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CN105123766A - Entomopathogenic nematode HbSD and insecticide thereof, preparation method and application - Google Patents

Entomopathogenic nematode HbSD and insecticide thereof, preparation method and application Download PDF

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Publication number
CN105123766A
CN105123766A CN201510653569.2A CN201510653569A CN105123766A CN 105123766 A CN105123766 A CN 105123766A CN 201510653569 A CN201510653569 A CN 201510653569A CN 105123766 A CN105123766 A CN 105123766A
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hbsd
entomopathogenic nematode
nematode
preparation
larvae
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CN105123766B (en
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白成
龙海波
岳建军
孙燕芳
刘丽平
彭正强
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CATAS Environment and Plant Protection Institute
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Abstract

本发明公开了昆虫病原线虫HbSD、其杀虫剂及制备方法和应用，其中悬浮液制剂的制备方法包括将HbSD感染末龄黄粉虫幼虫，第6天获得颜色变为红棕色的死虫，继续培养至第10天，HbSD从死虫尸体内爬入水中，即得悬浮液；往悬浮液加入Triton？X-100制得昆虫病原线虫HbSD悬浮液制剂；虫尸剂的制备方法包括将昆虫病原线虫HbSD感染末龄黄粉虫幼虫，第6天获得颜色变为红棕色的死虫，继续培养至第9天获得虫尸剂。本发明制备的悬浮液制剂和虫尸剂具有效果稳定、生产简易、成本低廉、无污染、杀害虫活性高等优点。

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 After culturing until the 10th day, HbSD climbed into the water from the corpse of the dead insect to obtain the suspension; add Triton? X-100 prepared entomopathogenic nematode HbSD suspension preparation; the preparation method of entomopathogenic nematode HbSD included infecting the last instar Tenebrio molitor larvae with entomopathogenic nematode HbSD, and obtained dead worms whose color changed to reddish brown on the 6th day, and continued to cultivate until the 9th day Days to get insect corpses. 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 and preparation method and application
Technical field
The present invention relates to biological insecticides, particularly relate to entomopathogenic nematode HbSD, its insecticide and preparation method and application.
Background technology
Entomopathogenic nematode is the entomogenous nematode carrying symbiotic bacteria in a class body, and such nematode, as biological insecticides novel in the world, has host range extensive; To vertebrate, plant and other non-target organism safety, be beneficial to environmental protection; Be easy to carry out live body or in vitro mass propgation, easy to use; Initiatively can find host, kill off the insect pests fast; Can recycle in the environment, can with the advantage such as number of chemical is mixed with biological insecticides, in the multiple soil pests of control and hidden insect, played important effect.
Entomopathogenic nematode is under the jurisdiction of animal kingdom's Nemathelminthes (Nematoda), phasmid (Secernentea), Rhabditida (Rhabditida), up to now, the known nematode parasitizing insect is about 40 Duo Ge sections, more desinsection nematode is wherein used to comprise Steinernema Carpocapsae (Steinernemacarpocapsae, Sc) and Heterorhabditis bacteriophora-NJ (Heterorhabditisbacteriophora, Hb).The bacterium of nematode and its parachorium acts synergistically to kill insect.Nematode for desinsection is considered to the biological insecticides to environment and mankind close friend.Current desinsection nematode has been widely used in dwelling property of soil insect, as chafer, wireworm, cutworm, mole cricket, root maggot, and the control of the insects such as leek maggot.Application process comprises the worm corpse after nematode spray suspension liquid and nematode infections; but because its spray suspension liquid need add 4-5 kind auxiliary agent and protectant (as anti-evaporating agent, water-loss reducer, adhesive, uv-protector); cause its production cost high, and its application of disadvantages affect such as prepared insecticide insecticide efficiency is low.
Summary of the invention
A kind of entomopathogenic nematode HbSD provided by the invention, improves insecticide efficiency.
On the other hand, suspension preparation provided by the invention solves biological insecticides production process need increase multiple auxiliary agent and protectant causes high in cost of production problem.
Entomopathogenic nematode HbSD of the present invention, by being placed in nematode by children yellow mealworm in age, making end yellow mealworm in age become the dead worm of rufous, filtering out nematode HbSD further behind some skies.
