CN117821332A - Saline-alkali-resistant azotobacter and application thereof - Google Patents
Saline-alkali-resistant azotobacter and application thereof Download PDFInfo
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- CN117821332A CN117821332A CN202410038998.8A CN202410038998A CN117821332A CN 117821332 A CN117821332 A CN 117821332A CN 202410038998 A CN202410038998 A CN 202410038998A CN 117821332 A CN117821332 A CN 117821332A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to saline-alkali resistant azotobacter and application thereof, in particular to streptomyces sp.SJA 2CGMCC No.28000. The strain of the invention has the following comprehensive excellent effects: the strain has stronger salt and alkali resistance, can secrete auxin IAA to promote plant growth, and can promote the chlorophyll content of plants to be improved and has good promotion effect in a saline-alkali environment. It also contains genes related to nitrogen fixation function, and can reduce nitrogen into molecular ammonia nitrogen by utilizing in-vivo nitrogen fixation enzyme. The strain solid preparation can obviously improve various agronomic indexes and chlorophyll content of plants in a saline-alkali environment, has good promotion effect, can be used as a candidate strain for improving a functional microbial inoculum in barren saline-alkali soil, is beneficial to industrial production, has strong stress resistance, can improve soil environment, has nitrogen fixation effect and promotes plant growth, and has wide application prospect.
Description
Technical Field
The invention relates to a saline-alkali resistant and nitrogen-fixing Streptomyces sp and application thereof, belonging to the technical field of biology.
Background
Soil salinization has become a worldwide problem limiting the utilization of cultivated land soil. The physical properties of the salinized soil are deteriorated, such as poor soil structure, dispersion of soil particles and low water conductivity, and at the same time, salt stress usually occurs along with alkali stress, which plays a role in inhibiting or even slowing the growth and development of living animals and plants and microorganisms therein, which are main causes of low productivity of the salinized soil.
Nitrogen is the most critical nutrient element in the plant growth process, but the available nitrogen elements of plants in soil are very limited, and the requirement of high yield of crops cannot be met. The free-living nitrogen fixing bacteria are bacteria which do not need to be symbiotic with plants in soil and independently fix nitrogen, and can convert nitrogen elements in the air into ammonia for plant absorption and utilization. Along with the continuous expansion of the cultivated area of the saline-alkali soil, the saline-alkali soil resource is urgently needed to be improved, and the application of saline-alkali resistant nitrogen fixation microorganisms can improve the plant rhizosphere soil environment, promote plant growth, lose weight and increase efficiency, so that the method is one of the important methods for improving the saline-alkali soil.
The microbial improvement scheme is a scheme with environmental protection benefit and economic benefit in the saline-alkali soil improvement measures. The nitrogen fixation microorganisms play an important role in the nitrogen fixation process of the land ecological system, and some salt-tolerant microorganisms can improve the rhizosphere environment and the physical and chemical properties of the soil of plants, reduce the inhibition of salt on the growth of crops and promote the growth of the plants, so that the screening of microorganisms which can endure extreme habitats and have the functions of nitrogen fixation, phosphorus and potassium dissolution and the like is urgently needed. At present, few reports are applied to research of nitrogen fixation microorganisms suitable for saline-alkali soil, and as the area of saline-alkali soil in China increases year by year, salt-tolerant nitrogen fixation microorganism resources are required to be fully excavated. The research and application of the salt-tolerant nitrogen-fixing microorganism mainly comprises the following three aspects, namely, the preparation of a salt-tolerant microbial fertilizer, wherein the microbial fertilizer is a substance containing living cells or dormant cells of beneficial microorganisms and has the characteristic of promoting the growth and development of plants. Has very wide application prospect. The nitrogen fixation microbial agent has obvious effect on maize growth promotion and has the effect of saving nitrogen fertilizer, and the use of nitrogen fixation microorganisms can improve soil environment, so the nitrogen fixation microbial agent has application for improving saline-alkali soil. The application is also directed to the application as a biological control agent, and can be used for preventing diseases and promoting growth. In conclusion, the method for excavating the salt-tolerant nitrogen-fixing microbial resource has important significance for improving saline-alkali soil and reducing weight and enhancing efficiency.
The research screens salt-tolerant microorganisms capable of fixing nitrogen from extreme saline-alkali soil of Shanxi, carries out strain identification through morphological characteristics and 16srDNA fragment (gene) sequence analysis, researches salt tolerance, alkali resistance and growth promotion characteristics of the microorganisms, verifies nitrogen fixation and growth promotion effects of the microorganisms through potting experiments, and hopefully provides strain resources for developing a composite microbial agent for extreme saline-alkali soil improvement.
Disclosure of Invention
The invention provides a Streptomyces sp. SJA2 resistant to saline and alkaline nitrogen fixation and application thereof, which is Streptomyces sp. SJA2 preserved in China general microbiological culture Collection center of China general microbiological culture Collection center (China general microbiological culture Collection center) for 7 months and 24 days in 2023, wherein the preservation number is: CGMCC No.28000, preservation address: beijing, china.
In some embodiments, SJA2 may be applied to microbial improvement of saline-alkali soil.
In some embodiments, SJA2 may be applied to microbial improvement of saline-alkali soil, and is an improvement of saline-alkali soil in extreme environments.
In some embodiments, SJA2 may be applied to microbial improvement of saline-alkali soil, and is an improvement of saline-alkali soil in extreme environments of salt stress and alkali stress.
In some embodiments, SJA2 may improve plant rhizosphere environment and soil physicochemical properties, reduce inhibition of crop growth by salt, and/or promote plant growth.
In some embodiments, SJA2 may increase plant biological nitrogen fixation.
In some embodiments, SJA2 may increase plant glutamine synthetase and/or nitrate reductase activity.
In some embodiments, SJA2 may promote plant growth.
In some embodiments, SJA2 may secrete auxin IAA to promote plant growth.
In some embodiments, SJA2 may promote increased chlorophyll content of plants in a saline-alkali environment.
In some embodiments, the plant is corn, preferably the corn is corn zhengdan 958.
In some embodiments, the streptomyces SJA2 can be used to prepare a biological fertilizer.
