Pantoea agglomerans B10 and application thereof in preparation of plant growth promoting agent

Abstract

The invention discloses Pantoea agglomerans B10 and application thereof in preparation of a plant growth promoting agent, experiments prove that the Pantoea agglomerans B10 has the preservation number of CGMCC No.31199, and also has the capability of producing IAA, the IAA yield is 31.97 mu g / mL, the relative yield of siderophores is 72.03%, the capabilities of dissolving inorganic phosphorus, fixing nitrogen and producing polyglutamic acid reach 443.76 mu g / mL, and the comprehensive growth promoting capability is relatively strong.

Description

Technical Field

[0001] The present invention relates to the technical field of strain separation and identification, and more specifically to a rhizosphere growth-promoting bacterium B10 having growth-promoting functions such as producing IAA and iron carriers, and applications thereof. Background Art

[0003] There is currently a large amount of evidence showing that planting halophytes in saline-alkali land can effectively improve saline-alkali soil. In addition, the application of some microbial agents such as biological fertilizers, plant stimulants, and rhizosphere regulators can change the physical and chemical composition of saline-alkali soil, reduce the threat of high salt in the soil to plant growth, and obtain better plant benefits. Because of their low cost and environmental friendliness, halophytes and microbial populations have gradually attracted the attention of researchers.

[0004] Plants secrete some special substances in their roots to select and gather specific microbial groups, and then improve their salt tolerance through interaction with microorganisms. These microorganisms are collectively referred to as rhizosphere growth-promoting bacteria. Rhizosphere growth-promoting bacteria are key factors that can determine plant productivity and stress tolerance. Microorganisms associated with plants growing under harsh environmental conditions can help plants reduce the harm of abiotic stress, thereby enabling plants to complete their growth cycle under abiotic stress. In this context, a better understanding of the microbial community in saline-alkali environments, especially the microbial community in the rhizosphere and inner layer of naturally occurring plants, will likely open up a new way to understand plant salt tolerance and how it is affected by related microorganisms, thereby achieving the goal of improving plant salt tolerance. Therefore, screening salt-tolerant bacteria with multiple plant growth-promoting properties to enhance crop tolerance to salt is an effective method, thereby providing an important resource for promoting agriculture based on saline-alkali soils.

[0005] 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.

[0006] Song Yang and others screened a strain of Enterobacter from the rhizosphere of saline-alkali plants and numbered it AD2-2. Experiments found that it had a good promoting effect on the root growth of Arabidopsis, rice and corn under saline-alkali stress. These experimental results show that Enterobacter bacteria have good growth-promoting ability and may have great potential in helping plants cope with abiotic stress and have good prospects for agricultural applications. However, there are still deficiencies in the current research on rhizosphere growth-promoting bacteria, and more beneficial rhizosphere growth-promoting bacteria need to be explored for application in agricultural production. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Pantoea agglomerans B10.

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

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

[0010] The invention discloses Pantoea agglomerans B10, which has the preservation number of CGMCC No.31199 and has the ability to promote plant growth.

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

[0012] Advantages of the present invention:

[0013] Experiments have shown that a kind of Pantoea agglomerans B10 of the present invention, with a preservation number of CGMCC No. 31199, (hereinafter referred to as strain B10) also has the ability to produce IAA, with an IAA yield of 31.97 μg / mL, a relative yield of iron carrier production of 72.03%, and the ability to decompose inorganic phosphorus, fix nitrogen, and produce polyglutamic acid is up to 443.76 μg / mL, and the comprehensive growth-promoting ability is strong. The IAA yield of strain B10 is high. The present invention further conducts potted experiments to verify the effect of the target strain on plant phenotype. In the present invention, after inoculation of B10, the morphological indicators of tobacco are significantly improved. As a strain with high IAA yield and multiple growth-promoting properties, B10 may act on tobacco through multiple pathways to promote its growth and development.

[0014] In the tobacco potted test, after inoculation with B10, the stem length, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight and underground dry weight of tobacco increased by 52.27%, 63.80%, 121.44%, 225.24%, 295.45% and 57.48% respectively compared with the control, indicating that strain B10 has excellent growth-promoting properties as well as outstanding growth-promoting potential and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the colony morphology of strain B10 on LB medium.

[0016] Figure 2 This is the phosphate solubilization circle of strain B10 on inorganic phosphorus solid culture medium.

[0017] Figure 3This is the nitrogen fixation circle of strain B10 on Asbhy nitrogen fixation medium.

[0018] Figure 4 The IAA synthesis content of strain B10 under different tryptophan concentrations.

[0019] Figure 5 This is a graph showing the experimental results of detecting the iron carrier content in strain B10.

[0020] Figure 6 This is the standard curve of polyglutamic acid.

[0021] Figure 7 This is the growth curve of strain B10 at different pH levels.

[0022] Figure 8 This is a diagram showing the growth-promoting effect of strain B10 on tobacco under saline-alkali stress. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1

[0025] Materials and methods

[0026] 1.1 Saline-alkali soil samples

[0027] Saline-alkali soil near Tianjin University Zhejiang Research Institute (Shaoxing).

[0028] 1.2 Culture medium

[0029] (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.

[0030] (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.

[0031] (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).

[0032] (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.

[0033] (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).

[0034] (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.

[0035] (7)CAS test solution:

[0036] 1) Dissolve 0.182 g CTAB (cetyltrimethylammonium bromide) in 50 ml deionized water;

[0037] 2) Dissolve 0.0054 g FeCl3·6H2O in 20 ml 10 mM HCl aqueous solution;

[0038] 3) Dissolve 0.0605 g of chrome azurol S in 50 ml of deionized water;

[0039] 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;

[0040] 5) Dissolve 4.307 g of anhydrous piperazine in 26 ml of deionized water and adjust the pH to 5.6 with concentrated HCl;

[0041] 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.

