CN112029675A - Screening and application of Antarctic low-temperature phosphate solubilizing bacteria - Google Patents
Screening and application of Antarctic low-temperature phosphate solubilizing bacteria Download PDFInfo
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- CN112029675A CN112029675A CN202010097150.4A CN202010097150A CN112029675A CN 112029675 A CN112029675 A CN 112029675A CN 202010097150 A CN202010097150 A CN 202010097150A CN 112029675 A CN112029675 A CN 112029675A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
Abstract
The invention obtains a low-temperature phosphate solubilizing bacterium from Antarctic penguin island soil by separation and screening, the name of the low-temperature phosphate solubilizing bacterium is Pseudomonas sp NJ01, the low-temperature phosphate solubilizing bacterium is preserved in China center for type culture Collection in 11-24 months in 2019, and the preservation number is CCTCC NO: m20191135. The low-temperature phosphate solubilizing bacteria NJ01 can adapt to low-temperature environment, can exert higher phosphate solubilizing effect under low-temperature condition, fill up the vacancy of a phosphate solubilizing bacteria database under low-temperature environment, convert insoluble or insoluble phosphorus in low-temperature soil into quick-acting phosphorus or soluble phosphorus to be stored in the soil, promote the germination, absorption and utilization of plants, effectively relieve the soil hardening caused by the fact that the plants cannot be utilized due to long-term application of phosphate fertilizer, and reduce environmental pollution.
Description
Technical Field
The invention belongs to the technical field of microorganism screening, and particularly relates to an Antarctic bacterium capable of efficiently dissolving phosphorus at a low temperature.
Background
Phosphorus is the second major macronutrient required for plant growth and development, and the elements of nucleic acid synthesis, photosynthesis and respiration, formation of fats, sugars and starches cannot be separated. Although there is a large amount of phosphorus in the soil, some of the phosphorus is not absorbed by plants, thus seriously affecting crop productivity. Therefore, in order to make up for the shortage of phosphorus in soil and ensure the maximum yield of crops, the excessive application of chemical phosphate fertilizers is the agricultural practice of various countries in the world at present. Globally, more than 1.755 million tons of fertilizer are used in agriculture each year to obtain the best yield of crops. However, only 5-25% of the applied phosphorus is absorbed by the plants, and the remaining 75-95% of the applied phosphorus is present in the soil in an insoluble form, which adversely affects the soil matrix and its ecological environment. Moreover, the continuous input of fertilizers not only reduces the nutritional status of the fertilizers, but also causes harm to sustainable crop production and environment.
Research results show that effective strategies for fertilizer, especially soil phosphorus management, are essential. In this regard, soil microorganisms play a significant role in improving crop productivity and soil health. Therefore, since the 50 s of the 20 th century, soil microorganisms have been used as microbial agents/biofertilizers as a substitute for chemical fertilizers. Because they perform various chemical and biological activities, improving crop health and productivity without causing any damage to the ecosystem. It has been reported worldwide that the growth and yield of many crops such as wheat, corn and sweet potato are improved by using phosphorus-dissolving bacterial agents. Under the influence of living environment and natural conditions, common phosphate solubilizing bacteria cannot fully exert the effect of converting insoluble phosphate into soluble phosphate. Especially under the influence of low temperature, the effect is more greatly reduced. Therefore, screening out a phosphate solubilizing bacterium which can adapt to low-temperature environment and can exert the phosphate solubilizing function has important significance. And the screening and application of the phosphate solubilizing bacteria from the south extremely low temperature provide convenience for solving the problem.
Disclosure of Invention
The invention aims to provide low-temperature phosphate solubilizing bacteria, which can solve the problems that the phosphate solubilizing bacteria are difficult to grow under the low-temperature condition, have poor phosphate solubilizing effect and are not suitable for low-temperature soil, so that the defects of the existing phosphate solubilizing bacteria resource are overcome, and the application range of the phosphate solubilizing bacteria is expanded.
