CN112522138B

CN112522138B - Multifunctional agrobacterium strain and application thereof

Info

Publication number: CN112522138B
Application number: CN202011337379.7A
Authority: CN
Inventors: 戴九兰; 何欢; 张律晴; 白利勇
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-11-25
Filing date: 2020-11-25
Publication date: 2022-06-17
Anticipated expiration: 2040-11-25
Also published as: CN112522138A

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to multifunctional agrobacterium tumefaciens and application thereof. A multifunctional Agrobacterium strain is Agrobacterium sp.SD307, which is preserved in China Center for Type Culture Collection (CCTCC) No. 11/12 in 2020 with the preservation number of CCTCC M2020730. The agrobacterium sp.SD307 strain provided by the invention can promote the seed germination and seedling growth of crops, and has multiple functions, such as nitrogen fixation and heavy metal resistance.

Description

Multifunctional agrobacterium strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to multifunctional agrobacterium tumefaciens and application thereof.

Background

Currently, in the agricultural production process, although the use of chemical fertilizers can significantly improve the yield of crops, in order to meet the demand for increasing the yield of agricultural products, the use amount of chemical fertilizers by farmers is increasingly large, which causes unreasonable use of chemical fertilizers. This agricultural practice of using excessive fertilizers for a long period of time leads to the continuous increase of salt content in the soil, especially the serious problem of nitrate accumulation, which causes a series of soil and environmental problems, such as secondary salinization of the surface layer of the soil, reduction of the organic matter content of the soil, destruction of the soil structure, etc. The problems seriously affect the germination of crop seeds and the growth of seedlings, cause incomplete development of crop root systems, obstruct the absorption of crops to soil nutrients, cause crop yield reduction and nutrition obstacle, and threaten the sustainable development of food safety production and green ecological agriculture in China.

Because some soil functional strains can improve the soil environment through the metabolites and the life activities of the strains, the germination rate and the seedling emergence speed of plant seeds are increased, the absorption of the plants on nutrient elements is promoted, and the plant production and the stress resistance environment growth resistance are increased. Meanwhile, the soil functional bacteria can also avoid a series of soil and water pollution problems caused by excessive use of chemical fertilizers. Therefore, the functional strains become the first choice fertilizer for developing green ecological agriculture.

The agrobacterium has the characteristics of short growth cycle, simple culture conditions, difficult pollution of infectious microbes, wide hosts, strong stability and the like, and can promote crops to generate hairy roots, promote the initial growth and development of crop seedlings, enable the crops to better absorb nutrient elements, improve the structure of soil aggregates, improve the quality of soil environment and achieve the purposes of reducing weight and improving efficiency of agricultural activities. Therefore, the preparation of the microbial inoculum by utilizing the agrobacterium is beneficial to improving the germination rate and the survival rate of crops, and has important significance and potential economic benefit for improving the quality of the crops.

The invention patent application with publication number CN109749950A discloses an agrobacterium microbial inoculum for promoting the rooting of green beans and a preparation method thereof, which discovers that the agrobacterium forms a symbiotic relationship with the green beans and stimulates the branching and root growth of the root of the green beans. The microbial inoculum has a promoting effect on the middle and later growth and development stages of a crop host, but how agrobacterium affects the germination of host seeds and the growth of seedlings at the early stage of the host is not described.

The invention patent application with publication number CN108056111A discloses a microbial agent for promoting nutrient absorption of wheat, which is prepared from Agrobacterium rhizogenes as a main raw material, and finds that the Agrobacterium rhizogenes preparation and wheat form a symbiotic relationship, thereby stimulating the growth of wheat root systems and enhancing the nutrient absorption capacity of wheat. The invention patent application with publication number CN107828700A discloses a microbial preparation for promoting tomato growth, wherein the main raw material of the microbial preparation is agrobacterium rhizogenes, and the microbial preparation is found to promote the growth of tomato roots, ensure the survival rate of plants and improve the quality and yield of tomatoes.

The inventions of the above patent documents relating to agrobacterium microbial agents have the following disadvantages and shortcomings: (1) only the symbiotic relation between the screened agrobacterium and the middle and later growth stages of the crop host is concentrated, and the germination of host seeds, the initial root development of the host and the growth of seedlings are not described; (2) the lack of development of other functions of the agrobacterium itself (such as nitrogen fixation function, seed germination promotion function, adversity growth function and the like) hinders the application range of the agrobacterium in the agricultural production process.

