CN110468073B - Stress-resistant nitrogen-fixing slow-growing rhizobium suitable for northeast regions and application thereof - Google Patents
Stress-resistant nitrogen-fixing slow-growing rhizobium suitable for northeast regions and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
- C05D9/02—Other inorganic fertilisers containing trace elements
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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Abstract
The invention discloses stress-resistant nitrogen-fixing slow rhizobium suitable for northeast and application thereof. The invention firstly discloses a Bradyrhizobium (Bradyrhizobium sp.) DBPB1, the preservation number of which in China general microbiological culture Collection center is CGMCC No. 17502. The invention further provides a microbial inoculum containing the slow rhizobium and application thereof. The Bradyrhizobium sp DBPB1 has growth promoting characteristics and strong adaptability, is a biologically safe strain, can obviously increase the characters of peanut plant height, root nodule number, overground fresh weight and the like in different development periods, greatly improves the yield of peanuts, provides a microbial germplasm resource with high-efficiency nitrogen fixation and stable growth promotion for one of main peanut planting areas in China, namely northeast areas, and is favorable for promoting the green, healthy and sustainable development of China agriculture.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to stress-resistant nitrogen-fixing slow rhizobium suitable for northeast and application thereof.
Background
Peanuts are one of the important economic crops and oil legumes in China. Peanuts are planted in various places in China and are mainly distributed in Huang-Huai areas, northeast, Shuangguang areas and other areas, the production of the peanuts in China mainly depends on chemical fertilizers, and the effect of a natural nitrogen fertilizer factory, namely nitrogen fixation by symbiosis of peanut rhizobium agents and the peanuts, is not utilized, so that the peanuts are very painful. The main reasons for excessive use of chemical fertilizers are as follows: 1. the substitute products of the fertilizer fall behind; 2. the fertilization equipment is poor, and the fertilizer loss is large; 3. aiming at leguminous crops, the chemical nitrogen fertilizer is completely relied on, so that great waste is caused; 4. aiming at the technical research and development lag of reducing the application and improving the efficiency of fertilizers of different planting systems, the enhancement of technical integration and the innovation of application modes are urgently needed. Therefore, the development of new green and environment-friendly technologies and new fertilizers such as organic fertilizers and biological fertilizers and the like, and the enhancement of the integrated innovation and application of the technologies are the key points for realizing the reduction and the improvement of the application amount of the fertilizers in China.
Rhizobia is a group of gram-negative bacteria that symbioses with legumes, forms nodules, and fixes nitrogen in the air for plant nutrition. The symbiotic system has the strongest nitrogen fixation capacity. In the root nodule, rhizobia absorbs carbohydrates, mineral salts and water from cortical cells of leguminous plant roots, and grows and multiplies. At the same time, they fix the free nitrogen in the air by the action of fixing nitrogen, and convert it into nitrogen-containing compound which can be used by plant for plant life. In this way, rhizobia forms an interdependent symbiotic relationship with the root. The rhizobia secretes some organic nitrogen to the soil in the life process, and the rhizobia can drop by oneself in the terminal stage of plant's growth to greatly improve the fertility of soil.
Although rhizobia agents have many advantages, nodulation and nitrogen fixation of rhizobia are also affected by many factors of soil environment such as moisture, pH, fertilizers, pesticides, and the like. In order to ensure the nitrogen fixation capability of the rhizobium japonicum with the maximum efficiency, the rhizobium japonicum strains which have the capabilities of being widely suitable for different ecological environments and have strong stress resistance and drug resistance and high-efficiency nitrogen fixation have to be screened so as to be suitable for the national conditions of wide application range, large environmental difference and common use of pesticides, herbicides and bactericides in leguminous plants in China, and relevant research reports in China are not found.
Therefore, it is necessary to develop a screening research work of stress-resistant and efficient nitrogen-fixing strains.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for efficiently fixing nitrogen and promoting plant growth.
In order to solve the problems, the invention firstly provides a slow rhizobium strain.
The Bradyrhizobium of the invention is Bradyrhizobium (Bradyrhizobium sp.) DBPB1, and the preservation number of the Bradyrhizobium is CGMCC No.17502 in the common microorganism center of China Committee for culture Collection of microorganisms.
The 16s rDNA sequence of the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 is shown as sequence 1 in the sequence table.
The Bradyrhizobium sp DBPB1 is stress-resistant and nitrogen-fixing Bradyrhizobium arachidis suitable for northeast regions.