The preparation method of entomopathogenic nematode HbSD suspension preparation, comprise and HbSD is infected end Yellow meal worm larva in age, within 6th day, obtain the dead worm that color becomes rufous, wet filter paper continues be cultured to the 10th flying worm HbSD infective stage larva climb out of in dead worm corpse, enter in water, obtain suspension, remain on 10-12 DEG C; Add TritonX-100 toward suspension during use and obtain HbSD suspension preparation.
Entomopathogenic nematode HbSD suspension preparation, comprises entomopathogenic nematode HbSD and TritonX-100.
Entomopathogenic nematode HbSD suspension preparation, the entomopathogenic nematode HbSD stoste being 900-1000 bar/mL by concentration during use is made into 27-30 bar/mL entomopathogenic nematode HbSD and volume accounting is the TritonX-100 of 0.05%.
The preparation method of entomopathogenic nematode HbSD worm corpse agent, uses entomopathogenic nematode HbSD to infect end Yellow meal worm larva in age, within the 6th day, obtains the dead worm that color becomes rufous, continue to be cultured to the 9th day and obtain worm corpse.
Entomopathogenic nematode HbSD suspension preparation for preventing and treating insect, insect be end age greater wax moth larva, 3 age Sugarcane Yellow snout moth's larva larva, end age Yellow meal worm larva, Brontispa longissima larva or end age Clania variegata Snellen larva.
The agent of entomopathogenic nematode HbSD worm corpse for preventing and treating insect, insect be end age greater wax moth larva, 3 age Sugarcane Yellow snout moth's larva larva, end age Yellow meal worm larva, end age Brontispa longissima larva or end age Clania variegata Snellen larva.。
The present invention obtains entomopathogenic nematode HbSD by screening, and finds that it has good effect to pest control.
Further, nematode suspension preparation of the present invention and the agent of worm corpse.These two kinds of preparations are as insecticide, and preparation method and application's method is all simpler.Compared with prior art, the preparation and application of nematode killer is simplified.
Entomopathogenic nematode HbSD suspension preparation of the present invention and the agent of worm corpse all have effect stability, produce simple and easy, with low cost, pollution-free, bactericidal activity advantages of higher.
Accompanying drawing explanation
Fig. 1 is the pesticidal experimental result pictures of 4 kinds of mass production nematodes to end greater wax moth larva in age;
Fig. 2 be entomopathogenic nematode HbSD infective stage larva respectively to 3 age Sugarcane Yellow snout moth's larva larva and end age Yellow meal worm larva pesticidal experimental result picture;
Fig. 3 is that the corpse agent of entomopathogenic nematode HbSD worm and entomopathogenic nematode HbSD suspension preparation are respectively to the control efficiency figure of Sugarcane Yellow snout moth's larva;
Fig. 4 is the experimental result picture that garden sprays that entomopathogenic nematode HbSD suspension preparation prevents and treats fragrant camphor tree Clania variegata Snellen insect.
Embodiment
By reference to the accompanying drawings the present invention is described in further detail below by embodiment.
(Hb is that heterorhabditis indica is called for short to entomopathogenic nematode HbSD of the present invention, SD is numbering) be a kind of heterorhabditis indica, by the side's of trapping acquisition in the wild, concrete grammar is: be placed in nylon wire by end Yellow meal worm larva in age, imbed in Field Soil, within 6th day, results color becomes the yellow mealworm of rufous, and the nematode parasitized in yellow mealworm is entomopathogenic nematode HbSD of the present invention.HbSD has good fecundity, also can obtain its offspring by artificial mass propgation.
Entomopathogenic nematode HbSD infective stage larva (IJs) is the insect larvae after infected insect pathogenic nematode HbSD.
