CN114196563A - Bacillus belgii and application thereof - Google Patents
Bacillus belgii and application thereof Download PDFInfo
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- CN114196563A CN114196563A CN202110979821.4A CN202110979821A CN114196563A CN 114196563 A CN114196563 A CN 114196563A CN 202110979821 A CN202110979821 A CN 202110979821A CN 114196563 A CN114196563 A CN 114196563A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention provides a Bacillus velezensis-GXMD-hs-L68 (Bacillus velezensis-GXMD-hs-L68)16S, which is preserved in Guangdong province microbial strain preservation center at 12/8/2021 with the preservation number GDMCC NO:61866, and the fermentation broth prepared from the Bacillus velezensis is applied to plant growth. Research shows that the GXMD-hs-L68 strain has strong phosphorus dissolving function, and the fermentation liquid has good growth promoting effect on crops. The discovery of the strain enriches available microbial resources in China, has the advantages of stable phosphorus dissolving function, high efficiency and environmental friendliness, and has good application prospect in the aspect of agricultural planting.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to bacillus beiLeisi and application thereof.
Background
Phosphorus is one of the most important nutrient elements in the growth process of crops. Although most of the soil contains abundant phosphorus elements, the phosphorus elements contained in the soil are easy to fix and form precipitates, so that the phosphorus elements actually utilized by plants in the soil are few, and generally account for 2% -3% of the total phosphorus. At present, the method of artificially applying phosphate fertilizer is mostly adopted. The method is a rapid means for solving the problem of phosphorus deficiency of plants, but the long-term application of the method can cause soil pollution and is not beneficial to the growth of the plants.
Recent studies have shown that phosphate solubilizing microorganisms can convert ineffective phosphorus in soil, which is difficult to be utilized by plants, into available phosphorus, which can be utilized by plants. Aiming at the current situation that the potential phosphorus source of soil is rich but available phosphorus is deficient, the development and utilization of phosphate solubilizing microorganisms are a research hotspot in the field of soil fertilizers and plant nutrition. The biological method for improving the bioavailability of the phosphorus by decomposing the indissolvable phosphorus in the soil by using the phosphorus-dissolving microorganisms is an effective way for solving the problem of phosphorus deficiency of crops.
The phosphorus-dissolving bacteria are important soil microorganisms in an agricultural ecosystem, can convert invalid phosphorus elements into soluble phosphorus which can be directly absorbed and utilized by plants, can improve the soil structure and improve saline-alkali soil, serve as environment-friendly biological factors, and play an important role in the development processes of white agriculture and green agriculture.
Therefore, the Bacillus belgii and the application thereof are provided.
Disclosure of Invention
It is an object of the present invention to solve or at least alleviate problems in the prior art.
In order to achieve the purpose, the invention is realized by the following technical scheme:
bacillus velezensis-GXMD-hs-L68, which has been deposited at 12/8/2021 with the deposit number GDMCC NO:61866 and is deposited at the Guangdong province collection center for microorganisms.
Optionally, the Bacillus velezensis-GXMD-hs-L68 grows on LB solid medium at 30 ℃ in a white concave-middle, viscous, irregular shape and peculiar smell.
Optionally, the Bacillus belgii is isolated from soil in Shanghai reservoir, Wen Yi City, Guangdong province.
A preparation method of Bacillus velezensis-GXMD-hs-L68 is characterized by comprising the following steps:
step one, material preparation; sampling a sample: the soil sampling place is a Shanghai reservoir in Ming Yi City, Maoming City, Guangdong province, surface soil is pulled out, a soil sample with the depth of 15cm below the surface layer is collected, stored in an incubator at 4 ℃, and taken back to a laboratory for next treatment.
Step two: separation and purification of bacterial strains
Adding 2g soil sample into 18ml sterile water to prepare soil suspension, shaking for 30min in a shaking table (180r/min), and diluting with sterile water to 10-1-10-6Taking 10 times of sample-5、10-6Two dilutions of 0.1ml of the sample were spread on screening medium andthe coated screening culture medium plate is placed in an incubator at the temperature of 32-37 ℃, and whether transparent circles appear on the plate or not is observed at intervals. And (4) selecting the strains with the transparent circles to an LB solid culture medium for streak culture to obtain single colonies.