The Antarctic low-temperature phosphate solubilizing bacteria NJ01 is named as Pseudomonas 1902 Pseudomonas sp 1902, is separated from Antarctic penguin island soil, is preserved in China center for type culture Collection in 12 months and 30 days in 2019, and has a preservation number of CCTCC NO: m20191135, deposit address: china, wuhan university.
The Antarctic low-temperature phosphate solubilizing bacteria NJ01 provided by the invention is applied to phosphate solubilizing.
The invention also provides a method for dissolving phosphorus in farmland, which is carried out by using the strain.
The invention enriches the resources of wild phosphate solubilizing bacteria and increases a backup library for whole genome breeding of phosphate solubilizing bacteria. In addition, the phosphate solubilizing bacteria NJ01 can adapt to a low-temperature environment, can exert a higher phosphate solubilizing effect under a low-temperature condition, fill up the vacancy of a phosphate solubilizing bacteria database under the low-temperature environment, can be used as a microbial inoculum to be applied to soil together with phosphate fertilizers in winter or early spring, convert insoluble or insoluble phosphorus in the soil into quick-acting phosphorus or soluble phosphorus to be stored in the soil, promote germination, absorption and utilization of plants, simultaneously effectively relieve soil hardening caused by incapability of the plants due to long-term application of the phosphate fertilizers, and reduce environmental pollution.
Drawings
FIG. 1: the invention discloses a phosphorus dissolving circle diagram of low-temperature phosphate solubilizing bacteria NJ 01.
FIG. 2: the invention relates to a flat-plate line drawing of low-temperature phosphate solubilizing bacteria NJ 01.
FIG. 3: the phosphate solubilizing effect diagram of the low-temperature phosphate solubilizing bacteria provided by the invention.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are provided for illustration and explanation only, and should not be construed as limiting the scope of the present invention in any way.
The strain is a low-temperature phosphate solubilizing bacterium NJ01, has a strain name of Pseudomonas 1902 Pseudomonas sp 1902 and is preserved in China center for type culture Collection in 12 months and 30 days in 2019, and the preservation number is CCTCC NO: m20191135, address: china, wuhan university.
Example 1: separation and screening of low-temperature resistant phosphate solubilizing bacteria NJ01
(1) Preparing an inorganic phosphorus solid culture medium: the following reagents, glucose 10g and Ca, were weighed out separately3(PO4)2 5g、FeSO4·7H2O 0.03g、MnSO4·4H2O 0.03g、MgSO4·7H2O 0.3g、NaCl 0.3g、KCl 0.3g、 (NH4)2SO40.5g of agar, 20g of agar and 1000g of distilled water, and adjusting the pH value to 7.0-7.5; sterilizing at 115 deg.C for 20 min.
(2) Taking 1g Antarctic penguin island soil in a sterilized conical flask, adding 90mL of sterile water, placing in a shaking table at 10 ℃ and uniformly mixing for 30min at the rotating speed of 150r/min, standing the mixed solution, taking supernatant and sequentially diluting by 10-3,10-5,10-7The multiple concentration gradient is evenly coated on the inorganic phosphorus solid culture medium plate. After incubation at 10 ℃ for 5-7 days, colonies with a phosphate solubilizing loop were re-streaked on LB solid medium for purification to give a single strain.
The obtained strains are spotted on an organophosphorus solid culture medium, the strains are cultured for 7 days at constant temperature of 10 ℃, 20 ℃ and 30 ℃ respectively to observe the ratio of the diameter (HD) of a phosphorus-dissolving ring to the diameter (CD) of a bacterial colony, and strains with larger ratio under the low-temperature condition are screened out and named as phosphate solubilizing bacteria NJ01 (figure 1).
As shown in Table 1, the HD/CD value of NJ01 was the largest at 10 ℃.
