CN104957122A - Rice leaf roller acquisition and biological assay method - Google Patents
Rice leaf roller acquisition and biological assay method Download PDFInfo
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- CN104957122A CN104957122A CN201510282297.XA CN201510282297A CN104957122A CN 104957122 A CN104957122 A CN 104957122A CN 201510282297 A CN201510282297 A CN 201510282297A CN 104957122 A CN104957122 A CN 104957122A
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Abstract
The invention discloses an assay method for field imago acquisition and ovum collection of rice leaf rollers and the virulence of pesticides to rice leaf rollers. During the imago occurrence period of the rice leaf rollers, rice filed imagoes are netted into a plastic cup which is sealed by a preservative film, cotton balls are fixed at the bottom in the cup through double faced adhesive tapes, sugar water or hydromel supplements nutrition to the imagoes, the imagoes spawn on the inner wall of the plastic cup, the ovum hatched larvae are directly subjected to bioassay, the larvae don't have to be bred indoors, and the rice seedling impregnation method for rice seedlings cultivated for 2-4 weeks (the height of 20-25cm) is adopted for determination. The method prevents larva breeding and overcomes the problem that the rice leaf rollers cannot be bred for large scale and medicament screening cannot be carried out; and moreover, the invention provides a simple and easy rice leaf roller virulence determination method for pesticide research and development organizations, so that the biological assay of the rice leaf rollers, the mixed formulation screening, and resistance monitoring are possible.
Description
Technical field
The invention belongs to research of agricultural science field, be specifically related to a kind of rice leaf roller and insecticide of gathering from field to the method for rice leaf roller toxicity test.
Background technology
Rice leaf roller is the important migratory pest of paddy fields, Asia, causes serious economic damage and loss to Rice Production throughout the year.Rice leaf roller takes food blade epidermis to the harm of paddy rice by leaf roll on blade, affects photosynthesis, causes the paddy rice underproduction, and when occurring serious, whole blade epidermis is eaten up, and rice field forms a slice white, causes paddy rice to have no harvest.In production for the control of rice leaf roller to use chemical insecticide, this just needs institution of scientific research and agricultural chemicals Corporation R & D to go out the efficient insecticide variety of rice leaf roller and mixture.
Indoor due to current rice leaf roller are raised on a large scale and are still had any problem, existing breeding technology is difficult to meet the requirement of insecticide screening to a large amount of standard examination worm, so considerably less to the research report of rice leaf roller pesticide control screening both at home and abroad, more rare to the report of the continuous continuously rearing of the drug-fast research of rice leaf roller and indoor.Though there is bibliographical information (Liao Huaijian etc., 2012) that corn seedling can be adopted to raise rice leaf roller, its raising scale is still difficult to meet the demand of insecticide biologicall test to a large amount of standard examination worm.Past, we once adopted rice seedlings to raise rice leaf roller larvae to 3 ages, measure the virulence of insecticide to rice leaf roller 3 instar larvae again, pharmacy variety (the Gao Zhongwen etc. recommended for preventing and treating rice leaf roller are filtered out from 29 kinds of insecticides, 2008), the method needs to utilize rice leaf to raise rice leaf roller larvae, need prepare a large amount of rice seedlings in advance, this just needs larger seedling cultivation of rice space; Rice leaf roller becomes worm's ovum to produce on rice seedling after hatching with rice leaf for food, and because rice leaf is less, larva grows to needs transfer multi-disc rice leaf to take food for 3 ages, larger to the consumption of rice leaf; In addition, larvae development to 3 needs after age to be removed by single head larva from leaf roll, and choose of the right age larva and carry out biologicall test, this needs at substantial manpower, takes a lot of work during special charges, causes the efficiency of rice leaf roller medicament biologicall test low especially.Also someone adopts the rice leaf roller larvae gathered from field directly to carry out biologicall test, but the larva collected from field is difficult to ensure that the physiological status of examination worm is consistent, and field larva may by natural enemy the parasitic or susceptibility of pathogenic bacterial infection impact to insecticide, therefore, gather field larva and directly carry out the method for biologicall test and inadvisable.
Summary of the invention
For current rice leaf roller indoor feeding difficulty, insecticide biologicall test to the difficult problem that technical requirement is higher, examination worm quantitative requirement is larger of standard examination worm, and the active demand of pesticide control research and development to easy, easy rice leaf roller biometric techniques, the invention provides one and gather rice leaf roller adult from field easily, and by the method for field acquisition adult speed delivery to biologicall test laboratory, and newly hatched larvae is utilized to carry out the biometric techniques of insecticide screening.
