CN105532583A - Living body breeding method for entomopathogenic nematodes - Google Patents
Living body breeding method for entomopathogenic nematodes Download PDFInfo
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- CN105532583A CN105532583A CN201511009910.7A CN201511009910A CN105532583A CN 105532583 A CN105532583 A CN 105532583A CN 201511009910 A CN201511009910 A CN 201511009910A CN 105532583 A CN105532583 A CN 105532583A
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- sponge block
- entomopathogenic
- nematode
- beet armyworm
- living body
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
Abstract
The invention relates to a living body breeding method for entomopathogenic nematodes. The method comprises the following steps that a cultivation container is taken, a sponge block is placed into the cultivation container, and sterile water is added to the sponge block dropwisely till the sponge block is completely wetted; a proper amount of entomopathogenic nematodes water solution is dropped on the surfaces of beet armyworm larvae which are starved for 12-24 h, and the beet armyworm larvae are placed on the sponge block after 1-3 min, the cultivation container is placed into an incubator at the temperature of 22-25 DEG C for constant temperature incubation, and the sponge block is constantly in the completely-wetting state in the cultivation process; after cultivation is performed for 10-15 d, entomopathogenic nematodes are distributed in the sponge block, sterile water is used for cleaning the sponge block and the needed entomopathogenic nematodes are collected. The cultivation device is simple, the living body cultivation cost is lowered, operation is simple, host insects are wide in source and convenient to collect, the sponge block is adopted as a breeding carrier, the water driving performance of the entomopathogenic nematodes is repeatedly utilized, it is ensured that the entomopathogenic nematodes effectively infect the beet armyworm larvae, and the yield of the entomopathogenic nematodes is further ensured.
Description
Technical field
The present invention relates to the breeding of entomopathogenic nematode, be specifically related 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 have host range wide, can initiatively find host, to people and animals and Environmental security nontoxic and can the advantage such as artificial mass propgation, can effectively prevent and treat agricultural, the soil of gardening plant dwells and borer pest, as peach fruit borer, various grub, longicorn, cutworm etc., be therefore called high potential biological control weapon by domestic and international expert.
Entomopathogenic nematode breeding is mainly divided into living body propagation and the breeding of in vitro synthetic medium, wherein in vitro synthetic medium breeding is divided into again solid culture and liquid culture, two methods all need plurality of raw materials and large-scale instrument and equipment, invest high, operating procedure is many, production cycle is long, and living body propagation method is easy, instrument equipment is little, with short production cycle, the nematode that the Pathogenicity of breeding is bred higher than in vitro synthetic medium.
Propagation method main at present has Whitetrap method, Lowtek method, liquid culture method (TSYS) and plate method etc., wherein Whitetrap method and Lowtek method generally adopt greater wax moth to be host insect, pathogenic nematode invades in insect bodies, with organic for application in body, nematode hydrotaxis is utilized to breed the method for pathogenic nematode, mainly there is following problem: (1) is host insect with greater wax moth, and field not easily gathers greater wax moth, host insect source is wideless; (2) collection method complex steps, intermediate demand multi-pass operation, wastes time and energy; (3) effectively cannot ensure that parasitic insect is effectively infected before putting into breeding vessel, thus effective breeding in later stage cannot be ensured.Liquid culture method and plate method are all the breedings utilizing synthetic medium to carry out pathogenic nematode, mainly there is following problem: (1) needs to use animal's liver, and cost is higher; (2), after continuous culture several generations, the active and adaptive capacity of infecting of pathogenic nematode all declines; (3) need the symbiotic bacteria cultivating pathogenic nematode, can not be infected by other miscellaneous bacterias in reproductive process, operation requirements is high.