Table 1: HD/CD value table of screened strains under different temperature conditions
Example 2: observation and identification of low-temperature resistant phosphate solubilizing bacteria NJ01
The physiological morphology of the low-temperature phosphate solubilizing bacteria NJ01 is that the surface of a bacterial colony is smooth, is light yellow, has neat edges, and is wet and viscous (shown in figure 2). The growth speeds of the low-temperature phosphate solubilizing bacteria NJ01 are different under different temperature conditions, the growth speed of the bacterial colony is higher at 10 ℃ and 20 ℃, the phosphorus-dissolving ring is not obviously attached to the edge of the bacterial colony at the initial culture stage, and the phosphorus-dissolving ring gradually increases and the bacterial colony does not increase after 3 days; colonies at 30 ℃ grew more slowly and had smaller phosphate rings than those at other conditions. The strain NJ01 is amplified by using the primers 27F and 1492R and subjected to 16S rDNA sequencing, and the obtained sequence is 1408bp in sequencing degree, and the specific sequence is SEQ ID NO 1. Alignment by BLAST with sequences in the GenBank database identified it as Pseudomonas sp NJ 01.
Example 3 measurement of phosphorus solubilizing ability of Low-temperature phosphate solubilizing bacterium NJ01
Preparing an inorganic phosphorus liquid culture medium: the following reagents, glucose 10g and Ca, were weighed out separately3(PO4)2 5g、FeSO4·7H2O 0.03g、MnSO4·4H2O 0.03g、MgSO4·7H2O 0.3g、NaCl 0.3g、 KCl 0.3g、(NH4)2SO40.5g, lecithin 0.2g and distilled water 1000 g; adding Ca3(PO4)2Separately sterilizing the mixture and the other components, mixing the sterilized mixture and the other components, and adjusting the pH value to 7.0-7.5.
After activating the low-temperature phosphate solubilizing bacteria NJ01 screened in example 1, 1mL of the activated low-temperature phosphate solubilizing bacteria NJ01 was added to 100mL of inorganic phosphate liquid medium, and the mixture was placed in a shaker at 10 ℃ and subjected to shaking culture at a rotation speed of 190r/min for 7 days, and 3 bottles of the repeat and control groups were set. And after 48 hours, pouring 5mL of culture solution into a centrifuge tube every 24 hours, centrifuging for 10min at 10000r/min, and measuring the content of soluble phosphorus in the supernatant by using a molybdenum-antimony anti-spectrophotometry method. And counting the phosphorus dissolving amount of each strain every day. As shown in FIG. 3, the phosphorus-dissolved amount of the low-temperature phosphate-solubilizing bacterium NJ01 was best on day 5, and the highest phosphorus-dissolved amount was 108.32 mg/L.
According to the study of the invalid phosphorus content in the farmland soil and the phosphate solubilizing capability of the low-temperature phosphate solubilizing bacteria NJ01 in the invention, the farmland inoculation amount of the low-temperature phosphate solubilizing bacteria NJ01 is estimated to be enlarged to 10-50L/m2Is reasonable and is preferably 20-30L/m2。
The above examples are only preferred results of the present invention and do not limit the technical scope of the present invention, so that any minor modifications, equivalent changes and modifications made to the above examples according to the technical spirit of the present invention are within the technical scope of the present invention.