Embodiment one: the preparation method of entomopathogenic nematode HbSD suspension preparation
The first entomopathogenic nematode HbSD insecticide is suspension preparation, and its preparation method comprises:
The preparation method of entomopathogenic nematode HbSD suspension preparation: put one deck filter paper at plastic box bottom, the Yellow meal worm larva 100 in last age selecting healthy growth is placed on filter paper, be added on the yellow mealworm in age of end with the HbSD nematode hanging drop that liquid-transfering gun absorption concentration is 1000/ml, cover, polypide color situation of change is checked: the 6th day after room temperature places 4-5 days, the yellow mealworm color of normal infection entomopathogenic nematode becomes rufous, continue to cultivate after discarding off-colour yellow mealworm, within the 10th day, results have infected the yellow mealworm of entomopathogenic nematode HbSD.By " white trapping " method, namely the hydrotaxis of HbSD infective stage larva (IJs) is utilized to collect nematode, nematode crawls into water in insect bodies, discarding of first day results, the infective stage larva suspension of 2-3 days is checked survival rate under the microscope, and the survival rate of nematode is greater than 95%; Then deposit in 10-15 DEG C of refrigerator to connect for next step experiment.
Before using, the nematode suspension concentration of storage is diluted to 900-1000 bar nematode/milliliter, is diluted to the entomopathogenic nematode HbSD of 27-30 bar/mL, adds TritonX-100 in the ratio of liquor capacity 0.05%.
TritonX-100 is Triton X-100, is a kind of nonionic surface active agent, has dispersed and effect that is thickness.
The present embodiment selects TritonX-100 as the single auxiliary agent of nematode HbSD suspension, and cost is low, insecticide efficiency is high and do not affect nematode and find parasitic ability.
Embodiment two: the preparation of entomopathogenic nematode HbSD worm corpse agent
The second entomopathogenic nematode HbSD insecticide is the agent of worm corpse, and its preparation method comprises:
The preparation method of entomopathogenic nematode HbSD worm corpse agent: infect end Yellow meal worm larva in age by with the entomopathogenic nematode HbSD infective stage larva of results on the 1st day.The yellow mealworm selecting color to become rufous on the 6th day, continues to cultivate, and the 9th day results worm corpse, is placed in 50-100mL centrifuge tube, saves backup under 10-14 DEG C of condition.
The survival rate of entomopathogenic nematode HbSD is greater than 95%, and the agent of worm corpse is HbSD metainfective end Yellow meal worm larva in age, and each worm corpse is at least containing 5000 entomopathogenic nematode HbSD.
Two kinds of HbSD nematode killer of the present invention: nematode suspension preparation and the agent of worm corpse.Preparation method and application's method is all simpler.Compared with prior art, the preparation and application of nematode killer is simplified.
Embodiment three: the insecticidal properties experiment of entomopathogenic nematode HbSD
For examination nematode strain: 4 kinds of Heterorhabditis bacteriophora-NJ belong to nematode HbSD, HbI, HbII and HbIII.
For examination insect: end greater wax moth larva in age
Method of testing: indoor bioassay method, namely measures the virulence of 4 kinds of mass production nematode suspension to end greater wax moth larva in age, adds up lethality after 6 days.Control group does not add nematode.
Results and analysis: 4 kinds of mass production nematodes to the pesticidal of end greater wax moth larva in age as shown in Figure 1, at four kinds in examination nematode strain (HbSD, HbI, HbII, HbIII), the insecticidal power of entomopathogenic nematode HbSD of the present invention is the highest, and fecundity is strong.HbSD is selected to be used for further toxicity test.
Embodiment four:
For examination nematode strain: entomopathogenic nematode HbSD infective stage larva
For examination insect: Sugarcane Yellow snout moth's larva (Tetramoeraschistaceana) 3 instar larvae and yellow mealworm linal-instar larvae
Indoor bioassay method, respectively by 3 age sugarcane borer larva or the number ratio of yellow mealworm linal-instar larvae and entomopathogenic nematode HbSD infective stage larva be that 1:100 puts into culture dish, respectively add 200ul water treatment, each process 36 sugarcane borer 3 instar larvaes or yellow mealworm linal-instar larvae, at room temperature place, control experiment, not add entomopathogenic nematode HbSD infective stage larva, only adds running water, adds up lethality after 6 days.
As shown in Figure 2, experimental result shows that entomopathogenic nematode HbSD all has significant virulence, the LC of Sugarcane Yellow snout moth's larva 3 instar larvae to Sugarcane Yellow snout moth's larva 3 instar larvae and end Yellow meal worm larva in age 50value is the LC of 9.00/head, yellow mealworm linal-instar larvae 50value is 16.80/head.
