CN116751709A - Burkholderia strain B2OP and application thereof - Google Patents

Burkholderia strain B2OP and application thereof Download PDF

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CN116751709A
CN116751709A CN202310641931.9A CN202310641931A CN116751709A CN 116751709 A CN116751709 A CN 116751709A CN 202310641931 A CN202310641931 A CN 202310641931A CN 116751709 A CN116751709 A CN 116751709A
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b2op
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burkholderia
burkholderia strain
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王志高
黄志远
李巧玲
江波
吴初平
钟哲科
张小平
姚良锦
焦洁洁
李婷婷
袁位高
朱锦茹
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Zhejiang Academy of Forestry
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Abstract

The application relates to the technical field of microbial preparations, in particular to a burkholderia strain B2OP and application thereof, wherein the burkholderia strain B2OP is preserved in China general microbiological culture collection center (CGMCC) in 11 months of 2022, the preservation number is CGMCC NO.26054, and the preservation address is North Chen West Lu No. 1 of the Korean region of Beijing, national academy of sciences microbiological study. Researches show that the burkholderia strain B2OP has the functions of nitrogen fixation, phosphorus dissolution and iron production, the nitrogen fixation rate is 120.19%, the organic phosphorus dissolution rate is 117.81%, and the inorganic phosphorus dissolution rate is 383.37%. Therefore, the burkholderia strain B2OP has strong nitrogen fixation and phosphorus decomposition functions, has an iron production function, and has wide application prospects in the aspects of increasing soil nutrients and promoting healthy growth of plants.

Description

Burkholderia strain B2OP and application thereof
Technical Field
The application relates to the technical field of microbial preparations, in particular to a burkholderia strain B2OP and application thereof.
Background
The nitrogen and phosphorus elements in the soil are core contents of soil nutrients, are important elements required by plant growth and development and physiological and ecological activities, and are the results of mutual influence of factors such as soil physics, chemistry, biology and the like. Iron is one of the essential trace elements of plants, is a main component of many enzymes, is a main condition for catalyzing synthesis of chlorophyll, and plays an important role in metabolism of nucleic acid.
Although the nitrogen content in the nature is very rich, most plants cannot be directly utilized generally, and the plants must depend on obtaining other nitrogen-containing substances from the environment for growth; the phosphorus in the soil is divided into inorganic phosphorus and organic phosphorus, wherein the inorganic phosphorus mainly exists in the forms of tricalcium phosphate, ferric phosphate and the like, has poor solubility and is difficult to be absorbed by plants; the iron in the soil is almost all Fe which is indissoluble in water 3+ The plant iron-deficiency type fertilizer is difficult to absorb and utilize by plants, and agricultural production is generally faced with the problem of iron deficiency of plants. But not manually adding nitrogen-containing and phosphorus-containing fertilizersOnly the environment is polluted, and the soil structure is destroyed. Therefore, the method is an effective way for screening safe and effective soil nitrogen fixation, phosphorus dissolution and iron production microorganisms, developing microbial fertilizers, increasing soil nitrogen and phosphorus nutrients and promoting plant growth, and can avoid a series of environmental problems caused by fertilizer use. Nitrogen-fixing microorganism uses nitrogen-fixing enzyme to make N in atmosphere 2 Is converted into plant effective nitrogen, thereby providing an additional nitrogen source for an ecological system, and most of the bioavailable nitrogen in the ecological system mainly comes from biological nitrogen fixation effect of soil nitrogen fixation microbial communities, for example, bacillus, arthrobacter, pseudomonas and the like are common nitrogen fixation functional bacteria. Phosphate-dissolving microorganisms are microorganisms capable of converting inorganic phosphate or mineralized organic phosphate which is difficult to be absorbed and utilized by plants into absorbable and utilized forms, so that plant growth is promoted. Currently, the phosphorus-decomposing bacteria found include phosphorus-decomposing bacteria, phosphorus-decomposing fungi, phosphorus-decomposing actinomycetes and the like. The iron-producing microorganism mainly secretes the iron-producing carrier, so that the absorption and transportation of the microorganism to iron ions are improved, the mobility of the iron ions in the soil environment is increased, and the plant growth is promoted.
The application of single-type and single-function microorganisms is limited, and the composite strains can realize functional complementation and mutual promotion, so that the application range is wider. In addition, the microbial fertilizer has certain requirements on different climatic regions and soil environments due to the characteristics of the microbial fertilizer. Therefore, the antagonism generated by mixing different types of microorganisms can be avoided by screening indigenous microorganisms with multiple functions, the manufacturing cost of the mixed bacterial manure of multiple microorganisms can be further reduced, and more strain selections are provided for expanding the application range of the microbial bacterial manure.
