CN112119980A - A kind of low-temperature propagation method of in vitro cultivation of Trichogramma - Google Patents

Abstract

The invention provides a low-temperature propagation method for in vitro culture of trichogramma. The method comprises the following steps: and (3) manufacturing an artificial egg card, placing the artificial egg card in a biological incubator, then parasitizing the trichogramma in the artificial egg, and placing the egg card with the trichogramma at 16-23 ℃ for culturing until the imago eclosion occurs. The trichogramma cultured by the method has longer delay time than that of natural trichogramma (trichogramma dendrolimi parasitizing tussah eggs). The method is more convenient for field application, prolongs the shelf life and reduces the cost on the basis of not influencing the quality and the application effect of the breeding bees.

Description

A kind of low-temperature propagation method of in vitro cultivation of Trichogramma

technical field

The invention belongs to the field of biotechnology, and in particular relates to a low-temperature propagation method for culturing Trichogramma in vitro.

Background technique

Trichogramma is an important egg-parasitic natural enemy insect. It has rich resources, wide distribution, large application area, and many pest control objects. It plays an important role in the biological control of pests in the production of food safety, pollution-free food and organic food. . The inoculation and propagation of Trichogramma is the key to biological control. At present, the most widely used is the use of tussah eggs for a large number of indoor breeding, which consumes a lot of manpower and material resources, and is limited by time, region, transportation and other factors in mass production and application. Not only the production cost is high, but also the problem of insufficient supply. Therefore, it is of great significance to extend the shelf life and reduce the cost of reproduction by developing a low-temperature propagation method for culturing Trichogramma in vitro.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a low-temperature propagation method for cultivating Trichogramma in vitro. When the method utilizes artificial eggs to multiply Trichogramma in large numbers, different low temperatures can be selected to adjust the cultivation time according to actual application needs, so as to prolong the shelf life and reduce reproduction. cost.

For achieving the above object, the present invention adopts the following technical scheme:

The invention relates to a low-temperature propagation method for cultivating Trichogramma in vitro.

Specifically, the low-temperature propagation method for cultivating Trichogramma in vitro includes the following steps: making an artificial egg card, placing the artificial egg card in a biological incubator, then parasitizing the Trichogramma in the artificial egg, attaching a The egg card of Trichogramma was cultivated at 16-23°C until adult eclosion.

Further, the egg liquid of the artificial egg is composed of: tussah silkworm pupa hemolymph 3mL, trehalose 0.1g, egg (take the yolk) 2.5mL, 10wt% skim milk 1mL, NS salt solution 1mL and sterile water 1.5mL.

The preparation steps of the described artificial egg card are: adopting the polyethylene-polypropylene composite film with a thickness of 30 μm, pressing the hemispherical artificial egg ( Diameter 2-3mm × height 2-3mm) 20 capsules, the film is placed under ultraviolet light for 30min to sterilize. Under aseptic conditions, 4 μL of Trichogramma culture solution was injected into each egg, with the convex surface of the hemisphere outside, and the sides of the film were sealed with a sealing machine. After making the egg card, use a soft brush to spread 10% polyvinyl alcohol aqueous solution on the surface of the egg card to induce Trichogramma to lay eggs. Each temperature treatment corresponds to 1 artificial egg card, and each treatment is repeated 3 times.

The temperature when the artificial egg card is placed in the biological incubator is 26-28°C, the relative air humidity is 70-80%, and the photoperiod is L:D=16:8h; Cultivation in artificial eggs is 24 hours after Trichogramma parasitized in the artificial eggs, and the egg card with Trichogramma was placed in a temperature of 16-23°C, a relative air humidity of 70-80%, and a photoperiod of L:D. =16:8h in a biological incubator until adult emergence.

The described parasitizing of Trichogramma in the artificial egg is that the ratio of bee-to-egg ratio is 8:1 to parasitize the Trichogramma in the artificial egg.

