CN111484948A - Root-promoting phosphate-solubilizing nitrogen-fixing bacillus and application thereof - Google Patents
Root-promoting phosphate-solubilizing nitrogen-fixing bacillus and application thereof Download PDFInfo
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Abstract
The invention provides a phosphate-solubilizing nitrogen-fixing bacillus for promoting rooting and application thereof, wherein the bacillus is Siamese bacillus FJAT-49376 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 16411. The bacillus of the invention can effectively degrade inorganic phosphorus, improve the content of soluble phosphorus in soil, obviously promote the growth of crops, develop root systems of the crops and enhance the stress resistance of the crops.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus strain capable of promoting growth, dissolving phosphorus and fixing nitrogen and application of the bacillus strain in plant growth.
Background
The phosphorus supply level of soil is one of the key factors influencing the growth of plants, 95 percent of phosphorus in the soil is in an invalid form, and the plants are difficult to directly absorb and utilize, so that the phosphorus deficiency phenomenon exists in 74 percent of cultivated land soil in China.
In a crop-microorganism interaction system, Plant growth-promoting rhizobacteria (PGPR) are colonized in the rhizosphere soil of crops, and can effectively decompose insoluble and fixed elements (phosphorus, potassium and the like) in the soil, promote the absorption of the crops on fertilizers and elements in the soil, and further promote the growth, yield increase, disease resistance and the like of the crops. Therefore, the microbial fertilizer with the efficient growth promoting function is screened and developed and applied to agricultural production, potential element resources of soil are fully utilized, and the microbial fertilizer has important significance for improving element shortage of soil such as phosphorus and potassium and the like, reducing environmental pollution and promoting agricultural sustainable development.
Disclosure of Invention
Therefore, a strain for promoting growth, dissolving phosphorus and fixing nitrogen is needed to be provided, and the problem that elements such as phosphorus, potassium and the like in soil cannot be absorbed and utilized by plants is solved.
In order to achieve the purpose, the inventor provides the following technical scheme:
a phosphorus-solubilizing nitrogen-fixing bacillus capable of promoting rooting is characterized in that: the Bacillus is Siamese Bacillus FJAT-49376 with the scientific name of Bacillus siemensis FJAT-49376, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC NO.16411, has the preservation date of 2018, 9 and 3 days and has the preservation address of the institute of microorganisms of China academy of sciences in Beijing, China.
The colony morphology of the bacillus FJAT-49376 is as follows: round, milky yellow, rough-edged, smooth-surfaced, flat, opaque, wet, medium-sized colonies.
Further, the phosphorus-dissolving nitrogen-fixing bacillus for promoting rooting is applied to degradation of insoluble phosphate.
Further, the application of the phosphorus-solubilizing nitrogen-fixing bacillus for promoting rooting in promoting plant seed germination is specifically characterized in that the bacillus fermentation liquor is prepared into the concentration of 1 × 105-1.5×105cfu/m L, soaking the strain for 2-3 days, and placing at 25-30 deg.C under illumination for 16-20 h/day.
Furthermore, the phosphorus-solubilizing nitrogen-fixing bacillus for promoting roots is applied to promoting the growth of plant roots.
Furthermore, the phosphorus-solubilizing nitrogen-fixing bacillus for promoting rooting is applied to plant nitrogen fixation.
Further, the phosphorus-solubilizing nitrogen-fixing bacillus for promoting rooting is applied to preparation of a phosphorus-solubilizing nitrogen-fixing composite microbial agent.
A plant growth promoting microbial inoculum comprises the bacillus.
The invention has the beneficial effects that:
(1) the bacillus of the invention can effectively degrade inorganic phosphorus, promote insoluble phosphate to release phosphorus, and improve the content of soluble phosphorus in soil, thereby obviously promoting the growth of crops, enabling the roots of the crops to be developed, and enhancing the stress resistance of the crops.
(2) The bacillus of the invention can improve the activity of plant seeds, promote the seeds to take root and sprout, and effectively shorten the growth cycle of plants.