A method for preparing fermentation liquid from Bacillus velezensis ((Bacillus velezensis-GXMD-hs-L68) is characterized in that the Bacillus velezensis ((Bacillus velezensis-GXMD-hs-L68)) is streaked on an LB solid culture medium, single colonies are picked to be inoculated into 1ml of an LB liquid culture medium for shaking culture overnight, 1ml of the liquid culture medium is inoculated into the liquid culture medium, and shaking culture is carried out on a shaking table (30-32 ℃, 150 and 200r/min) for one day to prepare the Bacillus velezensis ((Bacillus velezensis-GXMD-hs-L68) fermentation liquid.
An application of Bacillus velezensis-GXMD-hs-L68 in fermenting liquid to promote the growth of plant is disclosed.
A Bacillus velezensis-GXMD-hs-L68 fermentation liquid is used to prepare bacterial agent or to be added into organic fertilizer to form bacterial fertilizer.
The embodiment of the invention provides Bacillus belgii and application thereof. The method has the following beneficial effects:
the strain is simple to screen and easy to culture, and the inventor also establishes a corresponding culture method. Research shows that the GXMD-hs-L68 strain has strong phosphorus dissolving function, and the fermentation liquid has good growth promoting effect on crops. The discovery of the strain enriches available microbial resources in China, has the advantages of stable phosphorus dissolving function, high efficiency and environmental friendliness, and has good application prospect in the aspect of agricultural planting.
Drawings
FIG. 1 is a diagram showing the growth morphology of Bacillus belgii on LB solid medium.
FIG. 2 is a graph showing the size of the phosphate solubilizing circle for the primary screening of Bacillus belgii
FIG. 3 is a histogram of the increase in plant height of Eucalyptus
FIG. 4 is a histogram of the increase in the diameter of a eucalyptus tree
FIG. 5 is a histogram of the chlorophyll content of eucalyptus leaves
FIG. 6 is a histogram of eucalyptus dry/wet weights
FIG. 7 is a histogram of the increase in plant height of maize
FIG. 8 is a histogram of the increment of the ground diameter of corn
FIG. 9 is a histogram of corn leaf width increment
FIG. 10 is a long cylindrical plot of corn roots
FIG. 11 is a dry weight/wet weight bar graph of corn
FIG. 12 is a graph showing the results of strain electrophoresis (from the left: the first band is the strain of the present invention)
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Isolation and purification experiment of bacillus belgii:
1.1 Material preparation
Sampling a sample: the soil sampling place is a Shanghai reservoir in Ming Yi City, Maoming City, Guangdong province, surface soil is pulled out, a soil sample with the depth of 15cm below the surface layer is collected, stored in an incubator at 4 ℃, and taken back to a laboratory for next treatment.
Screening and preserving culture medium:
inorganic phosphorus liquid culture medium: 10g of glucose, (NH4)2SO40.5g, KCI0.3g, NaCl0.3g, FeSO4.7H2O 0.03g, MgSO4.7H2O 0.3g, MnSO4.4H2O 0.03g, Ca3(PO4)210g, 1000mL of distilled water and pH 7.0-7.5.
Inorganic phosphorus solid medium: 10g of glucose, (NH4)2SO40.5g, KCI0.3g, NaCl0.3g, FeSO4.7H2O 0.03g, MgSO4.7H2O 0.3g, MnSO4.4H2O 0.03g, Ca3(PO4)210g, 18g of agar powder, 1000mL of distilled water and pH 7.0-7.5.
LB liquid medium: 10.0g of tryptone, 5.0g of yeast powder, 5.0g of sodium chloride and 1000mL of distilled water.
LB solid medium: 10.0g of tryptone, 5.0g of yeast powder, 5.0g of sodium chloride, 15g of agar powder and 1000mL of distilled water.
