CN102577897B - Method for ecological safety evaluation on nilaparvata lugens by Bt genetically modified rice based on intervention of pesticides - Google Patents
Method for ecological safety evaluation on nilaparvata lugens by Bt genetically modified rice based on intervention of pesticides Download PDFInfo
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Abstract
The invention relates to a method for ecological safety evaluation on nilaparvata lugens by Bt genetically modified rice based on intervention of pesticides. The method provided by the invention comprises the following steps: selecting triazophos and validamycin to simulate a pesticide spraying method for rice fields to respectively spraying the farm chemicals to the Bt genetically modified rice plant and a parent comparison rice plant during the tillering stage of the rice plant, collecting lower rice plant stems with a same growing condition for raising lately hatched nymphs of the nilaparvata lugens in an illumination incubator, recording the growing parameters of the nilaparvata lugens population and analyzing and comparing the population generation indexes after the farm chemicals are sprayed for 24h; putting 100 nymphs in third instar on each potted rice plant, checking the survival rate of the nymphs every 24h, and observing the injury grade of the rice plants respectively at seventh day and fourteenth day after the rice plants receiving the nymphs; and comparing the oxalate contents of the two rice plants after the farm chemicals are sprayed for 7 days. According to the invention, the facticity of ecological safety harmfulness evaluation on non-target important nilaparvata lugens by the Bt genetically modified rice plant is increased, and the method is low in cost and easy to popularize.
Description
Technical field
The present invention relates to a kind of based on the method for Bt Transgenic Rice to the brown planthopper safety evaluatio after the agricultural chemicals intervention.
Background technology
Paddy rice is one of most important cereal crops of China, insect is the key factor that causes Rice Yield Loss Caused, the loss that causes as lepidoptera pest (striped rice borer, paddy stem borer, pink rice borer and rice leaf roller etc.) accounts for 2%～10% of Asia annual production, and the loss that China causes because of insect pest every year accounts for more than 5% of paddy rice gross yield.Along with the progress of biotechnology, anti-lepidoptera pest trans Bt gene (thuringiensis) rice varieties successfully comes out, and studies confirm that much they have obvious resistance to target pest.But Bt Transgenic Rice is not worldwide commercially produced widely, and this depends on their ecological environment security and foodsafety to a great extent.In the ecological environment security evaluation, be the important content of anti-lepidoptera pest Bt Transgenic Rice safety evaluatio to the evaluation of non-target pest occurrence risk.
Turn Cry2A gene (a kind of toxalbumin expressing gene) paddy rice T2A-1 from State Key Laboratory of Crop Genetic Improvent molecular biology locellus, the genes of interest of introducing in genetically modified plants be artificial reconstructed synthetic thuringiensis (
Bacillus thuringiensis, Bt).From 2005, transgenic pest-resistant rice T2A-1 has successively carried out pilot experiment and environment and has discharged in Hubei Province, result of the test shows, the T2A-1 inheritance stability, harmless and economical character and former kind bright extensive 63 are basically identical to non-target organism, paddy rice main harm snout moth's larva (striped rice borer and rice leaf roller) is had extremely strong and single-minded toxicity, but to non-lepidoptera pest all without any resistance, especially in recent years break out continuously as paddy rice the important pests brown planthopper that causes disaster.For this reason, domestic a lot of expert has set up some Bt Transgenic Rices to rice fulgorid impact assessment method, investigate this insect growth and development state as artificial feeding brown planthopper on Bt Transgenic Rice, or the Occurrence of this insect of field investigation on Bt Transgenic Rice.These methods can fundamental reaction Bt Transgenic Rices on the direct impact of brown planthopper, but do not consider the impact of other environmental factors in the Rice Production process, especially as the effect to brown planthopper of the agricultural chemicals of chemical control.In fact, control preferably effect although Bt Transgenic Rice can have the target lepidoptera pest, the field will continue to use for the chemical pesticide of the non-target pest of Bt Transgenic Rice undoubtedly.Therefore, set up a kind of agricultural chemicals and get involved rear Bt Transgenic Rice to the method for brown planthopper safety evaluatio, will provide important technical support for the effective prevention and control of brown planthopper in Bt Transgenic Rice more practically.