(3) The bacillus of the invention has double effects of dissolving phosphorus and fixing nitrogen, and can be used as a microbial organic fertilizer for improving soil fertility. Nitrogen and phosphorus are essential elements for plant growth, and the bacteria capable of fixing nitrogen and dissolving phosphorus can provide sufficient nutrition for plants in soil deficient in phosphorus and less in nitrogen and improve the soil nutrition structure. If different azotobacter and phosphate-solubilizing bacteria are prepared into bacterial manure, competition between two or more bacteria may exist, and the effects of bacterial strain colonization and phosphate-solubilizing and nitrogen-solubilizing are influenced. The bacterial manure prepared from the bacterial strain with the functions of dissolving phosphorus and fixing nitrogen can avoid the competition effect and better provide nutrition for the growth of plants.
Drawings
FIG. 1 shows colony morphology of Bacillus strain FJAT-49376, wherein the left image shows colony growth state of FJAT-49376 strain on a whole plate, and the right image shows a partial enlarged view of colonies in the left image.
FIG. 2 is a tree showing the results of identifying the 16S rRNA sequence of Bacillus FJAT-49376 according to an embodiment.
FIG. 3 shows the effect of Bacillus FJAT-49376 on the growth of tomato seeds according to an embodiment.
FIG. 4 shows the growth promoting effect of Bacillus FJAT-49376 on tomato seeds according to an embodiment.
Detailed Description
To explain technical contents, achieved objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in combination with specific embodiments.
EXAMPLE 1 phosphate solubilization of Bacillus
1. Test materials
1.1 test strains
Test strains: bacillus FJAT-49376, which is isolated from mango of fruit tree base of Putian institute of agricultural science and research, is frozen and preserved in-80 ℃ glycerol, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 16411.
1.2 culture Medium
L B solid culture medium (purchased from the manufacturer) formula, i.e. 10g tryptone, 10g sodium chloride, 5g yeast powder, 15g agar, 1000m L water, pH 7.0, (2) L B liquid culture medium (purchased from the manufacturer) formula, i.e. 10g tryptone, 10g sodium chloride, 5g yeast powder, 1000m L water, pH 7.0.
Inorganic phosphorus medium (NBRIP medium): glucose 10g, Ca3(PO4)25g，MgCl2·6H2O 5g，KCl0.2g，MgSO4·7H2O 0.25g，(NH4)2SO40.1g, distilled water 1000m L, natural pH.
The organic phosphorus growth medium comprises 10g of glucose, 0.5g of ammonium sulfate, 0.5g of yeast extract powder, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate, 0.03g of ferrous sulfate, 0.03g of manganese sulfate, 0.2g of lecithin, 1.0g of calcium carbonate, 1000m of distilled water L, 15g of agar and pH 7.0-7.5.
1.3 preparation of test reagents
153m L concentrated sulfuric acid (analytically pure, density 1.84g/m L) is measured, slowly added into 400m L distilled water, continuously stirred and cooled, 10g of ground ammonium molybdate is also weighed and poured into the solution, stirred and dissolved, 0.5% (5 g/L) of antimony potassium tartrate solution 100m L is added, after cooling, water is added to dilute the solution to 1000m L, the solution is shaken evenly and stored in a brown reagent bottle, and the stock solution contains 1% of ammonium molybdate and 2.75mo L/L of sulfuric acid.
The molybdenum-antimony color-developing agent is prepared by weighing 1.50g ascorbic acid and dissolving in 100m L molybdenum-antimony storage solution, wherein the solution has short effective period and is suitable for use.
5 mg/L phosphorus Standard solution 0.4394g of potassium dihydrogen phosphate (KH) dried at 50 deg.C2PO4Analytically pure), 100m L water, 5m L concentrated sulfuric acid (preservation), water to make the volume 1L, the concentration of phosphorus 100 mg/L, the solution can be preserved for a long time, the solution 5m L is absorbed into a 100m L volumetric flask, water to make the volume 5 mg/L phosphorus standard solution, the solution is not suitable for long-term preservation.
