CN114410471A - Method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil and culture medium used in method - Google Patents
Method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil and culture medium used in method Download PDFInfo
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 72
- 235000009566 rice Nutrition 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 47
- 239000001963 growth medium Substances 0.000 title claims abstract description 46
- 238000012216 screening Methods 0.000 title claims abstract description 29
- 240000007594 Oryza sativa Species 0.000 title 1
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 32
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 16
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
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- LMSDCGXQALIMLM-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;iron Chemical compound [Fe].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O LMSDCGXQALIMLM-UHFFFAOYSA-N 0.000 claims description 2
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- 238000003113 dilution method Methods 0.000 claims description 2
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- 239000011702 manganese sulphate Substances 0.000 claims description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- 235000015393 sodium molybdate Nutrition 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims 1
- 239000002609 medium Substances 0.000 description 13
- 238000004178 biological nitrogen fixation Methods 0.000 description 6
- 239000000618 nitrogen fertilizer Substances 0.000 description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 6
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- 230000012010 growth Effects 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 229930003756 Vitamin B7 Natural products 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
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- 239000011735 vitamin B7 Substances 0.000 description 2
- 235000011912 vitamin B7 Nutrition 0.000 description 2
- 241000203069 Archaea Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000001680 brushing effect Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- PASHVRUKOFIRIK-UHFFFAOYSA-L calcium sulfate dihydrate Chemical compound O.O.[Ca+2].[O-]S([O-])(=O)=O PASHVRUKOFIRIK-UHFFFAOYSA-L 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
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- 238000012851 eutrophication Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
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- FDEIWTXVNPKYDL-UHFFFAOYSA-N sodium molybdate dihydrate Chemical compound O.O.[Na+].[Na+].[O-][Mo]([O-])(=O)=O FDEIWTXVNPKYDL-UHFFFAOYSA-N 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
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Abstract
The invention discloses a method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil and a corresponding culture medium, wherein the screening method comprises the following steps: preparing a selective culture medium of a non-symbiotic nitrogen-fixing strain of rice rhizosphere soil, and naming the culture medium as an AVM culture medium; obtaining rice rhizosphere soil; separating strains; purifying the strain; extracting DNA of the strain, and carrying out PCR analysis and identification; acetylene reduction activity was measured for nitrogen fixation ability. The method can be used for screening nitrogen-fixing bacteria in the rice rhizosphere soil.
Description
Technical Field
The invention relates to a method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil and a culture medium used by the method.
Background
In recent years, the problem of food safety has become an agricultural problem of major global concern. In order to improve the grain yield, the environmental problems of nitrogen fertilizer utilization rate reduction, soil hardening, water eutrophication and the like caused by excessive use of nitrogen fertilizer are increased suddenly, and the relationship between how to treat the nitrogen fertilizer and agricultural production is a problem to be solved urgently. Biological nitrogen fixation is a natural process for converting nitrogen in the air into active nitrogen by nitrogen-fixing microorganisms under the catalysis of nitrogen-fixing enzyme, and is a main non-artificial input source of the active nitrogen in land and marine ecosystems. In soil microbiota there are a number of species with nitrogen fixing action, typically bacteria or archaea, which are capable of converting atmospheric nitrogen into a form more useful for plant growth. Before the widespread use of nitrogen fertilizers, biological nitrogen fixation was the primary source of active nitrogen input in agricultural production. With the increase of the application amount of the nitrogen fertilizer, people pay less attention to biological nitrogen fixation, however, the biological nitrogen fixation effect in the natural process is a potential way for gradually reducing the application amount of the nitrogen fertilizer. The azotobacter has great significance for improving the agricultural production, and is researched to be applied to the production of fertilizers, so that the fertilizer has bright prospect and has a certain effect on increasing the yield of crops.
The non-symbiotic nitrogen fixation is a main biological nitrogen fixation way in the non-leguminous crop cultivation process and plays a wide role in farmland nitrogen supply. In many cases, symbiotic nitrogen-fixing bacteria are isolated from legume nodules. Non-symbiotic nitrogen-fixing bacteria are nitrogen-fixing bacteria that do not need to enter the organism for nitrogen fixation, as opposed to symbiotic nitrogen-fixing bacteria. Therefore, the non-symbiotic azotobacter is separated from the soil, and the symbiotic azotobacter is separated from the plant root nodule; there are few reports on non-symbiotic azotobacteria. Therefore, a method for screening soil nitrogen-fixing bacterial strains is urgently needed, so that non-symbiotic nitrogen-fixing bacteria can be separated and screened.