Summary of the invention
The objective of the invention is for above-mentioned present situation, aim to provide a kind of can be conscientiously for Bt Transgenic Rice to the Occurrence of non-target important pests brown planthopper make safety evaluation, be easy to promote the use of get involved based on agricultural chemicals after Bt Transgenic Rice to the method for brown planthopper safety evaluatio.
Implementation of the present invention is that the method for Bt Transgenic Rice to the brown planthopper safety evaluatio after getting involved based on agricultural chemicals comprises the following steps:
1) selection of agricultural chemicals: select Hostathion, the former medicine of jinggangmeisu, concentration setting: Hostathion 10,20,40,80ppm; Jinggangmeisu 100,200,400,800ppm;
2) processing method: adopt pot rice to connect the worm method, the spray-on process dispenser;
3) spray medicine: spray pot rice in tillering stage, spray by pesticide concentration order from low to high during sprinkling, rice leaf and stem part are processed in spraying; Pot rice after processing covers guard, and each processing and contrast all arrange 5 repetitions, represents in contrast with water spray and does not use pesticide-treated;
4) safety is investigated:
1. Population of Rice Brown Planthopper life parameters: Bt Transgenic Rice and parent contrast paddy rice spray respectively medicine 24h simultaneously after, gather the consistent paddy rice middle and lower part cane of growing way, be placed in glass, two strain paddy rice/cups, rear every glass connects first 10 of the brown planthopper nymphs that incubate, cultivate in illumination box, each is processed 5 times and repeats; Adopt the indoor Population life-table method of brown planthopper, the life parameters of Population of Rice Brown Planthopper in record process-each survival rate and the length of time, adult eclosion rate, male and female ratio, length wing type ratio, female worm life-span, calculate trend index of population in length of time;
2. paddy rice mined grade: cover pot rice with 80 order gauzes after spray medicine 24h, 100 of every cave paddy rice accesses nymph in 3 age, 300, every basin, the contrast water sprays, all are grown under natural field for the rice of trying the water, each contrast and process 20 repetitions checked the survival condition of a nymph in every 24 hours, and did corresponding nymph and replenish.7d and 14d observe mined grade after paddy rice connects worm respectively;
3. rice plant oxalic acid: adopt titanium trichloride determination of color oxalic acid content, get after pesticide-treated the rice plant of 7 days, weigh rear with crossing leaching juice after the tissue mashing crusher machine, 30min at normal temperatures decolours to add active carbon (addition be Normal juice volume 1/4), then use 80-2 type centrifuge isolating active charcoal, centrifuge speed is 3000 r/min, after solution is colourless or is creamy white, getting 3ml rice juice, to add 0.15ml concentration be that 1% titanium trichloride is molten) colour developing, measuring light absorption value on spectrophotometer under 400 nm, prepare calibration curve with oxalic acid, obtain regression equation, obtain thus the content of oxalic acid.
The present invention shows, after jinggangmeisu, two kinds of agricultural chemicals of Hostathion use, there is significant difference in the impact that contrasts brown planthopper resistant content of material in the generation of Population of Rice Brown Planthopper on paddy rice and paddy rice body on turning Bt paddy rice and parent, has estimated out the ecological security of Bt Transgenic Rice to non-target important pests brown planthopper Occurrence more effectively.
The present invention based on agricultural chemicals field practical application concentration, around three important investigation indexs of brown planthopper Occurrence, can operate in indoor and half field and complete, more understandable grasp on operation sequence.Cost of the present invention aspect is cheap, as long as basic equipment that the insects such as some insectaries, illumination box raise and centrifuge, spectrophotometric determination instrument are arranged, is easy to promote the use of.