2. Test method
2.1 determination of phosphate solubilizing ability
2.1.1 activation of the test strains
Taking out the test strain from a refrigerator at the temperature of minus 80 ℃, after the test strain is warmed to room temperature, streaking the test strain in an ultra-clean bench into a L B solid agar medium plate, inversely placing the test strain in a biological incubator for 2 d.2d at the temperature of 30 ℃, observing the colony morphology, selecting a single colony for secondary streaking culture, ensuring that the activated colony morphology is single, selecting a proper amount of the single colony in a L B liquid medium, and performing shake culture at the temperature of 30 ℃ for 2d to obtain a seed solution.
2.1.2 liquid Shake flask fermentation
Diluting the seed liquid by 2 times, and detecting OD by an enzyme-labeling instrument600nmCombining with bacterial count under microscope, diluting properly, and adjusting bacterial density to 108cfu/m L (bacterial liquid OD diluted 2 times)600nmBetween 0.3 and 0.5), 200 μ L was inoculated into 50m L centrifuge tubes containing 10m L liquid culture medium of organophosphorus and inorganic phosphorus, respectively, and shake-cultured at 230rpm and 30 ℃ for 6d, and two replicates of each test bacterium were inoculated with 200 μ L sterile water as a control.
2.1.3 detection of effective phosphorus content in supernatant by MoSb antibody method
a. Preparation of supernatant
And centrifuging the fermentation liquor cultured for 6d at 1200rpm for 30min, taking supernatant, and discarding the precipitate.
b. Drawing of standard curve
Respectively and accurately sucking 0, 2, 4, 6, 8 and 10m L of 5 mg/L phosphorus standard solution into a 50m L volumetric flask, diluting the solution to a position with water with the total volume of about 3/5, adding 2 drops of 2, 6-dinitrophenol as an indicator, adjusting 50m L/L dilute sulfuric acid (or hydrochloric acid) and 10 percent sodium hydroxide until the solution is just yellowish, accurately adding 5m L molybdenum-antimony anti-color developing agent, shaking up, adding water to fix the volume to obtain a standard solution series with phosphorus contents of 0.0, 0.2, 0.4, 0.8 and 1.0 mg/L, respectively, shaking up, standing at room temperature of more than 15 ℃, measuring the absorbance at a wavelength of 700nm, taking the absorbance as a vertical coordinate and the phosphorus concentration (mg/L) as a horizontal coordinate, and drawing a standard curve.
c. Determination of available phosphorus content in supernatant
Transferring appropriate amount of supernatant into 50m L volumetric flask, diluting with water to total volume of about 3/5, adding 1-2 drops of dinitrophenol indicator, accurately adding 5m L molybdenum antimony anti-color developing agent, shaking, adding water to desired volume, standing at room temperature above 15 deg.C for 30min, and reading absorbance OD700nmThen, the corresponding phosphorus content is checked from the standard curve.
d. Calculating the effective dissolved phosphorus content in the supernatant
The effective phosphorus content p (mg/L) of the supernatant is × colorimetric volume of the supernatant, × times of division/total volume of fermentation liquor
Wherein, the concentration of the supernatant fluid is that the concentration of phosphorus mg/L is found from a standard curve;
the colorimetric volume is 50m L of constant volume;
dividing times are the total volume of the fermentation liquor/sampling volume.
Effective phosphorus-dissolving amount P (mg/L) ═ effective phosphorus content of strain supernatant-control supernatant phosphorus content
2.2 phospholytic bacteria morphology and 16s rRNA identification
And (3) the morphology of the phosphate solubilizing bacteria, namely inoculating the purified bacillus onto an L B plate by streaking, culturing at the constant temperature of 30 ℃ for 48h, and observing the characteristics of the size, the color, the edge uniformity, the wettability and the like of a bacterial colony after the bacterial colony grows out.
