CN109439563A - One bacillus licheniformis and its application in prevention and treatment grape root rot - Google Patents
One bacillus licheniformis and its application in prevention and treatment grape root rot Download PDFInfo
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Abstract
The invention discloses a bacillus licheniformis and its applications in prevention and treatment grape root rot.The bacterial strain is bacillus licheniformis (Bacillus licheniformis) 08220414, and deposit number is CGMCC No.15216.Experiment shows that lichem bacillus strain 08220414 of the invention has that the speed of growth is fast, sporulation quantity is big, Saline alkali tolerance is strong, it is good to prevent and treat grape root rot effect, grape rhizosphere can rapid, high volume colonize the features such as, therefore have a good application prospect.The biological control agent prepared by the bacillus licheniformis 08220414, can be used as biological pesticide or bio-feritlizer, and prevention and treatment grape root rot control efficiency is up to 90% or more.
Description
Technical field
The invention belongs to technical field of plant disease biological control.Specifically, the present invention relates to one plant of lichens gemma bars
Bacterium and its application in prevention and treatment grape root rot, and the biological control agent using bacillus licheniformis preparation.
Background technique
Grape root rot, also known as grape round spot root rot.European grape root starts i.e. by this evil of being critically ill after sprouting, generally from plant
Fibrous root morbidity, makes old complaint browning rot, and gradually spreads to meat lateral root, and form a bronzing round spot around fibrous root base portion,
Scab constantly expands, and often rots to reach xylem, and number spot is fused together, and makes cortex easily with woody part from old complaint is gradually
Become dark brown and dead.Since plant callus ability itself is strong, old complaint after death can also sprout new root out, and causing disease, this plays that
Volt.Overground part, which is sprouted, after the onset of usual grapevine seedling root postpones, and the part branch or complete stool after sprouting grow also weaker, leaf
It is small and color is light；When root morbidity is serious, 4~7 leaves of young sprout can wither suddenly, later withered death.
Grape root rot is mainly caused by three kinds of pathogens, respectively Fusarium solani, Fusarium oxysporum and bent angle reaping hook
Bacterium.These types of sickle-like bacteria is all soil inhabitant, saprophytic in the soil, and subterranean pest-insect, the soil to carry disease germs, garden tool set can bands
Disease is propagated.Prevention and treatment to grape root rot, the conventional method of use include traditional chemical prevention, cultivate disease-resistant variety and life
Object prevention and treatment.Chemical prevention such as Bordeaux mixture, improper use will lead to copper ion murder by poisoning or lime phytotoxicity, reduce fruit quality and production
Amount；And disease-resistant variety cultivation period is long, resistance is single, and investment is big；Biological control mainly utilizes beneficial microorganism, passes through life
Competition, antibacterial action, hyperparasitism, cross-protection and induced resistance between object etc. inhibit certain pathogens
Survival and activity, have the advantages that it is nontoxic, pollution-free, do not develop drug resistance, be efficient, show wide application prospect.
Bacillus licheniformis is a kind of Gram-positive bacillus, can produce endogenous spore, heat-resisting resistance is strong, in soil
Earth and plant surface are generally existing.Bacillus licheniformis is a kind of endophyte common in plant, nontoxic to people and animals, no
Pollute environment.The speed of growth is fast, and nutritional need is simple, is easy to survive on the surface of plant, colonizes and breed, production technology letter
Single, preparation stabilization is easy to use, and Storage period is long.Bacillus licheniformis is various to the mechanism of action and mode of phytopathogen.
It mainly include the several respects such as Competition, antibiosis, bacteriolysis and promotion plant growth.It therefore is a kind of ideal biological and ecological methods to prevent plant disease, pests, and erosion
Microorganism.
Summary of the invention
The object of the present invention is to provide one plant for efficiently preventing and treating new microorganism --- the lichens gemma of grape root rot
Bacillus (Bacillus licheniformis) bacterial strain 08220414.Lichem bacillus strain of the invention has the speed of growth
Fastly, yield is big, Saline alkali tolerance is strong, plant surface can rapid, high volume colonize the features such as, therefore have a good application prospect.
