CN114934000B - Salt-tolerant bacillus amyloliquefaciens and screening and application thereof - Google Patents
Salt-tolerant bacillus amyloliquefaciens and screening and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01B—SOIL WORKING IN AGRICULTURE OR FORESTRY; PARTS, DETAILS, OR ACCESSORIES OF AGRICULTURAL MACHINES OR IMPLEMENTS, IN GENERAL
- A01B79/00—Methods for working soil
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- 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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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C09K2101/00—Agricultural use
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract
The application relates to the technical field of microbial strain application, in particular to salt-tolerant bacillus amyloliquefaciens and screening and application thereof. The application provides salt-tolerant bacillus amyloliquefaciens, and screening and application thereof, and the strain can adapt to salt-tolerant environment, has a strong growth promoting function and has the characteristic of enabling plants to normally grow in salinized soil. The bacillus with the preservation number of CGMCC No.24545 used for improving the salinized soil has the advantages of being green, simple, convenient and low in cost, and has obvious advantages in practical application. The salinized agricultural biocontrol strain has the characteristics of salt tolerance, promoting crop growth and improving salt tolerance and stress resistance of crops, and compared with the non-inoculation, the plant growth promotion test shows that the strain can obviously promote seed germination, can improve the salt tolerance and stress resistance of crops, can normally grow at 28-32 ℃, can obviously promote plant growth, and can be used in actual production practice.
Description
Technical Field
The application relates to the technical field of microbial strain application, in particular to salt-tolerant bacillus amyloliquefaciens and screening and application thereof.
Background
With unreasonable utilization of chemical fertilizers, excessive use of macroelement fertilizers and unreasonable farmland irrigation, the soil salinization phenomenon is more and more aggravated. The reasons for the salinization in the current generation are mainly caused by improper production activities of human beings, mainly comprise geological changes and underground water source exploitation, and change the original natural water-salt balance state.
The general solution of salinization is mainly divided into 3 types, namely 3 types of water conservancy, chemistry and biology, wherein the water conservancy and chemistry methods are mainly treated by manually improving irrigation measures and methods, changing physicochemical properties and granular structures of soil, and the like. The biological measure is to repair soil by the action of plants and microorganisms, and the plants with good salt tolerance and salt absorption effect are used for absorbing salt in soil during plant growth, so that the salt content in the soil is reduced, and the salinization level of the soil is improved. The microbial measures mainly utilize the tolerance of microorganisms to soil salinity, and the microbial measures are synergistic with crops to improve the tolerance of the crops to the salinity, so that the adaptability of the crops to the salinized soil is improved, and some microorganisms can also self-solidify the salinity in the soil, such as trichoderma reesei and the like.
The chemical method has the problems of low efficiency, high consumption, harsh reaction conditions, chemical toxicity, secondary pollution and the like; the biological purification method is widely focused due to the advantages of high efficiency, low consumption, mild reaction conditions, small secondary pollution and the like, and the method for treating the salinized soil by utilizing microorganisms has few reports at home and abroad, and has the advantage that a small number of strains can simultaneously promote plant growth and repair the microorganisms of the salinized soil.
Disclosure of Invention
In view of the above, the application aims to provide salt-tolerant bacillus amyloliquefaciens, and screening and application thereof, wherein the strain has a proper salt-tolerant environment, a strong growth promoting function and the characteristic of enabling plants to normally grow in salinized soil.
The application provides bacillus with a preservation number of CGMCC No.24545.
The bacillus provided by the application has the advantages that the nutrition cells are in rod-shaped and round ends, the majority of the nutrition cells are in single, few pairs or chain arrangement, the cell walls are of gram positive structures, the surfaces of bacterial colonies in the nutrition agar culture medium are rough and opaque, dirty and white, and the edges of the nutrition cells are rough, thick or frozen.
The application is to identify the phylogenetic status of the strain, obtain the reference strain sequence from NCBI (GenBank) database, analyze the 16S rDNA sequence of the isolated strain and the reference strain by using software BioEdit and MEGA11, construct the phylogenetic tree of the isolated strain and the reference strain. Thus, the Bacillus strain was identified as a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) (FIG. 3) and designated BGB-95R.