Disclosure of Invention

The invention aims to provide a multifunctional agrobacterium tumefaciens and application thereof aiming at overcoming the defects in the prior art, and aims to solve the following technical problems: (1) the agrobacterium is utilized to promote the germination of crop seeds and the growth of seedlings, and the germination rate and the survival rate of plants are improved; (2) the multifunctional agrobacterium microbial inoculum is developed, and the utilization value of agrobacterium is improved.

In order to achieve the above object, the present invention provides the following technical solutions: a multifunctional Agrobacterium strain is Agrobacterium sp.SD307 strain, which is preserved in China Center for Type Culture Collection (CCTCC) No. 11/12 in 2020 with the preservation number of CCTCC M2020730.

The invention also provides application of the agrobacterium sp.SD307 strain, and the strain can be used for preparing products (microbial inoculum) for promoting crop seed germination and/or promoting crop plant seedling growth, and the like.

The invention further provides a microbial inoculum for promoting the germination of crop seeds and/or the growth of seedlings, wherein the microbial inoculum comprises the agrobacterium sp.SD307 strain.

Further preferably, the OD value of the agrobacterium sp.SD307 strain in the microbial inoculum is 0.8-1.2.

A microbial inoculum with nitrogen fixation function comprises the agrobacterium sp.SD307 strain.

A microbial inoculum for heavy metal contaminated soil comprises the agrobacterium sp.SD307 strain.

Compared with the prior art, the invention has the beneficial effects that:

screened by the present inventionAgrobacterium sp.sd307 (Agrobacterium) The strain has the advantages of short growth period, simple culture condition and the like. And the host of the strain is wide, so the strain has wide application prospect in the agricultural production process.

The agrobacterium sp.SD307 strain provided by the invention has a remarkable promoting effect on the seed germination rate and seedling development of crops (including pakchoi, wheat and soybean), and supplements the comprehensive cognition of agrobacterium on the growth and development of crops.

The agrobacterium sp.SD307 strain provided by the invention can promote crop seed germination and seedling growth, and has multiple functions, such as: the nitrogen fixation function of the strain can be applied to promoting the strain to play a role in weight-reducing and efficiency-increasing agricultural activities; the characteristic of heavy metal Cd resistance of the strain can be applied to agricultural soil polluted by heavy metal Cd.

Drawings

FIG. 1 shows 4 pure colonies obtained by culturing azotobacter;

FIG. 2 shows the state of the nitrogen-fixing Agrobacterium under a 60-fold microscope;

FIG. 3 is a schematic diagram of the tolerance of strain sp.SD307 at different Cd2 + concentrations;

fig. 4 is a schematic diagram of the tolerance curve of strain sp.sd307 to heavy metal Cd;

FIG. 5 shows the effect of bacterial liquid of strain sp.SD307 on the growth of plantlets of pakchoi;

FIG. 6 shows the effect of different concentrations of sp.SD307 bacterial liquid on soybean seedling production;

fig. 7 shows the effect of different concentrations of sp.sd307 bacterial liquid on wheat seedling production.

Detailed Description

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Screening of bacterial strains

1. Soil sample collection

The method comprises the steps of collecting wheat-corn saline-alkali farmland soil with 15 years of planting age in yellow river delta by a five-point sampling method, mixing, filling into a sterilization sealing bag, attaching a sampling land information label, and transporting a soil sample to a laboratory at low temperature.

2. Preparation of Selective Medium

And preparing a nitrogen-free culture medium Ashby for separating and screening the free-living nitrogen-fixing bacteria.

Ashby liquid medium: 2 g of sucrose, 0.04 g of monopotassium phosphate, 0.04 g of magnesium sulfate heptahydrate, 0.04 g of sodium chloride, 0.02 g of calcium sulfate dihydrate, 1 g of calcium carbonate and 200 ml of distilled water, wherein the pH value is 7.0.

Ashby solid medium: to the above 200 ml of Ashby liquid medium was added 4 g of agar to obtain a solid medium.

LB culture medium: 5g of yeast extract, 10g of peptone, 10g of sodium chloride and 1000ml of distilled water.

