CN116200292A - Agrobacterium 1502IPR-08 and application thereof - Google Patents

Abstract

The invention relates to the fields of microbiology and plant growth promoting bacteria, in particular to an Agrobacterium 1502IPR-08 and application thereof. The Agrobacterium 1502IPR-08 provided by the invention has the preservation number of CGMCC No.25883, is gram-negative bacteria, has good nitrogen fixation capability, can convert nitrogen in air into nitrogen which can be utilized by plants, increases soil nutrients, promotes plant growth, can successfully colonize plant rhizosphere soil, increases nitrogen content in the rhizosphere soil, improves glutamine enzyme activity of plant root systems, and has great application potential.

Description

Agrobacterium 1502IPR-08 and application thereof

Technical Field

The invention relates to the fields of microbiology and plant growth promoting bacteria, in particular to an Agrobacterium 1502IPR-08 and application thereof.

Background

Peanut is one of the important oil crops in China, is one of the export foreign exchange-creating bulk agricultural products in China, and plays an important role in promoting national economy.

The nitrogen is one of three nutritional elements necessary for the growth and development of crops, is a main component of protein, and participates in various physiological processes of the growth and development of plants. The growing process of crops requires high nitrogen demands and a large amount of nitrogen is taken away from the soil with the harvest of crops, so that sufficient nitrogen needs to be applied in the agricultural production process. However, farmers often overstock nitrogen fertilizer in pursuit of yield, creating a series of negative problems such as soil hardening, water eutrophication, groundwater pollution, global warming, photochemical smog, nitrate accumulation in drinking water and food, etc., severely hampering sustainable development of agriculture and human health. Therefore, on the premise of ensuring the production requirement of crops, reducing the application of chemical nitrogenous fertilizer is an important requirement for realizing the green development of agriculture.

Molecular nitrogen in the atmosphere is reduced to ammonia, a process known as nitrogen fixation. Without nitrogen fixation, molecular nitrogen in the atmosphere cannot be absorbed and utilized by plants. There are three routes of nitrogen fixation on earth: biological nitrogen fixation, industrial nitrogen fixation (nitrogen is converted into ammonia by high temperature, high pressure and chemical catalysis) and high-energy nitrogen fixation (such as high energy generated by high-altitude instant discharge such as lightning, etc., can combine nitrogen in air with hydrogen in water to form ammonia and nitric acid, and the ammonia and the nitric acid are brought to the ground by rainwater). The total amount of biological nitrogen fixation accounts for about 90% of the total amount of nitrogen fixation on the earth every year, and the biological nitrogen fixation plays a very important role in the nitrogen circulation of the earth. The strain with nitrogen fixation capability can be inoculated to plant seeds or roots to remarkably promote the growth of plants and increase the yield, and can improve the nutrition condition of the plants and biomass by improving soil fertility, antagonizing pathogenic bacteria and the like. When nitrogen is deficient, the plant can move through the root system, so that the contact area between the root system and the soil is increased; and (5) sucking nitrogen-fixing bacteria and colonizing the nitrogen-fixing bacteria to root systems to obtain available nitrogen. According to the characteristics of the nitrogen-fixing microorganism and the relationship with the host, the nitrogen-fixing microorganism can be classified into symbiotic nitrogen fixation, autogenous nitrogen fixation and combined nitrogen fixation. The nitrogen-fixing bacteria and plants exchange the nutrient substances needed by each other through symbiotic and non-symbiotic modes. More and more researches show that the living characteristics of the free-living nitrogen fixing bacteria lead the application range of the free-living nitrogen fixing bacteria to be wider, the free-living nitrogen fixing bacteria do not need to be colonized in plants, and nitrogen can be provided for crops only in rhizosphere soil. In plant rhizosphere soil, the nitrogen fixation efficiency of the free-living nitrogen fixation bacteria is far higher than that of non-rhizosphere soil. The beneficial interaction of the free-living nitrogen fixing bacteria-soil and plants can promote the improvement of nitrogen nutrition of plants and the improvement of crop yield. Therefore, the method for fixing nitrogen by utilizing microorganisms has important significance in reducing the application amount of nitrogen fertilizer, improving the crop yield, reducing the environmental pollution, promoting the sustainable development of agriculture and the like.

