CN105532583B - A kind of living body propagation method of entomopathogenic nematode - Google Patents
A kind of living body propagation method of entomopathogenic nematode Download PDFInfo
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- CN105532583B CN105532583B CN201511009910.7A CN201511009910A CN105532583B CN 105532583 B CN105532583 B CN 105532583B CN 201511009910 A CN201511009910 A CN 201511009910A CN 105532583 B CN105532583 B CN 105532583B
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- 241000244206 Nematoda Species 0.000 title claims abstract description 92
- 230000000967 entomopathogenic effect Effects 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 29
- 241000256247 Spodoptera exigua Species 0.000 claims abstract description 35
- 239000007864 aqueous solution Substances 0.000 claims abstract description 24
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 241000238631 Hexapoda Species 0.000 claims abstract description 15
- 239000008223 sterile water Substances 0.000 claims abstract description 6
- 238000011534 incubation Methods 0.000 claims abstract description 5
- 235000003642 hunger Nutrition 0.000 claims abstract description 3
- 230000037351 starvation Effects 0.000 claims abstract description 3
- 241001480223 Steinernema carpocapsae Species 0.000 claims description 7
- 229920003023 plastic Polymers 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 5
- 241000500097 Heterorhabditis indica Species 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 3
- 241000244173 Rhabditis Species 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 19
- 230000001488 breeding effect Effects 0.000 abstract description 19
- 238000009736 wetting Methods 0.000 abstract description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 241000255896 Galleria mellonella Species 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- 235000016068 Berberis vulgaris Nutrition 0.000 description 3
- 241000335053 Beta vulgaris Species 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 3
- 230000003071 parasitic effect Effects 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007918 pathogenicity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241001198505 Anarsia lineatella Species 0.000 description 1
- 241001481710 Cerambycidae Species 0.000 description 1
- 241000289763 Dasygaster padockina Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001523412 Heterorhabditis bacteriophora Species 0.000 description 1
- 241001414987 Strepsiptera Species 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 230000005110 hydrotaxis Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000001418 larval effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000027272 reproductive process Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000002689 soil Substances 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
Landscapes
- 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
The present invention relates to a kind of living body propagation method of entomopathogenic nematode, it comprises the following steps：Culture vessel is taken, sponge block is put into the culture vessel, sterilized water is added dropwise to sponge block complete wetting to sponge block；The appropriate entomopathogenic nematode aqueous solution is dropped in 12~24h of the starvation high instar larvae body surface of beet armyworm, the high instar larvae of the beet armyworm is placed on sponge block after 1~3min；Culture vessel is positioned over it is incubated in 22~25 DEG C of incubators, in incubation keep sponge block be continuously in complete moisture state；After culture 10~15 days, pathogenic nematode is distributed in sponge block, required entomopathogenic nematode is collected with sterile water wash sponge block.Present invention breeding equipment is simple, it is low to reduce living body propagation cost, it is simple to operate, host insect, source is wide, convenient collection, using sponge block as breeding carrier, the expelling water of nematode is reused, pathogenic nematode effectively infecting to the high instar larvae of beet armyworm is ensure that, is further ensured that the yield of pathogenic nematode.
Description
Technical field
The present invention relates to the breeding of entomopathogenic nematode, and in particular to a kind of living body propagation method of entomopathogenic nematode.
Background technology
Entomopathogenic nematode is a kind of nematode of special parasitic pest, it has, and host range is wide, can actively find host,
It is nontoxic to people and animals and Environmental security and can artificial mass propgation the advantages that, can effectively prevent and treat agricultural, the soil of gardening plant is dwelt and
Borer pest, such as small heart-eating peach worm, various grubs, longicorn, cutworm, therefore high potential biology is referred to as by domestic and international expert
Prevent and treat weapon.
Entomopathogenic nematode breeding is broadly divided into living body propagation and the breeding of in vitro synthetic medium, wherein in vitro artificial culture
Base breeding is divided into solid culture and Liquid Culture again, and two methods are required to plurality of raw materials and large-scale instrument and equipment, and investment is high, operation
Step is more, production cycle length, and living body propagation method is easy, instrument equipment is seldom, with short production cycle, and the nematode of breeding causes
Sick power is higher than the nematode of in vitro synthetic medium breeding.