The technical scheme that the present invention is taked for achieving the above object is, clap net is utilized to catch rice leaf roller adult from rice field net, the adult caught is transferred in transparent plastic cup, plastic cup preservative film seals, the interior cotton balls of cup provides extra-nutrition for adult, after adult takes food extra-nutrition in plastic cup, lay eggs on plastic cup inwall, plastic cup is taken back laboratory and is hatched, also plastic cup can be loaded in ventilative carton and be addressed to biologicall test laboratory by express delivery, the newly hatched larvae hatched in plastic cup is directly used in biologicall test, assay method adopts dipping seedling method.
Rice leaf roller adult collecting method described in the present invention is, early 6 ~ 8 time and afternoon 5 ~ 7 time catch rice leaf roller adult with clap net from rice field net, the adult caught in net transparent pipe (diameter 2 centimetres, long 15 ~ 20 centimetres, one end nylon wire seals, one end open) to transfer in transparent plastic cup (350 ~ 500 milliliters), plastic cup preservative film seals, and adult directly lays eggs on plastic cup inwall.
It is 5% ~ 20% syrup or hydromel that rice leaf roller described in the present invention supplements the nutrients, extra-nutrition supplying method pastes the double faced adhesive tape of a piece about 1 square centimeter, one little cotton balls double faced adhesive tape is fixed on plastic cup bottom, Additional nutrient solution is adsorbed onto in cotton balls.Cotton balls at the bottom of being fixed on glass can not move up and down in plastic cup, can reduce the harmful effect of moistening cotton balls to adult in cup.As preferably, rice leaf roller extra-nutrition working concentration is 10% syrup or hydromel.
Bioassay method described in the present invention is dipping seedling method, and concrete operating procedure is as follows:
1) preparation of biologicall test rice seedling: cultivate rice seedlings after disposal plastic cup (350 milliliters) fills soil, every glass of 15 ~ 35 strain rice seedlings, after 2 ~ 4 weeks, (about 20 ~ 25 centimetres high) can be used for biologicall test.
2) insecticide mother liquor preparation: the compound method of preparing protodrug mother liquor is solvability according to this medicament and the LC reported in the past
50the mother liquid concentration that value estimation will be prepared.Accurately take required former dose and put into volumetric flask, add solvent (acetone, dimethyl formamide or methyl-sulfoxide) and be settled to graduation mark.The mother liquor sealed membrane prepared seals bottleneck, and label indicates rearmounted 4 DEG C of concentration, medicine name, preparation time and medicament producer and saves backup.Carry out biologicall test according to preparation, then need not prepare mother liquor, but directly with the working solution of Formulation biologicall test.
3) biologicall test working solution preparation: by the mother liquor amount needed for pipette, extract or liquid formulation amount or the amount accurately taking solid pharmaceutical preparation, with the liquid 800 milliliters of the distilled water preparation biologicall test maximum concentration containing 1 ‰ Triton X-100, get 400 milliliters of use wherein and carry out doubling dilution containing the distilled water of 1 ‰ Triton X-100 and obtain required series concentration.
4) rice seedling dipping method: by cultivated 2 ~ 4 weeks in disposable cup rice seedling (20 ~ 25 centimetres high) gently at the bottom of inverted cup upwards, rice seedling makes rice seedling immerse completely in work liquid downwards 10 ~ 60 seconds, natural seasoning after rice seedling takes out.
5) worm processing method is tried: in culture dish (Φ 7 ~ 9 centimetres), put into 3 large little suitable filter paper, add water filter paper is fully moistening, the middle and upper part leaves and stems of 20 ~ 40 rice seedlings is divided and is cut into 5 centimeter length fragments, put on the filter paper of culture dish, rice leaf roller newly hatched larvae writing brush is chosen lightly on the rice leaf in culture dish, after water is moistening, cover culture dish with the pure cotton cloth of suitable size, then cover tightly with culture dish lid.Examination worm puts 25 DEG C, relative moisture 75%, cultivates in the incubator under illumination 16: 8.As preferably, try worm process and carry out 8 ~ 10 of the morning every day time.
6) result is observed: chemicals treatment 3 ~ 5 days observes rice leaf roller larvae survival condition afterwards, opens culture dish lid, tears the rice leaf rolled up and takes out larva, overturn gently by larva with fine, soft fur pen, belly upwards, with larva can not autonomic activities for death standard.5 ~ 8 series concentration established by each insecticide of said determination, with the distilled water containing 1 ‰ Triton X-100 for contrast, and each process repetition 3 ~ 6 times.
