CN114703081B - A strain of Brevundimonas ST3CS3 and its application - Google Patents

Abstract

The invention discloses a strain of BrevundimonasBrevundimonasolei) ST3CS3 with the preservation number of CGMCC No.24140 in the China general microbiological culture Collection center; the strain has the capabilities of fixing nitrogen and dissolving potassium, and has higher capabilities of dissolving phosphorus and producing indoleacetic acid; the strain is colonized in a plant body, can obviously promote plant growth, increase chlorophyll and nitrogen content of the plant, further reduce the use of chemical fertilizers, improve crop yield and have important significance in agricultural production.

Description

A strain of Brevundimonas ST3CS3 and its application

technical field

The invention belongs to the biological technical field of growth-promoting bacteria, and in particular relates to a strain of Brevundimonas and its application in promoting plant growth.

Background technique

With the development of my country's industrialization and modernization, the area of cultivated land has decreased sharply year by year. Since the 1960s, the use of fertilizers has guaranteed national food security and played an important role in increasing agricultural production and income in my country. However, the excessive use of chemical fertilizers will not only affect the quality of agricultural products, but also cause huge pressure on the rural ecological environment. In recent years, traditional conventional chemical fertilizers (such as nitrogen, phosphorus, and potassium compound fertilizers) have always had problems of low utilization and abuse. Because chemical fertilizers generally contain heavy metals, unreasonable application of chemical fertilizers in successive years will cause accumulation of heavy metals in the soil. It is harmful to the soil structure and the sustainable development of human society. Blindly reducing the application of conventional chemical fertilizers may cause a series of negative effects such as production reduction. It is imminent to rationally apply or even reduce the application of conventional chemical fertilizers.

Plant growth-promoting rhizobacteria (PGPR) refers to self-growing bacteria that are beneficial to crops. The effect of growth-promoting bacteria directly promoting crop growth means that some plant growth-promoting bacteria can synthesize certain substances that have a direct effect on crop growth and development (such as auxin, etc.) and/or change the form of some invalid elements in the soil , making it effective and beneficial to crop absorption (such as nitrogen fixation, phosphorus solubilization, etc.). Growth-promoting bacteria can also act indirectly on certain plants, inhibiting or alleviating the adverse effects of certain plant diseases on crop growth and yield. This has prompted scientists all over the world to actively explore and develop microbial fertilizers with microorganisms as the core to replace (or replace part of) the use of chemical fertilizers and pesticides.

Microbial fertilizer is a green and environmentally friendly new type of fertilizer with plant growth-promoting bacteria as the core. It has the characteristics of long-term, non-toxic, pollution-free, energy-saving, and low-cost. The nitrate content in crop products can improve crop disease resistance, improve soil fertility, reduce the use of chemical fertilizers, purify the environment and maintain ecological balance. Microbial fertilizers mainly rely on microbial life activities to produce enzymes and beneficial substances to play a role. Nitrogen, phosphorus and potassium are essential elements for plant growth. Nitrogen in the air, inorganic phosphorus in the soil and potassium in the form of silicate minerals are difficult for plants to directly absorb. Microbes produce enzymes and beneficial substances that can convert unusable substances into directly usable elements; indole acetic acid (IAA) is a plant endogenous auxin, which participates in the regulation and regulation of various physiological and biochemical processes such as plant cell growth. Control, the IAA produced by microorganisms plays an important role in the plant micro-ecological environment. Under the current situation that our country is facing energy crisis, resource shortage, and environmental pollution, the importance of microbial fertilizers to the development of modern agriculture in our country is self-evident. An important cornerstone, it is a real pollution-free new fertilizer, which is in line with my country's sustainable development concept. As people's awareness and demand for green and pollution-free agricultural products are getting higher and higher, microbial fertilizers must have broad application scenarios, and Play an important role in the sustainable development of agriculture in our country.

