CN103399126A - Indoor bioassay method of bradysia odoriphaga larvae - Google Patents
Indoor bioassay method of bradysia odoriphaga larvae Download PDFInfo
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Abstract
The invention discloses an indoor bioassay method of bradysia odoriphaga larvae, and the steps of the method are shown as follows: (1) drug-mixed artificial feeds are prepared according to a series of concentrations designed by experiments; (2) after filter papers are flatly laid on the bottom of a culture dish, quantitative drug liquids with a series of concentrations are respectively added so as to completely wet the filter papers; (3) drug-mixed artificial feeds with corresponding concentrations are cut into small pieces, and the small pieces are put on the wet filter papers in the culture dish; and (4) larvae of the appropriate age are accessed onto the drug-mixed artificial feeds, after a certain period of time, death situations of the sample larvae are checked, and virulence is calculated. The method is simple and economical, and compared with bioassay methods using poison fresh leek, times of addition of water and feeds are significantly reduced, the accuracy of results is high, the problem of seasonal shortages of leek can be solved, and the method can meet requirements of bioassay of the bradysia odoriphaga larvae.
Description
(1) technical field:
The present invention relates to a kind of indoor biometrics method of olaiphage larva in Chinese Chive, belong to Pesticide Science and agricultural entomology field.
(2) background technology:
Bradysia odoriphaga (Bradysia odoriphaga), be commonly called as the fragrant-flowered garlic maggot, is subordinate to Diptera, Nematocera, and the fungus gnat Superfamily, the eye Sciaridae, the eye Sciara, be a kind of important pests on northern vegetables produce late.Mainly be distributed in the ground such as North China, northeast, northwest and Shandong, Jiangxi, Taiwan, since the nineties in 20th century, this worm distributes more and more wider, comprises the provinces such as Hubei, Sichuan, Zhejiang, Jiangsu, and almost there is generation all parts of the country.
Bradysia odoriphaga is caused harm with larva in field, newly hatched larvae with bulb one side gradually inwardly eat into into, down in bulb, causing harm.Spring, mainly cause harm Plant Leaf sheath, young stem, bud of olaiphage larva in Chinese Chive, caused that young stem rot is rotten, blade is withered and yellow, then stem bitten broken and eaten in stem.Summer, larva was movable downwards, ate into into bulb, caused bulb to rot to cause the death of fragrant-flowered garlic clump.Bradysia odoriphaga is caused harm not only affects the leek quality, and causes the output decrease, and the killed strain rate 20%～30% in general plot is serious up to 100%, causes production loss 30%～80%.
In recent years, along with the large tracts of land of facility leek is planted year after year, Bradysia odoriphaga is caused harm and is increased the weight of gradually.The vegetable grower when control during this obstinate insect much amounts use high poison or hypertoxic chemical agent, such as 1605, thimet (3911), acephatemet, the different sulphur phosphorus of methyl, Furadan etc., cause " malicious leek " event to take place frequently, cause the extensive concern of society.The medicament of the control Bradysia odoriphaga of screening low toxicity, efficient, low-residual, become the important channel of current " malicious leek " problem of solution.Chemicals needs a kind of, fast and accurately indoor biometrics method easy for Bradysia odoriphaga.2000, the employing dip methods such as Dang Zhihong were measured the indoor virulence of phoxim to olaiphage larva in Chinese Chive; 2002, the employings such as Xue Ming were soaked the method that worm combines with stomach toxicity and have been measured the indoor virulence of the medicaments such as chlopyrifos, alphamethrin, phoxim, carbosulfan, diazinon, fluorine pyridine urea, cyromazine, Imidacloprid to Bradysia odoriphaga 4 instar larvaes; Mu Wei etc. 2002 adopt the stomach toxicity co-toxicity method of tagging to measure cyromazine, phonamiphos, diazinon, phoxim, chlopyrifos, carbosulfan and the 5a,6,9,9a-hexahydro-6,9-methano-2,4 indoor virulence to Bradysia odoriphaga 3 instar larvaes, and studied the toxicological effect of cyromazine to Bradysia odoriphaga, adopted the stomach toxicity co-toxicity method of tagging to measure the LC of 8 kinds of medicaments such as chlopyrifos, phoxim, Avermectin, chlorfenapyr, Benfuracard micro, Methomyl, carbosulfan and Imidacloprid to Bradysia odoriphaga 3 instar larvaes in 2005
20And LC
502011 years, the employing stomach toxicitys such as the Wang Ping co-toxicity method of tagging was measured natural pyrethrum, oxygen amarolide, Avermectin, the chlopyrifos indoor virulence to Bradysia odoriphaga 4 instar larvaes; 2012, Li Nana, Jianping YANG adopted dip method to measure the indoor virulence of botanical pesticide to olaiphage larva in Chinese Chive.