However, some beneficial microorganisms and products such as microbial inoculum and bacterial manure in the prior art have single functions, and cannot achieve the effects of nitrogen fixation, phosphorus dissolution and iron production. In addition, in the artificial management mode, crops need to rapidly root, colonize and grow, and in the practice of barren soil forestation, soil nutrient supply is one of key factors for improving crop yield and forestation survival rate.
Therefore, in order to promote the safety and high efficiency of nutrient supply in the production process of agriculture and forestry, reduce the use of pesticides and fertilizers, realize green development, search and screen more indigenous microorganisms with high safety and multiple functions, and the method is a technical work which needs to be continuously carried out by the technicians in the field.
The beneficial burkholderia has great application potential in promoting plant growth and maintaining plant health, but the current understanding of the species and functions of the burkholderia is insufficient, and the popularization and application are less. Therefore, the development of the screening, the function measurement and the popularization and the application of the burkholderia strain provide more microbial inoculum types for replacing chemical fertilizers, and are a problem which needs to be solved urgently by the technicians in the field.
Disclosure of Invention
The application aims to provide a burkholderia strain B2OP and application thereof, and the burkholderia strain B2OP has strong nitrogen fixation and phosphorus dissolution functions, has an iron production function, and has wide application prospects in the aspects of increasing soil nutrients and promoting healthy growth of plants.
The application provides a burkholderia strain B2OP, wherein the preservation number of the burkholderia strain B2OP is CGMCC NO.26054.
The Burkholderia strain B2OP of the application is obtained from the root soil of cypress (Cupressus funebris) in forest public garden land in mountain area of noon in Hangzhou, zhejiang province, and is identified as Burkholderia (Burkholderia) with the homology of more than 99% with fungus Burkholderia (Burkholderia fungorum) and fungus Burkholderia (Burkholderia fungorum) through morphological observation and sequencing and analysis of 16sRNA sequences by a biological engineering (Shanghai) stock limited company.
The Burkholderia strain B2OP of the application is preserved in China general microbiological culture Collection center (CGMCC) at 11-7-2022, the preservation number is CGMCC NO.26054, and the preservation address is North Chen West Lu No. 1 and 3 in the Korean region of Beijing city, and the microbiological study center of China academy of sciences.
Researches show that the burkholderia strain B2OP of the application has the nitrogen fixation rate of 120.19%, the organic phosphorus dissolution rate of 117.81% and the inorganic phosphorus dissolution rate of 383.37% and has the iron production capacity. Therefore, the burkholderia strain B2OP has strong nitrogen fixation and phosphorus decomposition functions, has an iron production function, and has wide application prospect in the aspects of increasing soil nutrients and promoting healthy growth of plants.
The Burkholderia strain B2OP is white, round, complete in edge, smooth and moist in surface, slightly convex in the middle, opaque in colony and 1-2mm in diameter. The gene sequence of the 16S rRNA is shown as SEQ ID NO:1, the similarity with fungus Burkholderia (Burkholderia fungorum) is highest, and the homology is more than 99%.
On the basis of the above, the application also provides the application of the Burkholderia strain B2OP in nitrogen fixation, phosphate dissolution and iron production of plants, and the application belongs to the protection scope of the application.
On the basis of the above, the application also provides the application of the Burkholderia strain B2OP in the fields of increasing soil nutrients and promoting healthy growth of plants, and the application belongs to the protection scope of the application.
The application also provides application of the burkholderia strain B2OP in preparing nitrogen fixation, phosphorus dissolution and iron production microbial agents.
And the microbial agent comprising the burkholderia strain B2OP should also fall within the protection scope of the application.
Preferably, the microbial agent is a bacterial suspension of a burkholderia strain B2OP, and the bacterial count of the burkholderia strain B2OP in the microbial agent is preferably 1 multiplied by 10 9 -2×10 9 And preferably 1.5X10 g/mL 9 And each mL.
The application also discloses a preparation method of the microbial agent, which comprises the steps of placing single bacterial colony of Burkholderia strain B2OP in liquid LB culture, and culturing at a constant temperature of 20-30 ℃ for 20-30h, wherein the obtained bacterial suspension is the microbial agent.
The application of the microbial agent or the microbial agent prepared by the preparation method in nitrogen fixation, phosphate dissolution and iron production of plants is also considered to belong to the protection scope of the application.
