CN117229985B - Biocontrol strain bacillus atrophaeus BGB-98R and application thereof - Google Patents
Biocontrol strain bacillus atrophaeus BGB-98R and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to the technical field of microorganisms and application thereof, and particularly discloses a biocontrol strain bacillus atrophaeus BGB-98R and application thereof. The bacillus atrophaeus BGB-98R is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27752 at 2023, 6 and 30. The application also provides a culture, a fermentation broth, a bacterial suspension, a bacterial agent and bacterial powder which comprise the bacterial strain; and the application of the strain and the culture, fermentation broth, bacterial suspension, bacterial agent and bacterial powder thereof in the fields of improving the soil microorganism structure and preventing and controlling pathogenic bacteria. The bacillus atrophaeus BGB-98R provided by the application has a stronger biocontrol function, can normally grow in drought-barren soil, can reduce the disease rate of potato rot, can obviously promote potato growth, improves potato yield, and can also improve the soil microbial community structure in field use.
Description
Technical Field
The application relates to the technical field of microorganisms and applications thereof, in particular to a biocontrol strain bacillus atrophaeus BGB-98R and applications thereof.
Background
Bacillus is widely present in nature and belongs to a class of aerobic or facultative anaerobic bacteria, and bacillus can produce spores with strong stress resistance in severe environments to pass through bad environmental conditions. The research shows that the bacillus has the most obvious biocontrol advantage, can grow in severe environment and can propagate in a large amount in certain environment, and has good inhibition effect on common pathogenic bacteria which are easy to cause rot diseases, such as fusarium graminearum, fusarium oxysporum, gray mold and the like.
With the development of scientific technology, more and more strains are discovered and cultivated, but some strains which can be utilized and are cultivated in laboratory have poor viability in practical production and application. In order to improve the viability of the screened strains, the strain screening is performed in an extreme environment, and the method has more and more practical significance. The soil body of the desert soil is thin, the moisture is less, a humus layer is lacked, and the strain living more actively in the desert can be more actively in actual production and application.
With the exploration of desert soil by humans, more and more desert soil is reformed, and crops or field crops are planted. However, as the planting time increases, these modified soils also become less suitable for planting, such as continuous cropping obstacles, etc., caused by planting potatoes in a desert. Thus, there is an urgent need to obtain microbial species that are capable of improving desert soil, making it suitable for planting.
Disclosure of Invention
The application provides biocontrol strain bacillus atrophaeus BGB-98R and application thereof. The bacillus atrophaeus BGB-98R has a stronger biocontrol function, can normally grow in drought and barren soil, can reduce the disease rate of potato rot, can obviously promote potato growth, improves potato yield, and can also improve the soil microbial community structure in field use.
In a first aspect, the application provides bacillus atrophaeus BGB-98R, which adopts the following technical scheme:
bacillus atrophaeus (latin Wen Xueming:Bacillus atrophaeus) BGB-98R is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27752 and the preservation address of the institute of microbiological culture, national academy of sciences, 1, 3, of Beijing, chaoyang, and Classification of China at 2023, 30Bacillus atrophaeus。
The strain is obtained by separating from a soil sample of red Yao county in Ningxia Guyuan city, which has continuous cropping obstacle and non continuous cropping obstacle areas. The vegetative cells of the strain are in rod-shaped and round ends, the majority are arranged singly, the minority are arranged in pairs or in chain, and the cell walls are of gram-positive structures. The optimal growth temperature is 28-32 ℃, the maximum growth temperature is 35 ℃, the minimum growth temperature is 18 ℃, and the anaerobic or facultative anaerobic is adopted. The colony surface cultured for 20h at 28 ℃ in nutrient agar culture medium is rough and opaque, dirty white or yellow, the edge forms wrinkles, and the colony surface is picked to be sticky or frozen.
Multiple reference strain sequences were obtained from NCBI (GenBank) database and the strains were isolated from the reference strain using software BioEdit and MEGA1116SrDNA sequence is analyzed, phylogenetic tree of the separated strain and the reference strain is constructed, thereby determining that the strain of the separated strain is bacillus atrophaeus @Bacillus atrophaeus) Is shown in FIG. 3) and designated BGB-98R.