At present, a large number of non-symbiotic azotobacter screening culture mediums are reported, such as an Ashby nitrogen-free culture medium, a Waksmen nitrogen-free culture medium and the like. However, there are disadvantages such as low efficiency in screening strains.
Disclosure of Invention
The invention aims to provide a method for screening non-symbiotic nitrogen-fixing strains in rhizosphere soil of rice and a corresponding culture medium.
In order to solve the technical problems, the invention provides a selective culture medium (AVM culture medium) for a non-symbiotic nitrogen-fixing strain of rice soil, which comprises the following components in percentage by weight: 3g/L of beef protein extract, 2.5g/L of DL-malic acid, 2.5g/L of potassium hydroxide, 1.5g/L of potassium dihydrogen phosphate, 1.1g/L of sodium chloride, 1g/L of dipotassium hydrogen phosphate, 0.2g/L of yeast extract, 0.2g/L of magnesium sulfate, 0.2g/L of calcium chloride, 66mg/L of Fe-EDTA, 10mg/L of manganese sulfate, 2mg/L of sodium molybdate, 0.2mg/L of biotin, 0.5mg/L of ammonium chloride, 15g/L of agar, and the balance of water (namely, the water constant volume is 1L), and the pH value is 6.6-7.0.
Biotin refers to vitamin H.
The AVM medium is subsequently sterilized, i.e.sterilized through a 0.22 μm filter. If the preparation is carried out by using sterile water, subsequent sterilization treatment can be omitted.
The invention also provides a screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice, which comprises the following steps:
1) preparing a selective culture medium-AVM culture medium of the non-symbiotic nitrogen-fixing strain of the rice soil:
2) obtaining rice rhizosphere soil;
3) separating strains;
4) purifying the strain;
5) DNA extraction, PCR analysis and identification of the strain.
As an improvement of the screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice, the method also comprises the step 6) of measuring the nitrogen-fixing capacity by acetylene reduction activity.
As a further improvement of the screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice, the step 3) is as follows:
cleaning the rice rhizosphere soil obtained in the step 2), and then separating the strains by adopting a dilution method:
respectively adding 10ml of sterile phosphate buffer solution (PBS buffer solution) into test tubes 1# to 7# and adding 2.0g of washed rice rhizosphere soil into the test tube 1# and uniformly mixing, adding 500ul of soil suspension obtained by taking the test tube 1# into the test tube 2# and uniformly mixing, and repeating the steps until 500ul of soil suspension obtained by taking the test tube 6# is added into the test tube 7# and uniformly mixing;
the sterile phosphate buffer solution is prepared by performing sterile filtration on PBS buffer solution by 2 channels of 0.22 um;
the formula of the PBS buffer solution is as follows: 8g/L of NaCl, 0.2g/L of KCl and KH2PO4:0.27g/L，Na2HPO41.44g/L, and the balance of water (namely, the water is fixed to the volume of 1L);
the following treatments were performed for test tubes # 3 to # 7:
firstly melting 200ml of AVM culture medium, pouring the molten AVM culture medium into a sterile flat plate while the AVM culture medium is hot, after the AVM culture medium is solidified, sucking 200ul of rice rhizosphere soil suspension by using a sterile suction pipe, adding the rice rhizosphere soil suspension into the AVM culture medium flat plate, quickly and uniformly coating the soil suspension in culture dishes by using a coating rod, drying the soil suspension, sealing each culture dish by using a sealing film, and inversely placing the culture dishes in an incubator at 32 ℃ for 2-3 days.
Description of the drawings: in this step, after culturing, the bacteria in the soil suspension grow and propagate from single cells to form colonies, and the growth of the bacteria is observed.
The medium, sterilized water, etc. during the operation were sterilized by conventional autoclaving (103.4kpa, 30 min).
The cleaning mode of the rice rhizosphere soil is as follows: putting the rice rhizosphere soil obtained in the step 2) into a NaCl sterile water solution with the mass concentration of 0.9%, stirring for 4-6 minutes, centrifuging (8000 +/-1000 g for 10 +/-2 min), and collecting the precipitate obtained by centrifuging to serve as the washed rice rhizosphere soil.