3. Separation, screening, purification and re-screening of free living nitrogen-fixing bacteria

(1) Separating and screening azotobacter: weighing 10g of farmland soil sample, adding 100 ml of sterile water, oscillating for 30 min on a 120 r/min shaking table, and standing for 1 h; taking the supernatant, sequentially diluting the supernatant into 10-1, 10-2, 10-3 and 10-4 soil diluents, respectively sucking 100 ul of the soil diluents, coating the soil diluents on an Ashby solid culture medium plate, coating 3 dishes at each concentration, inverting, culturing at the constant temperature of 28-30 ℃ for 2-4 days, selecting single colonies with different morphologies in repeated dishes when bacteria appear, transferring the single colonies into 1 ml of Ashby liquid culture medium, and culturing at the temperature of 28-30 ℃.

(2) And (3) purifying nitrogen-fixing bacteria: and (3) streaking a bacterial liquid in the Ashby liquid culture medium on an Ashby solid culture medium plate by using an inoculating loop, culturing at 28-30 ℃, and picking a single colony when a colony appears. The plate streaking separation procedure was repeated to obtain the following 4 different pure colonies of free-living nitrogen fixation, as shown in FIG. 1.

(3) Re-screening:

and (3) measuring the yield of the nitrogen-fixing bacteria IAA: 50 ml of LB liquid medium containing 100 ug/ml L-tryptophan was prepared, and the above purified strain was inoculated at an inoculum size of 1% and shake-cultured at 30 ℃ and 180 rpm for 24 hours. Then, the bacterial liquid is centrifuged at 8000 Xg for 10 min, 4 ml of the centrifuged supernatant is taken and mixed with 4 ml of Salkowski colorimetric liquid, the mixture is reacted in the dark for 30 min, and 4 ml of sterile culture medium is used for reaction under the same treatment to serve as negative control. The Salkowski colorimetric solution preparation used the first colorimetric technique, which was a 7.9 mol/l H2SO4 solution containing 12 g/l FeCl 3. After the reaction, the absorbance of the reaction solution was measured at 530 nm with an ultraviolet spectrophotometer. The reaction was carried out using 0, 5, 10, 15, 20 ug/ml IAA standards under the same treatment to draw a standard curve. The IAA standard curve was repeated 3 times in parallel and each sample 3 times in parallel.

II, identifying strains

1. LB amplification culture is carried out according to the strain which produces the largest IAA content (2.358 ug/ml, and the OD = 0.8-1) in the screened free-living azotobacter, and separation and identification are carried out.

2. Determination of azotobacter 16S: PCR is carried out by taking bacterial liquid of the strain as a template, and the primer is a 16S rDNA full-length amplification universal primer 27F: AGAGTTTGATCCTGGCTCAG, SEQ ID NO. 2; 1492R: GGTTACCTTGTTACGACTT, SEQ ID NO. 3. The PCR amplification system is as follows: bacterial suspension template 1 ul, Taq mix 10 ul, ddH2O 8 ul, primer 0.5 ul each. The reaction conditions are as follows: 94 ℃ for 5 min, 94 ℃ for 30 s, 54 ℃ for 30 s, 72 ℃ for 1 min for 30 s, cycle number 34, 72 ℃ for 10 min. The PCR product was sent to Wuhan Strongylocentron Biotechnology GmbH for 16S sequencing. Obtaining a 16S rDNA sequence successfully spliced by the strain, comparing the sequence with a known sequence in a Nucleotide stacking (nr/nt) database by utilizing a BLAST function on an NCBI website, and analyzing the homology. The results showed that the strain belongs to the Rhizobiaceae (Rhizobiaceae) genus Agrobacterium (Agrobacterium) of the phylum Proteobacteria (Proteobacteria), and the strain was namedAgrobacteriumSD307 and preservation of the strain with 50% glycerol at-80 ℃.

The 16S rDNA sequence of the strain Agrobacterium sp.SD307 is shown in SEQ ID NO. 1.