Above-mentioned living survey work provides great help for Chemicals and the field control of olaiphage larva in Chinese Chive really, but has problems.Dip method is applicable to the Toxicity Determination take action of contace poison as major pharmaceutical, for take stomach poison function as main medicament Toxicity Determination, causing very large systematic error.Stomach toxicity is tagged in co-toxicity method mensuration process, and the leek of soaking medicament is very easily rotten, non-being poisoned to death and the inaccuracy of result that can cause the fragrant-flowered garlic maggot.In the living test of above two kinds of methods is tested, give birth to the survey personnel for keeping the interior suitable humidity of ware need to add at least water one time every day, workload is large, complex operation, and in ware, humidity is not easy to control.Existing fragrant-flowered garlic maggot gives birth to the feed that survey technology adopts, be generally the natural fresh leek, so just caused seasonal feed shortage, and can't guarantee the leek non agricultural chemical residuum that uses in living test is tested, be unfavorable for that the fragrant-flowered garlic maggot gives birth to the process of carrying out and affect Chemicals of survey work.
(3) summary of the invention
The present invention is take existing research method as basis, for the existing deficiency that exists in the survey method of giving birth to, proposes, and purpose is to provide the assay method of a kind of easy, quick, accurate, economic pesticide to the olaiphage larva in Chinese Chive virulence.
To achieve these goals, living survey method provided by the invention comprises the following steps: (1) examination worm for the slow eye fungus gnat larva that gathers from field indoor expansion numerous after stable population or directly adopt the indoor feeding population in many generations; (2) with reference to the artificial synthetic diet formula (the artificial synthetic diet of a kind of fragrant-flowered garlic maggot, the patent No. 200810238714.0) of firm invention such as the people of grade, make the fragrant-flowered garlic maggot artificial synthetic diet of eye fungus gnat larva late, after carrying out, be kept in 4 ℃ of refrigerators stand-by; (3) artificial synthetic diet extremely melts with microwave-oven-heating before use, according to the experimental concentration designing requirement by preparing protodrug be diluted to a series of 10 times to the mother liquor of test design concentration, then mother liquor and the mass ratio of the artificial synthetic diet that has melted by 1:9 are mixed, make mixed medicine man-made feeds, after mixed medicine man-made feeds solidify, be cut into 0.5cm
3Fritter; (4) by after 10 times of a series of 10 times of mother liquors to experimental design concentration dilutions, drip on the filter paper of 1.2mL in the 9cm double dish, and filter paper is paved.The mixed medicine man-made feeds fritter that will cut again is placed in the double dish that is covered with identical liquid processing filter paper, then step (1) is expanded to the agglomerating larva of the consistent gathering of numerous rear growth and chooses gently to double dish feed fritter with writing brush; (5) above double dish again lid layer soaked the black cotton of water, to keep moistening in ware, and the fragrant-flowered garlic maggot is in dark, reduce creeping of fragrant-flowered garlic maggot, be conducive to the survival of fragrant-flowered garlic maggot.Double dish is placed under 20～27 ℃ and cultivates, kept as the case may be the humidity in feed and ware; (6) check result after certain hour, obtain the toxicity test result of certain medicament to olaiphage larva in Chinese Chive.
