CN111593001A - Bacillus megaterium with phosphate solubilizing capability and application thereof in dissolving phosphate rock powder - Google Patents
Bacillus megaterium with phosphate solubilizing capability and application thereof in dissolving phosphate rock powder Download PDFInfo
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Abstract
The invention relates to a bacillus megaterium with phosphate solubilizing capability and application thereof in dissolving phosphate rock powder, belonging to the technical field of agricultural biology. The yield of the phosphate fertilizer in China cannot meet the requirement of agricultural production, and a large amount of phosphate fertilizer is imported every year; lack of high-concentration high-quality phosphate fertilizer and the like. These problems seriously hinder agricultural production, so methods and ways for improving fertilizer quality and soil fertility should be found, for example, microorganisms are used to activate phosphate rock powder or insoluble phosphorus in soil to release ineffective phosphorus in soil, and the ineffective phosphorus is converted into phosphorus which can be used by plants to improve soil fertility. Research shows that the bacillus megaterium HX63 strain can secrete organic acid and produce phosphatase, can improve the germination rate of seeds, promote the growth and development of plants and the like, and can be used for dissolving insoluble phosphorus. Therefore, the bacillus megaterium HX63 strain can provide good strain resources for the development of microbial fertilizers, and has good development and application prospects.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a bacillus megaterium HX63 strain, which has a phosphate solubilizing capability and particularly has a good dissolving effect on phosphate which is difficult to dissolve in soil.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Phosphorus is an important organic compound component in crops, and the components of nucleic acid, nucleoprotein, phospholipid, phytosine, adenosine phosphate and a plurality of enzymes in crops contain phosphorus which is an important component element of nucleic acid. The normal supply of phosphorus is beneficial to cell division and proliferation and promotes the growth and development of crops. When the supply of phosphorus is insufficient, the synthesis of nucleic acid and nucleoprotein is influenced, the formation and proliferation of cells are inhibited, and the growth and development of crops are stopped, the root system is dysplastic and the plants are short. Phosphorus is a regulator of crop metabolic processes. The synthesis, decomposition, interconversion and transfer of carbohydrates in crops require the participation of phosphorus. In photosynthesis, phosphorus participates in photosynthetic phosphorylation and stores light energy in ATP while forming NADPH. The synthesis of sucrose and starch in crops also requires the participation of phosphorus. Since hexoses need to be phosphorylated in the crop to form hexose phosphates before sucrose and starch can be synthesized. Phosphorus also promotes carbohydrate transport. The running of the cane sugar is influenced by the lack of the phosphoric acid, so that the sugar is accumulated, and the formation of anthocyanin is facilitated; phosphorus is a component of various enzymes in the nitrogen metabolism process in plants, and can promote the processes of amination, deamination, transamination and the like; phosphorus is also beneficial to the transformation and utilization of nitrate nitrogen in plants; can change the phosphorus nutrition of leguminous crops, increase the nodulation quantity and the quality of a single root nodule, and is beneficial to improving the nitrogen fixation efficiency of leguminous plants. Besides, phosphorus can promote fat metabolism, and enhance the drought resistance, cold resistance, disease resistance and lodging resistance of crops, thereby enhancing the stress resistance of the crops.
Inorganic phosphorus compounds in soil are mainly classified into three types, one type is calcium phosphate and magnesium phosphate compounds, and the compounds mainly exist in alkaline or neutral soil; the other class is iron phosphate and aluminum compounds which are mainly present in acid soil; the third type is occluded phosphorus which is insoluble phosphate formed after phosphate is wrapped by an iron-aluminum compound adhesive film. There are several ways of phosphorus immobilization, which can be classified into two categories: precipitation and adsorption.
Phosphorus exists in various forms in powdered rock phosphate, such as Ex-P (Exchangeable/Loose boom), Al-P (Aluminum-boom), Fe-P (Iron-boom), Ca-P (organic and Ca-boom), De-P (tertiary and alternate), Or organic boom P. Wherein Ex-P represents loosely absorbed and exchangeable phosphorus, which is easily leached out due to its relatively loose binding; Al-P exists in rock mainly in a chemisorption form, the release of Al-P is susceptible to time, and the Al-P in the rock can be converted into easily soluble or weakly adsorbed phosphorus; Fe-P refers to phosphate or hydroxide coprecipitated by ferric oxide, and the content of the Fe-P is greatly influenced by the input of exogenous phosphorus; De-P is mainly from magma or metamorphic rock and is formed by coating a layer of ferric hydroxide colloid outside phosphate; Ca-P refers to autologous and biological apatite and calcium carbonate, including bone and algae of biological bone fragments such as fish; Occ-P (Occluded-bound phosphorus) refers to phosphorus in a scooped form and is not readily available to the organism; Or-P refers to phosphorus bound to organic matter.