The Bradyrhizobium sp DBPB1 of the invention is negative in hemolytic activity detected by a blood agar plate culture method, which indicates that the Bradyrhizobium sp DBPB1 is harmless to human and livestock.
The Bradyrhizobium sp DBPB1 disclosed by the invention is identified to have various functional characteristics, and mainly comprises the following components: (1) the function of promoting plant growth: promoting the increase of plant height, root nodule number, needle number of fruit needles, fruit number, yield and overground fresh weight of plants in different development periods; (2) stress resistance: the salt tolerance concentration is 4g/100ml (namely the concentration which can tolerate NaCl is 4g/100 ml); acid-resistant pH 6; alkali resistance pH 11; drought resistance (the tolerable concentration is 30g/100ml of PEG 6000, severe drought); (3) drug resistance: can tolerate the maximum using concentration of the bactericide azoxystrobin (250mg/L) and the insecticide imidacloprid (58.3mg/L) which are commonly used in agricultural application.
The invention further provides a microbial inoculum.
The microbial inoculum of the invention contains the Bradyrhizobium sp DBPB 1.
In the microbial inoculum, the microbial inoculum has at least one of the following characteristics:
A1) salt tolerance;
A2) acid resistance;
A3) alkali resistance;
A4) drought resistance;
A5) drug resistance;
A6) fixing nitrogen;
A7) promoting the growth of plants.
In the above-mentioned microbial inoculum, the active component of said microbial inoculum can be the above-mentioned Bradyrhizobium (Bradyrhizobium sp.) DBPB1, the culture of above-mentioned Bradyrhizobium (Bradyrhizobium sp.) DBPB1 or the metabolite of above-mentioned Bradyrhizobium (Bradyrhizobium sp.) DBPB1, and the active component of said microbial inoculum can also contain other biological components or/and non-biological components, and the other active components of said microbial inoculum can be determined by those skilled in the art according to the actual need of said invention.
The microbial inoculum contains active ingredients and auxiliary materials. The auxiliary materials can be selected according to needs.
Among the above-mentioned microbial agents, the culture of the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 is a substance, such as a fermentation broth, in a culture vessel obtained by culturing the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 in a microbial culture medium.
Among the above-mentioned bacterial agents, the metabolite of DBPB1 of the Bradyrhizobium (Bradyrhizobium sp.) can be obtained from the culture of DBPB1 of the Bradyrhizobium (Bradyrhizobium sp.).
In the microbial inoculum, the microbial culture medium is a YMA liquid culture medium.
In one embodiment of the present invention, the microbial inoculum is specifically a liquid microbial inoculum obtained by culturing the Bradyrhizobium (Bradyrhizobium sp) DBPB1 with a YMA liquid medium; the concentration of the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 in the liquid inoculum is 1 × 1010-11cfu/ml。
The invention further discloses application of the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 or the microbial inoculum in preparing products with at least one of the following characteristics:
A1) salt tolerance;
A2) acid resistance;
A3) alkali resistance;
A4) drought resistance;
A5) drug resistance;
A6) fixing nitrogen;
A7) promoting the growth of plants.
In the above application, the product may be a microbial fertilizer.
In the microbial inoculum or the application, the concentration of NaCl resistant to salt tolerance is 4g/100 ml; the acid resistant is tolerant with a pH of 6; the pH at which alkali resistance is tolerated is 11; the concentration of the drought-resistant and tolerant PEG 6000 is 30g/100ml (namely severe drought); the drug resistance is capable of tolerating bactericide azoxystrobin and insecticide imidacloprid which are commonly used in agricultural application, the maximum using concentration of the tolerant azoxystrobin is 250mg/L, and the maximum using concentration of the imidacloprid is 58.3 mg/L.
The invention further provides a using method of the microbial inoculum.
The using method of the microbial inoculum comprises the following steps:
s1) adding trace elements into the bacterial liquid of the Bradyrhizobium (Bradyrhizobium sp.) DBPB 1;
s2) spraying the bacterial liquid added with the trace elements obtained in the step S1) on the surface of the plant seeds, and drying in the shade;
s3) dressing and coating the seeds dried in the shade in the step S2) by using a sodium carboxymethyl cellulose solution (protective agent), and drying in the shade;
s4).
In the method for using the microbial inoculum, in step S1), the bacterial solution of the Bradyrhizobium (Bradyrhizobium sp) DBPB1 can be obtained by culturing a strain mother liquor (the concentration of the strain mother liquor can reach 1 × 10) by adopting a YM liquid culture medium10 -11cfu/m1) is obtained by diluting with clean tap water, and the mixture ratio of the two is 1 volume of strain mother liquor: 2 volumes of clean tap water.