Propagation method main at present has White trap methods, Lowtek methods, liquid culture method (TSYS) and solid training
The method of supporting etc., wherein White trap methods and Lowtek methods typically use greater wax moth as host insect, pathogenic nematode intrusion insect bodies
It is interior, using internal organic matter as application, the method that pathogenic nematode is bred using nematode hydrotaxis, it is primarily present problems with：(1) with
Greater wax moth is host insect, and field is not easy to gather greater wax moth, and host insect source is not wide；(2) collection method complex steps, it is middle
Multi-pass operation is needed, is wasted time and energy；(3) parasitic insect can not be effectively ensured effectively to have been infected before breeding vessel are put into, from
And effective breeding in later stage can not be ensured.Liquid culture method and plate method are to carry out pathogenic nematode using synthetic medium
Breeding, be primarily present problems with：(1) need to use animal's liver, cost is higher；(2) continuously after culture several generations, cause of disease line
Infect activity and the adaptability of worm decline；(3) symbiotic bacteria of culture pathogenic nematode is needed, can not be by it in reproductive process
He infects miscellaneous bacteria, and operation requires high.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of insect easy to operate, yield is high for prior art
The living body propagation method of pathogenic nematode.
Technical scheme is used by the present invention solves above-mentioned technical problem：A kind of living body propagation side of entomopathogenic nematode
Method, it is characterised in that comprise the following steps：
(1) culture vessel is taken, sponge block is put into the culture vessel, it is complete to sponge block that sterilized water is added dropwise to sponge block
Wetting；
(2) the entomopathogenic nematode aqueous solution is dropped in 12~24h of the starvation high instar larvae body surface of beet armyworm, 1~3min
The high instar larvae of the beet armyworm is placed on sponge block afterwards；Nature enemy wherein is carried out to the high instar larvae of beet armyworm, can be effective
The manure contamination culture vessel of the high instar larvae of beet armyworm is avoided, facilitates the collection of entomopathogenic nematode, while ensures to breed
Entomopathogenic nematode quality.
(3) culture vessel handled through the step (2) is positioned over incubated in 22~25 DEG C of incubators, cultivated
Sponge block is kept to be in complete moisture state in journey；
(4) after cultivating 10~15 days, pathogenic nematode is distributed with sponge block, is collected with sterile water wash sponge block required
Entomopathogenic nematode.
Preferably, the entomopathogenic nematode aqueous solution contains 4500~5000 entomiasises for every milliliter in above-mentioned steps (2)
The suspension of former nematode, and every high instar larvae of 1g beet armyworms matches 1/5~1/3 milliliter of the entomopathogenic nematode aqueous solution.It is high
The entomopathogenic nematode of density is advantageous to obtain the maximum number of infection period nematode within the most short time, but too high density meeting
The breeding rate of nematode is reduced, therefore the entomopathogenic nematode aqueous solution of above-mentioned concentration is selected in the application.
Further, the entomopathogenic nematode is Steinernema Carpocapsae or heterorhabditis indica, and when entomopathogenic nematode is Si Shi
During nematode, 1/5~1/3 milliliter of the entomopathogenic nematode aqueous solution is matched per the high instar larvae of 1g beet armyworms；When Insect Pathogenic line
When worm is heterorhabditis indica, 1/5~1/4 milliliter of the entomopathogenic nematode aqueous solution is matched per the high instar larvae of 1g beet armyworms.
Further to increase the yield of entomopathogenic nematode, preferably, the sponge block laterally fills up culture vessel bottom
Face.
Further, the culture vessel is any of transparent vial, glass case, plastic bottle or plastic casing.
Further, the culture vessel is circular discs, and its a diameter of 12~18cm, is highly 2.2~3cm, corresponding
Ground, a diameter of 12~18cm of the sponge block, it is highly 1~2cm.
Compared with prior art, the advantage of the invention is that：
(1) breed that equipment is simple, and it is low to reduce living body propagation cost, simple to operate, without repeatedly collecting nematode, once grasp
Work；
(2) host insect being used as using beet armyworm, source is wide, convenient to gather, and the high instar larvae activity of beet armyworm is good,
Entomopathogenic nematode can be made to obtain higher yield；
(3) using keeping the sponge block of moisture state to be used as breeding carrier, the expelling water of nematode is reused, ensure that disease
Former nematode is effectively infected to the high instar larvae of beet armyworm, is further ensured that the yield of pathogenic nematode.
Using the propagation method in the present invention, pathogenic nematode yield is high and can guarantee that the pathogenic nematode of breeding to host insect
The higher pathogenicity of holding.
Embodiment
The present invention is described in further detail with reference to embodiments.