Summary of the invention
Technical problem to be solved by this invention is a kind of living body propagation method providing easy to operate, entomopathogenic nematode that output is high for prior art.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of living body propagation method of entomopathogenic nematode, is characterized in that comprising the following steps:
(1) get culture vessel, in this culture vessel, put into sponge block, drip sterile water to sponge block complete wetting to sponge block;
(2) the entomopathogenic nematode aqueous solution is dropped in the beet armyworm height instar larvae body surface of hungry 12 ~ 24h, after 1 ~ 3min, this beet armyworm height instar larvae is placed on sponge block; Wherein Nature enemy is carried out to beet armyworm height instar larvae, effectively can avoid the manure contamination culture vessel of beet armyworm height instar larvae, facilitate the collection of entomopathogenic nematode, ensure the quality of the entomopathogenic nematode of breeding simultaneously.
(3) culture vessel processed through described step (2) is positioned over constant temperature culture in 22 ~ 25 DEG C of incubators, in incubation, keeps sponge block to be in complete moisture state;
(4) cultivate after 10 ~ 15 days, be distributed with pathogenic nematode in sponge block, collect required entomopathogenic nematode with sterile water wash sponge block.
As preferably, in above-mentioned steps (2), the entomopathogenic nematode aqueous solution is every milliliter of suspension containing 4500 ~ 5000 entomopathogenic nematodes, and the entomopathogenic nematode aqueous solution of every 1g beet armyworm height instar larvae proportioning 1/5 ~ 1/3 milliliter.Highdensity entomopathogenic nematode is conducive to the infection period nematode obtaining maximum number within the shortest time, but too high density can reduce the breeding rate of nematode, therefore selects the entomopathogenic nematode aqueous solution of above-mentioned concentration in the application.
Further, described entomopathogenic nematode is Steinernema Carpocapsae or heterorhabditis indica, and when entomopathogenic nematode is Steinernema Carpocapsae, the entomopathogenic nematode aqueous solution of every 1g beet armyworm height instar larvae proportioning 1/5 ~ 1/3 milliliter; When entomopathogenic nematode is heterorhabditis indica, the entomopathogenic nematode aqueous solution of every 1g beet armyworm height instar larvae proportioning 1/5 ~ 1/4 milliliter.
For increasing the output of entomopathogenic nematode further, as preferably, described sponge block laterally fills up culture vessel bottom surface.
Further, described culture vessel is any one in transparent vial, glass case, plastic bottle or plastic casing.
Further again, described culture vessel is circular discs, and its diameter is 12 ~ 18cm, is highly 2.2 ~ 3cm, and accordingly, the diameter of described sponge block is 12 ~ 18cm, is highly 1 ~ 2cm.
Compared with prior art, the invention has the advantages that:
(1) equipment of breeding is simple, reduces living body propagation cost low, simple to operate, without the need to repeatedly collecting nematode, and once-through operation;
(2) adopt beet armyworm as host insect, source is wide, convenient collection, and beet armyworm height instar larvae activity is good, and entomopathogenic nematode can be made to obtain higher output;
(3) adopt the sponge block of maintenance moisture state as breeding carrier, the expelling water of recycling nematode, ensure that pathogenic nematode effectively infecting beet armyworm height instar larvae, ensures the output of pathogenic nematode further.
Utilize the propagation method in the present invention, the pathogenicity that the high maintenance of pathogenic nematode to host insect that also can ensure to breed of pathogenic nematode output is higher.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1:
Culture vessel: transparent plastic preserving box, comprises discoid box body and lid, and the internal diameter of box body is 15cm, is highly 2.2cm;
For examination insect: beet armyworm height instar larvae, pick up from the large Ao vegetable fields of Ningbo Heng Xi, indoor feeding is to advanced age.
For examination nematode: Steinernema Carpocapsae (Steinernemacarpocapsae is purchased from Jiyuan, Henan white clouds Industrial Co., Ltd.)
Process of the test: cut-off footpath is 15cm, highly for the sponge block (original fresh sponge) of 1.5cm is placed in box body, and make sponge block laterally be paved with box bottom, drip sterile water to sponge block complete wetting to sponge block, box bottom has little water.