In the method for the invention, described step (3) further comprises mixed medicine man-made feeds.In manufacturing process, the ratio of medicament mother liquor and man-made feeds is 1:9.The medicament mother liquor is definite through test of many times with the ratio of artificial synthetic diet, and both mass ratioes only just can reach best test effect when 1:9.When ratio is too high, affects the fragrant-flowered garlic maggot to the taxis of artificial synthetic diet and take food; When ratio was too low, the molding effect of artificial synthetic diet was bad.Liquid preparation is that the medicinal acetone of raw insecticide (or the organic solvents such as methyl alcohol, dimethylbenzene, DMF) is dissolved, and with massfraction, is then that 0.1% Tween-80 aqueous solution directly is mixed with desired concn (if the material that preparing protodrug is indissoluble can add peaceful newborn 34#, xanthans etc. to become certain density aqueous suspension agent mother liquor to add through attrition process again).Artificial synthetic diet, fully mixes and makes mixed medicine man-made feeds with medicament mother liquor after dilution to after melting through microwave-oven-heating.Described step (4) further comprises tests the first bromine aqueous ammonium soaked overnight in the last of the ten Heavenly stems that the double dish (9cm) that uses is first used massfraction 0.5%, then with clear water, rinses, dries rear use after with liquid detergent, cleaning; With writing brush, provoke gently and assemble agglomerating and olaiphage larva in Chinese Chive of the same size in feed central authorities, 10～50, every ware.Described step (5) further comprises for common insecticides and part novel pesticide processes latter 48 hours or 72 hours check results, comprises at least organic phosphates, carbamates, pyrethroid, macrolide Avermectins, oxadiazine class, pyroles, plant source insecticides; The insect growth regulator, IGR insecticides is for checking the mortality ratio of larval mortality, percentage of pupation and a pupa every 24 hours after processing, until pupa is all sprouted wings or death, this type of medicament comprises moulting hormone analog, juvenoid at least.
Beneficial effect of the present invention
1) Chinese medicine mother liquor of the present invention is determined through test of many times with the ratio of artificial synthetic diet, and both mass ratioes only just can reach best test effect when 1:9.Stomach toxicity is tagged co-toxicity method and dip method while adopting leek to do experiment feed, needs in time rotten leek is chosen and changes fresh leek, and complex operation, the smell is awful; And mixed medicine man-made feeds are compared with it, have non-perishable characteristics, have greatly reduced the workload of test, have improved the accuracy that living test is tested.
2) man-made feeds that use in the present invention have good moisture retention, have reduced the probability that the examination worm is uncomfortable because of humidity, die unexpectedly, and have obviously reduced number of times and the water yield that adds water, and are time saving and energy saving.
3) the man-made feeds material that uses in the present invention is easily preserved, and can solve seasonal feed shortage problem, and for avoiding the fresh leek agricultural chemicals high residue impact that market is bought to accomplish " to the disposable Detecting Pesticide of feed material, repeatedly relieved use ".
4) herb liquid of the present invention is evenly distributed in man-made feeds, has strengthened the dispersion effect of liquid in man-made feeds, and then has improved the medicament Stability and veracity that action of contace poison is measured to examination worm stomach toxicity.
(4) embodiment
Below the embodiment of this research further described.
Embodiment 1: the purpose of this experiment is to adopt man-made feeds to mix tag co-toxicity method and dip method of medicine method, stomach toxicity and measures respectively the indoor virulence of the former medicine of chlopyrifos to the Bradysia odoriphaga four-age larva.