Description of drawings
Fig. 1 is that jinggangmeisu is processed forward and backward Bt Transgenic Rice (T2A-1) and the parent contrasts the upward variation diagram of the female egg laying amount of list of brown planthopper of paddy rice (MH63);
Fig. 2 is that Hostathion is processed forward and backward Bt Transgenic Rice (T2A-1) and the parent contrasts the upward variation diagram of the female egg laying amount of list of brown planthopper of paddy rice (MH63);
Fig. 3 is that jinggangmeisu is processed forward and backward Bt Transgenic Rice (T2A-1) and the parent contrasts the upward variation diagram of the trend index of population of brown planthopper of paddy rice (MH63);
Fig. 4 is that Hostathion is processed forward and backward Bt Transgenic Rice (T2A-1) and the parent contrasts the upward variation diagram of the trend index of population of brown planthopper of paddy rice (MH63);
Fig. 5 is the variation diagram that jinggangmeisu processing forward and backward Bt Transgenic Rice (T2A-1) and parent contrast paddy rice (MH63) stem stalk mesoxalic acid content;
Fig. 6 is the variation that Hostathion processing forward and backward Bt Transgenic Rice (T2A-1) and parent contrast paddy rice (MH63) stem stalk mesoxalic acid content.
Embodiment
The present invention selects the reason of Hostathion, jinggangmeisu to be: Hostathion is the chemical pesticide of rice field control commonly used snout moth's larva, but because Bt Transgenic Rice is not can be to all lepidoptera pests in the field, as pink rice borer, good prevention and control effect is arranged, so this medicine will use in the Bt Transgenic Rice field; On the other hand, the reproduction that the existing unreasonable use that studies confirm that Hostathion can stimulate brown planthopper namely promotes the Occurrence of this insect.Whether this potential risk exists on Bt Transgenic Rice or amplifies to some extent, and this is important content of the present invention.Jinggangmeisu is the conventional disease control chemical pesticide in rice field, is also common pesticides at Bt Transgenic Rice, to insect without direct toxic action, but may the generation of remote-effects brown planthopper by affect paddy rice " physique " (the interior against-plant hopper index of body).Therefore, the present invention selects Hostathion, two kinds of rice field common pesticides relevant to brown planthopper harm of jinggangmeisu.
1, the selection of material and using method:
The selection of agricultural chemicals: select Hostathion, jinggangmeisu, test agricultural chemicals used and be former medicine, jinggangmeisu is available from Yangzhou pioneer's chemical industry Co., Ltd, and Hostathion is available from Yangzhou agriculture green for a long time Co., Ltd.Concentration arranges: Hostathion 10,20,40,80ppm; Jinggangmeisu 100,200,400,800ppm.
Adopt pot rice to connect the worm method, pesticide-treated is pressed pesticide concentration order from low to high in rice plant of tillering stage, spraying rice leaf and stem part.Bt Transgenic Rice (T2A-1) and parent contrast paddy rice (MH63) (Han Y., Xu XL., Ma WH., et al. 2011. The Influence of Transgenic cry1Ab/cry1Ac, cry1C and cry2A Rice on Non-Target Planthoppers and Their Main Predators Under Field Conditions. Agricultural Sciences in China, 10 (11): 1739-1747.) (from State Key Laboratory of Crop Genetic Improvent molecular biology locellus, the depositary promises to undertake in 20 years and provides to the public.
Processing method: adopt pot rice to connect the worm method, the dispenser spray-on process is the spray-on process consistent with the field, and sprayer is the conical nozzle that a diameter 1mm is housed, pressure 45 psi, the simple and easy suitable device of flow 300mL/min.
The spray medicine: spray pot rice in tillering stage, spray by pesticide concentration order from low to high during sprinkling, rice leaf and stem part are processed in spraying; Pot rice after processing covers guard, in case other factors are disturbed.Each processing and contrast all arrange 5 repetitions, with water spray in contrast.
2, index is investigated in safety:
1) Population of Rice Brown Planthopper life parameters: after Bt Transgenic Rice and parent contrast paddy rice and spray respectively medicine 24h simultaneously, gather the consistent paddy rice middle and lower part cane of growing way, be placed in glass (two strain paddy rice/cups), rear every glass connects first 10 of the brown planthopper nymphs that incubate, cultivate in illumination box, each is processed 5 times and repeats.Adopt the indoor Population life-table method of brown planthopper, the life parameters of Population of Rice Brown Planthopper in record process (each survival rate and the length of time, adult eclosion rate, male and female ratio, length wing type ratio, female worm life-span etc., calculate trend index of population) in length of time.