In the using method of the microbial inoculum, in step S1), the trace elements are added into the microbial inoculum in the form of trace element liquid, and the content of each component of the trace element liquid is as follows: h3BO3 2.86g/L，MnSO4 1.81g/L，CuSO4·5H2O 0.80g/L，ZnSO4 0.22g/L，H2MoO40.02g/L, and the solvent is water; the amount of the trace element solution added to the diluted bacterial solution may be 1 μ L of the trace element solution per 3mL of the bacterial solution. Wherein the trace elements act to increase nutrition, enhance bacterial activity and promote nodule formation and nitrogen fixation.
In the method for using the microbial inoculum, in the step S2), the bacterial liquid is used in an amount that the surface of the seeds is wet.
In the method for using the microbial inoculum, in step S3), the sodium carboxymethyl cellulose solution (protective agent) is a sodium carboxymethyl cellulose aqueous solution with a final concentration of 10 g/L. The preparation method comprises the following steps: 10g of sodium carboxymethylcellulose (800-1200 mPas) is dissolved in 1L of deionized water, and the solution is stirred and dissolved in a water bath at 60 ℃ to form a transparent pasty glue solution. The dosage of the 1% sodium carboxymethyl cellulose solution (protective agent) can be 25mL per kg of seeds, and the effect of the protective agent is to keep the attachment of the microbial inoculum and the humidity of the seeds.
In the method for using the microbial inoculum, in the step S4), watering can be further included after the sowing, and the function of the watering is to ensure the humidity necessary for the activity of the strain.
The application of the method for using the microbial inoculum in promoting plant growth is also within the protection scope of the invention.
In the present invention, the plant growth promotion may be embodied as at least one of:
B1) promoting an increase in plant height of said plant at different developmental stages of said plant;
B2) promoting an increase in the number of fruits of the plant at different developmental stages of the plant;
B3) promoting an increase in the number of nodules in the plant;
B4) promoting an increase in the number of entries in the plant;
B5) promoting an above-ground fresh weight gain of said plant;
B6) promoting an increase in yield of said plant;
the different developmental stages are the full-bloom stage (about 60 days after sowing) and/or the mature harvest stage (about 120 days after sowing).
In the application, the plant growth promotion can be embodied as plant growth promotion in nitrogen-stressed soil. In one embodiment of the present invention, the nitrogen-stressed soil is specifically soil in the northeast China area (specifically, the town of the sea in the pear county of the city of quan, jilin province).
Further, the nitrogen stress is that the application amount of a nitrogen fertilizer is 60 percent of the conventional application amount, and the phosphate fertilizer and the potassium fertilizer are not changed. The conventional fertilizing amount is as follows: p (P) is2O5)∶K(K2O)＝9.6Kg·ha-1∶16Kg·ha-1∶12Kg·ha-1. In the present invention, the nitrogen fertilizer is specifically urea.
In the present invention, the plant may be a food crop, in particular a peanut, such as white sand 308.
The invention aims at the current situation of peanut production and soil in the northeast region, measures the environmental adaptability and other abilities of the high-efficiency nitrogen-fixing peanut rhizobia separated from the northeast region, separates and breeds Bradyrhizobium sp DBPB1 from the peanut rhizobia, is suitable for the northeast region, has the growth promoting characteristic, has strong adaptability such as drought resistance, pesticide resistance, acid and alkali resistance and the like, and is a biological safety strain. Under the condition of reducing the application amount of a conventional nitrogen fertilizer by 40 percent, the characters such as peanut plant height, root nodule number, fresh weight on the ground and the like in different development periods can be obviously increased by applying the microbial inoculum in the field compared with a control without applying the microbial inoculum, and the peanut yield is improved by 11.2 percent compared with the conventional fertilization treatment.
The invention provides a microbial germplasm resource with high nitrogen fixation efficiency and stable growth promotion for one of the main planting areas of the Chinese peanuts, namely the northeast area, and is beneficial to promoting the green, healthy and sustainable development of the Chinese agriculture. The strain DBPB1 of the Bradyrhizobium sp can reduce the input of nitrogen fertilizer and the pollution to the environment caused by excessive application of the nitrogen fertilizer, and meets the requirements of sustainable green agricultural development in China and the requirements of low carbon, environmental protection and ecological agriculture. The invention expands the quality resources of the growth-promoting strains of Chinese grain crops and provides a foundation for researching and developing efficient and stable microbial fertilizers and application technologies.
