CN113215048B - Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof - Google Patents
Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof Download PDFInfo
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Abstract
The invention discloses a Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof, and relates to the technical field of agricultural microorganisms; the Kluyveromyces sp AZ981 disclosed by the invention is preserved in China general microbiological culture Collection center (CGMCC) on 06 months 05 in 2021, and the preservation number is CGMCC No. 22269; the strain can obviously improve the nitrogen fixation enzyme activity and nitrogen fixation efficiency of the nitrogen-fixing bacteria under aerobic conditions. The implementation of the invention has important revelation effect on the field of biological nitrogen fixation, provides a new direction for further research and improvement of nitrogen fixation capability of nitrogen-fixing bacteria, provides a useful tool for green ecological agriculture, and has important revelation effect on the excavation and utilization work of nitrogen-fixing bacteria in the future. The invention is suitable for large-scale popularization and application in industry and has huge potential commercial value.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof.
Background
In a natural ecosystem, only nitrogen fixing microorganisms can convert nitrogen in the air into ammoniacal nitrogen through the action of nitrogen fixing enzymes, and the nitrogen fixing microorganisms are a core link in the global nitrogen circulation process. According to the interaction relationship between the nitrogen-fixing microorganisms and host plants, biological nitrogen fixation can be divided into self-generating nitrogen-fixing bacteria, symbiotic nitrogen fixation forming special organs, combined nitrogen fixation not forming special organs and the like. Compared with a symbiotic nitrogen fixation system, the relation between the combined nitrogen fixation bacteria and plants is not tight, and the combined nitrogen fixation bacteria and the plants do not form a specific structure similar to a nodule. According to the planting position of the combined nitrogen-fixing bacteria, the combined nitrogen-fixing bacteria can be divided into epiphytic nitrogen-fixing bacteria planted on the surface of a plant and endogenous nitrogen-fixing bacteria planted in the plant. They have the common characteristic that the secretion produced by the host plant is utilized to grow and reproduce, and the biological nitrogen fixation effect is carried out under proper conditions, so as to provide nitrogen fertilizer which can be directly utilized for the host plant, and the two can form good mutual-benefit symbiosis relationship. Therefore, the method optimizes the mutual relation between the combined nitrogen-fixing bacteria and crops, improves the nitrogen-fixing efficiency, and is a key technical means for reducing the application and improving the efficiency of the fertilizer in the green development of modern agriculture.
But the combined azotobacter can not form a specific structure similar to a nodule, so that the azotobacter is easily influenced by external conditions such as oxygen, ammonia concentration, plant growth condition and other microorganism competition and the like without the protection of specific organs, thereby reducing the azotobacter efficiency. The concentration of oxygen in the nitrogen fixation microenvironment is one of key factors influencing the effect of endogenous nitrogen fixation, the nitrogen fixation efficiency of nitrogen-fixing bacteria can reach the maximum value only under the condition of low oxygen partial pressure, and the excessive concentration of oxygen can inactivate the nitrogenase of most nitrogen-fixing microorganisms and inhibit the synthesis of the nitrogenase. However, most endogenous nitrogen-fixing bacteria are aerobic microorganisms, and energy needs to be provided through aerobic respiration. Therefore, how to solve the contradiction between the anaerobic nitrogen fixation process and the aerobic respiration of the combined nitrogen-fixing bacteria is a key point and a hotspot of the research of the combined nitrogen-fixing bacteria and is one of the important reasons that the effects of many nitrogen-fixing strains are poor in practical application at present.
In the current research, researchers often focus on screening more efficient nitrogen-fixing strains, and neglect the synergistic effect of non-nitrogen-fixing bacteria on nitrogen-fixing bacteria. In the process of separating and screening the azotobacter, the microorganism is primarily screened by a nitrogen-free culture medium, and potential synergistic non-azotobacter is eliminated at the initial stage of screening. Therefore, no screening and utilization research aiming at the non-nitrogen-fixing bacteria capable of improving the nitrogen fixing efficiency of the nitrogen-fixing bacteria is found in the field at present.
Disclosure of Invention
The invention aims to provide the Kluyveromyces AZ981 capable of improving the nitrogen fixation capacity of the nitrogen-fixing bacteria and the application thereof, so as to solve the problems in the prior art.
In order to achieve the above purpose, the present invention at least provides the following technical solutions:
one of the technical schemes provides the Kluyveromyces AZ981 for improving the nitrogen fixation capacity of the nitrogen-fixing bacteria, wherein the Kluyveromyces (Kluyvera sp.) AZ981 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No.22269 and the preservation date of 2021, 05 and 06 days.
According to the second technical scheme, a biological agent for improving the nitrogen fixation capacity of nitrogen-fixing bacteria is provided, and the biological agent contains the Kluyveromyces AZ 981.