Embodiment five:
For examination material: entomopathogenic nematode HbSD infective stage larva;
For examination insect: greater wax moth (Galleriamellonella.) linal-instar larvae, Brontispa longissima (Brontispalongissim) linal-instar larvae, Clania variegata Snellen (Cryptotheleavariegate) linal-instar larvae.Greater wax moth is cultivated, with fresh coconut leaf feeding Brontispa longissima with artificial feed; From leaf of Cortex cinnamomi camphorae feeding Clania variegata Snellen larva, be all placed in 25 DEG C of climatic cabinate cultivation.
The ratio of all insect infection entomopathogenic nematode infective stage larvas is the same, insect and pathogenic nematode infective stage larva is added bottom and is covered with bottom the culture dish of the graceful filter paper of shellfish, for examination insect Clania variegata Snellen 200, and Brontispa longissima 300, greater wax moth 400.Each process repetition 4 times, recorded lethality after 6 days.
The virulence of HbSD infective stage larva to Brontispa longissima linal-instar larvae, Clania variegata Snellen linal-instar larvae, greater wax moth linal-instar larvae is as shown in table 1, with 8.6-11.7 HbSD head/mL dosage, can kill the Brontispa longissima of 50%, Clania variegata Snellen, greater wax moth linal-instar larvae.
Table 1
Embodiment six:
For examination material: embodiment 1 prepares the entomopathogenic nematode HbSD worm corpse agent of entomopathogenic nematode HbSD suspension preparation and embodiment two preparation
For examination field: there is the sugarcane field that yellow snout moth's larva endangers
Adopt the spray method of suspension preparation, suspension spray amount is 1.02 × 10 4bar/m 2, within one week, spray once, continuous three weeks, each plot area 4m 2, the buffer strip that minizone is 5 meters, each process 6 replicated plots.
The infective stage larva in the worm corpse of embodiment two is counted, about containing 7.71 × 10 in every cephalont corpse under anatomical lens 4entomopathogenic nematode HbSD infective stage larva, worm corpse landfill is the lower 5-8 centimetre of depths of soil around sugarcane plant, hole, 85 centimetres, interval, landfill 2 cephalont corpse in each hole, 6 repetitions.Not add worm corpse for control group, within 30 days, distinguish investigation records result afterwards.Each heatable adobe sleeping platform (1 meter, interval) buries 2 HbSD nematode infections phase worm corpse, can reduce landfill number of times, improves insecticide efficiency, reaches the object of pest control.
Experimental result as Fig. 3 shows, HbSD worm corpse agent (the yellow mealworm corpse of HbSD nematode infection) and HbSD suspension preparation are to the control of Sugarcane Yellow snout moth's larva.After application HbSD suspension preparation process 30d, the relative survival rate of Sugarcane Yellow snout moth's larva larva is reduced to less than 30% than control group.After worm corpse agent process 30d, the relative survival rate of Sugarcane Yellow snout moth's larva larva is reduced to less than 61% than control group.Application HbSD suspension preparation and the agent of HbSD worm corpse all have good control efficiency to Sugarcane Yellow snout moth's larva.
Embodiment seven:
For examination material: entomopathogenic nematode HbSD suspension preparation prepared by embodiment one;
Test field: the area that fragrant camphor tree Clania variegata Snellen morbidity is more serious
The entomopathogenic nematode HbSD suspension preparation using embodiment one to prepare carries out blade back to the more serious fragrant camphor tree of morbidity and sprays; Within every 15 days, spray once, totally 3 times, within 3 months, " Invest, Then Investigate " prevents and treats result.
Garden sprays HbSD suspension preparation and prevents and treats fragrant camphor tree Clania variegata Snellen insect result as shown in Figure 4, and garden sprays HbSD suspension preparation can prevent and treat the outburst of fragrant camphor tree Clania variegata Snellen insect for 3 times effectively.
Above content is in conjunction with concrete embodiment further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made.