The invention also relates to a biological fertilizer prepared from the Streptomyces sp.SJA2, which can improve various agronomic indexes and chlorophyll content, promote crop yield increase and promote biological nitrogen fixation, and can be used as a functional microbial inoculum for improving barren saline-alkali soil.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention separates a strain of indigenous microorganism with the capability of resisting salt and alkali and fixing nitrogen, which can at least resist the salt and alkali environment with pH9 and NaCl concentration of 8 percent, and does not influence the growth promoting effect of plants under the stress of salt and alkali.
(2) The Streptomyces sp which is selected by the invention and has the capability of resisting salt and alkali and fixing nitrogen has the characteristic of secreting auxin IAA, the IAA concentration can reach 3.49 mug/mL after 48 hours, the effect of promoting the growth is good, and under the salt and alkali environment, the improvement of various agronomic indexes and chlorophyll content of an experimental group is most obvious, and the effect of promoting the growth is best.
(3) The invention collects the rhizosphere soil of the Shanxi saline-alkali soil plant, separates the strain SJA2 with higher saline-alkali resistance and nitrogen fixation capacity from the soil, takes the strain SJA2 as a candidate strain, and can still maintain stable excellent genetic characters after 3 generations of purification.
(4) The screening culture medium selected by the invention is an Abental Bei Modan culture medium, and 1.5% of NaCl is added to simulate a saline-alkali environment, so that the bacterial strain with nitrogen fixation property is screened. In the process of separation and purification, the strain obtained by primary screening is purified and cultured by a nitrogen fixation solid culture medium. The strain with good growth obtained by screening is selected to observe morphological characteristics, and a proper strain is selected as a test strain for subsequent experiments, so that the screening and purifying culture medium is simple and easy to operate.
(5) The Streptomyces sp. SJA2 has the related gene of nitrogen fixation function, can reduce nitrogen into molecular ammoniacal nitrogen by utilizing in-vivo nitrogen fixation enzyme, effectively plays a role in biological nitrogen fixation, and promotes the activity of glutamine synthetase and nitrate reductase.
(6) The salt-tolerant phosphate-solubilizing bacterium SJA2 can be applied to microbial fertilizers or microbial pesticides. After the bacterial liquid SJA2 is applied to the simulated saline-alkali soil, the influence on plant height is most obvious, the average plant height is increased by 3.8% compared with that of a control group, the influence on the leaf width and root length of the plant is obvious, the influence on the leaf width and root length of the plant is increased by 17.6% compared with that of the control group, and the chlorophyll content is increased by 117.92% compared with that of the control group. The salt-tolerant phosphate-solubilizing bacterium SJA2 solid preparation can improve stress resistance, improve soil environment, promote plant growth with nitrogen fixation effect, and facilitate industrial production.
Description of the drawings:
fig. 1: the experimental technical route of the research.
Fig. 2: screening results of the strains a, strain J1 b, strain SJA2c, strain N1 d, strain N2.
Fig. 3: PCR profile of 16SrDNA fragments of Maker and strains J1, SJA2, N1, N2.
Fig. 4: phylogenetic tree of strain J1 constructed based on the 16SrDNA sequence.
Fig. 5: phylogenetic tree of strain SJA2 constructed based on the 16SrDNA sequence.
Fig. 6: phylogenetic tree of strain N1 constructed based on the 16SrDNA sequence.
Fig. 7: phylogenetic tree of strain N2 constructed based on the 16SrDNA sequence.
Fig. 8: and judging the growth condition of each strain based on the OD value of the culture solution, wherein the growth condition shows the temperature resistance of the strain, the black color is the strain J1, the red color is the strain SJA2, the blue color is the strain N1, and the green color is the strain N2.
Fig. 9: the growth conditions of all the strains are judged based on the pH value of the culture solution, and the growth conditions indicate that the strain has alkali resistance, wherein black is the strain J1, red is the strain SJA2, blue is the strain N1 and green is the strain N2.
Fig. 10: the growth conditions of the strains are judged based on the NaCl concentration of the culture solution, so that the salt tolerance of the strains is shown, the black color is the strain J1, the red color is the strain SJA2, the blue color is the strain N1, and the green color is the strain N2.
Fig. 11: IAA concentration standard curve.
Fig. 12a: a color reaction diagram of the supernatant after the strain is centrifuged and the colorimetric solution is added; fig. 12b: plating of the strain onto phosphate solubilizing solid medium.
Fig. 13: colony PCR amplified nitrogen fixation enzyme gene results.
Fig. 14: graph of glutamine synthetase activity measurement results.
Fig. 15: a graph of the results of the nitrate reductase activity measurement.
Fig. 16: corn potting experiments, wherein the left is neutral soil, the right is simulated saline-alkali soil, and the following steps are sequentially carried out from left to right: control group CK, J1 fungus experimental group, SJA2 fungus experimental group, N1 fungus experimental group and N2 fungus experimental group.
Fig. 17: the main agronomic index changes after the potted corn grows for 28 days are plant height, stem thickness, leaf width, root length and biomass growth conditions of overground part and underground part of corn respectively.
Fig. 18: the main agronomic index changes under the saline-alkali environment of the potted corn are plant height, stem thickness, leaf width, root length and biomass growth conditions of the overground part and the underground part of the corn respectively.
Fig. 19: corn pot culture has average chlorophyll content in corn leaf under neutral soil condition and simulated saline-alkali soil condition.
The specific embodiment is as follows:
in order that the above-recited objects, features and advantages of the present invention will be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description. It should be noted that, in the case of no conflict, the embodiments of the present application and the features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, for example, using corn zhengdan 958 for testing and using specific application concentrations, however, the present invention may be practiced otherwise than as described herein, for example, using other application methods or for other plants. Therefore, the scope of the invention is not limited to the specific embodiments disclosed below.
EXAMPLE 1 isolation and purification of Streptomyces sp SJA2
1. Material
The present study uses commercially available maize Zhengdan 958 variety as the test material. Exemplary experimental materials include soil samples, LB solid/liquid medium, abaca Bei Modan medium, nitrogen fixation medium, and the like. There is also an electronic balance; an autoclave; an ultra-clean workbench; a biochemical incubator; an oven; a water bath kettle; shaking table; a PH meter; a refrigerator at 4 ℃; -80 ℃ refrigerator; a micropipette gun; various common strain identification uses experimental instruments, reagents and consumables.