In the plant growth process, the main functions are that seed germination can be promoted, plant growth can be promoted, the plant growth has a strong inhibition effect on various pathogenic bacteria such as fusarium in soil, the plant growth can normally grow in drought-barren soil, the disease rate of potato rot can be reduced, potato growth can be obviously promoted, potato yield can be improved, and the community structure of soil microorganisms can be improved. The strain is released into soil, is harmless to human, animals and plants, does not pollute the environment, can enrich the structure of natural flora under certain conditions, and improves the diversity of the natural flora.
In a second aspect, the present application provides a culture. The culture comprises the bacillus atrophaeus BGB-98R.
In the present application, the culture may be a fermentation broth and a fermentation supernatant obtained by using the fermentation broth.
In a third aspect, the present application provides a fermentation broth. The fermentation liquor comprises the bacillus atrophaeus BGB-98R.
In a fourth aspect, the present application provides a bacterial suspension. The bacterial suspension comprises the bacillus atrophaeus BGB-98R.
In a fifth aspect, the present application provides a microbial agent. The microbial inoculum comprises the bacillus atrophaeus BGB-98R.
In a sixth aspect, the present application provides a bacterial powder. The culture comprises the bacillus atrophaeus BGB-98R.
In a seventh aspect, the application provides an application of the bacillus atrophaeus BGB-98R, a culture, a fermentation broth, a bacterial suspension, a bacterial agent or bacterial powder in the field of improving a soil microbial structure and preventing and controlling pathogenic bacteria.
Optionally, the pathogenic bacteria include fusarium graminearum, fusarium oxysporum, and gray mold.
In an eighth aspect, the application provides an application of the bacillus atrophaeus BGB-98R, a culture, a fermentation broth, a bacterial suspension, a bacterial agent or bacterial powder in the fields of promoting seed germination and plant growth.
Optionally, the bacillus atrophaeus BGB-98R has a bacterial content of 1×10 7 -9×10 8 CFU/ml。
Alternatively, when the Bacillus atrophaeus BGB-98R has a bacterial content of 5×10 8 CFU/ml, germination rate of cucumber seeds can be improved by 12.2%, and root length of cucumber seedlings can be improved by 19.2%.
Alternatively, the bacillus atrophaeus BGB-98R can grow normally in drought-barren soil.
Optionally, the bacillus atrophaeus BGB-98R can reduce the disease rate of potato rot, remarkably promote potato growth and improve potato yield.
Optionally, the bacillus atrophaeus BGB-98R is capable of improving soil microbial community structure.
In summary, the present application has the following beneficial effects:
1. the application provides bacillus atrophaeus, which is named as bacillus atrophaeus BGB-98R, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27752 in the year of 2023 and the month of 6 and 30.
2. The bacillus atrophaeus BGB-98R has rod-shaped and round ends, most of the bacillus atrophaeus BGB-98R are arranged singly, in pairs or in chains, and the cell wall has a gram positive structure. The optimal growth temperature is 28-32 ℃, the maximum growth temperature is 35 ℃, the minimum growth temperature is 18 ℃, and the anaerobic or facultative anaerobic is adopted. The colony surface cultured for 20h at 28 ℃ in nutrient agar culture medium is rough and opaque, dirty white or yellow, the edge forms wrinkles, and the colony surface is picked to be sticky or frozen.
3. In the plant growth process, the main functions are that the germination of cucumber seeds and the growth of cucumber seedlings can be promoted, the growth of fusarium and other pathogenic bacteria in soil can be greatly inhibited, in the field application, the growth can be normally carried out in drought-barren soil, the disease rate of potato rot can be reduced, the growth of potato can be obviously promoted, the yield of potato can be improved, and the community structure of soil microorganisms can be improved. The strain is released into soil, is harmless to human, animals and plants, does not pollute the environment, can enrich the structure of natural flora under certain conditions, and improves the diversity of the natural flora.
