CN104531554A - Pantoea agglomerans with molluscicidal activity and application thereof - Google Patents
Pantoea agglomerans with molluscicidal activity and application thereof Download PDFInfo
- Publication number
- CN104531554A CN104531554A CN201410712468.3A CN201410712468A CN104531554A CN 104531554 A CN104531554 A CN 104531554A CN 201410712468 A CN201410712468 A CN 201410712468A CN 104531554 A CN104531554 A CN 104531554A
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- CN
- China
- Prior art keywords
- pantoea agglomerans
- oncomelania
- cctcc
- pantoea
- killing agent
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/205—Bacterial isolates
-
- 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
Abstract
The invention discloses pantoea agglomerans (Pantoea agglomerans C4) with the molluscicidal activity and application thereof. The biological preservation number of the pantoea agglomerans is CCTCC NO: M2014104; bacteria with the preservation number of CCTCC NO:M2014104 are pantoea bacteria and are screened from tee leaves; and oncomelania can be rapidly and effectively killed. The pantoea agglomerans has the advantages that the new bacteria for killing the oncomelania is provided; the pantoea agglomerans can be used as a biological oncomelania killing agent directly; and compared with a traditional chemical oncomelania killing agent, the biological oncomelania killing agent prepared with the bacteria with the preservation number of CCTCC NO: M2014104 is simple in preparation method, low in cost and environment-friendly.
Description
Technical field
What the present invention relates to is a kind of microorganism field, in particular a kind of pantoea agglomerans and application thereof with activity against snails.
Background technology
Oncomelania as the unique intermediate host of schistosomicide, its procreation and distribution closely related with the popular propagation of schistosomicide, so press down the spiral shell spiral shell that goes out to be considered to the important channel of prevention schistosomiasis propagation.Molluscacide main is at present chemicals class, and this kind of medicine easily causes irreversible breaking to environment.In plant, biological community structure is paid close attention to widely and is studied in recent years; Chinese scholars finds to there is a large amount of microorganisms in plant materials through research; these microorganisms all play an important role in the growth, growth, health etc. of animal; therefore; the direction of research be targeted by endophytic microflora by the present invention; wish to find the microbe snail-killing agent that a kind of selectivity is strong, have fewer environmental impacts, with alternative traditional chemical molluscacide, protection of the environment.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of pantoea agglomerans and the application thereof with activity against snails, to provide the new microbe snail-killing agent that a kind of selectivity is strong, have fewer environmental impacts.
The present invention is achieved by the following technical solutions:
Having a pantoea agglomerans for activity against snails, Pantoea agglomerans C4, is general campylobacter bacteria, and this bacterium is preserved in China typical culture collection center on March 26th, 2014 and (is called for short CCTCC; Address: Wuhan, China, Wuhan University; Postcode: 430072), its biological deposits number is CCTCC NO:M2014104, and Classification And Nomenclature is: Pantoea agglomerans C4 (pantoea agglomerans C4).
Described bacterium CCTCC NO:M2014104 screens and obtains from tea leaf, and its biological property is as follows:
(1) morphological feature: the dyeing of this bacterial Gram, for negative, is direct rod shape on this bacterium of the oily Microscopic observation of opticmicroscope 16 × 100, agglomerating distribution, peritrichous.
(2) cultural characteristic: well-grown in YEB solid medium, bacterium colony is mellow and full, in faint yellow, neat in edge, transparent, easily provoke, along with incubation time extends when cultivating in YEB liquid nutrient medium, nutrient solution is from the transparent light yellow yellow becoming muddiness.
The 16S rRNA sequence of described bacterium CCTCC NO:M2014104 is as shown in SEQ ID NO.1, wherein said 16S rRNA sequence has 480 bases, BLAST comparison process is carried out by sequence inputting GenBank database, found that, this 16S rRNA sequence and pantoea agglomerans SGS9 (Pantoea agglomerans, SGS9) 16S rRNA sequence (GenBank accession number: KJ529102.1) homology is 90%, combining form characteristic sum cultural characteristic identifies that this bacterium CCTCC NO:M2014104 is the pantoea agglomerans (Pantoea agglomerans) of general Pseudomonas.
Present invention also offers above-mentioned bacterium CCTCC NO:M2014104 and preparing the application in microbe snail-killing agent, the fermented liquid of described bacterium CCTCC NO:M2014104 is as microbe snail-killing agent Killing Oncomelania Hupensis.
