CN115975884B - Lekkera CYIJM6 and application thereof - Google Patents
Lekkera CYIJM6 and application thereof Download PDFInfo
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Abstract
The invention provides a Lekkera CYIJM6 strain and application thereof, and belongs to the technical field of microorganisms. The Lekkera CYIJM6 was stored in the China center for type culture collection (China, with a accession number of M20221561) at 10 months and 12 days of 2022. The strain provided by the invention has strong phosphorus dissolving capability, is suitable for activating phosphorus in soil in arid and semiarid regions, and can be used for remarkably improving the quick-acting phosphorus content of soil in arid and semiarid regions and reducing the application of phosphate fertilizer. The phosphate-dissolving strain provided by the invention has wide action substrate, can release organic acid and other micromolecule activated fixed inorganic phosphorus, can secrete phosphatase to hydrolyze organic phosphorus, and can remarkably improve the activation capability of soil phosphorus by utilizing the phosphate-dissolving strain.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a Lekkera CYIJM6 strain and application thereof.
Background
Phosphate application is one of the necessary measures to ensure crop yield, however 75-90% of the applied phosphate is fixed by the soil and not available. A large amount of phosphorus is accumulated in the soil in an invalid state, so that not only is the soil nutrient structure influenced, but also the risk of non-point source pollution is increased. More importantly, the main source of the phosphate fertilizer is non-renewable resources, and an effective soil phosphorus activation technology is needed, so that phosphorus accumulated in soil can be efficiently utilized, and agricultural production and sustainable utilization of phosphorus are considered.
The phosphorus-dissolving microorganism can effectively dissolve indissolvable phosphorus in soil, reduce fixation of phosphate fertilizer and increase phosphorus absorption of crops, thereby improving the utilization rate of phosphate fertilizer and the yield of crops, and is a safe, economic and effective biological measure currently accepted in the world. However, the high-efficiency phosphorus-dissolving microorganism strains which can be separated are limited in variety and quantity, and the screening capacity of the high-efficiency phosphorus-dissolving microorganism strains is poor in the adaptation to different environment soil. Especially in arid and semiarid regions, the soil phosphorus has extremely low effectiveness, and most of microbial activities and functions are greatly weakened due to water stress. Therefore, the specific drought-resistant phosphorus-dissolving microbial resource and the utilization thereof have important strategic significance for activating soil phosphorus, guaranteeing grain yield and reducing environmental pollution and energy waste.
Disclosure of Invention
In order to solve the problems, the invention provides the Lekkera CYIJM6 and the application thereof, and the strain provided by the invention has strong phosphorus dissolving capability, is suitable for activating phosphorus in soil in arid and semiarid regions, and can be used for remarkably improving the quick-acting phosphorus content of the soil in arid and semiarid regions and reducing the application of phosphate fertilizer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a strain of Leclericia sp.) CYIJM6 which is stored in China center for type culture Collection (China, with a storage number M20221561) at 10 and 12 of 2022.
The invention also provides application of the Lekkera CYIJM6 in dissolving phosphorus and dissolving phosphorus.
Preferably, the phosphorus in the phosphorus-dissolved phosphorus comprises organic phosphorus and/or inorganic phosphorus.
Preferably, the organic phosphorus comprises calcium phytate.
Preferably, the inorganic phosphorus comprises calcium phosphate.
The beneficial effects of the invention are as follows:
the strain provided by the invention has strong phosphorus dissolving capability, is suitable for activating phosphorus in soil in arid and semiarid regions, and can be used for remarkably improving the quick-acting phosphorus content of soil in arid and semiarid regions and reducing the application of phosphate fertilizer.
The phosphate-dissolving strain provided by the invention has wide action substrate, can release organic acid and other micromolecule activated fixed inorganic phosphorus, can secrete phosphatase to hydrolyze organic phosphorus, and can remarkably improve the activation capability of soil phosphorus by utilizing the phosphate-dissolving strain.
After the phosphorus-dissolving bacteria Leclericia sp.CYIJM 6 provided by the invention is cultured in a calcium phosphate (inorganic phosphorus) solid culture medium for 5 days, the phosphorus-dissolving index is 2.42, and the concentration of the soluble phosphorus reaches 430mg/L.