Compared with the prior art, the advantages of the present invention are: a kind of low-temperature breeding method for cultivating Trichogramma in vitro provided by the invention is to take the pine caterpillar Trichogramma dendrolimi as an object, and to use artificial eggs as host materials, The pine caterpillar Trichogramma was parasitized in the artificial eggs, and cultivated at 16-23° C. until adult emergence. The results show that compared with the control group in which the pine caterpillar Trichogramma was parasitized on the eggs of the tussah silkworm, the method of the present invention was used to cultivate the obtained product. Trichogramma has a longer delay than nature (the pine caterpillar Trichogramma parasitizing tussah eggs). The method of the invention is more convenient for field application, prolongs shelf life and reduces cost on the basis of not affecting the quality and application effect of bee breeding.

Detailed ways

The following examples are further illustrations of the present invention, rather than limitations of the present invention.

Example 1

1. Experimental materials

Insect: The pine caterpillar Trichogramma dendrolimi.

Host: artificial eggs, tussah eggs.

The egg liquid formula of artificial eggs: tussah silkworm pupa hemolymph 3mL, trehalose 0.1g, egg (take the yolk) 2.5mL, 10wt% skim milk 1mL, NS salt solution 1mL and sterile water 1.5mL, mix the components evenly to get Egg fluid from artificial eggs.

NS salt solution is physiological saline solution (0.9% sodium chloride aqueous solution).

2. Experimental method: The experiment set 4 low temperature temperatures, 23±1°C, 20±1°C, 16±1°C and 13±1°C, and the control temperature was 27±1°C. The artificial egg card was made using the tussah egg card and the preferred Trichogramma culture medium. Wherein, (1) the production method of the tussah egg card: 20 tussah eggs are pasted on the kraft paper coated with white latex to make the tussah egg card; each temperature treatment corresponds to one tussah egg card, and each treatment is repeated 3 times. (2) The preparation steps of the artificial egg card are as follows: using a polyethylene-polypropylene composite film with a thickness of 30 μm, a hemispherical artificial egg ( Diameter 2-3mm × height 2-3mm) 20 capsules, the film was placed under ultraviolet light for 30min to sterilize; under aseptic conditions, 4 μL of Trichogramma culture solution was injected into each egg, so that the convex surface of the hemisphere was outside, and a sealing machine was used. Seal the film tightly on all sides. After the egg card was made, a 10% polyvinyl alcohol aqueous solution was coated on the surface of the egg card with a soft brush to induce the pine caterpillar Trichogramma to lay eggs. Each temperature treatment corresponds to 1 artificial egg card, and each treatment is repeated 3 times. The egg card was placed in a biological incubator with a temperature of 27 ± 1 °C, a humidity of 75% ± 5% RH, and a photoperiod of 16:8h (L:D). 8 Trichogramma pine caterpillars were connected to 1 parasitic egg) Let Trichogramma pine caterpillars parasitize on artificial eggs and tussah eggs, and 24 hours after the bees were received, put the egg cards with Trichogramma pine caterpillars on 5 eggs respectively. At the set temperature, the incubator develops to adult eclosion in a biological incubator with a relative air humidity of 70-80% and a photoperiod of L:D=16:8h. The reproductive quality of Trichogramma pine caterpillars cultivated in vitro at different low temperatures was investigated from biological parameters such as pupation rate, emergence rate, female proportion and normal bee population. The experiment was set up in 3 replicates.

3. Experimental results

Table 1 is the reproductive quality parameters of the pine caterpillar Trichogramma cultivated with artificial eggs at the set temperature. As shown in Table 1, compared with the optimum temperature (27°C), at 13°C, 16°C, 20°C and 23°C, the development time of Trichogramma pine caterpillars cultivated with artificial eggs was prolonged by 21 days, 17 days, 8 days and 5 days. The pupation rate, eclosion rate, female proportion and normal bee population were not significantly different between 16℃, 20℃, 23℃ and 27℃. However, the developmental quality of artificial eggs at 13℃ was lower than other experimental temperatures. It shows that the Trichogramma cultured in vitro from artificial eggs can reproduce at a low temperature of 16℃-23℃ without affecting the quality.

