CN105123766B - Entomopathogenic nematode HbSD, its insecticide and preparation method and application - Google Patents

Abstract

The invention discloses entomopathogenic nematode HbSD, its insecticide, preparation method and application, wherein the preparation method of the suspension preparation includes infecting the last instar Tenebrio molitor larvae with HbSD, obtaining dead insects whose color changes to reddish brown on the 6th day, and continuing Cultivate to the 10th day, HbSD crawls into the water from the corpse of the dead insect to obtain the suspension; add Triton X-100 to the suspension to obtain the entomopathogenic nematode HbSD suspension preparation; the preparation method of the entomopathogenic nematode comprises the following steps: The last instar Tenebrio molitor larvae were infected with HbSD, and the dead larvae whose color changed to reddish brown were obtained on the 6th day, and the cadaveric agent was obtained by continuing to culture until the 9th day. The suspension preparation and insecticide prepared by the invention have the advantages of stable effect, simple production, low cost, no pollution, high insecticidal activity and the like.

Description

Entomopathogenic nematode HbSD, its insecticide, preparation method and application

technical field

The invention relates to biological insecticides, in particular to entomopathogenic nematode HbSD, its insecticides, preparation methods and applications.

Background technique

Entomopathogenic nematodes are a type of insect-parasitic nematodes that carry symbiotic bacteria in their bodies. As a new type of biopesticide in the world, this type of nematode has a wide range of hosts; it is safe for vertebrates, plants and other non-target organisms, and is conducive to environmental protection; It is easy to carry out large-scale cultivation in vivo or in vitro, and is convenient to use; it can actively find hosts and quickly kill pests; it can be recycled in the environment and can be mixed with a variety of chemical and biological pesticides. played an important role in concealing pests.

Entomopathogenic nematodes belong to the animal kingdom Nematoda, Secernentea, and Rhabditida. So far, there are more than 40 known nematodes that parasitize insects, and most of them are used Insecticidal nematodes include Steinernema carpocapsae (Sc) and Heterorhabditisbacteriophora (Hb). The nematodes work in tandem with the bacteria that live inside them to kill the insects. Nematodes used to kill insects are considered as environmentally and human-friendly biopesticides. At present, insecticidal nematodes have been widely used in the control of soil-dwelling pests, such as scarabs, needleworms, cutworms, mole crickets, root maggots, and leek maggots. Application methods include nematode spray suspensions and nematode-infected carcasses, but since the spray suspensions need to add 4-5 kinds of auxiliaries and protective agents (such as anti-evaporation agents, water retention agents, adhesives, and UV protection agents), The disadvantages such as high production cost and low insecticidal efficiency of the prepared insecticide affect its application.

Contents of the invention

The entomopathogenic nematode HbSD provided by the invention improves the insecticidal efficiency.

On the other hand, the suspension formulation provided by the invention solves the problems of high cost caused by the addition of various auxiliary agents and protective agents in the production process of biopesticides.

The entomopathogenic nematode HbSD of the present invention is further screened out by placing the young Tenebrio molitor in the nematode, and turning the last Tenebrio molitor into a reddish-brown dead insect several days later.

The preparation method of the entomopathogenic nematode HbSD suspension preparation comprises infecting the last instar Tenebrio molitor larvae with HbSD, obtaining the dead worm whose color turns reddish brown on the 6th day, and continuing to cultivate on the wet filter paper until the larvae of the HbSD infection period from the 10th day to The dead insects crawl out of the corpse and enter the water to obtain a suspension, which is kept at 10-12°C; when in use, add Triton X-100 to the suspension to prepare the HbSD suspension preparation.

Entomopathogenic nematode HbSD suspension preparations, including entomopathogenic nematode HbSD and Triton X-100.

For the entomopathogenic nematode HbSD suspension preparation, when used, the entomopathogenic nematode HbSD stock solution with a concentration of 900-1000/mL is prepared into 27-30 entomopathogenic nematode HbSD and a volume ratio of 0.05% Triton X-100.