The ovum that the present invention adopts rice leaf roller field adult to produce, after hatching, directly utilize newly hatched larvae to carry out biologicall test, screening for rice leaf roller pesticide control provides a kind of biometric techniques of simple and easy to do, result favorable reproducibility, and the method has following outstanding advantages:
1) larva raising link is removed from, time saving and energy saving save space: do not need to raise for examination worm to cultivate a large amount of rice seedlings, save the space needed for nursery; Worm does not need to raise in indoor in examination, dramatically reduces the time needed for the preparation of examination worm, decreases the manpower that examination worm live box needs.In the rice leaf roller field emergence period, the biologicall test completing 20 kinds of medicaments has prepared only to need 4 time-of-weeks from rice seedling.
2) measurement result favorable reproducibility: pilot scale worm of the present invention adopts newly hatched larvae, the physiological status of examination worm is very consistent, overcome in larva raising due to differences of Physiological between the examination worm individuality that causes of larvae development progress difference, and the differences of Physiological that different experiments room raising method is inconsistent produced, result of the test favorable reproducibility, between different experiments room, measurement result also has a comparativity.
3) cost is low: rice leaf roller adult provided by the invention gathers and extra-nutrition supplying method, rice leaf roller can be sent at a distance, the adult gathered in rice field, China paddy fields was addressed to domestic biologicall test laboratory by express delivery the same day, method adult survival rate provided by the invention is high, egg laying amount is large, namely carry out biologicall test after within 3 ~ 5 days, receiving, significantly reduce the acquisition cost of examination worm.Adopt newly hatched larvae to measure simultaneously, eliminate the process that examination worm is raised, can overspending be saved.
4) measurement result accurately can reflect the virulence of insecticide: the data adopting the inventive method to record are basically identical with its trend of result adopting 3 instar larvaes to measure.
Embodiment
In order to understand the present invention better, below in conjunction with embodiment, the invention will be further described.But the scope of protection of present invention is not limited to the scope that embodiment represents.
Embodiment 1: rice leaf roller adult gathers and extra-nutrition supplying method
In the rice leaf roller emergence period, every day early 6 ~ 8 time and afternoon 5 ~ 7 time catch rice leaf roller adult with clap net from rice field net, the adult caught transparent pipe (diameter 2 centimetres, long 15 ~ 20 centimetres, one end nylon wire seals) transfer in transparent plastic cup (350 ~ 500 milliliters), every glass 2 ~ 4 to female male imago, plastic cup preservative film seals, with the air-vent that cushion is little a bit on preservative film.Paste the double faced adhesive tape of a piece 1 square centimeter at plastic cup inner bottom part before adult gathers, one little cotton balls double faced adhesive tape is fixed on plastic cup inner bottom part, 10% syrup prepared in advance or hydromel are added in cup and supply rice leaf roller imago feeding in cotton balls, directly lay eggs in plastic cup after imago feeding.The plastic cup that adult is housed is brought back to laboratory hatch, after about 5 days, namely ovum hatches, and the larva hatched can be directly used in biologicall test.
Embodiment 2: rice leaf roller adult and ovum send method
The rice leaf roller adult collected by method described in embodiment 1 is sent by express delivery, the adult of sending should use the transparent plastic cup with higher-strength when gathering, fill with foam or paper scrap between plastic cup in case, to prevent extruding in mailing process and shock, anti-extrusion and the shatter-resistant intensity that packing case answers tool certain, casing surrounding leaves a small amount of air-vent.The adult that the night day before yesterday and morning on the same day gather can merge sends, and after laboratory receives adult and ovum, can put laboratory continue to hatch as ovum is not yet hatched, until can directly measure after hatching.
Embodiment 3: different insecticides is to the virulence of rice leaf roller.Adopt the dipping seedling method of introduction of the present invention to determine the virulence of 10 kinds of insecticides to Nanning, Changsha and ground, Nanjing three rice leaf roller population, the results are shown in Table 1.In the 10 kinds of insecticides measured, the highest with Avermectin, emamectin benzoate and the pleocidin virulence to rice leaf roller, three ground rice leaf roller population measured results are basically identical, its LC
50value all 0.02 milligram of active ingredient/liter below.Secondly the insecticide variety that virulence is higher is Rynaxypyr, insect amide, chlopyrifos and quinalphos, its LC
50value is between 0.3 ~ 2.0.Cupric sulfate and the virulence of Bt to rice leaf roller larvae minimum.