The phosphorus absorbed by crops (including inorganic phosphorus and organic phosphorus) is mainly inorganic phosphorusMainly comprises the following steps. Among inorganic phosphorus, orthophosphate is the main form absorbed by crops. In addition, the metaphosphate and pyrophosphate can be absorbed by crops and can be quickly hydrolyzed into orthophosphate in vivo for the crops to utilize. H2PO4 -Most easily absorbed by crops, HPO4 2-Then, PO4 3-Can only exist in strong alkaline medium, and is not suitable for the absorption of crops. Among the organic phosphorus compounds, hexose phosphate, sucrose phosphate, glycerophosphate, ribonucleic acid, phytochemicals and the like can be absorbed and utilized by crops, but part of organic phosphorus in the soil cannot be directly absorbed and utilized by the crops, so that the content of available phosphorus in the soil is increased, the yield of the crops is increased, and the increase of phosphate fertilizer is not feasible. The excessive application of the phosphate fertilizer makes the available phosphorus content of the soil and the dosage of the phosphate fertilizer in negative correlation. Not only increases the agricultural cost, but also causes the soil to accumulate too much ineffective phosphorus which can not be absorbed and utilized by crops, thereby causing soil hardening. Part of the water also enters the water body through runoff, so that the water body is eutrophicated. The phosphate solubilizing microorganisms can convert ineffective phosphorus in soil into effective phosphorus, and some can also secrete phosphatase to directly hydrolyze insoluble organophosphorus compounds into phosphorus which can be absorbed and utilized by plants, thereby promoting the growth and development of crops.
Microorganisms in nature participate in the geochemical cycle of phosphorus, the action is manifold, the decomposition action of the microorganisms on the phosphorus is stronger than the synthesis action, and the microorganisms can promote the effectiveness of the phosphorus in soil. The decomposition of phosphorus-containing compounds by microorganisms is divided into decomposition of organic phosphorus compounds and decomposition of inorganic phosphorus compounds. The former is mainly a result of involvement of various enzymes produced by microorganisms. In this complex of soil, the organic phosphorus changes very complexly and often forms some recalcitrant compounds. For example, organic phosphorus compounds in peat and humus can form complexes with certain organic substances. These complex compounds are only broken down by the action of the corresponding enzymes of the microorganisms. For example, bacillus hydrolysis of phytochemicals is an important source of rhizosphere available phosphorus, and bacterial phytase production hydrolyzes phytochemicals.
However, the inventor finds that: although the prior art has used phosphorus-dissolving microorganisms to solve the phosphorus deficiency of soil, the effect is not ideal.
Disclosure of Invention
Aiming at the blank in the prior art, the invention adopts a solid culture medium which takes inorganic tricalcium phosphate as the only phosphorus source, obtains the phosphate solubilizing strain by preliminary screening, and finally determines the phosphate solubilizing capability of the phosphate solubilizing microbial strain and the dissolving capability of various different insoluble phosphates by further utilizing a shake flask experiment and secondary screening. It was identified as Bacillus megaterium (Bacillus megaterium) using 16SrDNA and physiological and biochemical experiments. Further, the effect of the phosphorus-containing microbial strains on promoting the dissolution of the phosphate rock powder is determined through a shake flask experiment and a pot experiment, and the influence of the phosphorus-containing microbial strains on the growth of the plants after the phosphorus-containing microbial strains are mixed with the phosphate rock powder is verified through the pot experiment. The result shows that the phosphate solubilizing microorganism HX63 strain can be used as a microbial phosphate fertilizer to be applied to farmlands, and has important application value and practical significance for improving soil fertility and reducing the use amount of phosphate fertilizer.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention provides a Bacillus megaterium with phosphate-solubilizing capability, which is classified and named as Bacillus megaterium HX63 strain and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.19471, and the preservation date is as follows: and 3, 13 months in 2020, the preservation address is: the institute of microbiology, institute of Zhongkou institute of western-style No.1 Hospital, Beijing, Chaoyang, North Chen.
In a second aspect of the present invention, there is provided the use of the above-mentioned Bacillus megaterium having phosphate solubilizing ability for promoting phosphate solubilization.
The research finds that: when the HX63 strain takes insoluble phosphate as the only phosphorus source, organic acid and phosphatase are generated, thereby promoting the dissolution of the insoluble phosphorus by the HX63 strain.
In a third aspect of the invention, the application of the bacillus megaterium with the phosphate solubilizing capability in promoting the dissolution of organic phosphorus, lecithin or calcium phytate is provided.
In a fourth aspect of the invention, the application of the bacillus megaterium with the phosphate solubilizing capability in improving the fertilizer efficiency of the powdered rock phosphate is provided.
After the bacillus megaterium HX63 strain and the ground phosphate rock are mixed and applied, the bacillus megaterium HX63 strain can be well colonized on the surface of the ground phosphate rock, the release of phosphorus and other beneficial plant growth elements in the ground phosphate rock is effectively promoted, and the absorption and utilization of soil nutrient elements by plants are improved.
