CN109526884A - A method of culture Steinernema carpocapsae nematode - Google Patents
A method of culture Steinernema carpocapsae nematode Download PDFInfo
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- CN109526884A CN109526884A CN201811533466.2A CN201811533466A CN109526884A CN 109526884 A CN109526884 A CN 109526884A CN 201811533466 A CN201811533466 A CN 201811533466A CN 109526884 A CN109526884 A CN 109526884A
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- nematode
- carpocapsae
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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 present invention relates to a kind of culturesSteinernema carpocapsaeThe method of nematode, belongs to entomopathogenic nematode culture technique field, and the present invention willS.carpocapsaeInfective stage larva is inoculated into artificial eggs liquid, and for infective stage larva restoration ecosystem at adult, adult cherishes ovum, and egg hatching passes through second instar larvae, third-instar larvae, four-age larva at first-instar young, regrowth, and then regrowth obtains second generation adult.The adult of the second generation can continue to cultivate, and loop back and forth like this, and complete the alternation of generations.Pass through this method cultureS.carpocapsaeRestore and development is very fast, 2 d are observed that adult cherishes ovum, and small worm incubation can be observed in 3 d, and 6-7d is observed that 2nd generation adult cherishes ovum.Culture of the present inventionS.carpocapsaeThe method of nematode can not only makeS.carpocapsaeNematode completes the alternation of generations, recovery infective stage larva that can be faster more efficient, and its speed of growth is very fast, is commercialized for the quick of nematode, high-efficient culture and provides condition.
Description
Technical field
The invention belongs to entomopathogenic nematode culture technique fields, specifically, are related to a kind of culture S.carpocapsae
The method of nematode.
Background technique
It is novel in the world that Insect Pathogenic Si Shi, which belongs to Steinernema and different small bar category Heterorhabditis nematode,
Efficient biological insecticide (Georgis et al., 2006), is just used for the prevention and treatment of pest since the twentieth century thirties
(Smart, 1995) has killing ability strong, and insecticidal spectrum is wide, can actively search for host, the advantages that people and animals, Environmental security, easily
In mass propgation and product is prepared, and can be mixed with a variety of chemical pesticides, has special efficacy (Kaya and to brill moth and subterranean pest-insect
Gaugler 1993；Grewal et al., 2005；Georgis et al., 2006；Yan et al., 2013；Face Xun etc.,
2014)。
The history of life of entomopathogenic nematode originates in infective stage larva (Infective juveniles, IJ) and carries symbiosis
Bacterium enters in host insect body, and infective stage larva avoids the immune response of insect, and symbiotic bacteria is discharged in insect hemolymph
(Dowds and Peters, 2002).Symbiotic bacteria is proliferated in insect hemolymph, kills host insect, nematode trophobiosis
Bacterium is to breed.When insect endotrophic exhausts, 3 ages tolerance state infective stage larva is formed, and temperature wet enough in external environment
When degree is suitable for, infective stage larva carries symbiotic bacteria and leaves host insect (Brown and Gaugler, 1997), searches new
Host.Infective stage larva is the age that uniquely can freely survive in the entomopathogenic nematode history of life, and common sprayer can be used
It is administered with pest control.Infective stage larva can survive 1 to multiple moons, can carry out industrialization culture (Wright et al.,
2005)。
The industrialization culture of entomopathogenic nematode is the basis of entomopathogenic nematode popularization and commercial applications.Insect Pathogenic
Nematode can carry out In vivo culture by host insect, by solid medium or can also carry out liquid with fermentor in vitro
Culture.The in vitro culture of entomopathogenic nematode needs the participation of symbiotic bacteria, and symbiotic bacteria can be by the nutriment in culture medium
Ingredient (Dowds and Peters, 2002) needed for being converted to entomopathogenic nematode growth and development breeding.Carry out Insect Pathogenic
When the industrialization culture of nematode, Liquid Culture is most worthwhile, but solid culture and In vivo culture no less important, solid culture
It is more applicable in the countries and regions of cheap labor, because required early investment is few；In vivo culture is then suitable for testing
Indoor preserving seed and a small amount of experiments.
Summary of the invention
In order to solve the problems, such as background technique, the present invention provides a kind of culture S.carpocapsae nematodes
Method can not only make S.carpocapsae nematode complete the alternation of generations, recovery infective stage larva that can be faster more efficient,
And its speed of growth is very fast, provides condition for the quick of nematode, high-efficient culture commercialization.
To achieve the above object, the present invention is achieved through the following technical solutions:
The method of the culture Steinernema carpocapsae nematode, specifically includes the following steps:
1) tussah chrysalis hemolymph, trehalose, egg yolk, skimmed milk power, N ' s salting liquid and water deployed, mixed, obtained
To artificial eggs liquid；
2) S.carpocapsae infective stage larva is inoculated into artificial eggs liquid, infective stage larva restoration ecosystem at adult,
Adult cherishes ovum, and egg hatching passes through second instar larvae, third-instar larvae, four-age larva at first-instar young, regrowth, and then regrowth obtains
Second generation adult.