Embodiment 4: rice leaf roller is to the mensuration of resistance to insecticides.Within between 2010-2013 years continuous 4 years, carry out monitoring (see table 2) to the change tread of 8 kinds of insecticide resistances to Nanning, Changsha and Nanjing rice leaf roller population.Between 4 years rice leaf roller to the susceptibility of chlorine worm benzamide and worm hydrazides in continuous decline, this decline in Guangxi, there is same trend in Changsha, Nanjing.Compared with 2010, within 2013, Nanning, Changsha and Nanjing population have dropped 2.7 times, 7.1 times and 8.8 times respectively to the susceptibility of Rynaxypyr; 6.0 times, 34.4 times and 18.2 times be have dropped respectively to the susceptibility of worm hydrazides.The change list of other 6 kinds of Insecticide susceptibilities reveals the fluctuation between different regions, different year, does not find regular change.
Embodiment 5: Affirm (Merck Co.) and quinalphos synergistic formula screen, adopt bioassay method of the present invention, with the newly hatched larvae of Nanning rice leaf roller population for supplying examination insect, mensuration Affirm (Merck Co.) and quinalphos different proportion combine the synergistic effect (see table 3) to rice leaf roller.7 proportionings measured all have synergistic effect, and wherein the strongest with the synergistic effect of 1: 119 proportioning, co-toxicity coefficient reaches 535, are secondly that 1: 99 combination also has very high synergistic effect, minimum with the synergistic effect of 1: 169 proportioning.
| Proportioning | 1∶99 | 1∶119 | 1∶149 | 1∶169 | 1∶189 | 1∶199 | 1∶219 |
| Co-toxicity coefficient | 449 | 535 | 354 | 171 | 232 | 185 | 182 |
Embodiment 6: quinalphos and worm hydrazides compounded formula screen.With the newly hatched larvae of Nanning rice leaf roller population for supplying examination insect, measure quinalphos and worm hydrazides combine totally 7 fill a prescription to the synergistic effect (see table 4) of rice leaf roller, determination data shows that these combinations are all less than 80 to the co-toxicity coefficient of rice leaf roller, after quinalphos and worm hydrazides mixture, synergistic effect be there is no to rice leaf roller, there is antagonism on the contrary, be unsuitable for the complex preparation being developed to control rice leaf roller.
| Proportioning | 7∶1 | 5∶1 | 3∶1 | 1∶1 | 1∶3 | 1∶5 | 1∶7 |
| Co-toxicity coefficient | 31 | 29 | 24 | 10 | 13 | 13 | 10 |
Embodiment 7: chlopyrifos and worm hydrazides compounded formula screen.With the newly hatched larvae of Nanning rice leaf roller population for supplying examination insect, measure chlopyrifos and worm hydrazides combine totally 7 fill a prescription to the synergistic effect (see table 5) of rice leaf roller, determination data shows that these combinations are all less than 80 to the co-toxicity coefficient of rice leaf roller, after chlopyrifos and worm hydrazides mixture, synergistic effect be there is no to rice leaf roller, there is antagonism on the contrary, the complex preparation of control rice leaf roller should not be developed to.
| Proportioning | 7∶1 | 5∶1 | 3∶1 | 1∶1 | 1∶3 | 1∶5 | 1∶7 |
| Co-toxicity coefficient | 44 | 55 | 29 | 33 | 11 | 32 | 30 |
Embodiment 8: methylamino avermectin and indoxacarb compounded formula screen.With the newly hatched larvae of Nanjing rice leaf roller population for supplying examination insect, mensuration methylamino avermectin and indoxacarb combine the interaction (see table 6) of 3 proportionings to rice leaf roller, 3 proportionings all do not have synergistic effect to rice leaf roller, the proportioning of 1: 19 is summation action, and the proportioning of 1: 24 and 1: 39 is antagonism.
| Proportioning | 1∶19 | 1∶24 | 1∶39 |
| Co-toxicity coefficient | 116 | 59 | 68 |
Embodiment 9: indoxacarb and Rynaxypyr compounded formula screen.With the newly hatched larvae of Nanjing rice leaf roller population for supplying examination insect, mensuration indoxacarb and Rynaxypyr combine the interaction (see table 7) of 5 proportionings to rice leaf roller, 5 proportionings to the co-toxicity coefficient of rice leaf roller according between 80 ~ 120, there is no synergistic effect, only show summation action.