The preparation method of the entomopathogenic nematode HbSD cadaveric agent is to infect the last instar Tenebrio molitor larvae with the entomopathogenic nematode HbSD, obtain dead worms whose color changes to reddish brown on the 6th day, and continue culturing until the 9th day to obtain the larvae.

The entomopathogenic nematode HbSD suspension preparation is used to control insects, and the insects are the last instar larvae of Melonella mellonosa, the third instar larvae of sugarcane moth, the last larvae of Tenebrio molitor, the larvae of the cocoa beetle or the larvae of the last instar moth moth.

The entomopathogenic nematode HbSD insecticide is used to control insects, and the insects are the last instar larvae of the greater wax moth, the third instar sugarcane moth larvae, the last instar Tenebrio molitor larvae, the last instar larvae of the coconut heart beetle or the last instar larvae of the large bag moth. .

The invention obtains entomopathogenic nematode HbSD through screening, and finds that it has good effect on controlling pests.

Further, the nematode suspension preparation and nematode agent of the present invention. These two preparations are used as insecticides, and the preparation method and application method are relatively simple. Compared with the prior art, the method for preparing and using the nematode insecticide is simplified.

Both the entomopathogenic nematode HbSD suspension preparation and the nematode cadaver agent of the present invention have the advantages of stable effect, simple production, low cost, no pollution, high bactericidal activity and the like.

Description of drawings

Fig. 1 is the insecticidal experimental result figure of 4 kinds of Heterobacteria genus nematodes to the last instar wax moth larvae;

Fig. 2 is the result figure of the insecticidal experiment of entomopathogenic nematode HbSD infection stage larvae respectively to 3rd age sugarcane borer larvae and last age Tenebrio molitor larvae;

Fig. 3 is the control effect figure of entomopathogenic nematode HbSD insect corpse agent and entomopathogenic nematode HbSD suspension preparation to sugarcane yellow borer respectively;

Fig. 4 is a graph showing the experimental results of spraying the entomopathogenic nematode HbSD suspension preparation in the courtyard to control the pest of the camphor moth.

Detailed ways

The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

The entomopathogenic nematode HbSD of the present invention (Hb is the abbreviation of Heterobacterium nematode, and SD is the code number) is a kind of Heterobacterium nematode, which can be obtained by trapping in the wild. The specific method is: placing the last instar Tenebrio molitor larvae on a nylon net Inside, bury in the field soil, harvest the Tenebrio molitor that color turns reddish-brown on the 6th day, the nematode that parasitizes in Tenebrio molitor is entomopathogenic nematode HbSD of the present invention. HbSD has good reproductive ability, and its offspring can also be obtained through artificial mass culture.

Infectious stage larvae (IJs) of entomopathogenic nematode HbSD are insect larvae infected with entomopathogenic nematode HbSD.

Embodiment 1: the preparation method of entomopathogenic nematode HbSD suspension preparation

The first entomopathogenic nematode HbSD insecticide is a suspension preparation, and its preparation method comprises:

The preparation method of the entomopathogenic nematode HbSD suspension preparation: put a layer of filter paper at the bottom of the plastic box, select 100 of the last instar Tenebrio molitor larvae that grow healthy and place them on the filter paper, and use a pipette gun to absorb HbSD nematodes with a concentration of 1000 heads/ml Drop the suspension on the last instar Tenebrio molitor, cover it, and check the color change of the insect body after 4-5 days at room temperature: on the sixth day, the color of Tenebrio molitor normally infected with entomopathogenic nematodes turns reddish-brown, and discard the color. Normal Tenebrio molitors were continued to be cultured, and Tenebrio molitors infected with the entomopathogenic nematode HbSD were harvested on the 10th day. By "white trapping" method, that is, using the watertaxis of HbSD infecting larvae (IJs) to collect nematodes, nematodes climbed into the water from the insect corpses, and discarded the harvested on the first day, and the infecting larvae on the 2-3 day The survival rate of the suspension was checked under a microscope, and the survival rate of nematodes was greater than 95%; then it was stored in a refrigerator at 10-15° C. for the next experiment.