The burkholderia strain B2OP of the application has at least the following technical effects:
1. the burkholderia strain B2OP of the application has the nitrogen fixation rate of 120.19%, the organic phosphorus dissolution rate of 117.81% and the inorganic phosphorus dissolution rate of 383.37%, and has the iron production capability. Therefore, the burkholderia strain B2OP has strong nitrogen fixation and phosphorus decomposition functions and an iron production function, is a safe, green and efficient multifunctional microbial agent, and has wide application prospect in the aspects of increasing soil nutrients and promoting healthy growth of plants;
2. the burkholderia strain B2OP is obtained by separating naturally growing cypress rhizosphere soil, belongs to plant beneficial microorganisms, has wide application range, can reduce the use of chemical fertilizers and pesticides to a great extent, and has great potential in the field of microbial agent development.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows Burkholderia strain B2OP of the application;
FIG. 2 is a functional effect diagram of the Burkholderia strain B2OP phosphate-solubilizing nitrogen-fixing siderophores of the present application.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. 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 application 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 present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to 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.
The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Example 1
Isolation and screening of Burkholderia strain B2OP
Root system soil of cypress with good growth vigor is collected and placed in a sterile tube, and is temporarily stored in a refrigerator at the temperature of 4 ℃ in a laboratory.
Placing proper amount of root system into sterile culture dish, adding 10mL of sterile water, cleaning for three times, collecting cleaning solution, placing into 50mL sterile centrifuge tube, oscillating at 28deg.C and 180r/min for 0.5 hr, mixing thoroughly, and making into 10 -1 Concentration gradient, 10-fold dilution method -3 、10 -4 、10 -5 、10 -6 Each gradient of soil sample suspension is uniformly coated on an organic phosphorus culture medium (solid), an inorganic phosphorus screening culture medium (solid), a siderophore culture medium (solid) and an Abdominal shell nitrogen fixation culture medium (solid) by absorbing 100 mu L of suspension by each gradient, and 3 groups of the gradients are repeated; the plates were inverted and incubated in a constant temperature incubator at 28℃for 4d.
Placing the cleaned root system into a sterile mortar, adding sodium hypochlorite solution until the root system is completely submerged, standing for 8min, pouring the sodium hypochlorite solution, cleaning with sterile water for three times, shearing and grinding the root system with sterile scissors until the root system tissue is crushed, uniformly spreading the root system tissue on the three solid culture mediums, and repeating for three times; plates were placed in a constant temperature incubator at 28℃until bacterial colonies appeared.
According to the color change on the flat plate and the diameter of the phosphate dissolving ring, a bacterial colony is picked by a sterile inoculating loop, single bacterial colony is obtained by streaking, separating and purifying on a solid LB culture medium, the single bacterial colony is activated in the liquid LB culture medium, and the single bacterial colony is mixed with sterile glycerol according to the proportion of 7:3 and stored in a refrigerator at the temperature of minus 80 ℃.
Example 2
Identification of Burkholderia strain B2OP
1. Morphological features
The Burkholderia strain B2OP is white, round, complete in edge, smooth and moist in surface, slightly convex in the middle, opaque in colony and 1-2mm in diameter.
2. Functional features
Activating the strain stored on the inclined surface of the test tube, inoculating the strain to an LB liquid culture medium for activation, culturing for 48 hours, taking the strain as seed liquid, inoculating 10ul of seed liquid to a plate of the Abbe moschus manihot nitrogen fixation culture medium (0.2 g/L of monopotassium phosphate, 0.2g/L of sodium chloride, 0.2g/L of magnesium sulfate, 5.0g/L of calcium carbonate, 0.1g/L of calcium sulfate, 10g/L of mannitol, 15.0g/L of agar, pH value of 7.0-7.5, and sterilizing at 115 ℃ for 30 minutes under high pressure), and repeating the process for three times for 2 planting points per dish.
The strain was incubated at 37℃for 5 days, and a transparent ring was observed around the strain, indicating that the strain had nitrogen fixation ability (FIG. 2-1).
Activating the strain stored on the inclined surface of the test tube, inoculating the strain to an LB liquid culture medium for activation, culturing for 48 hours, taking the strain as seed liquid, inoculating 10ul of seed liquid to an organophosphorus culture medium plate (glucose 10.0g/L, ammonium sulfate 0.5g/L, sodium chloride 0.3g/L, magnesium sulfate 0.3g/L, manganese sulfate 0.03g/L, potassium sulfate 0.3g/L, ferrous sulfate 0.03g/L, lecithin 5.0g/L, agar 15.0g/L, pH value 7.0-7.5, and autoclaving at 115 ℃ for 30 min), and repeating the culture for 2 planting points per dish for three times.