Value information of biological material
The strain name is as follows: bradyrhizobium DBPB1
Latin name: bradyrhizobium sp.DBPB1
And (3) classification and naming: bradyrhizobium sp.
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
The preservation number is: CGMCC NO.17502
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
Drawings
FIG. 1 shows the colony morphology of strain DBPB1 on YMA solid medium.
FIG. 2 is a graph of the gram stain results of strain DBPB 1.
FIG. 3 is a colony plot of strain DBPB1 on acidic solid medium at pH6.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The media used and the preparation thereof used in the following examples are as follows:
TY culture medium (TY solid culture medium and TY liquid culture medium)
Tryptone 5.0g, yeast powder 3.0g, CaCl20.6g, 1L of deionized water, and the pH value is 6.8-7.2; sterilizing at 121 deg.C for 20 min. 15g-20g of agar (solid medium plus).
YMA medium (YMA solid medium and YMA liquid medium)
Mannitol 10.0g, K2HPO4 0.25g，KH2PO4 0.25g，MgSO40.1g, NaCl 0.1g, yeast powder 3.0g, deionized water 1L, pH 6.8-7.0, sterilizing at 121 deg.C for 20 min; 15g-20g of agar (solid medium plus).
Blood agar culture medium
Peptone 18g, yeast powder 1g, NaCl5g, agar 15g-20g, deionized water 1L, pH6.8-7.2. Sterilizing at 121 deg.C for 20min, cooling to 50 deg.C, adding 5% (5ml/100ml) defibrinated sanguis caprae seu ovis, mixing, and making into flat plate.
Salt-containing solid culture medium
Preparing TY solid culture medium, adding 1g, 4g and 8g of NaCl into warm 3 parts of TY solid culture medium respectively, and then pouring the mixture into a flat plate to obtain salt-containing solid culture medium with salt concentration of 1g/100ml, 4g/100ml and 8g/100 ml; sterilizing at 121 deg.C for 20 min.
Acid-base culture medium
Preparing TY solid culture medium, adjusting the pH of 5 parts of TY solid culture medium which is warm after sterilization to 5.0, 6.0, 7.0, 8.0 and 9.0 respectively by using sterile 0.1M HCl and NaOH, and then pouring the medium into a flat plate to obtain acid-base solid culture media with the pH values of 5.0, 6.0, 7.0, 8.0 and 9.0 (wherein, the pH values of 5.0 and 6.0 are acid solid culture media, the pH value of 7.0 is neutral solid culture media, and the pH values of 8.0 and 9.0 are alkaline solid culture media); sterilizing at 121 deg.C for 20 min.
Preparing TY liquid culture medium, adjusting the pH of 5 parts of TY liquid culture medium which is warm after sterilization to 3.0, 4.0, 7.0, 10.0 and 11.0 by using sterile 0.1M HCl and NaOH, and then subpackaging the mixture into sterile test tubes to obtain acid-base liquid culture medium with pH of 3.0, 4.0, 7.0, 10.0 and 11.0 (wherein, pH3.0 and 4.0 are acid liquid culture medium, pH7.0 is neutral liquid culture medium, and pH10.0 and 11.0 is alkaline liquid culture medium); sterilizing at 121 deg.C for 20 min.
Drought culture medium
YMA liquid medium was prepared, and 0g, 10g, 20g and 30g of PEG 6000 were added to 4 portions of the warmed YMA medium, respectively, so that the concentrations of PEG 6000 (in g/100m1) were: 0. 10, 20 and 30, four drought media were obtained with PEG 6000 concentrations of 0g/100ml (positive control), 10g/100ml (mild drought), 20g/100ml (moderate drought) and 30g/100ml (severe drought), respectively, corresponding to water potentials: 0, -0.185, -0.559, -1.122 MPa; sterilizing at 121 deg.C for 20 min.
Pesticide culture medium
Selecting common peanut herbicides, bactericides and insecticides, calculating the maximum dilution multiple and the minimum dilution multiple of each pesticide in practical application according to the dosage of the preparation on a pesticide specification, determining a proper dilution gradient in the interval, quickly injecting the pesticides with different volumes into a sterilized warm TY culture medium, uniformly mixing, and pouring a flat plate to obtain the pesticide solid culture medium with the corresponding dilution gradient.
