CN114317329A - Bacillus subtilis strain and screening method and application thereof - Google Patents
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Abstract
The invention provides a bacillus subtilis strain, a screening method and application thereof, wherein the preservation number of the strain is CCTCC NO: M20211416. The bacillus subtilis strain is screened from panicle bracts of rice in the booting stage, can be used for preventing and treating rice fungal diseases, and can be used for preventing and treating diseases caused by ustilaginoidea virens, pyricularia oryzae and rhizoctonia solani.
Description
Technical Field
The invention belongs to the technical field of microbial strains, and particularly relates to a bacillus subtilis strain and a screening method and application thereof.
Background
The rice false smut is a rice ear disease caused by Ustilaginoidea virens (Villospora virens). In recent years, the incidence of false smut is becoming more severe, and the main rice production areas all over the world are large-area rice blast and sheath blight, which are called as new three diseases of rice. The false smut not only causes serious yield loss of rice, but also pollutes rice grains by toxins generated by the germs, and has the risk of seriously harming the health and safety of people and livestock. Therefore, the environmental protection and effective control of false smut are related to the food safety and food safety of China. The occurrence of false smut seriously affects the yield and quality of rice.
The microbial community diversity has obvious influence on the plant health, and researches show that the disease resistance of the plant can be obviously improved by artificially utilizing the mixed microbial community to change the microbial diversity of the phyllosphere. A large number of reports show that part of phyllospheric microorganisms have higher inhibitory activity to rice blast, sheath blight and bacterial blight pathogenic bacteria. How to separate microorganisms with disease resistance from rice ears, and the microorganisms are used for preparing a disease inhibiting preparation, so that the use of chemical bactericides is reduced, and the occurrence of diseases can be effectively reduced in a green way.
However, the prior art does not disclose the use of rice for screening out strains capable of preventing and treating rice false smut.
Disclosure of Invention
In view of the above, the present invention provides a bacillus subtilis strain and a preparation method thereof, so as to solve the technical problems in the prior art.
In a first aspect, the invention provides a bacillus subtilis strain, wherein the preservation number of the strain is CCTCC NO: M20211416.
In a second aspect, the present invention also provides a screening method of the bacillus subtilis strain, comprising the following steps:
collecting spike bracts of disease-resistant rice products at the booting stage;
disinfecting spike bracts of disease-resistant products, peeling off spike husks to obtain glumes, putting the glumes into a sterile tube, adding potassium phosphate buffer solution, carrying out ultrasonic washing, carrying out vortex oscillation, collecting vortex eluent, carrying out vacuum suction filtration or centrifugal separation on the eluent to obtain precipitates, and then separating to obtain bacillus subtilis strains.
Preferably, the screening method of the bacillus subtilis strain adopts 75-80% of ethanol solution for disinfection.
Preferably, in the screening method of the bacillus subtilis strain, the pH value of the potassium phosphate buffer solution is 8-9.
Preferably, in the screening method of the bacillus subtilis strain, 8-12 mL of 0.08-0.12M potassium phosphate buffer solution is added to each g of glume.
In a third aspect, the invention also provides an application of the bacillus subtilis strain in preventing and treating rice fungal diseases.
Preferably, the rice fungal diseases comprise diseases caused by ustilaginoidea virens, pyricularia oryzae and rhizoctonia solani.
