CN106701623B - The atrophy bacillus of one plant of antagonism fructus lycii root rot and its application - Google Patents
The atrophy bacillus of one plant of antagonism fructus lycii root rot and its application Download PDFInfo
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- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses the atrophy bacillus of one plant of antagonism fructus lycii root rot, its classification naming is atrophy bacillus (Bacillus atrophaeus) GQJK17, China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC is preserved on November 10th, 2016, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), culture presevation number is CGMCC NO.13254.Isolated atrophy bacillus (Bacillus atrophaeus) GQJK17 of the present invention, the bacterial strain have stronger antagonism to fructus lycii root rot, provide new biocontrol microorganisms for the biological control of fructus lycii root rot, increase the type of biological prevention and control agent.
Description
Technical field
The present invention relates to the atrophy bacillus of one plant of antagonism fructus lycii root rot and its applications, belong to field of biotechnology.
Background technique
Fructus lycii is a kind of rare Chinese medicine, and Chinese wolfberry fruit has higher nutrition and health care and medical value, with Qiongzhen Ganoderma Lucidum, length
White recluse's ginseng, Donga donkey-hide gelatin are simultaneously known as " Chinese medicine four is precious ".Also increasingly recognize with social progress, people with the development of economy
To the value of fructus lycii, people is promoted to carry out extensive product development to fructus lycii, and achieves important achievement.Lycium barbarum is in
State cultivated area is maximum, as fructus lycii cultivated area constantly expands, old producing region and new producing region fructus lycii root rot disease incidence year after year on
It rises, gets worse, seriously affected local farmers income, there is certain destruction to entire industrial chain.Chinese holly
Qi root rot is a kind of system infections disease, and diseased plant root and rootstock occur to rot, stem vascular bundle browning color.Disease portion when moist
White or the mould layer (pathogen) of pink are grown once in a while.Due to root-rot, blade becomes smaller, and lacks gloss, and branch is downgraded, and fruit is thin
It is small；Or blade flavescence is wilted, plant is gradually withered when serious.People prevent and treat fructus lycii root rot and mainly spray insecticide at present, match
Side's fertilising, the mode of increasing application phosphorus potassium fertilizer.But these methods are time-consuming and laborious, are difficult to effectively prevent the generation of disease from the root, and
And cause serious pollution to the environment, also there is certain injury to human health, and biological control method is because it is with specificity and green ring
The advantages of guarantor, has been to be concerned by more and more people.There is bacillus for cotton wilt, potato sore using comparative maturity
The Biocontrol Effect of the plurality of plant diseases such as the red carninomatosis of scall, apple, root rotof flax, but without fructus lycii root rot biological control
The report of aspect, and the microorganism resource for finding antagonism fructus lycii root rot is crucial.
Atrophy bacillus (Bacillus atrophaeus) is a kind of mutation of bacillus subtilis, in agricultural, work
Industry, scientific research, medical treatment and health field etc. are all widely used.In countries and regions such as the U.S., Japan, European Union often by the bacterial strain
Bacterial strain is detected as quality control standard and Evaluation of Germicidal Efficacy tests indicator bacteria.But in terms of the bacterium is applied to controlling plant diseases
Report it is less, related research mainly has: prevention and treatment balsam pear gray mold (CN105670964A), prevention and treatment colorado potato bug larva
(CN104164378A), cotton boll blight (CN102747005A), prevention and treatment tomato seedling damping-off, capsicum early blight, cotton standing dead are prevented and treated
Sick (CN104531545A) etc., but do not have the report of the atrophy bacillus of prevention and treatment fructus lycii root rot also at present.Moreover, biological and ecological methods to prevent plant disease, pests, and erosion is micro-
Often exist between microbiologic population in biology and soil environment and interact, the introducing of external source Antagonistic Fungi can cause crop
The composition of rhizosphere soil microorganism group changes, and the field planting of different plant rhizosphere bacteriums of the same race and function have larger difference
Property；Moreover, can Biocontrol microorganism survive in soil environment and fast-growth is also the key for realizing preventive and therapeutic effect.Cause
This, screening can have highly important practical application value with the biocontrol bacterial strain of preventive and therapeutic effect to fructus lycii root rot.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide the atrophy bacillus of one plant of antagonism fructus lycii root rot and
It is applied.