After 5d incubation at 37℃a transparent ring was observed around the strain, indicating that the strain had the ability to cleave organophosphorus (FIG. 2-2).
Activating the strain stored on the inclined surface of the test tube, inoculating the strain on an LB liquid culture medium for activation, culturing for 24 hours, taking the strain as seed liquid, inoculating 10ul of seed liquid on an inorganic phosphorus culture medium flat plate (glucose 10.0g/L, ammonium sulfate 0.5g/L, sodium chloride 0.3g/L, magnesium sulfate 0.3g/L, manganese sulfate 0.03g/L, potassium sulfate 0.3g/L, ferrous sulfate 0.03g/L, calcium phosphate 5.0g/L, agar 15.0g/L, pH value 7.0-7.5, and sterilizing at 115 ℃ under high pressure for 30 min), and repeating the culture for 2 planting points per dish for three times.
The strain was incubated at 37℃for 5-7 days, and a transparent ring was observed around the strain, indicating that the strain had the ability to cleave organic phosphorus-free (FIGS. 2-3).
Activating strain stored on the inclined surface of a test tube, inoculating the strain to an LB liquid culture medium for activation, culturing for 24 hours, taking the strain as a seed solution, inoculating 10ul of the seed solution to a CAS solid culture medium (glucose 4.0g, peptone 5.0g/L, KCl 0.5g/L, mgSO4.7H2O 0.5g/L and pH of 7.0), adding 50mL of phosphoric acid buffer solution and 50mL of CAS dye solution, uniformly mixing, sterilizing at 115 ℃ for 20 minutes, and repeating for 1 planting point in each culture dish for 3 times.
The yellow halo was observed in the blue CAS medium at 30℃in an incubator for 3 days, with a deeper degree of discoloration indicating a stronger siderophore production (FIGS. 2-4).
3. 16S rRNA sequence analysis
Further 16S rRNA sequence analysis and alignment were performed on the isolated B2OP strain of Burkholderia.
Cloning and sequence determination were performed according to the conventional method, and the determination result was analyzed by comparison with the 16S rRNA sequence (accession number: DQ 513513) in the ribosome database (http:// rdp. Cme. Msu. Edu/index. Jsp) to achieve 99.5% gene homology. The strain was determined to be Burkholderia, a fungus of the genus Burkholderia (Burkholderia fungorum) based on Bergey' S Mannual of Determinative Bacteriology (Holt, J.G., gibbons, N.E., 1994) and the handbook of common bacterial System identification (Dongxil and Cai Miaoying et al, 2001), as well as morphological features and 16S rRNA sequence analysis.
Example 3
Nitrogen fixation efficiency
Under the aseptic condition, single colony burkholderia strain B2OP is selected, and after the single colony burkholderia strain B2OP is cultured for 24 hours in a liquid LB culture medium at constant temperature, 1mL of suspension is sucked for respective inoculationIn liquid Aju Bei Modan medium (Potassium dihydrogen phosphate 0.2g,NaCl 0.2g,MgSO) 4 .7H 2 0.2g of O, 5.0g of calcium carbonate, 10.0g of mannitol, 0.1g of calcium sulfate, 1000ml of distilled water, pH of 6.8-7.0 and sterilization at 121 ℃ for 20min for later use; 1mL of sterile water was added to the control group; each group was repeated three times.
Subsequently, the mixture was placed in a shaker 180r/min at 28℃for 72h; after the shaking is completed, the culture solution is centrifuged at 10000r/min for 5min, the supernatant is taken and the total nitrogen content is measured by an alkaline potassium persulfate oxidation method, and the nitrogen fixation capacity of the bacteria is determined by comparing with a control group. The results are detailed in Table 1.
TABLE 1 Nitrogen fixation after 72h culture of strain B2OP
As shown in Table 1, compared with the control group, after the Burkholderia B2OP of the application is cultured for 72 hours, the total nitrogen content in the culture medium is increased by 17.99mg/L, and the nitrogen fixation rate can reach 120.19%.
Example 4
Efficiency of organic phosphorus decomposition
Under the aseptic condition, single colony Burkholderia B2OP is picked up, cultured for 24 hours in a liquid LB culture medium at constant temperature, 1mL of suspension is sucked up and respectively inoculated into a liquid organophosphorus screening culture medium (10 g of glucose, (NH) 4 ) 2 SO 4 0.5g, yeast extract 0.5g,NaCl 0.3g,KCl 0.3g,MgSO 4 .7H 2 O 0.3g，FeSO 4 .7H 2 0 0.03g，MnSO 4 .H 2 0.03g of O, 2.0g of calcium phytate, 1000ml of distilled water, pH of 7.0-7.5, and sterilizing at 121 ℃ for 20 min); 1mL of sterile water was added to the control group; each group was repeated three times.