Before use, dilute the stored nematode suspension to 900-1000 nematodes/ml, dilute to 27-30 nematodes/mL entomopathogenic nematode HbSD, and add Triton X-100 at a ratio of 0.05% by volume of the solution.

Triton X-100 is polyethylene glycol octyl phenyl ether, a non-ionic surfactant with uniform dispersion and viscous effect.

In this example, Triton X-100 was selected as the single additive of the nematode HbSD suspension, which has low cost, high insecticidal efficiency and does not affect the ability of nematodes to find parasites.

Example 2: Preparation of Entomopathogenic Nematode HbSD Entomicide

The second entomopathogenic nematode HbSD insecticide is an insecticide, and its preparation method comprises:

The preparation method of entomopathogenic nematode HbSD carcasses: on the first day, the harvested larvae of entomopathogenic nematode HbSD infection stage are used to infect the last instar Tenebrio molitor larvae. On the 6th day, select the Tenebrio molitor whose color has changed to reddish brown, and continue to cultivate. On the 9th day, the dead insects are harvested, placed in a 50-100mL centrifuge tube, and stored at 10-14°C for later use.

The survival rate of the entomopathogenic nematode HbSD is greater than 95%. The carcasses are the last instar Tenebrio molitor larvae infected by HbSD, and each carcass contains at least 5000 entomopathogenic nematode HbSD.

Two kinds of HbSD nematode insecticides of the present invention: nematode suspension preparation and nematode agent. Both the preparation method and the application method are relatively simple. Compared with the prior art, the method for preparing and using the nematode insecticide is simplified.

Embodiment 3: The insecticidal property experiment of entomopathogenic nematode HbSD

The tested nematode strains: 4 species of bacteriophage Heterobacterium nematodes HbSD, HbI, HbII and HbIII.

Insects to be tested: Larvae of the last instar wax moth

Test method: Indoor bioassay method, that is, to test the toxicity of the suspension of 4 kinds of Heterobacteria genus nematodes to the last instar wax moth larvae, and count the mortality after 6 days. The control group received no nematodes.

Result and analysis: 4 kinds of Heterobradine nematodes are shown in Figure 1 to the insecticidal property of the last instar Mellonella mellonella larvae, in four kinds of test nematode strains (HbSD, HbI, HbII, HbIII), the present invention The entomopathogenic nematode HbSD has the highest insecticidal efficacy and strong reproductive ability. HbSD was chosen for further virulence assays.

Embodiment four:

Nematode strains tested: larvae of entomopathogenic nematode HbSD infection stage

Insects tested: 3rd instar larvae of sugarcane yellow borer (Tetramoera schistaceana) and last instar larvae of Tenebrio molitor

Indoor bioassay method, respectively put the 3rd instar sugarcane borer larvae or the last instar larvae of Tenebrio molitor and the larvae of entomopathogenic nematode HbSD infection stage at a ratio of 1:100 into a petri dish, add 200ul tap water for each treatment, 36 larvae for each treatment The 3rd instar larvae of sugarcane borer or the last instar larvae of Tenebrio molitor were placed at room temperature. In the control experiment, only tap water was added to the larvae at the infection stage without adding the entomopathogenic nematode HbSD, and the mortality was counted after 6 days.

As shown in Figure 2, _the experimental results show that the entomopathogenic nematode HbSD has significant toxicity to both the 3rd instar larvae and the last instar Tenebrio molitor larvae. The LC ₅₀ value of the last instar larvae was 16.80/head.

Embodiment five:

Test material: larvae of entomopathogenic nematode HbSD infection stage;

Insects to be tested: the last larvae of Galleria mellonella., the last larvae of Brontispalongissim, and the last larvae of Cryptothelea variegate. Melonella mellonella was cultured with artificial feed, Cocoa spp. was fed with fresh coconut leaves, and the larvae of Saccharomyces moth were fed with camphor leaves, all of which were cultured in an artificial climate chamber at 25°C.