To achieve the above object, the present invention adopts the following technical solutions:
According to the first aspect of the invention, the atrophy bacillus of one plant of antagonism fructus lycii root rot, classification naming are provided
For atrophy bacillus (Bacillus atrophaeus) GQJK17, China Microbiological is preserved on November 10th, 2016
Culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica), culture presevation number is CGMCC NO.13254.
The bacterial strain is isolated from fructus lycii rhizosphere soil, the bacterium colony and thallus feature of the bacterial strain are as follows: on LB plate
Bacterium colony is in subcircular after culture for 24 hours, and milky, bacterium colony is flat, the small dentation in edge, dry tack free, colony diameter 2.62mm.
Thallus is in elongated rod shape, produces gemma, Gram-positive.
The physiological and biochemical property of the bacterial strain are as follows: oxidizing ferment experiment is negative, and catalase experiment is positive, and citrate utilizes experiment
The positive, nitrate is negative using experiment, and Starch Hydrolysis experiment is positive, and M-R experiment is negative, and V-P experiment is positive, and glucose fermentation is real
The positive is tested, sucrose fermenting experiment is positive, and maltose fermenting experiment is negative, and arabinose fermenting experiment is positive, wood-sugar fermentation experiment
Feminine gender, cellobiose fermenting experiment is negative, and lactose fermentation experiment is negative, and sorbitol fermentation experiment is positive, mannose ferment experiment
It is positive.
According to the second aspect of the invention, a kind of biological prevention and control agent is provided, active constituent is above-mentioned atrophy bacillus
The tunning of (Bacillus atrophaeus) GQJK17.
Preferably, the biological prevention and control agent is liquid microbial inoculum.
Further, in the biological prevention and control agent atrophy bacillus (Bacillus atrophaeus) GQJK17 content
More than or equal to 2 × 108cfu/mL。
The present invention also provides the preparation method of above-mentioned biological prevention and control agent, include the following steps: the atrophy gemma bar for fermenting above-mentioned
Bacterium (Bacillus atrophaeus) GQJK17, obtains tunning.
In above-mentioned preparation method, the culture medium used that ferments is liquid bean sprouts medium, raw material composition are as follows: yellow bean sprout
100g, sucrose 50g, water 1000mL, pH are natural；
The liquid bean sprouts medium the preparation method comprises the following steps: yellow bean sprout boiling water boiling 30min, with four layers of filtered through gauze,
Sucrose constant volume is added to 1000mL.
In above-mentioned preparation method, the condition of fermentation are as follows: 35-40 DEG C, constant-temperature table culture 20-30h；Preferably, 37 DEG C, permanent
Warm shaking table culture is for 24 hours.
According to the third aspect of the invention we, above-mentioned atrophy bacillus (Bacillus atrophaeus) GQJK17 is provided
And/or application of the biological prevention and control agent in prevention and treatment fructus lycii root rot.
In above-mentioned application, the prevention and treatment fructus lycii root rot is embodied in the breaking-out for inhibiting fructus lycii root rot, reduces disease incidence.
According to the fourth aspect of the invention, above-mentioned atrophy bacillus (Bacillus atrophaeus) GQJK17 is provided
And/or biological prevention and control agent is promoting the application in fructus lycii volume increase.
Fructus lycii root rot, while the product increased production can be prevented and treated the present invention also provides a kind of, active constituent is above-mentioned withers
Contracting bacillus (Bacillus atrophaeus) GQJK17 or above-mentioned biological prevention and control agent.
Beneficial effects of the present invention:
(1) isolated atrophy bacillus (Bacillus atrophaeus) GQJK17 of the present invention, the bacterial strain is to Chinese holly
Qi root rot has stronger antagonism, provides new biocontrol microorganisms for the biological control of fructus lycii root rot, increases biological and ecological methods to prevent plant disease, pests, and erosion system
The type of agent.
