Growth-promoting rhizosphere strain W16 and application thereof

Abstract

The invention discloses a growth-promoting rhizosphere strain W16 and application thereof, the growth-promoting rhizosphere strain W16 has the preservation number of CGMCC No.31198 and has the capability of promoting plant growth, experiments prove that the strain W16 has the growth-promoting characteristics of IAA production, siderophore production, phosphate solubilization, nitrogen fixation and polyglutamic acid production at the same time, the comprehensive growth-promoting capability is high, and when the concentration of tryptophan in a culture medium is increased, the growth-promoting rhizosphere strain W16 can promote the growth of plants. The IAA generation capability of the strain W16 is enhanced, and the strain W16 has the phosphorus dissolving and nitrogen fixing capabilities at the same time; the relative content of the iron-producing carrier of the strain W16 is 65.52%; when the pH value is equal to 9, the strain W16 still has relatively strong bacterial activity and can grow relatively well; the strain W16 has a high polyglutamic acid production capability reaching 207.16 [mu] g / mL, a tobacco seedling pot experiment is carried out, and the result proves that the strain W16 can promote the growth of tobacco under saline-alkali stress.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a rhizosphere growth-promoting bacterium Enterobacterquasihormaechei, W16, with a preservation number of CGMCC No.31198. Background Art

[0002] In the rhizosphere of plants, there are many microorganisms that gather and interact with plant root cells in a continuous and complex manner. Some of them facilitate the growth and development of plants. It can be said that this complex microbial community is the second largest genome of plants and is of great significance to plant health. The rhizosphere of plants contains products of plant photosynthesis and different root secretions, which act on the microbial populations around the rhizosphere and have an important impact on the physical structure, chemical characteristics and biodiversity of the soil. In recent years, with the increasing severity of soil salinization, plant growth has been seriously threatened. In order to improve the survival ability of plants in salinized soils, the development of rhizosphere microorganisms has become an effective way. Among them, growth-promoting rhizobacteria (PGPR) can help plants resist abiotic stress and promote plant growth by symbiosis with plants. Its two main growth-promoting methods are to promote the absorption of plant nutrients and to affect the balance of plant hormones by regulating plant hormones. Rhizosphere growth-promoting bacteria refer to a group of microorganisms that can colonize the rhizosphere of plants. They promote plant growth, increase plant yields, improve soil fertility, and reduce pathogens by interacting with plants to help plants resist biological or abiotic stresses. In other words, bacteria that promote plant growth and development are collectively referred to as rhizosphere growth-promoting bacteria. For example, rhizosphere growth-promoting bacteria can promote plant growth and development through promoting phosphorus solubilization, nitrogen fixation, secretion of auxins, iron carriers, and other growth-promoting methods. At present, research on rhizosphere growth-promoting bacteria mainly focuses on promoting plant growth and development, crop disease prevention and control, soil improvement, and other aspects.

[0003] Zhu Zhiyu and others screened and isolated three salt-tolerant rhizosphere growth-promoting bacteria that produce auxins, and inoculated them into the rhizosphere of alfalfa under saline-alkali conditions, and found that they could significantly increase the dry weight and antioxidant enzyme activity of alfalfa plants. Biological nitrogen fixation is very important in improving soil productivity. Nitrogen deficiency affects plant growth and productivity, and nitrogen deficiency is very common in saline-alkali environments. Recent studies have found that inoculating rhizosphere growth-promoting bacteria can effectively improve the nitrogen fixation ability of alfalfa under saline-alkali conditions.

[0004] Han Xiao screened out a strain of Enterobacter bacteria in saline-alkali land and numbered it D2. On the 7th day, its phosphorus solubility was 521.81 mg / L, its IAA synthesis was 20.82 μg / mL, and its relative iron carrier content was 15.09%. When it was inoculated into the rhizosphere of rice under saline-alkali stress, it significantly promoted the growth and development of rice under saline-alkali stress.

[0005] Zhang Meng et al. screened out a strain of Enterobacter in the rhizosphere soil and numbered it Zm-123. Its maximum phosphorus solubility was 137.50 mg / L, IAA synthesis was 28.30 μg / mL, and the maximum iron carrier production was 82.34%. Inoculating it into the rhizosphere of rapeseed can significantly promote the growth and development of rapeseed.