In a fifth aspect of the invention, the application of the bacillus megaterium with phosphate solubilizing capability in improving the germination rate of seeds is provided.
In a sixth aspect of the invention, the application of the bacillus megaterium with phosphate solubilizing capability in promoting plant growth and development is provided.
After the bacillus megatherium HX63 and the phosphate rock powder are mixed and applied, the release of phosphorus and other plant growth beneficial elements in the phosphate rock powder is promoted, so that the absorption and utilization of soil nutrient elements by plants are improved, and the growth of the plants is promoted.
In a seventh aspect of the invention, the application of the bacillus megaterium with phosphate solubilizing capability in improving the tolerance of plants to heavy metals and antibiotics is provided.
The bacillus megaterium HX63 phosphate solubilizing strain can secrete organic acid and produce phosphatase, has resistance to partial heavy metals and antibiotics, and has good biological safety.
The invention has the beneficial effects that:
(1) the bacillus megaterium HX63 strain has a good dissolution promoting effect on soil insoluble phosphate, the inventor further determines a phosphorus dissolution mechanism of the strain, and the result shows that when the HX63 strain takes the insoluble phosphate as a unique phosphorus source, organic acid and phosphatase are generated, so that the dissolving of the HX63 strain on the insoluble phosphate is promoted, and the research result has a good theoretical guiding significance on the aspects of improving soil fertility, reducing chemical phosphate fertilizer application amount and the like when the bacillus megaterium P17 strain is used in fields.
(2) The bacillus megaterium HX63 phosphate solubilizing strain can secrete organic acid and produce phosphatase, has resistance to partial heavy metals and antibiotics, and has good biological safety.
(3) The bacillus megaterium HX63 phosphate solubilizing strain is simple in use method, good in solubilizing effect, strong in practicability and easy to popularize.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph of the seedling of the Shanghai green in the potting experiment of example 3.
FIG. 2 and FIG. 3 are the growth charts of the Shanghai green seedlings after the Hubei phosphate rock powder mixed in example 3 is transplanted.
FIG. 4 is a graph showing the growth of the plant of Haematococcus chinensis after the powdered rock phosphate from different sources and powdered rock phosphate are mixed in example 3.
FIG. 5 shows the dried and crushed plant samples of example 3.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The method comprises the steps of firstly separating a strain of phosphate solubilizing bacteria from soil at a phosphate rock powder gathering place, and carrying out plate separation, screening and purification on inorganic tricalcium phosphate culture medium to obtain the phosphate solubilizing bacteria; the strain is further identified and named as HX63 strain, the bacterial colony is small and wet, the edge is round, the color is milky white, the growth of the strain is consistent under the observation of a microscope, the strain is rod-shaped, and spores are generated at the later growth stage.
The strain is obtained, identified to be Bacillus megaterium, and prepared into freeze-dried powder for refrigerator preservation.
The inventors further studied the strain HX63 of the phosphate solubilizing microorganism obtained as described above, and the results were as follows:
the dissolving effect of the phosphate-solubilizing bacteria HX63 strain on the artificially synthesized insoluble inorganic phosphate is good, and the shaking experiment result shows that after inoculation treatment, the effective phosphorus content of fermentation liquor filtrate containing calcium phosphate, iron phosphate and aluminum phosphate is improved compared with that of an inoculation-free control; specific data are shown in table 1.
Respectively inoculating the Bacillus megaterium HX63 strain into corresponding culture media by using physiological and biochemical experiments, observing experimental phenomena, recording experimental results, and preliminarily identifying the Bacillus megaterium HX63 strain as Bacillus megaterium.
Preparing an LB culture medium, inoculating a phosphate solubilizing microorganism HX63 strain, then carrying out proper culture, extracting 16SrDNA of the strain, carrying out PCR amplification and sequencing by using a universal primer, comparing the sequence on an NCBI website, and determining the HX63 strain as Bacillus megaterium (Bacillus megaterium), wherein the specific results are shown in Table 2.
The biological characteristic research of the phosphate solubilizing bacteria HX63 strain shows that the Bacillus megaterium HX63 strain has good plant safety, can influence the germination rate of seeds, and has certain tolerance to heavy metals, antibiotics and the like.
And (3) further determining the dissolving effect of the bacillus megaterium HX63 strain on the phosphate rock powder and the influence on the growth and development of plants by using a pot experiment. As a result, it was found that: in a pot experiment, the bacillus megaterium HX63 strain can effectively activate insoluble phosphate in phosphate rock powder and better promote plant growth, the overground height of the plant is higher than that of the plant treated by only applying phosphate rock powder, other nitrogen fertilizers and potassium fertilizers, the number of leaves is more than that of the plant treated by mixing the bacillus megaterium HX63 strain and the phosphate rock powder, and the results are shown in Table 5. The demonstration shows that after the phosphate rock powder and the bacillus megatherium HX63 are mixed and applied, the bacillus megatherium HX63 bacterial strain can be well colonized on the surface of the phosphate rock powder, the release of phosphorus and other beneficial plant growth elements in the phosphate rock powder is effectively promoted, the absorption and utilization of soil nutrient elements by plants are improved, and the growth of the plants is promoted.