Preferably, the every 9ml of artificial eggs liquid prepares acquisition by the following method: by tussah chrysalis hemolymph 3ml, anhydrous seaweed
Sugared 0.1g, egg yolk 2.5ml, skimmed milk power 0.1g, N ' s salting liquid 1ml and sterile water 2.5ml are uniformly mixed.
Preferably, the formula of every liter of N ' s salting liquid is as follows: NaCl 7.5g/ liter, KCl 0.1g/ liter, CaCl2 0.2g/
It rises, NaHCO30.2g/ liter, surplus are water.
Preferably, S.carpocapsae infective stage larva is S.carpocapsae All infective stage larva.
The adult of the second generation can continue to cultivate, and loop back and forth like this, and complete the alternation of generations.
Beneficial effects of the present invention:
The present invention is using artificial eggs liquid as Incubating Solution culture S.carpocapsae infective stage larva, and development is very fast, 3d
It can be observed that adult cherishes ovum.The method of culture S.carpocapsae nematode of the invention can not only make S.carpocapsae line
Worm completes the alternation of generations, and compared to tussah chrysalis hemolymph, development that can be faster more efficient is nematode to bosom ovum adult stage
Rapidly and efficiently culture provide condition.
Detailed description of the invention
Fig. 1 is nematode recovery situation comparison diagram in embodiment 1 and comparative example 1-2.
Specific embodiment
It, below will be to preferred reality of the invention in order to keep the purpose of the present invention, technical scheme and beneficial effects clearer
It applies example to be described in detail, to facilitate the technical staff to understand.
Embodiment 1
1) entomopathogenic nematode: S.carpocapsae All collects infective stage larva from greater wax moth worm corpse
(Infective juveniles, IJ), filtering remove dead worm, and adjustment nematode concentration is 1000IJ/ml, and 15 DEG C, 100rpm is saved
It is spare.At room temperature nematode concentration is concentrated before measurement at 25 DEG C or so as 20000IJ/ml.
2) preparation of artificial eggs liquid: by tussah chrysalis hemolymph 3ml, anhydrous trehalose 0.1g, egg yolk 2.5ml, defatted milk
Powder 0.1g, N ' s salting liquid 1ml and sterile water 2.5ml are uniformly mixed, -20 DEG C save backup (N ' s salting liquid: by NaCl 7.5g,
KCl 0.1g, CaCl20.2g, NaHCO30.2g is added to 1000ml H2In O, 121 DEG C, 105KPa high pressure sterilization 30min, room
Temperature saves backup)；
3) 4 μ l Incubating Solutions are added in each hole of 384 orifice plates, the nematode that 2 μ l concentration are 20000IJ/ml is added in every hole
Liquid (contains about 40 infection phase nematodes), carries out renewal cultivation.
Comparative example 1
Step is same as Example 1, the difference is that Incubating Solution is tussah chrysalis hemolymph.
The preparation of tussah chrysalis hemolymph: cutting off cocoon, takes out tussah chrysalis, 65 DEG C of water-bath 10min, and 75% alcohol rub pupa table disappears
After poison, pupa tail portion is cut off in superclean bench, is squeezed out and is collected hemolymph, -20 DEG C save backup；
Comparative example 2
Step is same as Example 1, the difference is that Incubating Solution is tussah chrysalis hemolymph water mixed liquid.
The preparation of tussah chrysalis hemolymph water mixed liquid: tussah chrysalis hemolymph 3ml and sterile water 6ml is mixed, ready-to-use.
Comparative example 3
Step is same as Example 1, the difference is that Incubating Solution is basic liquid.
The preparation of basal liquid: by anhydrous trehalose 0.1g, egg yolk 2.5ml, skimmed milk power 0.1g, N ' s salting liquid 1ml and
Sterile water 5.5ml is uniformly mixed, and -20 DEG C save backup (N ' s salting liquid: by NaCl 7.5g, KCl 0.1g, CaCl20.2g,
NaHCO30.2g is added to 1000ml H2In O, 121 DEG C, 105KPa high pressure sterilization 30min, room temperature preservation are spare).
Experimental analysis
6 holes of each sample time sample, continuous sampling 7 days, 42 holes of each processing of every kind of nematode daily.In tally
Interior addition water, the nematode in each hole is drawn in each hole of tally, microscopy, records dead nematode, survival in each hole
The total quantity of nematode, while recording the nematode population of recovery.
S.carpocapsae All does not restore agensis in basal liquid.S.carpocapsae All infective stage larva exists
Recovery developmental state in 3 kinds of Incubating Solutions (artificial eggs liquid, tussah chrysalis hemolymph, tussah chrysalis hemolymph water mixed liquid) see Fig. 1 and
Table 1.