Compared with the prior art, the bacillus subtilis strain and the screening method thereof have the following beneficial effects:
(1) the bacillus subtilis strain is screened from panicle bracts of rice in the booting stage, can be used for preventing and treating rice fungal diseases, and can specifically prevent and treat diseases caused by ustilaginoidea virens, pyricularia oryzae and rhizoctonia solani;
(2) according to the screening method of the bacillus subtilis strain, the panicle bract at the booting stage of rice is selected as a microorganism extraction material, so that the colonization capacity of microorganisms at the disease-sensitive stage of a plant can be ensured; the ear buds of the rice at the booting stage are selected as microorganism extraction materials, so that competitive inhibition of microorganisms and pathogenic bacteria at the plant disease-sensitive stage can be ensured; the disease-resistant variety rice is selected as a pathogenic bacterium separation material, so that the screening efficiency can be improved, and more beneficial microorganisms capable of inhibiting the occurrence of diseases can be obtained; compared with biocontrol strains separated from soil, the microorganisms separated from the pathogen infection sites have the advantages of strong colonization ability and capability of plant immune synergy; according to the screening method, the small ultrasonic cleaning machine and the vortex instrument can be used for eluting microbial thalli from glumes to the greatest extent in the enrichment process, and the portable oil-free vacuum connection microbial limit detector can be used for enriching almost all microbes in suspension liquid, so that the microbes are prevented from being lost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph showing the results of staining Bacillus subtilis IR28-8 phenol red in example 1 of the present invention;
FIG. 2 is a diagram showing the growth state of Bacillus subtilis IR28-8 on LB solid medium in example 1 of the present invention;
FIG. 3 is a phylogenetic tree of MEGA7 in combination with a neighbor-joining method (16S sequence).
Detailed Description
In the following, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The embodiment of the application provides a bacillus subtilis strain, and the preservation number of the strain is CCTCC NO: M20211416.
Based on the same inventive concept, the embodiment of the application also provides a screening method of the bacillus subtilis strain, which comprises the following steps:
s1, collecting the panicle bracts of the disease-resistant rice at the booting stage;
s2, disinfecting spike bracts of disease-resistant products, peeling off spike hulls to obtain glumes, putting the glumes into a sterile tube, adding potassium phosphate buffer solution, performing ultrasonic washing, performing vortex oscillation, collecting vortex eluent, performing vacuum suction filtration or centrifugal separation on the eluent to obtain precipitates, and separating to obtain the bacillus subtilis strains.
According to the screening method of the bacillus subtilis strain, the booting stage is a key infection period of false smut, and the ears and buds of rice in the booting stage are selected as microorganism extraction materials, so that the colonization capacity of microorganisms in the disease-susceptible period of plants can be ensured; the ear buds of the rice at the booting stage are selected as microorganism extraction materials, so that competitive inhibition of microorganisms and pathogenic bacteria at the plant disease-sensitive stage can be ensured; the disease-resistant variety rice is selected as a pathogenic bacterium separation material, so that the screening efficiency can be improved, and more beneficial microorganisms capable of inhibiting the occurrence of diseases can be obtained; compared with biocontrol strains separated from soil, the microorganisms separated from the pathogen infection sites have the advantages of strong colonization ability and capability of plant immune synergy; according to the screening method, the small ultrasonic cleaning machine and the vortex instrument can be used for eluting microbial thalli from glumes to the greatest extent in the enrichment process, and the portable oil-free vacuum connection microbial limit detector can be used for enriching almost all microbes in suspension liquid, so that the microbes are prevented from being lost.
Specifically, in some embodiments, step S1 specifically includes: collecting the panicle bracts of the rice disease-resistant products in the stage of booting and when the panicle nodes are 5-10cm higher than the leaf pillows.
In some embodiments, ethanol solution with mass concentration of 75-80% is used for disinfection. Specifically, ethanol solution with mass concentration of 75% is adopted for disinfection.
In some embodiments, the pH of the potassium phosphate buffer is 8 to 9, and preferably, the pH of the potassium phosphate buffer is 8.
In some embodiments, 8-12 mL of 0.08-0.12M potassium phosphate buffer is added per g of glume, and preferably 10mL of 0.1M potassium phosphate buffer is added per g of glume.