Contents of the invention

The invention provides a plant endophytic bacterium Brevundimonas olei ST3CS3, which is classified and named as Brevundimonas olei , which was preserved in the General Microbiology Center of China Committee for Microbial Culture Collection on December 20, 2021, and the preservation number is CGMCC No.24140 , Preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Another object of the present invention is to provide a new application of the above-mentioned plant endophytic bacterium Brevundimonas ST3CS3, that is, to apply it in promoting plant growth, and to prepare biological bacteria agents or microbial fertilizers that promote plant growth. The present invention brevundimonas Bacteria ST3CS3 has the ability to fix nitrogen and dissolve potassium, and has high ability to dissolve phosphorus and produce IAA; colonizing the strain in plants can significantly promote plant growth, increase its chlorophyll and nitrogen content, and then reduce the use of chemical fertilizers.

In order to achieve the above object, the present invention takes the following technical measures:

1. Collect plant samples of Stellera chamaejasme L., an invasive plant in the Shangri-La pasture in Yunnan Province, and rinse them under tap water;

2. Divide the plant samples into the above-ground part and the underground part for surface disinfection respectively. First, soak in ethanol solution with a volume concentration of 75% for 3-5 minutes, rinse with sterile water for 3-5 times, and then use sodium hypochlorite with an available chlorine concentration of 5% Soak in the solution for 2-3 minutes, rinse with sterile water for 3-5 times, and place it on sterile filter paper to absorb the water after rinsing; use the mixed dilution plate method for bacterial isolation, and 1 g of plant samples sterilized on the surface Grind into powder in mortar, fully mix with 9mL sterile water, transfer the mixture to LB medium for culture after gradient dilution, culture at 37°C for 10 days, observe the next day, pick if there are colonies growing, and obtain more after separation and purification strain endophytic bacterial strains, and make endophytic bacterial strains into bacterial suspension;

3. Inoculate the isolated endophytic strains on the inorganic phosphorus medium, and use the hydrolysis circle to determine the phosphorus-dissolving ability of the strains;

4. Select the bacterial strain with strong phosphorus dissolving ability and store it on the LB slope for later use, and name the bacterial strain ST3CS3;

5. Identification of strain ST3CS3

Morphological characteristics of ST3CS3: colony diameter 2-3mm, colony color is yellow in the middle with white edges, opaque, moist surface, slightly raised, neat edges; Gram-negative short bacillus, with flagella, without spores;

Molecular identification: MoBio PowerSoil ^® DNA kit was used to extract the total DNA of the strain, after detection, it was sent to the sequencing company for sequence determination, and the sequencing results were compared with the sequence on NCBI;

Combined with the results of morphological characteristics and molecular identification, the strain was finally identified as Brevundimonas olei ; the medium used for the preservation and activation of the strain was LB medium.

The present invention carries out pot experiment with the Brevundimonas ST3CS3 isolated from Stylus chamaejasme, and explores its influence on the growth of tobacco seedlings, that is, the research on the influence of Brevundimonas ST3CS3 inoculation on the growth of potted tobacco is carried out, providing microbial fertilizers Bacterial species and theoretical research basis.

Compared with the prior art, the present invention has the following beneficial effects:

Brevundimonas ST3CS3 provided by the present invention is derived from Daphne chamaejasme, has the ability to fix nitrogen, dissolve phosphorus, dissolve potassium and produce IAA, and can effectively colonize in plants, effectively dissolve insoluble potassium in plant rhizosphere soil, The nitrogen content in the plant is increased, thereby promoting the growth of the plant, reducing the use of chemical fertilizers, and the cultivation operation is simple, the cost is low, the environment is safe, and it is suitable for industrial production and market promotion and application.