1.1 collecting and preserving soil samples to be tested
Soil samples are respectively collected in rhizosphere soil of mountain and western saline-alkali soil plants. The collected soil sample is put into a collection bag and is carried back to a laboratory, and is preserved in a refrigerator at the temperature of 4 ℃.
1.2 test Medium
(1) LB solid Medium (1L): tryptone 10g, yeast powder 5g, naCl 10g, agar 15g, ddH 2 O constant volume, natural pH 7.0-7.2.
(2) LB liquid Medium (1L): 10g of tryptone, 5g of yeast powder, 10g of NaCl and ddH 2 O constant volume, natural pH 7.0-7.2.
(3) Leptoradix Aristolochiae Bei Modan culture medium (Ashby 1L) KH 2 PO 4 0.2g、MgSO 4 ·7H 2 O 0.2g、NaCl 0.2g、CaCO 3 5.0g, mannitol 10g, caSO 4 ·2H 2 O0.1 g with ddH 2 O constant volume, and regulating pH to 7.0-7.2.
(4) Nitrogen fixation Medium (1L) KH 2 PO 4 0.2g、K 2 HPO 4 0.8g、MgSO 4 ·7H 2 O0.2 g, yeast extract 0.5g, mannitol 20g, caSO 4 ·2H 2 O 0.1g、FeCl 3 Trace amount of ddH 2 O constant volume, and regulating pH to 7.0-7.2.
(5) King's Medium (1L): peptone 20g, K 2 HPO 4 1.725g, glycerol 15mL, mgSO 4 ·7H 2 O1.5 g, tryptophan 0.1g, ddH 2 O constant volume, and regulating pH to 7.0-7.2.
(6) salkowski colorimetric solution: 0.45g FeCl 3 Dissolved in 100mL of perchloric acid.
(7) Phosphate solubilizing solid medium (1L): glucose 10g, yeast powder 0.5g, ca 3 (PO 4 ) 2 5g、(NH 4 ) 2 SO 4 0.5g、MgSO 4 0.1g, naCl 0.2g, agar 15g, ddH 2 O constant volume, and regulating pH to 7.0-7.2.
1.3 major instrumentation
An electronic balance; an autoclave; an ultra-clean workbench; a biochemical incubator; an oven; a water bath kettle; shaking table; a PH meter; a refrigerator at 4 ℃; -80 ℃ refrigerator; a micropipette gun; various common glassware and inoculation tools.
Other reagents and materials: 75% alcohol, carbonate buffer solution, corn seeds, flower pots, etc.
2. Experimental procedure
The whole thought and technical route of the research of the invention are shown in figure 1.
2.1 screening of saline-alkali tolerant Nitrogen fixing microorganisms in soil
1.0g of rhizosphere soil sample was weighed, crushed by a mortar, put into a conical flask containing 50ml of sterile physiological saline (0.9% NaCl), and shaken on a shaking table at a constant temperature of 28℃for 2 hours at a rotation speed of 170 r/min. After 10min of natural sedimentation, 100uL of central supernatant was taken in a centrifuge tube containing 900uL of sterile water to obtain 10 -1 Mixing the soil sample solution uniformly, taking 100uL of the solution with the concentration of 10 -1 Is the soil of (2)The sample solution is put into a centrifuge tube containing 900uL of sterile water to obtain 10 -2 A concentrated soil sample solution. Gradually diluting by concentration gradient method to set concentration gradient to 10 -1 、10 -2 、10 -3 、10 -4 . Each gradient was plated in20 μl of whisker Bei Modan medium and repeated three times. To screen for saline-alkali tolerant nitrogen-fixing microorganisms, 1.5% NaCl was added to the abaca Bei Modan medium to act as a salt stress, and the pH was adjusted to about 8 to simulate the co-presence of saline and alkaline earth under natural conditions.
The coated solid plate was cultured in a biochemical incubator at a constant temperature of 28℃for 3 days, and after 3 days, the colony growth condition on the plate was observed. Colony growth was observed, representing the screening of strains with nitrogen fixation capacity. It was picked and streaked onto new agaric Bei Modan medium, purified once every 3 days, purified 3 times total, and selected for strains with stable genetic traits.
2.2 preservation of saline-alkali tolerant Nitrogen fixing microorganisms in soil
The purified strain is placed in a refrigerator at 4 ℃ for standby. Short-term preservation: the strain with better salt-tolerant nitrogen fixation effect obtained by primary screening is inoculated into a nitrogen fixation solid culture medium, and is stored in a refrigerator at 4 ℃ after being cultured for 3 days at 28 ℃ and needs to be activated and stored again within 14 days. Long-term preservation: inoculating the salt-tolerant nitrogen-fixing strain obtained by primary screening onto LB solid culture medium, culturing at 28deg.C for 2 days, selecting appropriate amount of thallus into 2.5mL bacteria-preserving tube (containing 1.625mL LB liquid culture medium and 0.375mL 80% glycerol mixture), and storing in 3-80deg.C refrigerator of Shanghai university agricultural and biological college A building.
2.3 identification of species of saline-alkali tolerant Nitrogen-fixing microorganism in soil
2.3.1 morphological identification
Inoculating the bacteria to be detected to a nitrogen fixation liquid culture medium, carrying out shaking culture at a constant temperature of 28 ℃ for 24 hours at 170r/min, diluting the bacterial liquid, sucking 100uL, uniformly coating the bacterial liquid on the nitrogen fixation culture medium, and carrying out culture at the temperature of 28 ℃ for 24 hours. Morphological characteristics such as color, shape, texture, swelling state, edge, transparency, etc. of the colonies were observed.
2.3.2 16SrDNA identification
(1) Salt-tolerant azotobacter genome DNA extraction
(2) PCR amplification
(3) Salt-tolerant azotobacter PCR product detection and purification
(4) 16SrDNA sequencing
(5) And (3) strain identification: nucleotide alignment was performed on the sequenced nucleotide sequences using the BLAST function provided by the NCBI database.
The above procedures are routine procedures for those skilled in the art or modifications can be made by those skilled in the art according to the existing procedure manual, and the above extraction and sequencing of 16SrDNA is performed by Shanghai Biotechnology Co.