Disclosure of Invention

The invention aims to provide an agrobacterium with high-efficiency nitrogen fixation capability, improved plant nitrogen nutrition and increased nitrogen content in plant rhizosphere soil and application thereof.

The invention provides an Agrobacterium 1502IPR-08, wherein the preservation number of the Agrobacterium 1502IPR-08 is CGMCC No.25883.

The present invention provides a fermentation product, the fermentation broth of which comprises the Agrobacterium (sp.) 1502IPR-08.

The invention provides a microbial agent comprising 1502IPR-08 of said Agrobacterium or said fermentation product.

Preferably, the effective viable count of the Agrobacterium (Agrobacterium sp.) 1502IPR-08 in the microbial inoculum is not less than 1×10 ⁹ CFU/mL。

The invention provides application of the microbial inoculum in preparing a plant growth promoter, a plant photosynthesis improver or a plant nitrogen nutrition improver.

The invention provides a biofertilizer comprising said Agrobacterium 1502IPR-08 or said fermentation product or said microbial inoculum.

The invention also provides the use of said Agrobacterium 1502IPR-08 or said fermentation product or said microbial inoculum or said biofertilizer, said use comprising any one of the following methods:

(1) Promote the nitrogen in the air to be converted into nitrogen nutrition which can be effectively absorbed by plants;

(2) Promoting the nitrogen assimilation of plants in soil;

(3) Improving photosynthesis of plants;

(4) Promoting the growth and development of plants.

Preferably, the plants used in methods (1) - (4) comprise peanut and/or maize.

The beneficial effects are that:

the invention separates an Agrobacterium 1502IPR-08 from plant rhizosphere soil, and the preservation number is CGMCC No.25883. The agrobacterium 1502IPR-08 is gram-negative bacteria, has high-efficiency nitrogen fixation capability, can convert nitrogen in air into nitrogen which can be utilized by plants, and increases soil nutrients. Experiments show that the agrobacterium can increase nitrogen content in rhizosphere soil, improve glutamine enzyme activity of plant root systems, promote plant growth and development, and improve nitrogen nutrition of plants. Meanwhile, the plant growth promoter can successfully colonize plant rhizosphere soil, has no pollution and residue in the application process, is biological and environment-friendly, and has great application potential in the field of plant growth promotion.

Biological preservation description:

agrobacterium (Agrobacterium sp.) 1502IPR-08, deposited at China general microbiological culture Collection center (CGMCC, address: north Xielu No. 1, 3, the institute of microorganisms, china academy of sciences, post code 100101) at 10/09 of 2022, classified as Agrobacterium (Agrobacterium sp.) 1502IPR-08, accession number: CGMCC No.25883.

Drawings

In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.

FIG. 1 is a photograph showing the effect of Agrobacterium (Agrobacterium sp.) 1502IPR-08 on peanuts in example 2. The control group is on the left and the Agrobacterium 1502IPR-08 treatment is on the right.

FIG. 2 is a graph comparing the SPAD values of peanut leaves raised by Agrobacterium (Agrobacterium sp.) 1502IPR-08 of example 2.

FIG. 3 is a graph showing the comparison of the increase in peanut plant height by Agrobacterium (Agrobacterium sp.) 1502IPR-08 in example 2.

FIG. 4 is a graph showing the comparison of the improvement of the glutamine synthetase activity of peanut root system by Agrobacterium (Agrobacterium sp.) 1502IPR-08 in example 2.

FIG. 5 is a graph comparing the increase in nitrogen content of peanut rhizosphere soil by Agrobacterium (Agrobacterium sp.) 1502IPR-08 of example 2.

Detailed Description

The present invention provides an Agrobacterium 1502IPR-08.