4. When the bacillus atrophaeus BGB-98R has a bacterial content of 5×10 8 CFU/ml, germination rate of cucumber seeds can be improved by 12.2%, and root length of cucumber seedlings can be improved by 19.2%.
Drawings
FIG. 1 is the microscopic observation result (1000X) of the isolated strain (wherein A is the result of culturing the isolated strain for 15 hours-logarithmic growth phase; and B is the result of culturing the isolated strain for 40 hours-spore-producing phase).
FIG. 2 shows the growth of isolated strains on nutrient agar medium.
FIG. 3 is a phylogenetic tree of isolated strains.
FIG. 4 is a graph showing the results of inhibition of bacterial strain BGB-98R against pathogenic bacteria (wherein A is the result of inhibition of F.graminearum by bacterial strain BGB-98R; B is the result of inhibition of F.oxysporum by bacterial strain BGB-98R; C is the result of inhibition of F.griseofulva by bacterial strain BGB-98R).
FIG. 5 is a graph showing the result of the action of the strain BGB-98R on the growth promotion of cucumber seedlings.
FIG. 6 is an effect on soil microorganism (fungus) abundance structure in a microbial agent field test.
FIG. 7 is an effect on soil microorganism (bacteria) abundance structure in a microbial agent field test.
FIG. 8 shows the results of a field test of microbial agents-potato test.
Detailed Description
The application provides bacillus atrophaeusBacillus atrophaeus) BGB-98R, wherein the bacillus atrophaeus BGB-98R is preserved in China general microbiological culture Collection center (CGMCC) No.27752. In addition, the application also provides the separation, purification and cultivation of the strainA method for culturing. And provides a culture, fermentation liquor, bacterial suspension, bacterial powder and bacterial agent comprising the strain.
The bacillus atrophaeus BGB-98R provided by the application and the application of cultures, fermentation liquor, bacterial suspension, bacterial powder and bacterial agents comprising the bacillus atrophaeus BGB-98R in the fields of improving soil microbial structures and preventing and controlling pathogenic bacteria. It also can promote seed germination and plant growth. In experiments for seed germination and plant growth, the bacterial content of the bacillus atrophaeus BGB-98R was selected to be 1×10 7 -9×10 8 CFU/ml. When the bacterial content of the bacillus atrophaeus BGB-98R is 5 multiplied by 10 8 CFU/ml, germination rate of cucumber seeds can be improved by 12.2%, and root length of cucumber seedlings can be improved by 19.2%. Bacillus atrophaeus BGB-98R can normally grow in drought-barren soil, can reduce the disease rate of potato rot, can remarkably promote potato growth and improve potato yield.
The formula and preparation method of the culture medium used in the application are as follows:
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.
Nutrient agar 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.
Preparing nutrient soil and nutrient indexes: preparing nutrient soil by Wen Womei g of Beijing Jiabo (organic source soil conditioner), perlite, vermiculite and coconut husk according to the volume ratio of 3:1:2:4, and detecting conventional nutrition indexes (dry basis) after sterilization as follows: 65% of organic matters, 1.45% of nitrogen, 0.37% of phosphorus and 0.16% of potassium.
Liquid fermentation seed liquid culture medium: weighing 10g of peptone, 5g of beef extract, 10g of NaCl, 5g of yeast powder and 10g of corn flour, dissolving in 1L of pure water, and dissolving in pH:6.5-7.5.
Liquid fermentation medium: 5g/L of yeast powder, 10g/L of corn powder, 10g/L of molasses, 0.1g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 1g/L of calcium carbonate and 0.5g/L of defoamer are put into a liquid fermentation tank, dissolved in pure water and the pH value is that: 5.5-6.5.
For the purposes, technical solutions and advantages of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, are intended to be within the scope of the present application.
Reagents, solvents, and other test materials used in the examples below are all commercially available.