The molecular identification is that pure strains are inoculated to L B liquid culture medium, the shaking table is placed at 30 ℃, after the strains are cultured to a logarithmic phase, the Tris-saturated phenol method is adopted to extract the genome DNA of the strain FJAT-49376, 16SrRNA gene universal primers 27F and 1492R are adopted to carry out PCR amplification, the PCR reaction program refers to the literature of Zhengxuefang and the like (Zhengxuefang, Liubo, Zhuyan, and the like, the screening and identification of the tomato bacterial wilt biocontrol bacillus is [ J ]. Chinese biological control institute, 2016,32(5):657 and 665.), the PCR products are sent to Shanghai Boshang biotechnology Limited to carry out Sanger sequencing, EZBIOCou is adopted to complete sequence homology comparison, and the sequence is analyzed by MEGA 6.0.6 software and the system development tree is constructed.
3. Test results
3.1 determination of phosphate solubilizing ability
The phosphorus solubilizing ability of Bacillus FJAT-49376 on organic and inorganic phosphorus is shown in Table 1. Experimental results show that the bacillus FJAT-49376 has a relatively obvious phosphate solubilizing effect on inorganic phosphorus, and has a relatively poor phosphate solubilizing effect on organic phosphorus.
TABLE 1 phosphate solubilizing ability of Bacillus FJAT-49376
It can be seen that Bacillus FJAT-49376, a phosphate-solubilizing (inorganic phosphate-degrading) microorganism, promotes apatite Ca3(PO4)2The insoluble phosphate releases phosphorus, and the content of soluble phosphorus in the soil is improved, so that the growth of crops can be obviously promoted, the root system of the crops is developed, and the stress resistance of the crops is enhanced.
3.2 identification of the Strain
3.2.1 Strain morphology
The colony morphology of FJAT-49376 is: round, milky yellow, rough-edged, smooth-surfaced, flat, opaque, wet, medium-sized colonies. The colony morphology is shown in FIG. 1.
Identification of 3.2.216S rRNA Gene
The nucleic acid sequence of the 16S rRNA gene of strain FJAT-49376 is as follows:
ATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGAGCCAGCCGCCGA(SEQ ID NO:1)
comparing the 16S rRNA gene sequence SEQ ID NO:1 of the strain FJAT-49376 with an EZBioloud gene database, wherein the genetic relationship between the strain FJAT-49376 and Bacillus siamensis is nearest, the homology of the 16S rRNA gene is 99.86 percent, so that the strain FJAT-49376 belongs to Bacillus siamensis Siamese Bacillus. Downloading 16S rRNA gene sequences of strains with higher homology, carrying out comparative analysis, constructing a phylogenetic tree, and forming the phylogenetic tree as shown in figure 2 when a neighbor-Joining method is adopted and the Bootstrap value is 1000 times. In the constructed phylogenetic tree, the strain FJAT-49376 and Bacillus siamensis are gathered in the same branch.
EXAMPLE 2 growth promoting action of Bacillus
1. Tomato growth promotion test method
Selecting single colony of test strain, inoculating into 250m L conical flask containing 100m L L B liquid culture, and culturing at 30 deg.C for 48 hr (counting bacteria under microscope, bacterial density is 10%8cfu/m L above) the test strain fermentation broth was diluted 1000-fold and clear water was used as control (ck).
Selecting tomato seeds with relatively consistent growth vigor, placing the tomato seeds in a transparent culture box of 9cm with 2-3 layers of filter paper laid at the bottom, placing 15 mung bean seeds in a constant-temperature artificial climate box at 27 ℃, and illuminating for 16h and dark for 8 h. Tracking and observing the tomato germination condition, recording the germination number until no new germination grains appear in 3d continuously, measuring the germ length of the tomato germination radicle machine, and analyzing the promoting effect of the test bacteria on tomato seed germination.
The germination percentage (number of germinated seeds on specified days/number of test seeds) is × 100%
The germination index is ∑ (Gt/Dt), wherein Gt is the number of seeds to be germinated at t d, and Dt is the corresponding number of days to be germinated.