With reference to firm artificial synthetic diet formula (the artificial synthetic diet of a kind of fragrant-flowered garlic maggot that waits people's invention, the patent No. 200810238714.0) make eye fungus gnat larvae feeds late, and from approximately 250 of field collection olaiphage larva in Chinese Chives, at indoor feeding, to the third generation, with writing brush, provoke gently and assemble the agglomerating and examination worm suitable length of time of the same size and carry out this test.Accurately take the former medicine 1.0309g of 97% chlopyrifos, with acetone solution, be settled in the 100mL volumetric flask, be made into 10000mgL
-1The chlopyrifos mother liquor, with 0.1% Tween-80 aqueous solution by mother liquor be diluted to 400,200,80,40,20mgL
-1For dip method, stomach toxicity, tag co-toxicity method Chlorpyrifos to the Toxicity Determination of Bradysia odoriphaga 4 instar larvaes, with 0.1% Tween-80 aqueous solution by mother liquor be diluted to 4000,2000,800,400,200mgL
-1For the Toxicity Determination of the mixed medicine method Chlorpyrifos of man-made feeds to Bradysia odoriphaga 4 instar larvaes.Three kinds of Toxicity Determination method operations are as follows:
1 man-made feeds mix the medicine method
Take the artificial synthetic diet 4.5g that has melted, the liquid 0.5g that has diluted is evenly sneaked in the artificial synthetic diet of making, make mixed medicine man-made feeds, after feed solidifies, be cut into 0.5cm
3Fritter.Mother liquor is diluted respectively to 10 times, get 1.2mL and evenly drip on the filter paper in being laid in the 9cm double dish.The mixed medicine man-made feeds fritter that cuts is placed in and is covered with the double dish central authorities that the same concentrations liquid is processed filter paper, and the 4 agglomerating instar larvaes of consistent gathering of then growing are chosen to double dish feed fritter gently with writing brush; Every ware access examination 25 left and right of worm, every processing repeats 3 times, establishes simultaneously the feed blank that does not contain medicament.Above double dish again lid layer soaked the black cotton of water, to keep moistening in ware, and the fragrant-flowered garlic maggot is in dark, reduce creeping of fragrant-flowered garlic maggot, be conducive to the survival of fragrant-flowered garlic maggot.Putting into 25 ℃, relative humidity and be 60% unglazed incubator raises.72h check result after medicine, touch polypide with the writing brush point, and the motionless or health of larva obviously is punctured into death.Calculation correction mortality ratio and LC
50Value etc.
The 2 stomach toxicitys co-toxicity method of tagging
Filter paper is infiltrated in the liquid that has diluted, dry slightly with dripless and form and be as the criterion, be laid in the 9cm double dish.The white portion of leek stem foot is cut into to the 2cm segment, after in the variable concentrations liquid, soaking 30s, take out 4 sections leek stems, on thieving paper, suck unnecessary liquid and be placed on and be covered with identical liquid and process in the double dish of filter paper, then with writing brush, provoke gently and assemble agglomerating and 4 instar larvaes of the same size in double dish.Every ware examination 25 left and right of worm, repeat 3 times, establishes simultaneously without the medicine blank.Putting into 25 ℃, relative humidity and be 60% unglazed incubator raises.Result checks and calculates with 1.
3 dip methods
Filter paper is converted into to funnel-form, with writing brush, provokes gently and assemble agglomerating and 4 instar larvaes of the same size in the filter paper center, then drip in funnel with the pipette, extract liquid, larva is soaked 30s in liquid after, with thieving paper, suck unnecessary liquid in funnel.To soak the examination worm of liquid and choose and be placed in the double dish that is lined with clean filter paper (not processing with liquid) with writing brush, on filter paper, quantitative watering 1.2mL, with moisturizing, is put the long fresh leek stem of 4 sections 2cm and is made feed.Put into 25 ℃, relative humidity and be 60% unglazed incubator and raise, 25 left and right of every ware, every processing repeats 3 times.If without the medicine blank.Result checks and calculates with 1.
Test findings is in Table 1,2,3.