The invention provides a phosphorus-dissolving bacterium Leclericia sp.CYIJM 6 which is cultured in a calcium phytate (organic phosphorus) solid culture medium for 5 days, wherein the phosphorus-dissolving index is 2.85, and the phosphorus-dissolving amount is 228mg/L.
Description of biological preservation
Leclericia sp.) CYIJM6 was maintained at the China center for type culture collection (CCTCC NO: M20221561) at 10 and 12 days 2022, with the preservation number: university of martial arts.
Drawings
FIG. 1 shows the construction of a sequence phylogenetic tree by the method FastMinimum Evolution.
Detailed Description
The invention provides a strain of Leclericia sp.) CYIJM6 which is stored in China center for type culture Collection (China, with a storage number M20221561) at 10 and 12 of 2022.
The invention also provides application of the Lekkera CYIJM6 in dissolving phosphorus and dissolving phosphorus. In the present invention, the phosphorus in the phosphorus-dissolved phosphorus preferably includes organic phosphorus and/or inorganic phosphorus. In the present invention, the organic phosphorus preferably includes calcium phytate. In the present invention, the inorganic phosphorus includes calcium phosphate.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
1. Bacterial strain screening and preservation
(1) Collecting farmland plough layer soil in the double-tower area of the morning sun, and placing a sterile bag for low-temperature preservation. The dry and cold air of the Mongolia highland in the North of the morning is often invaded to form a semiarid semi-moist drought-prone region, so that the method is suitable for screening drought-resistant strains.
(2) 1g of the soil was placed in 100mL of an inorganic phosphorus screening liquid medium (glucose: 10g/L, ca) 3 (PO 4 ) 2 ：5g/L，MgCl 2 ：5g/L，MgSO 4 ·7H 2 O：0.25g/L，KCl：0.2g/L，(NH 4 ) 2 SO 4 :0.1 g/L), and placing the mixture in a constant temperature incubator at 28 ℃ for shake culture for 24 hours.
(3) 1mL of the culture suspension is inoculated into 100mL of freshly prepared inorganic phosphorus screening liquid culture medium, and the culture medium is placed in a constant temperature incubator at 28 ℃ for shake culture for 24 hours.
(4) Repeating (3) for 1 time, and taking culture suspension for gradient dilution 10 4 And 10 5 Double, 200 mu L of inorganic phosphorus-coated screening culture medium plate is taken and placed in a constant temperature incubator at 28 ℃ for culture for 24-48 hours.
(5) The strain with the larger transparent circle (i.e., the phosphate solubilizing circle) was selected and named as CYIJM6, and the phosphate solubilizing index (phosphate solubilizing circle diameter/colony diameter) was calculated. And the strain was purified on an inorganic phosphorus screening plate, and the monoclonal purified at least 3 times. The purified strain is mixed with fresh bacterial liquid by 50% glycerol in a ratio of 1:1, and stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃.
2. Identification of strains
The bacterial genome DNA rapid extraction kit is used for extracting DNA of a bacterial strain CYIJM6, and the concentration and purity of the extracted DNA are detected by Qubit2.0. The Polymerase Chain Reaction (PCR) was performed using the DNA as a template and 16S rRNA 27F/1429R as a primer. PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 56℃for 30s, extension at 72℃for 90s,30 cycles; extending at 72℃for 10min. After the quality of the PCR amplified product is detected by 1.5% agarose gel electrophoresis, the amplified fragment is sequenced by adopting a Sanger method, and the 16S rRNA sequence of the strain is shown as SEQ ID No. 1.
The sequences were aligned in the NCBI 16S rRNA database, and a sequence treeing was constructed by Fast Minimum Evolution method with the strain sequences having higher similarity (FIG. 1), and CYIJM6 was determined to be Leclericia.
TCGAACGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGTGGCGGACGGGTGAGTAACGTCTGGGAAACTGCCCGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGGGGGACCTTCGGGCCTCACACCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGGTGAAGTTAATAACTTCACCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTTCCCTTGAGGAGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGAC。
example 2
Determination of phosphorus activation ability (phosphorus-solubilizing amount):
(1) Preparing a bacterial suspension: 1 loop of the strain stored in example 1 was picked up by an inoculating loop, inoculated into a test tube containing LB medium, and cultured overnight at 28℃with shaking. Transferring the strain into a triangular flask filled with LB culture medium according to the inoculation amount of 2%, and culturing the strain at the constant temperature of 28 ℃ until the bacterial suspension OD600 apprxeq 1.