Vigor index (germination index × embryo root length (cm)
The test data adopts DPS software and a new double-polarization method (Duncan) to carry out the significance test of the data difference among treatments.
2. Garlic growth promotion test method
Inoculating a test strain into L B liquid culture medium, culturing for 48h in a shaking table at 30 ℃ and 170rpm to obtain fermentation liquor, diluting the fermentation liquor by 1000 times with clear water, then putting the diluted fermentation liquor into a 150m L conical flask, putting garlic on a bottle mouth to ensure that the bottom of the garlic is soaked by bacterial liquid, taking the clear water as a Control (CK), replenishing water at proper time, culturing for 14d in a laboratory greenhouse at 25 ℃, and measuring the length, fresh weight and dry weight of roots.
3. Test results
3.1 tomato growth promotion test results
The comparison between the test group and the ck group is shown in Table 2, FIG. 3 and FIG. 4. the experimental results show that the bacterial concentration of the Bacillus FJAT-49376 is about 1 × 10 when the Bacillus FJAT-49376 is diluted 1000 times compared with the growth effect of the tomato seeds of the ck control group5-1.5×105The cfu/m L has germination rate, germination index, root length, stem length and seed vitality index of 110.53%, 105.34%, 32.74%, 74.30% and 34.48% of ck control, and the five indexes are obviously different from the ck control, but only the germination rate and the germination index are higher than those of the ck control group.
TABLE 2 growth promoting ability of Bacillus FJAT-49376 on tomato seeds
Note: in the above table, ab represents significant difference (P <0.05)
3.2 Garlic growth promotion test results
The growth of garlic roots is shown in table 3. The experimental result shows that compared with the CK group, the fresh weight of the root of the garlic in the FJAT-49376 bacteria liquid soaking group is increased by 30.38%, and the dry weight of the root is increased by 30.38%. Experimental results show that the FJAT-49376 bacterial liquid has a promoting effect on the growth of plant roots.
TABLE 3 growth promoting action of Bacillus FJAT-49376 on garlic
Example 3 Nitrogen fixation of Bacillus
1. Nitrogen fixation efficiency determination method (national standard NY411-2000)
Adding 100m L nitrogen-free medium (formula: 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate heptahydrate, 0.2g of sodium chloride, 5g of calcium sulfate, 10g of mannitol, 0.1g of calcium sulfate dihydrate, 1000m L of water and pH 7.0) into a 500m L triangular flask, sterilizing at 121 ℃ for 30min, performing aseptic operation, adding two rings of strains to be detected (or fermentation liquor obtained by culturing 1m L for 3 d) into each flask, placing the flasks on a shaking bed, performing shaking culture at 30 ℃ for 5-7d (120r/min), taking out the solution, determining sugar and nitrogen, determining sugar by adopting an anthrone photoelectric colorimetry method, and determining nitrogen by adopting a micro photoelectric colorimetry.
1.1 Anthracene ketone photoelectric colorimetry
1.1.1 test treatment, namely taking 1-4m L (depending on the sugar content) of fermentation culture liquid, diluting to 100m L, taking 1.00m L of the diluted liquid in a colorimetric tube, adding water to 2m L, adding 4m L of anthrone in each tube, shaking up, boiling and heating for 15min, cooling, carrying out colorimetric determination at 620nm, recording absorbance, and simultaneously carrying out blank test.
1.1.2 Standard Curve is drawn, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00 and 20.00m L of 1.000mg/m L glucose standard solution is sucked into a 100m L volumetric flask, water is added to the volumetric flask to the scale, the solution contains 10, 20, 40, 60, 80, 100 and 200 mug/m L of sugar respectively, 1.00m L is sucked into a colorimetric tube, water is added to 2.00m L, the absorbance is recorded according to the method of 1.1.1, the abscissa is defined by the sugar content of the standard system, and the standard curve is drawn by the ordinate.