The former medicine of table 1 chlopyrifos is to Bradysia odoriphaga 4 instar larvae Toxicity Determination results
The former medicine of table 2 chlopyrifos is to Bradysia odoriphaga 4 instar larvae Toxicity Determination interpretations of result ()
From result of calculation, can find out: the degree of fitting of three kinds of living survey methods probit analysis model separately and level of significance p value thereof show that the mixed medicine method of man-made feeds of the present invention is better than stomach toxicity and tags that (the Chi-square Test value is respectively 0.0615,0.5557,0.7564 for co-toxicity method and dip method; The p value is respectively 0.996,0.8599,0.9065, all greater than 0.05).While adopting Toxicity Determination method of the present invention, it is good that each repeats collimation, LC
50Be worth with a high credibilityly, fiducial interval is little, and correlativity is high, and to give birth to survey methods suitable with other for slope error.In whole test 72h, the mixed medicine method of man-made feeds of the present invention only adds water once, and stomach toxicity tag co-toxicity method and dip method respectively add water 4 times, change feed 3 times, and leek is rotted obviously.From the number of times that adds water and replacing feed, can find out, method moisture retention of the present invention is good, really can reduce in test the input of manpower.
The former medicine of table 3 chlopyrifos is to Bradysia odoriphaga 4 instar larvae Toxicity Determination interpretations of result (two)
These three kinds of medicaments are carried out to the test of hypothesis of regression straight line collimation, and its result can find out from the results of analysis of variance, and significantly (F=3.0855, less than F=8.05 for the difference of three kinds of living survey methods; The p value is greater than 0.05), so there is close slope in these three tropic.From lethal dose (LC
50, LC
95) ratio measures, the LC of the former medicine of chlopyrifos
0, LC
95Ratio 95% fiducial interval all comprise 1, so the difference between the lethal dose of these three kinds living survey methods is not remarkable.
From the Toxicity Determination of the former medicine of above chlopyrifos, show that methodological science of the present invention is reliable, can reach and give birth to the requirement of surveying.
Embodiment 2: the purpose of this experiment is to adopt man-made feeds to mix tag co-toxicity method and dip method of medicine method, stomach toxicity and measures respectively the indoor virulence of the former medicine of clothianidin to the Bradysia odoriphaga four-age larva.
With reference to firm artificial synthetic diet formula (the artificial synthetic diet of a kind of fragrant-flowered garlic maggot that waits people's invention, the patent No. 200810238714.0) make fragrant-flowered garlic maggot eye fungus gnat feed late, and from approximately 250 of field collection olaiphage larva in Chinese Chives, at indoor feeding, to the third generation, with writing brush, provoke gently and assemble the agglomerating and examination worm suitable length of time of the same size and carry out this test.Accurately take the former medicine 1.0417g of 94% clothianidin, add peaceful newborn 34#3.0012g, xanthans 0.1502g, deionized water is supplied 100g, after add the 100mL abrasive media to grind 2 hours, the 10000mgL of system
-1The aqueous suspension agent mother liquor, with deionized water by mother liquor be diluted to 10,7.5,5,2.5,1mgL
-1For dip method, stomach toxicity, tag co-toxicity method Chlorpyrifos to the Toxicity Determination of Bradysia odoriphaga 4 instar larvaes, with deionized water by mother liquor be diluted to 100,50,25,12.5,10mgL
-1For the Toxicity Determination of the mixed medicine method Chlorpyrifos of man-made feeds to Bradysia odoriphaga 4 instar larvaes.Three kinds of Toxicity Determination method operations are as follows:
1 man-made feeds mix the medicine method
Take the artificial synthetic diet 4.5g that has melted, the liquid 0.5g that has diluted is evenly sneaked in the artificial synthetic diet of making, make mixed medicine man-made feeds, after feed solidifies, be cut into 0.5cm
3Fritter.Mother liquor is diluted respectively to 10 times, gets 1.2mL and evenly drip on the filter paper in being laid in the 9cm double dish.The mixed medicine man-made feeds fritter that cuts is placed in and is covered with the double dish central authorities that the same concentrations liquid is processed filter paper, and the 4 agglomerating instar larvaes of consistent gathering of then growing are chosen to double dish feed fritter gently with writing brush; Every ware access examination 25 left and right of worm, every processing repeats 3 times, establishes simultaneously the feed blank that does not contain medicament.Above double dish again lid layer soaked the black cotton of water, to keep moistening in ware, and the fragrant-flowered garlic maggot is in dark, reduce creeping of fragrant-flowered garlic maggot, be conducive to the survival of fragrant-flowered garlic maggot.Putting into 25 ℃, relative humidity and be 60% unglazed incubator raises.72h check result after medicine, touch polypide with the writing brush point, and the motionless or health of larva obviously is punctured into death.Calculation correction mortality ratio and LC
50Value etc.