The proportion of all insects infected with entomopathogenic nematode infection stage larvae is the same, and the insects and pathogenic nematode infection stage larvae are added to the bottom of the petri dish covered with Berman filter paper at the bottom, 200 insects for testing, 300 heads of the palm leaf beetle, and 400 heads of the wax moth. . Each treatment was repeated 4 times, and the mortality was recorded after 6 days.

The virulence of HbSD infection stage larvae to the last instar larvae of the cocoa beetle, the last instar larvae of Saccharomyces moth, and the last instar larvae of S. mellonella is shown in table 1, with 8.6-11.7 HbSD heads/mL dose, can kill 50% of the cocoa beetle, Last instar larvae of sack moth and greater wax moth.

Table 1

Embodiment six:

Test materials: the entomopathogenic nematode HbSD suspension preparation prepared in Example 1 and the entomopathogenic nematode HbSD insecticide prepared in Example 2

Field for test: sugarcane field with yellow borer damage

The spray method of suspension preparation is adopted, the spray volume of the suspension is 1.02×10 ⁴ /m ² , once a week for three consecutive weeks, the area of each plot is 4m ² , and there is a buffer zone of 5 meters between the plots, 6 for each treatment Repeat cell.

Count the infectious stage larvae in the carcass of embodiment two under the dissecting microscope, each carcass contains about 7.71× ¹⁰ Infection stage larvae of the entomopathogenic nematode HbSD, the carcass is buried 5-8 centimeters below the soil around the sugarcane plant In the depth, there are holes at an interval of 85 cm, and 2 insect corpses are filled in each hole, with 6 repetitions. Take no worm corpses as the control group, investigate and record the results after 30 days. Bury 2 corpses of HbSD nematode infection period in each pit (with an interval of 1 meter), which can reduce the number of landfills, improve the insecticidal efficiency, and achieve the purpose of pest control.

The experimental results shown in Figure 3 show that the HbSD insect corpse agent (the Tenebrio molitor corpse infected by the HbSD nematode) and the HbSD suspension preparation can prevent and control the sugarcane yellow borer. After treatment with HbSD suspension preparation for 30 days, the relative survival rate of the larvae of the sugarcane moth was reduced to less than 30% compared with the control group. After 30 days of treatment with the insecticide, the relative survival rate of the larvae of the sugarcane moth was reduced to less than 61% compared with the control group. The application of HbSD suspension preparation and HbSD insecticide has good control effect on sugarcane yellow borer.

Embodiment seven:

Test material: the entomopathogenic nematode HbSD suspension preparation prepared in Example 1;

Experimental field: the area where the occurrence of camphor tree big bag moth is more serious

Use the entomopathogenic nematode HbSD suspension preparation prepared in Example 1 to spray the leaf back of the camphor tree with severe disease; spray once every 15 days, a total of 3 times, and investigate the control results after 3 months.

The results of spraying HbSD suspension preparations in the courtyard to control the pests of the camphor moth are shown in Figure 4. Three times of spraying the HbSD suspension preparation in the courtyard can effectively control the outbreak of the camphor moth pests.

The above content is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. Those of ordinary skill in the technical field to which the present invention belongs can also make some simple deduction or replacement without departing from the concept of the present invention.

Claims (3)

1. A kind of 1. entomopathogenic nematode HbSD suspension preparations, it is characterised in that：Including entomopathogenic nematode HbSD and Triton X-100。
2. 2. entomopathogenic nematode HbSD suspension preparations according to claim 1, it is characterised in that：It is 27- including concentration The entomopathogenic nematode HbSD and volume accounting of 30/mL is 0.05% Triton X-100.
3. 3. the entomopathogenic nematode HbSD suspension preparations described in claim 1 or 2 are used for the application for preventing insect, the insect For last age greater wax moth larva, 3 age Sugarcane Yellow snout moth's larva larvas, last age Yellow meal worm larva, last age Brontispa longissima larva or last age Clania variegata Snellen Larva.