CN106701624B - The Te Jila bacillus of one plant of antagonism fructus lycii root rot and its application - Google Patents
The Te Jila bacillus of one plant of antagonism fructus lycii root rot and its application Download PDFInfo
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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
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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 Te Jila bacillus of one plant of antagonism fructus lycii root rot, its classification naming is Te Jila bacillus (Bacillus tequilensis) GQJK2, 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.13253.Isolated Te Jila bacillus (Bacillus tequilensis) GQJK2 of the present invention, the bacterial strain has stronger antagonism to fructus lycii root rot, new biocontrol microorganisms are provided 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 Te Jila bacillus of one plant of antagonism fructus lycii root rot and its applications, belong to biotechnology neck
Domain.
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.
Te Jila bacillus (Bacillus tequilensis) is one kind in bacillus, be can produce a variety of
Polypeptide antibiotic can generate antagonism to various plants pathogeny bacterium, and the biocontrol effect in relation to Te Jila bacillus is ground
Study carefully be concentrated mainly on prevention and treatment watermelon blight (CN105695368A), antagonism mulberry fruit core cane bacterium (CN105274021A), prevention and treatment
Wheat scab (CN104403962A) etc., but do not have the report of the Te Jila bacillus of prevention and treatment fructus lycii root rot also at present.
Moreover, often there is interaction, external source antagonism between microbiologic population in Biocontrol microorganism and soil environment
The introducing of bacterium can cause the composition of crop rhizosphere soils microbiologic population to change, and different plant rhizosphere bacteriums of the same race are determined
It plants and function has larger difference；Moreover, can Biocontrol microorganism survive in soil environment and fast-growth is also real
The key of existing preventive and therapeutic effect.Therefore, screening can have fructus lycii root rot with the biocontrol bacterial strain of preventive and therapeutic effect particularly significant
Practical application value.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide the Te Jila bacillus of one plant of antagonism fructus lycii root rot
And its application.
To achieve the above object, the present invention adopts the following technical solutions:
According to the first aspect of the invention, the Te Jila bacillus of one plant of antagonism fructus lycii root rot, classification life are provided
It is micro- to be preserved in China on November 10th, 2016 by entitled Te Jila bacillus (Bacillus tequilensis) GQJK2
Biological inoculum preservation administration committee common micro-organisms center (abbreviation CGMCC, address are as follows: BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, Institute of Microorganism, Academia Sinica), culture presevation number is CGMCC NO.13253.
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 milk yellow, bacterium colony is flat, the small dentation in edge, dry tack free, colony diameter 2.57mm.
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 negative, 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 positive, and arabinose fermenting experiment is positive, wood-sugar fermentation experiment
The positive, 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 Te Jila bacillus
The tunning of (Bacillus tequilensis) GQJK2.
Preferably, the biological prevention and control agent is liquid microbial inoculum.
Further, Te Jila bacillus (Bacillus tequilensis) GQJK2 contains in the biological prevention and control agent
Amount is greater than or equal to 3 × 108cfu/mL。
The present invention also provides the preparation method of above-mentioned biological prevention and control agent, include the following steps: the Te Jila gemma for fermenting above-mentioned
Bacillus (Bacillus tequilensis) GQJK2, 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 Te Jila bacillus (Bacillus tequilensis) is provided
The application of GQJK2 and/or 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 Te Jila bacillus (Bacillus tequilensis) is provided
GQJK2 and/or biological prevention and control agent are promoting the application in fructus lycii volume increase.
Fructus lycii root rot can be prevented and treated the present invention also provides a kind of, while promoting the product of volume increase, active constituent is above-mentioned
Te Jila bacillus (Bacillus tequilensis) GQJK2 or above-mentioned biological prevention and control agent.
Beneficial effects of the present invention:
(1) isolated Te Jila bacillus (Bacillus tequilensis) GQJK2 of the present invention, the bacterial strain pair
Fructus lycii 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
The type of preparation.
(2) studies have shown that under field condition, using Te Jila bacillus (Bacillus of the invention
Tequilensis) bacteria suspension of GQJK2 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；Te Jila bacillus (Bacillus of the invention
Tequilensis) GQJK2 can also improve the upgrowth situation of plant, improve the yield of fructus lycii, exist to the averagely volume increase of fructus lycii tree
550% or more.The above result of study shows that the bacillus of antagonism fructus lycii root rot of the invention has lycium barbarum root rot
There is certain control efficiency.
(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 Te Jila bacillus of fruit, due to the severe living environment in Ningxia, China, bacterial strain can survive and fast-growth at
For critical issue, therefore there may not be control efficiency to fructus lycii root rot.