The time that table 1.Steinernema carpocapsae All nematode restores and developed in 3 kinds of Incubating Solutions
S.carpocapsae All nematode 1d in artificial eggs liquid can restore, and 3d is observed that adult cherishes ovum.
S.carpocapsae goes out small worm and row in artificial eggs liquid and is slightly longer than at the time needed for the ovum adult of 2nd generation bosom in tussah chrysalis blood
Time in lymph and tussah chrysalis hemolymph water mixed liquid.
Method of the invention only needs a small amount of tussah chrysalis hemolymph to match with other substances, compared to tussah chrysalis blood strangury
Bar, development that can be faster more efficient to bosom ovum adult stage, and tests prove that, artificial eggs liquid of the invention is -20
DEG C save one month, effect is good as the effect just prepared.
Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention rather than limits, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (5)
1. a kind of cultureSteinernema carpocapsaeThe method of nematode, it is characterised in that: specifically includes the following steps:
1) tussah chrysalis hemolymph, trehalose, egg yolk, skimmed milk power, N ' s salting liquid and water deployed, mixed, obtain people
Work ovum liquid；
2) willS. carpocapsaeInfective stage larva is inoculated into artificial eggs liquid, infective stage larva restoration ecosystem at adult, at
Worm cherishes ovum, and egg hatching passes through second instar larvae, third-instar larvae, four-age larva at first-instar young, regrowth, and then regrowth obtains the
Two generation adults.
2. a kind of culture according to claim 1Steinernema carpocapsaeThe method of nematode, feature exist
In: every 9 ml of artificial eggs liquid prepares acquisition by the following method: by 3 ml of tussah chrysalis hemolymph, 0.1 g of anhydrous trehalose, egg
1 ml and 2.5 ml of sterile water of Huang 2.5 ml, skimmed milk power 0.1 g, N ' s salting liquid is uniformly mixed.
3. -2 described in any item a kind of cultures according to claim 1Steinernema carpocapsaeThe method of nematode,
Be characterized in that: the formula of every liter of N ' s salting liquid is as follows: 7.5 g/ liter of NaCl, 0.1 g/ liter of KCl, CaCl20.2 g/ liter,
NaHCO30.2 g/ liter, surplus are water.
4. a kind of culture according to claim 1Steinernema carpocapsaeThe method of nematode, feature exist
In:S. carpocapsaeInfective stage larva isS. carpocapsae All infective stage larva.
5. a kind of culture according to claim 1Steinernema carpocapsaeThe method of nematode, feature exist
In: the adult of the second generation can continue to cultivate, and loop back and forth like this, and complete the alternation of generations.
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| CN201811533466.2A CN109526884B (en) | 2018-12-14 | 2018-12-14 | Method for culturing Steinernema carpocapsae nematodes |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0191066A4 (en) * | 1984-08-14 | 1987-01-10 | Biotech Australia Pty Ltd | Liquid culture of nematodes. |
| CN102972446A (en) * | 2012-11-22 | 2013-03-20 | 北京市西山试验林场 | Preparation method of steinernema carpocapsae |
| CN103859210A (en) * | 2014-02-20 | 2014-06-18 | 广东省昆虫研究所 | Artificial medium of trichogramma |
| CN105831019A (en) * | 2016-04-21 | 2016-08-10 | 广东省昆虫研究所 | Method for quickly obtaining larvae of Steinernema carpocapsae at tidy age infection stage |
| CN106035238A (en) * | 2016-05-30 | 2016-10-26 | 中国科学院东北地理与农业生态研究所 | Nematode culture apparatus and simple breeding method for test entomopathogenic nematodes |
-
2018
- 2018-12-14 CN CN201811533466.2A patent/CN109526884B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0191066A4 (en) * | 1984-08-14 | 1987-01-10 | Biotech Australia Pty Ltd | Liquid culture of nematodes. |
| CN102972446A (en) * | 2012-11-22 | 2013-03-20 | 北京市西山试验林场 | Preparation method of steinernema carpocapsae |
| CN103859210A (en) * | 2014-02-20 | 2014-06-18 | 广东省昆虫研究所 | Artificial medium of trichogramma |
| CN105831019A (en) * | 2016-04-21 | 2016-08-10 | 广东省昆虫研究所 | Method for quickly obtaining larvae of Steinernema carpocapsae at tidy age infection stage |
| CN106035238A (en) * | 2016-05-30 | 2016-10-26 | 中国科学院东北地理与农业生态研究所 | Nematode culture apparatus and simple breeding method for test entomopathogenic nematodes |
Non-Patent Citations (1)
| Title |
|---|
| 颜珣等: "生物杀虫剂-昆虫病原线虫的培养技术", 《环境昆虫学报》 * |
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