The bacillus growth conditions provided by the application comprise: the pH value is 6-8, the temperature is 18-35 ℃, the rotating speed is 120-180 r/min, and a wide range of carbon sources and nitrogen sources can be utilized.
In some embodiments, the bacillus provided by the application has the following optimal growth conditions: the pH value is 7.0, the temperature is 28-32 ℃, the rotating speed is 160r/min, and a wide range of carbon sources and nitrogen sources can be utilized.
The bacillus disclosed by the application can promote seed germination, promote the normal growth of plants in salinized soil, and improve the stress resistance of the plants in salinization. The thalli are released into soil, are harmless to human, animals and plants, do not pollute the environment, can enrich the structure of the natural flora under certain conditions, and improve the diversity of the natural flora.
Therefore, the application also provides the application of the bacillus with the preservation number of CGMCC No.24545 in promoting plant growth and/or repairing salinized soil.
The use according to the application, wherein the promotion of plant growth comprises at least one of the following I) to III):
i) Promoting seed germination;
II) promoting plant root growth and/or development;
III), biomass accumulation is improved.
In the application of the application, the improvement of the seed germination rate comprises the improvement of the seed germination index, and particularly the improvement of the seed germination index to 5% -15%;
the growth of plant root systems is promoted to be improved to 20% -30%;
the biomass accumulation is improved to 60% -70%.
The use according to the application, wherein the plant comprises cucumber or rice.
The application of the application, wherein the salt content in the salinized soil is more than 2%.
In some embodiments, the application of the application, wherein the salt content in the salinized soil is 0% -4%.
In other embodiments, the use of the application, wherein the salinized soil has a salinity of 1%, 2%, 3%, 4%.
In some embodiments, the salinized soil has a salinity of 2%.
In other embodiments, naCl is used to simulate the salt concentration in the soil, and when the NaCl concentration is 2%, the concentration of the fermented bacteria is 64.3 hundred million CFU/ml at maximum, and when the NaCl concentration is 4%, the fermented bacteria can still reach 8.4 hundred million CFU/ml.
The application provides a product for promoting plant growth and/or repairing salinized soil, which comprises bacillus with a preservation number of CGMCC No.24545.
The product of the application, wherein the bacterial content of the product is 1 multiplied by 10 7 CFU/g～9×10 7 CFU/g。
The application also provides a preparation method of the product, which comprises the following steps: culturing bacillus with the preservation number of CGMCC No.24545, and collecting thalli.
The preparation method of the application further comprises the step of preparing bacterial suspension by re-suspending the bacterial after the bacterial is collected; or further comprises the steps of mixing thalli with a freeze-drying protective agent and preparing freeze-drying powder by freeze-drying.
The application also provides a method for promoting plant growth and/or repairing salinized soil, which comprises the application of the product or the product prepared by the preparation method.
The application uses the microorganism obtained by separating and screening the salinized soil, and is applied to the salinized soil and plant growth, can obviously promote the germination of plant seeds and the growth of crops, and has obvious practical application significance for the repair of the salinized soil.
Compared with the prior art, the application has the following effects that the bacillus with the preservation number of CGMCC No.24545 is used for improving the salinized soil, has the advantages of green, simplicity and low cost, and has obvious advantages in practical application; the salinized agricultural biocontrol strain has the characteristics of salt tolerance, promoting crop growth and improving salt tolerance and stress resistance of crops, and compared with the non-inoculation, the plant growth promotion test shows that the strain can obviously promote seed germination, can improve the salt tolerance and stress resistance of crops, can normally grow at 28-32 ℃, can obviously promote plant growth, and can be used in actual production practice.
Description of biological preservation
Biological material: BGB-95R, classified naming: bacillus amyloliquefaciens Bacillus amyloliquefaciens was deposited in China general microbiological culture Collection center, at a location of 2022, 03, 18: the collection number of the microbiological institute of China academy of sciences is CGMCC No.24545, and the North Chen Xili No. 1, 3 of the Chaoyang area of Beijing city.