Above-mentioned purpose of the invention is achieved through the following technical solutions: bacterial strain provided by the present invention is lichens gemma
Bacillus (Bacillus licheniformis) 08220414 is to separate to obtain from Laizhou City, Shandong Province Sanshan Island alkaline land soil
, China Committee for Culture Collection of Microorganisms's common micro-organisms center has been preserved on January 15th, 2018 (referred to as
CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number CGMCC
No.15216。
The cultural method or propagation method of bacillus licheniformis 08220414 of the present invention include:
(1) Nostoc commune Vanch, which saves, uses LB culture medium, formula are as follows: tryptone 10g, yeast extract 5g, sodium chloride
10g, 15~20g of agar, distilled water 1000mL, pH are adjusted to 7.0~7.2.
(2) laboratory fluids culture uses LB liquid medium, formula are as follows: tryptone 10g, yeast extract 5g, chlorine
Change sodium 10g, distilled water 1000mL, pH are adjusted to 7.0~7.2.
(3) seed liquor culture medium prescription: by mass percentage, beancake powder 3.00%, corn flour 4.00%, glucose
0.40%, fish meal 0.50%, potassium dihydrogen phosphate 0.03%, precipitated calcium carbonate 1.20%, ammonium sulfate 0.30%, sodium chloride
0.10%, remaining as water, pH is adjusted to 7.0-7.2.
(4) bulk fermentation culture medium prescription: with seed liquor culture medium prescription in (3).
The invention also discloses a kind of biological control agents including the bacillus licheniformis 08220414.
The preparation method of biological control agent of the present invention, comprising the following steps:
(1) seed liquor of bacillus licheniformis 08220414 is prepared: by bacillus licheniformis 08220414 from test tube slant
The middle a small amount of bacterium of picking, moves in seed liquid culture medium, and 26~30 DEG C of shaking table 20~28h of shaken cultivation, this is seed liquor；
(2) seed liquor prepared by step (1) is inoculated into an aseptic environment containing sterilized bulk fermentation culture medium
In fermentor, 40~45h of constant temperature incubation at 26-30 DEG C；
(3) carrier (attapulgite powder) will be added in the fermentation liquid of step (2) culture, be uniformly mixed, spray drying obtains
08220414 bacterium powder of bacillus licheniformis.
(4) 08220414 bacterium powder of bacillus licheniformis can further be added agriculturally common auxiliary agent such as wetting agent, fill out
It fills agent etc. and biological control agent is made.
Experiment shows that lichem bacillus strain 08220414 of the invention is fast with the speed of growth, sporulation quantity is big, promotes
Grape growth, Saline alkali tolerance are strong, plant surface can rapid, high volume colonize the features such as, therefore have a good application prospect.
The biological control agent prepared by the bacillus licheniformis can efficiently prevent and treat grape root rot, and preventive effect is up to 90% or more,
It is a kind of biological control agent of great application prospect.Since bacterial strain of the invention is from salt-soda soil, with stronger resistance to
Salt alkali ability can be adapted for salt-soda soil use.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are within the scope of the invention.
Embodiment one
1, the isolation and purification of bacillus licheniformis 08220414
Bacillus licheniformis 08220414 of the invention is from Laizhou City, Shandong Province Sanshan Island alkaline land soil using dilute
It releases flat band method and plate streak separates acquisition, separation method are as follows:
(1) separation of bacillus
Soil sample acquisition is from Laizhou City, Shandong Province Sanshan Island salt-soda soil, and using 5 point samplings, surrounding is in acquiring sample respectively
The soil of 10~20cm soil layer of centre, equivalent mix.1g soil sample is weighed in 100mL sterile water, is placed in 30 DEG C of shaking tables
150rpm shakes 10min, is subsequently placed in 60 DEG C of water-baths and is incubated for 30min, takes 100 μ L 10-2、10-3、10-4Dilution coating
In on LB culture medium flat plate, each gradient is coated with three parallel, different shapes after 30 DEG C of culture 2d on picking LB culture medium
Microbial bacteria fall within and cross on LB culture medium flat plate, bacterium colony growing state is observed in timing.Then plate streak is used,
Bacillus strain is purified, number saves respectively.