1.1.3 calculation of analytical results
The sugar content (X) in the 100m L solution was calculated according to the formula (C1):
in the formula: m is1- -the test sugar content, μ g/m L, found on the standard curve;
m2- -on the standard curveThe sugar content of the blank test is found to be μ g/m L;
m-suction fermentation volume, m L.
Tolerance difference: a) taking the arithmetic mean value of the parallel measurement as a measurement result;
b) the permissible difference of the results of the parallel measurement is not more than 0.005g/100m L.
1.2 azotobacter liquid total nitrogen colorimetric determination method
1.2.1 preparation and testing of samples, absorbing 1.00m L of azotobacteria liquid into a 30m L digestion tube, adding 3m L of sulfuric acid, adding 0.1g of catalyst and 5 drops of hydrogen peroxide for digestion until the solution is clear, adding 1-2 drops of hydrogen peroxide after cooling if the reaction solution is not well cooked, continuing to cook until the solution is colorless and transparent, taking down and cooling, adding a little distilled water, shaking up, adding 40% (m/V) of sodium hydroxide solution until copper hydroxide precipitation (about 11-12m L) appears, adding 20 drops of 50% (m/V) of potassium sodium tartrate solution to remove the calcium and magnesium hydroxide precipitate, transferring the test tube liquid into a 100m L volumetric flask, pouring the digestion tube into the volumetric flask together with the digestion solution, diluting to the scale, shaking up, filtering, placing the filtrate 10.00m 2 into a 493 tube, adding 2 mol/1.00 m L of sodium hydroxide, adding 1.00m 5 of sodium tartrate, adding 7.1g of Neishi reagent, 10.00m 2 into a 493 2, adding water, stirring and then pouring into a 5m of sodium hydroxide solution into a yellow bottle, stirring, standing still, adding a small amount of sodium hydroxide solution, dissolving the solution into a yellow wine bottle, adding water, standing and cooling, adding water, dissolving the solution before adding water, dissolving the solution into a yellow wine, standing until the solution with absorbance measuring, measuring the solution, adding water, stirring, standing and measuring the solution, adding water, stirring.
1.2.2 drawing of Standard Curve
Accurately weighing 0.4716g of high-grade reagent ammonium sulfate which is baked at 105 +/-5 ℃ for 1h till constant weight, dissolving in water, diluting to 100m L, wherein the solution contains nitrogen of 1mg/m L, taking 0.05, 0.10, 0.25, 0.50, 1.00 and 2.00m L of the solution, putting the solution into a 100m L volumetric flask, diluting to scale with water, wherein the nitrogen content is respectively 0.50, 1.00, 2.50, 5.00, 10.00 and 20.00 mu g/m L, taking 1m L of each standard solution, putting the standard solution into a colorimetric tube, adding water to 2m L, measuring according to a 1.2.1 method, and recording the absorbance.
And drawing a working curve by using the nitrogen content of the standard solution as an abscissa and the corresponding absorbance as an ordinate.
1.2.3 calculation of analytical results
The nitrogen (X) content, expressed in g/100m L, is calculated according to formula (C2):
in the formula: m is1- -the test nitrogen content found on the standard curve,. mu.g/m L;
m2- -the nitrogen content of the blank, μ g/m L, found on the standard curve;
m-suction fermentation volume, m L.
Tolerance difference: a) taking the arithmetic mean value of the parallel measurement as a measurement result;
b) the permissible difference of the results of the parallel measurement is not more than 0.005 g.
1.3 Nitrogen fixation efficacy calculation
Nitrogen-fixing bacteria take up milligrams of nitrogen from the air per 1g of carbohydrate (sugar) consumed, and nitrogen-fixing potency is expressed as mg nitrogen/g sugar.
2. Test results
The experiment shows that the sugar consumption of the bacillus FJAT-49376 is 92.80 mg/L, the nitrogen fixation amount is 2.39 mg/L, and the nitrogen fixation efficiency is 25.75 mg/g.