Instrumentation used in this application: incubator (Shanghai refined macro-DHG-9030A), low speed centrifuge (Sichuan Shuke-DD 5000), shaking incubator (Shanghai know Chu-ZQZY-78 BV), pH meter (Shanghai Lei Ci PHS-3E), ultraviolet visible spectrophotometer (Qingdao-Poly-Ind UV759 CRT), liquid fermenter system (Shanghai national institute of chemical engineering equipment-FUS-50L-300L), spray dryer (Shanghai Yuming Instrument Co., ltd. -YM-5L).
The present application is described in further detail below with reference to examples, figures and performance test results.
Examples
Example 1
The embodiment provides a new strain obtaining process. The method comprises the following steps:
(1) Soil sample collection: collecting the soil (main physicochemical properties of soil: 2.41g/kg of organic matter, 151mg/kg of alkaline hydrolysis nitrogen, 9mg/kg of quick-acting phosphorus, 36mg/kg of quick-acting potassium and pH 6.06) of potato planting base (103 degrees of east longitude 34 '42', 37 degrees of north latitude 35 '23') of agricultural group of Xuechuan of Xingvillage, yingn, gansu, and the like) with continuous cropping obstacle and non continuous cropping obstacle area of potato, registering date and other information, and preserving at 4 ℃ and delivering to a laboratory.
(2) Preliminary separation of target strains: weighing 10g of the collected soil sample, adding into a pre-sterilized triangular flask containing 100ml of physiological saline under aseptic condition, sufficiently oscillating for 30min at 28 ℃ and 160rpm, standing for 15min, and proportionally diluting to 10 -5 The dilution concentration was chosen to be 10 -3 、10 -4 、10 -5 Respectively sucking 0.1-0.2ml, respectively culturing on nutrient agar, PDA, gaoshi I solid cultureCoating the substrate with a plate, and culturing at 28deg.C for 15-30 hr. Selecting monoclonal on the 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 (i.e., isolated strain) was examined under a microscope and observed, and the results are shown in FIG. 1. The culture conditions of the above isolated strains for 15h (log phase) and 40h (spore phase) are shown in FIG. 1, respectively.
Based on the microscopic examination results of FIG. 1, the isolated strain was preliminarily determined to be Bacillus.
Example 2 Single Strain Properties16SrDNA sequencing
This example provides the single strain properties and the isolated strain obtained in example 116SSequencing results of rDNA sequence. The method comprises the following steps:
single strain Properties
(1) Morphological features
The isolated strain vegetative cells are in rod-shaped, round ends, mostly in single, few paired or chain-shaped arrangement, and the cell walls are in gram-positive structures. The optimal growth temperature is 28-32 ℃, the maximum growth temperature is 35 ℃, the minimum growth temperature is 18 ℃, and the anaerobic or facultative anaerobic is adopted. The colony surface cultured for 20h at 28 ℃ in nutrient agar medium is rough and opaque, dirty white or yellow, and wrinkled at the edge, and is picked into a sticky or frozen shape as shown in figure 2.
(2) Culture characteristics
The optimal growth conditions of the strain are as follows: ph=7.0, temperature 30 ℃, rotation speed 180r/min, a wide range of carbon and nitrogen sources can be utilized.
(3) Functional characteristics
In the plant growth process, the main functions are that the germination of cucumber seeds and the growth of cucumber seedlings can be promoted, the growth of fusarium and other pathogenic bacteria in soil can be greatly inhibited, the growth of the seedlings can be normally carried out in drought-barren soil, the disease rate of potato rot is reduced, the growth of potato can be obviously promoted, the yield of potato is improved, and the microbial structure of soil can be improved. The strain is released into soil, is harmless to human, animals and plants, does not pollute the environment, can enrich the structure of natural flora under certain conditions, and improves the diversity of the natural flora.
(II)16SrDNA sequencing
To identify the phylogenetic status of the strain, the isolated strain is subjected to16SSequencing rDNA sequence to obtain isolated strain16SrDNA sequence.