The Agrobacterium 1502IPR-08 is separated from peanut rhizosphere soil in the middle of a peanut and corn intercropping system, and is named 1502IPR-08, and is called 1502IPR-08 hereinafter. The strain is preserved in China general microbiological culture collection center (CGMCC) at 10-09 of 2022, and has a classification of Agrobacterium (Agrobacterium sp.) 1502IPR-08 and a preservation number of CGMCC No.25883.

The colonies and thalli of 1502IPR-08 strain are characterized by: colony round, central protrusion, yellowish. The microscopic observation shape is characterized by a rod shape, single cells, gram staining negative, and no spores.

Experiments of the invention find that the Agrobacterium 1502IPR-08 has the functions of high-efficiency nitrogen fixation, plant nitrogen nutrition improvement, plant rhizosphere soil nitrogen content increase and plant growth promotion. The growth promoting bacteria preparation can improve the activity of glutamine synthetase in plant root system and raise nitrogen content in plant rhizosphere soil.

Fermentation of the above-described Agrobacterium 1502IPR-08 yields fermentation products that are also within the scope of the present invention.

In a preferred embodiment of the invention, the medium used for fermentation is LB medium: 5g of yeast extract; 10g of peptone; 10g of sodium oxide; distilled water is added to fix the volume to 1000mL; ph=7.0; sterilizing at 121deg.C for 20min; the fermentation conditions are as follows: the temperature is 30 ℃ and the rotating speed is 150-200rpm. The fermentation product has the functions of high-efficiency nitrogen fixation, improvement of plant nitrogen nutrition, increase of nitrogen content in plant rhizosphere soil and promotion of plant growth.

The invention provides a microbial inoculum containing the Agrobacterium 1502IPR-08 or a fermentation product thereof. Preferably, the microbial inoculum is a plant growth promoter, a plant photosynthesis improver or a plant nitrogen nutrition improver.

Preferably, the microbial inoculum contains an effective viable count of Agrobacterium (Agrobacterium sp.) 1502IPR-08 of not less than 1X 10 ⁹ CFU/mL。

The present invention provides biofertilizer containing said Agrobacterium 1502IPR-08 or fermentation product thereof.

The invention provides an application of Agrobacterium 1502IPR-08 or a fermentation product thereof or a microbial inoculum or a biological fertilizer containing the same in promoting nitrogen absorption of plants in soil. The Agrobacterium 1502IPR-08 or its fermentation product or microbial inoculum or biofertilizer containing it can promote the conversion of nitrogen in air to nitrogen nutrients that can be efficiently absorbed by plants.

The invention provides an application of Agrobacterium 1502IPR-08 or a fermentation product thereof or a microbial inoculum or a biological fertilizer containing the same in improving nitrogen fixation capacity of plant roots. The Agrobacterium 1502IPR-08 or fermentation product thereof or microbial inoculum or biofertilizer containing the same can promote nitrogen assimilation in soil by plants.

The invention provides an application of Agrobacterium 1502IPR-08 or a fermentation product thereof or a microbial inoculum or a biological fertilizer containing the same in promoting plant growth. The Agrobacterium 1502IPR-08 or fermentation product thereof or microbial inoculum or biofertilizer containing the same can improve plant photosynthesis; has effect in promoting plant growth.

In the above application, the plant may be peanut and/or corn, preferably peanut. Those skilled in the art will appreciate that the 1502IPR-08 of the present invention can be used for nitrogen absorption and growth promotion in other crops, and also have similar growth promotion as described above.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the materials, reagents and the like used are all commercially available.

The culture medium used in the embodiment of the invention has the following formula unless specified otherwise: LB medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, the volume is fixed to 1L by distilled water, and the pH is adjusted to 7.0.

Example 1 identification of the growth promoting function of Agrobacterium (Agrobacterium sp.) 1502IPR-08

Identification of Agrobacterium (Agrobacterium sp.) 1502IPR-08 Nitrogen fixation Capacity:

and inoculating the strain to be detected in the logarithmic growth phase into an LB liquid culture medium, and shake culturing at 30 ℃ for overnight. After the OD600 value of the strain was measured, the cells were collected by centrifugation, washed 3 times with physiological saline, and suspended in a nitrogen-limited medium, and the OD 600=0.2 was adjusted. Each anaerobic tube is connected with 1mL of bacterial liquid, the anaerobic tube is in a sealed state, air is removed, 2.5mL of fresh acetylene is added by injecting high-purity argon, and the anaerobic tube is subjected to shaking culture at 30 ℃ for 72h. 100 mu L of gas is taken from each anaerobic tube by a microsyringe, and the gas chromatograph is used for measuring the ethylene content.