The research result shows that the bacillus megaterium HX63 strain has a good dissolution promoting effect on soil insoluble phosphate, the inventor further determines a phosphorus dissolution mechanism of the strain, and the result shows that when the HX63 strain takes the insoluble phosphate as a unique phosphorus source, organic acid and phosphatase are generated, so that the dissolving of the HX63 strain on the insoluble phosphate is promoted, and the research result has a good theoretical guiding significance on the aspects of improving soil fertility, reducing chemical phosphate fertilizer application amount and the like when the bacillus megaterium P17 strain is used in fields.
The inventor carries out biological preservation on a phosphate solubilizing microorganism HX63 strain, and the specific preservation information is as follows:
preservation time: year 2020, 3, 13;
the name of the depository: china general microbiological culture Collection center;
the preservation number is: CGMCC No. 19471;
the address of the depository: the institute of microbiology, Siro No.1 Hospital, Kyoto, Beijing, Chaoyang;
and (3) classification and naming: bacillus megaterium (Bacillus megaterium).
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
The culture medium and components used in the embodiment of the invention are as follows:
calcium phosphate solid culture medium containing glucose 10.0g and (NH)4)2SO40.5g、NaCl 0.3g、KCl 0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·H2O 0.03g、Ca3(PO4)25.0g、H21000ml of O, 15.0-20.0 g of agar powder, 7.0-7.5 of pHs, and sterilizing for 30min at 105 ℃.
Calcium phosphate liquid culture medium containing glucose 10.0g and (NH)4)2SO40.5g、NaCl 0.3g、KCl 0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·H2O 0.03g、Ca3(PO4)25.0g、H2O1000 ml, pH7.0-7.5, sterilizing at 105 deg.C for 30 min.
The solid culture medium of the ground phosphate rock comprises 10.0g of glucose and (NH)4)2SO40.5g、NaCl 0.3g、KCl 0.3g、MgSO4·7H2O 0.30g、FeSO4·7H2O 0.03g、MnSO4·H2O 0.03g、H21000ml of O, 5.0g of ground phosphate rock, 15.0-20.0 g of agar powder, pH 7.0-7.5, and sterilizing for 30min at 105 ℃.
The liquid culture medium of the phosphate rock powder comprises 10.0g of glucose and (NH)4)2SO40.5g、NaCl 0.3g、KCl 0.3g、MgSO4·7H2O 0.30g、FeSO4·7H2O 0.03g、MnSO4·H2O 0.03g、H21000ml of O, 5.0g of ground phosphate rock, pH 7.0-7.5, and sterilizing for 30min at 105 ℃.
Organic phosphorus (yolk) solid culture medium comprising peptone 10.0g, beef extract 3.0g, NaCl 5.0g, agar powder 15.0g, and H2O1000 ml, pH7.0, and sterilizing at 121 deg.C for 20 min. When in use, 3.0ml of fresh egg yolk liquid (the egg yolk is mixed with normal saline in equal proportion) is added into every 50ml of the egg yolk liquid.
Organic phosphorus (calcium phytate) solid culture medium, glucose 10.0g, (NH)4)2SO40.5g、NaCl 0.3g、KCl 0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·H20.03g of O, 1000ml of deionized water, 5.0g of calcium phytate, 15.0g of agar powder and pH of 7.0-7.5. Sterilizing at 105 deg.C for 30 min.
Organic phosphorus (calcium phytate) liquid culture medium, glucose 10.0g, (NH)4)2SO40.5g、NaCl 0.3g、KCl 0.3g、MgSO4·7H2O 0.3g、FeSO4·7H2O 0.03g、MnSO4·H20.03g of O, 1000ml of deionized water, 5.0g of calcium phytate and 15.0g of agar powderg. The pH value is 7.0-7.5. Sterilizing at 105 deg.C for 30 min.
LB solid culture medium comprises 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl and 15.0g of agar powder. Sterilizing at 121 deg.C for 30 min.
LB liquid culture medium comprises peptone 10.0g, yeast powder 5.0g, and NaCl 10.0 g. Sterilizing at 121 deg.C for 30 min.
HX63 strain seed medium: 10g of glucose, 0.5g of ammonium sulfate, 0.3g of potassium chloride, 0.03g of ferrous sulfate heptahydrate, 0.3g of sodium chloride, 0.3g of magnesium sulfate heptahydrate, 0.03g of manganese sulfate monohydrate, 2g of monopotassium phosphate and 1000ml of water, wherein the pH value is 7.2-7.4, and the mixture is sterilized at 105 ℃ for 30min for later use.