The sequences of 9 reference strains were obtained from NCBI (GenBank) database and isolated using software BioEdit and MEGA1116SrDNA sequence and reference strain16SrDNA sequence analysis was performed to construct phylogenetic tree separating strain and reference strain. Thus, the strain of the isolated strain is determined to be bacillus atrophaeus @Bacillus atrophaeus) Is shown in FIG. 3) and designated BGB-98R. The DNA sequence of the strain BGB-98R is shown as SEQ ID NO 1. Meanwhile, the application provides a group of primer groups aiming at the DNA sequences, wherein the upstream primer sequence is shown as SEQ ID NO 2, and the downstream primer sequence is shown as SEQ ID NO 3.
Compared with the related art, the isolated strain BGB-98R has the following effects: the strain BGB-98R has the characteristics of promoting growth and preventing diseases, and compared with the strain without inoculation, the strain can be used for obviously promoting seed germination, inhibiting the growth of various pathogenic bacteria in soil and normally growing at 28-32 ℃. The isolated strain BGB-98R can also grow normally in drought-barren soil, can reduce the disease rate of potato rot, can obviously promote potato growth, improves potato yield, improves soil microbial community structure, and can be used for actual production.
Bacillus atrophaeus @Bacillus atrophaeus) BGB-98R is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27752 in 2023, 6 and 30.
Example 3
The embodiment provides a fermentation broth of bacillus atrophaeus BGB-98R.
The preparation method of the fermentation broth specifically comprises the following steps:
the isolated strain obtained in example 1 was activated and transferred to a nutrient broth and cultured at 28℃for 20h to obtain a fermentation broth of B.atrophaeus BGB-98R.
Example 4
The embodiment provides fermentation supernatant of bacillus atrophaeus BGB-98R.
The preparation method of the fermentation supernatant specifically comprises the following steps:
activating the isolated strain obtained in the example 1, transferring the activated strain into a nutrient liquid culture medium, and culturing the strain for 20 hours at the temperature of 28 ℃ to obtain fermentation liquor of bacillus atrophaeus BGB-98R; and centrifuging the fermentation liquor to obtain a supernatant, namely the fermentation supernatant of the bacillus atrophaeus BGB-98R.
Example 5
The embodiment provides a bacterial suspension of bacillus atrophaeus BGB-98R.
The preparation method of the bacterial suspension specifically comprises the following steps:
activating the isolated strain obtained in the example 1, transferring the activated strain into a nutrient liquid culture medium, and culturing the strain for 24 hours at the temperature of 28 ℃ to obtain fermentation liquor of bacillus atrophaeus BGB-98R; centrifuging the fermentation liquor, reserving a precipitate, and re-suspending the precipitate with sterile water to obtain the bacterial suspension of the bacillus atrophaeus BGB-98R.
Performance test one-antagonistic pathogen test
And (3) performing an antagonistic pathogen test on the bacillus atrophaeus BGB-98R. The pathogenic bacteria to be tested are fusarium graminearum, fusarium oxysporum and botrytis cinerea. The method comprises the following steps:
(1) Strain BGB-98R: the strain BGB-98R obtained by the separation and screening is inoculated into a nutrient liquid culture medium for culture after being activated, the strain is cultured to a logarithmic phase (the growth condition of the strain is detected by a nucleic acid protein tester) at the temperature of 30 ℃ and the rpm, and the strain is transferred into a nutrient agar culture medium flat plate and is placed into a constant temperature incubator at the temperature of 30 ℃ for culture for 36 hours for standby.
Pathogenic bacteria: punching a fusarium graminearum PDA solid culture medium plate stored at 4 ℃, a fusarium oxysporum PDA solid culture medium plate and a gray mold PDA solid culture medium plate, respectively inoculating round bacterial cakes with the diameter of 5mm onto the PDA liquid culture medium plate, and culturing in a constant-temperature incubator at 28 ℃ for 3-4d for later use.