The third technical scheme provides the application of the Kluyveromyces AZ981 or the biological agent in improving the nitrogen fixation capacity of the nitrogen-fixing bacteria.
The fourth technical proposal provides the application of the Kluyveromyces AZ981 or the biological medicament in the preparation of the microbial nitrogen-fixing fertilizer.
In some preferred embodiments, the nitrogen-fixing bacteria are a single strain of nitrogen-fixing bacteria or a strain of nitrogen-fixing bacteria.
In some preferred embodiments, the nitrogen-fixing bacteria are corn endophytic nitrogen-fixing bacteria.
The fifth technical scheme provides a method for improving the nitrogen fixation capability of nitrogen-fixing bacteria, and the nitrogen-fixing bacteria and the Kluyveromyces AZ981 are mixed and cultured.
In some preferred embodiments, the ratio of said azotobacter to said Kluyveromyces AZ981 is 1-9: 1.
The invention discloses the following technical effects:
biological nitrogen fixation is a key technical means for reducing the application of agricultural nitrogen fertilizers, however, the inhibition effect of oxygen on the activity of nitrogen-fixing enzymes is one of the important reasons for poor effect of nitrogen-fixing bacteria in the practical application process. According to the invention, through research on the synergistic effect of non-nitrogen-fixing bacteria on nitrogen-fixing bacteria, a matched strain AZ981 for improving the nitrogen-fixing efficiency of the nitrogen-fixing bacteria under aerobic conditions is found, and the nitrogen-fixing enzyme activity of endogenous nitrogen-fixing bacteria can be greatly improved under aerobic conditions. The implementation of the invention has important revelation effect on the field of biological nitrogen fixation, provides a new direction for improving the nitrogen fixation capacity of the nitrogen-fixing bacteria, provides a new useful tool for green ecological agriculture, and has important revelation effect on the excavation and utilization work of the nitrogen-fixing bacteria in the future. The invention is suitable for large-scale popularization and application in industry and has huge potential commercial value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the colony morphology of Kluyveromyces AZ981 on R2A solid medium;
FIG. 2 shows the results of PCR detection of nifH gene of Kluyveromyces AZ 981;
FIG. 3 is a graph showing the results of the determination of the azotase activity of a composite strain of endogenous nitrogen-fixing bacteria in corn in combination with non-nitrogen-fixing bacteria under the condition of 1% microaerobic conditions; wherein, F is a nitrogen-fixing compound bacteria system F formed by mixing Klebsiella sp MNAZ1050, Citrobacter sp MNAZ1397 and Pseudomonas sp MNAZ228 in equal proportion; abcde is a non-nitrogen-fixing compound bacteria system abcde formed by mixing Acinetobacter azotobacter sp (ACZLY 512), Kluyvera sp (Kluyvera sp) AZ981, Escherichia coli (Escherichia coli) TOP10, Bacillus subtilis ACCC19374 and Pseudomonas fluorescens ACCC10190 in equal proportion;
FIG. 4 is a graph showing the results of the determination of the azotase activity of a composite strain of endogenous nitrogen-fixing bacteria in corn in combination with non-nitrogen-fixing bacteria under a condition of 21% normal air oxygen content; wherein, F is a nitrogen-fixing compound bacteria system F formed by mixing Klebsiella sp MNAZ1050, Citrobacter sp MNAZ1397 and Pseudomonas sp MNAZ228 in equal proportion; abcde is a non-nitrogen-fixing compound bacteria system abcde formed by mixing Acinetobacter azotobacter sp (ACZLY 512), Kluyvera sp (Kluyvera sp) AZ981, Escherichia coli (Escherichia coli) TOP10, Bacillus subtilis ACCC19374 and Pseudomonas fluorescens ACCC10190 in equal proportion;
FIG. 5 shows the effect of adding non-nitrogen-fixing bacterial strains in the ratio of 1: 1 of nitrogen-fixing bacteria to non-nitrogen-fixing bacteria on the activity of nitrogen-fixing enzyme of composite bacteria system for endogenous nitrogen-fixing of corn under the condition of 21% normal air oxygen content; wherein, F is a nitrogen-fixing compound bacteria system F formed by mixing Klebsiella sp MNAZ1050, Citrobacter sp MNAZ1397 and Pseudomonas sp MNAZ228 in equal proportion; a. b, c, d and e are respectively Acinetobacter azotobacter (Acinetobacter sp.) ACZLY512, Kluyvera sp AZ981, Escherichia coli (Escherichia coli) TOP10, Bacillus subtilis ACCC19374 and Pseudomonas fluorescens (Pseudomonas fluorescens) ACCC 10190; the abcde is a non-nitrogen-fixing compound bacteria system abcde formed by mixing the five non-nitrogen-fixing bacteria in equal proportion; indicates that the treatment is very different from the nitrogen-fixing complex strain F (p < 0.01);