Above-mentioned nematode sterile water is mixed with the entomopathogenic nematode aqueous solution, this entomopathogenic nematode aqueous solution is every milliliter of suspension containing 5000 entomopathogenic nematodes, get the beet armyworm height instar larvae body surface that the appropriate entomopathogenic nematode aqueous solution drops in hungry 24h, on average every 1 gram of beet armyworm height instar larvae proportioning 1/5 milliliter of entomopathogenic nematode aqueous solution, is positioned over sponge block top by beet armyworm height instar larvae after 1min.
Cover lid and keep certain air capacity of soils, the crisper that beet armyworm height instar larvae is housed is positioned over constant temperature culture in 25 DEG C of incubators, adding sterile water in incubation in good time and keep sponge block to be in complete moisture state.Cultivate after 10 days, beet armyworm height instar larvae is substantially dead, there is pathogenic nematode in sponge block, and collect required entomopathogenic nematode with sterile water wash sponge block, Breeding results is as shown in table 1.
Embodiment 2:
Culture vessel: transparent plastic preserving box, comprises discoid box body and lid, and the internal diameter of box body is 12cm, is highly 3cm;
For examination insect: beet armyworm height instar larvae, from the large Ao vegetable fields of Ningbo Heng Xi, indoor feeding is to advanced age.
For examination nematode: heterorhabditis indica (Heterorhabditisbacteriophora is purchased from Jiyuan, Henan white clouds Industrial Co., Ltd.).
Process of the test: cut-off footpath is 12cm, highly for the sponge block (original fresh sponge) of 2cm is placed in box body, and make sponge block laterally be paved with box bottom, drip sterile water to sponge block complete wetting to sponge block.
Above-mentioned nematode sterile water is mixed with the entomopathogenic nematode aqueous solution, this entomopathogenic nematode aqueous solution is every milliliter of suspension containing 4500 entomopathogenic nematodes, get the beet armyworm height instar larvae body surface that the appropriate entomopathogenic nematode aqueous solution drops in hungry 24h, on average every 1 gram of beet armyworm height instar larvae proportioning 1/4 milliliter of entomopathogenic nematode aqueous solution, is positioned over above sponge block equably by beet armyworm height instar larvae after 2min.
Cover lid and keep certain air capacity of soils, the crisper that beet armyworm height instar larvae is housed is positioned over constant temperature culture in 25 DEG C of incubators, in incubation, keeping sponge block to be in complete moisture state.Cultivate after 10 days, beet armyworm height instar larvae is substantially dead, there is pathogenic nematode in sponge block, and collect required entomopathogenic nematode with sterile water wash sponge block, Breeding results is as shown in table 1.
Embodiment 3:
Culture vessel: transparent plastic preserving box, comprises discoid box body and lid, and the internal diameter of box body is 18cm, is highly 2.2cm;
For examination insect: beet armyworm height instar larvae, from the large Ao vegetable fields of Ningbo Heng Xi, indoor feeding is to different larval instar.
For examination nematode: Steinernema Carpocapsae (Steinernemacarpocapsae is purchased from Jiyuan, Henan white clouds Industrial Co., Ltd.).
Process of the test: cut-off footpath is 18cm, highly for the sponge block (original fresh sponge) of 1cm is placed in box body, and make sponge block laterally be paved with box bottom, drip sterile water to sponge block complete wetting to sponge block.
Above-mentioned nematode sterile water is mixed with the entomopathogenic nematode aqueous solution, this entomopathogenic nematode aqueous solution is every milliliter of suspension containing 5000 entomopathogenic nematodes, get the beet armyworm height instar larvae body surface that the appropriate entomopathogenic nematode aqueous solution drops in hungry 12h, on average every 1 gram of beet armyworm height instar larvae proportioning 1/3 milliliter of entomopathogenic nematode aqueous solution, by beet armyworm height instar larvae after 3min, be positioned over equably above sponge block.
