CN109526884B

CN109526884B - Method for culturing Steinernema carpocapsae nematodes

Info

Publication number: CN109526884B
Application number: CN201811533466.2A
Authority: CN
Inventors: 颜珣; 吕欣; 谷星慧; 张立猛; 计思贵; 韩日畴
Original assignee: YUXI TABACOO Co OF YUNNAN PROVINCE; Guangdong Institute of Applied Biological Resources
Current assignee: YUXI TABACOO Co OF YUNNAN PROVINCE; Guangdong Institute of Applied Biological Resources
Priority date: 2018-12-14
Filing date: 2018-12-14
Publication date: 2022-07-26
Anticipated expiration: 2038-12-14
Also published as: CN109526884A

Abstract

The present invention relates to a cultureSteinernema carpocapsaeThe invention relates to a method for nematode, belonging to the technical field of entomopathogenic nematode cultureS.carpocapsaeInoculating the infected larva into artificial egg liquid, restoring the infected larva to grow into adult, breeding the adult, incubating the egg into first-instar larva, growing again to second-instar larva, third-instar larva and fourth-instar larva, and growing again to obtain the second-generation adult. The second generation of adults can be cultured continuously, and the steps are repeated in a circulating way to complete the generation alternation. Cultured by the methodS.carpocapsaeThe recovery and development are fast, the eggs of the imago can be observed in 2 days, the incubation of the small insects can be observed in 3 days, and the eggs of the 2 nd generation imago can be observed in 6-7 days. Culture of the inventionS.carpocapsaeThe method of nematode not only can makeS.carpocapsaeThe nematode completes the generation alternation, can recover the larvae in the infected stage more quickly and efficiently, has higher growth speed, and provides conditions for the quick and efficient culture and commercialization of the nematode.

Description

Method for culturing Steinernema carpocapsae nematodes

Technical Field

The invention belongs to the technical field of entomopathogenic nematode culture, and particularly relates to a method for culturing S.carpocapsae nematodes.

Background

Entomopathogenic Steinernema and Heterorhabditis nematodes are internationally novel high-efficiency biological insecticides (Georgis et al, 2006), are used for pest control from the beginning of the twenty-30 th century (Smart, 1995), have the advantages of strong insecticidal capacity, wide insecticidal spectrum, capability of actively searching hosts, safety to human and livestock, environment and the like, are easy to culture and prepare products in large quantities, can be mixed with various chemical pesticides, and have special effects on boring and underground pests (Kaya and Gaugler 1993; Grewal et al, 2005; Georgis et al, 2006; Yan et al, 2013; face 29667 and the like, 2014).

The life history of entomopathogenic nematodes begins with the introduction of symbiotic bacteria carried by the infected larvae (IJ) into the host insect, which evade the immune response of the insect and release the symbiotic bacteria in the insect hemolymph (downs and pets, 2002). The symbiotic bacteria proliferate in the hemolymph of the insect to kill the host insect, and the nematode feeds on the symbiotic bacteria to reproduce. When the insect is nutritionally depleted and 3-instar tolerant infective stage larvae develop, are sufficiently moist in the external environment and moderate in temperature, the infective stage larvae carry commensal bacteria away from the host insect (Brown and Gaugler, 1997) and search for new hosts. The infested larvae are the only free-living instar of the life history of entomopathogenic nematodes and can be applied using conventional sprayers to control pests. Larvae in the infected stage survive 1 to more months and can be cultured industrially (Wright et al, 2005).

The industrialized culture of entomopathogenic nematodes is the basis for the popularization and commercial application of entomopathogenic nematodes. The entomopathogenic nematodes can be cultured in vivo by host insects, or in vitro by solid culture medium or liquid culture in fermentors. In vitro culture of entomopathogenic nematodes requires the involvement of symbiotic bacteria that convert nutrients in the culture medium into components required for growth, development and reproduction of the entomopathogenic nematodes (Dowds and pets, 2002). In the case of industrial cultivation of entomopathogenic nematodes, liquid culture is most cost-effective, but solid culture is important as in vivo culture, and solid culture is more suitable in countries and regions where labor is low, because less early investment is required; the in vivo culture is suitable for germ plasm preservation and small amount experiment in laboratory.