(2) Culturing: 1mL of the bacterial suspension was aspirated, placed in a 1.5mL centrifuge tube, washed 3 times with sterile water, and resuspended in sterile water.
(3) Absorbing 0.2mL of the washed bacterial suspension (inoculum size: 1%) and transferring to 20mL of liquid culture medium (measuring inorganic phosphorus dissolution capacity: inorganic phosphorus screening liquid culture medium as in example 1; measuring organic phosphorus dissolution capacity: calcium phytate culture medium (glucose: 10g/L, calcium phytate: 3g/L, mgCl) 2 ：5g/L，MgSO 4 ·7H 2 O：0.25g/L，KCl：0.2g/L，(NH 4 ) 2 SO 4 :0.1 g/L), culturing at 28 ℃ for 7d with constant temperature shaking, setting 3 parallel samples every 24 hours; meanwhile, 0.2mL of sterile water was aspirated instead of the bacterial suspension as a negative control.
(4) Standing at room temperature for 15min, pouring the fermentation broth into a centrifuge tube, centrifuging at 1500rpm for 3min, and collecting supernatant A; part of the supernatant A was centrifuged again at 10000rpm for 10min, and the supernatant B was collected.
(5) Measuring the concentration of bacterial liquid: 100. Mu.L of supernatant A was aspirated, 100. Mu.L of 1M HCl was added, and the OD was measured by a spectrophotometer 600 Values.
(6) Determination of available phosphorus:
absorbing a proper amount of supernatant B into a 50mL volumetric flask, adding 8mL of molybdenum-antimony anti-chromogenic liquid (0.528 g of ascorbic acid is dissolved in 100mL of molybdenum-antimony sulfate stock solution, and uniformly mixing, wherein the solution is required to be prepared at present, the molybdenum-antimony sulfate stock solution is prepared by weighing 6.0g of ground ammonium molybdate and dissolving in 200mL of water, weighing 0.146g of potassium antimony tartrate and dissolving in 50mL of water, weighing 74mL of concentrated sulfuric acid, slowly adding 400mL of water, continuously stirring and cooling, slowly mixing the three solutions, cooling, adding water to dilute the solution to a constant volume of 1000mL, shaking the solution, and then storing the solution in a brown reagent bottle), fixing the volume to 50mL by deionized water, shaking the solution fully and uniformly, and measuring the light absorption value of the solution at 880nm by a spectrophotometer after color development.
Drawing a standard curve: accurately sucking 5mg/L of phosphorus standard solution 0, 1, 2, 4, 6, 8 and 10mL, respectively placing into 50mL volumetric flasks, adding 5mL of molybdenum-antimony anti-reagent, and fixing the volume to 50mL with water. Shaking thoroughly, and measuring the absorbance of the solution at 880nm by a spectrophotometer after color development.
Phosphate solubilizing amount (mg/L) =ρXV 2/V1
Wherein: ρ -the mass concentration of phosphorus in the test solution (mg/L);
v1-aspirate supernatant volume B (mL);
v2-volume of solution developed (mL).
(7) Measuring the pH value: the pH of the supernatant B was measured with a pH meter.
The results were as follows:
after the phosphorus-dissolving bacteria Leclericia sp.CYIJM 6 is cultured in a calcium phosphate (inorganic phosphorus) solid culture medium for 5 days, the phosphorus-dissolving index is 2.42, and the phosphorus-dissolving amount reaches 430mg/L.
After phosphorus-dissolving bacteria Leclericia sp.CYIJM 6 is cultured in a calcium phytate (organic phosphorus) solid culture medium for 5 days, the phosphorus dissolution index is 2.85, and the phosphorus dissolution amount is 228mg/L.
Therefore, the phosphorus-dissolving strain provided by the invention has wide action substrate, can release organic acid and other small molecules to activate fixed inorganic phosphorus, can secrete phosphatase to hydrolyze organic phosphorus, and can remarkably improve the phosphorus activation capability of soil by utilizing the phosphorus-dissolving strain.