Drawings
For a clearer description of embodiments of the application or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the application, and that, without the inventive effort, other drawings can be obtained from them to those skilled in the art:
FIG. 1 shows a photograph of B.amyloliquefaciens BGB-95R taken under an optical microscope X1000;
FIG. 2A shows the morphology of colonies of B.amyloliquefaciens BGB-95R, cultured at 28℃on nutrient agar medium; b in fig. 2 shows a colony map;
FIG. 3 shows a phylogenetic tree constructed based on the 16S rDNA sequence;
FIG. 4 shows a BGB-95R salt tolerant culture of Bacillus amyloliquefaciens;
a in fig. 5 shows that bacillus amyloliquefaciens BGB-95R promotes germination of cucumber seeds in a culture dish; b in fig. 5 shows a cucumber seed versus dipstick control graph of bacillus amyloliquefaciens BGB-95R promoting germination in a petri dish;
FIG. 6 shows the growth promoting effect of B.amyloliquefaciens BGB-95R on cucumber seedlings;
FIG. 7 shows that Bacillus amyloliquefaciens BGB-95R promotes germination of rice seeds under high salinity conditions;
FIG. 8A shows a control plot of B.amyloliquefaciens BGB-95R-promoted rice seedlings against a measuring scale; b in FIG. 8 shows a control plot of B.amyloliquefaciens BGB-95R-promoted-growth seedlings of rice planted in a flowerpot against a measuring scale;
FIG. 9 shows that B.amyloliquefaciens BGB-95R promotes rice seedling growth and improves salt tolerance in flowerpots, wherein T represents the treatment group and CK represents the control group.
Detailed Description
The application provides screening and application of salt-tolerant bacillus amyloliquefaciens, and a person skilled in the art can properly improve process parameters by referring to the content of the application. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present application. While the methods and applications of this application have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the application can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the application.
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
1. Isolation and purification of strains
1) Soil sample collection: salinized soil of mud flat saline land with 2% salt content is collected from Shangyang county of salt city of Jiangsu province, linhai county of sea town (120 degrees 23 '20' of east longitude, 33 degrees 58 '44' of north latitude), date and other information are registered, and basic physicochemical properties of a sample are detected.
2) Primary separation of target bacteria: weighing 10g of the collected sample, adding into a pre-sterilized triangular flask containing 100ml of physiological saline under aseptic condition, sufficiently oscillating at 28deg.C for 30min, and pressingDiluting to 10 according to the proportion -5 The dilution concentration was chosen to be 10 -3 、10 -4 、10 -5 Respectively sucking 0.1 ml-0.2 ml, coating on high-salinity nutrient agar (NaCl content is 30.5 g/L) (formula is shown in Table 1), and culturing at 28deg.C for 15-30 hr. Selecting a monoclonal on a plate, selecting different bacterial colonies according to the microbial forms on the plate, streaking, separating and purifying (culturing according to the culture conditions) for 2-3 times until the bacterial colonies are single, numbering, recording and preserving.
3) Preliminary identification: the isolated and purified strain was examined under a microscope and observed, FIG. 1.
Table 1 high salinity nutrient agar medium formulation
2. Properties of the Single Strain
1) Morphological features
Strains: the vegetative cells are in rod shape, round end, most of which are single, few of which are arranged in pairs or chains, and the cell walls are of gram positive structures. The optimal growth temperature is 28-32 ℃, the highest growth temperature is 35 ℃, the lowest growth temperature is 18 ℃, and the anaerobic or facultative anaerobic is adopted. The colony surface cultured in nutrient agar medium at 28deg.C for 18 hr is rough, opaque, dirty white, rough, thick or frozen (figure 2).
2) Culture characteristics
The optimal growth conditions of the strain are as follows: the pH=7.0, the temperature is 28-32 ℃, the rotating speed is 160r/min, and a wide range of carbon sources and nitrogen sources can be utilized.
3) Functional characteristics
In the plant growth process, the main functions are to promote seed germination, promote the normal growth of plants in salinized soil and improve the stress resistance of plants in salinization. The thalli are released into soil, are harmless to human, animals and plants, do not pollute the environment, can enrich the structure of natural flora under certain conditions, and improve the diversity of the natural flora.