Disclosure of Invention

In order to overcome the problems in the background art, the invention provides a method for culturing S.carpocapsae nematodes, which can not only complete the generation alternation of the S.carpocapsae nematodes, but also recover the larvae in the infected stage more quickly and efficiently, and has higher growth speed, thereby providing conditions for the quick and efficient culture and commercialization of the nematodes.

In order to realize the purpose, the invention is realized by the following technical scheme:

the method for culturing Steinernema carpocapsae nematodes specifically comprises the following steps:

1) blending and uniformly mixing tussah pupa hemolymph, trehalose, egg yolk, skimmed milk powder, N's salt solution and water to obtain artificial egg liquid;

2) inoculating S.carpocapsae infected larva into artificial egg liquid, restoring the infected larva to grow into adult, breeding the adult with eggs, incubating the eggs into first-instar larvae, growing again through second-instar larvae, third-instar larvae and fourth-instar larvae, and then growing again to obtain second-generation adult.

Preferably, every 9ml of the artificial egg liquid is prepared by the following method: 3ml of tussah pupa hemolymph, 0.1g of anhydrous trehalose, 2.5ml of egg yolk, 0.1g of skimmed milk powder, 1ml of N's salt solution and 2.5ml of sterile water are mixed uniformly.

Preferably, the formulation per liter of N's salt solution is as follows: NaCl 7.5 g/l, KCl 0.1 g/l, CaCl ₂ 0.2 g/l, NaHCO ₃ 0.2 g/liter, and the balance of water.

Preferably, the s.carpocapsae infected stage larvae are s.carpocapsae All infected stage larvae.

The second generation of adults can be cultured continuously, and the steps are repeated in a circulating way to complete the generation alternation.

The invention has the beneficial effects that:

the artificial egg liquid is used as an incubation liquid to culture S.carpocapsae infected larva, the development is fast, and adult eggs can be observed in 3 days. The method for culturing the S.carpocapsae nematodes can not only enable the S.carpocapsae nematodes to complete generation alternation, but also can develop to the brood egg adult stage more quickly and efficiently compared with tussah pupa hemolymph, thereby providing conditions for the quick and efficient culture of the nematodes.

Drawings

FIG. 1 is a graph comparing recovery of nematodes in example 1 and comparative examples 1-2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.

Example 1

1) Entomopathogenic nematodes: and S, carpocapsae All, collecting larvae (IJ) at an infection stage from insect corpses of the greater wax borer, filtering to remove dead insects, adjusting the concentration of nematodes to be 1000IJ/ml, and storing at 15 ℃ and 100rpm for later use. The nematode concentration was concentrated to 20000IJ/ml at room temperature around 25 ℃ before measurement.

2) Preparing artificial egg liquid: mixing 3ml of tussah pupa hemolymph, 0.1g of anhydrous trehalose, 2.5ml of egg yolk, 0.1g of skimmed milk powder, 1ml of N's salt solution and 2.5ml of sterile water uniformly, and storing at-20 deg.C (N's salt solution: NaCl 7.5g, KCl 0.1g, CaCl) ₂ 0.2g，NaHCO ₃ 0.2g of the suspension was added to 1000ml of H ₂ In O, 121 ℃, 10 ⁵ Sterilizing under kPa for 30min, and storing at room temperature for later use);

3) add 4. mu.l of the incubation solution to each well of a 384-well plate, and add 2. mu.l of a nematode solution (containing about 40 nematodes in the infection phase) at a concentration of 20000IJ/ml to each well, and perform recovery culture.

Comparative example 1

The procedure was the same as in example 1, except that the incubation liquid was tussah pupa hemolymph.