Claims (9)

1.昆虫病原线虫HbSD，其特征在于：所述昆虫病原线虫HbSD感染末龄黄粉虫幼虫，使末龄黄粉虫幼虫变为红棕色的死虫。1. The entomopathogenic nematode HbSD, characterized in that: the entomopathogenic nematode HbSD infects the last instar Tenebrio molitor larvae, turning the last instar Tenebrio molitor larvae into reddish-brown dead insects. 2.昆虫病原线虫HbSD悬浮液制剂，其特征在于：包括权利要求1所述的昆虫病原线虫HbSD和TritonX-100。2. The entomopathogenic nematode HbSD suspension preparation, characterized in that it comprises the entomopathogenic nematode HbSD and TritonX-100 according to claim 1. 3.根据权利要求2所述的昆虫病原线虫HbSD悬浮液制剂，其特征在于：包括浓度为27-30条/mL的昆虫病原线虫HbSD和体积占比为0.05％的TritonX-100。3. The entomopathogenic nematode HbSD suspension preparation according to claim 2, characterized in that it comprises entomopathogenic nematode HbSD at a concentration of 27-30/mL and TritonX-100 with a volume ratio of 0.05%. 4.根据权利要求2或3所述的昆虫病原线虫HbSD悬浮液制剂的制备方法，其特征在于：用昆虫病原线虫HbSD感染末龄黄粉虫幼虫，第6天获得颜色变为红棕色的死虫，继续培养至第10天，HbSD从死虫尸体内爬出并进入水中，即得悬浮液；往悬浮液加入TritonX-100制得昆虫病原线虫HbSD悬浮液制剂。4. according to the preparation method of the entomopathogenic nematode HbSD suspension preparation described in claim 2 or 3, it is characterized in that: with entomopathogenic nematode HbSD infects last instar Tenebrio molitor larvae, the 6th day obtains the dead worm that color changes into reddish-brown , continue culturing until the 10th day, HbSD climbed out from the dead insects and entered the water to obtain a suspension; add TritonX-100 to the suspension to prepare the entomopathogenic nematode HbSD suspension preparation. 5.昆虫病原线虫HbSD虫尸剂的制备方法，其特征在于：采用权利要求1所述的昆虫病原线虫HbSD感染末龄黄粉虫幼虫，第6天获得颜色变为红棕色的死虫，继续培养至第9天获得虫尸剂。5. the preparation method of entomopathogenic nematode HbSD insect corpse agent is characterized in that: adopt entomopathogenic nematode HbSD described in claim 1 to infect last instar Tenebrio molitor larvae, obtain the dead worm that color turns reddish brown on the 6th day, continue to cultivate To the 9th day to obtain the necrotic agent. 6.昆虫病原线虫HbSD虫尸剂，其特征在于：采用权利要求5所述的昆虫病原线虫HbSD虫尸剂的制备方法制得。6. Entomopathogenic nematode HbSD insecticide, characterized in that: it is prepared by the preparation method of entomopathogenic nematode HbSD insecticide according to claim 5. 7.根据权利要求6所述的昆虫病原线虫HbSD虫尸剂，其特征在于：所述虫尸剂中每个虫尸体内至少含有5000条昆虫病原线虫HbSD。7. The entomopathogenic nematode HbSD carcass agent according to claim 6, characterized in that: each carcass of the entomopathogenic nematode HbSD contains at least 5000 entomopathogenic nematode HbSD. 8.权利要求2或3所述的昆虫病原线虫HbSD悬浮液制剂用于防治昆虫，所述昆虫为末龄大蜡螟幼虫、3龄甘蔗黄螟幼虫、末龄黄粉虫幼虫、末龄椰心叶甲幼虫或末龄大袋蛾幼虫。8. the entomopathogenic nematode HbSD suspension preparation described in claim 2 or 3 is used for preventing and controlling insects, and described insect is last age greater wax moth larvae, 3 age sugarcane moth larvae, last age Tenebrio molitor larvae, last age coconut heart leaf beetle larvae or last instar larvae of the large bag moth. 9.权利要求6或7所述的昆虫病原线虫HbSD虫尸剂用于防治昆虫，所述昆虫为末龄大蜡螟幼虫、3龄甘蔗黄螟幼虫、末龄黄粉虫幼虫、末龄椰心叶甲幼虫或末龄大袋蛾幼虫。9. the entomopathogenic nematode HbSD insect corpse agent described in claim 6 or 7 is used for preventing and controlling insects, and described insect is last instar greater wax moth larvae, 3 instar sugarcane moth larvae, last instar Tenebrio molitor larvae, last instar coconut heart leaf beetle larvae or last instar larvae of the large bag moth.
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