-2

[0054] 1. Producing NH 3 Activity assay

[0055] Pure colonies of HH-2 were selected and inoculated into 10 mL of peptone water (10 g of peptone, 1000 mL of distilled water, pH = 7.6) containing 0, 1, 2, or 3 M NaCl. The culture was shaken at 180 r / min and 30 °C for 3 days. 0.5 mL of Nessler's reagent was added to each tube. If a yellow-brown precipitate appeared, it indicated that the bacteria had the ability to produce NH. 3 If there is no yellow-brown precipitate, it means that it does not produce NH 3 Activity. Figure 4 As shown in Table 1, strain HH-2 had the ability to produce NH under 0, 1, 2, and 3 M NaCl salt stress. 3 activity.

[0056] 2. Determination of IAA (indoleacetic acid) production capacity

[0057] Salkowski colorimetric solution: absorb 0.5 mol / L FeCl 3 Dissolve 1.5 mL in 50 mL ddH 2 O, and then add 30 mL of concentrated H 2 SO 4, stirring constantly during the process.

[0058] Determination of IAA standard curve: weigh 0.1g IAA powder and dissolve it in 50mL 50% ethanol, draw 5mL and add 95mL 50% ethanol to obtain 100μg / mL IAA standard solution; take 100μg / mL IAA standard solution to prepare 1, 3, 5, 10, 30, 50, 70, 90μg / mL IAA standard solution, use 50% ethanol as 0μg / mL standard solution, and mix each standard solution with colorimetric solution at a volume ratio of 1:1, place at room temperature away from light for 30min, and measure the OD of each concentration standard solution 530 The absorbance value was used to make a standard curve.

[0059] Pure colonies of HH-1 were picked and placed in 10 mL centrifuge tubes containing LB liquid medium containing tryptophan (200 mg / L). Each centrifuge tube contained 3 mL of medium and cultured at 30 °C and 180 r / min for 3 days. Then the bacterial suspension was centrifuged at 10000 r / min for 10 min. 2 mL of supernatant was added to 2 mL of Salkowski colorimetric solution and allowed to react for 30 min in the dark. The OD was measured. 530 The IAA standard curve equation was: y = 0.0970x-0.2083 (R 2 =0.9293). As shown in Table 1, the absorbance linear formula was used to calculate that the HH-1 strain could secrete 2.323 μg / mL of IAA.

[0060] 3. Determination of Siderophore Production Capacity

[0061] Pick pure colonies of HH-2 and inoculate them on CAS medium plates, culture at 30℃ for 7 days. If an orange-yellow transparent circle appears around the colony, it is positive. Measure the diameter of the transparent circle D (cm) and the colony diameter d (cm), and calculate the D / d value. The larger the ratio, the greater the activity of producing siderophores. Figure 5 As shown in Table 1, the results showed that the HH-2 strain had an obvious orange-yellow transparent circle around it, indicating that the strain had the ability to produce siderophores.

[0062] Table 1 Analysis results of growth-promoting properties of strain HH-1

[0063] Strain number <![CDATA[Producing NH 3 > IAA concentration (μg / ml) Siderophore production (D / d value) HH-2 + 2.323 3.41

[0064] Example 4

[0065] Experiment on the Salt Tolerance and Growth Promotion of Plants by Strain HH-2

[0066] The plant treated in this example is wheat.

[0067] 1. Experiment on promoting wheat seed germination by strain HH-2

[0068] Preparation of salt-tolerant bacterial agent: Pick pure colonies of HH-2 and inoculate them into LB liquid medium. After shaking and culturing at 180r / min and 30℃ for 24h, collect the bacteria by centrifugation at 4℃, wash them three times with sterile water and resuspend them in sterile water. Adjust the OD of the bacterial suspension with 0, 50, 100, and 150mM NaCl sterile solutions respectively. 600 The value is 1.0.

[0069] Select wheat seeds of uniform size and fullness, disinfect them with 75% alcohol for 5 minutes, rinse the seeds repeatedly with sterile water, place the seeds in a culture dish with filter paper, add 10ml of 0, 50, 100, 150mM NaCl sterile solution and 10ml of bacterial suspension, respectively, take the corresponding treatment without adding bacterial agent as control, and culture at 26℃ for 8 days. Figure 6 As shown in Table 2, under 50, 100, and 150 mM NaCl salt stress, strain HH-2 was able to promote the germination of wheat seeds and the growth of wheat sprouts and roots, and increase the accumulation of wheat seedling biomass.

[0070] Table 2 Growth indexes of wheat seeds treated with strain HH-2 for 8 days

[0071]

[0072] 2. Potted experiment of wheat growth promotion by strain HH-2

[0073] Preparation of salt-tolerant bacterial agent: Pick pure colonies of HH-2 and inoculate them into LB liquid medium. After shaking culture at 180r / min and 30℃ for 48h, collect the bacteria by centrifugation at 4℃, wash them three times with sterile water and resuspend them in sterile water. Adjust the OD of the bacterial suspension with 0, 50, 100, and 150mM NaCl sterile solutions respectively. 600 The value is 1.0.

[0074] Select wheat seeds of uniform size and fullness, disinfect them with 75% alcohol for 5 minutes, rinse the seeds repeatedly with sterile water for several times, germinate them in sterile water at 26°C for 1 day, place the seeds in sterilized vermiculite, soak the vermiculite with sterile water, and culture them at 4°C for 1 month. Replenish sterile water in time according to the water absorption. Select wheat seedlings with uniform growth and transplant them into pots filled with sterilized nutrient soil (8 seeds per pot). After watering with saline (150mM NaCl solution), drip 2.5mL of bacterial agent into the rhizosphere of each seedling. Use the control without bacterial agent. Figure 7 As shown, the growth-promoting effect of wheat inoculated with bacteria was obvious under 150 mM NaCl salt stress conditions.

[0075] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Kushnerella strain Kushneria avicenniae HH-2, characterized in that It was deposited in the China General Microbiological Culture Collection Center on April 11, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30312.

2. A microbial agent, characterized in that: Including the Kushnera HH-2 as claimed in claim 1.

3. Use of Kushnerella HH-2 as claimed in claim 1 or the microbial agent as claimed in claim 2 in promoting plant growth.

4. The use according to claim 3, characterized in that: The plants include wheat.

5. Use of the Kushnerella HH-2 according to claim 1 or the microbial agent according to claim 2 in the production of indoleacetic acid.

6. Use of the Kushnerella HH-2 according to claim 1 or the microbial agent according to claim 2 in producing NH3.

7. Use of the Kushnerella HH-2 according to claim 1 or the microbial agent according to claim 2 in producing siderophores.

Images