1.2 isolation and purification of the Strain
Adding 2g soil sample into 18ml sterile water to prepare soil suspension, shaking for 30min in a shaking table (180r/min), and diluting with sterile water to 10-1-10-6Taking 10 times of sample-5、10-60.1ml of samples with two dilution times are smeared on a screening culture medium, a flat plate of the screened culture medium after being smeared is placed in an incubator at the temperature of 32-37 ℃, and whether transparent rings appear on the flat plate or not is observed at intervals. And (4) selecting the strains with the transparent circles to an LB solid culture medium for streak culture to obtain single colonies.
1.3 preliminary screening of the Strain
Preparing a seed solution: selecting a ring of strains from a preservation culture medium, inoculating the strains on an LB culture medium, and performing activated culture at 28 ℃ for 24 hours; inoculating the activated thallus in 50mL of sterilized LB liquid culture medium, and shake culturing at 28 deg.C at 100r/min for 24h to obtain seed liquid.
A phosphorus dissolving ring method is adopted, 10uL of seed liquid is injected by a liquid transfer gun to be connected to an inorganic phosphorus culture medium, the size of a phosphorus dissolving ring is observed and measured when the phosphorus dissolving ring is cultured at the constant temperature of 28 ℃ for 7D and has no obvious change, the diameter (D) of the phosphorus dissolving ring and the diameter (D) of a bacterial colony are measured, D/D is calculated, and the strength of the phosphorus dissolving capacity is preliminarily judged. The results of the preliminary screening of the above strains are shown in Table 4 below and FIG. 2.
TABLE 4 preliminary screening data table for phosphorus dissolving function of Bacillus belgii
| Strain name | The diameter (D) of the phosphorus dissolving ring is mm | Diameter of bacterial colony (d) mm | D/d |
| L68 | 11.05 | 20.3 | 0.544334975 |
The results show that the Bacillus belgii has ideal phosphorus solubilizing function.
1.4 rescreening of the Strain
Preparing a seed solution: selecting a ring of strains from a preservation culture medium, inoculating the strains on an LB culture medium, and performing activated culture at 28 ℃ for 24 hours; inoculating the activated thallus in 50mL of sterilized LB liquid culture medium, and shake culturing at 28 deg.C at 100r/min for 24h to obtain seed liquid.
Inoculating the seed solution into 100mL of sterilized inorganic phosphorus liquid culture medium according to the inoculation amount of 2%, taking the culture medium with the same inoculation amount as a blank control, performing shake culture at 28 ℃ for 12d at 180r/min, taking fermentation liquor, and measuring the effective phosphorus content of the fermentation liquor by adopting a molybdenum-antimony anti-colorimetric method. The results of rescreening the above strains are shown in table 5 below.
TABLE 5 double-sifting data table for phosphorus dissolving function of Bacillus belgii
| Strain name | Effective phosphorus content (mg/L) |
| GXMD-hs-L68 | 130.3542 |
The results show that the phosphorus dissolving function of the bacillus beilesensis is good not only on primary screening, but also on secondary screening.
Example 2
Identification of Bacillus velezensis (Bacillus velezensis-GXMD-hs-L68)
The Bacillus velezensis-GXMD-hs-L68 is preserved in Guangdong province microbial strain preservation center with the preservation number GDMCC NO:61866 at 8/12/2021.
The Bacillus belgii has a 16S rRNA gene with a base sequence of a sequence table SEQ.ID.NO. 1.
The bacillus beleisi of the invention is combined with the attached figure 1, and has the following characteristics:
growing on LB solid culture medium at 30 deg.C, and making it white, concave, viscous, irregular, and smelly.
The optimal nitrogen source of the Bacillus belgii is yeast extract, and the optimal carbon source is sucrose.
The Bacillus belgii is used to convert phosphorus unavailable to crops in soil to available phosphorus.
The application of the Bacillus belgii in agricultural planting is provided.
The application consists in irrigating the crop with a fermentation broth of Bacillus belgii.