2.3.3 data processing
Experimental data were processed using origin2019 software and phylogenetic tree was constructed using MEGA11.0 software.
3. Experimental results
In order to obtain the microorganism with the capability of resisting the saline-alkali and fixing nitrogen, the acquired saline-alkali soil sample is prepared into a soil sample solution, diluted and coated on a higher-salinity Abental Bei Modan culture medium, and several strains of bacteria with different forms are found to grow on a flat plate. And (3) separating and purifying the screened azotobacter, wherein the strain grows slowly on an Abental Bei Modan culture medium, and inoculating the strain subjected to the primary screening on a nitrogen fixation culture medium plate for subsequent experiments for culture. Finally, strains with nitrogen fixation capacity are obtained, which are respectively numbered J1, SJA2, N1 and N2, as shown in FIG. 2.
And (3) streaking and culturing the excellent strains J1, SJA2, N1 and N2 obtained by screening on a nitrogen fixation solid culture medium to obtain single colonies, and observing morphological characteristics such as color, edge, shape, texture, swelling state, transparency and the like of the colonies. The morphological characteristics of each strain are shown in Table 1.
TABLE 1 morphological characteristics of strains
The 4 strains obtained by screening are purified and cultured and then sent to Shanghai workers for 16SrDNA sequencing. As shown in FIG. 3, the obtained 16SrDNA sequence was subjected to agarose gel electrophoresis, and all 4 strains showed a specific target fragment of about 1500 bp.
Homology comparison with other 16SrDNA was performed at NCBI website using BLAST database, phylogenetic tree construction was performed by MEGA11 software based on the neighbor-joining method (Neighbor Joining Method), and boottrap was set to 1000.
As a result of 16SrDNA sequencing of the strain J1, 1477bp of the sequence was found, and the strains having high similarity to J1 all belong to Streptomyces sp. Phylogenetic tree was constructed based on adjacency using MEGA11 software, as shown in fig. 4, strain J1 was closest to streptomyces sp. (JX 047066.1) relatedness. Based on morphological features and phylogenetic analysis results of strain J1, strain J1 was identified as Streptomyces sp.
As a result of 16SrDNA sequencing of the strain SJA2, 1427bp of sequence was found, and the strains having high similarity with SJA2 all belong to Streptomyces sp. Phylogenetic tree was constructed based on adjacency using MEGA11 software, as shown in fig. 5, strain SJA2 was closest to streptomyces sp. (MZ 960294.1). By combining morphological characteristics and phylogenetic analysis results of the strain SJA2, the strain SJA2 was identified as Streptomyces sp.
The 16SrDNA sequencing result of strain N1 is 1475bp sequence, and the comparison is made in the database of NCBI website, and the strain with high similarity with N1 belongs to mycelial genus (mycelial fungi sp.). Phylogenetic tree was constructed based on the adjacency method using MEGA11 software, and as shown in fig. 6, the relationship between strain N1 and Myceligenerans xiligouense strain (KF 463140.1) was closest, and strain N1 was assumed to be Myceligenerans xiligouense (chinese name is unknown). By combining morphological characteristics of the strain N1 with phylogenetic analysis results, the strain N1 is identified as a mycelial fungus (myceliophthora sp.) and identified as evidence that more physiological biochemistry is required.
The 16SrDNA sequencing result of strain N2 is 1465bp sequence, and the strains with high similarity to N2 belong to Streptomyces sp. Phylogenetic tree was constructed based on the adjacency method using MEGA11 software, as shown in fig. 7, strain N2 was closest to streptomyces sp. (MG 679512.1) with 100% similarity. By combining morphological characteristics of the strain N2 with phylogenetic analysis results, the strain N2 was identified as Streptomyces sp.
EXAMPLE 2 characterization of saline-alkali-tolerant Azotobacter in soil
1. Experimental procedure
1.1 alkali resistance of saline-alkali-resistant Azotobacter
And respectively inoculating the nitrogen-fixing bacteria obtained by separation and screening into a liquid nitrogen-fixing culture medium, and carrying out shake culture at 200r/min and 37 ℃ for 24 hours to serve as seed bacterial liquid for later use. According to the inoculum size of 5%, respectively inoculating seed bacterial liquid into liquid nitrogen fixation culture medium with pH of 7, 8, 9 and 10 and NaCl content of 0.6%, shake culturing at 37deg.C for 3d at 200r/min, and measuring OD 600 Values.
1.2 salt tolerance of saline-alkali-tolerant Azotobacter
And respectively inoculating the nitrogen-fixing bacteria obtained by separation and screening into a liquid nitrogen-fixing culture medium, and carrying out shake culture at 200r/min and 37 ℃ for 24 hours to serve as seed bacterial liquid for later use. Preparing liquid nitrogen fixation culture medium with pH of 9 and NaCl content of 0%, 2%, 4%, 6% and 8%, inoculating the seed bacterial liquid according to 5% inoculum size, shake culturing at 37deg.C for 3d at 200r/min, and measuring OD 600 Values.
1.3 temperature resistance of saline-alkali resistant azotobacter
Inoculating azotobacter into 5mL liquid LB culture medium according to 5% inoculum size, shake culturing at different temperatures (20deg.C, 40deg.C, 60deg.C) for 24 hr, and determining OD 600 Values.
1.4 determination of growth-promoting Properties of Strain
(1) The content of secreted auxin (IAA) of the strain was determined by Salkowski colorimetric method: the test strains were inoculated into 100mL of King's liquid medium, shaking culture was carried out at 200r/min and 37℃for 2d, the liquid medium was centrifuged to obtain a supernatant, a colorimetric solution was added at a ratio of 1:1, and the absorbance was measured at a wavelength of 530 nm.
Drawing an IAA standard curve: the method comprises the steps of preparing a 3-IAA (3-indoleacetic acid) standard sample, weighing 10mg of indoleacetic acid reference substance, diluting with 50% methanol, fixing the volume to 10mL, diluting with methanol to IAA standard solutions of 10 mug/mL, 20 mug/mL, 30 mug/mL, 40 mug/mL, 50 mug/mL and 100 mug/mL, taking the concentration of the standard substance as an abscissa, taking an OD value as an ordinate, and drawing an IAA concentration standard curve. The IAA concentration secreted by the test strain after two days of culture can be determined against the IAA concentration standard curve.