HX63 strain fermentation medium: respectively changing the types of a carbon source and a nitrogen source on the basis of a seed culture medium; initial pH value of the culture medium; the culture temperature; inoculating amount; sterilizing the culture medium with inorganic salt concentration at 105 deg.C for 30 min.
Example 1
Dissolution promoting effect of bacillus megatherium HX63 strain on insoluble phosphate and powdered rock phosphate
Inoculating slant strain of activated phosphate solubilizing bacteria HX63 strain in seed culture medium, culturing at 28 deg.C for 48hr, and counting the cell concentration to 108cfu ml-1And then is ready for use. Respectively setting 3 treatments of inoculating bacteria, inoculating killed bacteria and adding culture medium, repeating 3 treatments, respectively inoculating HX63 strain seed liquid in a fermentation culture medium containing calcium phosphate according to the inoculation amount of 10%, and culturing at 28 deg.C for 7 d. Sterilizing the fermentation broth at 121 deg.C for 40min, adding 6% H2O22 drops in a triangular flask, and 60 ℃ water bath for 48hr to destroy the thallus. The fermentation liquor is filtered by phosphorus-free filter paper, the volume is determined to be 55ml, and the content of available phosphorus in the filtrate is measured by a molybdenum-antimony colorimetric method. The dissolving effects of the HX63 strain on aluminum phosphate, iron phosphate and powdered rock phosphate from different sources are respectively detected by the same method. The results are shown in tables 1 to 3.
As can be seen from the results in tables 1 to 3, Bacillus megaterium HX63 strain was Ca-resistant3(PO4)2、AlPO4、FePO4Has different dissolution promoting effects, wherein the dissolution promoting effect on the ferric phosphate is the best. HX63 has solubilizing effect on insoluble phosphateBecause the HX63 strain probably produces more metabolites which are beneficial to dissolving phosphorus, the pH value drops quickly, so that more available phosphorus in the insoluble phosphate is dissolved out.
Example 2
Physiological and biochemical characteristics and 16SrDNA identification of Bacillus megaterium HX63 strain
Gram staining: sampling and smearing HX63 strain on logarithmic phase fermentation liquid, air-drying, fixing, staining with crystal violet for 1min, and washing with water; mordanting with Lugol iodine solution for 1min, washing with water, decolorizing with 95% ethanol for 20-25 s, re-staining with safranin, washing with water, air drying, observing thallus characteristics and gram staining condition by microscopic examination, and recording microscopic examination result.
And (4) spore observation: taking a ring of fermentation liquor of HX63 in the death period, dying the smear after airing, staining the smear with malachite green, and observing whether spores exist and the growth condition of the spores through microscopic examination.
V.p. reaction detection: and (3) taking each strain fermentation liquor with good growth in an acetyl methyl methanol (V-P) culture medium, adding a V.P. indicator into each fermentation liquor, and observing and recording the color change of the fermentation liquor.
Sugar fermentation experiment: grinding sugar fermentation tube with grinding wheel, inoculating fresh thallus Porphyrae into the fermentation tube with inoculating needle, culturing at proper temperature in inverted state, and observing whether the bacteria produce acid and gas after 48 hr.
Gelatin liquefaction experiment: inoculating thallus Porphyrae into fresh culture medium by puncture inoculation method, culturing at proper temperature, and observing whether gelatin is liquefied in ice bath.
Starch hydrolysis experiments: inoculating fresh lawn onto starch hydrolysis culture medium, and culturing at proper temperature. After culturing for several days, the iodine solution is dripped on the flat plate, the flat plate is blue-black, and a non-discoloring transparent ring is formed around the colony, which indicates that the starch is hydrolyzed positively; it was still negative in blue-black.
Citrate utilization: and (4) streaking and inoculating on the slant of the citrate culture medium, and culturing for 3-7 d at a proper temperature. The medium is alkaline (indicator blue or pink) and positive, otherwise negative.
Indole experiments: and slowly adding an indole reagent with the height of 3-5 mm into the culture solution cultured for 1, 2, 4 and 7 days along the tube wall, wherein the interface red of the liquid layer is a positive reaction. If not obvious, adding 4-5 drops of ether into the culture solution, shaking to disperse the ether, and observing.
And (3) contact enzyme determination: the strain is taken out and placed on a glass plate dripped with 3% of hydrogen peroxide, if bubbles are generated, the strain is positive, and if no bubbles are generated, the strain is negative.
And (3) salt resistance determination: inoculating the lawn in inorganic phosphorus culture medium containing NaCl of different concentrations, culturing at proper temperature for 48hr, and observing the growth condition of the lawn.
Determination of pH tolerance: inoculating thallus Porphyrae into culture medium with different pH, culturing at proper temperature for 48hr, and observing the growth condition of the thallus Porphyrae.
And (3) detecting oxidase: a piece of filter paper was placed in a clean petri dish, and a 1% aqueous solution of dimethyl-p-phenylenediamine was dropped to wet only the filter paper. Placing the lawn on wet filter paper, and making the lawn coated within 10s turn red.