The fermentation liquor of the Bacillus belgii is used for preparing a microbial inoculum or is added into an organic fertilizer to form a bacterial fertilizer.
The inventor separates the functional microorganism Bacillus belgii of the invention from the soil of a Shanghai reservoir of Michelia city, Michelia, Guangdong province, the preservation number is GDMCC NO:61866, and the classification name is Bacillus velezensis-GXMD-hs-L68.
Identification of Strain genes
Extracting genome DNA of the strain by using a TSINGKE plant DNA extraction kit (universal type), diluting an extracted DNA sample by a proper amount to be used as a PCR template, and amplifying by using a Scopheraceae 1 multiplied by TSE101 gold medal mix, wherein each component of an amplification system is shown in a table 1; the sequences of the universal primers are shown in Table 2.
TABLE 1 amplification System
TABLE 2 Universal primer sequences
The amplification procedure of the above amplification system is shown in Table 3.
TABLE 3 amplification procedure
The 16SrRNA amplified product was checked by electrophoresis on 1% agar gel, and the results are shown in FIG. 12. The qualified PCR product is subjected to sequencing and identification by Beijing Optimalaceae Biotechnology Limited, and the identification result shows that the strain with the phosphorus dissolving function and the obvious growth promoting effect on crops provided by the invention is Bacillus belgii.
Test example 1
Growth promoting effect of Bacillus beleisi GXMD-hs-L68 on eucalyptus
Preparation of the fermentation broth
Streaking the preserved strain on an LB solid culture medium, selecting a single strain to fall into 1ml of an LB liquid culture medium for shake culture overnight, inoculating 1ml of the strain into the liquid culture medium, and carrying out shake culture on a shaking table (30-32 ℃, 150-200r/min) for one day before pouring the experimental seedling.
Eucalyptus potting experiment
And (3) seedling recovering, namely transplanting the eucalyptus seedlings into a flowerpot filled with soil and a matrix (1:1) to ensure that the original soil nutrition is relatively consistent.
Selecting seedlings: picking out eucalyptus seedlings with relatively consistent growth vigor to carry out experiments. The experimental group and the control group were prepared by pouring the fermentation liquid of the above-mentioned strain and the control group with the same amount of LB liquid medium. And (5) after marking, randomly placing.
Pouring bacteria: 5ml of strain fermentation liquor is poured into one experimental seedling of the experimental group, and 5ml of LB liquid culture medium is poured into each experimental seedling of the control group. Pouring once a week.
Measurement: and (3) measuring indexes such as plant height, ground diameter, chlorophyll content and the like of the experimental seedlings by using a ruler, a vernier caliper, a chlorophyll measuring instrument and the like. Before the microbial inoculum is irrigated, measuring initial data, measuring relevant indexes again after two months, pulling out the experimental seedlings to measure the fresh weight and the dry weight, and calculating the dry-wet ratio.
The experimental result shows that the fermentation liquor of the strain has obvious growth promoting effect on eucalyptus. In two months, the height of eucalyptus and the diameter of land of the eucalyptus poured with the strain fermentation liquor are increased by 3.85% and 36.39% compared with the control group, and besides, the strain fermentation liquor also increases the chlorophyll of eucalyptus leaves by 10.52% and increases the dry weight/wet weight by 23.33% compared with the control group. The experimental results are shown in the attached FIGS. 3-6.
Test example 2
Growth promoting effect of the Bacillus beleisi L68 on corn potted plants
Preparation of the fermentation broth
Streaking the preserved strain on an LB solid culture medium, selecting a single strain to fall into 1ml of an LB liquid culture medium for shake culture overnight, inoculating 1ml of the strain into the liquid culture medium, and carrying out shake culture on a shaking table (30-32 ℃, 150-200r/min) for one day before pouring the experimental seedling.
Experiment of potted corn
Selecting seedlings: and planting corns in the flowerpot, and selecting the corns with relatively consistent growth vigor to carry out an experiment after the corns grow to 5 cm.