TABLE 1 concentration of pesticides used and corresponding pesticide content
Example 1 functional analysis of peanut Bradyrhizobium sp DBPB1
First, separation of strain DBPB1
The method collects peanut plants from northeast regions, separates and purifies strains from nodules of the peanut plants, and identifies the strains to obtain the strain DBPB 1.
Strain DBPB1 was cultured in YMA solid medium, colony morphology: the colonies were white, round, smooth and opaque on the surface (FIG. 1), gram-negative, rod-like, and spore-free (FIG. 2).
Second, analysis of Strain DBPB1
1. Security detection
The strain DBPB1 was inoculated into a blood agar plate medium, cultured at 37 ℃ for 7 days, and the presence or absence of a lysoloop was observed. The representative strain has hemolytic activity when a hemolytic cycle appears, and has potential threat to human and livestock, the strain should not be applied to microbial fertilizer; if no hemolytic cycle appears, the representative strain has no hemolytic activity, is a safe strain, and can be used as a microbial fertilizer for application of the strain.
The strain DBPB1 showed no lysoloop on blood agar plates and the hemolytic activity was negative.
2. Stress resistance detection
1) Salt tolerance detection
Will OD600The bacterial liquid of the strain DBPB1 with the value of about 0.3 is respectively inoculated on the saline solid culture media with the salt concentrations of 1g/100ml, 4g/100ml and 8g/100ml, the inoculation amount is 10 mu L, each treatment is repeated three times, the bacterial liquid is placed in an incubator at 28 ℃, and the growth condition of the strain DBPB1 on the saline solid culture media with the concentration gradients is observed and recorded.
The results show that the strain DBPB1 can grow on a saline solid culture medium with the salt concentration of 1g/100ml and 4g/100ml, and does not grow on a saline solid culture medium with the salt concentration of 8g/100ml, which indicates that the maximum salt tolerance concentration of the strain DBPB1 is 4g/100ml (shown in Table 2), and indicates that the salt tolerance of the strain DBPB1 is strong.
2) Acid and alkali resistance detection
Will OD600Respectively inoculating bacterial strains of the strain DBPB1 with the value of about 0.3 to the acid-base solid culture media with the pHs of 5.0, 6.0, 7.0, 8.0 and 9.0, wherein the culture medium with the pHs of 7.0 is a positive control group, the inoculation amount is 10 mu L, each treatment is repeated three times, the culture is carried out in an incubator at 28 ℃, and when the growth condition of the strain DBPB1 of the positive control group is good, the growth condition of the strain DBPB1 on each gradient acid-base solid culture medium is observed and recorded.
Will OD600Inoculating bacterial strain DBPB1 with the value of about 0.6 into acid-base liquid culture media with the pH values of 3.0, 4.0, 7.0, 10.0 and 11.0, wherein the culture medium with the pH value of 7.0 is a positive control group, the inoculation amount is 150 mu L (3% v/v inoculation amount), each treatment is repeated three times, shaking culture is carried out at 28 ℃ and 180r/min for 7d, then uniformly mixing and sampling are carried out, the OD value of the strain DBPB1 in each acid-base liquid culture medium is measured at 600nm, and the growth and reproduction conditions of the strain DBPB1 are evaluated according to the OD value.
The results show that the strain DBPB1 can grow well on an acid-base solid culture medium with the pH value of 6-9 (the growth condition of the strain DBPB1 is shown in figure 3 on the basis of the pH6 acid solid culture medium), the colony growth is similar to that of a positive control group, and the colony does not grow on an acid liquid culture medium and an acid solid culture medium with the pH value of 3-5 (namely OD)6000), absorbance (OD) of bacterial liquid grown on neutral liquid medium and alkaline liquid medium at pH7.0, 10.0 and 11.0600) The values are similar and are respectively 1.75, 1.35 and 1.75, which shows that the strain DBPB1 can grow well in the pH range of 6-11 and has certain acid resistance and strong alkali resistance (shown in Table 2).
3) Drought tolerance detection
Adopting polyethylene glycol (PEG 6000) to adjust water potential to artificially simulate drought conditions for identifying drought-resistant strains. Will OD600The strain DBPB1 with a value of about 0.6 was inoculated into drought medium of 0g/100ml (positive control), 10g/100ml (mild drought), 20g/100ml (moderate drought) and 30g/100ml (severe drought), each treatment was repeated three times, shaking culture was carried out at 28 ℃ and 180r/min for 7d, and the absorbance (OD) of the bacterial liquid was measured600) The average absorbance values were similar, 5.0, 5.2, 4.5 and 4.3, respectively. Strain DBPB1 was able to grow on three drought-strength media and, similar to the positive control, indicated that strain DBPB1 was able to tolerate severe drought, i.e., was able to tolerate PEG 6000 at a concentration of 30g/100ml (see Table 2).