FIG. 6 is a graph showing the effect of adding non-nitrogen-fixing bacterial strains in a ratio of 3: 1 of nitrogen-fixing bacteria to non-nitrogen-fixing bacteria on the activity of nitrogen-fixing enzymes of an endogenetic nitrogen-fixing complex bacterial system in corn under a 21% normal air oxygen content condition; wherein, F is a nitrogen-fixing compound bacteria system F formed by mixing Klebsiella sp MNAZ1050, Citrobacter sp MNAZ1397 and Pseudomonas sp MNAZ228 in equal proportion; a. b, c, d and e are respectively Acinetobacter azotobacter (Acinetobacter sp.) ACZLY512, Kluyvera sp AZ981, Escherichia coli (Escherichia coli) TOP10, Bacillus subtilis ACCC19374 and Pseudomonas fluorescens (Pseudomonas fluorescens) ACCC 10190; the abcde is a non-nitrogen-fixing compound bacteria system abcde formed by mixing the five non-nitrogen-fixing bacteria in equal proportion; indicates that the treatment is very different from the nitrogen-fixing complex strain F (p < 0.01);
FIG. 7 is a graph showing the effect of the addition of non-nitrogen-fixing bacterial strains on the activity of nitrogen-fixing enzymes in an endogenetic nitrogen-fixing complex bacterial system of corn in a ratio of nitrogen-fixing bacteria to non-nitrogen-fixing bacteria of 9:1 under a normal air oxygen content condition of 21%; wherein, F is a nitrogen-fixing compound bacteria system F formed by mixing Klebsiella sp MNAZ1050, Citrobacter sp MNAZ1397 and Pseudomonas sp MNAZ228 in equal proportion; a. b, c, d and e are respectively Acinetobacter azotobacter (Acinetobacter sp.) ACZLY512, Kluyvera sp AZ981, Escherichia coli (Escherichia coli) TOP10, Bacillus subtilis ACCC19374 and Pseudomonas fluorescens (Pseudomonas fluorescens) ACCC 10190; the abcde is a non-nitrogen-fixing compound bacteria system abcde formed by mixing the five non-nitrogen-fixing bacteria in equal proportion; represents that the treatment is significantly different from the nitrogen-fixing complex strain F (p < 0.05);
FIG. 8 shows the effect of the addition of the non-azotobacter strain AZ981 on the activity of the single strain azotobacter of the endogenous azotobacter of corn in the ratio of azotobacter to non-azotobacter of 1: 1 under the condition of 21% normal air oxygen content; wherein A is Klebsiella sp MNAZ1050, B is Citrobacter sp MNAZ1397, C is Pseudomonas sp MNAZ228, B is Kluyvera sp AZ 981; the activity of the corresponding azotobacter is changed very significantly after the strain AZ981 is added (p < 0.01);
FIG. 9 shows the effect of the addition of the non-Azotobacter strain AZ981 in the ratio of Azotobacter firmus to non-Azotobacter firmus of 5: 1 on the activity of the nitrogen-fixing enzyme of the single strain of Azotobacter firmus in maize under the condition of 21% normal air oxygen content; wherein A is Klebsiella sp MNAZ1050, B is Citrobacter sp MNAZ1397, C is Pseudomonas sp MNAZ228, B is Kluyvera sp AZ 981; the activity of the corresponding azotobacter is changed very significantly after the strain AZ981 is added (p < 0.01);
FIG. 10 shows the effect of the addition of the non-Azotobacter strain AZ981 in the ratio of Azotobacter firmus to non-Azotobacter firmus of 9:1 on the activity of the nitrogen-fixing enzyme of the single strain of Azotobacter firmus in maize under the condition of 21% normal air oxygen content; wherein A is Klebsiella sp MNAZ1050, B is Citrobacter sp MNAZ1397, C is Pseudomonas sp MNAZ228, B is Kluyvera sp AZ 981; represents that the azotobacter activity of the corresponding azotobacter is changed very significantly after the strain AZ981 is added (p < 0.01).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
Example 1 isolation, screening and identification of Strain AZ981
1.1 isolation and screening of Strain AZ981
(1) The strain AZ981 is derived from the stalk guide tissue juice of No. 13 corn variety Bao Dan. Selecting the 3 rd stem central position above the corn base part, cutting off the corn stem by pruning shears, leading the tissue juice of the corn stem to flow out from the cross section under the action of the corn root pressure, sucking the juice by using 0.5g of sterile absorbent cotton balls, filling the absorbent cotton balls into a 50ml sterile centrifuge tube, placing the sterile centrifuge tube on ice and bringing the sterile centrifuge tube back to a laboratory.