Sequence listing
<110> Shandong university of agriculture of first oceanographic institute of Nature resources department
<120> screening of Antarctic low-temperature phosphate solubilizing bacteria and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1408
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctgcagtcga gcggcagcac gggtacttgt acctggtggc gagcggcgga cgggtgagta 60
atgcctagga atctgcctgg tagtggggga taacgctcgg aaacggacgc taatacggca 120
tacgtcctac gggagaaagc aggggaccat cgggccttgc gctatcagat gagcctaggt 180
cggattagct agttggtgag gtaatggctc accaaagcga cgatccgtaa ctggtctgag 240
aggatgatca gtcacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300
gggaattttg gacaatgggc gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc 360
ttcggattgt aaagcacttt aagttgggag gaagggcatt tacctaatac gtaagtgttt 420
tgacgttacc gacagaataa gcaccggcta actctgtgcc agcagccgcg gtaatacaga 480
gggtgcaagc gttaatcgga attactgggc gtaaagcgcg cgtaggtggt tcgttaagtt 540
ggatgtgaaa tccccgggct caacctggga actgcattca aaactgtcga gctagagtat 600
ggtagagggt ggtggaattt cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac 660
cagtggcgaa ggcgagcacc tggactgata ctgacactga ggtgcgaaag tgtggggagc 720
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcaactag ccgttgggag 780
ccttgagctc ttagtggcgc agctaacgca ttaagttgac cgcctgggga gtacggccgc 840
aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttda 900
ttcgaagcaa cgcgaagaac cttaccaggc cttgacatcc tatgaatctg ctagagatag 960
cggagtgcct tcgggaacat tgagacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg taacgagcgc aacccttgtc cttagttacc agcacgtcat 1080
ggtgggcact caaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag 1140
acatcatggc ccttacggcc tgggctacac acgtgctaca atggtcggta ctgagggttg 1200
ccaagccgcg aggtggagct aatcccagaa aaccgatcgt agtccggatc gcagtctgca 1260
actcgactgc gtgaagtcgg aatcgctagt aatcgcgaat cagaatgtcg cggtgaatac 1320
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca ccagaagtag 1380
ctagtctaac cttcgggagg acggtaca 1408
Claims (3)
1. The Antarctic low-temperature phosphate solubilizing bacterium is characterized in that the preservation number of the Antarctic low-temperature phosphate solubilizing bacterium is CCTCC NO: m20191135.
2. The use of the antarctic low-temperature phosphate solubilizing bacterium of claim 1 in phosphate solubilizing.
3. A phosphate solubilizing method for farmlands, characterized in that the phosphate solubilizing method is to use the Antarctic low-temperature phosphate solubilizing bacterium of claim 1 for phosphate solubilizing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115873745A (en) * | 2022-08-05 | 2023-03-31 | 自然资源部第一海洋研究所 | Saline-alkali tolerant phosphate solubilizing bacteria and application thereof |
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2020
- 2020-02-17 CN CN202010097150.4A patent/CN112029675A/en active Pending
Patent Citations (3)
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| KR100973168B1 (en) * | 2008-05-08 | 2010-07-30 | 충북대학교 산학협력단 | Pseudomonas corrugata mutants promoting plant growth at low temperature and method for promoting plant growth using the same |
| CN105112319A (en) * | 2015-07-08 | 2015-12-02 | 南京农业大学 | Pseudomonas phosphate dissolving bacterium Y2, bioorganic fertilizer prepared through using bacterium, and application of fertilizer |
| CN108239610A (en) * | 2016-12-26 | 2018-07-03 | 北京有色金属研究总院 | One plant of low temperature resistant phosphate solubilizing bacteria and its method for carrying out farmland phosphorus decomposing |
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| GOVINDAN SELVAKUMAR 等: "Pseudomonas lurida M2RH3 (MTCC 9245), a psychrotolerant bacterium from the Uttarakhand Himalayas, solubilizes phosphate and promotes wheat seedling growth", 《WORLD J MICROBIOL BIOTECHNOL》 * |
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| PRATIBHA VYAS 等: "Stress Tolerance and Genetic Variability of Phosphate-solubilizing Fluorescent Pseudomonas from the Cold Deserts of the Trans-Himalayas", 《MICROB ECOL.》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115873745A (en) * | 2022-08-05 | 2023-03-31 | 自然资源部第一海洋研究所 | Saline-alkali tolerant phosphate solubilizing bacteria and application thereof |
| CN115873745B (en) * | 2022-08-05 | 2023-09-01 | 自然资源部第一海洋研究所 | Saline-alkali tolerant phosphate-dissolving bacterium and application thereof |
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