(2) studies have shown that under field condition, using atrophy bacillus (Bacillus of the invention
Atrophaeus) bacteria suspension of GQJK17 to fructus lycii root rot fall ill serious plot fructus lycii tree carry out root irrigation after, energy
The obvious breaking-out for inhibiting fructus lycii root rot, reduces disease incidence；Atrophy bacillus (Bacillus atrophaeus) of the invention
GQJK17 can also improve the upgrowth situation of plant, improve the yield of fructus lycii, to the averagely volume increase of fructus lycii tree 484% or more.More than
Result of study shows that the bacillus of antagonism fructus lycii root rot of the invention there is certain prevention and treatment to imitate lycium barbarum root rot
Fruit.
(3) root rot of different plants, pathogen is different, and pathogenesis can also have differences.To the root of other plant
Maize ear rot has the bacillus of control efficiency, can not directly deduce that it has control efficiency to fructus lycii root rot.We were testing
It finds there is the bacterial strain of antagonistic effect to phytopathogen in plate face-off experiment in journey, but does not ensure that it in crop field reality
Effect in testing.The result of field experiment also turns out, possesses the bacterial strain of preferable plate face-off experimental result, in field experiment
In, apparent inhibitory effect may not also be possessed to fructus lycii Pathogens Causing Root Rot Disease.Therefore, prevention and treatment imitates the root rot of other plant
For the bacillus of fruit, due to the severe living environment in Ningxia, China, can bacterial strain survive and fast-growth becomes key
Problem, therefore may not have control efficiency to fructus lycii root rot.
Detailed description of the invention
Fig. 1: the colonial morphology figure of atrophy bacillus (Bacillus atrophaeus) GQJK17；
Fig. 2: the phyletic evolution tree graph of atrophy bacillus (Bacillus atrophaeus) GQJK17.
Specific embodiment
Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection
Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..
Embodiment 1: separation, the screening of bacterial strain
(1) separation of strain
Pedotheque 5, healthy, dying, in heaven fructus lycii tree root border is acquired from the Zhongning County village Shi Laba, Ningxia Province.It weighs
10.0 grams of fructus lycii Rhizosphere Soils, in every 10 grams of soil add 90mL sterile water ratio be put into preprepared be equipped with bead triangle
In bottle, it is placed in 180rpm on 37 DEG C of constant-temperature tables and shakes 20-30min.Soil supension is diluted to 10 by gradient dilution method-2、10-3、10-4、10-5、10-6Etc. different gradients.The soil liquid 0.1mL for drawing appropriate dilution is placed in LB plating medium, is coated with
Uniformly (this experiment chooses 10-4、10-5、10-6Three gradients are coated).37 DEG C of constant incubator cultures for 24 hours, picking single bacterium
It falls.The bacterial strain that will acquire is purified with the method for three rides, until obtaining single bacterium colony, and bacterial strain is numbered, and is protected
It is spare to be stored in 4 DEG C of refrigerators.
(2) screening of Antagonistic Fungi
Primary election is carried out to Antagonistic Fungi using tablet face-off method.The bacterial strain of preservation is activated, it is spare.It will be stored on inclined-plane
Fructus lycii Pathogens Causing Root Rot Disease is activated using scribble method in PDA plate, 28 DEG C of cultures to covering with plate, with the blue electron gun head of sterilizing or
Above-mentioned pathogen fungus block under punch vertical cutting, the intermediate position of PDA plate is transferred to tweezers, is placed in 28 DEG C of incubator cultures
One day.With the activated Antagonistic Fungi of toothpick picking be inoculated into away from pathogen edge 2cm, be placed in 28 DEG C of incubators continue cultivate 2-
It 5 days, sees whether the appearance of antibacterial band, measures the width of antibacterial band.Bacteriostatic diameter is measured simultaneously, calculates bacteriostasis rate.
Bacteriostasis rate (%)=(colony diameter D- bacteriostatic diameter d)/colony diameter D × 100%.
2. test result:
The bacterial strain for selecting one plant of fungistatic effect best, strain number GQJK17, plate dual test the results are shown in Table
1。
Table 1: bacteriostasis rate measurement result
As can be seen from Table 1: bacterial strain GQJK17 has preferable bacteriostasis to the pathogen of fructus lycii root rot.
2. the form and physiological and biochemical property of bacterial strain GQJK17 measure
(1) colony characteristics and thalli morphology
The bacterium colony and thallus feature of the bacterial strain are as follows: bacterium colony is in subcircular, milky, bacterium colony after cultivating on LB plate for 24 hours
It is flat, the small dentation in edge, dry tack free, colony diameter 2.62mm.Thallus is in elongated rod shape, produces gemma, Gram-positive.
