CN108260567A - A kind of quick breeding of Bursaphelenchus xylophilus and separation method - Google Patents
A kind of quick breeding of Bursaphelenchus xylophilus and separation method Download PDFInfo
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- CN108260567A CN108260567A CN201810066132.2A CN201810066132A CN108260567A CN 108260567 A CN108260567 A CN 108260567A CN 201810066132 A CN201810066132 A CN 201810066132A CN 108260567 A CN108260567 A CN 108260567A
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- 241000243771 Bursaphelenchus xylophilus Species 0.000 title claims abstract description 43
- 238000000926 separation method Methods 0.000 title claims abstract description 23
- 238000009395 breeding Methods 0.000 title claims abstract description 12
- 230000001488 breeding effect Effects 0.000 title claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 49
- 241000244206 Nematoda Species 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 16
- 239000007921 spray Substances 0.000 claims abstract description 15
- 241000935931 Diplodia sapinea Species 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 12
- 239000008223 sterile water Substances 0.000 claims abstract description 9
- 235000015170 shellfish Nutrition 0.000 claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 8
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 102000002322 Egg Proteins Human genes 0.000 claims description 4
- 108010000912 Egg Proteins Proteins 0.000 claims description 4
- 238000004821 distillation Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 230000005110 hydrotaxis Effects 0.000 claims description 4
- 230000008595 infiltration Effects 0.000 claims description 4
- 238000001764 infiltration Methods 0.000 claims description 4
- 229910052742 iron Inorganic materials 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 210000004681 ovum Anatomy 0.000 claims description 4
- 238000005192 partition Methods 0.000 claims description 4
- 230000003068 static effect Effects 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 abstract 2
- 238000005406 washing Methods 0.000 abstract 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 7
- 241000018646 Pinus brutia Species 0.000 description 7
- 235000011613 Pinus brutia Nutrition 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 206010062701 Nematodiasis Diseases 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Quick breeding and separation method the invention discloses a kind of Bursaphelenchus xylophilus.This method includes the following steps：Sphaeropsis sapinea is inoculated in PDA culture medium, is cultivated 5 days in 26 DEG C of constant incubators, Bursaphelenchus xylophilus is inoculated with when mycelia covers with culture dish substantially；Suitable sterile water is uniformly sprayed in culture dish interior surface, 5 10ml aqua sterilisas are added in after 4 days and rinse culture dish lid, collect the water for rinsing culture dish lid and centrifugation, you can obtain a large amount of Bursaphelenchus xylophilus；Continue uniformly to spray suitable sterile water to culture dish interior surface after collection, repeat culture 23 times, Bursaphelenchus xylophilus can be obtained with aseptic water washing, collection, centrifugation after the completion of culture.Culture medium is cut into small pieces simultaneously and is put into the graceful funnel separation nematode of shellfish, the nematode for detaching acquisition is placed in 4 DEG C of refrigerators and is stored for future use.Compared with classical culture protocols, the present invention can rapid, high volume breeding Bursaphelenchus xylophilus, separation method is simple, can greatly save the time.
Description
Technical field
Quick breeding and separation method the invention belongs to nematode preparing technical field more particularly to a kind of Bursaphelenchus xylophilus.
Background technology
Pine nematode (Pine wilt disease) is one kind by Bursaphelenchus xylophilus Bursaphelenchus
Xylophilus (Steiner and Buhrer) quick withered death of pine tree caused by Nickle, the dangerous venereal disease destroyed in flakes
Evil.Paid much attention to due to endangering serious, prevention difficulty by countries in the world, be listed in important dangerous forest disease,
And by more than 40 a national legislations be emphasis quarantine object.Bursaphelenchus xylophilus initially originating from the U.S., through timber-trade be passed to Japan,
China, South Korea, Mexico, Portugal and Spain, and pine nematode has occurred in succession.By the end of China's pine in 2017
Nematodiasis has spread to Taiwan, Liaoning, Jiangsu, Zhejiang, Anhui, Fujian, Jiangxi, Shandong, Henan, Hubei, Hunan, Guangdong, wide
16 provinces such as west, Chongqing, Sichuan, Guizhou, Shaanxi, municipality directly under the Central Government or autonomous region, 246 counties (area), wherein newly-increased epidemic-stricken area in 2016
45 counties (area) destroy more than 30 ten thousand hectares of pine forest, bring about great losses to national economy.Traditional Reproduction In Bursaphelenchus Xylophilus culture
It is needed in the process by the graceful funnel method separation nematode of shellfish, complex steps, operation is time-consuming and laborious, and reproductive number is also relatively limited, limit
The research prevented Bursaphelenchus xylophilus is made.