(2) In a clean bench, 2mL of 0.9% sterile physiological saline was added to the centrifuge tube containing the absorbent cotton balls, incubated for 1 hour at 160rpm in a shaker at 28 ℃ and thoroughly mixed. Diluting the cultured solution by 10 times, spreading 100 μ L diluted solution on R2A solid culture medium, and culturing at 28 deg.C for 2-4 d. After the culture medium grows out, selecting bacteria, purifying the strain by a scribing method for 3 times, and storing the strain in 30% glycerol at-70 ℃.
R2A solid medium (g.L)-1) The components are as follows: 0.5g of yeast extract, 0.5g of peptone, 0.5g of casein hydrolysate, 0.5g of glucose, 0.5g of starch, 0.3g of sodium pyruvate, 0.3g of dipotassium hydrogen phosphate, 0.05g of magnesium sulfate and 15.0g of agar.
(3) The colony of the strain AZ981 on the R2A solid medium is light yellow, viscous, round and convex, and has neat edges (figure 1).
(4) 16S rDNA sequence analysis of Strain AZ 981: PCR amplification was performed using bacterial 16S rDNA universal primers F27/R1492. The F27/R1492 primer sequences are respectively:
AGAGTTTGATCCTGGCTCAG(SEQ ID NO：1)；
TACGGCTACCTTGTTACGACTT(SEQ ID NO：2)。
the PCR system was 25. mu.L, including 12.5. mu.L EasyTaq PCR Supermix (Beijing Quanjin Biotechnology Co., Ltd.), 1. mu.L (10. mu.M) of each of F27/R1492 primers, and 10.5. mu.L of sterile water, and a single colony was spotted directly into the PCR system using a tip for expansion. The PCR amplification conditions were: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 50 ℃ for 40 seconds, and 72 ℃ for 1 minute; finally 10 minutes at 72 ℃. After detection by 1% agarose gel electrophoresis, the purified product was subjected to sequencing by Biotechnology engineering (Shanghai) Ltd. The sequencing result and morphological characteristics show that the strain AZ981 is Kluyvero (Kluyvera sp.), and the 16SrDNA sequence is shown as SEQ ID NO: 3, respectively.
SEQ ID NO：3：AGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACTCCTCAAGGGAACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCCAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTAAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGGTGAAGCTATTAACTTCACCGCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCATTACCCCACCTACTAGCTAATCCCATCTGGGCACATCCGATGGTGTGAGGCCCGAAGGTCCCCCACTTTGGTCTTGCGACGTTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCGGGCAGTTTCCCAGACGTTACTCACCCGTCCGCCACTCGTCACCCGAGAGCAAGCTCTCTGTGCTACCGTTCGACTGC
1.2 identification of whether Strain AZ981 is a Nitrogen-fixing bacterium
1.2.1 validation Using Nitrogen-free Medium
The strain AZ981 was streaked onto Ashby solid nitrogen-free medium and passaged 2 times in succession, the first generation showed weak growth and the second generation showed no growth, indicating that the strain AZ981 could not grow on nitrogen-free medium.
Ashby solid nitrogen-free medium (g.L)-1) The components are as follows: 10g of mannitol, 0.2g of monopotassium phosphate, 0.2g of 7 hydrated magnesium sulfate, 0.2g of sodium chloride, 0.1g of 2 hydrated calcium sulfate, 5g of calcium carbonate and 18g of agar.
1.2.2 verification of amplification of nifH Gene by use of azotase nifH
The PCR front primer and the PCR rear primer are respectively:
Pol-F：TGCGAYCCSAARGCBGACTC(SEQ ID NO：4)；
Pol-R：ATSGCCATCATYTCRCCGGA(SEQ ID NO：5)。
the PCR system was 25. mu.L, including 12.5. mu.L EasyTaq PCR Supermix (Beijing Omegal gold Biotechnology Co., Ltd.), 1. mu.L Pol-F pre-primer (10. mu.M), 1. mu.L Pol-R post-primer (10. mu.M), and 10.5. mu.L sterile water, and a single colony was spotted directly into the PCR system using a tip for expansion. The PCR amplification conditions were: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute; finally 10 minutes at 72 ℃. The strain AZ981 was not amplified to produce a nifH gene band by 1% agarose gel electrophoresis (FIG. 2), indicating that the strain AZ981 does not contain the nifH gene.