CATGCGGAGCTTACCATGCAGTCGAACGCCCCGCAGGGGAGTGGCAGACGGGTGAGTAACGCGTGGGAACATACCCTTTCCTGCGGAATAGCTCCGGGAAACTGGAATTAATACCGCATACGCCCTACGGGGGAAAGATTTATCGGGGAAGGATTGGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGATGAAGATAATGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGATATTTAAGTCAGGGGTGAAATCCCGCAGCTCAACTGCGGAACTGCCTTTGATACTGGGTATCTTGAGTATGGAAGAGGTAAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCTCTTGACATTCGGGGTATGGGCATTGGAGACGATGTCCTTCAGTTAGGCTGGCCCCAGAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACAGCGATGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCACGTAGTCAGGAGT。

The strain is preserved in China Center for Type Culture Collection (CCTCC) No. 11 and 12 in 2020, and the preservation number is CCTCC M2020730. The address of the preservation unit is as follows: wuhan university in Wuchang Lojia mountain in Wuhan city, Hubei province.

3. And (3) observing the shape of the nitrogen-fixing agrobacterium: inoculating Agrobacterium sp.SD307 into LB liquid culture medium for overnight culture, sucking 1 ml of bacterial liquid, adding into a sterilized 1.5 ml centrifuge tube, and centrifuging at 4000 r/min at room temperature for 2 min. Sucking out the supernatant, adding sterile water to resuspend and precipitate, immediately coating an inoculating loop of bacterial liquid on a glass slide, naturally drying, dropwise adding a crystal violet staining solution to stain for 10 min, gently washing off the staining solution with distilled water, sucking surface water with absorbent paper, naturally drying, and observing the strain morphology with a microscope, wherein the strain morphology is shown in figure 2.

4. Azotobacteria agrobacterium pair Cd²⁺Tolerance test: activating strain Agrobacterium sp.SD307 with LB culture medium, and preparing into product containing Cd^2＋Solid LB medium at concentrations of 0mg/l, 10 mg/l, 20 mg/l, 50 mg/l, 100mg/l, 200 mg/l, 300 mg/l. Inoculating a loop to obtain a loop bacterial solution in different Cd^2＋The LB solid plate at the concentration was streaked and cultured at 30 ℃ until colonies appeared. The results show that the strain is in Cd^2＋Colonies grew on plates at concentrations of 0mg/l, 10 mg/l, 20 mg/l, 50 mg/l, and followed by Cd^2＋The less the colony grows at elevated concentrations, in Cd^2＋After a concentration of 100mg/l no colonies were growing on the plates. Therefore, the concentration of the heavy metal Cd resistant by the self-generated nitrogen-fixing agrobacterium is determined to be 0-100 mg/l. Then adding Cd^2＋Inoculating liquid LB culture medium with concentration of 0mg/l, 20 mg/l, 40 mg/l, 60 mg/l, 80 mg/l and 100mg/l into activated strain according to 1% inoculum size, performing shake cultivation at 30 ℃ and 180 rpm, taking bacterial liquid every 3 h to determine absorbance value OD600, and drawing a heavy metal Cd tolerance curve of the strain. Three replicates were set up for each culture. As can be seen from FIGS. 3 and 4, the strain is a heavy metal Cd^2＋The tolerance concentration is 100mg/L, which shows that the screened strain has extremely strong heavy metal tolerance and can be applied to heavy metal polluted farmlands.

Application of three sp.SD307 strain

Influence of the sp.SD307 Strain on crop seed Germination and seedling growth

(1) Preparation of Agrobacterium liquid (Agrobacterium sp.sd307): inoculating the agrobacterium strain plate after streak activation culture into LB sterilizing culture medium, and culturing for 12-18 hours at (28-30) DEG C by shaking table, so that the OD of the bacterial liquid is (0.8-1.2), and each dose is (40-60 ml). Wherein the LB sterilization medium is 1000ml, which contains 10g of peptone; 5g of yeast extract; NaCl10 g.

(2) Seed dip dyeing

2.1 seed selection: selecting Chinese cabbage seeds, wheat seeds and soybean seeds which are full, uniform and consistent in particle size and have no damage on the surfaces;

2.2 seed sterilization: soaking the selected seeds in 75% alcohol solution for sterilization for 5-10 min, and then washing with distilled water for 3 times;

2.3 soaking of seeds: the process can be omitted from the Chinese cabbage seed. For wheat and soybean seeds, the seeds are first soaked in distilled water for 5-12h, during which time the water is changed once;

2.4 bacterial liquid seed soaking: placing the seeds in a bacterial solution of agrobacterium sp.SD307, OD = (0.8-1.2) for soaking the seeds for 2-4h, and then fishing out and airing for later use;

2.5 for the Chinese cabbage seeds, a round culture dish is obtained by spreading filter paper. The seeds were spread on moistened filter paper, 30 seeds per dish. Then putting the culture dish into an illumination artificial climate box, and culturing at 25 ℃ in the daytime and 20 ℃ at night;

for wheat and soybean seeds, sprouting and cultivation are carried out by using a seedling tray for sprouting vegetables. The seeds were spread on a seed plate, and 30 seeds were placed in each small plate. Then the cultivation plate is put into an illumination artificial climate box, and the cultivation is carried out at 25 ℃ in the daytime and 20 ℃ in the evening.