Pouring bacteria: the experimental group and the control group were prepared by pouring the fermentation liquid of the above-mentioned strain and the control group with the same amount of LB liquid medium. And (5) after marking, randomly placing. One experimental corn seedling is watered with 5ml of strain fermentation liquor, and each experimental seedling of the control group is watered with 5ml of LB liquid culture medium. Pouring the mixture once every two weeks.
Measurement: and measuring the indexes of the experimental seedlings, such as plant height, ground diameter, leaf width, root length and the like by using instruments such as a ruler, a vernier caliper and the like. Before the microbial inoculum is irrigated, measuring initial data, measuring relevant indexes again after two months, pulling out the experimental seedlings to measure the root length, fresh weight and dry weight, and calculating the dry-wet ratio.
The experimental result shows that the fermentation liquor of the strain has obvious growth promoting effect on corn. In two months, compared with the corn of a control group, the height of the corn irrigated with the strain fermentation liquor is increased by 54.00%, the ground diameter is increased by 10.02%, and the leaf width is increased by 24.68%. Besides, the strain fermentation liquor can also make the root length of corn 100.73% longer than that of control group, and make dry weight/wet weight increase 19.05%. The experimental results are shown in FIGS. 7 to 11.
The gene sequence of Bacillus velezensis ((Bacillus velezensis-GXMD-hs-L68)16S rRNA:
it is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention 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 solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. Bacillus velezensis-GXMD-hs-L68, which has been deposited at 12/8/2021 with the deposit number GDMCC NO:61866 and is deposited at the Guangdong province collection center for microorganisms.
2. The Bacillus velezensis-GXMD-hs-L68 as claimed in claim 1, wherein the Bacillus velezensis ((Bacillus velezensis-GXMD-hs-L68) grows on LB solid medium at 30 ℃, and is white, concave, viscous, irregular, and smelly.
3. The Bacillus velezensis-GXMD-hs-L68 of claim 1, wherein the Bacillus velezensis is isolated from soil in Shang reservoir, Wen Yi City, Guangdong province.
4. The method for preparing Bacillus velezensis-GXMD-hs-L68 according to claim 1, comprising the steps of:
step one, material preparation; sampling a sample: the soil sampling place is a Shanghai reservoir in the West and Yi City of Maoming City, Guangdong province, surface soil is pulled out, a soil sample with the depth of 15cm below the surface layer is collected and stored in an incubator at 4 ℃, and the soil sample is taken back to a laboratory for next treatment;
step two: isolation of the strainsMelting; adding 2g soil sample into 18ml sterile water to prepare soil suspension, shaking for 30min in a shaking table (180r/min), and diluting with sterile water to 10-1-10-6Taking 10 times of sample-5、10-6And (3) coating 0.1ml of samples with two dilution multiples on a screening culture medium, placing a flat plate of the screening culture medium coated on the screening culture medium in an incubator at the temperature of 32-37 ℃, observing whether a transparent ring appears on the flat plate at intervals, and picking the strain with the transparent ring to an LB solid culture medium for streak culture to obtain a single colony.
5. A method for preparing a fermentation liquid from the Bacillus subtilis ((Bacillus velezensis-GXMD-hs-L68) as claimed in claim 1, which comprises streaking the Bacillus subtilis ((Bacillus velezensis-GXMD-hs-L68) on LB solid medium, shaking culturing the single strain in 1ml LB liquid medium overnight, inoculating 1ml of the above-mentioned strain liquid into the liquid medium, shaking culturing in a shaker (30-32 ℃, 150-200r/min) for one day to obtain a Bacillus subtilis ((Bacillus velezensis-GXMD-hs-L68) fermentation liquid.
6. Use of a Bacillus velezensis-GXMD-hs-L68 fermentation broth for promoting plant growth according to claim 4.
7. The Bacillus velezensis-GXMD-hs-L68 fermentation liquid of claim 4, which is used for preparing bacterial preparation or added into organic fertilizer to form bacterial fertilizer.
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