1.2.3 determination of nitrogenase Activity by acetylene reduction
The AZ981 single colony to be detected was picked from the R2A solid medium and inoculated into 4mL of R2A liquid medium, and cultured overnight at 28 ℃ with shaking at 160 rpm. The secondary suspension was centrifuged at 4 ℃ for 10min at 5000rpm, and the supernatant was removed. The cells were washed 2 times with an equivalent amount of 0.9% physiological saline and centrifuged for 10min under the same conditions to remove residual media, antibiotics and cell metabolites. The washed bacterial liquid OD600Adjusted to 1.0. Add 4.5mL DN nitrogen-free liquid Medium and 0.5mL OD to sterile 20mL headspace bottle6001.0 of AZ981 to obtain the initial OD of the culture600Is 0.1. 5 experimental replicates were made for each sample. Each headspace bottle was replaced with argon for 4min to purge the bottle of air. Oxygen gas in an amount of 1% of the internal volume of the flask and acetylene gas in an amount of 10% of the internal volume of the flask were respectively injected into each of the argon-filled headspace bottles. The gas-filled headspace bottles were incubated at 28 ℃ with shaking at 160 rpm. The time was recorded and after 12 hours the ethylene content was determined by gas chromatography and the nitrogenase activity was calculated. The results show that strain AZ981 is not able to convert acetylene to ethylene, indicating no nitrogenase activity.
DN nitrogen-free liquid medium (g.L)-1) The ingredients of the beverage comprise 10.0g of sucrose, 5g of malic acid, 0.2g of 1-hydrated dipotassium phosphate, 0.4g of 1-hydrated potassium dihydrogen phosphate, 0.1g of sodium chloride, 0.01g of ferric chloride, 0.002g of sodium molybdate, 0.02g of 7-hydrated magnesium sulfate and 0.002g of 1-hydrated calcium chloride.
1.2.4 conclusion
In conclusion, the strain AZ981 is a strain of maize endogenic non-nitrogen-fixing Kluyvero-Volville (Kluyvera sp.) AZ981 which is preserved in China general microbiological culture Collection center (China institute of microbiology, institute of sciences, China institute of sciences, No. 3, West Lu 1 institute of North Chen West Lu, Chao, Beijing) on 06 days 05.2021, and the preservation number is CGMCC No. 22269.
Example 2
Promoting effect of strain AZ981 on nitrogen fixation function of endophytic nitrogen-fixing bacteria
2.1 Nitrogen fixation composite bacterial system F for nitrogen fixation effect verification test and other 4 non-nitrogen fixation comparison bacterial strains
In order to compare the difference of the influence of the strain AZ981 and other non-nitrogen-fixing bacteria on the endogenous nitrogen-fixing bacteria, 3 strains of nitrogen-fixing bacteria are selected and mixed in equal proportion to form a nitrogen-fixing composite bacterial system F. In addition, other 4 non-nitrogen-fixing bacteria except the strain AZ981 were selected as comparative strains of the strain AZ981, and the influence of the addition of the non-nitrogen-fixing bacteria on the nitrogen fixing effect of the nitrogen-fixing complex bacteria F was studied. The method for verifying the activity of the nitrogenase was the same as in example 1 except that the amount of oxygen added was different.
2.1.1 Nitrogen-fixing Complex bacterial line F
The nitrogen-fixing compound bacterial system F is as follows: the 3 nitrogen-fixing bacteria are respectively Klebsiella sp MNAZ1050 with the preservation number of CGMCC No. 22270; the preservative is the citric acid bacillus (Citrobacter sp.) MNAZ1397 with the preservation number of CGMCC No. 22267; pseudomonas sp MNAZ228 with the preservation number of CGMCC No.22266 (three strains are preserved in China general microbiological culture Collection center at 06.05.2021. and the address is the institute of microbiology of China academy of sciences No. 3, North West Lu No. 1 institute of China, North Kogyo-Naja Cheng, Beijing).
Under the condition of 1% of micro-oxygen, the 3 strains can carry out nitrogen fixation, and are mixed in equal proportion to form a nitrogen fixation composite bacterial system F. The azotobacter complex system F has the azotobacter activity of 1439.0nmol C under the condition of 1% of micro oxygen2H4/mg pro hr (FIG. 3); under aerobic conditions (oxygen content of 21 percent, normal air oxygen content), the activity of the azotobacter complex system F is inhibited, and the azotobacter efficiency is only 92.8nmol C2H4Mg pro hr (FIG. 4).