Description of drawings

Fig. 1 is the colony morphology of Brevundimonas ST3CS3 on LB medium;

Fig. 2 is the growth situation of Brevundimonas ST3CS3 on the inorganic phosphorus solid medium;

Fig. 3 is the standard working curve of phosphorus standard solution;

Fig. 4 is the measurement result of brevundimonas ST3CS3 phosphorus dissolving ability;

Fig. 5 is the assay result of Brevundimonas ST3CS3 nitrogen fixation ability;

Fig. 6 is the assay result of brevundimonas ST3CS3 potassium solution ability;

Fig. 7 is the standard working curve of IAA standard solution;

Fig. 8 is the measurement result of IAA secretion ability of Brevundimonas ST3CS3;

Fig. 9 is the influence of Brevundimonas ST3CS3 on the growth of tobacco seedlings;

Figure 10 shows the effect of Brevundimonas ST3CS3 on the chlorophyll content of tobacco seedling leaves.

Detailed ways

The technical solutions of the present invention will be described in further detail below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions, manuals, etc. before the filing date of the present invention for implementation.

Embodiment 1: Isolation, screening and identification of Brevundimonas ST3CS3

(1) Collect plant samples of Daphne chamaejasma, the dominant plant in the Shangri-La pasture in Yunnan Province, and rinse them under tap water;

(2) Divide the plant samples into the above-ground part and the underground part and carry out surface disinfection respectively. First, soak in ethanol solution with a volume concentration of 75% for 5 minutes, rinse with sterile water for 5 times, and then soak in sodium hypochlorite solution with an available chlorine concentration of 5% for 2 minutes. , rinsed with sterile water for 4 times, after rinsing, place it on sterile filter paper to blot dry; use the mixed dilution plate method to separate bacteria, grind 1 g of plant samples sterilized on the surface into powder in sterile mortar, and mix with 9mL of sterile water was fully mixed, and the mixture was serially diluted to make dilutions of 10 ^-3 , 10 ^-4 , and 10 ^-5 , and transferred to LB medium (yeast extract 5g, tryptone 10g, sodium chloride 10g, agar 14g , distilled water 1L, pH 7.0-7.2), put it upside down in a biochemical incubator at 37°C for 10 days, observe the next day, pick a single colony if there are colonies growing out, and obtain multiple strains of endophytic bacteria after separation and purification. The endophytic bacterial strains are made into bacterial suspensions respectively;

(3) Inoculate the isolated endophytic strains into inorganic phosphorus medium (glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate heptahydrate 0.3g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate tetrahydrate 1g, tricalcium phosphate 5g, agar 14g, distilled water 1L, pH 7.0-7.5), the phosphorus-dissolving ability of the strain was determined by the hydrolysis circle;

(4) Select the strain with the strongest phosphorus-dissolving ability and save it on the LB slope for later use, and name the bacterial strain ST3CS3;

(5) Identification of strain ST3CS3

Morphological characteristics of ST3CS3: the diameter of the colony is 2-3 mm, the color of the colony is yellow in the middle and white at the edge, opaque, the surface is moist, slightly raised, and the edges are neat; Gram-negative short bacillus, with flagella, without spores (Figure 1);

Molecular identification: MoBio PowerSoil ^® DNA kit was used to extract the total DNA of the strain, and after detection, it was sent to the sequencing company for sequence determination. The sequencing result is shown in SEQ ID NO:1, and the sequencing result was compared with the sequence on NCBI; the binding morphology According to the results of biological characteristics and molecular identification, its homology with Brevundimonas olei reached 99%, and the strain was finally identified as Brevundimonas olei .

Embodiment 2: Determination of Phosphorus Solubilizing Ability of Brevundimonas ST3CS3

Phosphorus-dissolving circle: inoculate the isolated Brevundimonas ST3CS3 on the inorganic phosphorus solid medium, repeat three times at 4 inoculation points per plate, and culture at a constant temperature of 30°C for 5 days, observe and record the growth of the strain and the phosphorus-dissolving circle (D) and colony diameter (d). The larger the D/d, the stronger the phosphorus-dissolving ability. The results are shown in Figure 2. Brevundimonas ST3CS formed a more obvious phosphorus-dissolving circle on the inorganic phosphorus medium. The circle diameter is 0.75 cm, and the colony diameter is 0.3 cm.