3. Sequencing of the 16S rDNA sequence of the Strain
To identify the phylogenetic status of the strain, the 16S rDNA sequences of the isolated strains were sequenced. The reference strain sequences were obtained from NCBI (GenBank) database, and 16S rDNA sequences of the isolated strain and the reference strain were analyzed using software BioEdit and MEGA11 to construct phylogenetic trees of the isolated strain and the reference strain. Thus, the Bacillus strain was identified as a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) (FIG. 3) and designated BGB-95R.
Compared with the prior art, the salinized agricultural biocontrol strain has the following effects that the salinized agricultural biocontrol strain has the characteristics of salt tolerance, growth promotion and crop growth improvement, and plant growth promotion experiments show that compared with the plant growth promotion experiments, the salinized agricultural biocontrol strain can obviously promote seed germination, can improve the salt tolerance and stress resistance of crops, can normally grow at 28-32 ℃, can obviously promote plant growth, and can be used for actual production.
EXAMPLE 2 isolation and purification of strains
1. Isolation and screening of strains
After soil sample collection, the samples were stored in a laboratory at low temperature, diluted 100-fold with sterile water into triangular flasks, respectively, and allowed to stand for 15min after shaking at 28℃for 30min at 160 rpm. Plating in nutrient medium, gao's I and PDA medium, and culturing at 28 deg.C in constant temperature incubator. When single colonies were grown on the plate, the single colonies were numbered, and representative colonies were picked, isolated and purified.
2. Purification of strains
After the bacterial colony grows out of the flat plate, a small amount of bacterial colony is scraped from the flat plate by using a sterilized toothpick, diluted by sterile water and streaked on the flat plate again for culture. If no monoclonal colony appears, repeatedly scribing and purifying on the plate until the monoclonal colony appears, purifying for multiple times, and until the colony forms are uniform and no other colonies with different forms appear.
Strains: the vegetative cells are in rod shape, round end, most of which are single, few of which are arranged in pairs or chains, and the cell walls are of gram positive structures. The optimal growth temperature is 28-32 ℃, the highest growth temperature is 35 ℃, the lowest growth temperature is 18 ℃, and the anaerobic or facultative anaerobic is adopted. The colony surface cultured for 20h at 28deg.C in nutrient agar medium is rough, opaque, dirty white, and rough in edge, and is picked into viscous or frozen form (figure 2).
By using the separation and purification method, repeated purification is carried out for a plurality of generations, and streak culture is carried out in an improved nutrition solid culture medium (table 1), so that a single strain is obtained, and the strain has stronger salt tolerance after culture in a flat plate culture medium.
EXAMPLE 3 16S rDNA sequence sequencing of Strain Single Strain
The monoclonal bacteria liquid of the strain is subjected to PCR specific amplification, the primers are 27F (SEQ ID NO: 2) and 1492R (SEQ ID NO: 3), and 2 XStar Mix is used as enzyme. And detecting the PCR amplified product by using an electrophoresis imaging technology, observing whether the PCR amplified product has a band, and using the rest PCR amplified product for sequence determination. The sequencing result is shown as SEQ ID NO. 1. The PCR reaction system is as follows:
TABLE 2 16SrDNA 2 XStarMix enzyme reaction System
Multiple reference strain sequences were obtained from NCBI (GenBank) database, and the whole length of the 16S rDNA sequences of the isolated strain and the reference strain was analyzed by using software BioEdit and MEGA11 to construct a phylogenetic tree of the isolated strain and the reference strain (FIG. 3). Thus, the genus relationship of the strain was determined to be Bacillus amyloliquefaciens and designated as BGB-95R (Jiabo text No. 95).
EXAMPLE 4 salt tolerance growth-promoting test of Strain
1 test purpose: and detecting the salt tolerance and growth promotion effects of the strain.
2 test materials
2.1 test materials: cucumber seeds (No. 16 of Chinese farmers), and a Jiabo Wen Turang conditioner (the main materials are organic substances which are formed by sieving and decomposing kitchen waste and adding auxiliary materials), namely the kitchen waste is recycled into organic substances, perlite, vermiculite, coco coir and the like.