2.1.2 other 4 non-Nitrogen-fixing control strains
The other 4 non-nitrogen-fixing control strains were: book removing deviceIn addition to the obtained strain AZ981, in this example, other 4 non-nitrogen-fixing bacteria were selected as comparative strains, which are Acinetobacter (Acinetobacter sp.) ACZLY512 (the common microorganism center of the commission on preservation and management of chinese microorganisms, with the preservation number of CGMCC No.22268, shown by a in fig. 5-7), Escherichia coli (Escherichia coli) TOP10 (purchased from tiangen biochemical science and technology (beijing) limited, shown by c in fig. 5-7), Bacillus subtilis (ACCC 19374 (the collection and management center of chinese agricultural microorganisms, with the preservation number of ACCC19374, shown by d in fig. 5-7), Pseudomonas fluorescens (Pseudomonas fluorescens) ACCC10190 (the collection and management center of chinese agricultural microorganisms, with the preservation number of ACCC10190, shown by e in fig. 5-7), respectively. The strain AZ981 is indicated by b in FIGS. 5-10. The strain AZ981 and the 4 comparative strains have no azotase nifH gene and no azotase activity, and the azotase activity of the composite strain abcde formed by mixing the strain AZ981 and the comparative strains in equal proportion is 0nmol C under aerobic or micro-aerobic conditions2H4Mg pro hr (FIGS. 3-4).
2.2 under the condition of 21% normal air oxygen content
2.2.1 Strain AZ981 is added in the ratio of azotobacter to non-azotobacter 1: 1
As shown in FIG. 5, the azotobacter system F was added with the strain AZ981 in a ratio of 1: 1 (F + b) and its azotobacter activity was 633.9nmol C2H4Permg pro hr, compared with the azotobacteria F and F + b treatment azotobacteria activity is increased by 5.8 times, and the difference reaches an extremely significant level (p)<0.01). However, when any one of the Escherichia coli TOP10(c), Bacillus subtilis ACCC19374(d) and Pseudomonas fluorescens ACCC10190(e) is added into the nitrogen-fixing complex strain F at a ratio of 1: 1, the change of the nitrogen-fixing enzyme activity does not reach a statistically significant difference (p)>0.05), showing that the 3 non-nitrogen-fixing strains have no significant influence on the nitrogen-fixing enzyme activity of the nitrogen-fixing complex strain F.
2.2.2 Strain AZ981 was added in a ratio of 3: 1 of azotobacter to non-azotobacter
As shown in FIG. 6, the azotobacter system F was added with 3: 1 ratio of the strain Kluyveromyces AZ981 (F + b), and its azotase activity was 387.9nmol C2H4/mg pro hr，Compared with the azotobacteria F, the azotobacteria activity of F + b treatment is increased by 3.2 times, and the difference reaches a very significant level (p)<0.01). However, when any one of the Escherichia coli TOP10(c), Bacillus subtilis ACCC19374(d) and Pseudomonas fluorescens ACCC10190(e) is added into the nitrogen-fixing complex strain F at a ratio of 3: 1, the change of the nitrogen-fixing enzyme activity does not reach a statistically significant difference (p)>0.05), showing that the 3 non-nitrogen-fixing strains have no significant influence on the nitrogen-fixing enzyme activity of the nitrogen-fixing complex strain F.
2.2.3 Strain AZ981 was added in a ratio of 9:1 of azotobacter to non-azotobacter
As shown in FIG. 7, the azotobacter system F was added with 9:1 ratio of the strain Kluyveromyces AZ981 (F + b), and its azotase activity was 169.5nmol C2H4Permg pro hr, compared with the azotobacteria F and F + b treatment azotobacteria activity is increased by 0.8 times, and the difference reaches an extremely significant level (p)<0.01). However, when any one of the Escherichia coli TOP10(c), Bacillus subtilis ACCC19374(d) and Pseudomonas fluorescens ACCC10190(e) is added into the nitrogen-fixing complex strain F at a ratio of 9:1, the change of the nitrogen-fixing enzyme activity does not reach a statistically significant difference (p)>0.05), showing that the 3 non-nitrogen-fixing strains have no significant influence on the nitrogen-fixing enzyme activity of the nitrogen-fixing complex strain F.
2.3 conclusion
Under aerobic conditions (oxygen content is 21 percent, oxygen content in normal air), the nitrogen fixation effect of the nitrogen-fixing compound bacteria system is severely inhibited by oxygen, and when the strain AZ981 is added into the nitrogen-fixing compound bacteria system F according to the proportion of 1: 1, the nitrogen fixation activity of the nitrogen-fixing compound bacteria system F is remarkably improved by 5.8 times; when the strain AZ981 is added into the nitrogen-fixing compound strain F according to the proportion of 3: 1, the nitrogen-fixing activity of the nitrogen-fixing compound strain F is obviously improved by 3.2 times; when the strain AZ981 is added into the nitrogen-fixing compound strain F according to the proportion of 9:1, the nitrogen-fixing activity of the nitrogen-fixing compound strain F is obviously improved by 0.8 times.