Specifically, in step S2, the following steps are performed: disinfecting the spike bracts of the disease-resistant products by using 75% ethanol solution, peeling off the outer skins of the spikes to obtain glumes, putting the glumes into a sterile tube, adding 10mL of 0.1M potassium phosphate buffer (pH 8.0) into each gram of samples according to the weight, then putting the sample into an ultrasonic generator to wash for 1min, taking out the sample, and performing vortex oscillation for 10s, wherein the step is repeated for 2 times; pouring the ultrasonic waves and the vortex eluent into a collecting cup, adding new buffer solution again, and eluting microorganisms in the ear by using the ultrasonic waves and the vortex again; mixing the two eluents, connecting a microorganism limiting device to a filter flask and a filter pump, enriching the microorganisms on a filter membrane with the diameter of 0.22 mu m by using vacuum pumping (or centrifuging by 13000g for 10min, collecting the precipitate), re-eluting, and separating by using a plate separation method to obtain the bacillus subtilis strain.
Based on the same invention concept, the application of the bacillus subtilis strain in preventing and treating rice fungal diseases is provided. Specifically, the rice fungal diseases include diseases caused by ustilaginoidea virens, pyricularia grisea and rhizoctonia solani.
The method for screening a Bacillus subtilis strain of the present application is further described below in specific examples.
Example 1
The embodiment of the application also provides a screening method of the bacillus subtilis strain, which comprises the following steps:
s1, collecting the panicle bracts of the disease-resistant rice in the booting stage when the panicle nodes are 5-10cm higher than the leaf pillows;
s2, disinfecting spike bracts of disease-resistant products by using 75% ethanol solution, peeling off spike hulls to obtain glumes, putting the glumes into a sterile tube, adding 10mL of 0.1M potassium phosphate buffer (pH 8.0) into each gram of sample according to weight, then putting into an ultrasonic generator to wash for 1min, taking out the sample, and performing vortex oscillation for 10S, wherein the step is repeated for 2 times; pouring the ultrasonic waves and the vortex eluent into a collecting cup, adding new buffer solution again, and eluting microorganisms in the ear by using the ultrasonic waves and the vortex again; mixing the two eluents, connecting a microorganism limiting device to a filter flask and a filter pump, enriching microorganisms on a filter membrane with the diameter of 0.22 mu m by using vacuum pumping, and separating by using a flat plate separation method;
the bracts of a disease-resistant variety IR28 and a disease-susceptible variety WX98 are respectively selected as microorganism enrichment source materials, microorganisms in the bracts are enriched according to the method, samples are sent to carry out amplicon sequencing, a plate separation method is used for separating and culturing microbial communities, 16S-8F (the gene sequence of which is 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:1)) and 16S-1492R (the gene sequence of which is 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:2)) are used for amplifying 16S identification sequences by taking bacterial liquid as a template, and the preliminary result of microorganism identification is obtained by comparing the sequences through Blastn in an NCBI website. A phylogenetic tree was constructed using the analytical software MEGA7 in combination with the 16S sequence of a Bacillus standard strain and the IR28-8 strain was identified as Bacillus subtilis by phenol red staining and colony morphology observation. Subculturing and storing the microorganisms with candidate antibacterial activity.
Specifically, the sequencing results of the bacillus subtilis strain (code IR28-8)16S of the present application are:
CACCAGCGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTATGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAGACTGAAAACTCAAAGGGAATTGACGGGGGCCGCACAGCGTGGAGCATGTGGATTAATCCGAGCACGCGAGACTAACAAGTCTGACATCTTCTGACAATCCTAGAAGAATAGG(SEQ ID NO:3)
1. rice ear bud microorganism separation statistics
A total of 31 bacterial isolates were isolated from ears of disease-resistant rice variety IR28, and a total of 21 antagonistic bacterial isolates accounted for 67.7% of all bacterial isolates. 21 bacterial isolates, 2 antagonistic strains, were isolated from ear heads of the susceptible rice variety WX98, accounting for 9.5% of all bacterial isolates. Specifically, the table of the segregation statistics of rice panicle bud microorganisms of rice varieties IR28 and WX98 is shown in table 1 below.