The 2 stomach toxicitys co-toxicity method of tagging
Filter paper is infiltrated in the liquid that has diluted, dry slightly with dripless and form and be as the criterion, be laid in the 9cm double dish.The white portion of leek stem foot is cut into to the 2cm segment, after in the variable concentrations liquid, soaking 30s, take out 4 sections leek stems, on thieving paper, suck unnecessary liquid and be placed on and be covered with identical liquid and process in the double dish of filter paper, then with writing brush, provoke gently and assemble agglomerating and 4 instar larvaes of the same size in double dish.Every ware examination 25 left and right of worm, repeat 3 times, establishes simultaneously without the blank clear water contrast of medicine.Putting into 25 ℃, relative humidity and be 60% unglazed incubator raises.Result checks and calculates with 1.
3 dip methods
Filter paper is converted into to funnel-form, with writing brush, provokes gently and assemble agglomerating and 4 instar larvaes of the same size in the filter paper center, then drip in funnel with the pipette, extract liquid, larva is soaked 30s in liquid after, with thieving paper, suck unnecessary liquid in funnel.To soak the examination worm of liquid and choose and be placed in the double dish that is lined with clean filter paper (not processing with liquid) with writing brush, on filter paper, quantitative watering 0.5mL, with moisturizing, is put the long fresh leek stem of 4 sections 2cm and is made feed.Put into 25 ℃, relative humidity and be 60% unglazed incubator and raise, 25 left and right of every ware, every processing repeats 3 times.If without the medicine blank.Result checks and calculates with 1.
Test findings is in Table 4,5,6.
The former medicine of table 4 clothianidin is to Bradysia odoriphaga 4 instar larvae Toxicity Determination results
The former medicine of table 5 clothianidin is to Bradysia odoriphaga 4 instar larvae Toxicity Determination interpretations of result ()
From result of calculation, can find out: the degree of fitting of three kinds of living survey methods probit analysis model separately and level of significance p value thereof show that the mixed medicine method of man-made feeds of the present invention is better than stomach toxicity and tags that (the Chi-square Test value is respectively 0.8454,5.6355,5.9771 for co-toxicity method and dip method; The p value is respectively 0.8386,0.1308,0.1127, all greater than 0.05).While adopting Toxicity Determination method of the present invention, it is good that each repeats collimation, LC
50Be worth with a high credibilityly, fiducial interval is little, and correlativity is high, and to give birth to survey methods suitable with other for slope error.In whole test 72h, the mixed medicine method of man-made feeds of the present invention only adds water once, and tag co-toxicity method and dip method of stomach toxicity respectively adds water 5 times, change feed 2 times, and leek rotted obviously.From the number of times that adds water and replacing feed, can find out, method moisture retention of the present invention is good, and is not perishable, really can reduce in test the input of manpower.
The former medicine of table 6 clothianidin is to Bradysia odoriphaga 4 instar larvae Toxicity Determination interpretations of result (two)
These three kinds of medicaments are carried out to the test of hypothesis of regression straight line collimation, and its result can find out from the results of analysis of variance, and significantly (F=0.0697, less than F=8.05 for the difference of three kinds of living survey methods; The p value is greater than 0.05), so there is identical slope in these three tropic.From lethasl concentration (LC) ratio, measure, tag co-toxicity method and dip method of stomach toxicity records the LC of the former medicine of clothianidin
50Ratio 95% fiducial interval comprise 1, so these two kinds of methods record the LC of the former medicine of clothianidin
50Difference is not remarkable, but the mixed medicine method of this and man-made feeds of the present invention records the LC of the former medicine of clothianidin
5xSignificant difference.Three kinds of living survey methods record the LC of the former medicine of clothianidin
95Ratio 95% fiducial interval all comprise 1, so these three kinds living survey methods record the LC of the former medicine of clothianidin
95Between difference not remarkable.