Table 1 Breeding of Trichogramma pine caterpillar in vitro at different temperatures

*The same capital letters after the mean ± standard error of the same column indicate that the difference is not significant (Tukey's test: P>0.05).

Table 2 is the reproductive quality parameters of Trichogramma pine caterpillars cultivated from tussah eggs at the set temperature. As shown in Table 2, the quality of Trichogramma pine caterpillars reproduced from tussah eggs is equivalent to that of artificial eggs, but at 13°C, 16°C, 20°C, and 23°C, the development time was prolonged by 20 days, 15 days, and 20 days, respectively. 6 days and 3 days, which are shorter than the extended time of artificial egg cultivation. It can be seen that, compared with the control group in which the pine caterpillar Trichogramma was parasitized on the eggs of the tussah silkworm, the Trichogramma obtained by adopting the method of the present invention has a longer delay time than the natural (the pine caterpillar Trichogramma parasitizing the eggs of the tussah silkworm). Therefore, when using artificial eggs to breed Trichogramma in large numbers, different low temperatures can be selected to adjust the cultivation time according to the actual application needs, so as to prolong the shelf life and reduce the reproduction cost.

Table 2 Breeding of Trichogramma pine caterpillars parasitizing tussah eggs at different temperatures

*The same capital letters after the mean ± standard error of the same column indicate that the difference is not significant (Tukey's test: P>0.05).

The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limitations of the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (6)

1. A low-temperature breeding method for in vitro breeding trichogramma is characterized in that the trichogramma is parasitized in artificial eggs and cultivated at the temperature of 16-23 ℃.

2. The low-temperature propagation method for breeding trichogramma in vitro as claimed in claim 1, comprising the steps of: and (3) manufacturing an artificial egg card, placing the artificial egg card in a biological incubator, then parasitizing the trichogramma in the artificial egg, and placing the egg card with the trichogramma at 16-23 ℃ for culturing until the imago eclosion occurs.

3. The method for low-temperature reproduction of trichogramma in vitro culture according to claim 1 or 2, wherein the egg fluid of the artificial egg is composed of: 3mL of tussah pupa hemolymph, 0.1g of trehalose, 2.5mL of eggs, 1mL of 10 wt% skimmed milk, 1mL of NS salt solution and 1.5mL of sterile water.

4. The low-temperature propagation method for in vitro breeding trichogramma as claimed in claim 2, wherein the artificial egg card is prepared by the steps of: a polyethylene-polypropylene composite film with the thickness of 30 mu m is adopted, 20 hemispherical artificial eggs with the diameter of 2-3mm and the height of 2-3mm are pressed on the film with the size of 8 x 7cm through a special mould and a warm glass rod, the film is placed under ultraviolet light for irradiation for 30min for sterilization, 4 mu L of trichogramma culture solution is injected into each egg under the aseptic condition, the hemispherical convex surface is exposed, each side of the film is sealed by a sealing machine, after an egg card is manufactured, 10% polyvinyl alcohol aqueous solution is coated on the surface of the egg card by a soft brush pen to induce the trichogramma to lay eggs, each temperature treatment corresponds to 1 artificial egg card, and each treatment is repeated for 3 times.

5. The low-temperature propagation method for in vitro culture of trichogramma as claimed in claim 2, wherein the temperature of the artificial egg card in the biological incubator is 26-28 ℃, the relative humidity of air is 70-80%, and the photoperiod is L: D: 16:8 h; the trichogramma parasitizing in the artificial eggs is to culture the trichogramma parasitizing in the artificial eggs for 24 hours, and then place the egg cards with the trichogramma in a biological incubator with the temperature of 16-23 ℃, the relative air humidity of 70-80% and the photoperiod L: D: 16:8 hours until the imago eclosion.

6. The method for low-temperature breeding of trichogramma in vitro as claimed in claim 2, wherein the trichogramma parasitizing in the artificial eggs is carried out by parasitizing the trichogramma in the artificial eggs at a ratio of 8: 1.