Generally, the ratio of the rice rhizosphere soil to the 0.9% NaCl sterile water solution is 1 g/8-12 ml.
The screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice is further improved as follows: the purification of the strain in the step 4) comprises the following steps:
aiming at the bacteria cultured in the step 3), selecting different colonies with better growth vigor and higher growth speed according to colony morphology, taking a single colony of the bacteria by using an inoculating needle, streaking the single colony on an AVM culture medium plate, sealing the culture dish by using a sealing film after streaking, and performing sealed culture at 28 +/-0.5 ℃ until a complete colony visible to naked eyes is obtained (the bacteria start to grow under the culture condition and are generally cultured for 24 hours to obtain the complete colony); obtaining a purified strain;
adding the purified strain into LB liquid culture medium, sealing and culturing for 18 hours, centrifuging (10000 Xg, 10min), discarding supernatant, and taking bottom precipitate obtained by centrifugation as strain seed liquid;
the LB liquid culture medium has the formula: tryptone 2.5g/250mL, yeast extract 1.25g/250mL, sodium chloride 2.5g/250mL, water 250 mL.
The screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice is further improved as follows:
the step 6) is as follows:
inoculating 1ml of the strain seed solution obtained in the step 4) into a container (such as an erlenmeyer flask) containing 100ml of AVM medium, and performing rotary shaking flask (110rpm) sealed culture at 30 +/-0.5 ℃ until the strain grows to the middle exponential phase (about 48 h);
inoculating 1mL of the bacterial liquid which grows to the middle exponential phase into a container containing 10mL of AVM medium, and hermetically culturing at 30 +/-0.5 ℃ until the exponential phase (about 48 hours); then, 10% (volume%) of the headspace gas of the sealed vessel was replaced with acetylene, and sealed again at 30. + -. 0.5 ℃ for 6 hours; the ethylene produced was measured by gas chromatography.
As a further improvement of the screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice, the step 5) is as follows:
firstly, extracting DNA of the strain: carrying out the purification of the strain obtained in the step 4) according to a bacterial genome DNA extraction kit (Solarbio company);
PCR analysis of the strain: using a universal primer to carry out sequencing PCR amplification on the 16s rRNA gene full-length sequence of the strain genome DNA obtained in the step I;
the universal primer is as follows: 27F-GAGTTTGATCCTGGCTCAG, 1492R-TACGGYTACCTTGTTACGACTT
The amplification conditions were as follows:
an amplification system: DNA template 0.5 uL/sample, 2 XTaq PCR MasterMix 12.5uL, universal primer 1uL, double distilled water 10 uL;
mixing, and amplifying in a PCR instrument with the following PCR procedure of pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; keeping the temperature at 72 ℃ for 10 min;
after the PCR is finished, uniformly mixing 2uL reaction solution with 2uL DNA loading buffer, and carrying out agarose gel electrophoresis by taking 3uL DNA marker as a control to detect whether the PCR amplification is successful or not;
when the electrophoresis amplification obtains about 1500bp segments, the success of PCR amplification is judged;
otherwise, when the corresponding fragment is not obtained in the electrophoresis result, judging that the PCR amplification fails;
③ sequencing the DNA of the strain: the strains were sequenced using the universal primers (27F and 1492R) at Sangon; the sequencing results were analyzed using Bioedit and Mega4, and the obtained fasta-format results were subjected to 16S-rRNA bacterial identification on an EZ-biocloud website to confirm whether the bacteria were known or unknown species.
The screening method of the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice is further improved as follows:
in the step 2), the rice is directly pulled up with roots, loose soil around the roots is shaken off, and then the residual soil on the roots is lightly brushed off by a brush to obtain the rice rhizosphere soil.
The invention develops the selective culture medium-AVM culture medium of the non-symbiotic nitrogen-fixing strain of the rice soil, analyzes the proper nutrient composition of the non-symbiotic nitrogen-fixing strain, performs the element proportion of the selective culture medium, improves the content of potassium, redistributes the content proportion of phosphorus, potassium and calcium, and improves the separation and screening efficiency.
The traditional method for collecting the rice rhizosphere soil is a shaking-off method, and has great limitation.
The invention sets a specific strain purification method.
The method can effectively separate and screen the target bacterial strain, and the obtained bacterial strain has high nitrogen fixation efficiency.