Methyl red reaction: a drop of methyl red reagent is added into the bacterial liquid, red is positive reaction, and yellow is negative reaction.
And (3) urease reaction: culturing at moderate temperature after inoculation, and observing after 2hr, 4hr and overnight respectively. The slant is peach red positive, and the unchanged one is negative.
Malonic acid utilization: inoculating young strain, culturing at moderate temperature for 48hr, and making culture medium positive when the culture medium changes from green to blue; the medium was negative without changing color.
Tartrate utilization detection: inoculating the strain to fresh tartrate culture medium, observing color change every day, adding equal volume of lead acetate reagent 14 days after inoculation, and setting no-inoculation control. If the color of the red pigment is changed into yellow green, the red pigment is positive, and the blue or green pigment is negative.
L-tyrosine hydrolysis test: and (3) preparing a culture medium by using the L-tyrosine as a nitrogen source, and observing whether the strain to be detected can hydrolyze the L-tyrosine.
The results of the above physiological and biochemical experiments are shown in Table 3.
And simultaneously extracting the total DNA of the Bacillus megaterium HX63 strain, performing 16SrDNA amplification, and comparing the sequence of the 16SrDNA on NCBI to determine that the strain is the Bacillus megaterium. The sequence is shown in the specification sequence list.
Example 3
Bacillus megaterium HX63 strain is mixed with ground phosphate rock under the potting experiment condition, and the influence on the plant growth is researched
Preparing: selecting poor soil from farmland planting soil around the south of the Ji, detecting the contents of total phosphorus and available phosphorus in the soil, selecting soil with higher total phosphorus and lower available phosphorus, and taking a large number of samples for later use. The influence of different phosphate solubilizing microorganisms and two kinds of phosphate rock powder on plant growth and soil phosphorus content after mixed application is researched and determined by a pot experiment. Grinding soil, sieving with 20 mesh sieve, and loading into pot according to 0.5 kg/pot with pot mouth length and width of 10.0cm and height of 10.0 cm; each pot is provided with a pot support, which is beneficial to different treatments and watering.
Fertilizing: respectively adding 216mg/kg of N and K2The addition amounts of urea and potassium chloride are calculated to be 700mg/kg for O, namely 925.71mg/kg and 554.80mg/kg soil, so that the addition amounts of urea and potassium chloride are 0.4629g and 0.277g respectively per pot. Grinding the test phosphate rock powder, sieving the ground phosphate rock powder with a 100-mesh sieve, weighing 3.0g of the ground phosphate rock powder per basin, and fully and uniformly mixing the urea, the potassium chloride, the phosphate rock powder and the soil in the basin for later use.
Seedling culture: taking a plurality of culture dishes, filling filter paper with the same size at the bottom of the culture dish, and sterilizing. Adding a proper amount of deionized water. Placing the rape seeds into a sterilized culture dish according to 100 grains/dish, placing the rape seeds into a light incubator for 25-degree seedling culture, adding a proper amount of deionized water every 24 hours, and transferring the seedlings into a pot when transplanting. As shown in particular in figure 1.
Marking: transplanting the cultivated Chinese cabbage into a pot filled with soil; after the Shanghai green grows strong, seedlings with poor growth conditions are removed, and 3 plants/pot are reserved. Counting the inorganic phosphorus culture solution of the phosphate-solubilizing microorganism to 108CFU or spore/mL for later use, after the Shanghai green grows for 20 days, respectively mixing 1mL of 100-fold diluent of the phosphate-solubilizing microorganism fermentation broth into each pot marked with inoculation treatment; adding blank culture diluted by the same amount and with the same times into each pot marked with a 'control', and setting 3 times of the control and each treatment; the total number of the pots is 30. As shown in fig. 2 and 3.
Seedling watering: potting experiments were performed under constant illumination at 25 ℃. In total 30 basin, wherein the Hubei Xianglong phosphate rock powder and the Shikefeng phosphate rock powder respectively have 15 basins, are respectively provided with a non-inoculation contrast and inoculation treatment, and have 3 basins repeatedly. Proper amount of water is watered at the bottom of the pot every day to slightly moisten the soil in the pot. After the seedlings grow slightly and about 20 days later, 1mL of corresponding bacterial liquid (10 mL) is poured into the root of each plant in each pot for inoculation treatment8CFUmL-1Diluting the fermentation liquor to prepare); for the control, 1mL of basal medium diluted 100-fold was poured into the roots of the plants. Continuously watering the bottom of the pot every day to keep the soil moist and the vegetable leaves not to be wilted, and continuously repeating the operation after 20 days to insert each strain fermentation liquor. Then, proper amount of water is watered at the bottom of the pot every day to slightly moisten the soil in the pot.