(2) physiological and biochemical property
The physiological and biochemical property of the bacterial strain are as follows: oxidizing ferment experiment is negative, and catalase experiment is positive, and citrate utilizes experiment
The positive, nitrate is negative using experiment, and Starch Hydrolysis experiment is positive, and M-R experiment is negative, and V-P experiment is positive, and glucose fermentation is real
The positive is tested, sucrose fermenting experiment is positive, and maltose fermenting experiment is negative, and arabinose fermenting experiment is positive, wood-sugar fermentation experiment
Feminine gender, cellobiose fermenting experiment is negative, and lactose fermentation experiment is negative, and sorbitol fermentation experiment is positive, mannose ferment experiment
It is positive.
3. the 16S rDNA measurement and analysis of bacterial strain GQJK17
The 16S rDNA gene order of bacterial strain GQJK17, as shown in SEQ ID NO.1 in nucleotides sequence list.It will be surveyed
Sequence alignment in 16S rDNA sequence and GenBank database, the results showed that GQJK17 and Bacillus atrophaeus
(JCM 9070) is in together in a minimum branch, 16S rRNA gene order and Bacillus atrophaeus (NBRC
15539) strain sequence similarity is 99%, in conjunction with above-mentioned physiological and biochemical property as a result, identifying that GQJK17 is atrophy gemma bar
Bacterium (Bacillus atrophaeus).
Bacterial strain GQJK17, classification naming be atrophy bacillus (Bacillus atrophaeus) GQJK17, in
On November 10th, 2016 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address
Are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), culture presevation number is CGMCC
NO.13254。
Embodiment 2: the preparation of bacterial strain GQJK17 biological prevention and control agent
It is specific the preparation method is as follows:
Atrophy bacillus (Bacillus atrophaeus) GQJK17 for the antagonism fructus lycii root rot that inclined-plane is saved turns
It is connected in the test tube equipped with 5mL bean sprout juice fluid nutrient medium, is put in 37 DEG C, cultivates 12h in 180rpm constant-temperature table.Then it presses
1% inoculum concentration is inoculated into the liquid bean sprouts medium of 100mL/250mL, is put in 37 DEG C, is trained in 180rpm constant-temperature table
It supports for 24 hours, it is 2 × 10 that concentration, which is made,8The bacteria suspension of cfu/mL.
Liquid bean sprouts medium, raw material composition are as follows: yellow bean sprout 100g, sucrose 50g, water 1000mL, pH are natural；
The preparation method comprises the following steps: sucrose constant volume is added to 1000mL with four layers of filtered through gauze in yellow bean sprout boiling water boiling 30min.
Embodiment 3: field trial
1. test method:
The serious plot of fructus lycii root rot morbidity is selected, two processing are designed, respectively processing 1:GQJK17 bacterium solution (is implemented
It is prepared by example 2), every fructus lycii tree dosage is 25mL；Processing 2: it is compared with the clear water of equivalent.
3 repetitions are arranged in each processing, each to repeat to select 20 fructus lycii trees.
Specific test method: pouring root is carried out to fructus lycii tree after bacterium solution is diluted 200 times with clear water.The control clear water of equivalent
Pouring root.
2. test result:
(1) control efficiency of the bacterial strain GQJK17 to fructus lycii root rot
The results are shown in Table 2.
Table 2: fructus lycii tree morbidity statistical result in field experiment
As can be seen from Table 2:
1. disease incidence: in field experiment, the disease incidence of control is 90%, with atrophy bacillus (Bacillus
Atrophaeus) the fructus lycii tree disease incidence of GQJK17 bacterium solution processing is 41.67%, compared with the control, the Chinese holly handled with GQJK17
The disease incidence of Qi Shu reduces 48.33 percentage points.
2. relative control effect: atrophy bacillus (Bacillus atrophaeus) GQJK17 is opposite to fructus lycii root rot
Preventive effect is up to 63%.
(2) effect of increasing production of the bacterial strain GQJK17 to fructus lycii tree
It the results are shown in Table 3.