Preparing tussah pupa hemolymph: cutting cocoons, taking out tussah pupae, carrying out water bath at 65 ℃ for 10min, smearing pupae surface with 75% alcohol for disinfection, cutting pupae tail in a clean bench, extruding and collecting hemolymph, and storing at-20 ℃ for later use;

comparative example 2

The procedure is the same as in example 1, except that the incubation liquid is tussah pupa hemolymph water mixed liquid.

Preparing a tussah pupa hemolymph water mixed solution: 3ml of tussah pupa hemolymph and 6ml of sterile water are mixed uniformly and are ready to use.

Comparative example 3

The procedure was the same as in example 1 except that the incubation solution was used as a base solution.

Preparing a base liquid: mixing anhydrous trehalose 0.1g, ovum gallus Domesticus flavus 2.5ml, skimmed milk powder 0.1g, N's salt solution 1ml and sterile water 5.5ml, and storing at-20 deg.C (N's salt solution prepared from NaCl 7.5g, KCl 0.1g, CaCl) ₂ 0.2g，NaHCO ₃ 0.2g was added to 1000ml H ₂ In O, 121 ℃, 10 ⁵ Autoclaving for 30min under kPa, and storing at room temperature for use).

Analysis of experiments

Samples were taken at 6 wells per sampling time, daily, for 7 consecutive days, and 42 wells per nematode were treated. Adding water into the counting plate, sucking the nematodes in each hole into each hole of the counting plate, performing microscopic examination, recording the total number of dead nematodes and survival nematodes in each hole, and simultaneously recording the number of recovered nematodes.

Carpocapsae All did not recover from development in the basal fluid. The recovery development conditions of the larvae in the carpocapsae All infected stage in 3 kinds of incubation fluids (artificial egg fluid, tussah pupa hemolymph and tussah pupa hemolymph mixed solution) are shown in figure 1 and table 1.

TABLE 1 recovery and development time of Steinernema carpocapsae All nematodes in 3 incubations

And S, enabling the carpocapsae All nematodes to recover 1d in the artificial egg liquid, and observing adult eggs at 3 d. The time required for carpocapsae to grow small insects and to form generation 2 pregnant egg imagoes in the artificial egg liquid is slightly longer than that in the tussah pupa hemolymph and tussah pupa hemolymph water mixed liquid.

The method only needs a small amount of tussah pupa hemolymph to be matched with other substances, can develop to the imagination stage of the pregnant ovum more quickly and efficiently compared with the tussah pupa hemolymph, and tests prove that the artificial ovum liquid disclosed by the invention is preserved for one month at the temperature of-20 ℃, and the effect is as good as that of the just prepared artificial ovum liquid.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, while the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. Culture mediumSteinernema carpocapsaeA method of nematode worms, comprising: the method specifically comprises the following steps:

1) blending and uniformly mixing tussah pupa hemolymph, trehalose, egg yolk, skimmed milk powder, N's salt solution and water to obtain artificial egg liquid; the artificial egg liquid is prepared by the following method every 9 ml: 3ml of tussah pupa hemolymph, 0.1g of anhydrous trehalose, 2.5ml of egg yolk, 0.1g of skimmed milk powder, 1ml of N's salt solution and 2.5ml of sterile water are uniformly mixed; the formulation of N's salt solution per liter is as follows: NaCl 7.5 g/l, KCl 0.1 g/l, CaCl ₂ 0.2 g/l, NaHCO ₃ 0.2 g/liter, and the balance of water;

2) will be provided withS. carpocapsaeAnd inoculating the infected larva into the artificial egg liquid, recovering the growth of the infected larva into an adult, breeding the adult into eggs, incubating the eggs into first-instar larvae, growing the eggs into second-instar larvae, third-instar larvae and fourth-instar larvae, and then growing the eggs to obtain second-generation adults.

2. A culture according to claim 1Steinernema carpocapsaeA method of nematode worms, comprising:S. carpocapsaethe larvae in the infection stage areS. carpocapsae All infected stage larvae.

3. A culture according to claim 1Steinernema carpocapsaeA method of nematode infestation, characterized by: the second generation of adults can be cultured continuously, and the steps are repeated in a circulating way to complete the generation alternation.