Invention describes the quick methods bred and detach of Bursaphelenchus xylophilus, are the research of Bursaphelenchus xylophilus and pine line
The prevention of parasitosis provides more quickly and easily method.
Invention content
For problems of the prior art, quick breeding and separation side the present invention provides a kind of Bursaphelenchus xylophilus
Method.
The present invention uses following technical scheme：
A kind of quick breeding of Bursaphelenchus xylophilus and separation method, it includes the following steps：
（1）Sphaeropsis sapinea LYU151212 is inoculated in PDA culture medium, is cultivated 5 days in 26 DEG C of constant incubators, treats bacterium
When silk covers with culture dish substantially, it is inoculated with the Bursaphelenchus xylophilus suspension containing Benzylpenicillin sodium salt and streptomycin sulphate；The pinecone shell spore
Bacterium LYU151212 for the mould Sphaeropsis sapinea of pure green cyan (Sphaeropsis sapinea) LYU151212,In 2017 12
The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center and survives on the 21st, preserving number CGMCC
NO.14997；
（2）Culture dish interior surface after inoculation is sprayed into appropriate amounts of sterilized water, culture dish is wound with sealed membrane, then by culture dish
It is placed in 26 DEG C of constant incubators and cultivates, when culture dish covers sterile water water droplet and occurs a large amount of muddy after 4 days, add in 5-10ml and go out
Bacterium water rinses culture dish lid, is fully stirred with triangle spreading rod, collects the water for rinsing culture dish lid；It rinses 2-3 times, with
3000rpm centrifuges 1min, you can obtains a large amount of Bursaphelenchus xylophilus；
（3）Culture dish interior surface is cleaned, sprays appropriate amounts of sterilized water again, repeats step 2）I.e. 2-3 acquisition is largely being cultivated
Ware covers the Bursaphelenchus xylophilus of acquisition；
（4）It is after mycelia is depleted substantially by nematode, the separation of Bursaphelenchus xylophilus in culture dish bottom medium is graceful using shellfish
Funnel partition method；Its device be the funnel stand by diameter 7-10cm on iron stand, connect below one section about 5cm rubber tube,
The centrifuge tube of diameter 1.0cm is connect in the end of rubber tube, a spring clip is filled on rubber tube；Filter paper or face tissue are positioned over
In funnel, with distillation water infiltration filter paper, then culture medium chopping to be separated is positioned on filter paper；Add water submerged training to be separated
Base is supported, static separation is for 24 hours；Due to the hydrotaxis and the weight of itself of nematode, nematode leaves culture sill, wriggles in water,
Finally it is deposited in glass tube via the rubber tube of funnel end；Spring clip is closed, removes centrifuge tube, the line that will be collected into pipe
The nematode that worm is collected into culture dish upper cover is combined, spare；
（5）The Bursaphelenchus xylophilus being collected into and worm's ovum are disinfected, 4 DEG C store for future use.
Preferably, step（1）Described in Bursaphelenchus xylophilus suspension of the inoculation containing Benzylpenicillin sodium salt and streptomycin sulphate when, often
Ware is inoculated with 3000 nematodes.
Preferably, step（2）Described in culture dish interior surface sprinkling sterile water method be：Water spray 120-180 μ l,
Spray a diameter of 1-5mm of water droplet.