Phosphorus dissolving ability: After culturing Brevundimonas ST3CS in LB liquid medium on a shaker at 30°C and 180rpm for 15 hours, insert 4% inoculum into inorganic phosphorus liquid medium, and at the same time inoculate the same amount of LB liquid culture As a blank control, three replicates were set up for each treatment, and cultured on a shaker at 30°C and 180rpm for 6 days; the content of soluble phosphorus was determined by molybdenum antimony colorimetry (spectrophotometer wavelength 700nm), and the specific steps were as follows:

①Centrifuge the fermentation broth at 10,000rpm for 10min, take 2.5mL of the supernatant and place it in a 50mL volumetric flask, add 2 drops of 2,6-dinitrophenol indicator, and adjust the pH to the solution with 10% sodium hydroxide or 5% dilute sulfuric acid Just light yellow, add molybdenum antimony anti-color agent 5mL, then add deionized water to dilute to 50mL;

② After standing for 30 minutes, compare the color with a spectrophotometer at 700nm, and measure the blank control group at the same time;

③ At the same time as the experiment, draw a phosphorus standard curve, draw 0, 2, 4, 6, 8, and 10 mL of 5 μg/mL phosphorus standard solution into a 50 mL volumetric flask, add 2 drops of 2,6-dinitrophenol indicator, and use 10% Sodium hydroxide or 5% dilute sulfuric acid to adjust the pH until the solution is slightly yellow, add molybdenum antimony anti-color agent 5mL, add deionized water to constant volume, obtain 0, 0.2, 0.4, 0.6, 0.8, 1.0μg/mL phosphorus standard After standing still for 30 minutes, compare the color with a spectrophotometer at 700nm, draw a standard working curve (Figure 3), and then substitute the absorbance value measured in step ② into the standard working curve to obtain Brevundimonas ST3CS in inorganic phosphorus culture solution The amount of phosphorus dissolved in it was 51mg/L (Figure 4), indicating that Brevundimonas ST3CS has the ability to convert inorganic phosphorus into organic phosphorus, making it easier for plants to absorb phosphorus, thereby promoting plant growth.

Embodiment 3: Determination of Nitrogen Fixation Ability of Brevundimonas ST3CS3

Brevundimonas ST3CS3 was inoculated into Ashubei solid medium (disodium hydrogen phosphate 0.2g, magnesium sulfate heptahydrate 0.2g, sodium chloride 0.2g, calcium carbonate 5g, mannitol 10g, calcium sulfate 0.1g, agar 14g , distilled water 1L, pH 7.0), repeat three times, and culture at 30°C for 5 days; if the strain can grow on the medium, it proves that the strain has nitrogen-fixing ability, otherwise, it has no nitrogen-fixing ability.

The results are shown in Figure 5. It can be seen from the figure that Brevundimonas ST3CS3 can grow on the Ashubei medium, indicating that it has the ability to fix nitrogen. Brevundimonas _ST3CS3 can convert N in the air into available nitrogen, which has Help plants absorb nitrogen, thereby promoting plant growth.

Embodiment 4: Determination of Potassium-decomposing Ability of Brevundimonas ST3CS3

Brevundimonas ST3CS3 was inoculated into silicate solid medium (2g disodium hydrogen phosphate, 5g sucrose, 0.5g magnesium sulfate heptahydrate, 0.005g ferric chloride, 0.1g calcium carbonate, 1g potassium feldspar powder, 14g agar, Distilled water 1L, pH 7.0-7.5), 4 inoculation points per plate, repeated three times, 30 ℃ constant temperature culture for 5 days; if the strain forms a potassium-dissolving circle on it, it proves that it has the ability to dissolve potassium, otherwise, no Potassium dissolving ability.

The results are shown in Figure 6, Brevundimonas ST3CS forms a potassium-dissolving circle on the silicate solid medium, the diameter of the potassium-dissolving circle is 0.45cm, and the diameter of the colony is 0.35cm, indicating that Brevundimonas ST3CS3 can convert insoluble potassium into effective Potassium helps plants absorb potassium, which in turn promotes plant growth.