In conclusion, the invention can be used for screening nitrogen-fixing bacteria in the rice rhizosphere soil, improves the utilization rate of soil nitrogen, and can be used as potential material for manufacturing high-efficiency nitrogen fertilizer.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows nitrogen-fixing bacteria isolated from rice rhizosphere soil;
in FIG. 1, the left graph shows the morphology of colonies isolated from rice rhizosphere soil I, and the right graph shows the morphology of colonies isolated from rice rhizosphere soil II.
FIG. 2 is a colony morphology after streaking on a plate after purification of bacteria of the isolated Azo-1 to Azo-16 strains; from left to right, sequentially comprises Azo-1 to Azo-4, Azo-5 to Azo-8, Azo-9 to Azo-12 and Azo-13 to Azo-16.
FIG. 3 is a phylogenetic tree diagram of strain Azo-1.
FIG. 4 is a phylogenetic tree diagram of strain Azo-2.
FIG. 5 is a phylogenetic tree diagram of strain Azo-3.
FIG. 6 shows acetylene reduction activity of the strains.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
1) the formula of the selective culture medium (AVM) of the nitrogen-fixing strain in the rice soil is as follows:
in a 1L vessel, the components of Table 1 were added and water (sterile water) was added to bring the volume to 1000 mL.
TABLE 1 Selective Medium VM formulation
Biotin refers to vitamin H.
The AVM medium is subsequently sterilized, i.e.sterilized through a 0.22 μm filter. If the preparation is carried out by using sterile water, subsequent sterilization treatment can be omitted.
2) Collecting the rice rhizosphere soil:
using 371500 of Ningbo city in Zhejiang province, shaking off loose soil of rice rhizosphere by using rice soil in Jinrumu village in Yanxi village in State region as a sample, and using the soil still adhered to the surface of the root as rhizosphere soil, namely, slightly brushing off the residual adhered soil on the root by using a brush and collecting the soil, thereby obtaining the rice rhizosphere soil; the 2 rhizosphere soil samples (I and II) obtained were mixed and then subjected to the following step 3).
3) And (3) separating the strain:
5g of rice rhizosphere soil is put into 50ml of sterile 0.9% NaCl solution and slightly stirred for 5min, then 8000g of the rice rhizosphere soil is centrifuged for 10min, and precipitate obtained by centrifugation is collected to be used as the cleaned rice rhizosphere soil.
Seven test tubes were prepared, 10ml of sterile phosphate buffer solution was added to each test tube, numbered from 1 to 7, and 2.0g of the washed rice rhizosphere soil was added to test tube # 1 and mixed well. And taking 500ul of the soil suspension from the test tube No. 1, adding the soil suspension into the test tube No. 2, uniformly mixing, taking 500ul of the suspension from the test tube No. 2, adding the suspension into the test tube No. 3, uniformly mixing, and the like until 500ul of the suspension is taken out from the test tube No. 6, adding the suspension into the test tube No. 7, and uniformly mixing.
The sterile phosphate buffer solution is prepared by performing sterile filtration on PBS buffer solution through 2 channels of 0.22um, and the formula of the PBS buffer solution is as follows: 8g/L of NaCl, 0.2g/L of KCl and KH2PO4:0.27g/L，Na2HPO41.44g/L, and the balance of water (namely, the water is fixed to the volume of 1L).
The suspension of the rice rhizosphere soil in the test tube 3# to 7# is respectively treated as follows:
adding 200ul of rice rhizosphere soil suspension into a culture dish filled with AVM solid selective medium (about 200ml), uniformly coating the rice rhizosphere soil suspension in the culture dish by using a coating rod, keeping the rice rhizosphere soil suspension dry (sealing each culture dish by using a sealing film), placing the culture dish in an incubator at 32 ℃ for 2-3 days, and observing the growth of bacteria; when the single cell grows and breeds to form a macroscopic colony condition, the culture of the step is finished, so that the culture time is about 2-3 days.
Description of the drawings: and melting 200ml of AVM culture medium, pouring the molten AVM culture medium into a sterile flat plate while the AVM culture medium is hot, and solidifying to obtain the AVM solid selective culture medium.
The results obtained are shown in FIG. 1.