(2) Phosphorus dissolution test: the strain is inoculated on a phosphate solubilizing solid culture medium, and after the strain is cultured for a period of time at 37 ℃, the strain forms transparent circles around colonies, which is positive.
1.5 bacterial colony PCR detection of bacterial Strain nitrogen fixation functional Gene
N which can not be absorbed and utilized by plants in the air can be absorbed and utilized by nitrogen-fixing microorganisms 2 Conversion to NH for plant uptake 3 The nitrogen-fixing bacteria need catalysis of nitrogen-fixing enzyme for nitrogen-fixing. The nitrogen fixation enzyme generally consists of molybdenum protein and ferritin, wherein the nitrogen fixation enzyme ferritin is encoded by nifH genes, the nifH genes are the most conserved functional genes contained in all nitrogen fixation microorganisms, have higher relativity with 16SrRNA genes in evolution, are often used as the standard of nitrogen fixation microorganism analysis and species classification, and are one of the currently known nitrogen fixation related functional genes with the greatest sequence data.
According to NCBIhttps://www.ncbi.nlm.nih.gov/) The CDS region (the region of the sequence required to encode the protein) of the ferritin nifH gene of the enzyme published in the database was primed using Primer 5.0 software. The primer sequences are shown in Table 2 and were synthesized by Shanghai Biotechnology Co., ltd.
TABLE 2 primer sequences
Colony PCR: using SJA2 genomic DNA as a template, amplification was performed with nifH-F (forward primer)/nifH-R (reverse primer) and with primers, respectively, in a system of 20. Mu.L, as shown in Table 3.
TABLE 3 PCR reaction System
And (3) verifying the nitrogen fixation effect of the bacterial strain: leptoradix Aristolochiae Bei Modan culture medium (Ashby 1L) KH 2 PO 4 0.2g、MgSO 4 ·7H 2 O 0.2g、NaCl 0.2g、CaCO 3 5.0g, mannitol 10g, caSO 4 ·2H 2 O0.1 g with ddH 2 O constant volume, and regulating pH to 7.0-7.2.
Plate scribing: the single colony growing on the LB plate is selected, streaked and inoculated to Abbe's (Ashby) the culture medium is a culture medium lacking combined inorganic or organic nitrogen source, so that the free nitrogen in the air is utilized to synthesize nitrogen-containing organic matters required by the free nitrogen in the air under the condition of guaranteeing the supply of organic carbon source and inorganic nutrition, and most of other various microorganisms have no free nitrogen fixation capacity, thereby achieving the effect of enrichment and separation. The materials used for preparing the culture medium comprise: water, mannitol, KH 2 PO 4 、NaCl、CaSO 4 ·2H 2 O、CaCO 3 Distilled water and agar powder.
1.6 measurement of glutamine synthetase Activity
Glutamine synthetase is one of the key enzymes for ammonia assimilation in organisms, and catalyzes the synthesis of glutamine from ammonium ions and glutamic acid, so that excessive ammonium ions can be prevented from being toxic to organisms, and glutamine is also a main storage and transportation form of ammonia. At ATP and Mg 2+ In the presence of glutamine synthetase catalyzes the synthesis of glutamine from ammonium ions and glutamic acid; glutamine is further converted to gamma-glutamyl hydroxamic acid, forming a red complex with iron under acidic conditions; the complex has a maximum absorption peak at 540 nm.
Extraction buffer: 0.05mol/L Tris-HCl, pH8.0, containing 2mmol/L Mg 2+ 2mmol/L DTT,0.4mol/L sucrose. Tris (Trimethylol) is weighedMethyl amino methane) 1.5295g,0.1245g MgSO4.7H2O, 0.1543g DTT (dithiothreitol) and 34.25g sucrose, after dissolution in deionized water, adjusted to pH8.0 with 0.05mol/L HCl, and finally to a volume of 250ml. Reaction mixture A (0.1 mol/LTris-HCl buffer, pH 7.4): contains 80mmol/L Mg 2+ 20mmol/L glutamic acid sodium salt, 20mmol/L cysteine and 2mmol/L EGTA, and 3.0590g Tris,4.9795gMgSO were weighed 4 ·7H 2 O,0.8628g of sodium glutamate, 0.6057g of cysteine, 0.1920g of EGTA and deionized water are dissolved, and then the pH is adjusted to 7.4 by using 0.1mol/L HCI, and the volume is fixed to 250ml. Reaction mixture B (hydroxylamine hydrochloride, ph 7.4): the reaction mixture A was further added with 80mmol/L hydroxylamine hydrochloride, pH7.4. Color developer (0.2 mol/L TCA,0.37mol/L FeCl) 3 And 0.6mol/L HCl mixture): 3.3176g TCA (trichloroacetic acid), 10.1021g FeCl 3 ·6H 2 O, after deionized water is dissolved, 5ml of concentrated hydrochloric acid is added to fix the volume to 100ml.40mmol/L ATP solution: 0.1210g ATP was dissolved in 5ml deionized water (prepared just prior to use).
Sample processing: inoculating the strain SJA2 into LB liquid medium, shake culturing at 28deg.C at 150r/min, and standing for bacterial liquid OD 600 When the sample is 1, bacteria are collected into a centrifuge tube, and all supernatant liquid is taken out for testing after centrifugation. The weight of the bacterial cells is weighed, 1mL of extraction buffer solution is added every 0.1g, bacteria are crushed by using an ultrasonic cytoclasis instrument in an ice bath, the power is 300W, the ultrasonic waves are carried out for 3s, the interval is 10s, the repeated steps are carried out for 40 times), the bacterial cells are centrifuged for 10min at the temperature of 8000g at the temperature of 4 ℃, the supernatant is taken, and the bacterial cells are placed on ice for measurement. Each sample was replicated 3 times.