2.2 test strains: bacillus amyloliquefaciens BGB-95R strain
2.3 main test instruments and equipment: ultra-clean bench, autoclave, constant temperature shaking incubator, ultraviolet spectrophotometer, 500mL conical flask, tweezers, culture dish, glass rod, inoculating loop, 1.5mL centrifuge tube, puncher, coating rod, seedling tray, 2.5L flowerpot, etc.
2.4 test drugs and reagents
(1) Test drug: peptone, H 2 O, naCl Yeast powder, beef extract, agar, and sodium hypochlorite.
(2) Test reagent:
1) Nutrient liquid medium: 10g of peptone, 5g of beef extract and 10g of NaCl are weighed and dissolved in 1L of pure water, and the pH value is: 6.8-7.5, at 121 ℃, for 30min, and sterilizing.
2) Nutrient solid medium: weighing 10g of peptone, 5g of beef extract, 10g of NaCl and 20g of agar, dissolving in 1L of pure water, and dissolving in pH: 6.8-7.5, at 121 ℃, for 30min, and sterilizing.
3) Preparing nutrient soil and nutrient indexes: preparing nutrient soil by using a conditioning agent of cabo Wen Turang, perlite, vermiculite and coconut husk according to a volume ratio of 3:1:2:4, wherein the conventional nutrition indexes (dry basis) after sterilization are respectively as follows: 65% of organic matters, 1.45% of nitrogen, 0.37% of phosphorus and 0.16% of potassium.
3 test procedure
3.1 Strain culture
3 times activating test strain stored at-80deg.C, inoculating into nutrient liquid culture medium, culturing to logarithmic phase, detecting strain growth condition with ultraviolet spectrophotometer, estimating bacterial content with OD value, counting with dilution coating plate, diluting with sterile water to 1×10 9 CFU/ml～9×10 9 CFU/ml for use. And culturing the strain to be tested in a nutrient solid culture medium by streaking for later use.
3.2 salt tolerance experiments with strains
The BGB-95R strain plate obtained by culturing in the nutrition culture medium is perforated by a sterile 5mm puncher and placed in a sterilized nutrition liquid culture medium, wherein 50ml of nutrition liquid is respectively added into each 100ml of conical bottle, 1 piece of bacteria piece is placed in each bottle, the bottle is placed at 28 ℃, the bottle is subjected to shaking culture at a constant temperature of 180rpm, three times of treatment are repeated, blank treatment is carried out, OD value of the nutrition liquid is observed after 34h of culture, and the bacteria content is detected by a dilution coating plate method. The statistical results are shown in Table 3 below, and the culture results are shown in FIG. 4:
TABLE 3 cultivation of NaCl strains BGB-95R with different concentrations
| NaCl concentration% | OD value | Bacterial count hundred million CFU/ml |
| CK | 0.000±0.000 | 0.0±0.0 |
| 0 | 0.492±0.005 | 48.0±0.0 |
| 1 | 0.527±0.014 | 64.3±3.1 |
| 2 | 0.484±0.010 | 46.0±3.0 |
| 3 | 0.361±0.012 | 32.3±3.1 |
| 4 | 0.225±0.022 | 8.4±1.8 |
The result shows that the bacillus amyloliquefaciens can normally grow when the NaCl concentration is 0% -4%, the concentration of the fermentation bacteria is 64.3 hundred million CFU/ml at most when the NaCl concentration is 2%, and the fermentation bacteria amount can still reach 8.4 hundred million CFU/ml when the NaCl concentration is 4%. The bacillus amyloliquefaciens BGB-95R can normally grow in a high-concentration salt environment, has a certain salt environment resistance, and can be applied to soil with high salt content.
3.3 experiments of Strain promoting seed germination
Culturing BGB-95R with nutrient liquid culture medium according to salt tolerance test method, culturing for 34 hr, centrifuging the above fermentation broth at 8000rpm with centrifuge, collecting the lower precipitate, and diluting bacteria content to 1×10 with sterile water 9 CFU/ml～9×10 9 CFU/ml. Fresh and full cucumber seeds are taken, sterilized and washed for 15s by sodium hypochlorite with the solute mass fraction of 0.2%, and repeatedly washed for 2-3 times, so that the surface sterilization of the cucumber seeds is ensured to be complete without damaging the cucumber seeds.