(2) screening of the efficient antagonistic Bacillus of grape root rot
1. primary dcreening operation: using opposite culture method, PDA (Potato Dextrose Agar) plate is prepared, with punch in bud
It is the bacteria cake of 5mm that spore bacillus, grape root rot edge, which take diameter, is implanted in the opposite two sides center of plate, 25 DEG C of constant temperature respectively
Culture observes bacillus to the inhibiting effect of pathogen day by day.
2. secondary screening: the Bacillus strain with efficient antagonistic activity screened being carried out secondary screening, mainly by resistance to
Warm nature, resistance to acid and alkali, drug-resistant test screen the preferable Bacillus strain of patience, carry out field controling test.
The present inventor obtains one plant of bacillus licheniformis that can efficiently prevent and treat grape root rot by a large amount of screening operations
08220414.It is demonstrated experimentally that the bacillus licheniformis original powder shows very efficient prevention and treatment effect in prevention and treatment grape root rot
Fruit.Thus, bacillus licheniformis of the invention is the new strain of Bacillus licheniformis with wide application prospect, can be used for making
The biological control agent of standby prevention and treatment grape root rot.
2, bacterial strain is identified
(1) Microbiological Characteristics
Colonial morphology is flat in beef extract-peptone agar medium or LB culture medium, round or irregular shape, table
The coarse protrusion in face, it is opaque, it adheres, extension；In beef extract-peptone agar medium, cultivated at 28 DEG C two days later, microscopy,
Somatic cells are in the shape of a rod, and Dan Sheng not chaining can move.It is positive through Gram's staining (with Escherichia coli for control).Through gemma
Observe that gemma is oval or column after dyeing.
(2) molecular biological characteristic
The 16s rDNA result of gene sequence determination of the bacterial strain is following (SEQ-1):
TGCAGTCGAGCGGACCGACGGGAGCTTGCTCCCTTAGGTCAGCGGCGGACGGGTGAGT AACACGTGG
GTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTC
AATCATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT
CACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTA
CGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCCTGAGTGATGAAGGTTT
TCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCA
GAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGT
AAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGG
GAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGT
GGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTG
GTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAG
CACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCAT
GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCC
CCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA
CGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGA
AGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGG
CAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTG
AAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC
ACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCGTATG。
The bacterial strain is bacillus licheniformis (Bacillus licheniformis), is preserved in on January 15th, 2018
China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica), deposit number is CGMCC No.15216.
Embodiment two
1,08220414 fermentation process of bacillus licheniformis and its biological control agent preparation process
Seed liquor culture medium prescription: by mass percentage, beancake powder 3.00%, corn flour 4.00%, glucose
0.40%, fish meal 0.50%, potassium dihydrogen phosphate 0.03%, precipitated calcium carbonate 1.20%, ammonium sulfate 0.30%, sodium chloride
0.10%, remaining as water, pH is adjusted to 7.0-7.2.
Bulk fermentation culture medium prescription: with seed liquid culture medium formula.