The measuring step comprises the following steps: taking a 1.5mL centrifuge tube, adding 400 mu L of reaction mixture A,175 mu L of 40mmol/L ATP solution and 175 mu L of sample into a measuring tube in sequence; each sample was subjected to a control tube, to which 400. Mu.L of reaction mixture B, 175. Mu.L of 40mmol/L ATP solution, and 175. Mu.L of sample were added in this order. CK tube replaces the sample with LB liquid medium. After mixing, the mixture was subjected to a water bath at 25℃for 30 minutes, and 250. Mu.L of a color-developing agent was added. After mixing evenly and standing for 10min, centrifuging at 5000g at normal temperature for 10min, and taking all supernatant to measure the absorbance at 540 nm.
Calculating glutamine synthetase activity unit according to bacteria liquid volume, wherein the unit is defined as OD (optical density) per mL 600 1 bacterial liquid per mL of reaction systemThe absorbance change at 540nm at clock speed of 0.01 was defined as one enzyme activity unit. The formula is as follows:
GS (U/mL) =ΔA×V inverse total ≡ (V×V sample ≡V sample total) ≡0.01≡T.
Δa = assay tube absorbance-control tube absorbance.
V inverse total: the total volume of the reaction system is 1mL; v sample: add sample volume, 0.175mL; sample V total: adding the volume of the extracting solution and mL; t: the reaction time is 30min; v: the volume of bacteria was used, mL.
1.7 determination of nitrate reductase Activity
Nitrate reductase is a key enzyme for converting nitrate nitrogen into ammonia nitrogen in organisms, is also an inducer, has influence on crop yield and quality, can catalyze nitrate to reduce nitrite, and quantitatively generates red azo compounds with p-aminobenzenesulfonic acid (or p-aminobenzenesulfonamide) and a-naphthylamine under acidic conditions. The red azo compound has a maximum absorption peak at 540 nm.
Extraction buffer: 5mmol/L EDTA and 5mmol/L cysteine were dissolved in 0.025mol/L phosphate buffer pH 8.7. The electron donor NADH was 2mg/mL. Standard solution: 10 mu mol/mL sodium nitrite standard solution. Before use, the sodium nitrite standard solution with the concentration of 0.1 mu mol/mL is obtained by diluting the sodium nitrite standard solution with distilled water for 100 times. Sample processing: as above.
The measuring step comprises the following steps: the measurement tube was sequentially filled with 20. Mu.L of the sample extract and 75. Mu.L of 0.1mol/L KNO 3 25 mu LNADH; the control tube was sequentially added with 20. Mu.L of the sample extract, 75. Mu.L of distilled water, 25. Mu.L of NADH, and the CK tube replaced the sample with LB liquid medium; the standard tube was then charged with 20. Mu.L of 0.1. Mu. Mol/mL standard solution, 75. Mu.L of 0.1mol/L KNO 3 25. Mu.L NADH; the blank tube was sequentially filled with 95. Mu.L distilled water, 25. Mu.L ADH. After mixing, the mixture was reacted at 25℃for 30 minutes, and 50. Mu.L of 1% sulfanilamide was immediately added to neutralize the excess NADH to terminate the reaction. After the reaction, 50 mu L of 0.02% a-naphthylamine is added, the mixture is uniformly mixed, the color development is carried out at room temperature for 20min, and the absorbance value of the mixture at 540nm is measured by a spectrophotometer and is respectively recorded as A measurement, A control, A standard and A blank.
Calculating the activity unit of nitrate reductase according to the volume of bacteria liquidDefinition of units is OD per mL 600 The absorbance of the bacterial liquid 1 was changed by 0.01 per mL of the reaction system at 540nm per minute, and the absorbance was defined as one enzyme activity unit.
NR activity (U/mL) =Condition X (A assay-A control)/(A Condition-A blank) ×V sample/(V×V sample/(V extraction/(T×F).
C standard: sodium nitrite standard solution concentration, 0.1 mu mol/mL; v extraction: adding the volume of the extracting solution and mL; t: reaction time, 0.5h; v sample: the added sample volume, 0.02mL; v: using the volume of bacteria liquid, mL; f: sample dilution.
2. Experimental results
And (3) measuring the temperature resistance, alkali resistance and salt resistance of the 4 saline-alkali-resistant nitrogen-fixing bacteria obtained by screening.
2.1 determination of the suitable growth temperature of the Strain
Inoculating 4 saline-alkali-tolerant azotobacter (J1, SJA2, N1 and N2) into a liquid LB culture medium according to the same inoculation amount, and respectively carrying out shake culture at different temperatures for 48 hours. The growth of each strain was judged by OD value of the culture solution, and the experimental results are shown in FIG. 8. Each strain has no high temperature resistance, the optimal temperature is between 20 ℃ and 40 ℃, and the growth amount is obviously reduced after the temperature is higher than 40 ℃.
2.2 determination of alkali resistance of Strain
As shown in the results of FIG. 9, the 4 azotobacter strains have certain alkali resistance and can normally grow in the pH range of 7-9. Strain J1 reached its maximum OD at pH9, with an optimum pH around 9; while the optimum pH of the strains SJA2, N1 and N2 is around 8; when the pH value reaches about 10, the growth amount of 4 azotobacter strains is limited. In addition, the growth amount of the strain SJA2 is higher than that of other strains, the strain reaches the maximum under the condition of pH8, and the strain still keeps higher growth amount under the condition of pH9, so that the alkali resistance is stronger. In the comprehensive view, 4 azotobacter strains have certain alkali resistance, are obviously inhibited when the pH is close to 10, can adapt to extremely alkaline lands, and have stronger alkali resistance than SJA 2.
2.3 determination of salt tolerance of Strain
In the experiment, naCl with the concentration of 0-8% is respectively added into the nitrogen fixation culture medium with the pH value of 9 so as to simulate nutrient-barren saline-alkali soil. As shown in the results of FIG. 10, the 4 azotobacter strains all show a certain degree of salt tolerance, and can maintain a relatively rapid growth and propagation speed under the NaCl condition of 0% -2%. Wherein the salt tolerance of the strains J1 and SJA2 is higher than that of the strains N1 and N2.
2.4 analysis of growth-promoting Properties of Strain
(1) The strain to be tested is inoculated in 100mL of King's liquid culture medium, shaking culture is carried out for 2d at 200r/min and 37 ℃, the liquid culture medium is centrifuged to obtain supernatant, colorimetric solution is added according to the ratio of 1:1, the absorbance is measured at the wavelength of 530nm, and the content of auxin (IAA) secreted by the strain is measured by adopting a Salkowski colorimetric method.