Cover lid and keep certain air capacity of soils, the crisper that beet armyworm height instar larvae is housed is positioned over constant temperature culture in 25 DEG C of incubators, in incubation, keeping sponge block to be in complete moisture state.Cultivate after 15 days, beet armyworm height instar larvae is substantially dead, there is pathogenic nematode in sponge block, and collect required entomopathogenic nematode with sterile water wash sponge block, Breeding results is as shown in table 1.
Comparative example 1: as different from Example 1, adopts greater wax moth (5 instar larvaes pick up from Heng Xi land for growing field crops, Ningbo) as host insect in comparative example 1, together, Breeding results is as shown in table 1 for other experimental conditions and embodiment 1.
Comparative example 2: as different from Example 1, adopts the sponge block that filter paper replaces in embodiment 1 in comparative example 2, the diameter of filter paper is 15cm, highly is 1cm, and be laterally paved with box bottom, together, Breeding results is as shown in table 1 for other experimental conditions and embodiment 1.
The Breeding results of each embodiment of table 1, comparative example
From table 1, the propagation method in application the present invention, the effective parasitic rate (i.e. infection rate) of pathogenic nematode to host insect significantly improves, and go out nematode time shorten, the output of pathogenic nematode significantly increases.
The pathogenic nematode of breeding in embodiment 1 ~ 3 is infected beet armyworm height instar larvae 5min, and the parasitic rate of pathogenic nematode to beet armyworm height instar larvae of breeding reaches more than 85%, has very strong pathogenicity.
Claims (6)
1. a living body propagation method for entomopathogenic nematode, is characterized in that comprising the following steps:
(1) get culture vessel, in this culture vessel, put into sponge block, drip sterile water to sponge block complete wetting to sponge block;
(2) the appropriate entomopathogenic nematode aqueous solution is dropped in the beet armyworm height instar larvae body surface of hungry 12 ~ 24h, after 1 ~ 3min, this beet armyworm height instar larvae is placed on sponge block;
(3) culture vessel processed through described step (2) is positioned over constant temperature culture in 22 ~ 25 DEG C of incubators, in incubation, keeps sponge block to continue to be in complete moisture state;
(4) cultivate after 10 ~ 15 days, be distributed with pathogenic nematode in sponge block, collect required entomopathogenic nematode with sterile water wash sponge block.
2. living body propagation method as claimed in claim 1, it is characterized in that, in described step (2), the entomopathogenic nematode aqueous solution is every milliliter of suspension containing 4500 ~ 5000 entomopathogenic nematodes, and the entomopathogenic nematode aqueous solution of every 1g beet armyworm height instar larvae proportioning 1/5 ~ 1/3 milliliter.
3. living body propagation method as claimed in claim 2, it is characterized in that, described entomopathogenic nematode is Steinernema Carpocapsae or heterorhabditis indica, and when entomopathogenic nematode is Steinernema Carpocapsae, the entomopathogenic nematode aqueous solution of every 1g beet armyworm height instar larvae proportioning 1/5 ~ 1/3 milliliter; When entomopathogenic nematode is heterorhabditis indica, the entomopathogenic nematode aqueous solution of every 1g beet armyworm height instar larvae proportioning 1/5 ~ 1/4 milliliter.
4. the living body propagation method as described in claim 1 or 2 or 3, is characterized in that, described sponge block laterally fills up culture vessel bottom surface.
5. living body propagation method as claimed in claim 4, is characterized in that, described culture vessel is any one in transparent vial, glass case, plastic bottle, plastic casing.
6. living body propagation method as claimed in claim 5, it is characterized in that, described culture vessel is circular box, and its internal diameter is 12 ~ 18cm, is highly 2.2 ~ 3cm, and accordingly, the diameter of described sponge block is 12 ~ 18cm, is highly 1 ~ 2cm.
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Cited By (3)
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| 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 |
| 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 |
| 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
| 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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