The present invention has the following advantages compared to existing technology: the invention provides a kind of pantoea agglomerans and the application thereof with activity against snails, this bacterium CCTCC NO:M2014104 can as microbe snail-killing agent fast and effeciently Killing Oncomelania Hupensis, can directly use as microbe snail-killing agent, the microbe snail-killing agent prepared with this bacterium CCTCC NO:M2014104 is compared with traditional chemical molluscacide, preparation method is simple, cost is low, environmentally friendly.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
A kind of screening with the pantoea agglomerans CCTCC NO:M2014104 bacterial strain of activity against snails of the present embodiment, comprises the following steps:
1, the preparation of substratum:
1) YEB liquid nutrient medium: beef extract is 5.0g/L, yeast extract paste is 1.0g/L, and peptone is 5.0g/L, MgSO
47H
2o is 4g/L, and sucrose is 5.0g/L, pH7.0 ~ 7.5;
2) YEB solid medium: will add the agar of 15g/L in above-mentioned YEB liquid nutrient medium, obtains YEB solid medium.
2, step is screened:
1) aseptically, get fresh tea leaf, be 75% alcohol-pickled 1 minute by volume fraction, 0.1% mercuric chloride soaks 12 minutes, after stroke-physiological saline solution rinses 3 times, is ground by blade, obtains and organize broken end;
2) by step 1) broken end of organizing be inoculated in the YEB liquid nutrient medium of 100mL, 37 DEG C cultivate 12 ~ 16 hours, the rotating speed of cultivation is 180rpm, obtain nutrient solution;
3) by volume, by step 2) nutrient solution stroke-physiological saline solution to dilute successively be 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6with 10
-7, obtain diluent;
4) by step 3) diluent be applied on YEB solid medium, 37 DEG C cultivate 12 ~ 16 hours, picking list bacterium colony carries out purifying, namely obtains described bacterium CCTCC NO:M2014104.
3, Molecular Identification:
Bacterium CCTCC NO:M2014104 after above-mentioned purifying is inoculated on YEB solid medium, cultivate 12 ~ 16 hours for 37 DEG C, picking list bacterium colony carries out bacterium colony PCR, and the primer of described bacterium colony PCR is bacterial 16 S rRNA universal primer 27F and R518, and the sequence of described primer is as follows:
SEQ NO.2：27F：AGAGTTTGATCCTGGCTCAG
SEQ NO.3：R518：ATTACCGCGGCTGCTGG
The amplification program of described bacterium colony PCR is: 94 DEG C of denaturations 3 minutes, 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, and 72 DEG C extend 1 minute, circulates 30 times, and 72 DEG C are continued extension 7 minutes, acquisition pcr amplification product;
It is the agarose gel electrophoresis detection of 1.2% by pcr amplification product massfraction, purifying is carried out with PCR purification kit, obtain the 16S rRNA of bacterium CCTCC NO:M2014104, 16S rRNA is delivered to the order-checking of order-checking company, result obtains the sequence as shown in SEQID NO.1, by this sequence inputting GenBank database, to compare process with BLAST software, found that, this 16S rRNA sequence and pantoea agglomerans SGS9 (Pantoea agglomerans, SGS9) 16S rRNA sequence (GenBank accession number: KJ529102.1) homology is 90%, combining form characteristic sum cultural characteristic identifies that this bacterium CCTCC NO:M2014104 is the pantoea agglomerans of general Pseudomonas.
Embodiment 2
The present embodiment is the mensuration of described bacterium CCTCC NO:M2014104 activity against snails:
1, the preparation of molluscacide:
Bacterium CCTCC NO:M2014104 is inoculated in the YEB liquid nutrient medium of 100mL, cultivate 24 hours at 37 DEG C, the rotating speed cultivated is 180rpm, obtain seed liquor, 1mL seed liquor transferred in the YEB liquid nutrient medium of 100mL, cultivate 12 ~ 16 hours for 37 DEG C, the rotating speed of cultivation is 180rpm, obtain fermented liquid, be microbe snail-killing agent.
2, the preparation of molluscacide diluent: molluscacide is spent chlorine water and be mixed with the solution that volume fraction is 10%, be molluscacide diluent.