Detailed process:
1. strain seed liquor culture: by bacillus licheniformis 08220414 from test tube slant a small amount of bacterium of picking, move to
In seed liquid culture medium, for 24 hours, this is seed liquor to 28 DEG C of shaking table shaken cultivations；
2. bulk fermentation culture: bulk fermentation culture medium material is sterilized (121-124 DEG C of temperature, 30 points in the fermenter
Clock), aseptically, cultured seed liquor (by inoculation volume ratio 10%) is poured into fermentor, 28 DEG C of fermented and cultureds
42h.Turn spore rate up to 80% or more, bacterium number is up to 165~18,000,000,000/ml；
3. adding carrier (attapulgite powder) in the fermentation liquid of fermentation ends, it is uniformly mixed, the mass ratio with carrier: 1:
0.05,178 DEG C of spray-drying installation inlet air temperature, 170 DEG C of leaving air temp, fermentation liquid through centrifugal spray to the 10% of original weight amount,
Bacterium powder is obtained, wherein 08220414 content of bacillus licheniformis >=160,000,000,000 viable bacterias/g；
4. taking each raw material by following weight ratios: step 3. prepared 08220414 bacterium powder 10% of bacillus licheniformis；It forms sediment
Powder 8%；Glucose 10%；Lauryl sodium sulfate 8%；Pull open powder 2%；Bean cake powder 10%；Surplus is filled out with attapulgite powder
It fills；
5. above-mentioned raw materials are uniformly mixed, the biological control agent of prevention and treatment grape root rot is obtained, wherein bacillus licheniformis
08220414 content >=16,000,000,000 viable bacterias/g.
Embodiment three
In order to which the biological control agent of the clearly 08220414 bacterium powder containing bacillus licheniformis imitates the prevention and treatment of grape root rot
Fruit provides foundation for its popularization and application.We have carried out field experiment in Shandong Province Laixi City 3~April in 2017, examination
Result is tested to be reported as follows:
1 material to be tested and method
1.1 reagent agent
By the biological control agent (160 for the 08220414 bacterium powder 10% containing bacillus licheniformis that the embodiment of the present invention 2 produces
Hundred million viable bacterias/g)；50% multiple-effect spirit wettable powder (Weihai Hanfu Biochemical Pharmaceutical Co., Ltd.'s production, commercially available).
1.2 materials to be tested and controlling object
Material to be tested is grape, and controlling object is grape root rot.
1.3 experimental field situations
It is experimental field located at Shandong Province, the market day village town village Men Jiazhuan, Laixi, which occurred seriously in recent years.Examination
It tests and is carried out in grape institute, test soil is light clay loam, and the content of organic matter 0.98%, pH 6.2, all experimental plots plant
Training condition and management measure are consistent.
1.4 experimental designs and arrangement
This test totally 5 processing, respectively containing 08220414 biological control agent of lichem bacillus strain, 500 times of liquid,
750 times of liquid, 1000 times of liquid, 50% 800 times of liquid of multiple-effect spirit wettable powder, are not administered clear water and compare, 4 weights of each processing
It is multiple, each 50 plants of processing.In progress on April 5th, 2017 pouring root, every plant of 1.5Kg.
1.5 pilot surveies and calculation method
1.5.1 meteorological condition
Application on April 5, the same day is cloudy, and 2 grades of wind-force, the highest temperature is 14.4 DEG C, and the lowest temperature is 11.9 DEG C, relatively wet
Degree is 29%.
1.5.2 drug effect and security survey
Efficacy survey: investigation 1 time in total, carrying out for 90 days after administration, records each processing group grape root rot morbidity number,
Calculate diseased plant rate and control efficiency.
Drug effect following formula calculates:
1.6 result
(1) control efficiency of the reagent agent to grape root rot
90 days investigation diseased plant numbers after pouring root, table 1 the results show that clear water control diseased plant rate be up to 35.75%, each processing
Diseased plant rate be below clear water control.The biological control agent of the 08220414 bacterium powder containing bacillus licheniformis dilute respectively 500 times,
750 times, the diseased plant rate after 1000 times of pouring roots be respectively 2.68%, 6.72%, 9.32%, 50% multiple-effect spirit wettable powder dilution agent
The diseased plant rate of pouring root is 6.86% after 800 times；The each processing of the biological control agent of 08220414 original powder containing bacillus licheniformis
Control efficiency be respectively 92.50%, 81.20%, 73.93%, wherein dilution 500 times control efficiency it is best.Other processing
There is significant difference in the level of P < 0.05,50% multiple-effect spirit wettable powder is to the control efficiency of grape root rot
80.81%.The harm of grape root rot can be effectively controlled in 08220414 biological control agent of bacillus licheniformis.