2) paddy rice mined grade: cover pot rice with 80 order gauzes after spray medicine 24h, 100 3 nymphs in age (every basin 3 caves) of every cave paddy rice access, the contrast water sprays.All are grown under natural field for the rice of trying the water.Each contrast and process 20 repetitions checked the survival condition of a nymph in every 24 hours, and did replenishing of corresponding nymph.7d and 14d observe mined grade after paddy rice connects worm respectively.
3) rice plant oxalic acid content: in the paddy rice body, oxalic acid content is the important indicator that paddy rice is just estimated Brown Planthopper Resistance, and the higher resistance of content is stronger.
Oxalic acid content of the present invention is measured and is adopted the titanium trichloride development process, get after pesticide-treated the rice plant of 7 days, weigh rear with crossing leaching juice after the tissue mashing crusher machine, 30min at normal temperatures decolours to add active carbon (addition be Normal juice volume 1/4), then use 80-2 type centrifuge isolating active charcoal, centrifuge speed is 3000 r/min, and once decolouring not exclusively, carry out secondary decolourization with same method, treat that its solution is colourless or slightly is creamy white.The paddy rice juice 3ml that gets centrifugal decolouring adds 1% titanium trichloride solution (analyzing pure) 0.15ml colour developing, measuring light absorption value on spectrophotometer under 400 nm, prepares calibration curve with oxalic acid, obtains regression equation, obtains thus the content of oxalic acid.
4, evaluation result analysis
By above-mentioned processing method, after having obtained the conventional pesticide processing of two kinds, rice field, Bt Transgenic Rice and parent contrast paddy rice to the impact of brown planthopper Occurrence.The spy is analyzed as follows by brown planthopper hazard level and paddy rice cane oxalic acid content three aspects respectively from Population of Rice Brown Planthopper life parameters, paddy rice:
1) Population of Rice Brown Planthopper life parameters:
Pesticide-treated is forward and backward, and the population life parameters comparing result that the rear Bt Transgenic Rice (T2A-1) of Hostathion processing and parent contrast the upper brown planthopper of paddy rice (MH63) sees Table 1.The population life parameters comparing result that jinggangmeisu processing forward and backward Bt Transgenic Rice (T2A-1) and parent contrast the upper brown planthopper of paddy rice (MH63) sees Table 2.
From table 1 and table 2 as seen, in the situation that there is no pesticide-treated (CK), Bt Transgenic Rice and parent contrast Population of Rice Brown Planthopper life parameters no significant difference on paddy rice, this is consistent to Population of Rice Brown Planthopper impact assessment result with traditional Bt Transgenic Rice, i.e. Bt Transgenic Rice and the non-transgenic paddy rice no significant difference that affects on Population of Rice Brown Planthopper.
But after pesticide-treated, begin to occur difference between the Population of Rice Brown Planthopper growth in Population of Rice Brown Planthopper growth and conventional rice on Bt Transgenic Rice.Difference becomes with pesticide-treated concentration, especially is embodied in the reproduction aspect.When processing 80ppm as Hostathion on Bt Transgenic Rice the single female egg laying amount of brown planthopper be significantly higher than on the non-transgenic paddy rice and (see Fig. 1, Fig. 2), aspect Population of Rice Brown Planthopper occurrence tendency of future generation, during Hostathion 10ppm processes, Bt Transgenic Rice also is significantly higher than non-transgenic paddy rice (seeing figure, 3,4).Thereby confirm, jinggangmeisu, two kinds of pesticide-treated of Hostathion, Hostathion is more obvious on the impact of brown planthopper on Bt Transgenic Rice.Abscissa in Fig. 1,2 represents concentration, and ordinate represents single female egg laying amount (grain).