The enzyme activity calculation formula is as follows:

nitrogen fixation enzyme activity = ethylene peak area on recorder x (test tube gas volume/sample injection amount)/(1 nmol standard ethylene peak area x reaction time)

The results of the bacterial strain nitrogen fixation enzyme activity are shown in table 1. As shown by the determination result of the bacterial strain nitrogen fixation enzyme activity, the Agrobacterium (Agrobacterium sp.) 1502IPR-08 has stronger nitrogen fixation capacity, and reaches 1.24 mu mol/(h.ml).

Identification of the ability of Agrobacterium (Agrobacterium sp.) 1502IPR-08 to phosphate:

single colonies were inoculated into Meng Jinna inorganic phosphorus medium and shake-cultured at 28℃and 180rmp for two days. The bacterial liquid is centrifuged for 10min at 10000rpm, 5mL of bacterial liquid supernatant is taken, and the bacterial liquid is filtered by a filter membrane with a filter hole of 0.22 mu m and diluted. And 5mL of diluent is taken and added into a 50mL volumetric flask, then 5mL of molybdenum-antimony colorimetric solution is added, and deionized water is added to the scale mark. After standing for 30min, the absorbance OD880 at 880nm was measured. And drawing a phosphorus concentration standard curve, and calculating the phosphorus content dissolved out of the bacterial liquid according to the phosphorus standard curve and the OD880 value of the bacterial liquid.

The results of the determination of the phosphate solubilizing ability of the strain are shown in Table 1. As shown by the determination result of the phosphorus-dissolving capability of the strain, the Agrobacterium (Agrobacterium sp.) 1502IPR-08 has stronger phosphorus-dissolving capability, reaching 8.65 mug/mL.

Identification of the ability of Agrobacterium (Agrobacterium sp.) 1502IPR-08 to produce auxin:

tryptophan was sterilized separately and added to LB liquid medium to a final concentration of 100mg/L. Shaking culture was performed for 1 day after single colonies were inoculated, at 28℃and shaking speed of 180rpm. 1mL of bacterial liquid is centrifuged for 10min at 10000rpm, 100 mu L of supernatant is dripped on a white drip plate, a blank culture medium and 50mg/L IAA solution are respectively used as negative and positive controls, an equivalent amount of Salkowski chromogenic liquid is added, and the mixture is placed for 30min at room temperature in a dark place. 1mL of supernatant is taken and evenly mixed with an equal volume of Salkowski chromogenic solution, the chromogenic solution is placed in a water bath at 40 ℃ to react for 30min in a dark place, absorbance at 530nm wavelength is measured by a colorimetric method, and OD600 value of bacterial suspension is measured. And calculating the IAA amount generated by the bacterial liquid per unit volume when the bacterial liquid concentration OD600 = 1 by combining with an IAA concentration standard curve.

The results of the strain for its ability to produce auxin are shown in Table 1. As can be seen from the results of the auxin-producing ability measurement of the strain, agrobacterium (Agrobacterium sp.) 1502IPR-08 has a strong auxin secretion ability, reaching 3.08. Mu.g/mL.

EXAMPLE 2 Agrobacterium 1502IPR-08 activation of soil nitrogen to promote plant nitrogen assimilation and plant growth

The strain 1502IPR-08 of the invention is cultivated in LB liquid medium at 30 ℃ to 10 ⁹ Centrifuging CFU/ml in a high-speed centrifuge at 5000rpm for 10min, pouring out supernatant, adding sterilized water with equal amount to obtain bacterial suspension, and standing.