The single colonies obtained in this step were subjected to the following step 4):
4) and (3) purifying the strain:
using an inoculating needle to take the single bacterial colony on an AVM culture medium plate for streaking, sealing a culture dish by using a sealing film after streaking, and carrying out sealed culture at 28 ℃ until the bacteria start to grow until complete bacterial colonies can be seen by naked eyes (the culture time is about 24 hours); obtaining 16 purified strains (FIG. 2);
5) identification of the strains:
extracting the purified strain DNA obtained in the step 4), and performing PCR by using a 16s rRNA universal primer and then sequencing.
The method comprises the following specific steps:
firstly, extracting DNA of the strain: carrying out the purification of the bacterial strain obtained in the step 4) according to a bacterial genome DNA extraction kit purchased from Solarbio company;
PCR analysis of the strain: sequencing and PCR amplifying the 16s rRNA gene full-length sequence of the genomic DNA of the strain obtained in the step (I) by using universal primers (27F-GAGTTTGATCCTGGCTCAG, 1492R-TACGGYTACCTTGTTACGACTT);
the amplification conditions were as follows: DNA template 0.5 uL/sample, 2 XTaq PCR MasterMix 12.5uL, primer 1uL (concentration 15. mu. mol/L), double distilled water 10 uL;
mixing, and amplifying in a PCR instrument with the following PCR procedure of pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; keeping the temperature at 72 ℃ for 10 min;
after the PCR is finished, uniformly mixing 2uL reaction solution with 2uL DNA loading buffer, and carrying out agarose gel electrophoresis by taking 3uL DNA marker as a control to detect whether the PCR amplification is successful or not;
when the 1500bp segment is obtained by electrophoretic amplification, the success of PCR amplification is judged;
otherwise, when the electrophoresis result does not obtain the related segment, judging that the PCR amplification fails;
③ sequencing the DNA of the strain: the strains were sequenced in the Sangon using the universal primers (27F and 1492R). The sequencing results were analyzed using Bioedit and Mega4, and the obtained fasta-format results were subjected to 16S-rRNA bacterial identification on an EZ-biocloud website to confirm whether the bacteria were known or unknown species.
The 3 strains of the target strain obtained were Azo-1, Azo-2 and Azo-3 shown in FIGS. 3 to 5.
6) Acetylene reduction tests were performed on the isolated strains:
the acetylene reduction method is a common method for biological nitrogen fixation research, and is used for detecting that nitrogen-fixing microorganisms have the phenomenon of reducing acetylene into ethylene, and is equivalent to the nitrogen-fixing ability of reducing nitrogen into ammonia in the field of nitrogen fixation research.
The purified strains Azo-1, Azo-2 and Azo-3 were treated as follows:
inoculating a loop of purified strain, adding into LB liquid culture medium (10ml), culturing for 18 hr, centrifuging (10000 Xg, 10min), cleaning supernatant, and collecting the bottom precipitate obtained by centrifuging as strain seed solution;
the LB liquid culture medium has the formula: 2.5g/250mL of tryptone, 1.25g/250mL of yeast extract, 2.5g/250mL of sodium chloride and water to reach the constant volume of 250 mL.
Inoculating 1ml of strain seed solution into an Erlenmeyer flask containing 100ml of AVM liquid culture medium, performing rotary shaking flask (110rpm) sealed culture at 30 ℃, and growing to exponential metaphase (48 h);
inoculating 4mL of the bacterial liquid growing to the middle exponential phase into a sealed container containing 40mL of AVM liquid culture medium, and rotating the shake flask at 30 ℃ to culture in a sealed manner until the exponential phase. After the cultivation, acetylene (10% v/v) was used in place of the gas in the headspace of the sealed vessel, and the cultivation was sealed again at 30 ℃ for 6 hours; the ethylene produced was measured by gas chromatography. As shown in FIG. 6, all of the strains Azo-1, Azo-2, and Azo-3 exhibited acetylene reduction activity, and the strain Azo-2 exhibited the highest acetylene reduction activity.
The formula for the calculation of the ethylene generation rate is: ethylene production Rate (nmol mg)-1min-1) Ethylene concentration (nmol L)-1) X volume of container (L)/[ sealing time (min) × sample weight (mg)]。
The specific formula of the comparative example 1, which is obtained by changing the AVM culture medium into the conventional Ashby culture medium, is as follows: 0.2g/L potassium dihydrogen phosphate, 0.2g/L magnesium sulfate, 0.2g/L sodium chloride, 5.0g/L calcium carbonate, 10.0g/L mannitol, 0.1g/L calcium sulfate, 18.0g/L agar and 6.8-7.0 pH value.