| Proportioning | 1∶1 | 1∶2 | 1∶4 | 1∶30 | 1∶60 |
| Co-toxicity coefficient | 95 | 85 | 83 | 80 | 96 |
Embodiment 10: Rynaxypyr and chlorpyrifos mixture recipe determination.With the newly hatched larvae of Nanjing rice leaf roller population for supplying examination insect, mensuration Rynaxypyr and chlopyrifos combine the interaction (see table 8) of 4 proportionings to rice leaf roller, the co-toxicity coefficient of 1: 2 and 1: 3 proportioning is less than 80, for antagonism, the co-toxicity coefficient of 1: 4 and 1: 5 proportioning, between 80 ~ 120, shows as summation action.
| Proportioning | 1∶2 | 1∶3 | 1∶4 | 1∶5 |
| Co-toxicity coefficient | 57 | 48 | 86 | 81 |
Embodiment 11: ethyl pleocidin and indoxacarb compounded formula screen.With the newly hatched larvae of Nanjing rice leaf roller population for supplying examination insect, mensuration ethyl pleocidin and indoxacarb combine the interaction (see table 9) of 5 proportionings to rice leaf roller, ethyl pleocidin and indoxacarb are in 2: 1 ratio mixtures, co-toxicity coefficient is between 80 ~ 120, show as summation action, the co-toxicity coefficient of other 4 proportionings is all greater than 120, show as synergistic effect, especially by synergy during equal proportion mixture significantly, co-toxicity coefficient reaches 286 for ethyl pleocidin and indoxacarb.
| Proportioning | 3∶1 | 2∶1 | 1∶1 | 1∶2 | 1∶3 |
| Co-toxicity coefficient | 127 | 115 | 286 | 281 | 133 |
Claims (6)
1. one kind to gather rice leaf roller adult, collects Adult worms producting eggs, provides the method for extra-nutrition and utilize newly hatched larvae to carry out the method for biologicall test for adult from rice field, the adult caught from rice field net is it is characterized in that to be transferred in plastic cup, with the cotton balls being fixed on plastic cup bottom for adult in cup provides extra-nutrition, Adult worms producting eggs is on plastic cup inwall, and in cup, egg hatching is the biologicall test being directly used in insecticide after newly hatched larvae.
2. adult according to claim 1 gathers and ovum collecting method, and refer to and sooner or later catch rice leaf roller adult from rice field net, be transferred to by adult in transparent plastic cup, plastic cup preservative film seals, and Adult worms producting eggs is on the inwall of transparent plastic cup.
3. the Adult supplement nutrient supplying method according to any one of claim 1-2, it is characterized in that fritter double faced adhesive tape is pasted in the bottom in transparent plastic cup, cotton balls is fixed on bottom plastic cup, 5% ~ 20% syrup or hydromel are adsorbed in cotton balls, for the adult of rice leaf roller provides extra-nutrition.
4. the bioassay method according to any one of claim 1-3, is characterized in that utilizing field to gather the adult institute larva hatched that lays eggs and directly carries out biologicall test, do not raise in indoor to larva, larva after breathing out directly mensuration its to the reaction of medicament.
5. the bioassay method according to any one of claim 1-4, is characterized in that medicament processing method adopts dipping seedling method, the paddy rice seedling in 2 ~ 4 week age being flooded 10 ~ 60 seconds in liquid, for trying the process of worm after drying.
6. the adult collection according to any one of claim 1-5, ovum collecting and bioassay method can be used for the mensuration of rice leaf roller to drug response, the screening of rice leaf roller pesticide control, the screening of rice leaf roller mixture formula.
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Cited By (3)
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| CN106959352A (en) * | 2017-03-28 | 2017-07-18 | 浙江省农业科学院 | A kind of method acted on using the luxuriant behavior evaluation medicament of volume rice leaf roller |
| CN109328929A (en) * | 2018-11-01 | 2019-02-15 | 江西省农业科学院水稻研究所 | A kind of method of Natural infection and the anti-snout moth's larva rice material of screening |
| CN116076443A (en) * | 2022-07-29 | 2023-05-09 | 扬州大学 | Biological assay device, method and application of rice leaf roller larvae |
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| CN116076443A (en) * | 2022-07-29 | 2023-05-09 | 扬州大学 | Biological assay device, method and application of rice leaf roller larvae |
| CN116076443B (en) * | 2022-07-29 | 2024-04-16 | 扬州大学 | Biological assay device, method and application of rice leaf roller larvae |
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