Will have been diluted to 1X 10 9 CFU/ml～9×10 9 The CFU/ml bacterial suspension is diluted 100 times by sterile water for later use. Placing 1 piece of sterile filter paper on a 9cm sterile culture dish, placing 20 sterilized cucumber seeds with basically consistent size, adding 10ml of the diluted 100-time bacterial suspension, covering a culture dish cover, culturing for 48 hours in a 28 ℃ incubator in a dark place, counting the number of germinated seeds, and measuring the main root length one by a ruler. A blank test was performed using sterile water as a control.
The germination rate of seeds was calculated as follows.
The germination index G of seeds is expressed in percent and is calculated according to the formula:
G＝(A1*A2)/(B1*B2)
note that: a1-the number of germinated grains in the treated seeds accounts for the percentage of the total number of the grains; a2-average root length values for all seeds of the treatment group; b1-percentage of total number of germinated grains in seeds of the control group; average root length values for all seeds of the B2-treatment group.
3.4 Strain growth-promoting experiments
Placing nutrient soil in a seedling raising tray, thoroughly watering with sterile water, placing the treated seedling raising tray in a greenhouse at 28 ℃ for 12 hours, treating cucumber seeds with 0.2% sodium hypochlorite for 15 seconds, washing with sterile water for 3-5 times, planting in the seedling raising tray according to a hole sowing mode, germinating the cucumber seeds after 18 days, and transplanting cucumber seedlings. Placing the nutrient soil in another seedling tray, and diluting to bacterial content of 1×10 9 CFU/ml～9×10 9 The fermentation broth of CFU/ml BGB-95R is poured to be used as a treatment group (according to the quality of adding potting soil, the content of the potting soil bacteria is ensured to be 1 multiplied by 10) 9 CFU/ml～9×10 9 CFU/ml, the control group is thoroughly irrigated by using sterile water, and the consistent watering amount is ensured. Cucumber seedlings are transplanted into seedling raising trays of the treatment group and the control group respectively, and are placed in an artificial growth chamber for culture, wherein the growth chamber (the photoperiod is that day/night=16 h/8h, and the temperature is that day/night=30 ℃/24 ℃). And repeating for 10 times, culturing for 30 days, harvesting and detecting, and taking biological indexes such as fresh weight, root length and the like of plants as judging indexes of the BGB-95R growth promoting effect.
TABLE 4 influence of BGB-95R on germination index of cucumber seeds and seedling growth
| Experimental treatment | Germination index% | Root length cm | Fresh weight of whole plant g |
| Treatment group | 93.45±1.03 | 12.82±1.70 | 2.99±0.22 |
| Control group | 83.33±2.06 | 10.06±0.99 | 1.80±0.18 |
As shown in the experimental results in FIG. 5, FIG. 6 and Table 4, the germination of cucumber seeds can be promoted by using the BGB-95R fermentation broth, and compared with a control, the germination index is improved by about 10 percent. The BGB-95R fermentation broth can promote cucumber seedling growth in seedling stage, and compared with control, the fresh weight of cucumber in seedling stage can be increased by more than 65%, root length can be increased by more than 25%, and statistical data show that cucumber root length and fresh weight are obviously different.
Therefore, the BGB-95R can promote seed germination and plant growth in cucumber seeds and seedling stages, has remarkable promoting effect on cucumber growth, can normally grow in an environment with salt content of less than 4%, and has certain salt-tolerant growth capacity.
In conclusion, the bacillus amyloliquefaciens BGB-95R can be applied to soil with high salt content, can promote seed germination and plant growth in the environment, and has a certain promoting effect on improving salt tolerance of plants.
Example 5 test of growth promotion of saline-alkali soil Rice by bacterial strain
1 test purpose: and detecting the growth promoting effect of the strain on rice in saline-alkali soil.