[0006] These experimental results show that Enterobacter bacteria have good growth-promoting ability and may have great potential in helping plants cope with abiotic stresses, and have good prospects for agricultural applications. However, the above-mentioned rhizosphere growth-promoting bacteria often have fewer functions, so it is necessary to explore more rhizosphere growth-promoting bacteria with growth-promoting functions and apply them to agriculture in order to supplement microbial fertilizer resources. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a rhizosphere growth-promoting strain Enterobacterquasihormaechei W16.

[0008] The second object of the present invention is to provide an application of the above-mentioned rhizosphere growth-promoting strain W16 in the preparation of a plant growth-promoting agent.

[0009] The technical solution of the present invention is summarized as follows:

[0010] The rhizosphere growth-promoting strain Enterobacter quasihormaechei, W16, has a preservation number of CGMCC No. 31198 and has the ability to promote plant growth.

[0011] The above-mentioned rhizosphere growth-promoting strain is used in the preparation of a plant growth-promoting agent.

[0012] Advantages of the present invention: Experiments have shown that the rhizosphere growth-promoting strain Enterobacterquasihormaechei, W16, has a deposit number of CGMCC No.31198 (hereinafter referred to as strain W16) has the growth-promoting characteristics of producing IAA, siderophore, phosphate solubilization, nitrogen fixation, and polyglutamic acid, and has a strong comprehensive growth-promoting ability. When the tryptophan concentration in the culture medium increases, the ability of strain W16 to produce IAA is enhanced accordingly. When the tryptophan concentration in the culture medium is 500 mg / L, the IAA production capacity of strain W16 reaches 29.41 μg (ml*OD600). At the same time, strain W16 has the ability to solubilize phosphate and fix nitrogen; the relative content of siderophore produced by strain W16 is 65.52%; at pH = 9, strain W16 still has strong bacterial activity and can grow well; the polyglutamic acid ability of the strain was tested, and it was found that W16 has a high ability to produce polyglutamic acid, reaching 207.16 μg / mL. At the same time, a tobacco seedling pot experiment was carried out, and the results showed that strain W16 can promote the growth of tobacco under saline-alkali stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the colony morphology of strain W16 on LB medium.

[0014] Figure 2 This is the phosphate solubilization zone of strain W16 on PKO medium.

[0015] Figure 3 This is the nitrogen-fixing circle of strain W16 on Asbhy nitrogen-free medium.

[0016] Figure 4 The IAA synthesis content of strain W16 under different tryptophan concentrations.

[0017] Figure 5 This is a graph showing the experimental results of detecting the iron carrier content of strain W16.

[0018] Figure 6 This is the standard curve of polyglutamic acid.

[0019] Figure 7 This is the growth curve of strain W16 at different pH values.

[0020] Figure 8 This is a diagram showing the growth-promoting effect of strain W16 on tobacco under saline-alkali stress. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0022] Example 1

[0023] Materials and methods

[0024] 1.1 Saline-alkali soil samples

[0025] Saline-alkali soil near Tianjin University Zhejiang Research Institute (Shaoxing).

[0026] 1.2 Culture medium

[0027] (1) Potato dextrose medium (PDA): 200 g of potato slices were boiled for 30 min and filtered through double-layer gauze to obtain the filtrate. 20 g of glucose and 15 g of agar were added to make up to 1000 mL and the pH was adjusted to natural.

[0028] (2) PKO medium: weigh 0.25 g magnesium sulfate, 10.0 g glucose, 5.0 g magnesium chloride, 5.0 g tricalcium phosphate, 0.2 g potassium chloride, 0.1 g ammonium sulfate, and 16 g agar, add deionized water to make up to 1 L, and adjust the pH to 7.0.

[0029] (3) LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, add deionized water to make up to 1 L and sterilize at 115°C for 30 min; (LB solid medium (LB plate) requires the addition of 16 g of agar powder).

[0030] (4) Asbhy nitrogen-free medium: measure 5 g of glucose, 0.2 g of KH2PO4, 5 g of mannitol, 0.29 g of K2SO4·2H2O, 0.2 g of NaCl, 0.2 g of MgSO4·7H2O, and 5 g of CaCO3, add deionized water to make up to 1 L, and adjust the pH to 7.0.