Detailed description of the invention
Fig. 1: Te Jila bacillus (Bacillus tequilensis) GQJK2 colonial morphology figure；
The phyletic evolution tree graph of Fig. 2: Te Jila bacillus (Bacillus tequilensis) GQJK2.
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 bacterial 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 GQJK2, 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 GQJK2 has preferable bacteriostasis to the pathogen of fructus lycii root rot.
2. the form and physiological and biochemical property of bacterial strain GQJK2 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, milk yellow, bacterium colony after cultivating on LB plate for 24 hours
It is flat, the small dentation in edge, dry tack free, colony diameter 2.57mm.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 negative, 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 positive, and arabinose fermenting experiment is positive, wood-sugar fermentation experiment
The positive, 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 GQJK2
The 16S rDNA gene order of bacterial strain GQJK2, as shown in SEQ ID NO.1 in nucleotides sequence list.It will be surveyed
Sequence alignment in 16SrDNA sequence and GenBank database, the results showed that GQJK2 and Bacillus tequilensis
(10b) is in together in a minimum branch, the bacterial strain of 16S rRNA gene order and Bacillus tequilensis (10b)
Sequence similarity is 99%, in conjunction with above-mentioned physiological and biochemical property as a result, identifying that GQJK2 is Te Jila bacillus (Bacillus
tequilensis)。
Bacterial strain GQJK2, classification naming be Te Jila bacillus (Bacillus tequilensis) GQJK2, 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.13253。
Embodiment 2: the preparation of bacterial strain GQJK2 biological prevention and control agent
It is specific the preparation method is as follows:
Te Jila bacillus (Bacillus tequilensis) GQJK2 for the antagonism fructus lycii root rot that inclined-plane is saved
It is transferred 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 3 × 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:GQJK2 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 GQJK2 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 Te Jila bacillus (Bacillus
Tequilensis) the fructus lycii tree disease incidence of GQJK2 bacterium solution processing is 35%, compared with the control, the fructus lycii tree handled with GQJK2
Disease incidence reduce 55 percentage points.
2. relative control effect: phase of Te Jila bacillus (Bacillus tequilensis) GQJK2 to fructus lycii root rot
To preventive effect up to 75%.
(2) effect of increasing production of the bacterial strain GQJK2 to fructus lycii tree
It the results are shown in Table 3.
Effect of increasing production of 3: Te Jila bacillus of table (Bacillus tequilensis) GQJK2 to fructus lycii tree
As can be seen from Table 3: the fructus lycii tree average product of control treatment is 0.5kg/, with Te Jila bacillus
The yield of (Bacillus tequilensis) GQJK2 bacterium solution processing is 3.25kg/, compared with the control, is handled with GQJK2
Fructus lycii tree averagely increases production 550%.
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 Te Jila 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 GQJK2
<400> 1
ttcggcggct ggctcctaaa aggttacctc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggttt 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 aggggacgtc ctatctctag gattgtcaga 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 aaggtaccgc cctattcgaa cggtacttgt 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 tcaaacaacc atccggtatt agccccggtt 1320
tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc cgtccgccgc 1380
taacatcagg gagcaagctc ccatctgtcc gctcgactgc a 1421
Claims (10)
1. Te Jila bacillus (Bacillus tequilensis) GQJK2 of one plant of antagonism fructus lycii root rot, in 2016
On November 10, in 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 are CGMCC
NO.13253。
2. a kind of biological prevention and control agent, active constituent is Te Jila bacillus (Bacillus described in claim 1
Tequilensis) the tunning of GQJK2.
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 Te Jila bacillus in the biological prevention and control agent
The content of (Bacillus tequilensis) GQJK2 is greater than or equal to 3 × 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
Te Jila bacillus (Bacillus tequilensis) GQJK2 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. described in Te Jila bacillus (Bacillus tequilensis) GQJK2 described in claim 1 or claim 2
Biological prevention and control agent prevention and treatment fructus lycii root rot in application.
10. Te Jila bacillus (Bacillus tequilensis) GQJK2 described in claim 1 or claim 2 institute
The biological prevention and control agent stated is promoting the application in fructus lycii volume increase.
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|---|---|---|---|---|
| CN104403962A (en) * | 2014-10-31 | 2015-03-11 | 中国农业科学院农产品加工研究所 | Bacillus tequilensis and its use in prevention and control of fusarium graminearum |
| KR20160129369A (en) * | 2015-04-30 | 2016-11-09 | 한국생명공학연구원 | Composition for preventing plant storage disease and a control method of plant storage disease using the same |
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