The rice rhizosphere soil described in the above example 1 is repeated by using the culture medium, and in the formula, nitrogen-fixing strains cannot be obtained finally due to the content ratio of the nutrient elements of phosphorus, potassium and calcium.
Comparative example 2, the AVM medium was changed to the following medium: KH (Perkin Elmer)2PO4 0.2g，K2HPO4 0.8g，MgSO4.7H2O 0.2g，CaSO4.2H2O 0.1g，Na2MoO4.2H2O (trace), yeast extract 0.5g, mannitol 20g, FeCl3Trace, 1000ml of distilled water, 15g of agar, pH 7.2.
The rice rhizosphere soil described in example 1 above was repeated using this medium, and no nitrogen-fixing strain could be obtained.
Comparative example 3, the formulation of AVM medium of example 1 was changed to "potassium hydroxide 0.5g/L, potassium dihydrogenphosphate 1.5g/L, dipotassium hydrogenphosphate 1 g/L" to "potassium hydroxide 0.5g/L, potassium dihydrogenphosphate 0.5g/L, dipotassium hydrogenphosphate 1 g/L", and the rest was the same as in example 1.
The rice rhizosphere soil described in example 1 above was repeated using this medium, and no nitrogen-fixing strain could be obtained.
Comparative example 4, the formulation of AVM medium of example 1 was changed to "potassium hydroxide 2.5g/L, potassium dihydrogen phosphate 1.5 g/L" to "potassium hydroxide 1.0g/L, potassium dihydrogen phosphate 1.0 g/L", while eliminating "calcium chloride 0.2 g" from the formulation, and the rest was identical to example 1.
The rice rhizosphere soil described in example 1 above was repeated using this medium, and no nitrogen-fixing strain could be obtained.
Comparative example 5, a rice rhizosphere soil sample was obtained by a shake-off method; the rice rhizosphere soil described in example 1 above was repeated using this medium, and the results were obtained as follows: no nitrogen-fixing strain can be obtained.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Zhejiang university
<120> screening method of non-symbiotic nitrogen-fixing strain of rice rhizosphere soil and culture medium used by same
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tacggytacc ttgttacgac tt 22
Claims (9)
1. The selective culture medium of the non-symbiotic nitrogen-fixing strain of the rice soil is characterized in that:
the formula of the AVM culture medium is as follows: 3g/L of beef protein extract, 2.5g/L of DL-malic acid, 2.5g/L of potassium hydroxide, 1.5g/L of monopotassium phosphate, 1.1g/L of sodium chloride, 1g/L of dipotassium phosphate, 0.2g/L of yeast extract, 0.2g/L of magnesium sulfate, 0.2g/L of calcium chloride, 66mg/L of Fe-EDTA, 10mg/L of manganese sulfate, 2mg/L of sodium molybdate, 0.2mg/L of biotin, 0.5mg/L of ammonium chloride, 15g/L of agar, the balance of water, and the pH value of the beef extract is 6.6-7.0.
2. The screening method of the non-symbiotic nitrogen-fixing strain of the rice rhizosphere soil is characterized by comprising the following steps:
1) preparing a selective culture medium of a non-symbiotic nitrogen-fixing strain of rice rhizosphere soil, and naming the culture medium as an AVM culture medium;
2) obtaining rice rhizosphere soil;
3) separating strains;
4) purifying the strain;
5) DNA extraction, PCR analysis and identification of the strain.
3. The method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil according to claim 2, wherein the method comprises the following steps: further comprises the acetylene reduction activity of the step 6) to measure the nitrogen fixation capacity.
4. The method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil according to claim 3, wherein the step 3) comprises:
cleaning the rice rhizosphere soil obtained in the step 2), and then separating the strains by adopting a dilution method:
respectively adding 10ml of sterile phosphate buffer solution into test tubes 1# to 7#, adding 2.0g of washed rice rhizosphere soil into the test tube 1# and uniformly mixing, adding 500ul of soil suspension obtained by taking the test tube 1# into the test tube 2# and uniformly mixing, and then repeating the steps until 500ul of soil suspension obtained by taking the test tube 6# is added into the test tube 7# and uniformly mixing;
the following treatments were performed for test tubes # 3 to # 7:
firstly melting 200ml of AVM culture medium, pouring the molten AVM culture medium into a sterile flat plate while the AVM culture medium is hot, after the AVM culture medium is solidified, sucking 200ul of rice rhizosphere soil suspension by using a sterile suction pipe, adding the rice rhizosphere soil suspension into the AVM culture medium flat plate, uniformly coating the soil suspension in culture dishes by using a coating rod, drying the soil suspension, sealing each culture dish by using a sealing film, and inversely placing the culture dishes in an incubator at 32 ℃ for 2-3 days.