TABLE 2 identification of stress resistance of strain DBPB1
Note: + indicates strain growth.
In conclusion, the stress resistance of the strain DBPB1 is that the salt tolerance concentration is 4g/100ml NaCl; the acid resistance is pH 6; alkali resistance is pH 11; the drought resistance concentration is 30g/100ml PEG 6000, and severe drought is caused.
3. Drug resistance detection
Will OD600The bacterial solution of the strain DBPB1 with the value of about 0.6 was inoculated into the culture dishes containing the pesticide culture medium in Table 1, each treatment was repeated three times, the culture was carried out in a constant temperature incubator at 28 ℃, and the growth of the strain DBPB1 was observed after 7-10 days. The results are shown in table 3, where the strain DBPB1 did not grow on herbicide-containing medium, imidacloprid-containing medium, and azoxystrobin-containing medium and medium-low concentration, but did not grow on medium containing the highest concentration.
TABLE 3 identification of the resistance of strain DBPB1
Note: + indicates strain growth; -means that the strain did not grow.
Taken together, the results show that the resistance of strain DBPB1 is the maximum use concentration that can tolerate the fungicides azoxystrobin (250mg/L) and the insecticide imidacloprid (58.3mg/L) commonly used in agricultural applications.
4. 16s rDNA sequence sequencing identification
Using TreliefTMThe DNA of strain DBPB1 was extracted using the Plant Genomic DNA kit (TsingKe) and using the forward primer 16s rDNA P1 and the reverse primer 16s rDNA P6 (Table 4 and sequence listing)The middle sequence 2 and 3) carrying out PCR amplification on the 16s rDNA sequence to obtain a PCR amplification product; wherein the amplification reaction conditions are as follows: 5min at 95 ℃; 30 cycles of 94 ℃ for 1min, 60 ℃ for 30s and 72 ℃ for 90 s; final extension at 72 ℃ for 10 min. And after the PCR amplification product is qualified, sending the PCR amplification product to the biological medicine science and technology limited company of Meiji Sangge Beijing for sequencing.
TABLE 4 primer information
The double-end sequencing sequence is spliced by DNAMAN software to obtain a 1262bp 16s rDNA sequence, the nucleotide sequence of the sequence is shown as SEQ ID No.1, the obtained 16s rDNA sequence is subjected to BLAST comparison on an NCBI website, and the result shows that the strain has the highest similarity with BradyrhizobiumOTtawaense OO99 and the homology is 100%.
In view of the above colony morphology and sequencing identification of strain DBPB1, strain DBPB1 was determined to be a Bradyrhizobium (Bradyrhizobium sp.). The strain DBPB1 is preserved in China general microbiological culture Collection center (CGMCC) at 29.3.2019, and the preservation number is CGMCC No. 17502.
Example 2 field application study of peanut Bradyrhizobium sp DBPB1
Method for applying Bradyrhizobium sp DBPB1 in field
1. Strain activation and expanded culture: activating slow rhizobium (Bradyrhizobium sp.) DBPB1 strain stored in a refrigerator at-80 deg.C in YMA solid culture medium by streaking, culturing in a 28 deg.C constant temperature incubator, and separating single colony. Picking single colony to YMA liquid culture medium with sterile inoculating loop, performing amplification culture, placing on a shaker at 28 deg.C and 180 rpm, and performing shake culture for about 7 days until the concentration of bacterial liquid is 1 × 1010-11Around cfu/ml (OD)6001.2 or so), obtaining a mother solution of the microbial inoculum;
2. before use, adding clean tap water into the mother liquor of the microbial inoculum to dilute to three times of the volume to obtain bacterial liquid, mixing the bacterial liquid and the bacterial liquid uniformly, then dropwise adding a trace element liquid (the amount of the trace element liquid added into the diluted bacterial liquid is 1 mu L of the trace element liquid added into each 3mL of the bacterial liquid) into the bacterial liquid, and mixing the mixture uniformly to obtain a Bradyrhizobium (Bradyrhizobium sp.) DBPB1 liquid microbial inoculum.
Wherein the contents of the components of the trace element liquid are as follows: h3BO3 2.86g/L，MnSO4 1.81g/L，CuSO4·5H2O 0.80g/L，ZnSO4 0.22g/L，H2MoO40.02g/L, adding the components into a proper amount of water in sequence, dissolving all the components, and then complementing 1L of deionized water.