The beneficial effects of the invention are as follows：Compared with classical culture protocols, the present invention can rapid, high volume breeding Bursaphelenchus xylophilus, training
The nematode for supporting the separation of ware lid needs not move through the graceful funnel method of shellfish, can be directly over centrifuge and obtain nematode, greatly save the time.
Two to three times more than the nematode number that the amount for handling to obtain Bursaphelenchus xylophilus by two to water spray three times is obtained than conventional method.
Description of the drawings
Fig. 1 is the relationship of separation nematode number and nematode population after culture dish inner cover water spray.
Fig. 2 is the relationship of different training methods and nematode population.
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail.
Embodiment 1
A kind of quick breeding of Bursaphelenchus xylophilus and separation method, it includes the following steps：
（1）Sphaeropsis sapinea LYU151212 is inoculated in PDA culture medium, is cultivated 5 days in 26 DEG C of constant incubators, treats bacterium
When silk covers with culture dish substantially, the Bursaphelenchus xylophilus suspension containing Benzylpenicillin sodium salt and streptomycin sulphate is inoculated with, 3000 are inoculated with per ware
Head nematode；The Sphaeropsis sapinea LYU151212 is the mould Sphaeropsis sapinea of pure green cyanSphaeropsis sapinea
LYU151212,China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited on December 21st, 2017
And survive, preserving number is CGMCC NO.14997；
（2）Culture dish interior surface after inoculation is sprayed into appropriate amounts of sterilized water, culture dish is wound with sealed membrane, then by culture dish
It is placed in 26 DEG C of constant incubators and cultivates, when culture dish covers sterile water water droplet and occurs a large amount of muddy after 4 days, add in 5-10ml and go out
Bacterium water rinses culture dish lid, is fully stirred with triangle spreading rod, collects the water for rinsing culture dish lid；It rinses 2-3 times, with
3000rpm centrifuges 1min, you can obtains a large amount of Bursaphelenchus xylophilus；Water spray 120-180 μ l when wherein spraying sterile water, spray water
Drip a diameter of 1-5mm.
（3）Culture dish interior surface is cleaned, sprays appropriate amounts of sterilized water again, repeats step 2）I.e. 2-3 acquisition largely exists
Culture dish covers the Bursaphelenchus xylophilus of acquisition；
（4）After mycelia is depleted substantially by nematode（About 15 days）, by point of Bursaphelenchus xylophilus in culture dish bottom medium
From using the graceful funnel partition method of shellfish；Its device be the funnel stand by diameter 7-10cm on iron stand, connect below one section about 5cm
Rubber tube, the centrifuge tube of diameter 1.0cm is connect in the end of rubber tube, a spring clip is filled on rubber tube；By filter paper or face
Towel paper is positioned in funnel, and with distillation water infiltration filter paper, then culture medium chopping to be separated is positioned on filter paper；Add water logging
Do not have culture medium to be separated, static separation is for 24 hours；Due to the hydrotaxis and the weight of itself of nematode, nematode leaves culture sill,
It wriggles in water, is finally deposited in glass tube via the rubber tube of funnel end；Spring clip is closed, removes centrifuge tube, it will be in pipe
The nematode that the nematode being collected into is collected into culture dish upper cover is combined, spare；
（5）The Bursaphelenchus xylophilus being collected into and worm's ovum are disinfected, 4 DEG C store for future use.
Comparative example 1
（1）Sphaeropsis sapinea LYU151212 is inoculated in PDA culture medium, is cultivated 5 days in 26 DEG C of constant incubators, treats bacterium
When silk covers with culture dish substantially, the Bursaphelenchus xylophilus suspension containing Benzylpenicillin sodium salt and streptomycin sulphate is inoculated with, 3000 are inoculated with per ware
Head nematode；The Sphaeropsis sapinea LYU151212 is the mould Sphaeropsis sapinea of pure green cyanSphaeropsis sapinea
LYU151212,China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited on December 21st, 2017
And survive, preserving number is CGMCC NO.14997；
（2）Culture dish after inoculation is placed directly in 26 DEG C of constant incubators and is cultivated, does not spray the corresponding of sterile water process
Step.