5. The method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil according to claim 4, wherein the method comprises the following steps:
the sterile phosphate buffer solution is prepared by carrying out sterile filtration on PBS buffer solution for 2 times by 0.22 mu m;
the formula of the PBS buffer solution is as follows: NaCl 8g/L, KCl 0.2g/L, KH2PO4 0.27g/L，Na2HPO41.44g/L, and the balance of water.
6. The method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil according to any one of claims 2 to 5, wherein the method comprises the following steps:
the purification of the strain in the step 4) comprises the following steps:
aiming at the bacteria cultured in the step 3), taking a single bacterial colony of the bacteria by using an inoculating needle, streaking the single bacterial colony on an AVM culture medium plate, sealing a culture dish by using a sealing film after streaking, and performing sealed culture at 28 +/-0.5 ℃ until a complete bacterial colony visible to naked eyes is obtained; obtaining a purified strain;
adding the purified strain into an LB liquid culture medium, performing sealed culture for 18 hours, centrifuging, removing a supernatant, and taking a bottom precipitate obtained by centrifuging as a strain seed solution;
the LB liquid culture medium has the formula: 2.5g of tryptone, 1.25g of yeast extract, 2.5g of sodium chloride and 250mL of water.
7. The method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil according to claim 6, wherein the method comprises the following steps:
the step 6) is as follows:
inoculating 1ml of the strain seed solution obtained in the step 4) into a container containing 100ml of AVM culture medium, and carrying out rotary shaking sealed culture at the temperature of 30 +/-0.5 ℃ until the strain seed solution grows to the middle exponential phase;
inoculating 1mL of the bacterial liquid growing to the middle exponential phase into a container containing 10mL of AVM culture medium, and carrying out sealed culture at the temperature of 30 +/-0.5 ℃ until the exponential phase; then, 10% of the headspace gas in the sealed vessel was replaced with acetylene, and the culture was again carried out in a sealed condition at 30. + -. 0.5 ℃ for 6 hours; the produced ethylene was measured by gas chromatography.
8. The method for screening non-symbiotic nitrogen-fixing strains in rice rhizosphere soil according to claim 7, wherein the step 6) comprises:
firstly, extracting DNA of the strain: carrying out the purification of the bacterial strain obtained in the step 4) according to a bacterial genome DNA extraction kit;
PCR analysis of the strain: using a universal primer to carry out sequencing PCR amplification on the 16s rRNA gene full-length sequence of the strain genome DNA obtained in the step I;
the universal primer is as follows: 27F-GAGTTTGATCCTGGCTCAG, 1492R-TACGGYTACCTTGTTACGACTT;
an amplification system: DNA template 0.5 uL/sample, 2 XTaq PCR MasterMix 12.5uL, universal primer 1uL, double distilled water 10 uL;
mixing, and amplifying in a PCR instrument with the following PCR procedure of pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 90s, and 30 cycles; keeping the temperature at 72 ℃ for 10 min;
after the PCR is finished, uniformly mixing 2uL reaction solution with 2uL DNA loading buffer, and carrying out agarose gel electrophoresis by taking 3uL DNA marker as a control to detect whether the PCR amplification is successful or not;
when the 1.5kbp fragment is obtained by electrophoretic amplification, the success of PCR amplification is judged;
otherwise, when the electrophoresis result does not obtain the fragment, judging that the PCR amplification fails;
③ sequencing the DNA of the strain: the strains were sequenced using universal primers at Sangon; the sequencing results were analyzed using Bioedit and Mega4, and the obtained fasta-format results were subjected to 16S-rRNA bacterial identification on an EZ-biocloud website to confirm whether the bacteria were known or unknown species.
9. The method for screening the non-symbiotic nitrogen-fixing strain of the rhizosphere soil of the rice according to the claims 1 to 8, which is characterized by comprising the following steps:
in the step 2), the rice is directly pulled up with roots, loose soil around the roots is shaken off, and then the residual soil on the roots is lightly brushed off by a brush to obtain the rice rhizosphere soil.
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