2) paddy rice mined grade:
With reference to table 3 and table 4, taken food aspect the contrast of harm by brown planthopper in paddy rice, during without pesticide-treated (CK), be subjected to the rank of brown planthopper harm a little more than the non-transgenic paddy rice on Bt Transgenic Rice, but the statistical analysis there was no significant difference.After jinggangmeisu is processed, be subjected to the rank of brown planthopper harm without significant difference (seeing Table 3,4) on two kinds of paddy rice, but after Hostathion is processed, Bt Transgenic Rice is subjected to the more conventional contrast of the rank paddy rice of brown planthopper harm that the trend (seeing Table 3,4) of increase is arranged, and shows that namely the use of Hostathion may increase the risk that Bt Transgenic Rice is subjected to brown planthopper harm.Abscissa in table 3,4 represents concentration, and ordinate represents trend index of population I.
3) paddy rice cane oxalic acid content:
Oxalic acid is one of representative biochemical substances of brown planthopper resistant harm in the paddy rice body, and its content height is proportional with resistance.The higher resistance of content is stronger.Oxalic acid content is measured and is adopted the titanium trichloride development process, get after pesticide-treated the rice plant of 7 days, weigh rear with crossing leaching juice after the tissue mashing crusher machine, 30min at normal temperatures decolours to add active carbon (addition be Normal juice volume 1/4), then use 80-2 type centrifuge isolating active charcoal, centrifuge speed is 3000 r/min, and once decolouring not exclusively, carry out secondary decolourization with same method, treat that its solution is colourless or slightly is creamy white.The paddy rice juice 3ml that gets centrifugal decolouring adds 1% titanium trichloride solution (analyzing pure) 0.15ml colour developing, measuring light absorption value on spectrophotometer under 400 nm, prepares calibration curve with oxalic acid, obtains regression equation, obtains thus the content of oxalic acid.
With reference to Fig. 5,6, without pesticide-treated the time, in Bt Transgenic Rice and non-transgenic Culm of Rice, oxalic acid content is without significant difference, and namely the brown planthopper resistant of oxalic acid level endangers indifference.But after jinggangmeisu, two kinds of pesticide-treated of Hostathion, in two kinds of paddy rice bodies, oxalic acid content is totally on a declining curve, and wherein the overall contents level of Bt Transgenic Rice is substantially lower than the non-transgenic paddy rice.(see figure 5) during jinggangmeisu 200ppm processes, in the Bt Transgenic Rice body, oxalic acid content is significantly lower than the content in non-transgenic paddy rice body, be Bt Transgenic Rice to the resistance of brown planthopper significantly lower than the non-transgenic paddy rice, similarly significant difference is also shown in Hostathion 20ppm and 40ppm(sees Fig. 6).This shows, after adding jinggangmeisu, two kinds of pesticide-treated of Hostathion, Bt Transgenic Rice to brown planthopper from the resistance aspect of oxalic acid angle lower than the non-transgenic paddy rice, and this is unforeseeable in traditional assessment technique.
The present invention shows, jinggangmeisu, two kinds of agricultural chemicals of Hostathion all can obviously affect the content of the brown planthopper resistant material in growing of Population of Rice Brown Planthopper and paddy rice body, wherein Hostathion to brown planthopper harm on Bt Transgenic Rice than favourable on the non-transgenic paddy rice.These results can not reflect in " one to one " effect ecological evaluation technology of traditional Bt Transgenic Rice to brown planthopper, and the present invention is Bt Transgenic Rice has increased practical property to the Occurrence ecological security assessment of non-target important pests brown planthopper.
Aspect the production practical application, after the Bt Transgenic Rice use, chemical pesticide access times and the usage amount controlled for main lepidoptera pest will obviously reduce, and the continuation of this respect agricultural chemicals is used and will be played the double effect that Bt Transgenic Rice is controlled weak lepidoptera pest of controlling.The present invention also confirms, when the working concentration of Hostathion is low, and impact and the basic indifference of conventional rice that brown planthopper on Bt Transgenic Rice is occured and causes harm.Therefore, the present invention shows that turning the Hostathion working concentration of causing harm safer to brown planthopper on the Cry2A trans-genetic hybrid rice is 20ppm.