3. Coating a microbial inoculum: uniformly spraying a DBPB1 liquid microbial inoculum on the surface of peanut seeds until the surface of the seeds is wet, and drying in the shade;
4. coating a protective agent: mixing and coating seeds (25ml/kg) with a sodium carboxymethylcellulose solution (10 g/L sodium carboxymethylcellulose aqueous solution, the preparation method is that 10g sodium carboxymethylcellulose (800-1200 mPa.s) is dissolved in 1L deionized water, and the mixture is stirred and dissolved in a water bath at 60 ℃ to form a transparent pasty glue solution), keeping the attachment and humidity of the microbial inoculum, and drying in the shade;
5. manual or machine seeding: before sowing, making soil moisture and watering or sowing when the humidity is proper after rain, ensuring the necessary humidity of the bacterial strain to be beneficial to survival.
Examples of field applications of Bradyrhizobium sp DBPB1
The test site is arranged in the town of the Shanghai county of pear trees in Siping City of Jilin province; the selected peanut variety is the white sand 308 which is a variety commonly used in the local; the sowing date is 2018, 5 months and 23 days, and watering is carried out before sowing; the test field is divided into two zones: normal fertilization zones (nitrogen fertilizer, phosphate fertilizer and potassium fertilizer are applied at regular fertilization rates) and nitrogen reduction zones (nitrogen reduction is 40% but phosphate fertilizer and potassium fertilizer are applied at regular fertilization rates), each zone has an area of 60 square meters, and each zone is repeated three times. The nitrogen fertilizer is urea (the content of the effective component is 46 percent N), the phosphate fertilizer is diammonium phosphate (the content of the effective component is 18 percent N +46 percent P)2O5) The potassium fertilizer is potassium sulfate (effective component content is 50% K)2O); the conventional fertilizing amount is as follows: p (P) is2O5)∶K(K2O)＝9.6Kg·ha-1∶16Kg·ha-1∶12Kg·ha-1After being uniformly mixed, the fertilizer is used as a base fertilizer to be applied to soil at one time, and the specific application amount is shown in table 5; peanut seeds which are not treated are sown in a normal fertilization area to serve as a positive control group (CK), peanut seeds which are obtained in the first nitrogen-reducing area sowing step and are treated by Bradyrhizobium sp DBPB1 are sown in a nitrogen-reducing area to serve as a treatment group, and the biomass of the peanuts in the blooming period and the biomass of the peanuts in the harvesting period are observed.
As shown in Table 6, the biomass of peanuts in the full-bloom stage is compared with that of a positive control group (CK), the plant height and the fresh weight of the overground part of the plants in the full-bloom stage of the peanuts are obviously improved, and the total needle count (namely the sum of the fruit count and the needle count of the fruits which are not formed) and the fruit count are also improved.
The biomass of the peanuts in the harvest period is shown in the table 7, and compared with the positive control group (CK), the treated group has the advantages that the root nodule number is obviously increased, the plant height is increased, and the fruit number is increased. The peanut yield per mu of the treated group is 463kg, and is increased by about 11.3 percent compared with the positive control group (CK).
TABLE 5 Fertilizer consumption in Normal fertilization zone and fluorine reduction zone per plot
Note: the phosphate fertilizer in the nitrogen reduction treatment in the actual fertilizer dosage is also reduced by 8 percent.
TABLE 6 peanut full bloom biomass (60 days after sowing)
Note: different lower case letter representations are distinguished at the 0.05 level
TABLE 7 peanut harvest time Biomass (120 days after sowing)
Note: the different lower case letter representations differed significantly at the 0.05 level
The field experiment result shows that the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 can improve the biomass, the root nodule number and the total yield of peanut plants in different growth and development stages of peanuts, the growth promoting effect of the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 is obviously higher than that of a positive control group, and the Bradyrhizobium (Bradyrhizobium sp.) DBPB1 can be used for developing stable and efficient microbial fertilizer strain resources and is suitable for areas with similar environmental conditions. Or may not be limited to environmentally similar areas.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> university of agriculture in China
<120> stress-resistant nitrogen-fixing slow rhizobium suitable for northeast and application thereof
<130> GNCFY191573
<160> 3
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1262
<212> DNA
<213> Chroogonium sp (Bradyrhizobium sp.)