Example 3 promoting action of Strain AZ981 on Nitrogen fixation function of Nitrogen-fixing bacteria
In order to verify the influence of the strain AZ981 on the nitrogen fixation function of azotobacter monads, 3 azotobacter monads, namely Klebsiella sp MNAZ1050 (indicated by A in figures 8-10), Citrobacter sp MNAZ1397 (indicated by B in figures 8-10) and Pseudomonas sp MNAZ228 (indicated by C in figures 8-10), were respectively selected, and the influence of the addition of AZ981 on the azotobacter activity was verified.
3.1 measurement of nitrogenase Activity under the condition of 21% Normal air oxygen content
3.1.1 adding AZ981 strain in the ratio of azotobacter to non-azotobacter to 1: 1
The azotobacteria Klebsiella MNAZ1050(A) has the azotobacter activity of 22.9nmol C2H4Per mg pro hr, when the azotobacter Klebsiella MNAZ1050 is added into the strain AZ981 (A + a) in the ratio of 1: 1, the azotobacter activity is as high as 307.3nmol C2H4Per mg pro hr, compared with Klebsiella azotobacter MNAZ1050(A), the activity of the azotobacter treated by A + b is increased by 12.4 times, and the difference reaches a very significant level (p)<0.01) (fig. 8).
The azotobacter activity of the azotobacter MNAZ1397(B) is 18.7nmol C2H4Per mg pro hr, when azotobacteria, MNAZ1397, is added to strain AZ981 in a ratio of 1: 1 (B + a), its azotobacteria activity is as high as 486.5nmol C2H4Per mg pro hr, the azotobacter activity is increased by 25 times compared with the azotobacter MNAZ1397(B) treated by B + B, and the difference reaches a very significant level (p)<0.01) (fig. 8).
The azotobacteria pseudomonad MNAZ228(C) azotobacter activity is 13.6nmol C2H4Per mg pro hr, when the strain AZ981 is added into the pseudomonas azotoformans MNAZ228 in the ratio of 1: 1, (C + a), the azotoformase activity is as high as 501.7nmol C2H4Permg pro hr, compared with azotobacteria pseudomonad MNAZ228(C), the activity of azotobacteria treated by C + b is increased by 36.0 times, and the difference reaches a very significant level (p)<0.01) (fig. 8).
3.1.2 adding AZ981 strain in the ratio of azotobacter to non-azotobacter to 5: 1
The azotobacteria Klebsiella MNAZ1050(A) has the azotobacter activity of 22.9nmol C2H4Per mg pro hr, when the azotobacter Klebsiella MNAZ1050 is added into the strain AZ981 (A + a) in the ratio of 5: 1, the azotobacter activity is as high as 635.4nmol C2H4/mg pro hr, A + compared to nitrogen-fixing bacteria Klebsiella MNAZ1050(A)a treatment increases the nitrogenase activity by 26.7 times, and the difference reaches a very significant level (p)<0.01) (fig. 9).
The azotobacter activity of the azotobacter MNAZ1397(B) is 18.7nmol C2H4Per mg pro hr, when azotobacteria, MNAZ1397, is added to strain AZ981 in a ratio of 5: 1 (B + a), its azotobacteria activity is as high as 401.2nmol C2H4Per mg pro hr, the azotobacter activity was increased 20.5 times by B + B treatment compared to MNAZ1397(B) of Citrobacter azotobacter, and the difference reached a very significant level (p)<0.01) (fig. 9).
The azotobacteria pseudomonad MNAZ228(C) azotobacter activity is 13.6nmol C2H4Per mg pro hr, when the strain AZ981 is added into the pseudomonas azotoformans MNAZ228 in the ratio of 5: 1, (C + a), the azotoformase activity is as high as 184.5nmol C2H4Permg pro hr, compared with azotobacteria pseudomonad MNAZ228(C), the activity of azotobacteria treated by C + b is increased by 12.6 times, and the difference reaches a very significant level (p)<0.01) (fig. 9).
3.1.3 Strain AZ981 is added in the ratio of azotobacter to non-azotobacter 9:1
The azotobacteria Klebsiella MNAZ1050(A) has the azotobacter activity of 22.9nmol C2H4Per mg pro hr, when the azotobacter Klebsiella MNAZ1050 is added into the strain AZ981 (A + a) in the ratio of 9:1, the azotobacter activity is as high as 266.5nmol C2H4Per mg pro hr, compared with Klebsiella azotobacter MNAZ1050(A), the activity of the azotobacter treated by A + b is increased by 10.6 times, and the difference reaches a very significant level (p)<0.01) (fig. 10).