The present invention based on agricultural chemicals field practical application concentration, around three important investigation indexs of brown planthopper Occurrence, can operate in indoor and half field and complete, more understandable grasp on operation sequence.Cost of the present invention aspect is cheap, as long as the basic equipment (insectary, illumination box) and the conventional plant biochemical substances determining instrument (centrifuge, spectrophotometer) that have some insects to raise are easy to promote the use of.
Table 3 jinggangmeisu processing forward and backward Bt trans-genetic hybrid rice (T2A-1) and parent contrast paddy rice (MH63) and contrasted by the rank of brown planthopper harm
Annotate: different lowercases represent the significant difference on 5% level in longitudinal comparison (Fisher ' s PLSD test).
Table 4 Hostathion processing forward and backward Bt Transgenic Rice (T2A-1) and parent contrast paddy rice (MH63) and contrasted by the rank of brown planthopper harm
Annotate: different lowercases represent the significant difference on 5% level in longitudinal comparison (Fisher ' s PLSD test).
Claims (2)
1. the method for Bt Transgenic Rice to the brown planthopper safety evaluatio after getting involved based on agricultural chemicals is characterized in that:
1) selection of agricultural chemicals: the former medicine of Hostathion and jinggangmeisu, concentration setting: Hostathion 10,20,40,80ppm; Jinggangmeisu 100,200,400,800ppm;
2) processing method: adopt pot rice to connect the worm method, the spray-on process dispenser;
3) spray medicine: spray pot rice in tillering stage, spray by pesticide concentration order from low to high during sprinkling, rice leaf and stem part are processed in spraying; Pot rice after processing covers guard, and each processing and contrast all arrange 5 repetitions, represents in contrast with water spray and does not use pesticide-treated;
4) safety is investigated:
1. Population of Rice Brown Planthopper life parameters: Bt Transgenic Rice and parent contrast paddy rice spray respectively medicine 24h simultaneously after, gather the consistent paddy rice middle and lower part cane of growing way, be placed in glass, two strain paddy rice/cups, rear every glass connects first 10 of the brown planthopper nymphs that incubate, cultivate in illumination box, each is processed 5 times and repeats; Adopt the indoor Population life-table method of brown planthopper, the life parameters of Population of Rice Brown Planthopper in record process-each survival rate and the length of time, adult eclosion rate, male and female ratio, length wing type ratio, female worm life-span, calculate trend index of population in length of time;
2. paddy rice mined grade: cover pot rice with 80 order gauzes after spray medicine 24h, 100 of every cave paddy rice accesses nymph in 3 age, 300, every basin, the contrast water sprays, all are grown under natural field for the rice of trying the water, each contrast and process 20 repetitions checked the survival condition of a nymph in every 24 hours, and did corresponding nymph and replenish; 7d and 14d observe mined grade after paddy rice connects worm respectively;
3. rice plant oxalic acid: adopt titanium trichloride determination of color oxalic acid content, get after pesticide-treated the rice plant of 7 days, weigh rear with crossing leaching juice after the tissue mashing crusher machine, add active carbon, addition is 1/4 of Normal juice volume, 30min at normal temperatures decolours, then use 80-2 type centrifuge isolating active charcoal, centrifuge speed is 3000 r/min, after solution is colourless or is creamy white, getting 3ml rice juice, to add 0.15ml concentration be 1% titanium trichloride solution colour developing, measuring light absorption value on spectrophotometer under 400 nm, prepare calibration curve with oxalic acid, obtain regression equation, obtain thus the content of oxalic acid.
2. according to claim 1 based on the method for Bt Transgenic Rice to the brown planthopper safety evaluatio after the agricultural chemicals intervention, it is characterized in that the dispenser spray-on process is the spray-on process consistent with the field, sprayer is the conical nozzle that a diameter 1mm is housed, pressure 45 psi, the simple and easy suitable device of flow 300mL/min.
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