[0042] (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.

[0043] (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.

[0044] 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.

[0045] Example 2

[0046] Isolation and screening of strains

[0047] 2.1 Collect rhizosphere soil of common halophytes in saline-alkali land.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 2.2 Select the strains with uniformly dispersed colony growth and single colony on the plate for picking, PCR amplify the 16S region, and send 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:

[0052] Table 1 PCR reaction system

[0053]

[0054] Table 2 PCR reaction program

[0055]

[0056] 2.3 Compare the sequencing results on the NCBI website and detect the screened strains.

[0057] Comparison with the GenBank database showed that strain B10 had a similarity of 99.79% with strain Pantoea agglomerans strain JCM1236, and strain B10 was identified as Pantoea agglomerans.

[0058] 2.4 Strain preservation

[0059] The strain was classified and named Pantoea agglomerans, B10, and was deposited on July 8, 2024 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC NO.31199. The strain is hereinafter referred to as strain B10.

[0060] Example 3

[0061] Preliminary screening experiment on the phosphate solubilization characteristics of isolated strains

[0062] The isolated and purified strain B10 was inoculated on the PKO medium and cultured in a bacterial static incubator in the dark. After observation, the diameter of the phosphate dissolving zone (D) and the colony diameter (d) of the strain on the plate were measured with a caliper to preliminarily determine the phosphate dissolving ability of the obtained strain B10.

[0063] The results showed that the diameter of the phosphate dissolution zone (D) was 1.6 cm and the diameter of the colony (d) was 0.9 cm (see Figure 2 ), it was preliminarily determined that the phosphate solubilizing capacity of the obtained strain B10 was D / d=1.78 between 1.5-1.9, which had good phosphate solubilizing capacity.

[0064] Example 4 Preliminary screening experiment of nitrogen fixation properties of isolated strains

[0065] The Kim method for screening nitrogen-fixing strains was adopted. The strain B10 obtained by screening was spot-inoculated on an Asbhy nitrogen-free culture medium plate, and the plate was cultured statically. The growth of the strain was observed regularly.

[0066] The results showed that strain B10 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.

[0067] Example 5 Detection experiment of IAA (indoleacetic acid) production characteristics of isolated strains

[0068] The strain B10 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 600 Then 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 uninoculated culture supernatant and 4 mL of Salkowski colorimetric solution. After reacting at room temperature in the dark for 20 minutes, measure the OD 530nm .

[0069] 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 31.97 μg (ml*OD 600 ), indicating that strain B10 has a strong ability to produce IAA.

[0070] Example 6 Detection experiment of the siderophore production characteristics of isolated strains

[0071] The strain B10 was inoculated into MKB medium and cultured in a shaking incubator (180r rpm) at 28°C for 48h, and the fermentation product (bacterial liquid) was collected; the fermentation product was centrifuged at 1000rpm for 10min, and 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 1h, 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.

[0072] The results showed that the relative production of siderophore by strain B10 was 72.03%, indicating that the strain had a strong ability to produce siderophore (see Figure 5 ).

[0073] Example 7

[0074] Detection of the ability of isolated strains to produce polyglutamic acid (γ-PGA)

[0075] 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, and 40μg / mL γ-PGA solutions, respectively, and mix with 2g / L CTAB solution (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 ).

[0076] Determination of crude γ-PGA. The strain B10 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 a speed of 12000 r / min for 3 minutes, an equal volume of hexadecyltrimethylammonium bromide solution (solvent is 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 it into the standard curve.

[0077] The results showed that B10 had a higher ability to produce polyglutamic acid, reaching 433.76 μg / mL.

[0078] Example 8

[0079] Effects of different pH on strain growth

[0080] According to Han Xiao's experimental method, normal LB medium was prepared, and then the pH of the medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 with HCl and NaOH respectively, and the medium was sterilized with a sterilizer. After the strain was inoculated into 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-prepared liquid culture medium with different pH values, and culture at 30℃ and 180rpm for 12h, then measure OD 600 The absorbance value below.

[0081] The results showed that strain B10 still had strong bacterial activity at pH = 9, indicating that the strain had good salt and alkali tolerance (see Figure 7 ).

[0082] Example 9

[0083] 9.1 Tobacco seed germination experiment

[0084] 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.

[0085] 9.2 Inoculation experiment of rhizosphere growth-promoting bacteria

[0086] After 7 days of transplanting, the rhizosphere inoculation experiment was carried out on the uniformly growing tobacco seedlings. The strain was first cultured overnight, then centrifuged and resuspended in 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, and irrigated twice in total. To ensure saline-alkali conditions, 20 mL of 100 mM NaHCO3 solution was irrigated to each seedling pot 3 days after the first inoculation, and irrigated twice in total. The growth of the seedlings was observed, and 20 mL of sterile water was irrigated to each pot about 2-3 days later. The inoculation treatment and the control treatment were guaranteed to be a single variable.

[0087] 9.3 Tobacco seedling biomass determination

[0088] 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.

[0089] The results showed that after the experiment, the tobacco seedlings with different treatments were taken out with roots under 100mM NaHCO3 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 NaHCO3 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 B10 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 B10 were increased by 52.27%, 63.80%, 121.44%, 225.24%, 295.45% and 57.48%, respectively (see Figure 8 ), indicating that strain B10 has a strong growth-promoting ability.

[0090] Table 3 Effects of strain B10 on tobacco seedling biomass under stress

[0091]

Claims

1. A Pantoea agglomerans B10, with a deposit number of CGMCC No.31199, which has the ability to promote plant growth.

2. Use of the agglomerated Pantoea according to claim 1 in the preparation of a plant growth promoting agent.

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