（3）After mycelia is depleted substantially by nematode（About 15 days）, by Bursaphelenchus xylophilus in culture dish bottom medium
Separation use the graceful funnel partition method of shellfish；Its device be the funnel stand by diameter 7-10cm on iron stand, connect one section below about
For the rubber tube of 5cm, the centrifuge tube of diameter 1.0cm is connect in the end of rubber tube, a spring clip is filled on rubber tube；By filter paper or
Person's face tissue is positioned in funnel, and with distillation water infiltration filter paper, then culture medium chopping to be separated is positioned on filter paper；Add
Water submerged culture medium to be separated, static separation is for 24 hours；Due to the hydrotaxis and the weight of itself of nematode, nematode leaves culture base material
Material, wriggles, is finally deposited in glass tube via the rubber tube of funnel end in water；Spring clip is closed, removes centrifuge tube, it will
The nematode being collected into pipe is collected, spare；
（4）The Bursaphelenchus xylophilus being collected into and worm's ovum are disinfected, 4 DEG C store for future use.
The quantity situation of Bursaphelenchus xylophilus in preparation process in embodiment 1 and comparative example 1 is done into a statistical analysis, it is specific as schemed
Shown in 1-2.Wherein Fig. 1 is step in embodiment 1（3）In be repeated 3 times water spray and cultivate and the quantity statistics of nematode that detach, from figure
In it can be seen that three times separation nematode quantity there is no significant change.Fig. 2 is two kinds of distinct methods in embodiment 1 and comparative example 1
Cultivate the total amount statistical result of the nematode obtained.From figure 2 it can be seen that in identical incubation time, water spray handles to obtain
Nematode number obtain two to three times of nematode number for traditional approach.And the nematode that water spray is handled is easier to detach, significantly
Reduce culture and disengaging time.
The above content is combine specific preferred embodiment to the further description of the invention made, it is impossible to assert this
The specific implementation of invention is confined to these explanations, for those of ordinary skill in the art to which the present invention belongs, not
Under the premise of being detached from present inventive concept, several simple deduction or replace can also be made, the present invention should be all considered as belonging to and be carried
The protection domain that claims of friendship determine.
Claims (3)
1. a kind of quick breeding of Bursaphelenchus xylophilus and separation method, which is characterized in that it includes the following steps：
（1）Sphaeropsis sapinea LYU151212 is inoculated in PDA culture medium, is cultivated 5 days in 26 DEG C of constant incubators, treats bacterium
When silk covers with culture dish substantially, it is inoculated with the Bursaphelenchus xylophilus suspension containing Benzylpenicillin sodium salt and streptomycin sulphate；The pinecone shell spore
Bacterium LYU151212 for the mould Sphaeropsis sapinea of pure green cyan (Sphaeropsis sapinea )LYU151212,In 2017 12
The moon is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center and survives on the 21st, preserving number CGMCC
NO.14997；
（2）Culture dish interior surface after inoculation is sprayed into appropriate amounts of sterilized water, culture dish is wound with sealed membrane, then by culture dish
It is placed in 26 DEG C of constant incubators and cultivates, when culture dish covers sterile water water droplet and occurs a large amount of muddy after 4 days, add in 5-10ml and go out
Bacterium water rinses culture dish lid, is fully stirred with triangle spreading rod, collects the water for rinsing culture dish lid；It rinses 2-3 times, with
3000rpm centrifuges 1min, you can obtains a large amount of Bursaphelenchus xylophilus；
（3）Culture dish interior surface is cleaned, sprays appropriate amounts of sterilized water again, repeats step 2）I.e. 2-3 acquisition is largely being cultivated
Ware covers the Bursaphelenchus xylophilus of acquisition；
（4）It is after mycelia is depleted substantially by nematode, the separation of Bursaphelenchus xylophilus in culture dish bottom medium is graceful using shellfish
Funnel partition method；Its device be the funnel stand by diameter 7-10cm on iron stand, connect below one section about 5cm rubber tube,
The centrifuge tube of diameter 1.0cm is connect in the end of rubber tube, a spring clip is filled on rubber tube；Filter paper or face tissue are positioned over
In funnel, with distillation water infiltration filter paper, then culture medium chopping to be separated is positioned on filter paper；Add water submerged training to be separated
Base is supported, static separation is for 24 hours；Due to the hydrotaxis and the weight of itself of nematode, nematode leaves culture sill, wriggles in water,
Finally it is deposited in glass tube via the rubber tube of funnel end；Spring clip is closed, removes centrifuge tube, the line that will be collected into pipe
The nematode that worm is collected into culture dish upper cover is combined, spare；
（5）The Bursaphelenchus xylophilus being collected into and worm's ovum are disinfected, 4 DEG C store for future use.