<400> 2
gggaacatac cttttggttc ggaacaacac agggaaactt gtgctaatac cggataagcc 60
cttacgggga aagatttatc gccgaaagat tggcccgcgt ctgattagct agttggtgag 120
gtaatggctc accaaggcga cgatcagtag ctggtctgag aggatgatca gccacattgg 180
gactgagaca cggcccaaac tcctacggga ggcagcagtg gggaatattg gacaatgggg 240
gcaaccctga tccagccatg ccgcgtgagt gatgaaggcc ctagggttgt aaagctcttt 300
tgtgcgggaa gataatgacg gtaccgcaag aataagcccc ggctaacttc gtgccagcag 360
ccgcggtaat acgaaggggg ctagcgttgc tcggaatcac tgggcgtaaa gggtgcgtag 420
gcgggtcttt aagtcagggg tgaaatcctg gagctcaact ccagaactgc ctttgatact 480
gaagatcttg agttcgggag aggtgagtgg aactgcgagt gtagaggtga aattcgtaga 540
tattcgcaag aacaccagtg gcgaaggcgg ctcactggcc cgatactgac gctgaggcac 600
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaat 660
gccagccgtt agtgggttta ctcactagtg gcgcagctaa cgctttaagc attccgcctg 720
gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg 780
agcatgtggt ttaattcgac gcaacgcgca gaaccttacc agcccttgac atgtccagga 840
ccggtcgcag agatgtgacc ttctcttcgg agcctggaac acaggtgctg catggctgtc 900
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cccgtcctta 960
gttgctacca tttagttgag cactctaagg agactgccgg tgataagccg cgaggaaggt 1020
ggggatgacg tcaagtcctc atggccctta cgggctgggc tacacacgtg ctacaatggc 1080
ggtgacaatg ggatgctaag gggcgaccct tcgcaaatct caaaaagccg tctcagttcg 1140
gattgggctc tgcaactcga gcccatgaag ttggaatcgc tagtaatcgt ggatcagcac 1200
gccacggtga atacgttccc gggccttgta cacaccgccc gtcacaccat gggagttggt 1260
tt 1262
<210> 2
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agagtttgat cctggctcag aacgaacgct 30
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tacggctacc ttgttacgac ttcacccc 28
Claims (8)
1. A bradyrhizobium which is characterized in that: the Bradyrhizobium is Bradyrhizobium (Bradyrhizobium sp.) DBPB1, and the preservation number of the Bradyrhizobium in China general microbiological culture collection center is CGMCC No. 17502.
2. A microbial inoculum, which is characterized in that: the microbial inoculum comprises the bradyrhizobium of claim 1.
3. Use of the bradyrhizobium of claim 1 or the microbial inoculant of claim 2 in the preparation of a microbial fertilizer having at least one of the following characteristics:
A1) salt tolerance;
A2) acid resistance;
A3) alkali resistance;
A4) drought resistance;
A5) drug resistance;
A6) fixing nitrogen;
A7) promoting the growth of plants.
4. Use according to claim 3, characterized in that: the plant growth promotion is embodied as at least one of:
B1) promoting an increase in plant height of said plant at different developmental stages of said plant;
B2) promoting an increase in the number of fruits of the plant at different developmental stages of the plant;
B3) promoting an increase in the number of nodules in the plant;
B4) promoting an increase in the number of entries in the plant;
B5) promoting an above-ground fresh weight gain of said plant;
B6) promoting an increase in yield of said plant;
the different development stages are full-bloom stages and/or mature harvest stages.
5. Use according to claim 3, characterized in that: the plant is a grain crop.
6. The method for using the bacterial liquid is characterized by comprising the following steps of:
s1) adding trace elements to the bacterial liquid of the bradyrhizobium of claim 1;
s2) spraying the bacterial liquid added with the trace elements obtained in the step S1) on the surface of the plant seeds, and drying in the shade;
s3) dressing and coating the seeds dried in the shade in the step S2) with sodium carboxymethyl cellulose solution, and drying in the shade;
s4).
7. Use according to claim 6, characterized in that: in the step S1), the trace elements are added to the bacterial liquid in the form of a trace element liquid, and the content of each component of the trace element liquid is as follows: h3BO32.86 g/L，MnSO41.81 g/L，CuSO4·5H2O 0.80 g/L，ZnSO40.22 g/L，H2MoO40.02g/L, and the solvent is water;
and/or the sodium carboxymethyl cellulose solution is a sodium carboxymethyl cellulose aqueous solution with the final concentration of 10 g/L.
8. Use according to claim 6 or 7, characterized in that: the plant is a grain crop.
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