[0031] (5) Salkowski colorimetric solution: Take 37.5 mL of 0.5 M FeCl3 and 150 mL of H2SO4, add deionized water to make up to 250 mL, mix well and set aside for use (prepare immediately before use).

[0032] (6) MKB medium (1 L): 5 g of acid-hydrolyzed casein, 15 mL of glycerol, 2.5 g of K2HPO4, 2.5 g of MgSO4·7H2O, pH = 7.2.

[0033] (7)CAS test solution:

[0034] 1) Dissolve 0.182 g CTAB (cetyltrimethylammonium bromide) in 50 ml deionized water;

[0035] 2) Dissolve 0.0054 g FeCl3·6H2O in 20 ml 10 mM HCl aqueous solution;

[0036] 3) Dissolve 0.0605 g of chrome azurol S in 50 ml of deionized water;

[0037] 4) mixing 1.5 ml of the solution obtained in step 2) with 7.5 ml of the solution obtained in step 3), and then mixing with 6 ml of the solution obtained in step 1) to obtain a mixed solution;

[0038] 5) Dissolve 4.307 g of anhydrous piperazine in 26 ml of deionized water and adjust the pH to 5.6 with concentrated HCl;

[0039] 6) Mix the mixed solution obtained in step 4) and the solution obtained in step 5) and make up the volume to 100 ml with deionized water.

[0040] (8) Seed culture medium: 10 g / L peptone, 3 g / L beef extract powder, 20 g / L glucose, 5 g / L sodium chloride, and the balance is deionized water.

[0041] Seed culture: liquid volume 50 / 250mL, temperature 37℃, rotation speed 150r / min, shaker culture for 18h.

[0042] (9) Fermentation medium: 40 g / L sodium glutamate, 5 g / L peptone, 50 g / L glucose, 10 g / L sodium chloride, and the balance is deionized water.

[0043] Fermentation culture: inoculation volume 2% (V / V), liquid volume 50 / 250 mL, temperature 37°C, rotation speed 150 r / min, shaking culture for 48 h.

[0044] Example 2 Isolation and screening of strains

[0045] 2.1 Collection of rhizosphere soil of common halophytes in saline-alkali land

[0046] Rhizosphere soil of halophytes from saline-alkali land near Zhejiang Research Institute of Tianjin University (Shaoxing) was collected and brought back to the laboratory at 4°C for bacterial strain isolation.

[0047] After shaking off the large pieces of soil on the roots of the halophytes, the fibrous roots and the remaining soil attached to the roots were placed in a 100 mL Erlenmeyer flask filled with glass beads and saline. After shaking for 20 minutes, the flask was left to stand for 2 minutes, and the supernatant was taken and diluted with saline in different gradients. 150 μL of each gradient of dilution was taken and spread on an LB plate containing 3% NaCl and pH 8.0 with a spreading rod to isolate the rhizosphere salt-alkali tolerant strains.

[0048] The above plate was inverted and cultured in a static incubator, and the growth of the strains on the plate was observed at any time. The colonies with good growth and different morphological characteristics on the plate were selected and purified multiple times (see Figure 1 ) and then cultured in LB medium for liquid culture, and the prepared glycerol bacteria were stored in a -20°C refrigerator.

[0049] 2.2 Select the strains with uniformly dispersed colony growth and single colonies on the plate for picking, PCR amplify the 16S region, and send it to Qingke for 16S rDNA sequencing (SEQ ID NO.1). The PCR reaction system is shown in Table 1, and the procedure is as follows:

[0050] Table 1 PCR reaction system

[0051]

[0052] Table 2 PCR reaction program

[0053]

[0054] 2.3 Compare the sequencing results on the NCBI website and detect the screened strains.

[0055] Comparison with the GenBank database showed that strain W16 had a similarity of 99.72% with strain Enterobacterquasihormaechei strain WCHEs120003, and strain W16 was identified as Enterobacterquasihormaechei strain.

[0056] 2.4 Strain preservation

[0057] The classification name is Enterobacter quasihormaechei, W16, which was deposited on July 8, 2024 at the General Microbiology Center of China Culture Collection Administration, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit code CGMCC No.31198. The strain is hereinafter referred to as strain W16.