Drawing an IAA standard curve: the method is characterized in that a 3-IAA (3-indoleacetic acid) standard sample is adopted for preparation, 10mg of indoleacetic acid reference substance is weighed, 50% methanol is used for dilution and volume fixing to 10mL, then methanol is used for dilution to IAA standard solutions of 10 mug/mL, 20 mug/mL, 30 mug/mL, 40 mug/mL, 50 mug/mL and 100 mug/mL, the concentration of the standard substance is taken as an abscissa, the OD value is taken as an ordinate, and an IAA concentration standard curve is drawn, as shown in FIG. 11.
After centrifugation of the culture medium, a colorimetric solution was added in a ratio of 1:1, and a clear color reaction was observed as shown in FIG. 12a, indicating that the strain had the property of secreting auxin IAA. Strain J1 can produce IAA 3.12 μg/mL against the IAA concentration standard curve; the strain SJA2 can produce IAA of 3.49 mug/mL, and 4 strains have the characteristic of secreting auxin IAA in combination, wherein the strain SJA2 can produce more IAA than the strain J1.
(2) Phosphorus dissolution test: the bacterial strain is inoculated on a phosphate-dissolving solid culture medium, after the bacterial strain is cultured for a period of time at 37 ℃, the bacterial strain forms transparent rings around the bacterial colony, and as shown in fig. 12b, the salt-tolerant nitrogen-fixing bacteria which are screened do not form transparent rings, are all negative, and do not have phosphate-dissolving capability.
2.5 determination of Nitrogen fixation Gene and measurement of glutamine synthetase, nitrate reductase Activity
The designed specific primer is used for amplifying the genome DNA of the strain, so that a DNA band can be amplified, the SJA2 strain can normally grow in an Ashby nitrogen-free culture medium, and as shown in figure 13, the strain has genes related to nitrogen fixation function, and nitrogen can be reduced into molecular ammonia nitrogen by utilizing the in-vivo nitrogen fixation enzyme.
Glutamine synthetase is one of key enzymes for ammonia assimilation in organisms, and is an important physiological index for measuring nitrogen assimilation level of plants. Nitrate reductase is a key enzyme for converting nitrate nitrogen into ammonia nitrogen in organisms, is positioned at a key position of plant nitrogen metabolism and is related to plant nitrogen absorption and utilization of nitrogen fertilizer, and as shown in fig. 14 and 15, the activity of glutamine synthetase and nitrate reductase of SJA2 strain can reach 2.5U/mL.
EXAMPLE 3 Effect of saline-alkali tolerant Azotobacter in soil on plants
1. Experimental procedure
(1) Preparation of zhengdan 958 corn seeds and some neutral alkaline soil 5 treatments were set up in a sunlight greenhouse: control group (CK, 200mL of sterile water was poured), 4 experimental groups (J1, SJA2, N1, N2, respectively representing 4 azotobacter treatment groups, 200mL of bacterial suspension was poured), 3 replicates, 3 corn seeds were sown per pot (13 cm. Times.13 cm). After emergence, the growth condition of the corn is continuously observed, and a series of agronomic characters such as plant height, stem thickness, root length, maximum leaf width, fresh weight of the overground and underground parts, chlorophyll content (the last 1, 2 and 3 leaves) and the like of the corn are measured after 28 days.
Pretreatment of corn seeds: sterilizing with 75% ethanol surface for 1min, washing with sterile water for several times, and germinating on a culture dish for 3-5d.
Regarding the treatment of bacterial suspensions: culturing 4 strains respectively with LB culture solution to reach bacterial liquid OD 600 0.8, the bacterial liquid is collected and adjusted to 0.5 for standby. 2mL of the mixed bacteria solution was added to 200mL of sterile water and mixed with the soil (about 600 g) in the culture pot, and the control was replaced with sterile water.
(2) And (3) pouring a carbonate buffer solution with pH of 9 and NaCl of 0.6% into experimental soil to simulate an extreme saline-alkali environment. Thereafter, 5 treatments were set up in a solar greenhouse as in the experimental method described above. After 28 days, a series of agronomic traits such as plant height, stem thickness, root length, maximum leaf width, fresh weight of overground and underground parts, chlorophyll content (1, 2 and 3 leaves) and the like of the corn are measured. And detecting nitrogen fixation and promotion effects of the screened salt-tolerant nitrogen fixation bacteria in a saline-alkali environment.
2. Experimental results
The corn potting experiment was performed in a greenhouse for 28 days, and the agronomic traits of the corn of the nitrogen fixing bacteria-applied experimental group were significantly changed compared with the control group not treated with the bacterial suspension, as shown in fig. 16, in order from left to right: control group CK, J1 fungus experimental group, SJA2 fungus experimental group, N1 fungus experimental group and N2 fungus experimental group.
As shown in fig. 17, the main agronomic indicators changed after 28 days of corn growth. The strain with the best comprehensive performance is SJA2 bacteria, the average plant height of corn of an experimental group with the nitrogen fixing bacteria SJA2 poured reaches 53.9cm, and the average plant height is increased by 9.5 percent compared with the average plant height of a control group by 49.2 cm; the average stem thickness of the corn in the SJA2 experimental group reaches 0.8cm, which is increased by 60% compared with the average stem thickness of the corn in the control group by 0.5 cm; the average leaf width of the corn of the SJA2 experimental group reaches 2.2cm, and is increased by 10 percent compared with that of the corn of the control group.
The biomass accumulation is the comprehensive manifestation of the light energy utilization capability and the environment adaptation capability of the corn, and the strains J1, SJA2 and N1 can obviously promote the growth of the overground part and the underground part (root system) of the corn. Among them, the most remarkable effect is SJA2 experimental group, compared with control group, the biomass of the overground part and the underground part is respectively improved by 70.5% and 66.7%, and the difference reaches remarkable level.