The disease incidence and control efficiency of grape root rot after each processing of table 1
Note: with there is same letter to indicate that difference is not significant (P < 0.05) after Duncan Multiple range test after data line.
(2) grape security survey: it is observed that each chemicals treatment district, compared with check plot, grape is generated without phytotoxicity, explanation
08220414 biological control agent of bacillus licheniformis is safe to grape for examination concentration.
Therefore, from the point of view of disease incidence and control efficiency, the biological control of the invention containing bacillus licheniformis 08220414
Preparation has preferable control efficiency to grape root rot, and control efficiency is up to 90% or more after diluting 500 times of applications, hence it is evident that good
In comparison medicament, significant difference.In terms of grape growing way, 08220414 biological control agent of bacillus licheniformis does not have grape growth
There is phytotoxicity, securely and reliably.
SEQUENCE LISTING
<110>Qingdao Calendula Bell bioengineering Co., Ltd
<120>one bacillus licheniformis and its application in prevention and treatment grape root rot
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1422
<212> DNA
<213>the 16s rDNA gene order of bacillus licheniformis (Bacillus licheniformis) 08220414
<400> 1
tgcagtcgag cggaccgacg ggagcttgct cccttaggtc agcggcggac gggtgagtaa 60
cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120
tgcttgattg aaccgcatgg ttcaatcata aaaggtggct tttagctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcctgagt 360
gatgaaggtt ttcggatcgt aaaactctgt tgttagggaa gaacaagtac cgttcgaata 420
gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggcgcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttagag ggtttccgcc ctttagtgct gcagcaaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
ccctagagat agggcttccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggcagaa 1200
caaagggcag cgaagccgcg aggctaagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380
cccgaagtcg gtgaggtaac ctttggagcc agccgccgta tg 1422
Claims (6)
1. bacillus licheniformis (Bacillus licheniformis) bacterial strain 08220414, the deposit number of the bacterial strain are
CGMCC No.15216。
2. purposes of the lichem bacillus strain 08220414 described in claim 1 in prevention and treatment grape root rot.
3. a kind of biological control agent contains lichem bacillus strain 08220414 described in claim 1.
4. the preparation method of biological control agent as claimed in claim 3, characterized in that the following steps are included:
(1) seed liquor of lichem bacillus strain 08220414 is prepared；
(2) seed liquor prepared by step (1) is inoculated into the fermentor of bulk fermentation culture medium, constant temperature incubation at 26-30 DEG C
40~45h；
(3) carrier will be added in the fermentation liquid of step (2) culture, be uniformly mixed, spray drying obtains bacillus licheniformis
08220414 bacterium powder is added auxiliary agent and is prepared into biological control agent.
5. the preparation method of biological control agent as claimed in claim 4, characterized in that
Prepare the seed liquid culture medium of seed liquor are as follows: by mass percentage, beancake powder 3.00%, corn flour 4.00%, grape
Sugar 0.40%, fish meal 0.50%, potassium dihydrogen phosphate 0.03%, precipitated calcium carbonate 1.20%, ammonium sulfate 0.30%, sodium chloride
0.10%, remaining as water, pH is adjusted to 7.0-7.2；
Bulk fermentation culture medium is the same as seed liquid culture medium.
6. purposes of the biological control agent as claimed in claim 3 in prevention and treatment grape root rot.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117535169A (en) * | 2022-08-10 | 2024-02-09 | 河北省科学院生物研究所 | Bacillus licheniformis ZLP-81 and application thereof |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117535169A (en) * | 2022-08-10 | 2024-02-09 | 河北省科学院生物研究所 | Bacillus licheniformis ZLP-81 and application thereof |
| CN117535169B (en) * | 2022-08-10 | 2024-06-11 | 河北省科学院生物研究所 | Bacillus licheniformis ZLP-81 and application thereof |
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Application publication date: 20190308 |