Example 4: Determination of IAA Secretion Ability of Brevundimonas ST3CS3

Brevundimonas ST3CS3 was cultured in LB liquid medium (containing L-tryptophan 0.1mg/mL) in the dark at 30°C and 120rpm for 24h on a shaker, and 1.5mL of the bacterial liquid was centrifuged at 12000rpm for 10min to remove the precipitate, and 0.5mL was taken Add the same amount of Salkowski's reaction solution (1mL 0.5mol/L ferric chloride, 49mL 35% perchloric acid) to the supernatant, react in the dark for 30min, measure the absorbance at 530nm, repeat three times for each group, and culture without inoculation The above-mentioned treatment was used as the control and zeroed; the IAA standard solution with a concentration of 0, 5.5, 11, 22, 44, and 88 μg/mL was measured by the above method and the standard curve was drawn (Figure 7), and then the experiment inoculated with Brevundimonas Substituting the absorbance value of the group into the standard curve, the amount of IAA secreted by Brevundimonas ST3CS3 was 145.6 mg/L (Figure 8). Brevundimonas ST3CS3 synthesized plant hormone IAA with L-tryptophan as a precursor to stimulate plant cell growth and multiplication, effectively absorb water and nutrients, and regulate the life activities of plants at the same time.

Embodiment 5: the growth-promoting experiment of brevundimonas ST3CS3 to tobacco seedling

This embodiment aims to prove the promoting effect of Brevundimonas ST3CS3 on plant growth; with tobacco ( Nicotiana tabacum L.) as the test plant, the experimental process is as follows:

A. Preparation of sterile tobacco seedlings: Randomly select tobacco seeds (MSK326), soak them in ethanol solution with a volume concentration of 75% for 2 minutes, rinse with sterile water for 4 times; then soak them in NaClO solution with an available chlorine concentration of 5% for 1 minute 1. Rinse 5 times with sterile water, place it on sterile filter paper to absorb moisture, and set aside; then put the tray containing Canadian peat and perlite (volume ratio 7:3) into an autoclave (121°C, 15psi , 15min; 3 times), and then put the tobacco seeds into the tray to germinate at 25°C and a relative humidity of 60%. In order to maintain moisture, the plants were poured with distilled water once every 3 days;

B. Preparation of endophytic bacteria inoculant and inactivator: inoculate Brevundimonas ST3CS3 into a 250mL Erlenmeyer flask, and culture it in a constant temperature shaker at 28°C and 180rpm for 15h; divide the bacterial solution into parts A and B , Part A acts as an inoculant for endophytic bacteria,

Part B is sterilized at 121°C and 15psi for 15 minutes to kill endophytes and act as an inactivator;

C. Pot experiment: 30 days after the tobacco seeds were planted, 20 seedlings with the same growth were selected and transplanted into flowerpots (10.5×8.5cm; 1 seedling/pot), each pot containing 600g field soil and perlite (7:3, v/v) and 3 g calcium phosphate. The pots were randomly divided into two groups (Group I and Group II), with 10 pots in each group, and then the endophytic bacteria inoculant and inactivator in step B were sprayed on the leaf surfaces and roots of the seedlings in Group I and Group II, respectively. , each plant was inoculated with 3mL until wet (Group I: E+, ST3CS3 inoculated; Group II: E-, ST3CS3 not inoculated). Inoculate the bacterial solution on the 1st and 9th day after transplantation, and inoculate twice in total. Place the seedlings at room temperature (18-25°C) for natural light cultivation, and pour sterile water every 3 days during the cultivation period, watering 100mL per pot ( It is advisable to irrigate the soil with water without overflowing the bottom of the pot), and closely observe the growth status of the tobacco seedlings in each group during the experiment. To determine bacterial colonization in inoculated plants, 3 plants were randomly collected from the E+ and E- groups 5 days after the last inoculation, respectively, cleaned and surface-sterilized under running water; then, the seedlings were homogenized by grinding, and diluted The suspension was cultured in LB medium to detect the inoculated strain. Re-isolation of the strain from E+ plants, but not from E- plants, confirmed the colonization of Brevundimonas. After culturing for 25 days, the plants were harvested, and the height, dry weight, root length, leaf width, leaf length and chlorophyll content of the plants were measured, as well as the nitrogen, phosphorus and potassium contents in the plants and soil.