Embodiment 1：
Culture vessel：Transparent plastic preserving box, including discoid box body and lid, the internal diameter of box body is 15cm, high
Spend for 2.2cm；
For trying insect：The high instar larvae of beet armyworm, pick up from the big Ao vegetable fields of Ningbo Heng Xi, indoor feeding to advanced age.
For trying nematode：Steinernema Carpocapsae (Steinernema carpocapsae, is purchased from the limited public affairs of Jiyuan, Henan white clouds industry
Department)
Experiment process：Take a diameter of 15cm, be highly placed in box body for 1.5cm sponge block (original fresh sponge)
In, and sponge block is laterally paved with box bottom, sterilized water is added dropwise to sponge block complete wetting to sponge block, has on box bottom
Little water.
Above-mentioned nematode is configured to the entomopathogenic nematode aqueous solution with sterilized water, the entomopathogenic nematode aqueous solution is per milli
The suspension containing 5000 entomopathogenic nematodes is risen, takes the appropriate entomopathogenic nematode aqueous solution to drop in hungry 24h beet night
The high instar larvae body surface of moth, average every high instar larvae of 1 gram of beet armyworm match 1/5 milliliter of entomopathogenic nematode aqueous solution, after 1min
The high instar larvae of beet armyworm is positioned over sponge block top.
Cover lid and keep certain aeration, the crisper equipped with the high instar larvae of beet armyworm is positioned over 25 DEG C of trainings
Incubated in foster case, addition sterilized water keeps sponge block to be in complete moisture state in good time in incubation.After culture 10 days,
The high instar larvae of beet armyworm is substantially dead, pathogenic nematode in sponge block be present, is collected required with sterile water wash sponge block
Entomopathogenic nematode, Breeding results are as shown in table 1.
Embodiment 2：
Culture vessel：Transparent plastic preserving box, including discoid box body and lid, the internal diameter of box body is 12cm, high
Spend for 3cm；
For trying insect：The high instar larvae of beet armyworm, from the big Ao vegetable fields of Ningbo Heng Xi, indoor feeding to advanced age.
For trying nematode：Heterorhabditis indica (Heterorhabditis bacteriophora, is purchased from Jiyuan, Henan white clouds reality
Industry Co., Ltd).
Experiment process：Take a diameter of 12cm, be highly placed in box body for 2cm sponge block (original fresh sponge)
In, and sponge block is laterally paved with box bottom, sterilized water is added dropwise to sponge block complete wetting to sponge block.
Above-mentioned nematode is configured to the entomopathogenic nematode aqueous solution with sterilized water, the entomopathogenic nematode aqueous solution is per milli
The suspension containing 4500 entomopathogenic nematodes is risen, takes the appropriate entomopathogenic nematode aqueous solution to drop in hungry 24h beet night
The high instar larvae body surface of moth, average every high instar larvae of 1 gram of beet armyworm match 1/4 milliliter of entomopathogenic nematode aqueous solution, after 2min
The high instar larvae of beet armyworm is equably positioned over above sponge block.
Cover lid and keep certain aeration, the crisper equipped with the high instar larvae of beet armyworm is positioned over 25 DEG C of trainings
It is incubated in foster case, keep sponge block to be in complete moisture state in incubation.After culture 10 days, beet armyworm advanced age children
Worm is substantially dead, pathogenic nematode in sponge block be present, and required entomopathogenic nematode is collected with sterile water wash sponge block, numerous
It is as shown in table 1 to grow result.
Embodiment 3：
Culture vessel：Transparent plastic preserving box, including discoid box body and lid, the internal diameter of box body is 18cm, high
Spend for 2.2cm；
For trying insect：The high instar larvae of beet armyworm, from the big Ao vegetable fields of Ningbo Heng Xi, indoor feeding to different larval instar.
For trying nematode：Steinernema Carpocapsae (Steinernema carpocapsae, is purchased from the limited public affairs of Jiyuan, Henan white clouds industry
Department).
Experiment process：Take a diameter of 18cm, be highly placed in box body for 1cm sponge block (original fresh sponge)
In, and sponge block is laterally paved with box bottom, sterilized water is added dropwise to sponge block complete wetting to sponge block.
Above-mentioned nematode is configured to the entomopathogenic nematode aqueous solution with sterilized water, the entomopathogenic nematode aqueous solution is per milli
The suspension containing 5000 entomopathogenic nematodes is risen, takes the appropriate entomopathogenic nematode aqueous solution to drop in hungry 12h beet night
The high instar larvae body surface of moth, average every high instar larvae of 1 gram of beet armyworm match 1/3 milliliter of entomopathogenic nematode aqueous solution, after 3min
Beet armyworm high instar larvae is equably positioned over above sponge block.