TABLE 1 statistical table of the isolation of rice panicle bud microorganisms
The strain in Table 1 is designated IR28-8, which is the Bacillus subtilis strain of the present application.
As shown in FIG. 1, the bacterial cells of the strain are short rod-shaped and have a size of 0.7 to 0.8X 2 to 6 μm.
As shown in FIG. 2, the strain was grown on nutrient agar plates in which the colonies were white and showed irregular wrinkles.
As shown in FIG. 3, it was concluded that IR28 was Bacillus subtilis by phylogenetic analysis using a clinical approach.
2. Detection of bacteriostatic activity of main diseases of rice
Activity assay for inhibiting rice blast and sheath blight
Inoculating rice blast or rhizoctonia solani cakes in a PDA culture medium, culturing at the constant temperature of 28 ℃ for 3 days (the step is not carried out on rhizoctonia solani), placing filter paper sheets with the diameter of 5mm at four points around the bacterial colonies, dripping bacillus onto the filter paper sheets, repeating each treatment for three times, measuring the diameter when the control bacterial colonies grow full of the culture medium, and calculating the bacteriostasis rate.
The percent inhibition ratio (%) is the treated strain diameter/control pathogen growth diameter × 100%
Determination of inhibition of Ustilaginoidea virens Activity
Mixing ustilaginoidea virens conidia into PSA culture medium to make the culture medium sporeThe concentration of the seed reaches 1 × 106CFU/mL. Placing a sterile filter paper sheet in a four-point cross shape of the culture medium, dripping 5mL of bacteria solution to be detected, and repeating each treatment for three times. And (4) calculating the bacteriostasis rate when the green smut bacteria grow over the flat plate by taking a filter paper sheet dripped into an LB culture medium as a control.
Through experiments, the obtained 8 strains have an inhibiting effect on three main rice diseases. Inoculating Ustilaginoidea virens to the center of the plate, wherein strains IR28-8 and IR28-9 have strong bacteriostatic activity on Ustilaginoidea virens, and the inhibition rate is over 50%. The strains with the rice blast fungus inhibition rate of more than 60 percent comprise IR28-4, IR28-6 and IR 28-8. Specifically, the following table 2 shows.
TABLE 2 bacteriostatic activity of Bacillus on 3 rice pathogenic fungi
Control effect of pot culture test and control effect of next year field test
The plastic barrels are filled with soil, and the susceptible variety WX98 is planted, 2 plants are planted in each barrel. And (3) shaking and culturing the activated antagonistic bacteria in a culture medium at the temperature of 28 ℃ at 150r/min for 48 h. The rice ear is inoculated by injection, and the ustilaginoidea virens spore suspension is injected after 24h, each treatment is repeated three times, and LB culture solution is used as a control. The pot culture control test result shows that the 8 screened strains have certain control effect on false smut, wherein the control effect of IR28-8 (namely the bacillus subtilis strain of the application) is the best, the control effect is over 60 percent and is obviously higher than that of other strains. The control trend of the overall field test and the pot test is basically consistent, the control effect of IR28-8 and IR28-9 in the field is over 60 percent, and is obviously higher than that of other candidate strains, and the control effect is specifically shown in the following table 3.