In summary, Bacillus FJAT-49376 has the functions of phosphate-dissolving and growth-promoting, and in the practical application process, fermentation liquor diluted 1000 times, namely concentrate 1 × 10 can be used5Soaking plant seeds at cfu/m L or more to promote seed germination, or diluting 1000 times of fermentation liquid, i.e. bacterial concentration 1 × 105-2×105On the other hand, the bacillus FJAT-49376 has better phosphate-solubilizing effect and nitrogen-fixing efficiency, can be directly applied as a microbial organic fertilizer to improve soil fertility and better provide nutrition for plant growth, or can be freeze-dried to prepare a solid microbial organic fertilizer for convenient storage and transportation and can be used after being dissolved in water, and the bacillus FJAT-49376 can be mixed with other functional strains to prepare a composite microbial inoculum to further prepare the composite microbial inoculumImproving the effect of dissolving phosphorus and fixing nitrogen.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Sequence listing
<110> institute of agricultural biological resources of academy of agricultural sciences of Fujian province
<120> phosphate solubilizing and nitrogen fixing bacillus for promoting rooting and application thereof
<130>49376
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1424
<212>DNA
<213> Siamese Bacillus (Bacillus siamensis)
<400>1
atacatgcag tcgagcggac agatgggagc ttgctccctg atgttagcgg cggacgggtg 60
agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc ggggctaata 120
ccggatggtt gtctgaaccg catggttcag acataaaagg tggcttcggc taccacttac 180
agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc 240
gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300
gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt 360
gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtgccgttca 420
aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag 480
ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540
gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact 600
ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga 660
gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720
gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780
gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc 840
tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt 900
ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 960
acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc 1020
gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1080
gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg 1140
gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200
agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg 1260
gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat 1320
gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1380
aacacccgaa gtcggtgagg taacctttat gagccagccg ccga 1424
Claims (8)
1. A phosphorus-solubilizing nitrogen-fixing bacillus capable of promoting rooting is characterized in that: the Bacillus is Siamese Bacillus FJAT-49376 with the scientific name of Bacillus siemensis FJAT-49376, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC NO.16411, has the preservation date of 2018, 9 and 3 days and has the preservation address of the institute of microorganisms of China academy of sciences in Beijing, China.
2. Use of the root-promoting Bacillus phosphorus-solubilizing and nitrogen-fixing bacterium of claim 1 for degrading a sparingly soluble phosphate.
3. Use of the root-promoting Bacillus azotobacter phosphate-solubilizing bacterium of claim 1 for promoting germination of plant seeds.
4. The application of the phosphorus-solubilizing nitrogen-fixing bacillus for promoting roots to germinate in the plant seeds as claimed in claim 3, wherein the bacillus fermentation liquid is prepared to have a concentration of 1 × 105-1.5×105cfu/m L, soaking the strain for 2-3 days, and placing at 25-30 deg.C under illumination for 16-20 h/day.
5. Use of the root-promoting Bacillus azotobacter phosphate-solubilizing bacterium of claim 1 for promoting root growth in plants.
6. The use of the root-promoting Bacillus phosphorus-solubilizing and nitrogen-fixing bacterium of claim 1 in nitrogen fixation in plants.
7. The application of the phosphorus-solubilizing nitrogen-fixing bacillus for promoting rooting according to claim 1 in preparation of a phosphorus-solubilizing nitrogen-fixing composite microbial agent.
8. A plant growth promoting microbial inoculum is characterized in that: the microbial inoculum comprises the bacillus of claim 1.
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| CN114940958A (en) * | 2022-05-23 | 2022-08-26 | 贵州大学 | Bacillus siamensis, prepared microbial agent and application thereof |
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| CN114940958A (en) * | 2022-05-23 | 2022-08-26 | 贵州大学 | Bacillus siamensis, prepared microbial agent and application thereof |
| CN114940958B (en) * | 2022-05-23 | 2023-06-27 | 贵州大学 | Siamese bacillus, microbial agent prepared from same and application of microbial agent |
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