Cover lid and keep certain aeration, the crisper equipped with the high instar larvae of beet armyworm is positioned over 25 DEG C of trainings
It is incubated in foster case, keep sponge block to be in complete moisture state in incubation.After culture 15 days, beet armyworm advanced age children
Worm is substantially dead, pathogenic nematode in sponge block be present, and required entomopathogenic nematode is collected with sterile water wash sponge block, numerous
It is as shown in table 1 to grow result.
Comparative example 1：As different from Example 1, Ningbo Heng Xi (5 instar larvaes, it is big to be picked up from using greater wax moth in comparative example 1
Field) host insect is used as, other experimental conditions are same with embodiment 1, and Breeding results are as shown in table 1.
Comparative example 2：As different from Example 1, the sponge block in embodiment 1, filter paper are replaced using filter paper in comparative example 2
A diameter of 15cm, be highly 1cm, be laterally paved with box bottom, other experimental conditions and embodiment 1 are same, Breeding results such as table 1
It is shown.
1 each embodiment of table, the Breeding results of comparative example
From table 1, using the propagation method in the present invention, pathogenic nematode (is invaded effective parasitic rate of host insect
Dye rate) significantly improve, go out the shortening of nematode time, the yield of pathogenic nematode dramatically increases.
The pathogenic nematode bred in embodiment 1~3 is infected into the high instar larvae 5min of beet armyworm, the pathogenic nematode pair of breeding
The parasitic rate of the high instar larvae of beet armyworm reaches more than 85%, has very strong pathogenicity.
Claims (5)
1. a kind of living body propagation method of entomopathogenic nematode, it is characterised in that comprise the following steps：
(1) culture vessel is taken, sponge block is put into the culture vessel, sterilized water is added dropwise to sponge block moistens completely to sponge block
It is wet；
(2) the appropriate entomopathogenic nematode aqueous solution is dropped in 12~24h of the starvation high instar larvae body surface of beet armyworm, 1~3min
The high instar larvae of the beet armyworm is placed on sponge block afterwards, wherein, the above-mentioned entomopathogenic nematode aqueous solution is every milliliter and contains 4500
The suspension of~5000 entomopathogenic nematodes, and every high instar larvae of 1g beet armyworms matches 1/5~1/3 milliliter of Insect Pathogenic
The nematode aqueous solution；
(3) culture vessel handled through the step (2) is positioned over it is incubated in 22~25 DEG C of incubators, in incubation
Sponge block is kept to be continuously in complete moisture state；
(4) after cultivating 10~15 days, pathogenic nematode is distributed with sponge block, required elder brother is collected with sterile water wash sponge block
Worm pathogenic nematode.
2. living body propagation method as claimed in claim 1, it is characterised in that the entomopathogenic nematode is Steinernema Carpocapsae or different
Rhabditis axei, and when entomopathogenic nematode is Steinernema Carpocapsae, 1/5~1/3 milliliter is matched per the high instar larvae of 1g beet armyworms
The entomopathogenic nematode aqueous solution；When entomopathogenic nematode is heterorhabditis indica, every high instar larvae proportioning 1/5 of 1g beet armyworms~
1/4 milliliter of the entomopathogenic nematode aqueous solution.
3. living body propagation method as claimed in claim 1 or 2, it is characterised in that the sponge block laterally fills up culture vessel
Bottom surface.
4. living body propagation method as claimed in claim 3, it is characterised in that the culture vessel is transparent vial, glass
Any of glass box, plastic bottle, plastic casing.
5. living body propagation method as claimed in claim 4, it is characterised in that the culture vessel is circular box, and its internal diameter
It is highly 2.2~3cm for 12~18cm, a diameter of 12~18cm of the sponge block, is highly 1~2cm accordingly.
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| CN106070091A (en) * | 2016-08-18 | 2016-11-09 | 浙江绿神天敌生物技术有限公司 | A kind of entomopathogenic nematode one-level kind source preparation method |
| CN106035250A (en) * | 2016-08-18 | 2016-10-26 | 浙江绿神天敌生物技术有限公司 | Entomopathogenic nematode culture process |
| CN107027715A (en) * | 2017-05-11 | 2017-08-11 | 中国科学院东北地理与农业生态研究所 | A kind of entomopathogenic nematode living body propagation method |
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