TABLE 3 prevention and treatment effects of the strains on ustilaginoidea virens
| Bacterial strains | Potted plant control effect (%) | Control effect in field (%) | |
| IR28-4 | Bacillus belgii | 57.69±0.13c | 56.42±0.60c |
| IR28-6 | Bacillus tequilensis | 57.14±0.32c | 55.64±0.34c |
| IR28-7 | Bacillus belgii | 45.67±1.34e | 43.97±0.26f |
| IR28-8 | Bacillus subtilis | 64.76±1.02a | 63.41±0.38a |
| IR28-9 | Bacillus subtilis | 59.65±0.61b | 61.88±0.95b |
| IR28-11 | Bacillus marinus | 55.32±0.15d | 51.52±1.17d |
| IR28-15 | Bacillus marinus | 58.11±0.81c | 46.43±0.69e |
| IR28-18 | Bacillus marinus | 14.87±0.62h | 12.06±0.67h |
In table 3, the same letter indicates that the difference at the 0.05 level is not significant.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> institute of soil and fertilizer for plant protection of academy of agricultural sciences of Hubei province
<120> bacillus subtilis strain and screening method and application thereof
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aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata aaaggtggct 180
tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240
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gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600
gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720
ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttatggg gtttccgccc cttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggtcg cagactgaaa actcaaaggg aattgacggg 900
ggccgcacag cgtggagcat gtggattaat ccgagcacgc gagactaaca agtctgacat 960
cttctgacaa tcctagaaga atagg 985
Claims (7)
1. The bacillus subtilis strain is characterized in that the preservation number of the strain is CCTCC NO: M20211416.
2. A method of screening a bacillus subtilis strain according to claim 1, comprising the steps of:
collecting spike bracts of disease-resistant rice products at the booting stage;
disinfecting spike bracts of disease-resistant products, peeling off spike husks to obtain glumes, putting the glumes into a sterile tube, adding potassium phosphate buffer solution, carrying out ultrasonic washing, carrying out vortex oscillation, collecting vortex eluent, carrying out vacuum suction filtration or centrifugal separation on the eluent to obtain precipitates, and then separating to obtain bacillus subtilis strains.
3. The method for screening a Bacillus subtilis strain according to claim 2, wherein the sterilization is carried out by using an ethanol solution having a mass concentration of 75 to 80%.
4. The method for screening a Bacillus subtilis strain according to claim 2, wherein the pH of the potassium phosphate buffer is 8 to 9.
5. The method for screening a Bacillus subtilis strain according to claim 2, wherein 8 to 12mL of 0.08 to 0.12M potassium phosphate buffer is added per g of glume.
6. Use of the bacillus subtilis strain of claim 1 for controlling fungal diseases of rice.
7. The use as claimed in claim 6, wherein the fungal diseases of rice include those caused by Ustilago virens, Pyricularia oryzae and Rhizoctonia solani.
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| CN202111488564.0A CN114317329A (en) | 2021-12-07 | 2021-12-07 | Bacillus subtilis strain and screening method and application thereof |
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| CN102816725A (en) * | 2012-09-07 | 2012-12-12 | 江苏省农业科学院 | Bacillus subtilis and application thereof |
| CN103355290A (en) * | 2013-07-18 | 2013-10-23 | 江苏省农业科学院 | Bacillus subtilis dry suspension agent and preparation method thereof |
| CN103627659A (en) * | 2013-11-27 | 2014-03-12 | 福建省农业科学院植物保护研究所 | Bacillus subtilis and application in prevention of rice ustilaginoidea virens |
| CN104542713A (en) * | 2014-12-22 | 2015-04-29 | 江苏省农业科学院 | Bacillus subtilis composition and application thereof |
| CN106190920A (en) * | 2016-08-08 | 2016-12-07 | 湖南农业大学 | Bacillus subtilis YN145 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102816725A (en) * | 2012-09-07 | 2012-12-12 | 江苏省农业科学院 | Bacillus subtilis and application thereof |
| CN103355290A (en) * | 2013-07-18 | 2013-10-23 | 江苏省农业科学院 | Bacillus subtilis dry suspension agent and preparation method thereof |
| CN103627659A (en) * | 2013-11-27 | 2014-03-12 | 福建省农业科学院植物保护研究所 | Bacillus subtilis and application in prevention of rice ustilaginoidea virens |
| CN104542713A (en) * | 2014-12-22 | 2015-04-29 | 江苏省农业科学院 | Bacillus subtilis composition and application thereof |
| CN106190920A (en) * | 2016-08-08 | 2016-12-07 | 湖南农业大学 | Bacillus subtilis YN145 and application thereof |
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