From the Toxicity Determination of the former medicine of above clothianidin, show that methodological science of the present invention is reliable, can reach and give birth to the requirement of surveying.
Embodiment 3: the purpose of this experiment is to adopt man-made feeds to mix tag co-toxicity method and dip method of medicine method, stomach toxicity and measures respectively the indoor virulence of the former medicine of Nylar to the Bradysia odoriphaga four-age larva.
With reference to firm artificial synthetic diet formula (the artificial synthetic diet of a kind of fragrant-flowered garlic maggot that waits people's invention, the patent No. 200810238714.0) make fragrant-flowered garlic maggot eye fungus gnat feed late, and from approximately 250 of field collection olaiphage larva in Chinese Chives, at indoor feeding, to the third generation, with writing brush, provoke gently and assemble the agglomerating and examination worm suitable length of time of the same size and carry out this test.Accurately take the former medicine 1.0204g of 98% Nylar, with acetone solution, be settled in the 100mL volumetric flask, be made into 10000mgL
-1The Nylar mother liquor, with 0.1% Tween-80 aqueous solution by mother liquor be diluted to 400,200,80,40,20mgL
-1For dip method, stomach toxicity, tag co-toxicity method Chlorpyrifos to the Toxicity Determination of Bradysia odoriphaga 4 instar larvaes, with 0.1% Tween-80 aqueous solution by mother liquor be diluted to 4000,2000,800,400,200mgL
-1For the Toxicity Determination of the mixed medicine method Chlorpyrifos of man-made feeds to Bradysia odoriphaga 4 instar larvaes.Three kinds of Toxicity Determination method operations are as follows:
1 man-made feeds mix the medicine method
Take the artificial synthetic diet 4.5g that has melted, the liquid 0.5g that has diluted is evenly sneaked in the artificial synthetic diet of making, make mixed medicine man-made feeds, after feed solidifies, be cut into 0.5cm
3Fritter.Mother liquor is diluted respectively to 10 times, gets 1.2mL and evenly drip on the filter paper in being laid in the 9cm double dish.The mixed medicine man-made feeds fritter that cuts is placed in and is covered with the double dish central authorities that the same concentrations liquid is processed filter paper, and the 4 agglomerating instar larvaes of consistent gathering of then growing are chosen to double dish feed fritter gently with writing brush; Every ware access examination 25 left and right of worm, every processing repeats 3 times, establishes simultaneously the feed blank that does not contain medicament.Above double dish again lid layer soaked the black cotton of water, to keep moistening in ware, and the fragrant-flowered garlic maggot is in dark, reduce creeping of fragrant-flowered garlic maggot, be conducive to the survival of fragrant-flowered garlic maggot.Putting into 25 ℃, relative humidity and be 60% unglazed incubator raises.After medicine, every 24 hours, check the mortality ratio of larval mortality, percentage of pupation and a pupa, until pupa is all sprouted wings or death, with the writing brush point, touch polypide, the motionless or health of larva obviously is punctured into death.The calculation correction mortality ratio.
The 2 stomach toxicitys co-toxicity method of tagging
Filter paper is infiltrated in the liquid that has diluted, dry slightly with dripless and form and be as the criterion, be laid in the 9cm double dish.The white portion of leek stem foot is cut into to the 2cm segment, after in the variable concentrations liquid, soaking 30s, take out 4 sections leek stems, on thieving paper, suck unnecessary liquid and be placed on and be covered with identical liquid and process in the double dish of filter paper, then with writing brush, provoke gently and assemble agglomerating and 4 instar larvaes of the same size in double dish.Every ware examination 20 left and right of worm, repeat 3 times, establishes simultaneously without the medicine blank.Putting into 25 ℃, relative humidity and be 60% unglazed incubator raises.Result checks and calculates with 1.