2.6 spraying distilled water 2-4 times a day to keep the surface of the seeds moist, and recording the germination rate of the seeds and the length of the seedlings.

TABLE 1 Effect of sp.SD307 inoculum of the present invention on crop seed germination percentage and seedling growth

。

As can be seen from table 1, when the pakchoi seeds are cultivated for 12h, the pakchoi seeds soaked in the bacterial liquid sp.sd307 (OD = 0.8-1.0) group have a better seed germination rate. After the seeds are cultivated for 36 hours in the later period, the germination rate of the seeds reaches 100 percent.

For wheat seeds, wheat seeds soaked in sp.SD307 bacterial liquid OD = (0.8-1.0) group have higher seed germination rate all the time, which is higher than that of other treatments.

For soybean seeds, after the seeds are cultivated for 36 hours, the soybean seeds soaked in sp.SD307 bacterial liquid OD = (0.8-1.0) group have higher seed germination rate.

Influence of the bacterial liquid on growth of seedlings of the pakchoi, the wheat and the soybean:

as shown in fig. 5, after the pakchoi seeds are cultivated for 5 days, the seedling growth height of the seeds soaked in the agrobacterium sp.sd307 bacterial liquid OD = (0.8-1) group is significantly higher than that of the seeds soaked in other treatments.

As shown in fig. 6, after soybean seedlings were cultivated for 5 days, the seedling growth height of the seeds soaked in the group of agrobacterium sp.sd307 strain OD = (0.8-1) was significantly higher than that of the group of sterile water and agrobacterium sp.sd307 strain OD = (0.4-0.5). After soybean seedlings are cultivated for 6 days, the seedling growth height of the seeds soaked in the agrobacterium sp.SD307 bacterial liquid OD = (0.8-1) group is obviously higher than that of the seeds soaked in the agrobacterium sp.SD307 bacterial liquid OD = (0.4-0.5) group.

As shown in fig. 7, after 6 days of wheat seed cultivation, the seedling growth height of the seed soaked in agrobacterium sp.sd307 strain OD = (0.8-1) group is significantly higher than that of the other treatments.

In conclusion, the sp.SD307 strain provided by the invention can be prepared into a microbial inoculum for promoting the germination rate of plant seeds and the survival rate of seedlings. And can be prepared into a microbial inoculum with a nitrogen fixation function, improves the nitrogen fixation capability of crops, and can be applied to weight-reducing and synergistic agricultural activities. In addition, the strain has excellent capacity of tolerating heavy metal Cd, so that the microbial inoculum prepared by the strain can be applied to agricultural soil polluted by the heavy metal Cd to promote the growth of crops.

Sequence listing

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Claims

1. Multifunctional agrobacteria (A)Agrobacteriumsp.) A strain characterized by: the strain is an agrobacterium sp.SD307 strain, is preserved in China Center for Type Culture Collection (CCTCC) No. 12 at 11/2020, and has a preservation number of CCTCC M2020730.

2. Use of a multifunctional agrobacterium strain according to claim 1, characterized in that: the agrobacterium sp.SD307 strain is applied to the preparation of products for promoting the germination of crop seeds and/or promoting the growth of crop plant seedlings.

3. A microbial inoculum for promoting crop seed germination and/or seedling growth, which is characterized in that: the microbial inoculum comprises an agrobacterium sp.sd307 strain as defined in claim 1; the OD value of the agrobacterium sp.SD307 strain in the microbial inoculum is 0.8-1.0.

4. Heavy metal Cd with nitrogen fixation effect^2＋The microbial inoculum in the polluted soil is obtained by the following steps,the method is characterized in that: the microbial inoculum comprises the agrobacterium sp.SD307 strain as claimed in claim 1.