And (3) detection: the root-carrying of the rape plant in the pot is taken out together with the root from the soil, the rape plant is tiled on a desktop, and the length of the root-carrying of the plant and the length of the root are directly measured by a ruler with scales. The rooted plants were washed with deionized water and wiped dry, and the number of leaves was counted. Weighing fresh weight of each plant, drying to constant weight in an oven at 105 ℃, and weighing dry weight. In addition, the contents of plant total phosphorus and soil phosphorus are respectively detected by a molybdenum-antimony colorimetric-resistance method, the contents of plant total nitrogen and soil nitrogen are detected by an automatic azotometer method, and the contents of plant total potassium and soil potassium are detected by a flame photometer method.
In this example, the Hailaqing is a research object, and a potting experiment is adopted to investigate the influence of the inoculated Bacillus megaterium HX63 strain and different phosphate rock powders on the growth and development of the Hailaqing from the viewpoints of plant nutrition, plant physiology and soil science, and the Hailaqing undergoes the stages of seedling raising, seedling transplanting, growth and the like in the growth process of the Hailaqing.
The results after the detection are shown in tables 5, 6 and 7.
As can be seen from the data in Table 5, the powdered rock phosphate can effectively promote the growth of plants after being mixed with the phosphate solubilizing microorganism HX63 strain. For the phosphate rock powder provided by the Fengsheng, the bacillus megaterium HX63 strain can better promote the growth of Shanghai green plants, the overground height, the number of leaves, the fresh weight of the plants, the dry weight of the plants and the like of the Shanghai green plants are more than those of the control, which shows that the bacillus megaterium can better colonize the surface of the Fengsheng phosphate rock powder, and can better promote the release of phosphorus and other plant growth beneficial elements in the Fengsheng phosphate rock powder, so that the growth of the Shanghai green plants is promoted. For Hubei Xianglong phosphate rock powder, the bacillus megaterium HX63 strain can effectively improve the absorption and utilization of nutrient elements of soil by Shanghai green plants after being mixed with the strain, thereby promoting the growth of the plants.
As can be seen from the data in Table 6, the Bacillus megaterium HX63 strain has different degrees of influence on the total nitrogen, the total phosphorus and the total potassium of the Shanghai green plants after being respectively mixed with the Hubei Xianglong phosphate rock powder and the Shikefeng phosphate rock powder. Therefore, after the phosphate solubilizing microorganism HX63 strain and the powdered rock phosphate are mixed, the total nitrogen, total phosphorus and total potassium contents of the Shanghai green plant can be effectively improved, and the reason for the content can be that metabolites such as organic acid, enzyme and the like secreted by the phosphate solubilizing microorganism HX63 strain can better promote the growth of beneficial microorganisms of the root system of the Shanghai green plant, and can effectively improve the absorption of the plant to soil nutrient elements, so that the quality of the plant is improved.
The data in Table 7 show that the bacillus megaterium HX63 strain has the influence on the contents of total nitrogen, total potassium, quick-acting potassium and pH value of soil after being mixed with Hubei Xianglong phosphate rock powder and Shikefeng phosphate rock powder. Compared with the mineral powder without phosphorus, the indexes of the soil for planting Shanghai green plants after the addition of the nitrogen fertilizer (urea), the potassium fertilizer (potassium chloride) and the Hubei phosphate rock powder and the application of the Harvest phosphate rock powder are changed, and the pH value of the soil is reduced due to the action of microorganisms and organic acid secreted by plant roots. Compared with the ground phosphate rock inoculation-free contrast, the total nitrogen, the total phosphorus and the total potassium of the soil treated by the microorganisms are slightly increased, so that the proliferation of root system microorganisms is promoted, the microorganisms are massively propagated after utilizing organic matters and the like in the soil, and the total nitrogen, the total phosphorus and the total potassium of the soil are increased due to the increase of the content of the nitrogen, the phosphorus and the potassium in the biomass of the microorganisms. After inoculation, the contents of available phosphorus and available potassium in each soil sample are increased to different degrees, and organic acid, phosphatase and the like secreted by microorganisms possibly release nitrogen, phosphorus and potassium complexed with organic matters in the ground phosphate rock and the soil, promote the weathering of various mineral substances and enhance the absorption of various nutrients by plants.