2. the quick breeding of Bursaphelenchus xylophilus according to claim 1 and separation method, which is characterized in that step（1）Middle institute
When stating Bursaphelenchus xylophilus suspension of the inoculation containing Benzylpenicillin sodium salt and streptomycin sulphate, 3000 nematodes are inoculated with per ware.
3. the quick breeding of Bursaphelenchus xylophilus according to claim 1 and separation method, which is characterized in that step（2）Middle institute
State culture dish interior surface sprinkling sterile water method be：Water spray 120-180 μ l, a diameter of 1-5mm of sprinkling water droplet.
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| CN201810066132.2A CN108260567A (en) | 2018-01-24 | 2018-01-24 | A kind of quick breeding of Bursaphelenchus xylophilus and separation method |
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| CN201810066132.2A Pending CN108260567A (en) | 2018-01-24 | 2018-01-24 | A kind of quick breeding of Bursaphelenchus xylophilus and separation method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112237169A (en) * | 2020-10-23 | 2021-01-19 | 河北省农林科学院植物保护研究所 | Method for culturing and separating pratylenchus praecox |
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| WO2002057440A2 (en) * | 2001-01-18 | 2002-07-25 | Cambria Biosciences, Llc | Screens and assays for agents useful in controlling parasitic nematodes |
| CN102084849A (en) * | 2010-11-19 | 2011-06-08 | 南京林业大学 | Method for preparing bacteria-free pine wood nematodes |
| CN103004592A (en) * | 2012-12-10 | 2013-04-03 | 华南农业大学 | Method for breeding, culturing and preserving paratylenchus by using carrot callus |
| CN105613425A (en) * | 2014-10-27 | 2016-06-01 | 申茂军 | Pine wood nematode rapid preparation method |
| CN106755554A (en) * | 2017-03-21 | 2017-05-31 | 四川农业大学 | Pine wood nematode SSR label primer is used for the purposes of pine wood nematode detection and the PCR detection method of pine wood nematode |
-
2018
- 2018-01-24 CN CN201810066132.2A patent/CN108260567A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002057440A2 (en) * | 2001-01-18 | 2002-07-25 | Cambria Biosciences, Llc | Screens and assays for agents useful in controlling parasitic nematodes |
| CN102084849A (en) * | 2010-11-19 | 2011-06-08 | 南京林业大学 | Method for preparing bacteria-free pine wood nematodes |
| CN103004592A (en) * | 2012-12-10 | 2013-04-03 | 华南农业大学 | Method for breeding, culturing and preserving paratylenchus by using carrot callus |
| CN105613425A (en) * | 2014-10-27 | 2016-06-01 | 申茂军 | Pine wood nematode rapid preparation method |
| CN106755554A (en) * | 2017-03-21 | 2017-05-31 | 四川农业大学 | Pine wood nematode SSR label primer is used for the purposes of pine wood nematode detection and the PCR detection method of pine wood nematode |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112237169A (en) * | 2020-10-23 | 2021-01-19 | 河北省农林科学院植物保护研究所 | Method for culturing and separating pratylenchus praecox |
| CN112237169B (en) * | 2020-10-23 | 2022-03-08 | 河北省农林科学院植物保护研究所 | Method for culturing and separating pratylenchus praecox |
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Application publication date: 20180710 |