And (3) pouring a carbonate buffer solution with pH of 9 and NaCl of 0.6% into experimental soil to simulate an extreme saline-alkali environment. As shown in FIG. 18, the average plant height of the corn of the experimental group on which the azotobacter SJA2 is poured reaches 46.15cm, which is increased by 3.8% compared with the average plant height of 44.75cm of the control group; the average stem thickness of the corn in the SJA2 experimental group reaches 0.60cm, which is increased by 20% compared with the average stem thickness of 0.50cm in the control group; the average leaf width of the corn of the SJA2 experimental group reaches 2.25cm, which is increased by 15.4 percent compared with the average leaf width of the corn of the control group by 1.95 cm; the average maximum root length of the SJA2 experimental group corn is 25.1cm, which is increased by 17.6 percent compared with the average maximum root length of the control group corn which is 21.35 cm. The SJA2 experimental group has obvious effect on increasing the biomass of corn, the fresh weight of the overground part is increased by 42.0% compared with 5.60g of the control group, the fresh weight of the underground part (root system) is increased by 13.8% compared with 3.12g of the control group, and the strain SJA2 can obviously promote the growth of corn in a saline-alkali environment.
Compared with SJA2 strain, the experimental group on which the azotobacter N1 and N2 are poured does not show obvious growth promotion effect in a saline-alkali environment, and various agronomic indexes of corn are not obviously improved compared with a control group, and only root biomass is obviously increased. The biomass of the root system of the corn of the N1 experimental group is increased by 46.5 percent compared with 3.12g of the control group. The root biomass of the corn of the N2 experimental group is increased by 24.7 percent compared with 3.12g of the corn of the control group.
As shown in fig. 19, the average chlorophyll content after 28 days of corn growth was measured, 0.1g of each of the first last, second last and third last leaves of each corn was measured according to the method of the plant chlorophyll extraction kit, and then the average chlorophyll content of corn of each experimental group was calculated.
As shown in the graph, under the condition of neutral soil, the chlorophyll content of corn leaves of an experimental group of pouring azotobacter J1 and SJA2 is obviously increased, the chlorophyll content measured by the SJA2 experimental group is 30.38mg/g, and is improved by 12.56% compared with 26.99mg/g of a control group. The chlorophyll content of corn leaves of the experimental group to which nitrogen-fixing bacteria N1 and N2 are applied is not increased.
Under the condition of saline-alkali soil, the chlorophyll content of corn leaves is obviously reduced, the chlorophyll content of corn leaves in a control group is reduced from 26.99mg/g under the condition of pH7 to 18.25mg/g under the condition of pH9, and the chlorophyll content is reduced by 32.38%. The chlorophyll content of the experimental group of the nitrogen fixing bacteria J1 is 20.64mg/g, which is improved by 13.10% compared with the control group, but is lower than that of the same treatment group under the condition of pH7. The chlorophyll content of the experimental group of the nitrogen-fixing bacteria SJA2 applied by pouring is 39.77mg/g, which is 117.92 percent higher than that of the control group, and even higher than that of the same treatment group under the condition of pH7. The chlorophyll content of the experimental group for pouring nitrogen-fixing bacteria N1 is 37.99mg/g, which is 108.16% higher than that of the control group. The chlorophyll content of the experimental group for pouring nitrogen-fixing bacteria N2 is 27.02mg/g, which is improved by 48.05% compared with the control group. In summary, under the saline-alkali soil condition, the chlorophyll content of the corn leaves is obviously reduced, and the nitrogen-fixing bacteria screened by the experiment can be poured to improve the chlorophyll content of the corn leaves, so that the photosynthetic efficiency of corn is improved, the stress resistance of the corn under the saline-alkali environment is improved, and the nitrogen-fixing bacteria SJA2 has the best effect under the saline-alkali soil condition.
To sum up, in this study, 4 salt-tolerant azotobacter strains were separated and screened from saline-alkali soil, the numbers were J1, SJA2, N1 and N2, the species identification results were that the strains J1, SJA2 and N2 were Streptomyces, and the strain N1 was mycelium. And (3) carrying out the property measurement of temperature resistance, alkali resistance, salt resistance and nitrogen fixation genes on 4 saline-alkali resistance nitrogen fixation bacteria obtained by screening. The temperature-resistant experimental results show that each strain has no high temperature resistance, the optimal temperature is between 20 ℃ and 40 ℃, and the growth is obviously reduced when the temperature is higher than 40 ℃. The alkali-resistant experiment results show that the 4 azotobacter strains have certain alkali resistance, can be obviously inhibited when the pH value is close to 10, can be suitable for extremely alkaline lands, and has stronger alkali-resistant capability of SJA 2. Colony PCR showed that SJA2 contained a nitrogen fixation gene. The experimental result of the potted corn shows that azotobacter J1 and SJA2 have better effect on the promotion of corn, and under the saline-alkali environment, the experimental group corn of the strain SJA2 has the most obvious improvement of various agronomic indexes and chlorophyll content and the best effect on the promotion.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A soil saline-alkali resistant azotobacter is characterized in that the soil saline-alkali resistant azotobacter is Streptomyces sp.SJA 2, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 24 days in 2023, wherein the preservation number is: CGMCC No.28000.
2. A biologic, characterized in that: a method comprising the soil salt-tolerant nitrogen-fixing bacterium of claim 1, a bacterial suspension of the soil salt-tolerant nitrogen-fixing bacterium, a metabolite of the soil salt-tolerant nitrogen-fixing bacterium, a fermentation broth of the soil salt-tolerant nitrogen-fixing bacterium, a filtrate of a cell culture of the soil salt-tolerant nitrogen-fixing bacterium, and/or a mixture of one or more thereof.
3. Use of the soil salt tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 for improving saline-alkali soil.
4. Use of the soil salt and alkali tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 for improving nitrogen fixation capacity of plants.
5. Use of the soil salt tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 for increasing glutamine synthetase activity of plants.
6. Use of the soil salt tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 to increase plant nitrate reductase activity.
7. Use of the soil salt tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 to promote plant growth.
8. Use of the soil salt tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 for promoting secretion of auxin IAA.
9. Use of the soil salt tolerant nitrogen fixing bacterium of claim 1 or the biological agent of claim 2 for increasing chlorophyll content of a plant, preferably the plant is maize zhengdan 958.
10. A biological fertilizer prepared using the soil saline-alkali tolerant nitrogen fixing bacteria of claim 1 or the biological agent of claim 2.
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