Example 3
And (3) preparation of a microbial inoculum:
the monoclonal colonies on the purification plates were inoculated into 5mL of LB liquid medium and shake-cultured overnight at 28℃at 180 rpm. Transferring into 100mL LB culture medium according to 1% inoculum size, shake culturing at 28deg.C at 180rpmTo the logarithmic phase of growth. After centrifugation at 8000rpm for 10min, the supernatant was discarded and the cells were collected. The bacteria were resuspended in sterile water and centrifuged at 8000rpm for 10min, and this step was repeated 2-3 times. The bacterial cells are finally resuspended in sterile water to OD 600 Bacterial suspension=1.0, bacterial concentration about 10 9 /mL。
Example 4
Application of strain in farmland ecosystem in semiarid region
Positioning tests (120.594192E, 41.720583N) are set in the peach blossom town in the double-tower region of the morning sun, and the region belongs to an easily arid region, and the mass water content of the soil in spring is about 12% -14%. The soil is subjected to traditional cultivation of one-year-one-harvest for many years before the test starts, and the planting mode is mainly corn. The test adopts random granule design, mainly comprises conventional fertilization (NPK; N:195 kg/hectare, P) 2 O 5 :75 kg/hectare, K 2 O:90 kg/hectare), phosphate fertilizer decrement application 20% (NP 80K; n:195 kg/hectare, P 2 O 5 :60 kg/hectare, K 2 O:90 kg/hectare) and 20% of a dosing fungicide (NP 80KL, N:195 kg/hectare, P 2 O 5 :60 kg/hectare, K 2 O:90 kg/hectare, the microbial inoculum prepared in example 3: 40-50L/hectare) 3 treatments, each treatment being repeated 4 times. Each cell area is 70m 2 (7 m×10m), the planted crop is corn. Crop yield and soil available phosphorus content were measured during harvest.
1. Crop yield determination: and (3) weighing the representative area S (3 ridges width multiplied by 5m length is a sampling range, recording the ridge distance calculation area) in the mature period of the corn, taking 10 ears of the representative corn according to an average ear weight method, calculating the seed yield and the water content, and calculating the actual yield according to the area of the sample points, the number of the ears, the actual fresh weight, the seed yield and the water content. Yield (14% moisture content) (kg/mu) = (666.7/(spot area) × spot total spike number x average grain fresh weight per spike sample× (1-sample grain moisture content)/(1-14%) ×0.85 (note: 0.85 is measurement coefficient for correcting human error).
2. Soil sample collection, namely, after surface impurities are removed in each plot treated by the test, soil samples of soil layers with the diameters of 0-10 cm and 10-20cm are collected by using a soil drill with the diameter of 3cm according to a five-point sampling method. Mixing soil samples of 5 sampling points of the same soil layer, removing impurities such as stones and plant roots, sieving with a 2mm sieve, uniformly mixing to form a soil sample, and naturally air-drying to determine the effective phosphorus content of the soil.
The results are shown in Table 1:
table 1 experimental results
| NPK | NP80K | NP80KL | |
| Yield (kg/mu) | 658.3±17.1 | 630.6±6.8 | 647.6±24.7 |
| Available phosphorus (mg/kg) | 37.4±1.6 | 36.5±4.4 | 40.6±5.2 |
After the bacterial strain is used for preparing the microbial inoculum, the bacterial strain can be used as a phosphorus synergist, an activating agent and an agricultural microbial inoculum. The phosphorus-dissolving bacteria and the chemical fertilizer are applied in a matched manner, so that the effective phosphorus content of soil and the fertilizer utilization efficiency can be improved, the application of the phosphate fertilizer is reduced by 20%, the stable yield of crops can be ensured, and the effective phosphorus content of the soil is improved by 8.6%.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (2)
1. Leclerosis (Leclericia sp.) CYIJM6 was maintained at the China center for type culture collection (CCTCC NO) at 10 and 12 days 2022: m20221561.
2. Use of the luxuriant bacillus CYIJM6 according to claim 1 for dissolving phosphorus;
the phosphorus in the phosphorus-dissolving and phosphorus-dissolving is calcium phytate and/or calcium phosphate.
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