(2) Punching the nutrient agar culture medium plate of the cultured strain BGB-98R, and inoculating a circular bacterial cake with the diameter of 5mm to a position, which is 2.5cm away from the center of the plate, of a new PDA solid culture medium plate. And similarly, punching the cultured PDA liquid culture medium plates of fusarium graminearum, fusarium oxysporum and gray mold, and taking circular bacterial cakes with the diameter of 5mm to inoculate the positions of 2.5cm from the center of the new PDA solid culture medium plates respectively as a treatment group. The positions of pathogenic bacteria and bacterial strain BGB-98R in the novel PDA solid culture medium plate are symmetrical. Meanwhile, a PDA solid culture medium plate inoculated with fusarium graminearum, fusarium oxysporum and gray mold is used as a control group. Culturing in a constant temperature incubator at 28deg.C until pathogenic bacteria grow on the whole plate, observing antibacterial effect, and calculating antibacterial rate.
The calculation formula of the bacteriostasis rate is as follows:
antibacterial ratio (%) = (Rp-Rt)/rp×100%.
Where Rp represents the radius of growth of the mycelia of the pathogenic bacteria of the control group, and Rt represents the radius of growth of the mycelia of the pathogenic bacteria of the treatment group (near one side of strain BGB-98R).
(3) The test results are shown in FIG. 4 and Table 1.
TABLE 1 inhibition of pathogenic bacteria by BGB-98R
As can be seen from Table 1 and FIG. 4, bacillus atrophaeus BGB-98R has remarkable inhibiting effect on Fusarium graminearum, fusarium oxysporum and Proteus gray mold. Wherein, the antibacterial rate to fusarium graminearum is 44.39%, the antibacterial rate to fusarium oxysporum is 54.33%, and the antibacterial rate to gray mold is 53.90%.
Performance test II-seed germination promotion and plant growth promotion test
The bacillus atrophaeus BGB-98R was tested for promoting seed germination and plant growth. The test material was cucumber seed (middle farmer No. 16). The method comprises the following steps:
(1) Strain BGB-98R: the strain BGB-98R obtained by the separation and screening is inoculated into a nutrient liquid culture medium for culture after being activated, and is cultured to a logarithmic phase (the growth condition of the strain is detected by a nucleic acid protein tester) under the conditions of 30 ℃ and 220 rpm. The OD value was used to estimate the bacterial load and the dilution coating plate was used to count, and the broth was diluted with sterile water to a bacterial load of 1X 10 7 -9×10 8 CFU/ml, ready for use.
The sterilized nutrient soil is placed in a seedling tray, and a treatment group and a control group are respectively arranged. Treatment group: the content of the utilized bacteria is 1 multiplied by 10 7 -9×10 8 The nutrient soil is thoroughly poured by the fermentation broth of CFU/ml (the nutrient soil bacteria content in the seedling tray is 1 multiplied by 10) 7 -9×10 8 CFU/g fermentation broth was applied to the nutrient soil). In the present application, the fermentation broth is diluted with sterile water to a bacterial content of 5X 10 8 CFU/ml was tested. And the control group is completely irrigated with the nutrient soil by using sterile water, and the use volume of the sterile water in the control group is the same as that of the fermentation broth in the treatment group.
(2) Promotion of seed germination test: the nursery pots of the treatment group and the control group were placed in a greenhouse at 28℃for 12 hours, cucumber seeds were treated with 0.2% sodium hypochlorite for 2min, and washed with sterile water 5-10 times. Planting cucumber seeds in a seedling tray according to a hole sowing mode, and observing germination conditions of the cucumber seeds after 8-12 d. The treatment group and the control group were each set with 3 replicates.
(3) Promotion of plant growth test: after the cucumber seedlings emerge, transplanting the cucumber seedlings into seedling raising pots of a treatment group and a control group respectively, placing the seedling raising pots in an artificial growth room for culturing, harvesting after 30 days, and detecting fresh weight and root length of plants. The photoperiod of the artificial growth chamber is set as follows: day/night = 16h/8h; the temperature is set as follows: day/night = 30 ℃/24 ℃. The treatment group and the control group were each set with 10 replicates.
(4) The test results are shown in fig. 5 and table 2.
Germination rate (%) =number of seed germination/total number of seeds×100%.