[0058] Example 3 Preliminary screening experiment of phosphate solubilization characteristics of isolated strains

[0059] The isolated and purified strain W16 was inoculated on the PKO medium and cultured in a bacterial static incubator in the dark. After observation, the diameter of the phosphate solubilization zone (D) and the colony diameter (d) of the strain on the medium were measured with a caliper to preliminarily determine the phosphate solubilization ability of the obtained strain W16.

[0060] The results showed that the diameter of the phosphate dissolution zone (D) was 1.3 cm and the diameter of the colony (d) was 0.6 cm (see Figure 2 ), it was preliminarily determined that the phosphate-dissolving capacity of the obtained strain W16 was D / d=2.17>1.9, which had a strong phosphate-dissolving capacity.

[0061] Example 4 Preliminary screening experiment of nitrogen fixation properties of isolated strains

[0062] The Kim method for screening nitrogen-fixing strains was adopted, and the strain W16 obtained by screening was spot-inoculated on Asbhy nitrogen-free medium plates, and static culture was carried out, and the growth status of the strain was observed regularly.

[0063] The results showed that strain W16 was able to produce nitrogen fixation circles when cultured in Asbhy nitrogen-free medium (see Figure 3 ), indicating that the strain has the ability to fix nitrogen.

[0064] Example 5 Detection experiment of IAA (indoleacetic acid) production characteristics of isolated strains

[0065] The strain W16 was inoculated into 5 mL LB liquid medium containing 0, 100, 250, and 500 mg / L of L-tryptophan. The LB liquid medium without bacteria was used as a blank control. After culturing for 2 days, the OD 600Then centrifuge the remaining bacterial suspension, take 1 mL of the culture supernatant, add 4 mL of Salkowski colorimetric solution and mix well. The control condition is to add 1 mL of uninoculated culture supernatant and 4 mL of Salkowski colorimetric solution. After reacting at room temperature in the dark for 20 minutes, measure the OD 530 .

[0066] The results showed that when the tryptophan concentration in the culture medium increased, the strain's ability to produce IAA increased accordingly (see Figure 4 ), when the tryptophan concentration in the culture medium was 500 mg / L, its IAA production capacity reached 29.41 μg (ml*OD 600 ), indicating that strain W16 has a strong ability to produce IAA.

[0067] Example 6 Detection experiment of the siderophore production characteristics of isolated strains

[0068] The strain W16 was inoculated into MKB medium, cultured in a shaking incubator (180 rpm) at 28°C for 48 hours, and the fermentation product (bacterial liquid) was collected; the fermentation product was centrifuged at 1000 rpm for 10 minutes, the supernatant was taken, and CAS detection solution was added to the supernatant at a volume ratio of 1:1, and mixed thoroughly. After standing for 1 hour, the absorbance value (A) was measured at 630nm, and the Ar measured by mixing equal volumes of deionized water and CAS detection solution was used as the control. SU = (1-A / Ar) * 100% represents the relative yield of iron carriers in the sample. The higher the value, the higher the iron carrier yield.

[0069] The results showed that the relative production of siderophore by strain W16 was 65.52%, indicating that the strain had a strong ability to produce siderophore (see Figure 5 ).

[0070] Example 7 Detection experiment of the ability of isolated strains to produce polyglutamic acid (γ-PGA)

[0071] Accurately weigh 0.0100g of γ-PGA standard, dilute to 50mL with deionized water, and prepare 200μg / mL γ-PGA mother solution. Dilute the mother solution with deionized water to prepare 5, 10, 15, 20, 25, 30, 35, 40μg / mL γ-PGA solutions, respectively, and mix with 2g / L CTAB solution (the solvent is 5% NaOH aqueous solution) at a volume ratio of 1:1, react for about 3min, measure its absorbance at a wavelength of 250nm, and draw a standard curve with γ-PGA mass concentration as the horizontal axis and absorbance as the vertical axis. (See Figure 6 )

[0072] Determination of crude γ-PGA. The strain W16 was inoculated into the seed medium for overnight culture, and then transferred to the fermentation medium for 48 hours the next day. After the culture was completed, the fermentation broth was diluted, and 2 mL of the diluted fermentation broth was added to a centrifuge tube. After centrifugation at 12000 rpm for 3 minutes, an equal volume of 2 g / L hexadecyltrimethylammonium bromide (CTAB) solution (the solvent is a 5% NaOH aqueous solution) was added to react to form a suspension, and the absorbance was measured at a wavelength of 250 nm for about 3 minutes of complexation, and the actual yield was obtained by substituting the standard curve.