And (3) transplanting peanuts in a greenhouse of a Beijing university of China agriculture resource and environmental college in 2022, wherein the peanut variety adopts the Luhua 14. Bacterial liquid is added at a rate of 50 ml/strain every week after the first week of thinning, and the bacterial liquid concentration in the bacterial adding treatment: 1X 10 ⁹ CFU/ml, the control group was added with an equal amount of an equal concentration of sterile saline solution. Two sets of treatments each treated 4 pots, 6 biological replicates per pot of peanuts, 3 biological replicates per pot of corn. And (5) collecting samples three months after peanut transplanting. And measuring the SPAD value of the peanut leaf blade by using a SPAD instrument one week before sample collection to characterize the photosynthetic capacity of the peanut leaf blade, and drying the plant sample after sample collection.

The results are shown in FIGS. 1-5. Figures 1-5 show that the application of Agrobacterium 1502IPR-08 significantly increases the nitrogen content of peanut rhizosphere soil, promotes nitrogen assimilation by peanuts, improves nitrogen-deficient yellowing of peanuts, and promotes peanut growth. The main manifestations are: the nitrogen content of the peanut rhizosphere soil of the control group is 0.047g/kg, the nitrogen content of the peanut rhizosphere soil of the agrobacterium-applied treatment group is 0.053g/kg, and compared with the control group, the nitrogen content of the peanut rhizosphere soil after the agrobacterium is applied is improved by 12.77%; the activity of the glutamine synthetase of the peanut root system of the control group is 6.00U/g, the activity of the glutamine synthetase of the peanut root system of the treated group of the application of the agrobacterium is 10.48U/g, and compared with the control group, the activity of the glutamine synthetase of the peanut root system after the application of the agrobacterium is improved by 74.7 percent; the SPAD value of the peanut leaf blade of the control group is 40.05, the SPAD value of the peanut leaf blade of the treated group by applying the agrobacterium is 45.57, compared with the control group, the SPAD value of the peanut leaf blade after applying the agrobacterium is improved by 13.8%, the peanut plant height of the control group is 30.06cm, the peanut plant height of the treated group by applying the agrobacterium is 33.58cm, and compared with the control group, the peanut plant height after applying the agrobacterium is improved by 11.7%, and the remarkable level is achieved. From the above results, it can be concluded that applying Agrobacterium 1502IPR-08 to plant rhizosphere can effectively improve nitrogen nutrition absorption and utilization of plants, improve photosynthetic capacity of plants, and promote plant growth.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims (8)

1. The method is characterized in that the preservation number of the Agrobacterium (Agrobacterium sp.) 1502IPR-08 is CGMCC No.25883.

2. A fermentation product, wherein the fermentation bacteria of the fermentation product comprise Agrobacterium (Agrobacterium sp.) 1502IPR-08 of claim 1.

3. A microbial agent comprising the Agrobacterium (Agrobacterium sp.) 1502IPR-08 of claim 1 or the fermentation product of claim 2.

4. A bacterial agent according to claim 3, wherein the bacterial agent has an effective viable count of Agrobacterium (Agrobacterium sp.) 1502IPR-08 of not less than 1 x 10 ⁹ CFU/mL。

5. Use of the microbial inoculum of claim 3 or 4 for the preparation of a plant growth promoter, a plant photosynthesis improver or a plant nitrogen nutrition improver.

6. Biofertilizer characterized in that it comprises the Agrobacterium (Agrobacterium sp.) 1502IPR-08 of claim 1 or the fermentation product of claim 2 or the microbial inoculum of claim 3 or 4.

7. Use of Agrobacterium (sp.) 1502IPR-08 according to claim 1 or the fermentation product according to claim 2 or the microbial inoculum according to claim 3 or 4 or the biofertilizer according to claim 6, characterized in that the use comprises any one of the following methods:

(1) Promote the nitrogen in the air to be converted into nitrogen nutrition which can be effectively absorbed by plants;

(2) Promoting the nitrogen assimilation of plants in soil;

(3) Improving photosynthesis of plants;

(4) Promoting the growth and development of plants.

8. The use according to claim 7, wherein the plants of (1) - (4) comprise peanut and/or maize.

Images