TABLE 2 promotion effect of the seed BGB-98R on germination and plant growth of cucumber seeds
As can be seen from a combination of fig. 5 and table 2, the germination rate of the treated group was significantly different from that of the control group. The bacillus atrophaeus BGB-98R can promote germination of cucumber seeds, and the germination rate of the treated group is improved by 12.2% compared with that of the control group.
In addition, the root length and the whole fresh weight of the treated group were significantly different from those of the control group. The bacillus atrophaeus BGB-98R can promote cucumber seedling growth, and the root length of the treated group is improved by 19.2% compared with that of the control group.
Therefore, the bacillus atrophaeus BGB-98R can promote germination of cucumber seeds and growth of cucumber plants, and has remarkable promotion effect on growth of cucumbers.
Example 6
The embodiment provides a bacillus atrophaeus BGB-98R microbial inoculum.
The preparation process of the microbial inoculum concretely comprises the following steps:
the activated strain BGB-98R plate is utilized, a puncher is utilized to punch and inoculate in a 500mL conical flask with liquid fermentation seed liquid culture medium, and the culture is carried out for 24 hours in a constant temperature shaking table at 30 ℃ and 180rpm, thus obtaining the cultured seed liquid for later use.
Inoculating the cultured seed liquid into a 300L fermentation tank, wherein the volume of fermentation liquid in the fermentation tank is 70% of the total volume of the fermentation tank, the fermentation inoculation amount is 1%, the rotating speed is 200rpm, the temperature is 28 ℃, the ventilation amount is 1vvm, 20g/L of sterilized molasses water is added for 20h of fermentation, the fermentation is continued until the spore rate reaches more than 90%, and the fermentation tank is discharged.
Centrifuging the fermentation liquor by using a barrel type centrifuge according to 9:1, adding a protective agent (the weight ratio of the components in the protective agent, and the weight ratio of dextrin, a dispersing agent and sodium sulfite is 40:9:1) into the slurry to obtain the bacillus atrophaeus BGB-98R microbial inoculum.
Example 7
The embodiment provides bacillus atrophaeus BGB-98R bacterial powder.
The preparation process of the bacterial powder specifically comprises the following steps: and (3) carrying out spray drying on the microbial inoculum prepared in the example 6, and drying to obtain microbial powder, namely the microbial powder of the bacillus atrophaeus BGB-98R, wherein the microbial content of the microbial powder is 2000 hundred million/g.
Example 8
The embodiment provides a microbial agent. The microbial agent comprises a microbial agent prepared by utilizing bacillus atrophaeus BGB-98R.
The preparation process of the microbial agent specifically comprises the following steps:
the microbial inoculum prepared in the example 6 is compounded with Wen Womei g of organic source soil conditioner (microbial inoculum carrier) in the example 3, and the weight ratio of the microbial inoculum to the soil conditioner is 1:400, and the microbial agent product after mixing has a bacterial content of 5 hundred million/g.
Performance test III-field test Using microbial inoculant
The microbial agents provided in example 8 were subjected to field trials. The method comprises the following steps:
(1) Test field
Test field: the potato field blocks of the planting base are planted in the potatoes of the agricultural group of the snow river in the new village of the Xingvillage of the Qingzhen county of Gansu Gulang and the field blocks with even land condition are selected.
The test method is as follows: microbial inoculum is applied into soil by ploughing in 4 months and 15 days of 2023, 40 kg/mu is uniformly mixed, leveled and spring sowing of field potatoes is carried out after 7 days. Other planting practices were performed in accordance with farming practices without application of the sterilant product. The experiments were performed in 2 independent potato-planted areas (GB and GBS) and control experiments (GBC and GBSC) were set up without microbial inoculum, with equal amounts of inoculum carrier and protectant added. Each treatment was set at 2 mu and repeated 3 times. After 30d of the microbial inoculum provided by the application, surface soil sampling is carried out in the field, the surface soil sampling is placed in an ice box, and the surface soil sampling is brought back to a laboratory for sample pretreatment. Sampling by 5-point sampling method, each point taking 2m 2 Soil sampleThe potato quality was examined in the field (traits such as stem thickness, branch number, number of plants, etc.) during the expansion period at 2023, 7 and 21, and the yield was measured during the harvest period at 2023, 8 and 21.