2 test materials
2.1 test materials: rice seeds (Xuedao No. 10), salinized soil collected from the salt city of Jiangsu province, guanyang county in the coastal town in the temporary village, etc. The nutrient content of the salinized soil: 0.23% of total nitrogen, 69.20mg/kg of quick-acting phosphorus, 204mg/kg of quick-acting potassium, pH 7.81 and 20.12g/kg of total salt.
2.2 test strains: bacillus amyloliquefaciens BGB-95R strain
2.3 main test instruments and equipment: ultra-clean bench, autoclave, constant temperature shaking incubator, ultraviolet spectrophotometer, 500mL conical flask, tweezers, culture dish, glass rod, inoculating loop, 1.5mL centrifuge tube, puncher, coating rod, seedling tray, 2.5L flowerpot, etc.
2.4 test drugs and reagents
(1) Test drug: peptone, H 2 O, naCl yeast powder, beef extract, agar, sodium hypochlorite, etc.
(2) Test reagent:
1) Nutrient liquid medium: 10g of peptone, 5g of beef extract and 10g of NaCl are weighed and dissolved in 1L of pure water, and the pH value is: 6.8-7.5, at 121 ℃, for 30min, and sterilizing.
2) Nutrient solid medium: weighing 10g of peptone, 5g of beef extract, 10g of NaCl and 20g of agar, dissolving in 1L of pure water, and dissolving in pH: 6.8-7.5, at 121 ℃, for 30min, and sterilizing.
3) Soil sterilization and matrix configuration: sterilizing the salinized soil at 121 ℃ for 40min, and placing the sterilized soil in a 2.5L flowerpot which is sterilized by sodium hypochlorite for later use.
3 test procedure
3.1 Strain culture
In the same manner as in example 4, the strain to be tested stored at-80℃was subjected to 3-time activation and then transferred to a nutrient broth to be cultured until the logarithmic phase, the growth of the strain was detected by an ultraviolet spectrophotometer, the bacterial content was estimated by using the OD value, and the strain was counted by using a dilution-coated plate and diluted to 1X 10 with sterile water 9 CFU/ml～9×10 9 CFU/ml for use. And culturing the strain to be tested in a nutrient solid culture medium by streaking for later use.
3.2 experiments of promoting germination of Rice seeds by Strain
In the same manner as in example 4, BGB-95R was cultured in a nutrient broth according to the salt tolerance test method, after 34 hours of culture, the above fermentation broth was centrifuged at 8000rpm with a centrifuge, and the lower layer of precipitate was removed and diluted with sterile waterReleasing bacteria to 1X 10 9 CFU/ml～9×10 9 CFU/ml. Fresh full rice seeds are taken, sterilized and washed for 15s by sodium hypochlorite with the solute mass fraction of 0.2 percent, and repeatedly washed for 2 to 3 times, so that the surface sterilization of the rice seeds is ensured to be complete without damaging the rice seeds.
Will have been diluted to 1X 10 9 CFU/ml～9×10 9 The CFU/ml bacterial suspension is diluted 100 times by sterile water for later use. Placing 1 piece of sterile filter paper on a 9cm sterile culture dish, placing 20 sterilized rice seeds with basically consistent size on the sterile filter paper, adding 10ml of the diluted 100-time bacterial suspension, covering a culture dish cover, culturing for 48 hours in a 28 ℃ incubator in a dark place, counting the grain number of germinated seeds, and measuring the main root length and the bud length one by using a ruler. Wherein the control group is: 10ml of sterile water+1.5g of sterile NaCl, the experimental group is: 10ml of bacterial suspension+1.5 g of sterile NaCl, blank: 10ml of sterile water.
The germination rate of seeds was calculated as follows.
The germination index G of seeds is expressed in percent and is calculated according to the formula:
G＝(A1*A2)/(B1*B2)
note that: a1-the number of germinated grains in the treated seeds accounts for the percentage of the total number of the grains; a2-average root length values for all seeds of the treatment group; b1-percentage of total number of germinated grains in seeds of the control group; average root length values for all seeds of the B2-treatment group.