TABLE 1 available phosphorus, Water-soluble phosphorus and Total phosphorus content of poorly soluble phosphates
TABLE 2 solubilizing Effect of Bacillus megaterium HX63 Strain on various poorly soluble phosphates
TABLE 3 solubilizing effect of Bacillus megaterium HX63 strain on different sources of ground phosphate rock
TABLE 4 summary of physiological and biochemical characteristics of Bacillus megaterium HX63 Strain
Note: + indicates that the microorganism is able to grow or that the test result is positive; -indicating that the microorganism is unable to grow or that the test result is negative;
TABLE 5 influence of powdered rock phosphate from different sources on the green plants in Shanghai after mixed application of Bacillus megaterium HX63
TABLE 6 influence of mixed application of powdered rock phosphate from different sources and Bacillus megaterium HX63 on NPK of Shanghai green plants
TABLE 7 influence of ground phosphate rock from different sources on NPK in soil after mixed application with Bacillus megaterium HX63 strain
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Qingdao and Xiezhi Biotechnology Limited, Shandong architecture university
<120> bacillus megaterium with phosphate solubilizing capability and application thereof in dissolving phosphate rock powder
<130>2020
<160>1
<170>PatentIn version 3.5
<210>1
<211>1425
<212>DNA
<213> native sequence
<400>1
gcagtcgagcgaactgatttagaagcttgctttctatgacgttagcggcggacgggtgag 60
taacacgtgggcaacctgcctgtaagactgggataacttcgggaaaccgaagctaatacc 120
ggataggatcttctccttcatgggagatgattgaaagatggtttcggctatcacttacag 180
atgggcccgcggtgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcat 240
agccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgg 300
gaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtga 360
gtgatgaaggctttcgggtcgtaaaactctgttgttagggaagaacaagtacgagagtaa 420
ctgctcgtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccg 480
cggtaatacgtaggtggcaagcgttatccggaattattgggcgtaaagcgcgcgcaggcg 540
gtttcttaagtctgatgtgaaagcccacggctcaaccgtggagggtcattggaaactggg 600
gaacttgagtgcagaagagaaaagcggaattccacgtgtagcggtgaaatgcgtagagat 660
gtggaggaacaccagtggcgaaggcggctttttggtctgtaactgacgctgaggcgcgaa 720
agcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgagtgct 780
aagtgttagagggtttccgccctttagtgctgcagctaacgcattaagcactccgcctgg 840
ggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtgga 900
gcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgaca 960
actctagagatagagcgttccccttcgggggacagagtgacaggtggtgcatggttgtcg 1020
tcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttag 1080
ttgccagcatttagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtgg 1140
ggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatg 1200
gtacaaagggctgcaagaccgcgaggtcaagccaatcccataaaaccattctcagttcgg 1260
attgtaggctgcaactcgcctacatgaagctggaatcgctagtaatcgcggatcagcatg 1320
ccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgta 1380
acacccgaagtcggtggagtaaccgtaaggagctagccgcctaag 1425
Claims (10)
1. The Bacillus megaterium HX63 is deposited in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.19471 and the preservation date of the CGMCC is as follows: and 3, 13 months in 2020, the preservation address is: the institute of microbiology, institute of Zhongkou institute of western-style No.1 Hospital, Beijing, Chaoyang, North Chen.
2. The bacillus megaterium having phosphate solubilizing capability of claim 1, wherein the bacillus megaterium having phosphate solubilizing capability promotes dissolution of organic phosphorus, lecithin, or calcium phytate.
3. The bacillus megaterium having phosphate solubilizing capability of claim 1, wherein the bacillus megaterium having phosphate solubilizing capability improves a phosphate rock powder fertilizer efficiency.
4. The Bacillus megaterium having phosphate solubilizing ability according to claim 1, wherein the Bacillus megaterium having phosphate solubilizing ability increases seed germination rate.
5. The bacillus megaterium having phosphate solubilizing capability of claim 1, wherein the bacillus megaterium having phosphate solubilizing capability promotes plant growth and development.
6. The phosphate solubilizing Bacillus megaterium of claim 1, wherein the phosphate solubilizing Bacillus megaterium increases plant tolerance to heavy metals and antibiotics.
7. The Bacillus megaterium having phosphate solubilizing ability according to claim 5 or 6, wherein the plant is Haematococcus Shanghai.
8. Use of the bacillus megaterium having phosphate solubilizing ability of any one of claims 1 to 7 for promoting phosphate solubilization.
9. The use of claim 8, wherein the phosphorus comprises sparingly soluble inorganic and organic phosphates.
10. The use of claim 8, wherein said sparingly soluble inorganic phosphate comprises a synthetic and naturally occurring sparingly soluble inorganic phosphate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175880A (en) * | 2020-10-21 | 2021-01-05 | 山西大地生态环境技术研究院有限公司 | Saline-alkali-resistant phosphate solubilizing bacterium and application thereof |
| CN112920978A (en) * | 2021-04-15 | 2021-06-08 | 河南科技学院 | Bacillus megaterium LP17 and application thereof |
-
2020
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175880A (en) * | 2020-10-21 | 2021-01-05 | 山西大地生态环境技术研究院有限公司 | Saline-alkali-resistant phosphate solubilizing bacterium and application thereof |
| CN112175880B (en) * | 2020-10-21 | 2023-09-12 | 山西大地生态环境技术研究院有限公司 | Salt-tolerant alkaline-hydrolysis phosphorus bacteria and application thereof |
| CN112920978A (en) * | 2021-04-15 | 2021-06-08 | 河南科技学院 | Bacillus megaterium LP17 and application thereof |
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