Microorganism detection method and results: the samples were pretreated and sent to Beijing Nodejingyuan technologies Co., ltd for 16S and ITS soil microbial diversity library sequencing, and the sequencing results were analyzed. In the species relative abundance bar charts in fig. 6 and 7, the genus is taken as an analysis unit, and the microorganism with higher abundance in the first 20 positions is taken as an analysis object.
As can be seen from the results of FIGS. 6 and 7, the application of the microbial agent 30d provided in the present application, especially the highly abundant pathogenic bacteriaFusarium(Fusarium) the abundance level was significantly reduced relative to the control group, and the Fusarium abundance was reduced, thereby reducing the occurrence of rot disease corresponding thereto. In the treated group of soil compared to the controlBacillusThe increased abundance level of (bacillus) indicates that the use of the microbial agents provided herein can significantly increase the abundance level of bacillus in soil. Therefore, the microbial agent provided by the application can significantly improve the structural level of microorganisms in soil.
The potato field test results are shown in table 3 and fig. 8.
TABLE 3 growth promoting and biocontrol effects of microbial inoculants on potatoes in field trials
The results in Table 3 and FIG. 8 show that the average stem thickness of the potatoes is increased by 5.69% and 8.8%, the average plant height is increased by 10.28% and 12.02%, the average branch number of the individual plants is increased by 5.17% and 8.20%, the potato yield of the individual plants is increased by 7.32% and 7.69%, and the rotten disease plant rate is reduced by 77.28% and 79.31%, respectively, compared with the control group, after the microbial inoculum provided by the application is applied. Based on the above, compared with a control group, the microbial agent provided by the application can obviously promote the growth of potatoes and obviously reduce the disease rate of potato rot.
In addition, the potato field yield measurement results show that compared with a control group, the yield per mu of the potato by applying the microbial inoculum provided by the application is respectively increased by 7.65% and 6.15%. Therefore, based on the above, the application of the microbial agent provided by the application can significantly improve the potato yield.
From the above, the strain BGB-98R provided by the application has the characteristic of obviously inhibiting the growth of pathogenic bacteria. Meanwhile, germination of cucumber seeds and growth of cucumber seedlings can be effectively promoted. In field experiments, the strain BGB-98R can normally grow in drought-barren soil, can reduce the disease rate of potato rot, can obviously promote potato growth, and improves potato yield.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and are not limiting thereof; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (12)
1. Bacillus atrophaeus strainBacillus atrophaeus) The strain BGB-98R is characterized in that the preservation number of the strain BGB-98R is CGMCC No.27752.
2. A culture comprising the strain BGB-98R of claim 1.
3. A fermentation broth comprising the strain BGB-98R of claim 1.
4. A bacterial suspension comprising the strain BGB-98R of claim 1.
5. A microbial agent comprising the strain BGB-98R of claim 1.
6. A bacterial powder comprising the strain BGB-98R of claim 1.
7. Use of the strain BGB-98R of claim 1, the culture of claim 2, the microbial agent of claim 5, the microbial powder of claim 6 for improving soil microbial structure and fusarium graminearum, fusarium oxysporum, and gray mold prevention and control fields.
8. Use of the strain BGB-98R of claim 1, the culture of claim 2, the microbial inoculum of claim 5, the microbial powder of claim 6 for promoting germination of cucumber seeds, and for promoting growth of cucumber plants and potato plants.
9. The use according to claim 8, wherein the BGB-98R strain has a bacterial content of 1X 10 7 -9×10 8 CFU/ml。
10. The use according to claim 8, wherein the strain BGB-98R is capable of normal growth in drought-barren soil.
11. The use according to claim 8, wherein the strain BGB-98R is capable of reducing potato rot disease rate, promoting potato growth, and increasing potato yield.
12. The use according to claim 8, wherein the strain BGB-98R is capable of improving soil microbial community structure.
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