Subsequently, the mixture was placed in a shaker 180r/min at 28℃for 72h; after the shaking is completed, the culture solution is centrifuged for 5min at 10000r/min, and the supernatant is taken and the effective phosphorus content of the supernatant is measured by adopting a molybdenum-antimony colorimetric method. The phosphorus-dissolving capacity of the bacteria was determined by comparison with the control group, and the results are shown in Table 2.
TABLE 2 amount of dissolved organic phosphorus after 72h of cultivation of Strain B2OP
As shown in Table 2, compared with the control group, after the Burkholderia B2OP of the application is cultured for 72 hours, the phosphorus content in the culture medium is increased by 92.90mg/L, and the rate of dissolved organic phosphorus can reach 117.81 percent.
Example 5
Efficiency of inorganic phosphorus decomposition
Under the aseptic condition, single colony burkholderia strain B2OP is selected, cultured for 24 hours in a liquid LB culture medium at constant temperature, 1mL of suspension is sucked and respectively inoculated in a liquid inorganic phosphorus screening culture medium (10 g of glucose, (NH) 4 ) 2 SO 4 0.5g, yeast extract 0.5g,NaCl 0.3g,KCl 0.3g,MgSO 4 .7H 2 O 0.3g，FeSO 4 .7H 2 0 0.03g，MnSO 4 .H 2 0.03g of O, 5.0g of tricalcium phosphate, 1000ml of distilled water, pH of 7.0-7.5, and sterilizing at 121 ℃ for 20 min); the control group was added with 1mL of sterile water and each group was repeated three times.
Subsequently, the mixture was placed in a shaker 180r/min at 28℃for 72h; after the shaking is completed, the culture solution is centrifuged for 5min at 10000r/min, and the supernatant is taken and the effective phosphorus content of the supernatant is measured by adopting a molybdenum-antimony colorimetric method. The phosphorus-solubilizing ability of the bacteria was determined by comparison with the control group, and the results are shown in Table 3.
TABLE 3 amount of inorganic phosphorus dissolved after 72h culture of strain B2OP
As shown in Table 3, compared with the control group, after the Burkholderia B2OP is cultured for 72 hours, the phosphorus content in the culture medium is increased by 6.90mg/L, and the inorganic phosphorus dissolving rate can reach 383.37 percent.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims (10)

1. The burkholderia strain B2OP is characterized in that the preservation number of the burkholderia strain B2OP is CGMCC NO.26054.
2. The burkholderia strain B2OP of claim 1, wherein the gene sequence of the 16S rRNA of the burkholderia strain B2OP is as set forth in SEQ ID NO: 1.
3. Use of the burkholderia strain B2OP according to claim 1 for nitrogen fixation, phosphate dissolution and iron production in plants.
4. Use of the burkholderia strain B2OP of claim 1 in the field of increasing soil nutrients and promoting healthy growth of plants.
5. Use of the burkholderia strain B2OP according to claim 1 for preparing a nitrogen fixation and phosphorus decomposition iron-producing microbial agent.
6. A microbial agent comprising burkholderia strain B2OP of claim 1.
7. The microbial agent of claim 6, wherein the microbial agent is a bacterial suspension of burkholderia strain B2OP.
8. The microbial agent according to claim 6, wherein the microbial agent is burkholderiaThe bacterial count of the strain B2OP is 1 multiplied by 10 9 -2×10 9 And each mL.
9. A preparation method of a microbial agent is characterized in that single colony of a burkholderia strain B2OP is placed in liquid LB culture, and the microbial agent is obtained by culturing at a constant temperature of 20-30 ℃ for 20-30 hours.
10. Use of the microbial agent of any one of claims 6-8 or the microbial agent prepared by the preparation method of claim 9 for nitrogen fixation, phosphorus dissolution and iron production of plants.
CN202310641931.9A 2023-05-31 2023-05-31 Burkholderia strain B2OP and application thereof Pending CN116751709A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117229983A (en) * 2023-11-14 2023-12-15 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) Nitrosogenic paraburkholderia PX-418, microbial inoculum and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117229983A (en) * 2023-11-14 2023-12-15 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) Nitrosogenic paraburkholderia PX-418, microbial inoculum and application thereof
CN117229983B (en) * 2023-11-14 2024-01-30 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) Nitrosogenic paraburkholderia PX-418, microbial inoculum and application thereof

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