Table 3: effect of increasing production of atrophy bacillus (Bacillus atrophaeus) GQJK17 to fructus lycii tree
As can be seen from Table 3: the fructus lycii tree average product of control treatment is 0.5kg/, with atrophy bacillus
The yield of (Bacillus atrophaeus) GQJK17 bacterium solution processing is 2.92kg/, compared with the control, is handled with GQJK17
Fructus lycii tree averagely increase production 484%.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
It is any to be familiar with those skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its invent
Design is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
SEQUENCE LISTING
<110>Shandong Agricultural University
The atrophy bacillus of<120>one plants of antagonism fructus lycii root rot and its application
<130> 2016
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1421
<212> DNA
<213>bacterial strain GQJK17
<400> 1
cttcggcggc tggctcataa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggtct cgctgccctt tgttctgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgcccccga aggggaagcc ctatctctag gggtgtcaga ggatgtcaag 480
acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa 600
tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc atcgtttacg 660
gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc ctcagcgtca 720
gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta cgcatttcac 780
cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt tccaatgacc 840
ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc gagcccttta 900
cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960
ttagccgtgg ctttctggtt aggtaccgtc aaggtgccgc cctatttgaa cggcacttgt 1020
tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc ggcgttgctc 1080
cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc 1140
cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgttgccttg 1200
gtgagccatt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa gtggtagccg 1260
aagccacctt ttatgtttga accatgcggt tcaaacaagc atccggtatt agccccggtt 1320
tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc cgtccgccgc 1380
taacatcagg gagcaagctc ccatctgtcc gctcgactgc a 1421
Claims (10)
1. atrophy bacillus (Bacillus atrophaeus) GQJK17 of one plant of antagonism fructus lycii root rot, in 2016
November 10 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, and address is Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica, culture presevation number are CGMCC NO.13254.
2. a kind of biological prevention and control agent, active constituent is atrophy bacillus (Bacillus described in claim 1
Atrophaeus) the tunning of GQJK17.
3. biological prevention and control agent as claimed in claim 2, which is characterized in that the biological prevention and control agent is liquid bacterial agent.
4. biological prevention and control agent as claimed in claim 3, which is characterized in that atrophy bacillus in the biological prevention and control agent
The content of (Bacillus atrophaeus) GQJK17 is greater than or equal to 2 × 108cfu/mL。
5. the preparation method of the described in any item biological prevention and control agents of claim 2-4, which comprises the steps of: fermentation
Atrophy bacillus (Bacillus atrophaeus) GQJK17 described in claim 1, obtains tunning.
6. preparation method as claimed in claim 5, which is characterized in that the culture medium used that ferments is liquid bean sprout juice culture
Base, raw material composition are as follows: yellow bean sprout 100g, sucrose 50g, water 1000mL, pH are natural.
7. preparation method as claimed in claim 5, which is characterized in that the condition of fermentation are as follows: 35-40 DEG C, constant-temperature table culture
20-30h。
8. preparation method as claimed in claim 7, which is characterized in that the condition of fermentation are as follows: 37 DEG C, constant-temperature table culture is for 24 hours.
9. atrophy bacillus (Bacillus atrophaeus) GQJK17 or as claimed in claim 2 described in claim 1
Application of the biological prevention and control agent in prevention and treatment fructus lycii root rot.
10. described in atrophy bacillus (Bacillus atrophaeus) GQJK17 described in claim 1 or claim 2
Biological prevention and control agent promote fructus lycii volume increase in application.
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| CN109810918B (en) * | 2019-01-26 | 2020-09-25 | 武威市林业科学研究院 | Bacillus atrophaeus with effect of preventing wolfberry leaf blight, biological agent and application of biological agent |
| CN111662837B (en) * | 2019-03-07 | 2023-01-24 | 宁夏大学 | Bacillus atrophaeus and application thereof |
| CN112625958B (en) * | 2020-12-28 | 2022-02-01 | 中国林业科学研究院森林生态环境与保护研究所 | Bacillus atrophaeus, microbial inoculum and application thereof |
| CN113699075B (en) * | 2021-09-03 | 2023-03-28 | 山东农业大学 | Bacillus atrophaeus capable of producing protease and decomposing potassium and application thereof |
| CN114196565B (en) * | 2021-10-14 | 2024-03-01 | 河南农业大学 | Bacillus atrophaeus XZRKZ-23 and application thereof |
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