[0073] The results showed that strain W16 had a higher ability to produce polyglutamic acid, reaching 207.16 μg / mL.

[0074] Example 8 Effect of different pH on strain growth

[0075] The pH of the LB liquid medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 with HCl aqueous solution and NaOH aqueous solution, respectively, and the medium was sterilized with an autoclave. After the strain W16 was inoculated into a normal LB medium and cultured overnight, it was diluted with sterile water to OD 600 =0.5, pipette appropriate amount of bacterial suspension into the above-configured LB liquid culture medium with different pH values, and culture at 30°C, 180 rpm for 12 h, then measure OD 600 The absorbance value below.

[0076] The results showed that strain W16 still had strong bacterial activity at pH = 9, indicating that the strain had good salt and alkali tolerance (see Figure 7 ).

[0077] Example 9

[0078] 9.1 Germination experiment of tobacco seeds (commercial products)

[0079] Take an appropriate amount of uniform tobacco seeds and sterilize them with anhydrous ethanol, rinse them with sterile water for 5 times, and evenly pipette the seeds into the soil of a small pot, cover with plastic wrap, and let it germinate. When the tobacco seeds grow 2-3 cotyledons, transplant the tobacco plants, carefully pull out the tobacco with roots with tweezers, and transplant them into a new small pot filled with sterilized soil, with 1-2 tobacco seedlings transplanted into each pot.

[0080] 9.2 Inoculation experiment of rhizosphere growth-promoting bacteria

[0081] Seven days after transplanting, the W16 strain was tested on uniformly growing tobacco seedlings. The strain W16 was cultured overnight in LB liquid medium, then resuspended by centrifugation with sterile water, and the OD 600=0.6, 20 mL of water was irrigated at the root zone of each seedling pot, and the same amount of sterile water was irrigated at the control treatment. The second inoculation irrigation was carried out every 7 days, with the same method and dosage, for a total of 2 irrigations. To ensure saline-alkali conditions, 20 mL of 100 mM NaCl solution was irrigated to each seedling pot 3 days after the first inoculation, for a total of 2 irrigations, and the growth of the seedlings was observed. 20 mL of sterile water was irrigated to each pot about 2-3 days later, and the inoculation treatment and the control treatment were guaranteed to be a single variable.

[0082] 9.3 Tobacco seedling biomass determination

[0083] 45 days after the second inoculation, the tobacco seedlings with different treatments were slowly poured out of the pots with their roots, and the soil carried by the rhizosphere of the seedlings was slowly rinsed with running water. After cleaning, the plant height and root length of the tobacco seedlings were measured, and then the root fresh weight (underground fresh weight), stem fresh weight, leaf fresh weight (stem fresh weight and leaf fresh weight are collectively referred to as aboveground fresh weight) and root dry weight (underground dry weight), stem dry weight, and leaf dry weight (stem dry weight and leaf dry weight are collectively referred to as aboveground dry weight) were measured respectively.

[0084] The results showed that after the experiment, the tobacco seedlings with different treatments were taken out with roots under 100mM NaCl stress, and the plant height and root length were measured. Then, the aboveground fresh weight, root fresh weight, aboveground dry weight and root dry weight of the tobacco seedlings were measured. Under 100mM NaCl stress, the stem length, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight of the seedlings inoculated with strain W16 were significantly improved compared with the uninoculated seedlings. The stem length, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight of the seedlings inoculated with strain W16 were increased by 73.29%, 99.51%, 112.46%, 641.43%, 60.87% and 308.72%, respectively (see Figure 8 ), indicating that strain W16 has a strong growth-promoting ability.

[0085] Table 3 Effects of strain W16 on tobacco seedling biomass under stress

[0086]

Claims

1. The rhizosphere growth-promoting strain Enterobacter quasihormaechei, W16, has a deposit number of CGMCC No.31198 and has the ability to promote plant growth.

2. Use of the rhizosphere growth-promoting strain of claim 1 in the preparation of a plant growth-promoting agent.

Images