The results are shown in Table 1, Table 2, Figures 9 and 10. The results showed that Brevundimonas ST3CS3 had a significant promoting effect on tobacco seedlings, and there were significant differences between the experimental group and the control group (p<0.01, t test). At 25 days, compared with the control, plant height, leaf length, leaf width, aboveground dry weight, underground dry weight, plant nitrogen content and chlorophyll content increased by 43.36%, 27.63%, 25.17%, 56.90%, 74.84%, respectively, 20.20%, 15.06%;

Table 1 Effect of Brevundimonas ST3CS3 on tobacco biomass

Note: Taking a as the standard, b means there is a significant difference with a (p<0.01, t test);

Table 2 Effects of Brevundimonas ST3CS3 on the content of nitrogen, phosphorus and potassium in tobacco and its soil

Note: Taking a as the standard, b means there is a significant difference with a (p<0.01, t test);

The results of the foregoing examples illustrate that the Brevundimonas ST3CS3 isolated and obtained in the present invention has the ability to fix nitrogen, dissolve phosphorus and dissolve potassium, thereby promoting the absorption of nitrogen, phosphorus, and potassium elements by plants. It also has the ability to produce IAA, providing plant hormones to promote plant growth. Pot experiments have verified that inoculation of Brevundimonas ST3CS3 can improve the growth of host plants, and can be widely used in the production of microbial fertilizers to reduce the use and pollution of chemical fertilizers.

sequence listing

<110> Kunming University of Science and Technology

<120> A strain of Brevundimonas ST3CS3 and its application

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 1020

<212> DNA

<213> Stellera chamaejasme L.

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atgtgccctt cgggggaaag atttatcgcc attagagcgg cccgcgtctg attagctagt 180

tggtgaggta acggctcacc aaggcgacga tcagtagctg gtctgagagg atgatcagcc 240

acactgggac tgagacacgg cccggactcc tacgggaggc agcagtgggg aatcttgcgc 300

aatgggcgaa agcctgacgc agccatgccg cgtgaatgat gaaggtctta ggattgtaaa 360

attctttcac cggggacgat aatgacggta cccggagaag aagccccggc taacttcgtg 420

ccagcagccg cggtaatacg aagggggcta gcgttgctcg gaattactgg gcgtaaaggg 480

cgcgtaggcg gatcgttaag tcaggggtga aatcccgggg ctcaacctcg gaattgccct 540

tgatactggc gatcttgagt atgagagagg tatgtggaac tccgagtgta gaggtgaaat 600

tcgtagatat tcggaagaac accagtggcg aaggcgacat actggctcat tactgacgct 660

gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 720

gatgattgct agttgtcggg ctgcatgcag ttcggtgacg cagctaacgc attaagcaat 780

ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg gcccgcacaa 840

gcggtggagc atgtggttta attcgaagca acgcgcagaa ccttaccacc ttttgacatg 900

cctggaccgc cacggagacg tggctttccc ttcggggact aggacacagg tgctgcatgg 960

ctgtcgccag ctcgggcctg ggaatgttgg gttaagtccc gaacgagcgc accctcgcct 1020

Claims (3)

1. Brevundimonas strainBrevundimonasolei) ST3CS3 is a plant endophytic bacterium separated from stellera chamaejasme, and the preservation number of the plant endophytic bacterium in the China general microbiological culture Collection center is CGMCC No.24140.

2. Use of Brevundimonas ST3CS3 according to claim 1 for promoting plant growth, wherein: brevundimonas ST3CS3 has the capabilities of phosphate and potassium dissolving, nitrogen fixing and indoleacetic acid production.

3. The use according to claim 2, characterized in that: brevundimonas ST3CS3 is used for preparing biological microbial agents or fertilizers for promoting plant growth.

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