3 dip methods
Filter paper is converted into to funnel-form, with writing brush, provokes gently and assemble agglomerating and 4 instar larvaes of the same size in the filter paper center, then drip in funnel with the pipette, extract liquid, larva is soaked 30s in liquid after, with thieving paper, suck unnecessary liquid in funnel.To soak the examination worm of liquid and choose and be placed in the double dish that is lined with clean filter paper (not processing with liquid) with writing brush, on filter paper, quantitative watering 1.2mL, with moisturizing, is put the long fresh leek stem of 4 sections 2cm and is made feed.Put into 25 ℃, relative humidity and be 60% unglazed incubator and raise, 25 left and right of every ware, every processing repeats 3 times.If without the medicine blank.Result checks and calculates with 1.
Test findings is in Table 7.
The former medicine of table 7 Nylar is to Bradysia odoriphaga 4 instar larvae Toxicity Determination results
In giving birth to the survey process, the mixed medicine method of man-made feeds of the present invention remains on two days and adds water one time, the feed that adds during beginning can be maintained to larvae pupation, and tag co-toxicity method and dip method of stomach toxicity adds water 1～2 time every day and change leek section feed once, and leek is rotted obviously.From the number of times that adds water and replacing feed, can find out, method moisture retention of the present invention is good, and is not perishable, simple to operate, really can reduce in test the input of manpower.From result of calculation, can find out: three kinds of living survey method mensuration Nylars are basically identical to the indoor virulence result of slow eye fungus gnat larva, also illustrated that methodological science of the present invention is reliable, can reach and give birth to the requirement of surveying.
Claims (5)
1. the pesticide indoor biometrics method to olaiphage larva in Chinese Chive is characterized in that: comprise the following steps:
(1), according to the experimental concentration design, make the mixed medicine man-made feeds of series concentration pesticide;
(2), after filter paper being laid at the bottom of double dish, add respectively quantitative series concentration liquid complete wetting filter paper;
(3) will mix the medicine man-made feeds and be cut into small pieces, and put into and be covered with the above-mentioned double dish that the corresponding concentration liquid is processed filter paper;
(4) by on the mixed medicine man-made feeds of of the right age larva access, after certain hour, check death condition, calculate the virulence data.
2. method according to claim 1, it is characterized in that: wherein method step 1) described mixed medicine man-made feeds are in manufacturing process, the proportional range of mother liquor and man-made feeds is 1:9; The liquid preparation is by the medicinal acetone of raw insecticide or methyl alcohol or dimethylbenzene or DMF organic solvent dissolution, with massfraction, is then that 0.1% Tween-80 aqueous solution directly is mixed with desired concn: if the material that preparing protodrug is indissoluble can add peaceful newborn 34#, xanthans etc. to add after attrition process becomes certain density aqueous suspension agent mother liquor; Every concentration repeats 3～4 times; Artificial synthetic diet, fully mixes and makes the mixed medicine man-made feeds of series concentration pesticide with medicament mother liquor after dilution to after melting through microwave-oven-heating.
3. method according to claim 1, it is characterized in that: wherein method step 2) double dish that described test is used, first use the first bromine aqueous ammonium soaked overnight in the last of the ten Heavenly stems of massfraction 0.5%, then with clear water, rinse, dry rear use after with liquid detergent, cleaning.
4. method according to claim 1, it is characterized in that: wherein method step 3) described mixed medicine man-made feeds block sizes is 0.5～1cm
3, add 4～6.
5. method according to claim 1, it is characterized in that: wherein method step 4) described for common insecticides and the processing of part novel pesticide latter 48 hours or 72 hours check results, comprise at least organic phosphates, carbamates, pyrethroid, Avermectins, oxadiazine class, pyroles, macrolides, plant source insecticides; The insect growth regulator, IGR insecticides is for checking the mortality ratio of larval mortality, percentage of pupation and a pupa every 24 hours after processing, until pupa is all sprouted wings or death, this type of medicament comprises moulting hormone analog or juvenoid at least.
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