The azotobacter activity of the azotobacter MNAZ1397(B) is 18.7nmol C2H4Per mg pro hr, when azotobacteria, MNAZ1397, is added to strain AZ981 in a ratio of 9:1 (B + a), its azotobacteria activity is as high as 173.6nmol C2H4Per mg pro hr, the azotobacter activity is increased by 8.3 times compared with MNAZ1397(B) of azotobacter treated by B + B, and the difference reaches a very significant level (p)<0.01) (fig. 10).
The azotobacteria pseudomonad MNAZ228(C) azotobacter activity is 13.6nmol C2H4Per mg pro hr, after adding strain AZ981 to Pseudomonas azotoformans MNAZ228 in a ratio of 9:1 (C + a)The azotase activity is as high as 395.2nmol C2H4Permg pro hr, compared with azotobacteria pseudomonad MNAZ228(C), the activity of azotobacteria treated by C + b is increased by 28.1 times, and the difference reaches a very significant level (p)<0.01) (fig. 10).
3.2 conclusion
Under aerobic conditions (oxygen content of 21 percent and normal oxygen content in air), the nitrogen fixation effect of the nitrogen-fixing bacteria such as Klebsiella MNAZ1050, Citrobacter MNAZ1397, Pseudomonas MNAZ228 and the like is seriously inhibited by oxygen, and the activity of the nitrogen-fixing enzyme is less than 23nmol C2H4/mg pro hr. When the strain AZ981 is added in the ratio of 1: 1, the nitrogen-fixing activity of nitrogen-fixing bacteria such as Klebsiella MNAZ1050, Citrobacter MNAZ1397, Pseudomonas MNAZ228 and the like is obviously increased, and the nitrogen-fixing activity is 307.3-501.7nmol C2H4Per mg pro hr, the activity is increased by 12.4-36.0 times; when the strain AZ981 is added in the ratio of 5: 1, the nitrogen-fixing activity of nitrogen-fixing bacteria such as Klebsiella MNAZ1050, Citrobacter MNAZ1397, Pseudomonas MNAZ228 and the like is obviously increased, and the nitrogen-fixing activity is 184.5-635.4nmol C2H4Activity increases by 12.6-26.7 times between/mg pro hr; when the strain AZ981 is added according to the ratio of 9:1, the nitrogen-fixing activity of nitrogen-fixing bacteria such as Klebsiella MNAZ1050, Citrobacter MNAZ1397, Pseudomonas MNAZ228 and the like is obviously increased, and the nitrogen-fixing activity is 173.6-395.2nmol C2H4Per mg pro hr, the activity increases 8.3-28.1 times.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> institute of agricultural resources and agricultural regionalism of Chinese academy of agricultural sciences
<120> Kluyveromyces AZ981 for improving nitrogen fixation capacity of nitrogen-fixing bacteria and application thereof
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Claims (8)
1. The Kluyveromyces AZ981 for improving the nitrogen fixation capability of the nitrogen-fixing bacteria is characterized in that the Kluyveromyces AZ981 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No.22269 and the preservation date of 2021, 05 and 06 months.
2. A biological agent for improving the nitrogen fixation ability of nitrogen-fixing bacteria, which is characterized by comprising the kluyveromyces AZ981 in claim 1.
3. Use of the biopharmaceutical of claim 1 or claim 2 wherein said AZ981 or said biopharmaceutical is for enhancing the nitrogen-fixing ability of nitrogen-fixing bacteria.
4. Use of a biopharmaceutical according to claim 1 or claim 2 and AZ981 or claim 2 in the preparation of a microbial nitrogen-fixing fertilizer.
5. The use of claim 3, wherein said azotobacter is a single strain of azotobacter or a azotobacter line.
6. The use of claim 5, wherein said nitrogen-fixing bacteria are endogenous nitrogen-fixing bacteria of maize.
7. A method for improving the nitrogen fixation ability of a nitrogen-fixing bacterium, characterized in that the nitrogen-fixing bacterium is cultured in combination with the Kluyveromyces AZ981 as claimed in claim 1.
8. The method of claim 7, wherein the ratio of said azotobacter to said Kluyveromyces AZ981 is 1-9: 1.
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| CN106967633A (en) * | 2017-03-25 | 2017-07-21 | 云南农业大学 | One plant of Kluyvera ascorbata and its application |
| CN110331153A (en) * | 2019-06-24 | 2019-10-15 | 浙江工业大学 | A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application |
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| CN106967633A (en) * | 2017-03-25 | 2017-07-21 | 云南农业大学 | One plant of Kluyvera ascorbata and its application |
| CN110331153A (en) * | 2019-06-24 | 2019-10-15 | 浙江工业大学 | A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application |
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