3, the mensuration of activity against snails:
After the oncomelania of field acquisition is cultivated 3 days at 25 DEG C, the oncomelania that selection vigor is good spends chlorine water and rinses 3 times, oncomelania is divided into three groups, often organize three bags, every bag of 20 oncomelanias, every group selection one bag is placed in the above-mentioned molluscacide diluent configured and soaks 2 days respectively, 3 days, 4 days, spend chlorine water after taking-up to rinse well, room temperature recovers raising 3 days, clear water soaks 2 hours, take out observation oncomelania to stretch out with or without software, if there is software to stretch out, it is movable snail, that stretches out without software knocks oncomelania housing with hammering method, judge whether death, dechlorination water belongs with yin control group is set simultaneously.
4, result:
The activity against snails measurement result of bacterium CCTCC NO:M2014104 is as shown in table 1 below:
Table 1: the activity against snails measurement result table of bacterium CCTCC NO:M2014104
As can be seen from Table 1, the efficiency of this bacterium CCTCC NO:M2014104 Killing Oncomelania Hupensis increases along with the increase of time, when process 2 days, the average mortality of oncomelania is 46.67%, when process 3 days, the average mortality of oncomelania reaches 78.33%, when process 4 days, the average mortality of oncomelania reaches 93.33%, can find out, this bacterium CCTCC NO:M2014104 has the effect of good Killing Oncomelania Hupensis, and the efficiency of its Killing Oncomelania Hupensis increases along with the prolongation of time.
Claims (4)
1. have a pantoea agglomerans for activity against snails, Pantoea agglomerans C4, is characterized in that, the biological deposits number of described pantoea agglomerans is CCTCC NO:M2014104.
2. a kind of pantoea agglomerans with activity against snails according to claim 1, is characterized in that, the 16S rRNA sequence of described pantoea agglomerans is as shown in SEQ ID NO.1.
3. a pantoea agglomerans as claimed in claim 1 with activity against snails is preparing the application in microbe snail-killing agent.
4. a kind of pantoea agglomerans with activity against snails according to claim 3 is preparing the application in microbe snail-killing agent, and it is characterized in that, the fermented liquid of described pantoea agglomerans is as microbe snail-killing agent Killing Oncomelania Hupensis.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410712468.3A CN104531554B (en) | 2014-11-27 | 2014-11-27 | Pantoea agglomerans with molluscicidal activity and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410712468.3A CN104531554B (en) | 2014-11-27 | 2014-11-27 | Pantoea agglomerans with molluscicidal activity and application thereof |
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|---|---|
| CN104531554A true CN104531554A (en) | 2015-04-22 |
| CN104531554B CN104531554B (en) | 2017-05-17 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597930A (en) * | 2004-08-18 | 2005-03-23 | 杨建明 | Yellow bacillus and its snail killing agent |
| CN102533611A (en) * | 2012-02-10 | 2012-07-04 | 中国农业科学院农业资源与农业区划研究所 | Efficient phosphorus-dissolution promotion bacteria, microbial agent prepared from same and application of microbial agent |
| CN103602618A (en) * | 2013-11-18 | 2014-02-26 | 安徽农业大学 | Oncomelania killing bacteria and application thereof |
-
2014
- 2014-11-27 CN CN201410712468.3A patent/CN104531554B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1597930A (en) * | 2004-08-18 | 2005-03-23 | 杨建明 | Yellow bacillus and its snail killing agent |
| CN102533611A (en) * | 2012-02-10 | 2012-07-04 | 中国农业科学院农业资源与农业区划研究所 | Efficient phosphorus-dissolution promotion bacteria, microbial agent prepared from same and application of microbial agent |
| CN103602618A (en) * | 2013-11-18 | 2014-02-26 | 安徽农业大学 | Oncomelania killing bacteria and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GUO D等: "Screening of molluscicidal strain against Oncomelania hupensis from the rhizosphere of medicinal plant Phytolacca acinosa Roxb", 《PHARMACOGN MAG.》 * |
| 孙富艳 等: "成团泛菌感染的研究近况", 《中国感染与化疗杂志》 * |
| 杨建明 等: "微生物灭钉螺研究现状", 《湖北大学学报（自然科学版）》 * |
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| CN104531554B (en) | 2017-05-17 |
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Granted publication date: 20170517 Termination date: 20171127 |