3.3 test of growth promotion of seedlings of Rice by Strain
Using the sterilized salinized soil to obtain a bacterial content of 1×10 9 CFU/ml～9×10 9 The CFU/ml bacterial suspension is uniformly mixed with salinized soil according to the mass ratio of 1:100, and the bacterial content of the soil is 1 multiplied by 10 7 CFU/ml～9×10 7 CFU/ml, and no-bacteria blank control was set. Selecting rice seedlings with similar hollow white group growth vigor in the step 3.2, transplanting the rice seedlings into the seedling raising trays of the treatment group and the control group respectively, and placing the rice seedlings into an artificial growth chamber for culture, wherein the growth chamber (photoperiod is that day/night=16 h/8h, and the temperature is that day/night=30 ℃/24 ℃). 3 plants are arranged in each pot, 4 pots are treated in each pot, total 12 plants are obtained, and the plants are harvested after 20 days of cultureAnd detecting, wherein biological indexes such as fresh weight and plant height of plants are used as judging indexes of the BGB-95R growth promoting and salt tolerance effects.
TABLE 5 influence of BGB-95R on germination index of Rice seeds and seedling growth
| Experimental treatment | Germination rate% | Bud length cm | Germination index% | Plant height cm | Fresh weight of whole plant g |
| Treatment group | 96.7 | 2.06±0.23 | 92.00 | 26.61±0.81 | 0.59±0.01 |
| Control group | 83.3 | 1.72±0.24 | 65.06 | 21.75±3.45 | 0.37±0.01 |
| Blank group | 96.7 | 2.25±0.27 | - | - | - |
As shown in FIG. 7 and Table 5, the BGB-95R fermentation broth can promote germination of rice seeds, and compared with the control, the germination index of the seeds is improved by about 27%, the germination rate is improved by about 13%, and the growth of the buds and the root length is obviously increased. Compared with the control group, the BGB-95R can obviously improve the germination rate and the seed germination index of rice seeds when the salt content is about 1.5 percent.
As shown in fig. 8, 9 and table 5, the BGB-95R fermentation broth was used to promote rice seedling growth at the seedling stage, and the fresh weight of rice at the seedling stage was improved by more than 37% as compared with the control, and the statistical data showed that the treated group showed significant differences in both rice plant height and fresh weight as compared with the control group.
Therefore, BGB-95R can promote seed germination and plant growth in rice seeds and seedling stage, improve salt tolerance of rice in salinized soil, has remarkable promoting effect on rice growth, can normally grow in an environment with salt content lower than 2%, and can improve salt tolerance of plants.
In conclusion, the bacillus amyloliquefaciens BGB-95R can be applied to soil with high salt content, can promote seed germination and plant growth in the environment, has a certain promoting effect on planting and growing rice in saline-alkali soil and improving salt tolerance of the rice, and can have a certain promoting effect on improving salt tolerance of plants.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
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.
Sequence listing
<110> Beijing Jiabo Biotech Co., ltd
<120> salt-tolerant bacillus amyloliquefaciens, screening and application thereof
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Claims (6)
1. The bacillus amyloliquefaciens is characterized in that the bacillus amyloliquefaciens with the preservation number of CGMCC No.24545Bacillus amyloliquefaciens)。
2. Use of bacillus amyloliquefaciens according to claim 1 for promoting the growth of rice in salinized soil, said salinized soil having a salt content of less than 2%.
3. The product for promoting the growth of rice in salinized soil is characterized by comprising bacillus amyloliquefaciens with the preservation number of CGMCC No. 24545; the content of the bacillus amyloliquefaciens in the product is 1 multiplied by 10 7 CFU/g ~9×10 7 CFU/g。
4. A method of preparing a product according to claim 3, comprising: culturing bacillus with the preservation number of CGMCC No.24545, and collecting thalli.
5. The method according to claim 4, further comprising the step of preparing a bacterial suspension by re-suspending the bacterial cells after the bacterial cells are collected; or further comprises the steps of mixing thalli with a freeze-drying protective agent and preparing freeze-drying powder by freeze-drying.
6. A method for promoting rice growth in salinized soil comprising applying the product of claim 3 or the product of claim 4 or 5.
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